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Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella)
Accepted Manuscript Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella) Hui-Jun Xu, Wei-Dan Jiang, Lin Feng, Yang Liu, Pei Wu, Jun Jiang, Sheng-Yao Kuang, Ling Tang, Wu-Neng Tang, Yong-An Zhang, Xiao-Qiu Zhou PII:
S1050-4648(16)30077-8
DOI:
10.1016/j.fsi.2016.02.033
Reference:
YFSIM 3845
To appear in:
Fish and Shellfish Immunology
Received Date: 14 December 2015 Revised Date:
22 February 2016
Accepted Date: 29 February 2016
Please cite this article as: Xu H-J, Jiang W-D, Feng L, Liu Y, Wu P, Jiang J, Kuang S-Y, Tang L, Tang W-N, Zhang Y-A, Zhou X-Q, Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella), Fish and Shellfish Immunology (2016), doi: 10.1016/ j.fsi.2016.02.033. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Dietary vitamin C deficiency depresses theMANUSCRIPT growth, head kidney and spleen immunity ACCEPTED
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and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK
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signaling in young grass carp (Ctenopharyngodon idella)
4 a,1
, Lin Feng
a,b,c
, Yang Liu a,b,c , Pei Wu
a,b,c
Hui-Jun Xu
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Kuang d, Ling Tang d, Wu-Neng Tang d, Yong-An Zhang e, Xiao-Qiu Zhou a,b,c,*
, Jun Jiang
a,b,c
, Sheng-Yao
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, Wei-Dan Jiang
a,b,c,1
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7 a
Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China
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b
Fish Nutrition and safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural
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University, Chengdu 611130, China
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c
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Agricultural University, Chengdu 611130, China
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d
Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China
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Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
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Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan
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* Co-corresponding authors. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130,
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Sichuan, China. E-mail: [email protected], [email protected] (X.-Q. Zhou).
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These two authors contributed to this work equally
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ACCEPTED MANUSCRIPT
Abstract:
This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin
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immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp
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(Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels
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of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge
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test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The
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results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg
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diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4
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(C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed
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antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related
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factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor
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(TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated
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pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12
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P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ),
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target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and
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spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head
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kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C
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supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide
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dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione
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S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2,
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Claudin-b, -c, -3c, -7a, -7b, B-cell lymphoma-2, inhibitor of apoptosis protein, NF-E2-related factor 2
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mRNA levels (P < 0.05), increased reactive oxygen species (ROS), malondialdehyde (MDA) and protein
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carbonyl contents (P < 0.05), and up-regulated Claudin-12, 15a, -15b, Fas ligand, mitogen-activated protein
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kinase kinase 6, p38 mitogen-activated protein kinase, B-cell lymphoma protein 2 associated X protein, ACCEPTED MANUSCRIPT
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apoptotic protease activating factor-1, caspase-3, -7, -8, -9, Kelch-like ECH-associating protein (Keap) 1a
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and Keap 1b mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila,
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suggesting that vitamin C deficiency could decrease fish head kidney and spleen structural integrity through
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depression of antioxidative ability, induction of apoptosis and disruption of tight junctional complexes. In
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addition, except the activities of ACP and MnSOD, and mRNA expression levels of TGF-β1, Occludin and
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MnSOD, the effect of vitamin C on fish head kidney, spleen and skin immunity and structural integrity other
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indicators model are similar under infection of A. hydrophila. Finally, the vitamin C requirement for the
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growth performance (PWG) of young grass carp was estimated to be 92.8 mg/kg diet. Meanwhile, the
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vitamin C requirement for against skin lesion morbidity of young grass carp was estimated to be 122.9
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mg/kg diet. In addition, based on the biochemical indices [immune indices (LA activity in the head kidney
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and C4 content in the spleen) and antioxidant indices (MDA content in the head kidney and ROS content in
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the spleen)] the vitamin C requirements for young grass carp were estimated to be 131.2, 137.5, 135.8 and
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129.8 mg/kg diet, respectively.
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Keywords: Vitamin C; Immunity; Structural integrity; Head kidney; Spleen; Young grass carp
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(Ctenopharyngodon idella)
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1. Introduction
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Vitamin C is an essential nutrient for the normal growth of fish [1]. It was reported that vitamin C
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deficiency could lead to spinal deformation in juvenile cobia (Rachycentron canadum) [2], and scoliosis,
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lordosis and caudal fin erosion in Japanese seabass (Lateolabrax japonicas) [3]. Meanwhile, study from our
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lab showed that vitamin C deficiency decreased growth performance in juvenile Jian carp (Cyprinus carpio
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var. Jian) [4]. Fish growth is often related to disease resistance [5], which often relies on their immunity [6].
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Up to now, limited studies demonstrated that vitamin C deficiency depressed serum lysozyme activity (LA)
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in juvenile cobia (Rachycentron canadum) [2]. Further investigation is required to study the effects of
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vitamin C on immunity and it possible regulation mechanism in fish.
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Fish immunity is related to the structure and function of its immune organs [7]. Skin is an important
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mucosa immune organ in zebrafish [8]. It was reported that vitamin C deficiency led to skin lesion in Nile
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tilapia (Oreochromis niloticus L.) [9]. Fish easily infected by A. hydrophila after skin lesion [10], and the
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infection ultimately spread to the lymphatic immune organs [11]. Head kidney and spleen are two important
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lymphatic immune organs in fish [12]. In fish, the immune function of head kidney, spleen and skin are
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related to antibacterial compounds and cytokines [13-15]. In human, cytokines could be regulated by the
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inhibitor of κBα/nuclear factor κB (IκBα/NF-κB) signaling pathway [16] and target of rapamycin/ribosomal
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protein S6 kinase 1 (TOR/S6K1) signaling pathway [17]. However, up to now, no study concerns the effects
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of vitamin C on antibacterial compounds, cytokines and the IκBα/NF-κB and TOR/S6K1 signaling pathways
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in the immune organs of fish. It was reported that vitamin C could enhance the proliferative response of
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lymphocytes in hen [18]. In fish, lymphocytes could secrete the humoral compounds (such as LA and C3)
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[19]. Meanwhile, it was reported that vitamin C could improve intestinal iron uptake in human [20]. Dietary
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iron could up-regulate the mRNA level of hepcidin in rat [21]. In addition, vitamin C could elevate red blood
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cell glutathione in human [22]. Rahman et al. [23] reported that glutathione could inhibit NF-κB signaling
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pathway activation in THP-1 cells. ACCEPTED Meanwhile, vitamin C could decrease the total plasma cholesterol MANUSCRIPT
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content in fish [24]. In human HUVECs cells, cholesterol could activate mTOR signaling pathway [25].
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Accordingly, there may be a possible relationship between vitamin C and humoral compounds, antimicrobial
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peptides, cytokines and IκBα/NF-κB and TOR/S6K1 signaling pathways in fish, which remains to be
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elucidated.
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In fish, the immunity is correlated with structural integrity of immune organ [26]. Structural integrity of
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fish immune organ is associated with organ cellular membrane integrity, which can be destructed by
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oxidative injury and cell apoptosis [27]. However, no study has addressed the effects of vitamin C on
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oxidative injury, apoptosis and their possible signaling pathway in the head kidney, spleen and skin of fish.
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Moreover, a previous study showed that oxidation injury could combat by non-enzymatic compounds and
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antioxidant enzymes [28]. Vitamin C is an important non-enzymatic compound in green-lipped mussels (P.
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viridis) [29]. It was reported that dietary vitamin C increased the vitamin C concentration of large yellow
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croaker (Pseudosciaena crocea) head kidney [30] and rainbow trout spleen [31], and antioxidant enzyme
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activities of juvenile cobia (Rachycentron canadum) serum [2] and Wuchang bream (Megalobrama
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amblycephala Yih) liver [32]. Antioxidant enzyme activities were partly dependent on antioxidant enzymes
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gene transcriptions [33], which were regulated by NF-E2-related factor 2/Kelch-like-ECH-associated protein
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1 (Nrf2/keap1) signaling pathway in fish [34]. However, no study has conducted the effects of vitamin C on
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skin vitamin C concentration and immune organs (such as head kidney, spleen and skin) antioxidant ability
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and related molecular mechanisms in fish. In mice, vitamin C could up-regulate the PPARα expression [35].
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Toyama et al. [36] reported that up-regulated PPARα could increase the antioxidant enzyme catalase (CAT)
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and superoxide dismutase (SOD) mRNA levels. In human, vitamin C could enhance the synthesis of nitric
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oxide (NO) [37]. Liu et al. [38] reported that NO could up-regulate Nrf2 gene expression in rat vascular
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smooth muscle cells. In addition, vitamin C could inhibit the production of monocytes tumor necrosis factor
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α (TNF-α) in human [39]. Wang et ACCEPTED al. [40] reported that TNF-α could activate apoptosis-related factor MANUSCRIPT
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caspase-8 in HEK293T cells. Accordingly, there may be a possible relationship between vitamin C and
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antioxidant enzymes, Nrf2/keap1 signaling pathway, and apoptosis and its possible signaling pathway in fish,
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which warrants further investigation. In fish, the structural integrity of immune organ is also associated with the tight junctional complexes
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(TJs) [Occludin, Claudins and zonula occludens (ZO)] in intercellular, which are regulated by myosin light
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chain kinase (MLCK) [41, 42]. However, no study has focused on the effects of vitamin C on TJs and its
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possible signaling pathway in fish. In pig, vitamin C could decrease the plasma cortisol content [43].
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Chasiotis and Kelly [44] demonstrated that cortisol could down-regulate the expression of ZO-1 and
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Claudin-c in the gill of goldfish. It was reported that vitamin C could suppress p38 mitogen-activated protein
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kinase (p38 MAPK) in human [45]. In mice, inhibition of p38 MAPK could prevent MLCK expression [46].
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Therefore, these data suggest a possible correlation between vitamin C and TJs and its possible signaling
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pathway in fish, which remains to be elucidated.
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Grass carp is widely cultivated in the world [47]. The commercial rearing of grass carp relies heavily
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on the use of formulated feed, which is based on the complete information of the nutrient requirement for
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this species [48]. In fish, growth stages include juvenile, young, adult and so on [49]. Up to now, the
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requirements for vitamin C only investigated the juvenile stage and adult stage of grass carp, and mainly
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based on the growth performance. However, vitamin C requirements of fish may vary with different growth
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stage and different indices. The vitamin C requirement in juvenile stage grass carp [50] is higher than that in
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adult stage grass carp [51]. Meanwhile, our lab study has found that the riboflavin requirement for young
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grass carp based on gill LA was higher than that based on percent weight gain (PWG) [52]. Therefore, it is
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valuable to investigate the dietary vitamin C requirements of young grass carp based on growth and other
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indices.
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Here we firstly hypothesis that vitamin C deficiency could depress the immunity of fish head kidney ACCEPTED MANUSCRIPT
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and spleen by aggravation inflammatory response and disturbed the structural integrity. To test this
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hypothesis, we conducted the challenged test with A. hydrophila after feeding trial. Furthermore, we were
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for the first time to test the hypothesis that vitamin C deficiency could aggravate inflammation which was
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related to NF-κB and TOR signaling pathways in the head kidney and spleen of fish. And we were also for
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the first time to test the hypothesis that vitamin C deficiency could disrupt the structural integrity partly
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related the disruption of the integrity of cellular structure and cell-cell TJs these modulated by Nrf2 and
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MLCK signaling in the head kidney and spleen of fish. Meanwhile, the effects of vitamin C on the skin
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immunity are also estimated. In addition, the vitamin C requirements of young grass carp were also
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determined in this study, which may provide partial theoretical evidence for the commercial feed production
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of grass carp.
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2. Materials and methods
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2.1. Experimental diets preparation
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Formulation of the basal diet is shown in Table 1. Fish meal, casein and soybean protein concentrate
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were used as dietary protein sources. Fish oil and soybean oil were used as dietary lipid sources. The dietary
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protein level was fixed at 300 g/kg diet, which was reported to be optimum for the growth of grass carp, as
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described by Khan et al. [47]. Vitamin C was added to the basal diet to provide graded concentrations of 0
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(un-supplemented), 45, 90, 135, 180 and 225 mg vitamin C/kg diet, and the amount of corn starch was
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reduced to compensate, according to our lab study [4]. The vitamin C concentrations of the six diets were
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2.9 (un-supplemented), 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet, determined by the method of Wan et
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al. [53]. After being prepared completely, the diets were stored at -20 °C according to Liu et al. [4].
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(Table 1 inserted here) 2.2. Feeding trial 7
The procedures used in this study were approved by the University of Sichuan Agricultural Animal ACCEPTED MANUSCRIPT
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Care Advisory Committee. Grass carp were obtained from fisheries (Sichuan, China). Prior to the
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experiment, fish were acclimatized to the experimental environment for 2 weeks. The basal diet
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(un-supplemented with vitamin C diet) was fed to all fish during the conditioning period, according to Zhou
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et al. [2]. Then, 540 fish (mean weight 264.37 ± 0.66 g) were randomly assigned to 18 experimental cages
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(1.4 L × 1.4 W × 1.4 H m), resulting in 30 fish per cage. Each cage was equipped with a disc of 100 cm
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diameter in the bottom to collect the uneaten feed, according to Wu et al. [54]. Fish were fed with their
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respective diets to apparent satiation four times per day, according to our lab study [55]. After feeding 30
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min, uneaten feed was collected, dried and weighed to calculate the feed intake (FI), according to our lab
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study [55]. During the experimental period, dissolved oxygen was maintained higher than 6.0 mg/L
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throughout the experimental period. The water temperature was averaged at 28 ± 2 °C, pH value was
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maintained at 7.0 ± 0.2. The experimental units were under natural light and dark cycle, according to our lab
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study [56]. The feeding trial lasted for 10 weeks.
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At the initiation and termination of the feeding trial, fish from each cage were weighed for calculate
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PWG. Eighteen fish from each treatment were anaesthetized in a benzocaine bath as described by Affonso et
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al. [57], then the head kidney and spleen of fish were quickly removed and weighed for calculate the index
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of the head kidney and spleen.
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2.3. Challenge test
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After a 10 weeks feeding trial, a challenge test was conducted to study the effect of dietary vitamin C
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on the immune responses of young grass carp. A. hydrophila strain was obtained from College of Veterinary
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Medicine, Sichuan Agricultural University, China. Fifteen fish with a similar body weight collected from
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each treatment group were moved to labeled cages according to Liu et al. [58]. Fish were infected by
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intraperitoneal injection with A. hydrophila at a concentration of 2.5×108 CFU 1ml/fish, which is similar to 8
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Chen et al. [59]. During the infectious trial, the experimental conditions were the same as those in the ACCEPTED MANUSCRIPT
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feeding trial. The challenge test was conducted for 14 days according to Nya and Austin [60]. Dead fish
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were removed and counted from each tank daily.
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2.4. Sample collection and analysis after challenge test At the end of the challenge test, fish were anaesthetized in a benzocaine bath as described by Affonso et
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al. [57]. To evaluate the severity of skin lesion, a scoring system and skin lesion morbidity was specifically
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checked and calculated according to the methods described by Séguin et al. [61] and Steenland and
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Armstrong [62], respectively. Then sacrificed, quickly obtained the head kidney, spleen and skin and frozen
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in liquid nitrogen, and then stored at -80 °C until analysis as described by Huang et al. [63]. The head kidney,
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spleen and skin samples were homogenized on ice in 10 volumes (w/v) of ice-cold physiological saline and
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centrifuged at 6000 g at 4ºC for 20 min, then the collected supernatant was stored for the related parameters
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analysis as described by Safari et al. [64]. The vitamin C concentrations of the head kidney, spleen and skin
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were determined as described by Wan et al. [53]. LA and acid phosphatase (ACP) activities were assayed as
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Wu et al. [65]. The contents of C3 and C4 were assayed according to Welker et al. [66]. The reactive oxygen
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species (ROS) content was determined according to Yang et al. [67]. The contents of malondialdehyde
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(MDA), protein carbonyl (PC) and glutathione (GSH) were determined according to Ji et al. [68]. The CAT
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activity, anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) capacity were measured as described
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by Kuang et al. [69]. The total superoxide dismutase (SOD) and MnSOD activities were assayed as
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described by Durrant et al. [70]. The CuZnSOD, glutathione peroxidase (GPx), CAT, glutathione
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S-transferase (GST) and glutathione reductase (GR) activities were assayed as described by Wen et al. [57],
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Zhang et al. [71], Deng et al. [72], Lushchak et al.[73], and Lora et al. [74], respectively.
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Total RNA samples were isolated from the head kidney, spleen and skin using RNAiso Plus Kit (Takara,
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Dalian, China), then quantity and quality were assessed using spectrophotometry, as described by Luo et al. 9
[55]. The fist-strand cDNA was synthesized using a PrimeScripte RT reagent Kit, according to the ACCEPTED MANUSCRIPT
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manufacturer’s instructions as described by Gan et al. [75]. Specific primers were designed according to the
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sequences cloned in our laboratory or the published sequences of grass carp (Table 2) for quantitative
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real-time PCR. To verify the specificity and purity of all PCR products, melt curve analysis was performed
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after amplification. According to the results of our preliminary experiment concerning the evaluation of
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internal control genes (data not shown), β-actin was used as a reference gene to normalize cDNA loading.
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The mRNA levels of all the genes were performed on the CFX96TM Real-Time PCR Detection System
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(Bio-Rad, Laboratories, Inc.) according to standard protocols of the primers. The gene expression results
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were analyzed using the 2−∆∆CT method according to Luo et al. [55].
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(Table 2 inserted here) 2.5. Calculations and statistical analysis
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Data on initial body weight (IBW), final body weight (FBW) and feed intake (FI) were used to
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calculate PWG and feed efficiency (FE), as described by Wen et al. [56], and specific growth rate (SGR), as
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described by Deng et al. [76]. The weight of head kidney and spleen were used to calculate the index of head
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kidney and spleen, respectively, as described by Chen et al. [59]. Survival rate was calculated as described
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by Wu et al. [65].
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PWG =100 × [FBW (g/fish) – IBW (g/fish)]/IBW (g/fish);
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FE = 100 × [FBW (g/fish) – IBW (g/fish)]/FI (g/fish);
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SGR = 100 × Ln [final body weight (g)/initial body weight (g)]/feeding period (day)
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Head kidney index = (g head kidney weight/g body weight)/100;
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Spleen index = (g spleen weight/g body weight)/100;
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Survival rate (%) = (final fish number)/(initial fish number)/100.
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All data were subjected to one-way analysis of variance followed by Duncan’s multiple range tests to
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determine significant differences among treatment groups using the software SPSS 18.0 (SPSS Inc., Chicago, ACCEPTED MANUSCRIPT
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IL, USA) at a level of P < 0.05, as described by Gan et al. [75]. The results are presented as the means ± SD.
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Broken-line model was used to estimate the optimal dietary level of vitamin C for young grass carp
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according to Liu et al. [4].
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3. Results
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3.1. Growth performance, head kidney and spleen growth, head kidney, spleen and skin vitamin C
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concentration, and survival rate and skin lesion morbidity after infection with A. hydrophila
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As shown in Table 3, fish FBW, PWG, SGR, FI and FE were all significantly improved with dietary
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vitamin C levels up to 89.1mg/kg diet (P ˂ 0.05), and then plateaued. The head kidney weight, index and
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vitamin C concentration, spleen weight, index and vitamin C concentration, and skin vitamin C
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concentration were significantly increased with dietary vitamin C levels up to 89.1, 44.2, 179.4, 89.1, 44.2,
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89.1 and 133.8 mg/kg diet (P < 0.05), respectively, and then plateaued. Survival rate (Fig. 1A) after infection
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with A. hydrophila was significantly increased with dietary vitamin C levels up to 44.2 mg/kg diet (P < 0.05),
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and then plateaued. The skin lesion morbidity (Fig. 1B) after infection with A. hydrophila was significantly
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decreased with dietary vitamin C levels up to 133.8 mg/kg diet (P < 0.05), and then plateaued. Compared
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with optimal vitamin C supplementation, deficiency of vitamin C led to an obvious skin lesion symptom
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(Fig. 2) after infection with A. hydrophila in young grass carp.
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(Table 3 inserted here)
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(Fig. 1 inserted here)
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(Fig. 2 inserted here)
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3.2. The immune response-related parameters in the head kidney and spleen after infection with A.
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hydrophila
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The LA and ACP activities, C3 and C4 contents in the head kidney and spleen of young grass carp are 11
presented in Table 4. In the head kidney, LA and ACP activities, and C3 and C4 contents were significantly ACCEPTED MANUSCRIPT
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increased with dietary vitamin C levels up to 133.8, 179.4, 133.8 and 133.8 mg/kg diet (P < 0.05),
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respectively, and then plateaued. In the spleen, LA and ACP activities, C3 and C4 contents were
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significantly increased with dietary vitamin C levels up to 44.2, 44.2, 89.1 and 133.8 mg/kg diet (P < 0.05),
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respectively, and then plateaued. The mRNA levels of antimicrobial peptides in the head kidney and spleen
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of young grass carp are displayed in Fig. 3. In the head kidney, the liver expressed antimicrobial peptide 2A
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(LEAP-2A), liver expressed antimicrobial peptide 2B (LEAP-2B) and hepcidin mRNA levels were
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up-regulated with dietary vitamin C levels up to 89.1, 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, and
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then plateaued. The mRNA level of β-defensin was up-regulated with dietary vitamin C levels up to 133.8
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mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05). In the spleen, the mRNA levels of LEAP-2A,
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LEAP-2B and hepcidin were up-regulated with dietary vitamin C levels up to 44.2, 89.1 and 44.2 mg/kg diet
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(P < 0.05), respectively, and then plateaued. The β-defensin mRNA level was increased with dietary vitamin
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C levels up to 133.8 mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05).
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(Table 4 inserted here)
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(Fig. 3 inserted here)
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3.3. The inflammatory response-related parameters in the head kidney and spleen after infection with A.
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hydrophila
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The mRNA levels of anti-inflammatory cytokines, pro-inflammatory cytokines, and inflammatory
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cytokines-related signaling molecules in the head kidney and spleen of young grass carp are displayed in Fig.
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4, Fig.5 and Fig. 6, respectively. In the head kidney, the interleukin 4/13A (IL-4/13A), interleukin 4/13B
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(IL-4/13B), interleukin 10 (IL-10), interleukin 11 (IL-11), transforming growth factor β1 (TGF-β1),
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transforming growth factor β2 (TGF-β2), IκBα and 4EB-P1 mRNA levels were up-regulated with dietary
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vitamin C levels up to 89.1, 89.1, 89.1, 44.2, 89.1, 89.1, 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, 12
and then plateaued. The mRNA levelsACCEPTED of TNF-α, interferon γ2 (IFN-γ2), interleukin 1β (IL-1β), interleukin 6 MANUSCRIPT
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(IL-6), interleukin (IL-8), interleukin 17D (IL-17D), NF-κB p65, IKKα, IKKβ, IKKγ, TOR and S6K1 were
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down-regulated with dietary vitamin C levels up to 89.1, 179.4, 89.1, 44.2, 133.8, 89.1, 89.1, 44.2, 89.1,
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133.8, 44.2, and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA levels of
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interleukin 12 P40 (IL-12 P40) and interleukin 15 (IL-15) were down-regulated with dietary vitamin C
274
levels up to 179.4 and 133.8 mg/kg diet (P < 0.05), respectively, and increased thereafter (P < 0.05).
275
However, dietary vitamin C had no effect on the mRNA levels of interleukin 12 P35 (IL-12 P35), NF-κB
276
p52 and 4EB-P2 in the head kidney (P > 0.05). In the spleen, the mRNA levels of IL-4/13A, IL-10, TGF-β1,
277
TGF-β2, IκBα and 4EB-P1 were up-regulated with dietary vitamin C levels up to 44.2, 89.1, 89.1, 179.4,
278
89.1 and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA levels of TNF-α, IFN-γ2,
279
IL-1β, IL-6, IL-8, IL-12 P35, IL-12 P40, IL-15, IL-17D, NF-κB p65, IKKα, IKKγ, TOR and S6K1 were
280
down-regulated with dietary vitamin C levels up to 89.1, 89.1, 89.1, 89.1, 179.4, 89.1, 89.1, 89.1, 89.1, 89.1,
281
133.8, 133.8, 89.1 and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA level of
282
IKKβ was decreased with dietary vitamin C level (P < 0.05). The IL-11 mRNA level was increased with
283
dietary vitamin C levels up to 133.8 mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05). However,
284
dietary vitamin C had no effect on the mRNA levels of IL-4/13B, NF-κB p52 and 4EB-P2 in the spleen (P >
285
0.05).
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(Fig. 4 inserted here)
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(Fig. 5 inserted here)
288
(Fig. 6 inserted here)
289
3.4. Head kidney and spleen structural integrity after infection of A. hydrophila
290
3.4.1. Antioxidant-related parameters in the head kidney and spleen
291
The ROS, MDA, PC and GSH contents, ASA, AHR and antioxidant enzyme activities in the head 13
kidney and spleen are showed in Table 5. In the MANUSCRIPT head kidney, the ROS, MDA and PC contents were ACCEPTED
293
significantly decreased with dietary vitamin C levels up to 89.1, 133.8 and 44.2 mg/kg diet (P < 0.05),
294
respectively, and then plateaued. The AHR, ASA, T-SOD, CuZnSOD, MnSOD, CAT, GPx, GST and GR
295
activities and GSH content were significantly increased with dietary vitamin C levels up to 89.1, 133.8, 89.1,
296
89.1, 44.2, 133.8, 44.2, 89.1, 89.1 and 179.4 mg/kg diet (P < 0.05), respectively, and then plateaued. In the
297
spleen, the ROS, MDA and PC contents were significantly decreased with dietary vitamin C levels up to
298
133.8, 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, and then plateaued. The AHR, ASA, T-SOD,
299
CuZnSOD, MnSOD, CAT, GPx, GST and GR activities and GSH content were increased with dietary
300
vitamin C levels up to 179.4, 44.2, 133.8, 179.4, 133.8, 89.1, 179.4, 44.2, 44.2 and 179.4 mg/kg diet (P <
301
0.05), respectively, and then plateaued.
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The mRNA levels of antioxidant enzymes, and Nrf2, Keap1a and Keap1b in the head kidney and spleen
303
are showed in Fig. 7 and Fig. 8, respectively. In the head kidney, the CuZnSOD, MnSOD, CAT, GPx1a,
304
GPx1b, GPx4a, GPx4b, GSTR, GSTP1, GSTP2, GR and Nrf2 mRNA levels were up-regulated with dietary
305
vitamin C levels up to 89.1, 89.1, 89.1, 89.1, 44.2, 89.1, 89.1, 44.2, 89.1, 89.1, 89.1 and 44.2 mg/kg diet (P <
306
0.05), respectively, and then plateaued. The mRNA levels of Keap1a and Keap1b were significantly
307
down-regulated with dietary vitamin C levels up to 133.8 and 89.1 mg/kg diet (P < 0.05), respectively, and
308
then plateaued. However, dietary vitamin C had no effect on the mRNA levels of GSTO1 and GSTO2 in the
309
head kidney (P > 0.05). In the spleen, the CuZnSOD, MnSOD, CAT, GPx1a, GPx1b, GPx4b, GSTR, GSTP1,
310
GSTP2, GR and Nrf2 mRNA levels were up-regulated with dietary vitamin C levels up to 89.1, 89.1, 89.1,
311
89.1, 44.2, 44.2, 89.1, 44.2, 89.1, 89.1 and 44.2 mg/kg diet (P < 0.05), and then plateaued. The Keap1a and
312
Keap1b mRNA levels were all significantly down-regulated with dietary vitamin C level up to 89.1 mg/kg
313
diet (P < 0.05), and then plateaued. The mRNA level of GPx4a was up-regulated with dietary vitamin C
314
levels up to 133.8 mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05). However, dietary vitamin C
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had no effect on the mRNA levels of GSTO1 and GSTO2 in the spleen (P > 0.05). ACCEPTED MANUSCRIPT
316
(Table 5 inserted here)
317
(Fig. 7 inserted here)
318
(Fig. 8 inserted here) 3.4. 2. Apoptosis-related parameters mRNA levels in the head kidney and spleen
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The effects of dietary vitamin C on apoptosis-related gene expression in the head kidney and spleen are
321
showed in Fig. 9. In the head kidney, the mRNA levels of B-cell lymphoma-2 (Bcl-2) and inhibitor of
322
apoptosis protein (IAP) were up-regulated with dietary vitamin C levels up to 133.8 and 89.1 mg/kg diet (P
323
< 0.05), respectively, and then plateaued. The mRNA levels of Fas ligand (FasL), mitogen-activated protein
324
kinase kinase 6 (MPKK 6), p38 MAPK, B-cell lymphoma protein 2 associated X protein (Bax), apoptotic
325
protease activating factor-1 (Apaf-1), caspase-3, -7, -8 and caspase-9 were down-regulated with dietary
326
vitamin C levels up to 89.1, 89.1, 133.8, 89.1, 133.8, 89.1, 89.1, 133.8 and 44.2 mg/kg diet (P < 0.05),
327
respectively, and then plateaued. However, dietary vitamin C had no effect on the mRNA levels of c-Jun
328
N-terminal kinases (JNK), myeloid cell leukemia-1 (Mcl-1) and caspase-2 in the head kidney (P > 0.05). In
329
the spleen, the mRNA levels of Bcl-2 and IAP were all up-regulated with dietary vitamin C levels up to
330
89.1mg/kg diet (P < 0.05), and then plateaued. The mRNA levels of FasL, MAPKK 6, p38 MAPK, Bax,
331
Apaf-1, caspase-2, -3, -7, -8 and caspase-9 were down-regulated with dietary vitamin C levels up to 89.1,
332
133.8, 89.1, 89.1, 44.2, 44.2, 89.1, 133.8, 133.8 and 133.8 mg/kg diet (P < 0.05), respectively, and then
333
plateaued. However, dietary vitamin C had no effect on the mRNA levels of JNK and Mcl-1 in the spleen
334
(P > 0.05).
335 336 337
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(Fig. 9 inserted here) 3.4.3. TJs and MLCK mRNA levels in the head kidney and spleen The effects of dietary vitamin C on mRNA levels of head kidney and spleen TJs and MLCK are shown 15
in Fig. 10. In the head kidney, the mRNA levels ofMANUSCRIPT ZO-1, ZO-2, Claudin-b, -c, -3c and Claudin-7a were ACCEPTED
339
up-regulated with dietary vitamin C levels up to 44.2, 89.1, 44.2, 44.2, 89.1 and 89.1 mg/kg diet (P < 0.05),
340
respectively, and then plateaued. The mRNA levels of Claudin-12, -15a, -15b and MLCK were
341
down-regulated with dietary vitamin C levels up to 89.1, 44.2, 89.1 and 44.2 mg/kg diet (P < 0.05),
342
respectively, and then plateaued. The mRNA level of Claudin7b was increased with dietary vitamin C levels
343
up to 133.8 mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05). However, dietary vitamin C had no
344
effect on the mRNA level of Occludin in the head kidney (P > 0.05). In the spleen, the mRNA levels of
345
ZO-1, ZO-2, Claudin-b, -c, -3c and Claudin-7b were up-regulated with dietary vitamin C levels up to 44.2,
346
89.1, 89.1, 44.2, 44.2 and 44.2 mg/kg diet (P < 0.05), respectively, and then plateaued. The Claudin-12, -15a
347
and Claudin-15b mRNA levels were down-regulated with dietary vitamin C levels up to 44.2, 89.1 and
348
133.8 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA level of Claudin-7a was
349
up-regulated with dietary vitamin C levels up to 133.8 mg/kg diet (P < 0.05), and down-regulated thereafter
350
(P < 0.05). However, dietary vitamin C had no effect on the mRNA levels of Occludin and MLCK in the
351
spleen (P > 0.05).
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(Fig. 10 inserted here)
3.5. Skin immune response-related parameters and structural integrity-related parameters
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As shown in Table 6, the LA activity and C3 and C4 contents in the skin were increased with dietary
355
vitamin C levels up to 89.1, 44.2 and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. However,
356
dietary vitamin C had no effect on the ACP activity in the skin (P > 0.05). The mRNA levels of cytokines
357
and related signaling molecules in the skin were shown in Fig. 11. The mRNA levels of IL-10 and IκBα
358
were up-regulated with dietary vitamin C levels up to 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, and
359
then plateaued. The mRNA levels of TNF-α, IFN-γ2, IL-1β, IL-8, NF-κB p65, IKKα, IKKβ, IKKγ, TOR and
360
S6K1 were down-regulated with dietary vitamin C levels up to 89.1, 89.1, 44.2, 44.2, 133.8, 89.1, 133.8,
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89.1, 44.2 and 44.2 mg/kg diet (P < 0.05), respectively, and then plateaued. However, dietary vitamin C had ACCEPTED MANUSCRIPT
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no effect on the TGF-β1 mRNA level in the skin (P > 0.05). (Table 6 inserted here)
364
(Fig. 11 inserted here)
365
The activities and mRNA levels of antioxidant enzymes, and Nrf2, Keap1a and Keap1b in the skin
366
were shown in Table 7 and Fig. 12, respectively. The ROS, MDA and PC contents were significantly
367
decreased with dietary vitamin C levels up to 133.8, 133.8 and 179.4 mg/kg diet (P < 0.05), respectively,
368
and then plateaued. The AHR, ASA, T-SOD, CuZnSOD, CAT, GPx, GST and GR activities and GSH
369
content were significantly increased with dietary vitamin C levels up to 89.1, 89.1, 133.8, 133.8, 44.2, 133.8,
370
44.2, 44.2 and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA levels of CuZnSOD,
371
CAT, GPx1a, GSTR, GR and Nrf2 were up-regulated with dietary vitamin C levels up to 44.2, 44.2, 89.1,
372
44.2, 44.2 and 44.2 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA levels of The
373
Keap1a and Keap1b mRNA levels were down-regulated with dietary vitamin C level up to 133.8 and 44.2
374
mg/kg diet (P < 0.05), and then plateaued. As shown in Fig. 13, the mRNA levels of caspase-3, -8 and
375
caspase-9 were down-regulated with dietary vitamin C levels up to 89.1, 44.2 and 44.2 mg/kg diet (P < 0.05),
376
respectively, and then plateaued. As shown in Fig. 14, the mRNA levels of Occludin, ZO-1, Claudin-b, -c
377
and Claudin-3c were up-regulated with dietary vitamin C levels up to 89.1, 44.2, 89.1, 89.1 and 89.1 mg/kg
378
diet (P < 0.05), respectively, and then plateaued. The mRNA levels of Claudin-12 and Claudin-15a were
379
down-regulated with dietary vitamin C levels up to 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, and
380
then plateaued.
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(Table 7 inserted here)
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(Fig. 12 inserted here)
383
(Fig. 13 inserted here) 17
384
ACCEPTED MANUSCRIPT
(Fig. 14 inserted here)
385
4. Discussion
386
4.1. Vitamin C deficiency decreased growth performance and survival rate after infection with A. hydrophila
387
in fish The present study showed that, compared with optimal vitamin C supplementation, dietary vitamin C
389
deficiency resulted in poor PWG, SGR, FI, FE, and the head kidney and spleen weight, index, and vitamin C
390
concentrations in of young grass carp. Fish growth and immune organ (such as head kidney and spleen) size
391
are often related to its disease resistance [5, 77, 78], which could be reflect by survival rate after challenging
392
[79]. Our data indicated that, compared with appropriate dietary vitamin C supplementation, vitamin C
393
deficiency significantly decreased the survival rate of young grass carp under infection of A. hydrophila,
394
suggesting that vitamin C deficiency could decrease disease resistance in young grass carp. Meanwhile, we
395
observed that vitamin C deficiency caused the highest the skin lesion morbidity (53.3%) of young grass carp
396
after infection of A. hydrophila, whereas optimal vitamin C decreased the skin lesion morbidity (13.3%).
397
Based on the broken-line analysis of PWG (Fig. 15A) and to against skin lesion morbidity (Y = - 0.2847X +
398
49.215, R2 = 0.893) the dietary vitamin C requirements of young grass carp were estimated to be 92.8 and
399
122.9 mg/kg diet, respectively. Fish disease resistance is related to the immunity of head kidney and spleen
400
[66]. Thus, we further investigated the effects of vitamin C on the head kidney and spleen immunity of
401
young grass carp as well as its potential mechanisms.
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(Fig. 15 inserted here)
403
4.2. Vitamin C deficiency decreased the head kidney and spleen immunity in fish under infection of A.
404
hydrophila
405
The immunity of fish is largely rely on humoral components like LA, ACP, C3, C4 and antimicrobial
406
peptides like LEAP-2 and hepcidin [80]. In this study, vitamin C deficiency decreased the LA and ACP 18
activities, C3 and C4 contents and down-regulated LEAP-2B, hepcidin and β-defensin mRNA ACCEPTED LEAP-2A, MANUSCRIPT
408
levels in the head kidney and spleen of young grass carp, but which were increased with the optimal dietary
409
vitamin C supplementation under infection of A. hydrophila, suggesting that vitamin C deficiency depressed
410
the immunity of fish. In addition, immunity is also associated with inflammation, which is primarily
411
mediated by inflammatory cytokines in fish [81]. We then examined the effects of vitamin C on
412
inflammation in the head kidney and spleen of young grass carp.
413
4.3. Vitamin C deficiency aggravated inflammation and the potential molecular mechanisms in the head
414
kidney and spleen of fish under infection of A. hydrophila
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Studies have shown that up-regulation of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-8
416
in vaccinated pink (Oncorhynchus gorbuscha) [82], and down-regulation of anti-inflammatory cytokines,
417
such as IL-10 and TGF-β in mice [83], initiated the inflammatory process. In this study, compared with
418
optimal vitamin C supplementation, vitamin C deficiency resulted in up-regulating pro-inflammatory
419
cytokines TNF-α, IFN-γ2, IL-1β, IL-6, IL-8, IL-12 P40, IL-15 and IL-17D mRNA levels in the head kidney
420
and spleen, and IL-12 P35 mRNA level in the spleen, and down-regulating anti-inflammatory cytokines
421
IL-4/13A, IL-10, IL-11, TGF-β1 and TGF-β2 mRNA levels in the head kidney and spleen, and IL4/13B
422
mRNA level in the head kidney of young grass carp under infection of A. hydrophila. However, vitamin C
423
deficiency had no effect on the mRNA level of IL-12 P35 in the head kidney and IL4/13B mRNA level in
424
the spleen of young grass carp. The un-changed mRNA level of IL-12 P35 in the head kidney and IL4/13B
425
mRNA level in the spleen might be partially explained by them had distinct expression pattern in fish
426
different tissue. The IL-12 P35 expression level in the head kidney is lower than that of in the spleen of
427
Atlantic salmon [84], while, the IL4/13B shows very low constitutive expression in the spleen than that of in
428
the head kidney of zebrafish [85].
429
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In rat, NF-κB is clearly one of the most important regulators of pro-inflammatory gene expression [86]. 19
In multiple myeloma cells, NF-κB could be activate by two distinct mechanisms: the classical NF-κB ACCEPTED MANUSCRIPT
431
pathway and alternative NF-κB pathway, and NF-κB p65 and NF-κB p52 reflect the activities of those two
432
pathways, respectively [87]. It was reported that up-regulated NF-κB p65 expression could up-regulate the
433
mRNA levels of pro-inflammatory cytokines IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts [16],
434
and down-regulate the mRNA levels of anti-inflammatory cytokines IL-4 in human [88] and IL-10 and
435
TGF-β in mouse [89]. In this study, compared with optimal vitamin C supplementation, vitamin C
436
deficiency up-regulated NF-κB p65 mRNA level, but had no impact on mRNA level of NF-κB p52 in the
437
head kidney and spleen of young grass carp under infection of A. hydrophila. The no significant impact in
438
the NF-κB p52 mRNA expression in present study may be attributed to the activation of classical NF-κB
439
pathway (NF-κB p65), rather than alternative NF-κB pathway (NF-κB p52). However, further investigation
440
should be conducted to support our hypothesis. Correlation analysis indicated that pro-inflammatory
441
cytokines IL-6, IL-8, IL-12 P40, IL-15 and IL-17D mRNA levels were positively related to NF-κB p65
442
mRNA level, whereas anti-inflammatory cytokines IL-4/13A, IL-10, IL-11, TGF-β1 and TGF-β2 mRNA
443
levels were negatively related to NF-κB p65 mRNA level in the head kidney and spleen of young grass carp
444
(Table 8). These results indicated that vitamin C deficiency up-regulated those pro-inflammatory cytokines
445
mRNA levels and down-regulated those anti-inflammatory cytokines mRNA levels may be partly by
446
up-regulating NF-κB p65 mRNA level (rather than NF-κB p52) in the head kidney and spleen of fish. In
447
addition, IκB was an NF-κB p65-binding protein, which prevents NF-κB p65 translocation to the nucleus in
448
RAW 264.7 cells [90]. It was reported that down-regulation IκBα could promote the nuclear translocation of
449
NF-κB p65 in mice [91]. In this study, compared with optimal vitamin C supplementation, deficiency of
450
vitamin C decreased IκBα mRNA level in the head kidney and spleen of young grass carp under infection of
451
A. hydrophila. Correlation analysis indicated that NF-κB p65 mRNA level was negatively correlated with
452
IκBα mRNA level in the head kidney and spleen of young grass carp (Table 8), indicating that vitamin C
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20
deficiency up-regulated NF-κB p65 nuclear translocation may be partly ascribe to down-regulate IκBα gene ACCEPTED MANUSCRIPT
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expression in fish. Besides, it was reported that IκB kinase (IKK) (including IKKα, IKKβ and IKKγ three
455
subunits) is a key factor in NF-κB activation through regulation phosphorylating IκBα in HeLa cells [92]. In
456
this study, compared with optimal vitamin C supplementation, vitamin C deficiency significantly
457
up-regulated IKKα, IKKβ and IKKγ mRNA levels in the head kidney and spleen of young grass carp under
458
infection of A. hydrophila. Correlation analysis indicated that mRNA level of IκBα was negatively related to
459
IKKα, IKKβ and IKKγ mRNA levels in the head kidney and spleen of grass carp (Table 8). These data
460
demonstrated that vitamin C deficiency down-regulated IκBα mRNA expression may be partly by
461
up-regulating IKK (IKKα, IKKβ and IKKγ) mRNA levels in fish.
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In mouse microglial cells, TOR is a serin/threonin protein kinase with a central role in the regulation of
463
cytokines [93]. S6K1 and 4E-BP are downstream molecules of mTOR in human [94]. In Drosophila
464
Schneider 2 cells, TOR could up-regulate S6K1 and down-regulate 4E-BP expression [95]. It was reported
465
that down-regulated TOR could down-regulate the mRNA levels of pro-inflammatory cytokines IL-1β and
466
TNF-α in human [96]. In this study compared with optimal vitamin C supplementation, vitamin C deficiency
467
up-regulated TOR mRNA level in the head kidney and spleen of young grass carp under infection of A.
468
hydrophila. Correlation analysis showed that mRNA levels of pro-inflammatory cytokines TNF-α, IFN-γ2
469
and IL-1β were positively related to TOR in the head kidney and spleen of young grass carp (Table 8),
470
suggesting that vitamin C down-regulated pro-inflammatory cytokines TNF-α, IFN-γ2 and IL-1β mRNA
471
levels may be partly through up-regulating of TOR mRNA transcript abundance in fish. However, vitamin C
472
deficiency down-regulated 4E-BP1 mRNA level, whereas had no impact on the mRNA level of 4E-BP2 in
473
the head kidney and spleen of young grass carp under infection of A. hydrophila. The un-changed mRNA
474
level of 4E-BP2 might be partially explained by p38 MAPK. It was reported that, activation p38 MAPK
475
could down-regulate the expression of 4E-BP1 but have no effect on the expression of 4E-BP2 in human
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476
[97]. In the present study, vitamin C ACCEPTED deficiency up-regulated the p38 MAPK expression level in the head MANUSCRIPT
477
kidney and spleen of young grass carp under infection of A. hydrophila. However, further investigation
478
should be conducted to support our hypothesis. (Table 8 inserted here)
480
As stated above, our data firstly together corroborate the idea that, compared with optimum vitamin C
481
supplementation, vitamin C deficiency aggravated inflammation by up-regulation of pro-inflammatory
482
cytokines [IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15 and IL-17D] and down-regulation of
483
anti-inflammatory cytokines [IL-4/13A, IL-4/13B (only in head kidney), IL-10, IL-11 and TGF-β1] mRNA
484
levels may be involved in NF-κB p65 (rather than NF-κB p52)/IκBα/IKK signaling pathway of fish under
485
infection of A. hydrophila. Meanwhile, vitamin C deficiency up-regulated pro-inflammatory cytokines
486
TNF-α, IFN-γ2 and IL-1β mRNA levels may, in part, due to activate the TOR/S6K1/4E-BP1 (rather than
487
4E-BP2) signaling pathway in the head kidney and spleen of fish under infection of A. hydrophila. In
488
addition, the immunity of immune organs relies on their structural integrity in mice [98]. Structural integrity
489
of fish immune organ is associated with organ cellular membrane integrity, which can be destructed by
490
oxidative injury and cell apoptosis [27]. Thus we further investigated the effects of vitamin C on oxidative
491
injury and cell apoptosis in the head kidney and spleen of young grass carp.
492
4.4. Vitamin C deficiency induced oxidative injury, decreased antioxidant capacity and aggravated cell
493
apoptosis in the head kidney and spleen of fish under infection of A. hydrophila
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The major immune organs, head kidney and spleen, contain various immune cells [99], which could
495
generate reactive oxygen species (ROS) [100]. However, excessive ROS (mainly including superoxide
496
radical and hydroxyl radical) could lead to oxidative injury to lipids and proteins in rat [101]. In this study,
497
compared with optimal vitamin C supplementation, vitamin C deficiency increased ROS, MDA and PC
498
contents, and decreased anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) activities in the head 22
kidney and spleen of young grass carp under infection of A. hydrophila. In fish, ROS can be removed by ACCEPTED MANUSCRIPT
500
enzymatic antioxidants like SOD, CAT and glutathione-dependent enzymes and non-enzymatic antioxidant
501
GSH [102]. Our results clearly showed that compared with optimal vitamin C supplementation, vitamin C
502
deficiency decreased the T-SOD, CuZnSOD, MnSOD, CAT, GPx, GST and GR activities and GSH and
503
vitamin C contents in the head kidney and spleen of young grass carp under infection of A. hydrophila. In
504
fish antioxidant enzyme activities were partly dependent on antioxidant enzymes gene transcriptions [32],
505
which were regulated by Nrf2 signaling pathway [103]. Hence, we further elucidated the effects of vitamin
506
C on antioxidant enzymes mRNA expression and Nrf2 signaling pathway in the head kidney and spleen of
507
fish.
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Results from our study showed that dietary vitamin C limitation down-regulated the CuZnSOD,
509
MnSOD, CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTP1, GSTP2 and GR mRNA levels, whereas
510
optimal vitamin C supplementation could reverse these down-regulate in the head kidney and spleen of
511
young grass carp under infection of A. hydrophila. Correlation analysis revealed that CuZnSOD, MnSOD,
512
CAT and GR activities were positively related to their corresponding mRNA levels in the head kidney and
513
spleen (Table 9), suggesting that vitamin C limitation decreased those antioxidant enzyme activities may be
514
partly related to the down-regulation of their corresponding mRNA levels in the head kidney and spleen of
515
fish. Meanwhile, GPx activity in the head kidney and spleen was positively related to GPx1a, GPx1b, GPx4a
516
and GPx4b mRNA levels in the head kidney and GPx1b and GPx4b in the spleen (Table 9), suggesting that
517
vitamin C limitation decreased the GPx activity in the head kidney and spleen may be partly related to the
518
down-regulation GPx1a, GPx1b, GPx4a and GPx4b mRNA levels in the head kidney and GPx1b and GPx4b
519
in the spleen of fish, respectively. While GST activity was positively related to GSTR, GSTP1 and GSTP2
520
mRNA levels in head kidney and spleen of young grass carp (Table 9), suggesting that vitamin C limitation
521
decreased the GST activity may be partly related to the down-regulation GSTR, GSTP1 and GSTP2 in the
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head kidney and spleen of fish. Interestingly, vitamin C deficiency had no impact on mRNA levels of ACCEPTED MANUSCRIPT
523
GSTO1 and GSTO2 in the head kidney and spleen of young grass carp. The un-changed mRNA levels of
524
GSTO1 and GSTO2 might relate to its function. It was reported that, the major function of GSTO1 and
525
GSTO2 is reduction of dehydrogenation ascorbic acid to ascorbic acid, and this may be critical in the
526
maintenance of ascorbic acid levels in human [104]. While, in this study, we added ascorbic acid level in
527
fish head kidney and spleen through dietary supplement vitamin C, leading a no need for GTTO1 and
528
GSTO2. However, further investigation should be conducted to support our hypothesis. In fish, antioxidant
529
enzyme gene expression is regulated by an Nrf2 signaling pathway [103]. In the current study, vitamin C
530
deficiency caused a decline of Nrf2 mRNA level in head kidney and spleen of grass carp, whereas optimal
531
vitamin C supplementation could prevent this decline under infection of A. hydrophila. Correlation analysis
532
revealed that CuZnSOD, MnSOD, CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTP1, GSTP2 and GR
533
mRNA levels were positively related to the mRNA level of Nrf2 in the head kidney and spleen of young
534
grass carp (Table 9), supporting that vitamin C deficiency down-regulated antioxidant enzyme mRNA levels
535
may be partly related to the down-regulation of Nrf2 gene transcription in fish. In fish, Keap1 is the Nrf2
536
cytosolic repressor, which possesses two isoforms Keap1a and Keap1b [105]. In human, Keap1
537
overexpression could down-regulate Nrf2 mRNA level [106]. In this study, compared with optimal vitamin
538
C supplementation, fish fed vitamin C-deficient diet had a higher Keap1a and Keap1b mRNA levels in the
539
head kidney and spleen of young grass carp under infection of A. hydrophila. Correlation analysis showed
540
that the mRNA level of Nrf2 was negatively correlated with Keap1a and Keap1b in the head kidney and
541
spleen of young grass carp (Table 9), suggesting that vitamin C deficiency down-regulated Nrf2 mRNA
542
level may be partly attributed to up-regulated Keap1a and Keap1b transcript abundance in fish.
AC C
EP
TE D
M AN U
SC
RI PT
522
543
(Table 9 inserted here)
544
As stated above, our data firstly together corroborate the idea that vitamin C deficiency decreased 24
545
T-SOD, CuZnSOD, MnSOD, CAT, GPx, GST and GR activities by down-regulating CuZnSOD, MnSOD, ACCEPTED MANUSCRIPT
546
CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTP1, GSTP2 (rather than GTTO1 and GSTO2) and GR
547
mRNA levels, whereas optimal vitamin C supplementation could reverse these down-regulate, which may
548
be involved in Nrf2/Keap1 signaling pathway of fish under infection of A. hydrophila. Apoptosis is related to caspases, which generally be divided into two categories, the initiator caspases
550
(include caspase-2, -8 and caspase-9) and the effector caspases (include caspase-3 and caspase-7) [107].
551
Study showed that increased expression of caspase-3 could induce apoptosis in fish [108]. The present study
552
showed that compared with appropriate vitamin C level, vitamin C deficiency up-regulated the caspase-3, -7,
553
-8 and caspase-9 mRNA levels in the head kidney and spleen of young grass carp, indicating that vitamin C
554
deficiency at least in part increased the cell apoptosis in the head kidney and spleen of fish under infection
555
of A. hydrophila. However, vitamin C deficiency up-regulated caspase-2 mRNA expression in the spleen,
556
but had no impact on mRNA level of caspase-2 in the head kidney of young grass carp under infection of A.
557
hydrophila. This kind of phenomenon may relate to IL-12. It was reported that IL-12 could active caspase-2,
558
IL-12 P35 is a subunit of IL-12 in human [109, 110]. In the present study, vitamin C deficiency up-regulated
559
the IL-12 P35 expression level in the spleen, whereas had no impact on mRNA level of IL-12 P35 in the
560
head kidney of young grass carp under infection of A. hydrophila. However, further investigation should be
561
conducted to support our hypothesis.
AC C
EP
TE D
M AN U
SC
RI PT
549
562
In human, Fas death receptor pathway [mediated by Fas ligand (FasL)] is a major apoptosis pathway
563
[111]. In Jurkat T cells, up-regulation FasL could up-regulate caspase-8 expression [112]. In this study
564
compared with optimal vitamin C supplementation, vitamin C deficiency significantly up-regulated FasL
565
mRNA level in the head kidney and spleen of young grass carp under infection of A. hydrophila. Correlation
566
analysis showed that mRNA level of caspase-8 was positively related to FasL mRNA level in head kidney
567
and spleen of young grass carp (Table 10), suggesting that vitamin C deficiency up-regulated caspase-8 25
mRNA level may be partly through up-regulating mRNA transcript abundance in fish. In human, p38 ACCEPTEDFasL MANUSCRIPT
569
MAPK and JNK are MAPK family members, playing a key role in apoptosis [113]. It was reported that
570
down-regulation p38 MAPK expression could down-regulate FasL mRNA level in human ovarian
571
carcinoma cells [114]. In this study, compared with optimal vitamin C supplementation, vitamin C
572
deficiency significantly up-regulated p38 MAPK mRNA level, whereas had no impact on mRNA level of
573
JNK in the head kidney and spleen of young grass carp under infection of A. hydrophila. The reason that
574
vitamin C deficiency had no impact on mRNA level of JNK may relate to GSTO1. In HeLa cells, GSTO1
575
could inhibit JNK [115]. However, our study demonstrated that vitamin C had no impact on mRNA level of
576
GSTO1. Correlation analysis showed that mRNA level of FasL was positively related to p38 MAPK mRNA
577
level in head kidney and spleen of young grass carp (Table 10), suggesting that vitamin C deficiency
578
up-regulated FasL mRNA level may be partly by up-regulating p38 MAPK (rather than JNK) mRNA level
579
in the head kidney and spleen of fish. In mice, MAPKK 6 is an upstream kinase of the p38 MAPK pathway
580
[116]. Enslen et al. [117] reported that MAPKK 6 could up-regulate p38 MAPK gene expression in rat. In
581
this study, vitamin C deficiency significantly up-regulated MAPKK 6 mRNA level in head kidney and
582
spleen of young grass carp, whereas optimal vitamin C supplementation could prevent this up-regulation
583
under infection of A. hydrophila. Correlation analysis showed that mRNA level of p38 MAPK was
584
positively related to MAPKK 6 expression in head kidney and spleen of young grass carp (Table 10),
585
suggesting that vitamin C deficiency up-regulated p38 MAPK mRNA level may be partly through
586
up-regulating MAPKK 6 mRNA level in fish.
AC C
EP
TE D
M AN U
SC
RI PT
568
587
In K562 cells, mitochondria pathway plays an important role in apoptosis [118]. In human,
588
up-regulated Apaf-1 could increase the mRNA expression of caspase-9 [119]. Meanwhile, down-regulated
589
IAP could increase the expression of caspase-3 [120]. In this study, compared with optimal vitamin C
590
supplementation, vitamin C deficiency significantly up-regulated Apaf-1 mRNA level, and down-regulated 26
IAP mRNA level in the head kidneyACCEPTED and spleen ofMANUSCRIPT young grass carp under infection of A. hydrophila.
592
Correlation analysis showed that mRNA levels of caspase-3, -7 and caspase-9 were positively related to
593
Apaf-1 mRNA level, whereas negatively related to IAP mRNA level in the head kidney and spleen of young
594
grass carp (Table 10), respectively, suggesting that vitamin C deficiency up-regulated caspase-3, -7 and
595
caspase-9 mRNA levels may be partly through up-regulating Apaf-1 mRNA level and down-regulating IAP
596
mRNA level in fish. In 3T3 MEFs cells, Apaf-1 expression could be up-regulated by inhibition of Bcl-2 and
597
activation of Bax expression [121]. Bcl-2 family includes anti-apoptotic members (such as Bcl-2 and Mcl-1)
598
and pro-apoptotic members (such as Bax) in human [122]. In this study, compared with optimal vitamin C
599
supplementation, vitamin C deficiency significantly down-regulated Bcl-2 mRNA level and up-regulated
600
Bax mRNA level, whereas had no impact on mRNA level of Mcl-1 in the head kidney and spleen of young
601
grass carp under infection of A. hydrophila. The reason that vitamin C deficiency had no impact on mRNA
602
level of Mcl-1 may relate to JNK. In mice, inhibited JNK could down-regulate Mcl-1 expression [123].
603
However, our study demonstrated that vitamin C had no impact on mRNA level of JNK under infection of A.
604
hydrophila. Correlation analysis indicated that Apaf-1 mRNA level was negatively related to anti-apoptotic
605
member Bcl-2 mRNA level, whereas positively related to pro-apoptotic member Bax mRNA level in the
606
head kidney and spleen of young grass carp (Table 10). These results indicated that vitamin C deficiency
607
up-regulated Apaf-1 mRNA level may be partly through down-regulating Bcl-2 (rather than Mcl-1) and
608
up-regulating Bax mRNA levels in the head kidney and spleen of fish.
AC C
EP
TE D
M AN U
SC
RI PT
591
609
(Table 10 inserted here)
610
All together, our data firstly together corroborate the idea that vitamin C deficiency aggravated cell
611
apoptosis, whereas optimal vitamin C supplementation could prevent this aggravation, which may be partly
612
related to MAPKK 6/p38 MAPK (rather than JNK)/FasL/caspase-8 signaling and Bcl-2 (rather than Mcl-1),
613
Bax/Apaf-1, IAP/caspase-3, -7, -9 signaling in the head kidney and spleen of fish under infection of A. 27
hydrophila. In fish, the structural integrity of immune organ is also associated with the TJs [Occludin, ACCEPTED MANUSCRIPT
615
Claudins and zonula occludens (ZO)] in intercellular [42, 43]. Therefore, we next investigated the
616
relationship between vitamin C and TJs and its possible signaling pathway in the head kidney and spleen of
617
young grass carp.
618
4.5 Vitamin C deficiency disturbed the TJs in the head kidney and spleen of fish under infection of A.
619
hydrophila
RI PT
614
It was reported that ZO-1, Occludin, Claudin-b, -c and Claudin-3c are barrier-forming TJs, and
621
Claudin-12 and Claudin-15a are pore-forming TJs in fish [124]. In the present study, compared with the
622
optimal vitamin C supplementation, vitamin C deficiency down-regulated the mRNA levels of ZO-1, ZO-2,
623
Claudin-b, -c, -3c, -7a and Claudn-7b, and up-regulated the mRNA levels of Claudin-12, -15a and
624
Claudin-15b in the head kidney and spleen of fish under infection of A. hydrophila. These findings for the
625
first time implied that vitamin C deficiency might disturb TJs of head kidney and spleen in fish. Moreover,
626
MLCK was an important regulatory element of TJs in mice intestinal epithelium cells [125]. Study showed
627
that up-regulation of MLCK could down-regulate the mRNA levels of ZO-1 and Occludin in human Caco-2
628
cells [126]. Results from our study demonstrated that compared with optimal vitamin C supplementation,
629
vitamin C deficiency up-regulated the mRNA level of MLCK in the head kidney of young grass carp under
630
infection of A. hydrophila. Correlation analysis showed that barrier-forming TJs ZO-1, ZO-2, Claudin-b, -c,
631
-3c, -7a and Claudn-7b mRNA levels were negatively correlated with MLCK mRNA level, whereas
632
pore-forming TJs Claudin-12, -15a and Claudin-15b mRNA levels were positively related to MLCK mRNA
633
level in the head kidney of young grass carp (Table 11), suggesting that dietary vitamin C deficiency
634
down-regulated ZO-1, ZO-2, Claudin-b, -c, -3c, -7a and Claudn-7b mRNA levels and up-regulated
635
Claudin-12, -15a and Claudin-15b mRNA levels may be partly by up-regulating MLCK mRNA level in the
636
head kidney of fish. However, current study found that vitamin C deficiency had no impact on mRNA level
AC C
EP
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M AN U
SC
620
28
of MLCK in the spleen of young grassACCEPTED carp under infection of A. hydrophila. The possible reason for which MANUSCRIPT
638
vitamin C had no impact on spleen MLCK is that vitamin C may be partly through cytokines to regulate TJs,
639
rather than MLCK. Studies showed that TNF-α could down-regulate the expression of ZO-1 in BREC cells
640
and Claudin-7 in rat [127, 128] and up-regulated the expression of Claudin-12 and Claudin-15 in T84 cells
641
[129]. As mentioned above, spleen TNF-α mRNA level significantly up-regulated in vitamin C-limited diet
642
group under infection of A. hydrophila. Further correlation analysis showed that ZO-1, ZO-2, Claudin-b, -c,
643
-3c, -7a and Claudin-7b mRNA levels were negatively correlated with TNF-α mRNA level, Claudin-12, -15a
644
and Claudin-15b mRNA levels were positively related to TNF-α mRNA level in the spleen of young grass
645
carp (Table 11). Those results indicated that dietary vitamin C deficiency down-regulated ZO-1, ZO-2,
646
Claudin-b, -c, -3c, -7a and Claudin-7b mRNA levels, and up-regulated Claudin-12, -15a and Claudin-15b
647
mRNA levels be partly by up-regulating the gene transcription of TNF-α (rather than MLCK) in the spleen
648
of fish.
M AN U
SC
RI PT
637
(Table 11 inserted here)
650
As stated above, our data firstly together corroborate the idea that compared with optimal vitamin C
651
supplementation, vitamin C deficiency disturbed head kidney and spleen TJs through down-regulating ZO-1,
652
ZO-2, Claudin-b, -c, -3c, -7a and Claudin-7b mRNA levels, and up-regulating Claudin-12, -15a and
653
Claudin-15b mRNA levels, which may be partly regulated by MLCK signaling in head kidney and TNF-α
654
(rather than MLCK) in spleen of fish under infection of A. hydrophila. However, the underlying mechanism
655
needs to be further investigated.
656
4.6. Interesting results: high levels of vitamin C impaired some indicators that differed from the traditional
657
growth results
AC C
EP
TE D
649
658
Vitamin C as a water-solution vitamin; no negative effects on the growth performances were observed
659
in this study and our lab previous study in juvenile Jian carp when high doses were administered [4]. 29
Interestingly, our study founded that,ACCEPTED compared with optimal vitamin C supplementation, high levels of MANUSCRIPT
661
vitamin C (224.5 mg/kg diet) down-regulated the mRNA expression levels of β-defensin in the head kidney
662
and spleen, Claudin-7b in the head kidney, and IL-11, Claudin-7a and GPx4a in the spleen, and up-regulated
663
the mRNA expression levels of IL-12 P40 and IL-15 in the head kidney under infection of A. hydrophila.
664
The possible reasons are (1) High levels of vitamin C down-regulated β-defensin mRNA level in the head
665
kidney and spleen of fish may partly relate to vitamin C have a similar function to it. In carp, β-defensin
666
have a strong antibacterial activity to Escherichia coli (E. coli) [130]. While, study from our lab showed that
667
vitamin C significantly depressed intestinal E. coli in Jian carp (Cyprinus carpio var. Jian) [4]. (2) High
668
levels of vitamin C up-regulated IL-12 P40 and IL-15 mRNA expression levels in the head kidney may be
669
partly attributed to down-regulate β-defensin mRNA level. In human myeloid dendritic cells, β-defensin
670
could inhibit pro-inflammatory cytokines IL-12 P40 and IL-15 [131]. In this study, compared with optimal
671
vitamin C supplementation, high levels of vitamin C significantly down-regulated head kidney β-defensin
672
mRNA level in young grass carp. (3) High levels of vitamin C down-regulated IL-11 mRNA level in the
673
spleen of fish may be partly related to vitamin C has a similar function to it. It was reported that IL-11
674
played a role of anti-inflammatory through reducing TNF-α mRNA level in mice [132]. However, our study
675
indicated that vitamin C down-regulated TNF-α mRNA expression. (4) High levels of vitamin C
676
down-regulated the mRNA levels of Claudin-7b in the head kidney and Claudin-7a in the spleen may be
677
partly ascribed to vitamin C could enhance the TJs by increasing collagen content in fish. It was reported
678
that, vitamin C increased bone collagen in large yellow croaker (Pseudosciaena crocea) [30]. Collagen
679
could enhance the TJs in A6 cells [133]. In rat, Claudin-7 is a barrier-forming tight junction protein [128].
680
These may support our hypothesis, but need to be further investigated. (5) High levels of vitamin C
681
down-regulated GPx4a mRNA level in the spleen of fish may partly relate to vitamin C have a similar
682
function to it. In Coho salmon, the major function of GPx4a is reducing lipid peroxides [134]. However,
AC C
EP
TE D
M AN U
SC
RI PT
660
30
683
vitamin C played an important role of ACCEPTED reduction lipid MANUSCRIPT peroxides in rat [135].
684
4.7. Vitamin C deficiency reduced the immunity and damaged the structural integrity in the skin of fish:
685
compared with head kidney and spleen. Our results showed that except the activities of ACP and MnSOD, and mRNA expression levels of
687
TGF-β1, Occludin and MnSOD model were different, the effect of vitamin C on other indices model were
688
similar among the head kidney, spleen and skin in young grass carp under infection of A. hydrophila. Our
689
study demonstrated that, compared with optimal vitamin C supplementation, vitamin C limitation decreased
690
ACP and MnSOD activities and down-regulated the mRNA levels of TGF-β1 and MnSOD in young grass
691
carp head kidney and spleen, but had no significant effect on that of skin under infection of A. hydrophila.
692
This result may be partly attributed to the vitamin C concentration difference in each tissue. Our study
693
showed that vitamin C concentration in the skin was lower than that of head kidney and spleen in young
694
grass carp. Thus, the different ACP and MnSOD activities and mRNA levels of TGF-β2 and MnSOD model
695
may be partly related to the low vitamin C concentration in fish skin. Moreover, compared with optimal
696
vitamin C supplementation, vitamin C deficiency up-regulated the mRNA level of Occludin in the skin, but
697
had no significant effect on its mRNA level in the head kidney and spleen of young grass carp under
698
infection of A. hydrophila. This result may be partly attributed to the function of Occludin and its high
699
expression level in fish skin. In rainbow trout, Occludin could regulate salt and water balance, and its mRNA
700
was particularly abundant in the skin (which involved in the regulation of salt and water balance) but rare in
701
the head kidney and spleen [136]. Therefore, the different mRNA expression model of Occludin in fish skin
702
may be partly related to its function and high expression level in fish skin.
703
4.8. Compare the vitamin C requirements for growth performance with that for immunity and antioxidant of
704
young grass carp
705
AC C
EP
TE D
M AN U
SC
RI PT
686
Based on our study, vitamin C deficiency could decrease growth performance, increase skin lesion 31
morbidity and impair the immunity and structural integrity of head kidney and spleen in fish under infection ACCEPTED MANUSCRIPT
707
of A. hydrophila. Thus, evaluation of vitamin C requirement is quite necessary in fish. In this study, the
708
vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8
709
mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp
710
was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA
711
activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head
712
kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to
713
be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively. The requirements for against skin lesion
714
morbidity and enhancement immune and antioxidant indices were higher than that for the growth
715
performance, indicating that more vitamin C is required for the development of immunity and antioxidant of
716
fish. Similarly, the requirement of riboflavin based on immunity and antioxidant were higher than that on
717
growth requirement of young grass carp [52].
718
5. Conclusions
TE D
M AN U
SC
RI PT
706
In summary (summarized in Fig. 16), we report seven primary, novel, and interesting results. Compared
720
with optimal vitamin C supplementation, dietary vitamin C deficiency (1) decreased immunity partly
721
through decreasing LA and ACP activities and C3 and C4 contents in the head kidney and spleen of fish
722
under infection of A. hydrophila; (2) aggravated the inflammatory response by down-regulating
723
anti-inflammatory cytokines [IL-4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, TGF-β1 and TGF-β2],
724
and up-regulating pro-inflammatory cytokines [TNF-α, IFN-γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in
725
spleen), IL-12 P40, IL-15 and IL-17D] mRNA expression levels in the head kidney and spleen of fish under
726
infection of A. hydrophila. The regulation of above-mentioned cytokines may be involved in the NF-κB
727
pathway [including signal molecules NF-κB p65 (rather than NF-κB p50), IκBα, IKKα, IKKβ and IKKγ]
728
and TOR pathway [including signal molecules TOR, S6K1 and 4E-BP1 (rather than 4E-BP2)] in fish; (3)
AC C
EP
719
32
impaired head kidney and spleen TJsACCEPTED by up-regulating mRNA levels of Claudin-12, -15a and Claudin-15b, MANUSCRIPT
730
and down-regulating mRNA levels of ZO-1, ZO-2, Claudin-b, -c, -3c, -7a and Claudin-7b in the head kidney
731
and spleen of fish under infection of A. hydrophila, which may be partly regulated by up-regulating MLCK
732
mRNA level in the head kidney and TNF-α mRNA level in the spleen, respectively; (4) induced apoptosis by
733
up-regulating caspase-3, -7, -8 and caspase-9 mRNA levels in the head kidney and spleen of fish under
734
infection of A. hydrophila, which may be involved in Fas death receptor pathway [mediated by MAPKK
735
6/p38 MAPK (rather than JNK)] and mitochondria pathway [mediated by Bcl-2 (rather than Mcl-1),
736
Bax/Apaf-1, IAP]; (5) decreased antioxidant capacity by decreasing the antioxidant enzymes activities and
737
mRNA levels in the head kidney and spleen of fish under infection of A. hydrophila, which could be
738
regulated by the Nrf2 signaling pathway. (6) The effect of vitamin C on fish head kidney, spleen and skin
739
immunity and structural integrity are similar, except the activities of ACP and MnSOD, and mRNA
740
expression levels of TGF-β1, Occludin and MnSOD of fish under infection of A. hydrophila. (7) The
741
vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8
742
mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp
743
was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA
744
activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head
745
kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to
746
be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively.
748
SC
M AN U
TE D
EP
AC C
747
RI PT
729
(Fig. 16 inserted here) Acknowledgements
749
This research was financially supported by the National Basic Research Program of China (973
750
Program) (2014CB138600), the Specialized Research Fund for the Doctoral Program of Higher Education
751
of China (20135103110001), Science and Technology Support Program of Sichuan Province of China 33
(2014NZ0003), Major Scientific and Technological Achievement Transformation Project of Sichuan ACCEPTED MANUSCRIPT
753
Province of China (2012NC0007; 2013NC0045), the Demonstration of Major Scientific and Technological
754
Achievement Transformation Project of Sichuan Province of China (2015CC0011), Natural Science
755
Foundation for Young Scientists of Sichuan Province (2014JQ0007) and Sichuan Province Research
756
Foundation for Basic Research (2013JY0082). The authors would like to thank the personnel of these teams
757
for their kind assistance.
RI PT
752
758
SC
759
M AN U
760 761 762 763
TE D
764 765 766
EP
767
AC C
768 769 770 771 772 773
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774
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[112] S. M. Uriarte, S. Joshi-Barve, Z. Song, R. Sahoo, L. Gobejishvili, V. Jala, et al., Akt inhibition
1070
upregulates FasL, downregulates c-FLIPs and induces caspase-8-dependent cell death in Jurkat T
1071
lymphocytes, Cell Death Differ. 12 (2005) 233-242.
1072
1073
[113] E. K. Kim, E. J. Choi, Pathological roles of MAPK signaling pathways in human diseases, BBA-Mol. Basis Dis. 1802 (2010) 396-405. 47
1074
[114] A. Mansouri, L.D. Ridgway, A.L. Korapati, Q. Zhang, L. Tian, Y. Wang, et al., Sustained activation ACCEPTED MANUSCRIPT
1075
of JNK/p38 MAPK pathways in response to cisplatin leads to Fas ligand induction and cell death in
1076
ovarian carcinoma cells, J. Biol. Chem. 278 (2003) 19245-19256. [115] S. Piaggi, C. Raggi, A. Corti, E. Pitzalis, M.C. Mascherpa, M. Saviozzi, et al., Glutathione
1078
transferase omega 1-1 (GSTO1-1) plays an anti-apoptotic role in cell resistance to cisplatin toxicity,
1079
Carcinogenesis 31 (2010) 804-811.
RI PT
1077
[116] R. A. Thandavarayan, K. Watanabe, M. Ma, N. Gurusamy, P.T. Veeraveedu, T. Konishi, et al.,
1081
Dominant-negative p38α mitogen-activated protein kinase prevents cardiac apoptosis and
1082
remodeling after streptozotocin-induced diabetes mellitus, Am. J. Physiol.-Heart C. 297 (2009)
1083
H911-H919.
M AN U
SC
1080
[117] H. Enslen, J. Raingeaud, R.J. Davis, Selective activation of p38 mitogen-activated protein (MAP)
1085
kinase isoforms by the MAP kinase kinases MKK3 and MKK6, J. Biol. Chem. 273 (1998)
1086
1741-1748.
TE D
1084
[118] H. Jiang, C. Hou, S. Zhang, H. Xie, W. Zhou, Q. Jin, et al., Matrine upregulates the cell cycle protein
1088
E2F-1 and triggers apoptosis via the mitochondrial pathway in K562 cells, Eur. J. Pharmacol. 559
1089
(2007) 98-108.
EP
1087
[119] A. Haga, T. Funasaka, Y. Niinaka, A. Raz, H. Nagase, Autocrine motility factor signaling induces
1091
tumor apoptotic resistance by regulations Apaf-1 and Caspase-9 apoptosome expression, Int. J.
1092
cancer 107 (2003) 707-714.
AC C
1090
1093
[120] K. Yamamoto, S. Abe, Y. Nakagawa, K. Suzuki, M. Hasegawa, M. Inoue, et al., Expression of IAP
1094
family proteins in myelodysplastic syndromes transforming to overt leukemia, Leukemia Res. 28
1095
(2004) 1203-1211.
1096
[121] R. E. Molestina, T. M. Payne, I. Coppens, A. P. Sinai, Activation of NF-κB by Toxoplasma gondii 48
1097
correlates with increased expression of antiapoptotic genes and localization of phosphorylated IκB to ACCEPTED MANUSCRIPT
1098
the parasitophorous vacuole membrane, J. cell Sci. 116 (2003) 4359-4371. [122] S.N. Willis, L. Chen, G. Dewson, A. Wei, E. Naik, J.I. Fletcher, et al., Proapoptotic Bak is
1100
sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins, Gene. Dev.
1101
19 (2005) 1294-1305.
RI PT
1099
[123] Y. Kodama, K. Taura, K. Miura, B. Schnabl, Y. Osawa, D.A. Brenner, Antiapoptotic effect of c-Jun
1103
N-terminal Kinase-1 through Mcl-1 stabilization in TNF-induced hepatocyte apoptosis,
1104
Gastroenterology 136 (2009) 1423-1434.
SC
1102
[124] B. Wang, L. Feng, W. D. Jiang, P. Wu, S. Y. Kuang, J. Jiang, et al., Copper-induced tight junction
1106
mRNA expression changes, apoptosis and antioxidant responses via NF-κB, TOR and Nrf2 signaling
1107
molecules in the gills of fish: Preventive role of arginine, Aquat. Toxicol. 158 (2015) 125-137.
1108
[125] T. W. Costantini, W. H. Loomis, J. G. Putnam, L. Kroll, B. P. Eliceiri, A. Baird, et al., Pentoxifylline
1109
modulates intestinal tight junction signaling after burn injury: effects on myosin light chain kinase, J.
1110
Traum. 66 (2009) 17.
TE D
1112
[126] L. Nahidi, S. T. Leach, D. A. Lemberg, A. S. Day, Osteoprotegerin exerts its pro-inflammatory effects through nuclear factor-κB activation, Digest. Dis. Sci. 58 (2013) 3144-3155.
EP
1111
M AN U
1105
[127] C. A. Aveleira, C. M. Lin, S. F. Abcouwer, A. F. Ambrósio, D. A. Antonetti, TNF-α signals through
1114
PKCζ/NF-κB to alter the tight junction complex and increase retinal endothelial cell permeability,
1115
Diabetes 59 (2010) 2872-2882.
AC C
1113
1116
[128] M. Amasheh, I. Grotjohann, S. Amasheh, A. Fromm, J.D. Söderholm, M. Zeitz, et al., Regulation of
1117
mucosal structure and barrier function in rat colon exposed to tumor necrosis factor alpha and
1118
interferon gamma in vitro: a novel model for studying the pathomechanisms of inflammatory bowel
1119
disease cytokines, Scand. J. Gastroentero. 44 (2009) 1226-1235. 49
1120
[129] C.R. Weber, D.R. Raleigh, L.ACCEPTED Su, L. Shen, E.A. Sullivan, Y. Wang, et al., Epithelial myosin light MANUSCRIPT
1121
chain kinase activation induces mucosal interleukin-13 expression to alter tight junction ion
1122
selectivity, J. Biol. Chem. 285 (2010) 12037-12046.
1123
[130]
Li H, Guo H, Shan S, et al. Characterization and expression pattern of a novel β-defensin in common carp (Cyprinus carpio L.): implications for its role in mucosal immunity[J]. Biosci. Biotech. Bioch.
1125
2014, 78(3): 430-437.
RI PT
1124
[131] L.C. Pingel, K.G. Kohlgraf, C.J. Hansen, C.G. Eastman, D.E. Dietrich, K.K. Burnell, et al., Human
1127
β-defensin 3 binds to hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas
1128
gingivalis, and attenuates a pro-inflammatory cytokine response, Immunol. cell Biol. 86 (2008)
1129
643-649.
M AN U
SC
1126
[132] K. Shimizu, K. Shiratori, T. Sawada, M. Kobayashi, N. Hayashi, H. Saotome, et al., Recombinant
1131
human interleukin-11 decreases severity of acute necrotizing pancreatitis in mice, Pancreas 21 (2000)
1132
134-140.
1133
1134
TE D
1130
[133] M. Jaeger, G. Kalinec, V. Dodane, B. Kachar, A collagen substrate enhances the sealing capacity of tight junctions of A6 cell monolayers, Jo. membrane Biol. 159 (1997) 263-270. [134] L. Wang, S.M. Harris, H.M. Espinoza, V. McClain, E.P. Gallagher, Characterization of phospholipid
1136
hydroperoxide glutathione metabolizing peroxidase (gpx4) isoforms in Coho salmon olfactory and
1137
liver tissues and their modulation by cadmium, Aquat. Toxicol. 114 (2012) 134-141.
AC C
EP
1135
1138
[135] L.M.G. Antunes, J.D.A.C. Darin, M.D.L.P. Bi, Protective effects of vitamin C against
1139
cisplatin-induced nephrotoxicity and lipid peroxidation in adult rats: a dose-dependent study,
1140
Pharmacol. Res. 41 (2000) 405-411.
1141
[136] H. Chasiotis, C.M. Wood, S.P. Kelly, Cortisol reduces paracellular permeability and increases
1142
occludin abundance in cultured trout gill epithelia, Mol. cell. Endocrinol. 323 (2010) 232-238. 50
1143
Table 1 Composition and nutrients content of basal diet. ACCEPTED MANUSCRIPT
1144
1145
1
1146
L-a-tocopherol acetate (50%), 12.58; menadione (22.9%), 0.83; thiamine nitrate (98%), 0.11; calcium-D-pantothenate
1147
(98%), 2.56; pyridoxine hydrochloride (98%), 0.62; cyanocobalamin (1%), 0.94; niacin (99%), 2.58; D-biotin (2%), 0.75;
1148
meso-inositol (98%),19.39; folic acid (95%), 0.42; riboflavin (80%), 0.63. All ingredients were diluted with corn starch to 1
1149
kg.
1150
2
1151
Fe), 24.5700; ZnSO4.H2O (34.5% Zn), 8.2500; CuSO4.5H2O (25.0% Cu), 0.960; KI (76.9% I), 0.0668g; Na2SeO3 (44.7%
1152
Se), 0.0168. All ingredients were diluted with corn starch to 1 kg.
1153
3
Vitamin C premix: premix was added to obtain graded level of vitamin C.
1154
4
Crude protein and crude lipid contents were measured value.
1155
5
Available phosphorus, n-3 and n-6 contents were calculated according to NRC (2011).
RI PT
M AN U
SC
Mineral premix (g/kg premix): MnSO4.H2O (31.8% Mn), 1.8900; MgSO4.H2O (15.0% Mg), 200.0000; FeSO4.H2O (30.0%
TE D
1156
Table 2 Real-time PCR primer sequences 1.
1158
EP
1157
Vitamin premix (g/kg premix): retinyl acetate (500,000 IU/g), 2.10; cholecalciferol (500,000 IU/g), 0.40; D,
1159
1
1160
interleukin; TGF-β: transforming growth factor β; NF-κB: nuclear factor kappa B; IκBα: inhibitor of κBα;
1161
IKK: IκB kinase; TOR: target of rapamycin; S6K1: ribosomal protein S6 kinase 1; 4E-BP: eIF4E-binding
1162
protein; ZO: zonula occludens; MLCK: myosin light chain kinase; FasL: Fas ligand; MAPKK 6:
1163
mitogen-activated protein kinase kinase 6; p38 MAPK: p38 mitogen-activated protein kinases; JNK: c-Jun
1164
N-terminal kinases; Bcl-2: B-cell lymphoma-2; Mcl-1: myeloid cell leukemia-1; Bax: B-cell lymphoma
1165
protein 2 associated X protein; Apaf-1: apoptotic protease activating factor-1; IAP: inhibitor of apoptosis
AC C
LEAP-2: liver expressed antimicrobial peptide 2; IFN-γ2: interferon γ2; TNF-α: tumor necrosis factor α; IL:
51
1166
protein; CuZnSOD: copper, zinc superoxide dismutase; MnSOD: manganese superoxide dismutase; GST: ACCEPTED MANUSCRIPT
1167
glutathione-S-transferase; CAT: catalase; GPx, glutathione peroxidase; GR: glutathione reductase; Nrf2:
1168
NF-E2-related factor 2; Keap1: Kelch-like-ECH-associated protein 1.
1169
Table 3 Growth performance1, weight (g/fish), index (%) and vitamin C concentration (mg/kg tissue) of
1171
head kidney and spleen, and skin vitamin C concentration in young grass carp (Ctenopharyngodon idella)
1172
fed the diets with graded levels of vitamin C for 10 weeks.
SC
1173
RI PT
1170
1174
1
1175
rate (%/day); FI: feed intake (g/fish); FE: feed efficiency (%).
1176
2
1177
are significantly different (P < 0.05).
1178
3
Values are means ± SD (n = 18), and different superscripts in the same row are significantly different (P < 0.05).
1179
4
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05).
M AN U
Values are means ± SD for three replicate groups, with 30 fish in each group, and different superscripts in the same row
TE D
1180
IBW: Initial body weight (g/fish); FBW: final body weight (g/fish); PWG: percent weight gain (%), SGR: specific growth
Table 4 Immune parameters in the head kidney and spleen of young grass carp (Ctenopharyngodon idella)
1182
fed diets containing different vitamin C levels after infection with A. hydrophila for 14 days 1.
1183
AC C
EP
1181
1184
1
1185
Lysozyme activity (U/mg protein); ACP: acid phosphatase (U/mg protein); C3: complement component 3 (mg/g protein);
1186
C4: complement component 4 (mg/g protein).
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05). LA:
1187
1188
Table 5 Antioxidant status related parameters in the head kidney and spleen of young grass carp 52
1189
(Ctenopharyngodon idella) fed diets ACCEPTED containing different levels of vitamin C levels after infection with A. MANUSCRIPT
1190
hydrophila for 14 days 1.
1191
1192
1
1193
reactive oxygen species (% DCF florescence); MDA: malondialdehyde (nmol/mg protein); PC: protein carbonyl (nmol/mg
1194
protein); ASA: anti-superoxide anion (U/mg protein); AHR: anti-hydroxyl radical (U/mg protein); T-SOD: total superoxide
1195
dismutase (U/mg protein); SOD1: copper/zinc superoxide dismutase (U/mg protein); SOD2: manganese superoxide
1196
dismutase (U/mg protein); CAT: catalase (U/mg protein); GPx: glutathione peroxidase (U/mg protein); GR, glutathione
1197
reductase (U/mg protein); GSH, glutathione (mg/g protein).
M AN U
SC
RI PT
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05). ROS:
1198
1199
Table 6 Immune parameters in the skin of young grass carp (Ctenopharyngodon idella) fed diets containing
1200
different vitamin C levels after infection with A. hydrophila for 14 days 1.
1202
1
TE D
1201
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05).
1203
Table 7 Antioxidant status related parameters in the skin of young grass carp (Ctenopharyngodon idella) fed
1205
diets containing different levels of vitamin C levels after infection with A. hydrophila for 14 days 1.
1206
1207
1208
1
AC C
EP
1204
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05).
1209
Table 8 Correlation coefficients of NF-κB p65 with IL-4/13A, IL-6, IL-8, IL-10, IL-11, IL-12 P40, IL-15,
1210
IL-17D and TGF-β1, IκBα with NF-κB p65, IKKα, IKKβ and IKKγ, TOR with IFN-γ2, TNF-α and IL-1β in
1211
the head kidney and spleen. 53
ACCEPTED MANUSCRIPT
1213
Table 9 Correlation coefficients of CuZnSOD, MnSOD, CAT and GR activities with their mRNA levels in
1214
the head kidney and spleen, GPx activity with GPx1a, GPx1b, GPx4a and GPx4b mRNA levels in the head
1215
kidney and GPx1b and GPx4b mRNA levels in the spleen, GST activity with GSTR, GSTP1 and GSTP2
1216
mRNA levels in the head kidney and spleen, Nrf2 with CuZnSOD, MnSOD, CAT, GPx1a, GPx1b, GPx4a,
1217
GPx4b, GSTR, GSTP1, GSTP2, GR, Keap1a and Keap1b mRNA levels in the head kidney and spleen.
RI PT
1212
1218
Table 10 Correlation coefficients of FasL with caspase-8 and p38 MAPK, MAPKK6 with p38 MAPK,
1220
Apaf-1 with caspase-3,-7,-9, Bcl-2 and Bax, IAP with caspase-3,-7,-9 in the head kidney and spleen.
M AN U
SC
1219
1221
Table 11 Correlation coefficients of MLCK with ZO-1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, -12, -15a and
1223
Claudin-15b in the head kidney, and TNF-α with ZO-1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, -12, -15a and
1224
Claudin-15b in the spleen.
1227
1228
1229
1230
1231
EP
1226
AC C
1225
TE D
1222
1232
1233
1234
1235
54
1236
ACCEPTED MANUSCRIPT
Table 1
1237
1239
Nutrients content
g/kg
Fish meal
85.80
Crude protein 4
298.57
Casein
131.60
Crude lipid 4
45.40
Soybean protein concentrate
200.00
n-3 5
4.60
n-6 5
240.00
Corn starch
187.30
10.00
Available phosphorus 5
M AN U
α-starch
5.00
SC
DL-Met 99%
RI PT
g/kg
5.30
Soybean oil
19.30
Cellulose
50.00
Ca(H2PO4)2
30.60
Mineral premix 2 Vitamin C premix 3 Choline chloride (60%)
10.00 20.00
EP
Vitamin premix 1
TE D
Fish oil
AC C
1238
Ingredients
Ethoxyquin (30%)
10.00 5.00 0.50
1240
1241
1242 55
8.40
1243
ACCEPTED MANUSCRIPT
Table 2
1244 Primer sequence Forward (5’→3’)
Primer sequence Reverse (5’→3’)
Temperature (°C)
Accession number
Hepcidin
AGCAGGAGCAGGATGAGC
GCCAGGGGATTTGTTTGT
59.3
JQ246442.1
LEAP-2A
TGCCTACTGCCAGAACCA
AATCGGTTGGCTGTAGGA
59.3
FJ390414
LEAP2-B
TGTGCCATTAGCGACTTCTGAG
ATGATTCGCCACAAAGGGG
59.3
KT625603
β-defensin
TTGCTTGTCCTTGCCGTCT
AATCCTTTGCCACAGCCTAA
58.4
KT445868
IFN-γ2
TGTTTGATGACTTTGGGATG
TCAGGACCCGCAGGAAGAC
60.4
JX657682
TNF-α
CGCTGCTGTCTGCTTCAC
CCTGGTCCTGGTTCACTC
58.4
HQ696609
IL-1β
AGAGTTTGGTGAAGAAGAGG
TTATTGTGGTTACGCTGGA
57.1
JQ692172
IL-6
CAGCAGAATGGGGGAGTTATC
CTCGCAGAGTCTTGACATCCTT
62.3
KC535507.1
IL-8
ATGAGTCTTAGAGGTCTGGGT
ACAGTGAGGGCTAGGAGGG
60.3
JN663841
IL-15
CCTTCCAACAATCTCGCTTC
AACACATCTTCCAGTTCTCCTT
61.4
KT445872
IL-17D
GTGTCCAGGAGAGCACCAAG
GCGAGAGGCTGAGGAAGTTT
62.3
KF245426.1
IL-4/13A
CTACTGCTCGCTTTCGCTGT
CCCAGTTTTCAGTTCTCTCAGG
55.9
KT445871
IL-4/13B
TGTGAACCAGACCCTACATAACC
TTCAGGACCTTTGCTGCTTG
55.9
KT625600
IL-10
AATCCCTTTGATTTTGCC
GTGCCTTATCCTACAGTATGTG
61.4
HQ388294
IL-11
GGTTCAAGTCTCTTCCAGCGAT
TGCGTGTTATTTTGTTCAGCCA
57.0
KT445870
IL-12 P35
TGGAAAAGGAGGGGAAGATG
AGACGGACGCTGTGTGAGTGTA
55.4
KF944667.1
IL-12 P40
ACAAAGATGAAAAACTGGAGGC
GTGTGTGGTTTAGGTAGGAGCC
59.0
KF944668.1
TGF-β1
TTGGGACTTGTGCTCTAT
AGTTCTGCTGGGATGTTT
55.9
EU099588
TGF-β2
TACATTGACAGCAAGGTGGTG
TCTTGTTGGGGATGATGTAGTT
55.9
KM279716
NF-κB p52
TCAGTGTAACGACAACGGGAT
ATACTTCAGCCACACCTCTCTTAG
58.4
KM279720
NF-κB p65
GAAGAAGGATGTGGGAGATG
TGTTGTCGTAGATGGGCTGAG
62.3
KJ526214
IκBα
TCTTGCCATTATTCACGAGG
TGTTACCACAGTCATCCACCA
62.3
KJ125069
IKKα
GGCTACGCCAAAGACCTG
CGGACCTCGCCATTCATA
60.3
KM279718
IKKβ
GTGGCGGTGGATTATTGG
GCACGGGTTGCCAGTTTG
60.3
KP125491
IKKγ
AGAGGCTCGTCATAGTGG
CTGTGATTGGCTTGCTTT
58.4
KM079079
AC C
EP
TE D
M AN U
SC
RI PT
Target gene
56
TCCCACTTTCCACCAACT
ACACCTCCACCTTCTCCA
61.4
JX854449
S6K1
TGGAGGAGGTAATGGACG
ACATAAAGCAGCCTGACG
54.0
EF373673
4E-BP1
GCTGGCTGAGTTTGTGGTTG
CGAGTCGTGCTAAAAAGGGTC
60.3
KT757305
4E-BP2
CACTTTATTCTCCACCACCCC
TTCATTGAGGATGTTCTTGCC
60.3
KT757306
Occludin
TATCTGTATCACTACTGCGTCG
CATTCACCCAATCCTCCA
59.4
KF193855
ZO-1
CGGTGTCTTCGTAGTCGG
CAGTTGGTTTGGGTTTCAG
59.4
KJ000055
ZO-2
TACAGCGGGACTCTAAAATGG
TCACACGGTCGTTCTCAAAG
Claudin-b
GAGGGAATCTGGATGAGC
ATGGCAATGATGGTGAGA
Claudin-c
GAGGGAATCTGGATGAGC
CTGTTATGAAAGCGGCAC
Claudin-3c
ATCACTCGGGACTTCTA
CAGCAAACCCAATGTAG
Claudin-7a
ACTTACCAGGGACTGTGGATGT
Claudin-7b
ACCEPTED MANUSCRIPT
RI PT
TOR
KM112095
57.0
KF193860
59.4
KF193859
57.0
KF193858
CACTATCATCAAAGCACGGGT
59.3
KT625604
CTAACTGTGGTGGTGATGAC
AACAATGCTACAAAGGGCTG
59.3
KT445866
Claudin-12
CCCTGAAGTGCCCACAA
GCGTATGTCACGGGAGAA
55.4
KF998571
Claudin-15a
TGCTTTATTTCTTGGCTTTC
CTCGTACAGGGTTGAGGTG
59.0
KF193857
Claudin-15b
AGTGTTCTAAGATAGGAGGGGAG
AGCCCTTCTCCGATTTCAT
62.3
KT757304
MLCK
GAAGGTCAGGGCATCTCA
GGGTCGGGCTTATCTACT
53.0
KM279719
FasL
AGGAAATGCCCGCACAAATG
AACCGCTTTCATTGACCTGGAG
61.4
KT445873
MAPKK 6
GAGCATCTCCACAGCAACCT
CTTCGCCACTGAATCCACAA
57.1
KT445869
p38 MAPK
TGGGAGCAGACCTCAACAAT
TACCATCGGGTGGCAACATA
60.4
KM112098
JNK
ACAGCGTAGATGTGGGTGATT
GCTCAAGGTTGTGGTCATACG
62.3
KT757312
Bcl-2
AGGAAAATGGAGGTTGGGAT
CTGAGCAAAAAAGGCGATG
60.3
JQ713862.1
Mcl-1
TGGAAAGTCTCGTGGTAAAGCA
ATCGCTGAAGATTTCTGTTGCC
58.4
KT757307
Bax
CATCTATGAGCGGGTTCGTC
TTTATGGCTGGGGTCACACA
60.3
JQ793788.1
Apaf-1
AAGTTCTGGAGCCTGGACAC
AACTCAAGACCCCACAGCAC
61.4
KM279717
IAP
CACAATCCTGGTATGCGTCG
GGGTAATGCCTCTGGTGCTC
58.4
FJ593503.1
Caspase-2
CGCTGTTGTGTGTTTACTGTCTCA
ACGCCATTATCCATCTCCTCTC
60.3
KT757313
Caspase-3
GCTGTGCTTCATTTGTTTG
TCTGAGATGTTATGGCTGTC
55.9
JQ793789
Caspase-7
GCCATTACAGGATTGTTTCACC
CCTTATCTGTGCCATTGCGT
57.1
KT625601
AC C
EP
TE D
M AN U
SC
60.3
57
1247
1248
TCCATCTGATGCCCATACAC
59.0
KM016991
CTGTGGCGGAGGTGAGAA
GTGCTGGAGGACATGGGAAT
59.0
JQ793787
CuZnSOD
CGCACTTCAACCCTTACA
ACTTTCCTCATTGCCTCC
61.5
GU901214
MnSOD
ACGACCCAAGTCTCCCTA
ACCCTGTGGTTCTCCTCC
60.4
GU218534
CAT
GAAGTTCTACACCGATGAGG
CCAGAAATCCCAAACCAT
58.7
FJ560431
GPx1a
GGGCTGGTTATTCTGGGC
AGGCGATGTCATTCCTGTTC
61.5
EU828796
GPx1b
TTTTGTCCTTGAAGTATGTCCGTC
GGGTCGTTCATAAAGGGCATT
GPx4a
TACGCTGAGAGAGGTTTACACAT
CTTTTCCATTGGGTTGTTCC
GPx4b
CTGGAGAAATACAGGGGTTACG
CTCCTGCTTTCCGAACTGGT
GSTR
TCTCAAGGAACCCGTCTG
CCAAGTATCCGTCCCACA
GSTP1
ACAGTTGCCCAAGTTCCAG
GSTP2
RI PT
Caspase-9
ACCEPTED MANUSCRIPT
KT757315
60.4
KU255598
60.3
KU255599
58.4
EU107283
CCTCACAGTCGTTTTTTCCA
59.3
KM112099
TGCCTTGAAGATTATGCTGG
GCTGGCTTTTATTTCACCCT
59.3
KP125490
GSTO1
GGTGCTCAATGCCAAGGGAA
CTCAAACGGGTCGGATGGAA
58.4
KT757314
GSTO2
CTGCTCCCATCAGACCCATTT
TCTCCCCTTTTCTTGCCCATA
61.4
KU245630
GR
GTGTCCAACTTCTCCTGTG
ACTCTGGGGTCCAAAACG
59.4
JX854448
Nrf2
CTGGACGAGGAGACTGGA
ATCTGTGGTAGGTGGAAC
62.5
KF733814
Keap1a
TTCCACGCCCTCCTCAA
TGTACCCTCCCGCTATG
63.0
KF811013
Keap1b
TCTGCTGTATGCGGTGGGC
CTCCTCCATTCATCTTTCTCG
57.9
KJ729125
β-actin
GGCTGTGCTGTCCCTGTA
GGGCATAACCCTCGTAGAT
61.4
M25013
TE D
M AN U
SC
60.3
EP
1246
ATCTGGTTGAAATCCGTGAA
AC C
1245
Caspase-8
1249
1250
1251
1252 58
1253
ACCEPTED MANUSCRIPT
Table 3
1254 Dietary vitamin C levels (mg/kg diet)
44.2
89.1
133.8
179.4
224.5
IBW2
264.56±0.84
264.67±0.67
264.11±0.38
264.00±0.33
264.33±1.20
264.56±0.51
FBW2
784.67±8.50a
874.00±19.67b
1024.00±18.03c
1031.33±6.35c
1025.33±14.29c
1025.67±29.30c
PWG2
196.60±3.29a
230.22±6.97b
287.71±6.29c
290.66±2.84c
287.88±3.64c
287.69±10.61c
SGR2
1.55±0.02a
1.71±0.03b
1.94±0.02c
1.95±0.01c
1.94±0.01c
1.94±0.04c
FI2
728.95±0.43a
811.95±0.42b
943.98±0.71c
946.36±0.23c
FE2
71.35±1.16a
75.04±2.36b
Weight3
1.412±0.104a
1.864±0.137b
Index3
0.181±0.014a
0.213±0.018b
VC concentration4
7.25±0.61a
14.53±0.58b
Weight3
1.107±0.080a
Index3
0.141±0.007a
Skin
SC
M AN U
80.43±1.42c
80.42±3.04c
2.200±0.137c
2.212±0.248c
2.211±0.152c
2.209±0.152c
0.214±0.015b
0.214±0.019b
0.215±0.014b
0.215±0.012b
18.16±0.84c
19.16±1.54c
23.04±1.38d
23.44±1.37d
1.340±0.091b
1.573±0.122c
1.593±0.110c
1.587±0.083c
1.587±0.092c
0.153±0.008b
0.153±0.006b
0.154±0.007b
0.155±0.007b
0.155±0.007b
TE D
EP
VC concentration4
946.44±0.24c
81.08±0.70c
AC C
VC concentration4
946.22±0.46c
80.50±1.92c
Head kidney
Spleen
RI PT
2.9
13.81±1.05a
28.79±1.05b
42.20±3.35c
42.75±2.83c
42.10±2.49c
42.96±2.36c
3.47±0.25a
5.09±0.39b
8.37±0.47c
9.24±0.52d
8.97±0.27d
9.50±0.63d
Regression
YFI = 2.5010X + 714.7500
Ymax = 945.75
R2 = 0.989
P = 0.07
YFE = 0.1064X + 70.8010
Ymax = 80.61
R2 = 0.992
P = 0.06
59
Ymax = 1.94 ACCEPTED MANUSCRIPT
YSGR = 0.0045X + 1.5275
R2 = 0.988
P = 0.07
YVC concentration in head kidney = 0.0814X + 9.1096
Ymax = 23.24
R2 = 0.915
P
0.05
Yspleen weight = 0.0054X + 1.0947
Ymax = 1.585
R2 = 0.999
P
0.05
YVC concentration in spleen = 0.3289X + 13.3340
Ymax = 42.50
R2 = 0.997
P
0.05
YVC concentration in skin = 0.0470X + 3.3667
Ymax = 9.24
SC
1258
1259
1260
1267
1268
EP AC C
1266
TE D
1261
1265
R2 = 0.957
M AN U
1257
1264
RI PT
Ymax = 2.208
1256
1263
P = 0.05
Yhead kidney weight = 0.0091X + 1.4114
1255
1262
R2 = 0.994
1269
1270
1271
60
P
0.05
1272
ACCEPTED MANUSCRIPT
Table 4
1273 Dietary vitamin C levels (mg/kg diet)
2.9
44.2
89.1
133.8
LA
133.83±13.01a
182.07±12.49b
200.17±5.46c
251.36±11.43d
ACP
545.42±16.32a
567.81±24.21a
607.23±20.80b
637.00±20.72c
C3
53.18±3.23a
62.41±4.83b
83.24±6.76c
95.92±7.67d
C4
3.05±0.22a
3.84±0.29b
4.57±0.42c
5.05±0.48d
LA
92.68±6.38a
239.99±17.38b
241.21±16.56b
ACP
575.51±25.83a
624.99±14.44b
C3
101.91±5.96a
119.54±9.15b
C4
7.81±0.56a
9.37±0.78b
179.4
224.5
RI PT
Head kidney 240.99±13.94d
651.99±21.35c
650.51±23.08c
97.81±8.88d
100.77±7.31d
SC
244.72±13.07d
5.22±0.21d
238.30±17.34b
248.78±15.71b
254.75±19.48b
611.33±31.06b
624.96±20.14b
612.67±26.39b
621.23±30.32b
132.28±4.12c
133.02±11.27c
130.63±9.45c
130.64±4.53c
11.00±0.52cd
11.46±0.56d
11.43±0.54d
YACP in head kidney = 0.7188X + 540.8400
Ymax = 651.25
R2 = 0.993
P
0.01
YC3 in head kidney = 0.3412X + 50.6580
Ymax = 98.17
R2 = 0.984
P
0.01
YC3 in spleen = 0.3513X + 101.9600
Ymax = 131.64
R2 = 0.986
P = 0.07
YC4 in head kidney = 0.0153X + 3.0914
Ymax = 5.17
R2 = 0.985
P
TE D
Spleen
M AN U
5.23±0.42d
AC C
EP
Regression
10.41±0.60c
1274
1275
1276
1277 61
0.01
1278
ACCEPTED MANUSCRIPT
Table 5
1279 Dietary vitamin C levels (mg/kg diet)
2.9
44.2
89.1
133.8
179.4
ROS
100.00±5.26c
81.21±7.83b
64.18±5.65a
61.02±3.60a
MDA
5.01±0.41d
4.53±0.30c
4.17±0.12b
3.63±0.19a
PC
5.88±0.18b
4.30±0.22a
4.12±0.31a
4.19±0.18a
AHR
151.23±12.51a
160.76±13.09a
175.50±14.41b
180.50±11.24b
ASA
50.98±4.09a
59.82±2.96b
67.28±4.53c
T-SOD
4.74±0.13a
6.82±0.33b
CuZnSOD
3.00±0.06a
MnSOD
224.5
RI PT
Head kidney 59.84±4.87a
3.66±0.23a
3.61±0.21a
4.21±0.14a
4.38±0.13a
SC
60.36±5.14a
177.46±9.17b
77.20±4.46d
76.61±5.30d
78.71±4.82d
7.62±0.33c
7.63±0.30c
7.44±0.40c
7.40±0.30c
3.84±0.22b
4.32±0.14c
4.40±0.14c
4.27±0.18c
4.33±0.19c
1.74±0.13a
2.97±0.28b
3.29±0.23b
3.23±0.30b
3.17±0.31b
3.07±0.29b
CAT
0.86±0.06a
0.91±0.04ab
0.96±0.09b
1.09±0.10c
1.07±0.02c
1.09±0.04c
GPx
187.76±14.80a
225.94±16.45b
223.97±17.21b
234.01±15.18b
227.09±19.73b
231.79±20.80b
GST
35.92±3.16a
36.27±2.00a
40.66±3.59b
41.98±3.78b
42.63±3.23b
42.40±1.59b
GR
13.85±0.71a
15.52±0.41b
17.24±0.42c
17.81±0.77c
18.19±1.62c
17.91±0.77c
AC C
EP
TE D
M AN U
176.97±4.86b
0.98±0.08a
0.99±0.09a
1.25±0.12b
1.24±0.11b
1.40±0.09c
1.39±0.09c
100.00±7.43d
84.91±7.10c
74.86±6.92b
67.90±6.38ab
66.39±6.48a
66.20±3.28a
MDA
5.97±0.27c
4.85±0.92b
4.30±0.31a
4.37±0.23a
4.39±0.38a
4.41±0.10a
PC
4.01±0.19b
3.62±0.26a
3.64±0.09a
3.59±0.17a
3.53±0.24a
3.68±0.41a
AHR
165.53±13.32a
171.05±13.37a
167.56±12.96a
166.03±8.85a
193.13±19.29b
193.89±9.65b
GSH
Spleen
ROS
62
86.53±3.79 85.15±2.84 ACCEPTED MANUSCRIPT
82.07±5.87b
b
T-SOD
4.38±0.15a
5.15±0.11b
6.45±0.25c
CuZnSOD
2.63±0.08a
2.91±0.10b
MnSOD
1.74±0.11a
CAT
b
84.42±7.09b
86.75±1.56b
7.79±0.25d
7.97±0.32d
7.90±0.13d
3.52±0.24c
4.06±0.10d
4.32±0.11e
4.34±0.20e
2.23±0.18b
2.92±0.20c
3.73±0.25d
3.66±0.24d
3.56±0.20d
0.48±0.03a
0.50±0.04a
0.67±0.05b
0.65±0.05b
0.67±0.03b
0.68±0.04b
GPx
182.37±11.82a
199.20±13.73b
203.37±14.46b
211.67±13.24b
240.97±18.51c
241.23±4.07c
GST
26.70±2.11a
29.96±2.86b
30.99±2.90b
31.14±1.50b
31.34±2.56b
31.06±2.55b
GR
18.32±1.04a
19.39±1.14b
20.27±0.74b
20.18±0.59b
20.14±0.71b
20.18±0.82b
GSH
1.31±0.10a
1.34±0.05ab
1.45±0.13ab
1.46±0.12b
RI PT
64.61±5.80a
SC
ASA
1.98±0.15c
Ymin = 61.35
R2 = 0.997
P
Ymin = 4.37
R2 = 0.953
P = 0.14
Ymax = 77.51
R2 = 0.997
P
0.01
Ymax = 7.89
R2 = 0.990
P
0.01
Ymax = 4.33
R2 = 0.968
P=0.12
Ymax = 4.33
R2 = 0.982
P
0.01
Ymax = 3.65
R2 = 0.992
P
0.01
YCAT in head kidney = 0.0017X + 0.8405
Ymax = 1.08
R2 = 0.940
P
0.05
YGPx in spleen = 0.2933X + 181.1500
Ymax = 241.10
R2 = 0.909
P
0.05
YGR in head kidney = 0.0393X + 13.7520
Ymax = 17.79
R2 = 0.999
P
0.05
YGSH in head kidney = 0.0025X + 0.9502
Ymax = 1.40
R2 = 0.899
P
0.05
YGSH in spleen = 0.0031X + 1.2179
Ymax = 1.96
R2 = 0.744
P = 0.06
M AN U
1.94±0.13c
Regression
YROS in head kidney = -0.415X + 100.6400 YMDA in spleen = -0.0193X + 5.9149
YT-SOD in spleen = 0.0264X + 4.1606 YCuZnSOD in head kidney = 0.0152X + 3.0278
AC C
YMnSOD in spleen = 0.0152X + 1.6261
EP
YCuZnSOD in spleen = 0.0102X + 2.5679
TE D
YASA in head kidney = 0.1967X + 50.5400
1280
63
0.05
1281
ACCEPTED MANUSCRIPT
Table 6
1282 Dietary vitamin C levels (mg/kg diet)
44.2
89.1
133.8
179.4
224.5
LA
91.94±6.28a
91.58±5.47a
148.59±5.59b
150.32±8.00b
143.53±7.29b
144.31±8.19b
ACP
248.65±12.62a
249.43±12.80a
259.48±6.91a
262.66±18.03a
C3
12.18±0.90a
15.44±0.87b
16.35±1.22b
16.07±0.99b
C4
2.74±0.27a
2.95±0.23a
3.36±0.29b
3.39±0.30b
1284
1285
1286
EP AC C
1290
TE D
1287
1289
267.41±17.13a
262.24±15.84a
15.20±1.23b
16.11±0.73b
3.35±0.20b
3.38±0.14b
SC M AN U
1283
1288
RI PT
2.9
64
1291
ACCEPTED MANUSCRIPT
Table 7
1292
1294
44.2
89.1
133.8
179.4
224.5
ROS
100.00±8.30d
67.19±4.85c
39.22±2.60b
33.55±2.83a
32.16±2.12a
32.55±3.14a
MDA
11.98±0.69c
11.69±0.88c
7.11±0.51b
4.61±0.38a
PC
4.48±0.12d
4.44±0.38d
3.66±0.21c
2.93±0.22b
AHR
106.33±4.33a
116.43±9.41b
147.15±9.48c
145.31±5.26c
ASA
59.49±4.39a
65.64±4.14b
80.66±5.51c
84.47±7.04c
T-SOD
16.85±0.15a
17.04±0.18ab
17.24±0.36b
CuZnSOD
9.26±0.38a
9.33±0.30a
MnSOD
7.58±0.29a
CAT
RI PT
2.9
4.70±0.37a
2.37±0.20a
2.60±0.19a
150.94±8.77c
146.27±5.06c
81.16±5.57c
80.30±3.63c
18.36±0.16c
18.17±0.21c
18.28±0.21c
9.39±0.36a
10.59±0.55b
10.75±0.25b
10.89±0.67b
7.71±0.36a
7.85±0.37a
7.77±0.56a
7.41±0.36a
7.39±0.64a
1.70±0.11a
1.82±0.17ab
1.88±0.10b
1.90±0.11b
1.97±0.12b
1.95±0.08b
GPx
88.76±7.82a
106.13±6.49b
119.08±9.33c
130.48±10.79cd
133.78±11.96d
136.64±11.52d
GST
44.91±2.89a
51.00±3.05b
51.10±4.24b
52.58±4.94b
53.79±4.65b
53.72±2.68b
GR
31.94±2.39a
35.85±3.23b
38.64±3.37b
38.11±1.51b
39.00±2.01b
39.08±2.74b
GSH
6.97±0.35a
7.12±0.69a
10.12±0.44b
10.43±0.87b
10.38±0.54b
10.15±0.40b
M AN U
TE D
EP
SC
4.75±0.43a
AC C
1293
Dietary vitamin C levels (mg/kg diet)
1295
1296
1297
1298 65
1299
ACCEPTED MANUSCRIPT
Table 8
1300 Correlation coefficients
NF-κB p65
IL-4/13A
-0.973
0.01
IL-10
-0.905
0.05
IL-11
-0.991
0.01
TGF-β1
-0.963
0.01
TGF-β2
-0.976
0.01
RI PT
Independent parameters
SC
Head kidney
Dependent parameters
IL-8
0.990
M AN U
IL-6
0.01
IL-12 P40
0.947
0.01
IL-15
0.904
0.05
0.992
0.01
NF-κB p65
-0.957
0.01
IKKα
-0.980
0.01
IKKβ
-0.989
0.01
IKKγ
-0.911
0.05
IL-1β
0.938
0.01
TNF-α
0.972
0.01
IFN-γ2
0.884
0.05
IL-4/13A
-0.968
0.01
IL-10
-0.994
0.01
IL-11
-0.843
0.05
TE D EP
AC C TOR
Spleen
NF-κB p65
0.01
0.967
IL-17D IκBα
P
66
1303
1304
1305
1306
TGF-β2
-0.935
0.01
IL-6
0.983
0.01
IL-8
0.927
0.01
IL-12 P40
0.971
0.01
IL-15
0.981
0.01
IL-17D
0.955
0.01
NF-κB p65
-0.948
0.01
SC
RI PT
0.01
IKKβ IKKγ TOR
IL-1β
TE D
TNF-α
-0.919
M AN U
IKKα
IFN-γ2
EP
1302
IκBα
-0.960
AC C
1301
TGF-β1 ACCEPTED MANUSCRIPT
1307
1308
1309
67
0.01
-0.845
0.05
-0.909
0.05
0.977
0.01
0.989
0.01
0.994
0.01
1310
ACCEPTED MANUSCRIPT
Table 9
1311 Correlation coefficients
CuZnSOD activity
CuZnSOD mRNA level
0.944
0.01
MnSOD activity
MnSOD mRNA level
0.852
0.05
CAT activity
CAT mRNA level
0.863
0.05
GPx activity
GPx1a mRNA level
0.866
0.05
GPx1b mRNA level
0.934
0.01
0.838
0.05
0.889
0.05
GSTR mRNA level
0.923
0.01
GSTP1 mRNA level
0.960
0.01
GSTP2 mRNA level
0.941
0.01
GR mRNA level
0.957
0.01
CuZnSOD
0.973
0.01
MnSOD
0.975
0.01
CAT
0.976
0.01
GPx1a
0.984
0.01
GPx1b
0.996
0.01
GPx4a
0.936
0.01
GPx4b
0.982
0.01
GSTR
0.977
0.01
GSTP1
0.987
0.01
EP
Nrf2
P
GPx4b mRNA level
TE D
GST activity
GR activity
SC
M AN U
GPx4a mRNA level
RI PT
Independent parameters
AC C
Head kidney
Dependent parameters
68
GSTP2 ACCEPTED MANUSCRIPT GR
0.932
0.01
Keap1a
-0.941
0.01
Keap1b
-0.994
0.01
CuZnSOD activity
CuZnSOD mRNA level
0.958
0.01
MnSOD activity
MnSOD mRNA level
0.923
0.01
CAT activity
CAT mRNA level
0.941
0.01
GPx activity
GPx1a mRNA level
0.754
SC
0.05
0.861
0.05
GSTR mRNA level
0.943
0.01
GSTP1 mRNA level
0.998
0.01
GSTP2 mRNA level
0.951
0.01
GR mRNA level
0.993
0.01
CuZnSOD
0.981
0.01
MnSOD
0.949
0.01
CAT
0.969
0.01
GPx1a
0.959
0.01
GPx1b
0.983
0.01
GPx4a
0.900
0.05
GPx4b
0.989
0.01
GSTR
0.968
0.01
GSTP1
0.964
0.01
EP
Nrf2
0.08
GPx4b mRNA level
TE D
GST activity
GR activity
0.813
M AN U
GPx1b mRNA level
RI PT
0.01
AC C
Spleen
0.957
69
GSTP2 ACCEPTED MANUSCRIPT
0.966
0.01
GR
0.950
0.01
Keap1a
-0.988
0.01
Keap1b
-0.944
0.01
RI PT
1312
1313
1314
SC
1315
M AN U
1316
1317
1318
1319
1323
1324
1325
1326
1327
EP
1322
AC C
1321
TE D
1320
1328
1329
1330
70
1331
ACCEPTED MANUSCRIPT
Table 10
1332 Correlation coefficients
FasL
caspase-8
0.950
0.01
p38 MAPK
0.977
0.01
MAPKK 6
p38 MAPK
0.968
0.01
Apaf-1
caspase-3
0.959
0.01
caspase-7
0.859
0.05
0.849
0.05
RI PT
Independent parameters
SC
Head kidney
Dependent parameters
Bcl-2
-0.987
0.01
0.941
0.01
caspase-3
-0.991
0.01
caspase-7
-0.979
0.01
caspase-9
-0.974
0.01
caspase-8
0.961
0.01
p38 MAPK
0.984
0.01
p38 MAPK
0.956
0.01
caspase-3
0.995
0.01
caspase-7
0.966
0.05
caspase-9
0.944
0.01
Bcl-2
-0.966
0.01
Bax
0.996
0.01
caspase-3
-0.986
0.01
Bax
EP
FasL
MAPKK 6
AC C
Spleen
TE D
IAP
M AN U
caspase-9
Apaf-1
IAP
P
71
caspase-7 ACCEPTED MANUSCRIPT caspase-9
-0.981
0.01
-0.946
0.01
1333
1334
RI PT
1335
1336
1337
SC
1338
M AN U
1339
1340
1341
1342
1346
1347
1348
1349
1350
EP
1345
AC C
1344
TE D
1343
1351
1352
1353
72
1354
ACCEPTED MANUSCRIPT
Table 11
1355 Correlation coefficients
MLCK
ZO-1
-0.983
0.01
ZO-2
-0.986
0.01
Claudin-b
-0.924
0.05
Claudin-c
-0.951
0.01
Claudin-3c
-0.853
0.05
RI PT
Independent parameters
SC
Head kidney
Dependent parameters
-0.929
M AN U
Claudin-7a
0.01
-0.943
0.01
Claudin-12
0.976
0.01
Claudin-15a
0.980
0.01
Claudin-15b
0.947
0.01
ZO-1
-0.993
0.01
ZO-2
-0.993
0.01
Claudin-b
-0.937
0.01
Claudin-c
-0.998
0.01
Claudin-3c
-0.991
0.01
Claudin-7a
-0.891
0.05
Claudin-7b
-0.999
0.01
Claudin-12
0.927
0.01
Claudin-15a
0.997
0.01
Claudin-15b
0.957
0.01
TE D
Claudin-7b
EP
TNF-α
AC C
Spleen
P
73
1356
Fig.1. Effects of dietary vitamin C ACCEPTED levels on survival rate (A) and skin lesion mrobidity (B) of young MANUSCRIPT
1357
grass carp (Ctenopharyngodon idella) after infection with A. hydrophila for 14 days.
1358
Fig.2. Deficiency of vitamin C led to obviously skin lesion, compared with optimal vitamin C
1360
supplementation, after infection with A. hydrophila in young grass carp.
RI PT
1359
1361
Fig.3. Relative expression of LEAP-2A, LEAP-2B, Hepcidin and β-defensin in the head kidney (A)
1363
and spleen (B) of young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of
1364
vitamin C after infection with A. hydrophila for 14 days. Data represent means of six fish in each group,
1365
error bars indicate S.D. Values having different letters are significantly different (P < 0.05). LEAP-2: liver
1366
expressed antimicrobial peptide 2.
1367
M AN U
SC
1362
Fig.4. Relative expression of IL-4/13A, IL-4/13B, IL-10, IL-11, TGF-β1 and TGF-β2 in the head
1369
kidney (A) and spleen (B) of young grass carp (Ctenopharyngodon idella) fed diets containing graded
1370
levels of vitamin C after infection with A. hydrophila for 14 days. Data represent means of six fish in
1371
each group, error bars indicate S.D. Values having different letters are significantly different (P < 0.05). IL:
1372
interleukin; TGF-β: transforming growth factor β.
EP
AC C
1373
TE D
1368
1374
Fig.5. Relative expression of IFN-γ2, TNF-α, IL-1β, IL-6, IL-8, IL-12 P35, IL-12 P40, IL-15 and
1375
IL-17D in the head kidney (A) and spleen (B) of young grass carp (Ctenopharyngodon idella) fed diets
1376
containing graded levels of vitamin C after infection with A. hydrophila for 14 days. Data represent
1377
means of six fish in each group, error bars indicate S.D. Values having different letters are significantly
1378
different (P < 0.05). IFN-γ2: interferon γ2; TNF-α: tumor necrosis factor α. 74
Fig.6. Relative expression of NF-κBACCEPTED p65, NF-κB p52, IκBα, IKKα, IKKβ, IKKγ, TOR, S6K1, 4E-BP1 MANUSCRIPT
1380
and 4E-BP2 in the head kidney (A) and spleen (B) of young grass carp (Ctenopharyngodon idella) fed
1381
diets containing graded levels of vitamin C after infection with A. hydrophila for 14 days. Data
1382
represent means of six fish in each group, error bars indicate S.D. Values having different letters are
1383
significantly different (P < 0.05). NF-κB: nuclear factor kappa B; IκBα: inhibitor of κBα; IKK: IκB kinase;
1384
TOR: target of rapamycin; S6K1: ribosomal protein S6 kinase 1; 4E-BP: eIF4E-binding protein.
RI PT
1379
1385
Fig.7. Relative expression of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR,
1387
GSTP1, GSTP2, GSTO1, GSTO2 and GR in the head kidney (A) and spleen (B) of young grass carp
1388
(Ctenopharyngodon idella) fed diets containing graded levels of vitamin C after infection with A.
1389
hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate S.D. Values
1390
having different letters are significantly different (P < 0.05). CuZnSOD: copper, zinc superoxide dismutase;
1391
MnSOD: manganese superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GST: glutathione
1392
S-transferase; GR: glutathione reductase.
M AN U
TE D
1393
SC
1386
Fig.8. Relative expression of Nrf2, Keap1a and Keap1b in the head kidney (A) and spleen (B) of young
1395
grass carp (Ctenopharyngodon idella) fed diets containing graded levels of vitamin C after infection
1396
with A. hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate S.D.
1397
Values having different letters are significantly different (P < 0.05). Nrf2: NF-E2-related factor 2; Keap1:
1398
Kelch-like-ECH-associated protein 1.
AC C
EP
1394
1399
1400
Fig.9. Relative expression of FasL, MAPKK 6, p38 MAPK, JNK, Bcl-2, Mcl-1, Bax, Apaf-1, IAP,
1401
caspase-2, caspase-3, caspase-7, caspase-8 and caspase-9 in the head kidney (A) and spleen (B) of 75
young grass carp (Ctenopharyngodon idella) fedMANUSCRIPT diets containing graded levels of vitamin C after ACCEPTED
1403
infection with A. hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate
1404
S.D. Values having different letters are significantly different (P < 0.05). FasL: Fas ligand; MAPKK 6:
1405
mitogen-activated protein kinase kinase 6; p38 MAPK: p38 mitogen-activated protein kinases; JNK: c-Jun
1406
N-terminal kinases; Bcl-2: B-cell lymphoma-2; Mcl-1: myeloid cell leukemia-1; Bax: B-cell lymphoma
1407
protein 2 associated X protein; Apaf-1: apoptotic protease activating factor-1; IAP, inhibitor of apoptosis
1408
protein.
RI PT
1402
SC
1409
Fig.10. Relative expression of Occludin, ZO-1, ZO-2, Claudin -b, -c, -3c, -7a, -7b, -12, -15a, 15b and
1411
MLCK in the head kidney (A) and spleen (B) of young grass carp (Ctenopharyngodon idella) fed diets
1412
containing graded levels of vitamin C after infection with A. hydrophila for 14 days. Data represent
1413
means of six fish in each group, error bars indicate S.D. Values having different letters are significantly
1414
different (P < 0.05). ZO: zonula occludens; MLCK: myosin light chain kinase.
TE D
M AN U
1410
1415
Fig.11. Relative expression of IL-10, TGF-β1, IFN-γ2, TNF-α, IL-1β, IL-8 and NF-κB p65, IκBα,
1417
IKKα, IKKβ, IKKγ, TOR and S6K1 in the skin of young grass carp (Ctenopharyngodon idella) fed
1418
diets containing graded levels of vitamin C after infection with A. hydrophila for 14 days. Data
1419
represent means of six fish in each group, error bars indicate S.D. Values having different letters are
1420
significantly different (P < 0.05).
AC C
1421
EP
1416
1422
Fig.12. Relative expression of CuZnSOD, MnSOD, CAT, GPx1a, GSTR, GR, Nrf2, Keap1a and
1423
Keap1b in the skin of young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of
1424
vitamin C after infection with A. hydrophila for 14 days. Data represent means of six fish in each group, 76
1425
error bars indicate S.D. Values havingACCEPTED different lettersMANUSCRIPT are significantly different (P < 0.05).
1426
Fig.13. Relative expression of caspase-3, -8 and caspase-9 in the skin of young grass carp
1428
(Ctenopharyngodon idella) fed diets containing graded levels of vitamin C after infection with A.
1429
hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate S.D. Values
1430
having different letters are significantly different (P < 0.05).
RI PT
1427
1431
Fig.14. Relative expression of Occludin, ZO-1, Claudin -b, -c, -3c, -12 and Ccaudin-15a in the skin of
1433
young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of vitamin C after
1434
infection with A. hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate
1435
S.D. Values having different letters are significantly different (P < 0.05).
M AN U
SC
1432
1436
Fig.15. Broken-line analysis of PWG (A), LA activity (B) and MDA content (C) in head kidney and C4
1438
content (D) and ROS production (E) in spleen for young grass carp (Ctenopharyngodon idella) fed
1439
diets containing graded levels of vitamin C.
EP
1440
TE D
1437
Fig.16. The potential action pathways of vitamin C in the head kidney and spleen immunity and
1442
structural integrity of fish.
1443
1444
AC C
1441
1445
1446
1447
77
ACCEPTED MANUSCRIPT
Fig.1
RI PT
1448
SC
1449
1453
1454
1455
1456
TE D EP
1452
AC C
1451
M AN U
1450
1457
1458
1459
78
ACCEPTED MANUSCRIPT
SC
RI PT
Fig. 2
M AN U
1460
1464
1465
1466
1467
1468
EP
1463
AC C
1462
TE D
1461
1469
1470
1471
79
ACCEPTED MANUSCRIPT
RI PT
Fig. 3
SC
1472
M AN U
1473
1476
EP AC C
1475
TE D
1474
1477
1478
1479
80
ACCEPTED MANUSCRIPT
RI PT
Fig. 4
SC
1480
M AN U
1481
1484
EP AC C
1483
TE D
1482
1485
1486
1487
81
ACCEPTED MANUSCRIPT
RI PT
Fig. 5
SC
1488
M AN U
1489
1492
EP AC C
1491
TE D
1490
1493
1494
1495
82
ACCEPTED MANUSCRIPT
RI PT
Fig. 6
SC
1496
M AN U
1497
1500
EP AC C
1499
TE D
1498
1501
1502
1503
83
ACCEPTED MANUSCRIPT
RI PT
Fig. 7
SC
1504
M AN U
1505
1508
EP AC C
1507
TE D
1506
1509
1510
1511
84
ACCEPTED MANUSCRIPT
RI PT
Fig. 8
SC
1512
M AN U
1513
1516
EP AC C
1515
TE D
1514
1517
1518
1519
85
ACCEPTED MANUSCRIPT
RI PT
Fig. 9
SC
1520
M AN U
1521
1524
EP AC C
1523
TE D
1522
1525
1526
1527
86
ACCEPTED MANUSCRIPT
RI PT
Fig. 10
SC
1528
M AN U
1529
1532
EP AC C
1531
TE D
1530
1533
1534
1535
87
ACCEPTED MANUSCRIPT
RI PT
Fig. 11
SC
1536
M AN U
1537
1538
1539
1543
1544
1545
1546
1547
EP
1542
AC C
1541
TE D
1540
1548
1549
1550
1551 88
ACCEPTED MANUSCRIPT
RI PT
Fig. 12
SC
1552
M AN U
1553
1554
1555
1559
1560
1561
1562
1563
EP
1558
AC C
1557
TE D
1556
1564
1565
1566
1567 89
ACCEPTED MANUSCRIPT
RI PT
Fig. 13
SC
1568
M AN U
1569
1570
1571
1575
1576
1577
1578
1579
EP
1574
AC C
1573
TE D
1572
1580
1581
1582
1583 90
ACCEPTED MANUSCRIPT
RI PT
Fig. 14
SC
1584
M AN U
1585
1586
1587
1591
1592
1593
1594
1595
EP
1590
AC C
1589
TE D
1588
1596
1597
1598
1599 91
ACCEPTED MANUSCRIPT
Fig. 15
RI PT
1600
M AN U
SC
1601
1604
1605
EP AC C
1603
TE D
1602
1606
1607
1608
1609 92
1614
1615
1616
Vitamin C deficiency
Optimal vitamin C
Inhibition signaling
LA and
receptor pathway:
signaling
than NF-κB P52) and TOR
FasL
molecule:
ACP
Mitochondria
MLCK (only in
activities
pathway: activation
head kidney)
, C3 and
Pro-inflammatory cytokines:
Bcl2 (rather than
C4
TNT-α, IFN-γ2, IL-1β, IL-6,
Mcl-1), and
contents
IL-8, IL-12 P35 (only in spleen),
inhibition Bax
IL 12-P40, IL-15, IL-17D
junctional
Anti-inflammatory cytokines:
complexes
SC
Tight
caspase-2 (only in spleen),
IL-4/13A, IL-4/13B (only in
caspase-3, caspase-7,
M AN U
head kidney), IL-10, IL-11,
caspase-8, caspase-9
TGF-β1, TGF-β2
Antioxidant enzymes mRNA
RI PT
molecules: NF-κB P65 (rather
Activation signaling molecule: Nrf2
Inhibition death
Inhibition
expression: CuZnSOD,
MnSOD, CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTP (rather than GSTO), GR
Antioxidant enzymes activities: CuZnSOD, MnSOD, CAT, GPx, GST and GR
Head kidney and spleen structural integrity
Head kidney and spleen immunity
Fish disease resistance
TE D
1613
Fish
Fish growth performance
EP
1611 1612
ACCEPTED MANUSCRIPT
Fig. 16
AC C
1610
93
ACCEPTED MANUSCRIPT Highlights Compared with optimal vitamin C supplementation:
Vitamin C deficiency decreased fish head kidney, spleen and skin immunity under injection of A. hydrophila. Vitamin C deficiency aggravated inflammation in the head kidney, spleen and skin of fish under injection of A. hydrophila.
RI PT
Vitamin C deficiency induced oxidative injury via disruption of antioxidant system in the head kidney, spleen and skin of fish under injection of A.
SC
hydrophila.
Vitamin C deficiency disturbed tight junctional complexes in the head kidney,
M AN U
spleen and skin of fish under injection of A. hydrophila.
AC C
EP
TE D
Vitamin C regulated NF-κB, TOR, Nrf2, apoptosis and MLCK signaling.
S1050-4648(16)30077-8
DOI:
10.1016/j.fsi.2016.02.033
Reference:
YFSIM 3845
To appear in:
Fish and Shellfish Immunology
Received Date: 14 December 2015 Revised Date:
22 February 2016
Accepted Date: 29 February 2016
Please cite this article as: Xu H-J, Jiang W-D, Feng L, Liu Y, Wu P, Jiang J, Kuang S-Y, Tang L, Tang W-N, Zhang Y-A, Zhou X-Q, Dietary vitamin C deficiency depresses the growth, head kidney and spleen immunity and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK signaling in young grass carp (Ctenopharyngodon idella), Fish and Shellfish Immunology (2016), doi: 10.1016/ j.fsi.2016.02.033. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Dietary vitamin C deficiency depresses theMANUSCRIPT growth, head kidney and spleen immunity ACCEPTED
2
and structural integrity by regulating NF-κB, TOR, Nrf2, apoptosis and MLCK
3
signaling in young grass carp (Ctenopharyngodon idella)
4 a,1
, Lin Feng
a,b,c
, Yang Liu a,b,c , Pei Wu
a,b,c
Hui-Jun Xu
6
Kuang d, Ling Tang d, Wu-Neng Tang d, Yong-An Zhang e, Xiao-Qiu Zhou a,b,c,*
, Jun Jiang
a,b,c
, Sheng-Yao
RI PT
, Wei-Dan Jiang
a,b,c,1
5
7 a
Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China
9
b
Fish Nutrition and safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural
SC
8
University, Chengdu 611130, China
11
c
12
Agricultural University, Chengdu 611130, China
13
d
Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China
14
e
Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
M AN U
10
TE D
Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan
15
* Co-corresponding authors. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130,
17
Sichuan, China. E-mail: [email protected], [email protected] (X.-Q. Zhou).
19
20
1
These two authors contributed to this work equally
AC C
18
EP
16
1
21
ACCEPTED MANUSCRIPT
Abstract:
This study investigated the effects of dietary vitamin C on the growth, and head kidney, spleen and skin
23
immunity, structural integrity and related signaling molecules mRNA expression levels of young grass carp
24
(Ctenopharyngodon idella). A total of 540 grass carp (264.37 ± 0.66 g) were fed six diets with graded levels
25
of vitamin C (2.9, 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet) for 10 weeks. Subsequently, a challenge
26
test was conducted by injection of Aeromonas hydrophila and the survival rate recorded for 14 days. The
27
results indicated that compared with optimal vitamin C supplementation, vitamin C deficiency (2.9 mg/kg
28
diet) decreased lysozyme (LA) and acid phosphatase (ACP) activities, and complement 3 and complement 4
29
(C4) contents (P < 0.05), down-regulated the mRNA levels of antimicrobial peptides [liver expressed
30
antimicrobial peptide (LEAP) 2A, LEAP-2B, hepcidin, β-defensin] and anti-inflammatory cytokines-related
31
factors, interleukin (IL) 4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, transforming growth factor
32
(TGF) β1, TGF-β2, inhibitor of κBα and eIF4E-binding protein 1 (P < 0.05), and up-regulated
33
pro-inflammatory cytokines-related factors, tumor necrosis factor α, interferon γ2, IL-1β, IL-6, IL-8, IL-12
34
P35 (only in spleen), IL-12 P40, IL-15, IL-17D, nuclear factor κB p65, IκB kinases (IKKα, IKKβ, IKKγ),
35
target of rapamycin and ribosomal protein S6 kinase 1 mRNA levels (P < 0.05) in the head kidney and
36
spleen under injection fish of A. hydrophila, suggesting that vitamin C deficiency could decrease fish head
37
kidney and spleen immunity and cause inflammation. Meanwhile, compared with optimal vitamin C
38
supplementation, vitamin C deficiency decreased the activities and mRNA levels of copper/zinc superoxide
39
dismutase, manganese superoxide dismutase (MnSOD), catalase, glutathione peroxidase, glutathione
40
S-transferases and glutathione reductase (P < 0.05), and down-regulated zonula occludens (ZO) 1, ZO-2,
41
Claudin-b, -c, -3c, -7a, -7b, B-cell lymphoma-2, inhibitor of apoptosis protein, NF-E2-related factor 2
42
mRNA levels (P < 0.05), increased reactive oxygen species (ROS), malondialdehyde (MDA) and protein
43
carbonyl contents (P < 0.05), and up-regulated Claudin-12, 15a, -15b, Fas ligand, mitogen-activated protein
AC C
EP
TE D
M AN U
SC
RI PT
22
2
kinase kinase 6, p38 mitogen-activated protein kinase, B-cell lymphoma protein 2 associated X protein, ACCEPTED MANUSCRIPT
45
apoptotic protease activating factor-1, caspase-3, -7, -8, -9, Kelch-like ECH-associating protein (Keap) 1a
46
and Keap 1b mRNA levels (P < 0.05) in the head kidney and spleen under injection fish of A. hydrophila,
47
suggesting that vitamin C deficiency could decrease fish head kidney and spleen structural integrity through
48
depression of antioxidative ability, induction of apoptosis and disruption of tight junctional complexes. In
49
addition, except the activities of ACP and MnSOD, and mRNA expression levels of TGF-β1, Occludin and
50
MnSOD, the effect of vitamin C on fish head kidney, spleen and skin immunity and structural integrity other
51
indicators model are similar under infection of A. hydrophila. Finally, the vitamin C requirement for the
52
growth performance (PWG) of young grass carp was estimated to be 92.8 mg/kg diet. Meanwhile, the
53
vitamin C requirement for against skin lesion morbidity of young grass carp was estimated to be 122.9
54
mg/kg diet. In addition, based on the biochemical indices [immune indices (LA activity in the head kidney
55
and C4 content in the spleen) and antioxidant indices (MDA content in the head kidney and ROS content in
56
the spleen)] the vitamin C requirements for young grass carp were estimated to be 131.2, 137.5, 135.8 and
57
129.8 mg/kg diet, respectively.
58
Keywords: Vitamin C; Immunity; Structural integrity; Head kidney; Spleen; Young grass carp
59
(Ctenopharyngodon idella)
61
SC
M AN U
TE D
EP
AC C
60
RI PT
44
3
62
1. Introduction
ACCEPTED MANUSCRIPT
Vitamin C is an essential nutrient for the normal growth of fish [1]. It was reported that vitamin C
64
deficiency could lead to spinal deformation in juvenile cobia (Rachycentron canadum) [2], and scoliosis,
65
lordosis and caudal fin erosion in Japanese seabass (Lateolabrax japonicas) [3]. Meanwhile, study from our
66
lab showed that vitamin C deficiency decreased growth performance in juvenile Jian carp (Cyprinus carpio
67
var. Jian) [4]. Fish growth is often related to disease resistance [5], which often relies on their immunity [6].
68
Up to now, limited studies demonstrated that vitamin C deficiency depressed serum lysozyme activity (LA)
69
in juvenile cobia (Rachycentron canadum) [2]. Further investigation is required to study the effects of
70
vitamin C on immunity and it possible regulation mechanism in fish.
SC
RI PT
63
Fish immunity is related to the structure and function of its immune organs [7]. Skin is an important
72
mucosa immune organ in zebrafish [8]. It was reported that vitamin C deficiency led to skin lesion in Nile
73
tilapia (Oreochromis niloticus L.) [9]. Fish easily infected by A. hydrophila after skin lesion [10], and the
74
infection ultimately spread to the lymphatic immune organs [11]. Head kidney and spleen are two important
75
lymphatic immune organs in fish [12]. In fish, the immune function of head kidney, spleen and skin are
76
related to antibacterial compounds and cytokines [13-15]. In human, cytokines could be regulated by the
77
inhibitor of κBα/nuclear factor κB (IκBα/NF-κB) signaling pathway [16] and target of rapamycin/ribosomal
78
protein S6 kinase 1 (TOR/S6K1) signaling pathway [17]. However, up to now, no study concerns the effects
79
of vitamin C on antibacterial compounds, cytokines and the IκBα/NF-κB and TOR/S6K1 signaling pathways
80
in the immune organs of fish. It was reported that vitamin C could enhance the proliferative response of
81
lymphocytes in hen [18]. In fish, lymphocytes could secrete the humoral compounds (such as LA and C3)
82
[19]. Meanwhile, it was reported that vitamin C could improve intestinal iron uptake in human [20]. Dietary
83
iron could up-regulate the mRNA level of hepcidin in rat [21]. In addition, vitamin C could elevate red blood
84
cell glutathione in human [22]. Rahman et al. [23] reported that glutathione could inhibit NF-κB signaling
AC C
EP
TE D
M AN U
71
4
pathway activation in THP-1 cells. ACCEPTED Meanwhile, vitamin C could decrease the total plasma cholesterol MANUSCRIPT
86
content in fish [24]. In human HUVECs cells, cholesterol could activate mTOR signaling pathway [25].
87
Accordingly, there may be a possible relationship between vitamin C and humoral compounds, antimicrobial
88
peptides, cytokines and IκBα/NF-κB and TOR/S6K1 signaling pathways in fish, which remains to be
89
elucidated.
RI PT
85
In fish, the immunity is correlated with structural integrity of immune organ [26]. Structural integrity of
91
fish immune organ is associated with organ cellular membrane integrity, which can be destructed by
92
oxidative injury and cell apoptosis [27]. However, no study has addressed the effects of vitamin C on
93
oxidative injury, apoptosis and their possible signaling pathway in the head kidney, spleen and skin of fish.
94
Moreover, a previous study showed that oxidation injury could combat by non-enzymatic compounds and
95
antioxidant enzymes [28]. Vitamin C is an important non-enzymatic compound in green-lipped mussels (P.
96
viridis) [29]. It was reported that dietary vitamin C increased the vitamin C concentration of large yellow
97
croaker (Pseudosciaena crocea) head kidney [30] and rainbow trout spleen [31], and antioxidant enzyme
98
activities of juvenile cobia (Rachycentron canadum) serum [2] and Wuchang bream (Megalobrama
99
amblycephala Yih) liver [32]. Antioxidant enzyme activities were partly dependent on antioxidant enzymes
100
gene transcriptions [33], which were regulated by NF-E2-related factor 2/Kelch-like-ECH-associated protein
101
1 (Nrf2/keap1) signaling pathway in fish [34]. However, no study has conducted the effects of vitamin C on
102
skin vitamin C concentration and immune organs (such as head kidney, spleen and skin) antioxidant ability
103
and related molecular mechanisms in fish. In mice, vitamin C could up-regulate the PPARα expression [35].
104
Toyama et al. [36] reported that up-regulated PPARα could increase the antioxidant enzyme catalase (CAT)
105
and superoxide dismutase (SOD) mRNA levels. In human, vitamin C could enhance the synthesis of nitric
106
oxide (NO) [37]. Liu et al. [38] reported that NO could up-regulate Nrf2 gene expression in rat vascular
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smooth muscle cells. In addition, vitamin C could inhibit the production of monocytes tumor necrosis factor
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α (TNF-α) in human [39]. Wang et ACCEPTED al. [40] reported that TNF-α could activate apoptosis-related factor MANUSCRIPT
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caspase-8 in HEK293T cells. Accordingly, there may be a possible relationship between vitamin C and
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antioxidant enzymes, Nrf2/keap1 signaling pathway, and apoptosis and its possible signaling pathway in fish,
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which warrants further investigation. In fish, the structural integrity of immune organ is also associated with the tight junctional complexes
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(TJs) [Occludin, Claudins and zonula occludens (ZO)] in intercellular, which are regulated by myosin light
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chain kinase (MLCK) [41, 42]. However, no study has focused on the effects of vitamin C on TJs and its
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possible signaling pathway in fish. In pig, vitamin C could decrease the plasma cortisol content [43].
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Chasiotis and Kelly [44] demonstrated that cortisol could down-regulate the expression of ZO-1 and
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Claudin-c in the gill of goldfish. It was reported that vitamin C could suppress p38 mitogen-activated protein
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kinase (p38 MAPK) in human [45]. In mice, inhibition of p38 MAPK could prevent MLCK expression [46].
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Therefore, these data suggest a possible correlation between vitamin C and TJs and its possible signaling
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pathway in fish, which remains to be elucidated.
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Grass carp is widely cultivated in the world [47]. The commercial rearing of grass carp relies heavily
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on the use of formulated feed, which is based on the complete information of the nutrient requirement for
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this species [48]. In fish, growth stages include juvenile, young, adult and so on [49]. Up to now, the
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requirements for vitamin C only investigated the juvenile stage and adult stage of grass carp, and mainly
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based on the growth performance. However, vitamin C requirements of fish may vary with different growth
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stage and different indices. The vitamin C requirement in juvenile stage grass carp [50] is higher than that in
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adult stage grass carp [51]. Meanwhile, our lab study has found that the riboflavin requirement for young
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grass carp based on gill LA was higher than that based on percent weight gain (PWG) [52]. Therefore, it is
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valuable to investigate the dietary vitamin C requirements of young grass carp based on growth and other
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indices.
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Here we firstly hypothesis that vitamin C deficiency could depress the immunity of fish head kidney ACCEPTED MANUSCRIPT
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and spleen by aggravation inflammatory response and disturbed the structural integrity. To test this
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hypothesis, we conducted the challenged test with A. hydrophila after feeding trial. Furthermore, we were
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for the first time to test the hypothesis that vitamin C deficiency could aggravate inflammation which was
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related to NF-κB and TOR signaling pathways in the head kidney and spleen of fish. And we were also for
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the first time to test the hypothesis that vitamin C deficiency could disrupt the structural integrity partly
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related the disruption of the integrity of cellular structure and cell-cell TJs these modulated by Nrf2 and
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MLCK signaling in the head kidney and spleen of fish. Meanwhile, the effects of vitamin C on the skin
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immunity are also estimated. In addition, the vitamin C requirements of young grass carp were also
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determined in this study, which may provide partial theoretical evidence for the commercial feed production
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of grass carp.
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2. Materials and methods
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2.1. Experimental diets preparation
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Formulation of the basal diet is shown in Table 1. Fish meal, casein and soybean protein concentrate
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were used as dietary protein sources. Fish oil and soybean oil were used as dietary lipid sources. The dietary
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protein level was fixed at 300 g/kg diet, which was reported to be optimum for the growth of grass carp, as
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described by Khan et al. [47]. Vitamin C was added to the basal diet to provide graded concentrations of 0
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(un-supplemented), 45, 90, 135, 180 and 225 mg vitamin C/kg diet, and the amount of corn starch was
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reduced to compensate, according to our lab study [4]. The vitamin C concentrations of the six diets were
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2.9 (un-supplemented), 44.2, 89.1, 133.8, 179.4 and 224.5 mg/kg diet, determined by the method of Wan et
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al. [53]. After being prepared completely, the diets were stored at -20 °C according to Liu et al. [4].
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(Table 1 inserted here) 2.2. Feeding trial 7
The procedures used in this study were approved by the University of Sichuan Agricultural Animal ACCEPTED MANUSCRIPT
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Care Advisory Committee. Grass carp were obtained from fisheries (Sichuan, China). Prior to the
156
experiment, fish were acclimatized to the experimental environment for 2 weeks. The basal diet
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(un-supplemented with vitamin C diet) was fed to all fish during the conditioning period, according to Zhou
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et al. [2]. Then, 540 fish (mean weight 264.37 ± 0.66 g) were randomly assigned to 18 experimental cages
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(1.4 L × 1.4 W × 1.4 H m), resulting in 30 fish per cage. Each cage was equipped with a disc of 100 cm
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diameter in the bottom to collect the uneaten feed, according to Wu et al. [54]. Fish were fed with their
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respective diets to apparent satiation four times per day, according to our lab study [55]. After feeding 30
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min, uneaten feed was collected, dried and weighed to calculate the feed intake (FI), according to our lab
163
study [55]. During the experimental period, dissolved oxygen was maintained higher than 6.0 mg/L
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throughout the experimental period. The water temperature was averaged at 28 ± 2 °C, pH value was
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maintained at 7.0 ± 0.2. The experimental units were under natural light and dark cycle, according to our lab
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study [56]. The feeding trial lasted for 10 weeks.
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At the initiation and termination of the feeding trial, fish from each cage were weighed for calculate
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PWG. Eighteen fish from each treatment were anaesthetized in a benzocaine bath as described by Affonso et
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al. [57], then the head kidney and spleen of fish were quickly removed and weighed for calculate the index
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of the head kidney and spleen.
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2.3. Challenge test
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After a 10 weeks feeding trial, a challenge test was conducted to study the effect of dietary vitamin C
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on the immune responses of young grass carp. A. hydrophila strain was obtained from College of Veterinary
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Medicine, Sichuan Agricultural University, China. Fifteen fish with a similar body weight collected from
175
each treatment group were moved to labeled cages according to Liu et al. [58]. Fish were infected by
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intraperitoneal injection with A. hydrophila at a concentration of 2.5×108 CFU 1ml/fish, which is similar to 8
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Chen et al. [59]. During the infectious trial, the experimental conditions were the same as those in the ACCEPTED MANUSCRIPT
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feeding trial. The challenge test was conducted for 14 days according to Nya and Austin [60]. Dead fish
179
were removed and counted from each tank daily.
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2.4. Sample collection and analysis after challenge test At the end of the challenge test, fish were anaesthetized in a benzocaine bath as described by Affonso et
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al. [57]. To evaluate the severity of skin lesion, a scoring system and skin lesion morbidity was specifically
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checked and calculated according to the methods described by Séguin et al. [61] and Steenland and
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Armstrong [62], respectively. Then sacrificed, quickly obtained the head kidney, spleen and skin and frozen
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in liquid nitrogen, and then stored at -80 °C until analysis as described by Huang et al. [63]. The head kidney,
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spleen and skin samples were homogenized on ice in 10 volumes (w/v) of ice-cold physiological saline and
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centrifuged at 6000 g at 4ºC for 20 min, then the collected supernatant was stored for the related parameters
188
analysis as described by Safari et al. [64]. The vitamin C concentrations of the head kidney, spleen and skin
189
were determined as described by Wan et al. [53]. LA and acid phosphatase (ACP) activities were assayed as
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Wu et al. [65]. The contents of C3 and C4 were assayed according to Welker et al. [66]. The reactive oxygen
191
species (ROS) content was determined according to Yang et al. [67]. The contents of malondialdehyde
192
(MDA), protein carbonyl (PC) and glutathione (GSH) were determined according to Ji et al. [68]. The CAT
193
activity, anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) capacity were measured as described
194
by Kuang et al. [69]. The total superoxide dismutase (SOD) and MnSOD activities were assayed as
195
described by Durrant et al. [70]. The CuZnSOD, glutathione peroxidase (GPx), CAT, glutathione
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S-transferase (GST) and glutathione reductase (GR) activities were assayed as described by Wen et al. [57],
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Zhang et al. [71], Deng et al. [72], Lushchak et al.[73], and Lora et al. [74], respectively.
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Total RNA samples were isolated from the head kidney, spleen and skin using RNAiso Plus Kit (Takara,
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Dalian, China), then quantity and quality were assessed using spectrophotometry, as described by Luo et al. 9
[55]. The fist-strand cDNA was synthesized using a PrimeScripte RT reagent Kit, according to the ACCEPTED MANUSCRIPT
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manufacturer’s instructions as described by Gan et al. [75]. Specific primers were designed according to the
202
sequences cloned in our laboratory or the published sequences of grass carp (Table 2) for quantitative
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real-time PCR. To verify the specificity and purity of all PCR products, melt curve analysis was performed
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after amplification. According to the results of our preliminary experiment concerning the evaluation of
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internal control genes (data not shown), β-actin was used as a reference gene to normalize cDNA loading.
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The mRNA levels of all the genes were performed on the CFX96TM Real-Time PCR Detection System
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(Bio-Rad, Laboratories, Inc.) according to standard protocols of the primers. The gene expression results
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were analyzed using the 2−∆∆CT method according to Luo et al. [55].
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(Table 2 inserted here) 2.5. Calculations and statistical analysis
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Data on initial body weight (IBW), final body weight (FBW) and feed intake (FI) were used to
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calculate PWG and feed efficiency (FE), as described by Wen et al. [56], and specific growth rate (SGR), as
213
described by Deng et al. [76]. The weight of head kidney and spleen were used to calculate the index of head
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kidney and spleen, respectively, as described by Chen et al. [59]. Survival rate was calculated as described
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by Wu et al. [65].
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PWG =100 × [FBW (g/fish) – IBW (g/fish)]/IBW (g/fish);
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FE = 100 × [FBW (g/fish) – IBW (g/fish)]/FI (g/fish);
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SGR = 100 × Ln [final body weight (g)/initial body weight (g)]/feeding period (day)
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Head kidney index = (g head kidney weight/g body weight)/100;
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Spleen index = (g spleen weight/g body weight)/100;
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Survival rate (%) = (final fish number)/(initial fish number)/100.
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All data were subjected to one-way analysis of variance followed by Duncan’s multiple range tests to
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determine significant differences among treatment groups using the software SPSS 18.0 (SPSS Inc., Chicago, ACCEPTED MANUSCRIPT
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IL, USA) at a level of P < 0.05, as described by Gan et al. [75]. The results are presented as the means ± SD.
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Broken-line model was used to estimate the optimal dietary level of vitamin C for young grass carp
226
according to Liu et al. [4].
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3. Results
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3.1. Growth performance, head kidney and spleen growth, head kidney, spleen and skin vitamin C
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concentration, and survival rate and skin lesion morbidity after infection with A. hydrophila
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As shown in Table 3, fish FBW, PWG, SGR, FI and FE were all significantly improved with dietary
231
vitamin C levels up to 89.1mg/kg diet (P ˂ 0.05), and then plateaued. The head kidney weight, index and
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vitamin C concentration, spleen weight, index and vitamin C concentration, and skin vitamin C
233
concentration were significantly increased with dietary vitamin C levels up to 89.1, 44.2, 179.4, 89.1, 44.2,
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89.1 and 133.8 mg/kg diet (P < 0.05), respectively, and then plateaued. Survival rate (Fig. 1A) after infection
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with A. hydrophila was significantly increased with dietary vitamin C levels up to 44.2 mg/kg diet (P < 0.05),
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and then plateaued. The skin lesion morbidity (Fig. 1B) after infection with A. hydrophila was significantly
237
decreased with dietary vitamin C levels up to 133.8 mg/kg diet (P < 0.05), and then plateaued. Compared
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with optimal vitamin C supplementation, deficiency of vitamin C led to an obvious skin lesion symptom
239
(Fig. 2) after infection with A. hydrophila in young grass carp.
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(Table 3 inserted here)
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(Fig. 1 inserted here)
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(Fig. 2 inserted here)
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3.2. The immune response-related parameters in the head kidney and spleen after infection with A.
244
hydrophila
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The LA and ACP activities, C3 and C4 contents in the head kidney and spleen of young grass carp are 11
presented in Table 4. In the head kidney, LA and ACP activities, and C3 and C4 contents were significantly ACCEPTED MANUSCRIPT
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increased with dietary vitamin C levels up to 133.8, 179.4, 133.8 and 133.8 mg/kg diet (P < 0.05),
248
respectively, and then plateaued. In the spleen, LA and ACP activities, C3 and C4 contents were
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significantly increased with dietary vitamin C levels up to 44.2, 44.2, 89.1 and 133.8 mg/kg diet (P < 0.05),
250
respectively, and then plateaued. The mRNA levels of antimicrobial peptides in the head kidney and spleen
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of young grass carp are displayed in Fig. 3. In the head kidney, the liver expressed antimicrobial peptide 2A
252
(LEAP-2A), liver expressed antimicrobial peptide 2B (LEAP-2B) and hepcidin mRNA levels were
253
up-regulated with dietary vitamin C levels up to 89.1, 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, and
254
then plateaued. The mRNA level of β-defensin was up-regulated with dietary vitamin C levels up to 133.8
255
mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05). In the spleen, the mRNA levels of LEAP-2A,
256
LEAP-2B and hepcidin were up-regulated with dietary vitamin C levels up to 44.2, 89.1 and 44.2 mg/kg diet
257
(P < 0.05), respectively, and then plateaued. The β-defensin mRNA level was increased with dietary vitamin
258
C levels up to 133.8 mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05).
259
(Table 4 inserted here)
260
(Fig. 3 inserted here)
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3.3. The inflammatory response-related parameters in the head kidney and spleen after infection with A.
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hydrophila
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The mRNA levels of anti-inflammatory cytokines, pro-inflammatory cytokines, and inflammatory
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cytokines-related signaling molecules in the head kidney and spleen of young grass carp are displayed in Fig.
265
4, Fig.5 and Fig. 6, respectively. In the head kidney, the interleukin 4/13A (IL-4/13A), interleukin 4/13B
266
(IL-4/13B), interleukin 10 (IL-10), interleukin 11 (IL-11), transforming growth factor β1 (TGF-β1),
267
transforming growth factor β2 (TGF-β2), IκBα and 4EB-P1 mRNA levels were up-regulated with dietary
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vitamin C levels up to 89.1, 89.1, 89.1, 44.2, 89.1, 89.1, 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, 12
and then plateaued. The mRNA levelsACCEPTED of TNF-α, interferon γ2 (IFN-γ2), interleukin 1β (IL-1β), interleukin 6 MANUSCRIPT
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(IL-6), interleukin (IL-8), interleukin 17D (IL-17D), NF-κB p65, IKKα, IKKβ, IKKγ, TOR and S6K1 were
271
down-regulated with dietary vitamin C levels up to 89.1, 179.4, 89.1, 44.2, 133.8, 89.1, 89.1, 44.2, 89.1,
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133.8, 44.2, and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA levels of
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interleukin 12 P40 (IL-12 P40) and interleukin 15 (IL-15) were down-regulated with dietary vitamin C
274
levels up to 179.4 and 133.8 mg/kg diet (P < 0.05), respectively, and increased thereafter (P < 0.05).
275
However, dietary vitamin C had no effect on the mRNA levels of interleukin 12 P35 (IL-12 P35), NF-κB
276
p52 and 4EB-P2 in the head kidney (P > 0.05). In the spleen, the mRNA levels of IL-4/13A, IL-10, TGF-β1,
277
TGF-β2, IκBα and 4EB-P1 were up-regulated with dietary vitamin C levels up to 44.2, 89.1, 89.1, 179.4,
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89.1 and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA levels of TNF-α, IFN-γ2,
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IL-1β, IL-6, IL-8, IL-12 P35, IL-12 P40, IL-15, IL-17D, NF-κB p65, IKKα, IKKγ, TOR and S6K1 were
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down-regulated with dietary vitamin C levels up to 89.1, 89.1, 89.1, 89.1, 179.4, 89.1, 89.1, 89.1, 89.1, 89.1,
281
133.8, 133.8, 89.1 and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA level of
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IKKβ was decreased with dietary vitamin C level (P < 0.05). The IL-11 mRNA level was increased with
283
dietary vitamin C levels up to 133.8 mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05). However,
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dietary vitamin C had no effect on the mRNA levels of IL-4/13B, NF-κB p52 and 4EB-P2 in the spleen (P >
285
0.05).
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(Fig. 4 inserted here)
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(Fig. 5 inserted here)
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(Fig. 6 inserted here)
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3.4. Head kidney and spleen structural integrity after infection of A. hydrophila
290
3.4.1. Antioxidant-related parameters in the head kidney and spleen
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The ROS, MDA, PC and GSH contents, ASA, AHR and antioxidant enzyme activities in the head 13
kidney and spleen are showed in Table 5. In the MANUSCRIPT head kidney, the ROS, MDA and PC contents were ACCEPTED
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significantly decreased with dietary vitamin C levels up to 89.1, 133.8 and 44.2 mg/kg diet (P < 0.05),
294
respectively, and then plateaued. The AHR, ASA, T-SOD, CuZnSOD, MnSOD, CAT, GPx, GST and GR
295
activities and GSH content were significantly increased with dietary vitamin C levels up to 89.1, 133.8, 89.1,
296
89.1, 44.2, 133.8, 44.2, 89.1, 89.1 and 179.4 mg/kg diet (P < 0.05), respectively, and then plateaued. In the
297
spleen, the ROS, MDA and PC contents were significantly decreased with dietary vitamin C levels up to
298
133.8, 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, and then plateaued. The AHR, ASA, T-SOD,
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CuZnSOD, MnSOD, CAT, GPx, GST and GR activities and GSH content were increased with dietary
300
vitamin C levels up to 179.4, 44.2, 133.8, 179.4, 133.8, 89.1, 179.4, 44.2, 44.2 and 179.4 mg/kg diet (P <
301
0.05), respectively, and then plateaued.
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The mRNA levels of antioxidant enzymes, and Nrf2, Keap1a and Keap1b in the head kidney and spleen
303
are showed in Fig. 7 and Fig. 8, respectively. In the head kidney, the CuZnSOD, MnSOD, CAT, GPx1a,
304
GPx1b, GPx4a, GPx4b, GSTR, GSTP1, GSTP2, GR and Nrf2 mRNA levels were up-regulated with dietary
305
vitamin C levels up to 89.1, 89.1, 89.1, 89.1, 44.2, 89.1, 89.1, 44.2, 89.1, 89.1, 89.1 and 44.2 mg/kg diet (P <
306
0.05), respectively, and then plateaued. The mRNA levels of Keap1a and Keap1b were significantly
307
down-regulated with dietary vitamin C levels up to 133.8 and 89.1 mg/kg diet (P < 0.05), respectively, and
308
then plateaued. However, dietary vitamin C had no effect on the mRNA levels of GSTO1 and GSTO2 in the
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head kidney (P > 0.05). In the spleen, the CuZnSOD, MnSOD, CAT, GPx1a, GPx1b, GPx4b, GSTR, GSTP1,
310
GSTP2, GR and Nrf2 mRNA levels were up-regulated with dietary vitamin C levels up to 89.1, 89.1, 89.1,
311
89.1, 44.2, 44.2, 89.1, 44.2, 89.1, 89.1 and 44.2 mg/kg diet (P < 0.05), and then plateaued. The Keap1a and
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Keap1b mRNA levels were all significantly down-regulated with dietary vitamin C level up to 89.1 mg/kg
313
diet (P < 0.05), and then plateaued. The mRNA level of GPx4a was up-regulated with dietary vitamin C
314
levels up to 133.8 mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05). However, dietary vitamin C
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had no effect on the mRNA levels of GSTO1 and GSTO2 in the spleen (P > 0.05). ACCEPTED MANUSCRIPT
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(Table 5 inserted here)
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(Fig. 7 inserted here)
318
(Fig. 8 inserted here) 3.4. 2. Apoptosis-related parameters mRNA levels in the head kidney and spleen
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The effects of dietary vitamin C on apoptosis-related gene expression in the head kidney and spleen are
321
showed in Fig. 9. In the head kidney, the mRNA levels of B-cell lymphoma-2 (Bcl-2) and inhibitor of
322
apoptosis protein (IAP) were up-regulated with dietary vitamin C levels up to 133.8 and 89.1 mg/kg diet (P
323
< 0.05), respectively, and then plateaued. The mRNA levels of Fas ligand (FasL), mitogen-activated protein
324
kinase kinase 6 (MPKK 6), p38 MAPK, B-cell lymphoma protein 2 associated X protein (Bax), apoptotic
325
protease activating factor-1 (Apaf-1), caspase-3, -7, -8 and caspase-9 were down-regulated with dietary
326
vitamin C levels up to 89.1, 89.1, 133.8, 89.1, 133.8, 89.1, 89.1, 133.8 and 44.2 mg/kg diet (P < 0.05),
327
respectively, and then plateaued. However, dietary vitamin C had no effect on the mRNA levels of c-Jun
328
N-terminal kinases (JNK), myeloid cell leukemia-1 (Mcl-1) and caspase-2 in the head kidney (P > 0.05). In
329
the spleen, the mRNA levels of Bcl-2 and IAP were all up-regulated with dietary vitamin C levels up to
330
89.1mg/kg diet (P < 0.05), and then plateaued. The mRNA levels of FasL, MAPKK 6, p38 MAPK, Bax,
331
Apaf-1, caspase-2, -3, -7, -8 and caspase-9 were down-regulated with dietary vitamin C levels up to 89.1,
332
133.8, 89.1, 89.1, 44.2, 44.2, 89.1, 133.8, 133.8 and 133.8 mg/kg diet (P < 0.05), respectively, and then
333
plateaued. However, dietary vitamin C had no effect on the mRNA levels of JNK and Mcl-1 in the spleen
334
(P > 0.05).
335 336 337
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(Fig. 9 inserted here) 3.4.3. TJs and MLCK mRNA levels in the head kidney and spleen The effects of dietary vitamin C on mRNA levels of head kidney and spleen TJs and MLCK are shown 15
in Fig. 10. In the head kidney, the mRNA levels ofMANUSCRIPT ZO-1, ZO-2, Claudin-b, -c, -3c and Claudin-7a were ACCEPTED
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up-regulated with dietary vitamin C levels up to 44.2, 89.1, 44.2, 44.2, 89.1 and 89.1 mg/kg diet (P < 0.05),
340
respectively, and then plateaued. The mRNA levels of Claudin-12, -15a, -15b and MLCK were
341
down-regulated with dietary vitamin C levels up to 89.1, 44.2, 89.1 and 44.2 mg/kg diet (P < 0.05),
342
respectively, and then plateaued. The mRNA level of Claudin7b was increased with dietary vitamin C levels
343
up to 133.8 mg/kg diet (P < 0.05), and decreased thereafter (P < 0.05). However, dietary vitamin C had no
344
effect on the mRNA level of Occludin in the head kidney (P > 0.05). In the spleen, the mRNA levels of
345
ZO-1, ZO-2, Claudin-b, -c, -3c and Claudin-7b were up-regulated with dietary vitamin C levels up to 44.2,
346
89.1, 89.1, 44.2, 44.2 and 44.2 mg/kg diet (P < 0.05), respectively, and then plateaued. The Claudin-12, -15a
347
and Claudin-15b mRNA levels were down-regulated with dietary vitamin C levels up to 44.2, 89.1 and
348
133.8 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA level of Claudin-7a was
349
up-regulated with dietary vitamin C levels up to 133.8 mg/kg diet (P < 0.05), and down-regulated thereafter
350
(P < 0.05). However, dietary vitamin C had no effect on the mRNA levels of Occludin and MLCK in the
351
spleen (P > 0.05).
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(Fig. 10 inserted here)
3.5. Skin immune response-related parameters and structural integrity-related parameters
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As shown in Table 6, the LA activity and C3 and C4 contents in the skin were increased with dietary
355
vitamin C levels up to 89.1, 44.2 and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. However,
356
dietary vitamin C had no effect on the ACP activity in the skin (P > 0.05). The mRNA levels of cytokines
357
and related signaling molecules in the skin were shown in Fig. 11. The mRNA levels of IL-10 and IκBα
358
were up-regulated with dietary vitamin C levels up to 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, and
359
then plateaued. The mRNA levels of TNF-α, IFN-γ2, IL-1β, IL-8, NF-κB p65, IKKα, IKKβ, IKKγ, TOR and
360
S6K1 were down-regulated with dietary vitamin C levels up to 89.1, 89.1, 44.2, 44.2, 133.8, 89.1, 133.8,
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89.1, 44.2 and 44.2 mg/kg diet (P < 0.05), respectively, and then plateaued. However, dietary vitamin C had ACCEPTED MANUSCRIPT
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no effect on the TGF-β1 mRNA level in the skin (P > 0.05). (Table 6 inserted here)
364
(Fig. 11 inserted here)
365
The activities and mRNA levels of antioxidant enzymes, and Nrf2, Keap1a and Keap1b in the skin
366
were shown in Table 7 and Fig. 12, respectively. The ROS, MDA and PC contents were significantly
367
decreased with dietary vitamin C levels up to 133.8, 133.8 and 179.4 mg/kg diet (P < 0.05), respectively,
368
and then plateaued. The AHR, ASA, T-SOD, CuZnSOD, CAT, GPx, GST and GR activities and GSH
369
content were significantly increased with dietary vitamin C levels up to 89.1, 89.1, 133.8, 133.8, 44.2, 133.8,
370
44.2, 44.2 and 89.1 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA levels of CuZnSOD,
371
CAT, GPx1a, GSTR, GR and Nrf2 were up-regulated with dietary vitamin C levels up to 44.2, 44.2, 89.1,
372
44.2, 44.2 and 44.2 mg/kg diet (P < 0.05), respectively, and then plateaued. The mRNA levels of The
373
Keap1a and Keap1b mRNA levels were down-regulated with dietary vitamin C level up to 133.8 and 44.2
374
mg/kg diet (P < 0.05), and then plateaued. As shown in Fig. 13, the mRNA levels of caspase-3, -8 and
375
caspase-9 were down-regulated with dietary vitamin C levels up to 89.1, 44.2 and 44.2 mg/kg diet (P < 0.05),
376
respectively, and then plateaued. As shown in Fig. 14, the mRNA levels of Occludin, ZO-1, Claudin-b, -c
377
and Claudin-3c were up-regulated with dietary vitamin C levels up to 89.1, 44.2, 89.1, 89.1 and 89.1 mg/kg
378
diet (P < 0.05), respectively, and then plateaued. The mRNA levels of Claudin-12 and Claudin-15a were
379
down-regulated with dietary vitamin C levels up to 89.1 and 44.2 mg/kg diet (P < 0.05), respectively, and
380
then plateaued.
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381
(Table 7 inserted here)
382
(Fig. 12 inserted here)
383
(Fig. 13 inserted here) 17
384
ACCEPTED MANUSCRIPT
(Fig. 14 inserted here)
385
4. Discussion
386
4.1. Vitamin C deficiency decreased growth performance and survival rate after infection with A. hydrophila
387
in fish The present study showed that, compared with optimal vitamin C supplementation, dietary vitamin C
389
deficiency resulted in poor PWG, SGR, FI, FE, and the head kidney and spleen weight, index, and vitamin C
390
concentrations in of young grass carp. Fish growth and immune organ (such as head kidney and spleen) size
391
are often related to its disease resistance [5, 77, 78], which could be reflect by survival rate after challenging
392
[79]. Our data indicated that, compared with appropriate dietary vitamin C supplementation, vitamin C
393
deficiency significantly decreased the survival rate of young grass carp under infection of A. hydrophila,
394
suggesting that vitamin C deficiency could decrease disease resistance in young grass carp. Meanwhile, we
395
observed that vitamin C deficiency caused the highest the skin lesion morbidity (53.3%) of young grass carp
396
after infection of A. hydrophila, whereas optimal vitamin C decreased the skin lesion morbidity (13.3%).
397
Based on the broken-line analysis of PWG (Fig. 15A) and to against skin lesion morbidity (Y = - 0.2847X +
398
49.215, R2 = 0.893) the dietary vitamin C requirements of young grass carp were estimated to be 92.8 and
399
122.9 mg/kg diet, respectively. Fish disease resistance is related to the immunity of head kidney and spleen
400
[66]. Thus, we further investigated the effects of vitamin C on the head kidney and spleen immunity of
401
young grass carp as well as its potential mechanisms.
SC
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402
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(Fig. 15 inserted here)
403
4.2. Vitamin C deficiency decreased the head kidney and spleen immunity in fish under infection of A.
404
hydrophila
405
The immunity of fish is largely rely on humoral components like LA, ACP, C3, C4 and antimicrobial
406
peptides like LEAP-2 and hepcidin [80]. In this study, vitamin C deficiency decreased the LA and ACP 18
activities, C3 and C4 contents and down-regulated LEAP-2B, hepcidin and β-defensin mRNA ACCEPTED LEAP-2A, MANUSCRIPT
408
levels in the head kidney and spleen of young grass carp, but which were increased with the optimal dietary
409
vitamin C supplementation under infection of A. hydrophila, suggesting that vitamin C deficiency depressed
410
the immunity of fish. In addition, immunity is also associated with inflammation, which is primarily
411
mediated by inflammatory cytokines in fish [81]. We then examined the effects of vitamin C on
412
inflammation in the head kidney and spleen of young grass carp.
413
4.3. Vitamin C deficiency aggravated inflammation and the potential molecular mechanisms in the head
414
kidney and spleen of fish under infection of A. hydrophila
SC
RI PT
407
Studies have shown that up-regulation of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-8
416
in vaccinated pink (Oncorhynchus gorbuscha) [82], and down-regulation of anti-inflammatory cytokines,
417
such as IL-10 and TGF-β in mice [83], initiated the inflammatory process. In this study, compared with
418
optimal vitamin C supplementation, vitamin C deficiency resulted in up-regulating pro-inflammatory
419
cytokines TNF-α, IFN-γ2, IL-1β, IL-6, IL-8, IL-12 P40, IL-15 and IL-17D mRNA levels in the head kidney
420
and spleen, and IL-12 P35 mRNA level in the spleen, and down-regulating anti-inflammatory cytokines
421
IL-4/13A, IL-10, IL-11, TGF-β1 and TGF-β2 mRNA levels in the head kidney and spleen, and IL4/13B
422
mRNA level in the head kidney of young grass carp under infection of A. hydrophila. However, vitamin C
423
deficiency had no effect on the mRNA level of IL-12 P35 in the head kidney and IL4/13B mRNA level in
424
the spleen of young grass carp. The un-changed mRNA level of IL-12 P35 in the head kidney and IL4/13B
425
mRNA level in the spleen might be partially explained by them had distinct expression pattern in fish
426
different tissue. The IL-12 P35 expression level in the head kidney is lower than that of in the spleen of
427
Atlantic salmon [84], while, the IL4/13B shows very low constitutive expression in the spleen than that of in
428
the head kidney of zebrafish [85].
429
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In rat, NF-κB is clearly one of the most important regulators of pro-inflammatory gene expression [86]. 19
In multiple myeloma cells, NF-κB could be activate by two distinct mechanisms: the classical NF-κB ACCEPTED MANUSCRIPT
431
pathway and alternative NF-κB pathway, and NF-κB p65 and NF-κB p52 reflect the activities of those two
432
pathways, respectively [87]. It was reported that up-regulated NF-κB p65 expression could up-regulate the
433
mRNA levels of pro-inflammatory cytokines IL-6 and IL-8 in rheumatoid arthritis synovial fibroblasts [16],
434
and down-regulate the mRNA levels of anti-inflammatory cytokines IL-4 in human [88] and IL-10 and
435
TGF-β in mouse [89]. In this study, compared with optimal vitamin C supplementation, vitamin C
436
deficiency up-regulated NF-κB p65 mRNA level, but had no impact on mRNA level of NF-κB p52 in the
437
head kidney and spleen of young grass carp under infection of A. hydrophila. The no significant impact in
438
the NF-κB p52 mRNA expression in present study may be attributed to the activation of classical NF-κB
439
pathway (NF-κB p65), rather than alternative NF-κB pathway (NF-κB p52). However, further investigation
440
should be conducted to support our hypothesis. Correlation analysis indicated that pro-inflammatory
441
cytokines IL-6, IL-8, IL-12 P40, IL-15 and IL-17D mRNA levels were positively related to NF-κB p65
442
mRNA level, whereas anti-inflammatory cytokines IL-4/13A, IL-10, IL-11, TGF-β1 and TGF-β2 mRNA
443
levels were negatively related to NF-κB p65 mRNA level in the head kidney and spleen of young grass carp
444
(Table 8). These results indicated that vitamin C deficiency up-regulated those pro-inflammatory cytokines
445
mRNA levels and down-regulated those anti-inflammatory cytokines mRNA levels may be partly by
446
up-regulating NF-κB p65 mRNA level (rather than NF-κB p52) in the head kidney and spleen of fish. In
447
addition, IκB was an NF-κB p65-binding protein, which prevents NF-κB p65 translocation to the nucleus in
448
RAW 264.7 cells [90]. It was reported that down-regulation IκBα could promote the nuclear translocation of
449
NF-κB p65 in mice [91]. In this study, compared with optimal vitamin C supplementation, deficiency of
450
vitamin C decreased IκBα mRNA level in the head kidney and spleen of young grass carp under infection of
451
A. hydrophila. Correlation analysis indicated that NF-κB p65 mRNA level was negatively correlated with
452
IκBα mRNA level in the head kidney and spleen of young grass carp (Table 8), indicating that vitamin C
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20
deficiency up-regulated NF-κB p65 nuclear translocation may be partly ascribe to down-regulate IκBα gene ACCEPTED MANUSCRIPT
454
expression in fish. Besides, it was reported that IκB kinase (IKK) (including IKKα, IKKβ and IKKγ three
455
subunits) is a key factor in NF-κB activation through regulation phosphorylating IκBα in HeLa cells [92]. In
456
this study, compared with optimal vitamin C supplementation, vitamin C deficiency significantly
457
up-regulated IKKα, IKKβ and IKKγ mRNA levels in the head kidney and spleen of young grass carp under
458
infection of A. hydrophila. Correlation analysis indicated that mRNA level of IκBα was negatively related to
459
IKKα, IKKβ and IKKγ mRNA levels in the head kidney and spleen of grass carp (Table 8). These data
460
demonstrated that vitamin C deficiency down-regulated IκBα mRNA expression may be partly by
461
up-regulating IKK (IKKα, IKKβ and IKKγ) mRNA levels in fish.
M AN U
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453
In mouse microglial cells, TOR is a serin/threonin protein kinase with a central role in the regulation of
463
cytokines [93]. S6K1 and 4E-BP are downstream molecules of mTOR in human [94]. In Drosophila
464
Schneider 2 cells, TOR could up-regulate S6K1 and down-regulate 4E-BP expression [95]. It was reported
465
that down-regulated TOR could down-regulate the mRNA levels of pro-inflammatory cytokines IL-1β and
466
TNF-α in human [96]. In this study compared with optimal vitamin C supplementation, vitamin C deficiency
467
up-regulated TOR mRNA level in the head kidney and spleen of young grass carp under infection of A.
468
hydrophila. Correlation analysis showed that mRNA levels of pro-inflammatory cytokines TNF-α, IFN-γ2
469
and IL-1β were positively related to TOR in the head kidney and spleen of young grass carp (Table 8),
470
suggesting that vitamin C down-regulated pro-inflammatory cytokines TNF-α, IFN-γ2 and IL-1β mRNA
471
levels may be partly through up-regulating of TOR mRNA transcript abundance in fish. However, vitamin C
472
deficiency down-regulated 4E-BP1 mRNA level, whereas had no impact on the mRNA level of 4E-BP2 in
473
the head kidney and spleen of young grass carp under infection of A. hydrophila. The un-changed mRNA
474
level of 4E-BP2 might be partially explained by p38 MAPK. It was reported that, activation p38 MAPK
475
could down-regulate the expression of 4E-BP1 but have no effect on the expression of 4E-BP2 in human
AC C
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21
476
[97]. In the present study, vitamin C ACCEPTED deficiency up-regulated the p38 MAPK expression level in the head MANUSCRIPT
477
kidney and spleen of young grass carp under infection of A. hydrophila. However, further investigation
478
should be conducted to support our hypothesis. (Table 8 inserted here)
480
As stated above, our data firstly together corroborate the idea that, compared with optimum vitamin C
481
supplementation, vitamin C deficiency aggravated inflammation by up-regulation of pro-inflammatory
482
cytokines [IL-6, IL-8, IL-12 P35 (only in spleen), IL-12 P40, IL-15 and IL-17D] and down-regulation of
483
anti-inflammatory cytokines [IL-4/13A, IL-4/13B (only in head kidney), IL-10, IL-11 and TGF-β1] mRNA
484
levels may be involved in NF-κB p65 (rather than NF-κB p52)/IκBα/IKK signaling pathway of fish under
485
infection of A. hydrophila. Meanwhile, vitamin C deficiency up-regulated pro-inflammatory cytokines
486
TNF-α, IFN-γ2 and IL-1β mRNA levels may, in part, due to activate the TOR/S6K1/4E-BP1 (rather than
487
4E-BP2) signaling pathway in the head kidney and spleen of fish under infection of A. hydrophila. In
488
addition, the immunity of immune organs relies on their structural integrity in mice [98]. Structural integrity
489
of fish immune organ is associated with organ cellular membrane integrity, which can be destructed by
490
oxidative injury and cell apoptosis [27]. Thus we further investigated the effects of vitamin C on oxidative
491
injury and cell apoptosis in the head kidney and spleen of young grass carp.
492
4.4. Vitamin C deficiency induced oxidative injury, decreased antioxidant capacity and aggravated cell
493
apoptosis in the head kidney and spleen of fish under infection of A. hydrophila
AC C
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479
494
The major immune organs, head kidney and spleen, contain various immune cells [99], which could
495
generate reactive oxygen species (ROS) [100]. However, excessive ROS (mainly including superoxide
496
radical and hydroxyl radical) could lead to oxidative injury to lipids and proteins in rat [101]. In this study,
497
compared with optimal vitamin C supplementation, vitamin C deficiency increased ROS, MDA and PC
498
contents, and decreased anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) activities in the head 22
kidney and spleen of young grass carp under infection of A. hydrophila. In fish, ROS can be removed by ACCEPTED MANUSCRIPT
500
enzymatic antioxidants like SOD, CAT and glutathione-dependent enzymes and non-enzymatic antioxidant
501
GSH [102]. Our results clearly showed that compared with optimal vitamin C supplementation, vitamin C
502
deficiency decreased the T-SOD, CuZnSOD, MnSOD, CAT, GPx, GST and GR activities and GSH and
503
vitamin C contents in the head kidney and spleen of young grass carp under infection of A. hydrophila. In
504
fish antioxidant enzyme activities were partly dependent on antioxidant enzymes gene transcriptions [32],
505
which were regulated by Nrf2 signaling pathway [103]. Hence, we further elucidated the effects of vitamin
506
C on antioxidant enzymes mRNA expression and Nrf2 signaling pathway in the head kidney and spleen of
507
fish.
M AN U
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499
Results from our study showed that dietary vitamin C limitation down-regulated the CuZnSOD,
509
MnSOD, CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTP1, GSTP2 and GR mRNA levels, whereas
510
optimal vitamin C supplementation could reverse these down-regulate in the head kidney and spleen of
511
young grass carp under infection of A. hydrophila. Correlation analysis revealed that CuZnSOD, MnSOD,
512
CAT and GR activities were positively related to their corresponding mRNA levels in the head kidney and
513
spleen (Table 9), suggesting that vitamin C limitation decreased those antioxidant enzyme activities may be
514
partly related to the down-regulation of their corresponding mRNA levels in the head kidney and spleen of
515
fish. Meanwhile, GPx activity in the head kidney and spleen was positively related to GPx1a, GPx1b, GPx4a
516
and GPx4b mRNA levels in the head kidney and GPx1b and GPx4b in the spleen (Table 9), suggesting that
517
vitamin C limitation decreased the GPx activity in the head kidney and spleen may be partly related to the
518
down-regulation GPx1a, GPx1b, GPx4a and GPx4b mRNA levels in the head kidney and GPx1b and GPx4b
519
in the spleen of fish, respectively. While GST activity was positively related to GSTR, GSTP1 and GSTP2
520
mRNA levels in head kidney and spleen of young grass carp (Table 9), suggesting that vitamin C limitation
521
decreased the GST activity may be partly related to the down-regulation GSTR, GSTP1 and GSTP2 in the
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23
head kidney and spleen of fish. Interestingly, vitamin C deficiency had no impact on mRNA levels of ACCEPTED MANUSCRIPT
523
GSTO1 and GSTO2 in the head kidney and spleen of young grass carp. The un-changed mRNA levels of
524
GSTO1 and GSTO2 might relate to its function. It was reported that, the major function of GSTO1 and
525
GSTO2 is reduction of dehydrogenation ascorbic acid to ascorbic acid, and this may be critical in the
526
maintenance of ascorbic acid levels in human [104]. While, in this study, we added ascorbic acid level in
527
fish head kidney and spleen through dietary supplement vitamin C, leading a no need for GTTO1 and
528
GSTO2. However, further investigation should be conducted to support our hypothesis. In fish, antioxidant
529
enzyme gene expression is regulated by an Nrf2 signaling pathway [103]. In the current study, vitamin C
530
deficiency caused a decline of Nrf2 mRNA level in head kidney and spleen of grass carp, whereas optimal
531
vitamin C supplementation could prevent this decline under infection of A. hydrophila. Correlation analysis
532
revealed that CuZnSOD, MnSOD, CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTP1, GSTP2 and GR
533
mRNA levels were positively related to the mRNA level of Nrf2 in the head kidney and spleen of young
534
grass carp (Table 9), supporting that vitamin C deficiency down-regulated antioxidant enzyme mRNA levels
535
may be partly related to the down-regulation of Nrf2 gene transcription in fish. In fish, Keap1 is the Nrf2
536
cytosolic repressor, which possesses two isoforms Keap1a and Keap1b [105]. In human, Keap1
537
overexpression could down-regulate Nrf2 mRNA level [106]. In this study, compared with optimal vitamin
538
C supplementation, fish fed vitamin C-deficient diet had a higher Keap1a and Keap1b mRNA levels in the
539
head kidney and spleen of young grass carp under infection of A. hydrophila. Correlation analysis showed
540
that the mRNA level of Nrf2 was negatively correlated with Keap1a and Keap1b in the head kidney and
541
spleen of young grass carp (Table 9), suggesting that vitamin C deficiency down-regulated Nrf2 mRNA
542
level may be partly attributed to up-regulated Keap1a and Keap1b transcript abundance in fish.
AC C
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522
543
(Table 9 inserted here)
544
As stated above, our data firstly together corroborate the idea that vitamin C deficiency decreased 24
545
T-SOD, CuZnSOD, MnSOD, CAT, GPx, GST and GR activities by down-regulating CuZnSOD, MnSOD, ACCEPTED MANUSCRIPT
546
CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTP1, GSTP2 (rather than GTTO1 and GSTO2) and GR
547
mRNA levels, whereas optimal vitamin C supplementation could reverse these down-regulate, which may
548
be involved in Nrf2/Keap1 signaling pathway of fish under infection of A. hydrophila. Apoptosis is related to caspases, which generally be divided into two categories, the initiator caspases
550
(include caspase-2, -8 and caspase-9) and the effector caspases (include caspase-3 and caspase-7) [107].
551
Study showed that increased expression of caspase-3 could induce apoptosis in fish [108]. The present study
552
showed that compared with appropriate vitamin C level, vitamin C deficiency up-regulated the caspase-3, -7,
553
-8 and caspase-9 mRNA levels in the head kidney and spleen of young grass carp, indicating that vitamin C
554
deficiency at least in part increased the cell apoptosis in the head kidney and spleen of fish under infection
555
of A. hydrophila. However, vitamin C deficiency up-regulated caspase-2 mRNA expression in the spleen,
556
but had no impact on mRNA level of caspase-2 in the head kidney of young grass carp under infection of A.
557
hydrophila. This kind of phenomenon may relate to IL-12. It was reported that IL-12 could active caspase-2,
558
IL-12 P35 is a subunit of IL-12 in human [109, 110]. In the present study, vitamin C deficiency up-regulated
559
the IL-12 P35 expression level in the spleen, whereas had no impact on mRNA level of IL-12 P35 in the
560
head kidney of young grass carp under infection of A. hydrophila. However, further investigation should be
561
conducted to support our hypothesis.
AC C
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SC
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549
562
In human, Fas death receptor pathway [mediated by Fas ligand (FasL)] is a major apoptosis pathway
563
[111]. In Jurkat T cells, up-regulation FasL could up-regulate caspase-8 expression [112]. In this study
564
compared with optimal vitamin C supplementation, vitamin C deficiency significantly up-regulated FasL
565
mRNA level in the head kidney and spleen of young grass carp under infection of A. hydrophila. Correlation
566
analysis showed that mRNA level of caspase-8 was positively related to FasL mRNA level in head kidney
567
and spleen of young grass carp (Table 10), suggesting that vitamin C deficiency up-regulated caspase-8 25
mRNA level may be partly through up-regulating mRNA transcript abundance in fish. In human, p38 ACCEPTEDFasL MANUSCRIPT
569
MAPK and JNK are MAPK family members, playing a key role in apoptosis [113]. It was reported that
570
down-regulation p38 MAPK expression could down-regulate FasL mRNA level in human ovarian
571
carcinoma cells [114]. In this study, compared with optimal vitamin C supplementation, vitamin C
572
deficiency significantly up-regulated p38 MAPK mRNA level, whereas had no impact on mRNA level of
573
JNK in the head kidney and spleen of young grass carp under infection of A. hydrophila. The reason that
574
vitamin C deficiency had no impact on mRNA level of JNK may relate to GSTO1. In HeLa cells, GSTO1
575
could inhibit JNK [115]. However, our study demonstrated that vitamin C had no impact on mRNA level of
576
GSTO1. Correlation analysis showed that mRNA level of FasL was positively related to p38 MAPK mRNA
577
level in head kidney and spleen of young grass carp (Table 10), suggesting that vitamin C deficiency
578
up-regulated FasL mRNA level may be partly by up-regulating p38 MAPK (rather than JNK) mRNA level
579
in the head kidney and spleen of fish. In mice, MAPKK 6 is an upstream kinase of the p38 MAPK pathway
580
[116]. Enslen et al. [117] reported that MAPKK 6 could up-regulate p38 MAPK gene expression in rat. In
581
this study, vitamin C deficiency significantly up-regulated MAPKK 6 mRNA level in head kidney and
582
spleen of young grass carp, whereas optimal vitamin C supplementation could prevent this up-regulation
583
under infection of A. hydrophila. Correlation analysis showed that mRNA level of p38 MAPK was
584
positively related to MAPKK 6 expression in head kidney and spleen of young grass carp (Table 10),
585
suggesting that vitamin C deficiency up-regulated p38 MAPK mRNA level may be partly through
586
up-regulating MAPKK 6 mRNA level in fish.
AC C
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568
587
In K562 cells, mitochondria pathway plays an important role in apoptosis [118]. In human,
588
up-regulated Apaf-1 could increase the mRNA expression of caspase-9 [119]. Meanwhile, down-regulated
589
IAP could increase the expression of caspase-3 [120]. In this study, compared with optimal vitamin C
590
supplementation, vitamin C deficiency significantly up-regulated Apaf-1 mRNA level, and down-regulated 26
IAP mRNA level in the head kidneyACCEPTED and spleen ofMANUSCRIPT young grass carp under infection of A. hydrophila.
592
Correlation analysis showed that mRNA levels of caspase-3, -7 and caspase-9 were positively related to
593
Apaf-1 mRNA level, whereas negatively related to IAP mRNA level in the head kidney and spleen of young
594
grass carp (Table 10), respectively, suggesting that vitamin C deficiency up-regulated caspase-3, -7 and
595
caspase-9 mRNA levels may be partly through up-regulating Apaf-1 mRNA level and down-regulating IAP
596
mRNA level in fish. In 3T3 MEFs cells, Apaf-1 expression could be up-regulated by inhibition of Bcl-2 and
597
activation of Bax expression [121]. Bcl-2 family includes anti-apoptotic members (such as Bcl-2 and Mcl-1)
598
and pro-apoptotic members (such as Bax) in human [122]. In this study, compared with optimal vitamin C
599
supplementation, vitamin C deficiency significantly down-regulated Bcl-2 mRNA level and up-regulated
600
Bax mRNA level, whereas had no impact on mRNA level of Mcl-1 in the head kidney and spleen of young
601
grass carp under infection of A. hydrophila. The reason that vitamin C deficiency had no impact on mRNA
602
level of Mcl-1 may relate to JNK. In mice, inhibited JNK could down-regulate Mcl-1 expression [123].
603
However, our study demonstrated that vitamin C had no impact on mRNA level of JNK under infection of A.
604
hydrophila. Correlation analysis indicated that Apaf-1 mRNA level was negatively related to anti-apoptotic
605
member Bcl-2 mRNA level, whereas positively related to pro-apoptotic member Bax mRNA level in the
606
head kidney and spleen of young grass carp (Table 10). These results indicated that vitamin C deficiency
607
up-regulated Apaf-1 mRNA level may be partly through down-regulating Bcl-2 (rather than Mcl-1) and
608
up-regulating Bax mRNA levels in the head kidney and spleen of fish.
AC C
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SC
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591
609
(Table 10 inserted here)
610
All together, our data firstly together corroborate the idea that vitamin C deficiency aggravated cell
611
apoptosis, whereas optimal vitamin C supplementation could prevent this aggravation, which may be partly
612
related to MAPKK 6/p38 MAPK (rather than JNK)/FasL/caspase-8 signaling and Bcl-2 (rather than Mcl-1),
613
Bax/Apaf-1, IAP/caspase-3, -7, -9 signaling in the head kidney and spleen of fish under infection of A. 27
hydrophila. In fish, the structural integrity of immune organ is also associated with the TJs [Occludin, ACCEPTED MANUSCRIPT
615
Claudins and zonula occludens (ZO)] in intercellular [42, 43]. Therefore, we next investigated the
616
relationship between vitamin C and TJs and its possible signaling pathway in the head kidney and spleen of
617
young grass carp.
618
4.5 Vitamin C deficiency disturbed the TJs in the head kidney and spleen of fish under infection of A.
619
hydrophila
RI PT
614
It was reported that ZO-1, Occludin, Claudin-b, -c and Claudin-3c are barrier-forming TJs, and
621
Claudin-12 and Claudin-15a are pore-forming TJs in fish [124]. In the present study, compared with the
622
optimal vitamin C supplementation, vitamin C deficiency down-regulated the mRNA levels of ZO-1, ZO-2,
623
Claudin-b, -c, -3c, -7a and Claudn-7b, and up-regulated the mRNA levels of Claudin-12, -15a and
624
Claudin-15b in the head kidney and spleen of fish under infection of A. hydrophila. These findings for the
625
first time implied that vitamin C deficiency might disturb TJs of head kidney and spleen in fish. Moreover,
626
MLCK was an important regulatory element of TJs in mice intestinal epithelium cells [125]. Study showed
627
that up-regulation of MLCK could down-regulate the mRNA levels of ZO-1 and Occludin in human Caco-2
628
cells [126]. Results from our study demonstrated that compared with optimal vitamin C supplementation,
629
vitamin C deficiency up-regulated the mRNA level of MLCK in the head kidney of young grass carp under
630
infection of A. hydrophila. Correlation analysis showed that barrier-forming TJs ZO-1, ZO-2, Claudin-b, -c,
631
-3c, -7a and Claudn-7b mRNA levels were negatively correlated with MLCK mRNA level, whereas
632
pore-forming TJs Claudin-12, -15a and Claudin-15b mRNA levels were positively related to MLCK mRNA
633
level in the head kidney of young grass carp (Table 11), suggesting that dietary vitamin C deficiency
634
down-regulated ZO-1, ZO-2, Claudin-b, -c, -3c, -7a and Claudn-7b mRNA levels and up-regulated
635
Claudin-12, -15a and Claudin-15b mRNA levels may be partly by up-regulating MLCK mRNA level in the
636
head kidney of fish. However, current study found that vitamin C deficiency had no impact on mRNA level
AC C
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620
28
of MLCK in the spleen of young grassACCEPTED carp under infection of A. hydrophila. The possible reason for which MANUSCRIPT
638
vitamin C had no impact on spleen MLCK is that vitamin C may be partly through cytokines to regulate TJs,
639
rather than MLCK. Studies showed that TNF-α could down-regulate the expression of ZO-1 in BREC cells
640
and Claudin-7 in rat [127, 128] and up-regulated the expression of Claudin-12 and Claudin-15 in T84 cells
641
[129]. As mentioned above, spleen TNF-α mRNA level significantly up-regulated in vitamin C-limited diet
642
group under infection of A. hydrophila. Further correlation analysis showed that ZO-1, ZO-2, Claudin-b, -c,
643
-3c, -7a and Claudin-7b mRNA levels were negatively correlated with TNF-α mRNA level, Claudin-12, -15a
644
and Claudin-15b mRNA levels were positively related to TNF-α mRNA level in the spleen of young grass
645
carp (Table 11). Those results indicated that dietary vitamin C deficiency down-regulated ZO-1, ZO-2,
646
Claudin-b, -c, -3c, -7a and Claudin-7b mRNA levels, and up-regulated Claudin-12, -15a and Claudin-15b
647
mRNA levels be partly by up-regulating the gene transcription of TNF-α (rather than MLCK) in the spleen
648
of fish.
M AN U
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(Table 11 inserted here)
650
As stated above, our data firstly together corroborate the idea that compared with optimal vitamin C
651
supplementation, vitamin C deficiency disturbed head kidney and spleen TJs through down-regulating ZO-1,
652
ZO-2, Claudin-b, -c, -3c, -7a and Claudin-7b mRNA levels, and up-regulating Claudin-12, -15a and
653
Claudin-15b mRNA levels, which may be partly regulated by MLCK signaling in head kidney and TNF-α
654
(rather than MLCK) in spleen of fish under infection of A. hydrophila. However, the underlying mechanism
655
needs to be further investigated.
656
4.6. Interesting results: high levels of vitamin C impaired some indicators that differed from the traditional
657
growth results
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649
658
Vitamin C as a water-solution vitamin; no negative effects on the growth performances were observed
659
in this study and our lab previous study in juvenile Jian carp when high doses were administered [4]. 29
Interestingly, our study founded that,ACCEPTED compared with optimal vitamin C supplementation, high levels of MANUSCRIPT
661
vitamin C (224.5 mg/kg diet) down-regulated the mRNA expression levels of β-defensin in the head kidney
662
and spleen, Claudin-7b in the head kidney, and IL-11, Claudin-7a and GPx4a in the spleen, and up-regulated
663
the mRNA expression levels of IL-12 P40 and IL-15 in the head kidney under infection of A. hydrophila.
664
The possible reasons are (1) High levels of vitamin C down-regulated β-defensin mRNA level in the head
665
kidney and spleen of fish may partly relate to vitamin C have a similar function to it. In carp, β-defensin
666
have a strong antibacterial activity to Escherichia coli (E. coli) [130]. While, study from our lab showed that
667
vitamin C significantly depressed intestinal E. coli in Jian carp (Cyprinus carpio var. Jian) [4]. (2) High
668
levels of vitamin C up-regulated IL-12 P40 and IL-15 mRNA expression levels in the head kidney may be
669
partly attributed to down-regulate β-defensin mRNA level. In human myeloid dendritic cells, β-defensin
670
could inhibit pro-inflammatory cytokines IL-12 P40 and IL-15 [131]. In this study, compared with optimal
671
vitamin C supplementation, high levels of vitamin C significantly down-regulated head kidney β-defensin
672
mRNA level in young grass carp. (3) High levels of vitamin C down-regulated IL-11 mRNA level in the
673
spleen of fish may be partly related to vitamin C has a similar function to it. It was reported that IL-11
674
played a role of anti-inflammatory through reducing TNF-α mRNA level in mice [132]. However, our study
675
indicated that vitamin C down-regulated TNF-α mRNA expression. (4) High levels of vitamin C
676
down-regulated the mRNA levels of Claudin-7b in the head kidney and Claudin-7a in the spleen may be
677
partly ascribed to vitamin C could enhance the TJs by increasing collagen content in fish. It was reported
678
that, vitamin C increased bone collagen in large yellow croaker (Pseudosciaena crocea) [30]. Collagen
679
could enhance the TJs in A6 cells [133]. In rat, Claudin-7 is a barrier-forming tight junction protein [128].
680
These may support our hypothesis, but need to be further investigated. (5) High levels of vitamin C
681
down-regulated GPx4a mRNA level in the spleen of fish may partly relate to vitamin C have a similar
682
function to it. In Coho salmon, the major function of GPx4a is reducing lipid peroxides [134]. However,
AC C
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30
683
vitamin C played an important role of ACCEPTED reduction lipid MANUSCRIPT peroxides in rat [135].
684
4.7. Vitamin C deficiency reduced the immunity and damaged the structural integrity in the skin of fish:
685
compared with head kidney and spleen. Our results showed that except the activities of ACP and MnSOD, and mRNA expression levels of
687
TGF-β1, Occludin and MnSOD model were different, the effect of vitamin C on other indices model were
688
similar among the head kidney, spleen and skin in young grass carp under infection of A. hydrophila. Our
689
study demonstrated that, compared with optimal vitamin C supplementation, vitamin C limitation decreased
690
ACP and MnSOD activities and down-regulated the mRNA levels of TGF-β1 and MnSOD in young grass
691
carp head kidney and spleen, but had no significant effect on that of skin under infection of A. hydrophila.
692
This result may be partly attributed to the vitamin C concentration difference in each tissue. Our study
693
showed that vitamin C concentration in the skin was lower than that of head kidney and spleen in young
694
grass carp. Thus, the different ACP and MnSOD activities and mRNA levels of TGF-β2 and MnSOD model
695
may be partly related to the low vitamin C concentration in fish skin. Moreover, compared with optimal
696
vitamin C supplementation, vitamin C deficiency up-regulated the mRNA level of Occludin in the skin, but
697
had no significant effect on its mRNA level in the head kidney and spleen of young grass carp under
698
infection of A. hydrophila. This result may be partly attributed to the function of Occludin and its high
699
expression level in fish skin. In rainbow trout, Occludin could regulate salt and water balance, and its mRNA
700
was particularly abundant in the skin (which involved in the regulation of salt and water balance) but rare in
701
the head kidney and spleen [136]. Therefore, the different mRNA expression model of Occludin in fish skin
702
may be partly related to its function and high expression level in fish skin.
703
4.8. Compare the vitamin C requirements for growth performance with that for immunity and antioxidant of
704
young grass carp
705
AC C
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Based on our study, vitamin C deficiency could decrease growth performance, increase skin lesion 31
morbidity and impair the immunity and structural integrity of head kidney and spleen in fish under infection ACCEPTED MANUSCRIPT
707
of A. hydrophila. Thus, evaluation of vitamin C requirement is quite necessary in fish. In this study, the
708
vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8
709
mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp
710
was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA
711
activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head
712
kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to
713
be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively. The requirements for against skin lesion
714
morbidity and enhancement immune and antioxidant indices were higher than that for the growth
715
performance, indicating that more vitamin C is required for the development of immunity and antioxidant of
716
fish. Similarly, the requirement of riboflavin based on immunity and antioxidant were higher than that on
717
growth requirement of young grass carp [52].
718
5. Conclusions
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In summary (summarized in Fig. 16), we report seven primary, novel, and interesting results. Compared
720
with optimal vitamin C supplementation, dietary vitamin C deficiency (1) decreased immunity partly
721
through decreasing LA and ACP activities and C3 and C4 contents in the head kidney and spleen of fish
722
under infection of A. hydrophila; (2) aggravated the inflammatory response by down-regulating
723
anti-inflammatory cytokines [IL-4/13A, IL-4/13B (only in head kidney), IL-10, IL-11, TGF-β1 and TGF-β2],
724
and up-regulating pro-inflammatory cytokines [TNF-α, IFN-γ2, IL-1β, IL-6, IL-8, IL-12 P35 (only in
725
spleen), IL-12 P40, IL-15 and IL-17D] mRNA expression levels in the head kidney and spleen of fish under
726
infection of A. hydrophila. The regulation of above-mentioned cytokines may be involved in the NF-κB
727
pathway [including signal molecules NF-κB p65 (rather than NF-κB p50), IκBα, IKKα, IKKβ and IKKγ]
728
and TOR pathway [including signal molecules TOR, S6K1 and 4E-BP1 (rather than 4E-BP2)] in fish; (3)
AC C
EP
719
32
impaired head kidney and spleen TJsACCEPTED by up-regulating mRNA levels of Claudin-12, -15a and Claudin-15b, MANUSCRIPT
730
and down-regulating mRNA levels of ZO-1, ZO-2, Claudin-b, -c, -3c, -7a and Claudin-7b in the head kidney
731
and spleen of fish under infection of A. hydrophila, which may be partly regulated by up-regulating MLCK
732
mRNA level in the head kidney and TNF-α mRNA level in the spleen, respectively; (4) induced apoptosis by
733
up-regulating caspase-3, -7, -8 and caspase-9 mRNA levels in the head kidney and spleen of fish under
734
infection of A. hydrophila, which may be involved in Fas death receptor pathway [mediated by MAPKK
735
6/p38 MAPK (rather than JNK)] and mitochondria pathway [mediated by Bcl-2 (rather than Mcl-1),
736
Bax/Apaf-1, IAP]; (5) decreased antioxidant capacity by decreasing the antioxidant enzymes activities and
737
mRNA levels in the head kidney and spleen of fish under infection of A. hydrophila, which could be
738
regulated by the Nrf2 signaling pathway. (6) The effect of vitamin C on fish head kidney, spleen and skin
739
immunity and structural integrity are similar, except the activities of ACP and MnSOD, and mRNA
740
expression levels of TGF-β1, Occludin and MnSOD of fish under infection of A. hydrophila. (7) The
741
vitamin C requirement for the growth performance (PWG) of young grass carp was estimated to be 92.8
742
mg/kg diet. Meanwhile, the vitamin C requirement for against skin lesion morbidity of young grass carp
743
was estimated to be 122.9 mg/kg diet. In addition, based on the biochemical indices [immune indices (LA
744
activity in the head kidney and C4 content in the spleen) and antioxidant indices (MDA content in the head
745
kidney and ROS content in the spleen)] the vitamin C requirements for young grass carp were estimated to
746
be 131.2, 137.5, 135.8 and 129.8 mg/kg diet, respectively.
748
SC
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TE D
EP
AC C
747
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(Fig. 16 inserted here) Acknowledgements
749
This research was financially supported by the National Basic Research Program of China (973
750
Program) (2014CB138600), the Specialized Research Fund for the Doctoral Program of Higher Education
751
of China (20135103110001), Science and Technology Support Program of Sichuan Province of China 33
(2014NZ0003), Major Scientific and Technological Achievement Transformation Project of Sichuan ACCEPTED MANUSCRIPT
753
Province of China (2012NC0007; 2013NC0045), the Demonstration of Major Scientific and Technological
754
Achievement Transformation Project of Sichuan Province of China (2015CC0011), Natural Science
755
Foundation for Young Scientists of Sichuan Province (2014JQ0007) and Sichuan Province Research
756
Foundation for Basic Research (2013JY0082). The authors would like to thank the personnel of these teams
757
for their kind assistance.
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760 761 762 763
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768 769 770 771 772 773
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1143
Table 1 Composition and nutrients content of basal diet. ACCEPTED MANUSCRIPT
1144
1145
1
1146
L-a-tocopherol acetate (50%), 12.58; menadione (22.9%), 0.83; thiamine nitrate (98%), 0.11; calcium-D-pantothenate
1147
(98%), 2.56; pyridoxine hydrochloride (98%), 0.62; cyanocobalamin (1%), 0.94; niacin (99%), 2.58; D-biotin (2%), 0.75;
1148
meso-inositol (98%),19.39; folic acid (95%), 0.42; riboflavin (80%), 0.63. All ingredients were diluted with corn starch to 1
1149
kg.
1150
2
1151
Fe), 24.5700; ZnSO4.H2O (34.5% Zn), 8.2500; CuSO4.5H2O (25.0% Cu), 0.960; KI (76.9% I), 0.0668g; Na2SeO3 (44.7%
1152
Se), 0.0168. All ingredients were diluted with corn starch to 1 kg.
1153
3
Vitamin C premix: premix was added to obtain graded level of vitamin C.
1154
4
Crude protein and crude lipid contents were measured value.
1155
5
Available phosphorus, n-3 and n-6 contents were calculated according to NRC (2011).
RI PT
M AN U
SC
Mineral premix (g/kg premix): MnSO4.H2O (31.8% Mn), 1.8900; MgSO4.H2O (15.0% Mg), 200.0000; FeSO4.H2O (30.0%
TE D
1156
Table 2 Real-time PCR primer sequences 1.
1158
EP
1157
Vitamin premix (g/kg premix): retinyl acetate (500,000 IU/g), 2.10; cholecalciferol (500,000 IU/g), 0.40; D,
1159
1
1160
interleukin; TGF-β: transforming growth factor β; NF-κB: nuclear factor kappa B; IκBα: inhibitor of κBα;
1161
IKK: IκB kinase; TOR: target of rapamycin; S6K1: ribosomal protein S6 kinase 1; 4E-BP: eIF4E-binding
1162
protein; ZO: zonula occludens; MLCK: myosin light chain kinase; FasL: Fas ligand; MAPKK 6:
1163
mitogen-activated protein kinase kinase 6; p38 MAPK: p38 mitogen-activated protein kinases; JNK: c-Jun
1164
N-terminal kinases; Bcl-2: B-cell lymphoma-2; Mcl-1: myeloid cell leukemia-1; Bax: B-cell lymphoma
1165
protein 2 associated X protein; Apaf-1: apoptotic protease activating factor-1; IAP: inhibitor of apoptosis
AC C
LEAP-2: liver expressed antimicrobial peptide 2; IFN-γ2: interferon γ2; TNF-α: tumor necrosis factor α; IL:
51
1166
protein; CuZnSOD: copper, zinc superoxide dismutase; MnSOD: manganese superoxide dismutase; GST: ACCEPTED MANUSCRIPT
1167
glutathione-S-transferase; CAT: catalase; GPx, glutathione peroxidase; GR: glutathione reductase; Nrf2:
1168
NF-E2-related factor 2; Keap1: Kelch-like-ECH-associated protein 1.
1169
Table 3 Growth performance1, weight (g/fish), index (%) and vitamin C concentration (mg/kg tissue) of
1171
head kidney and spleen, and skin vitamin C concentration in young grass carp (Ctenopharyngodon idella)
1172
fed the diets with graded levels of vitamin C for 10 weeks.
SC
1173
RI PT
1170
1174
1
1175
rate (%/day); FI: feed intake (g/fish); FE: feed efficiency (%).
1176
2
1177
are significantly different (P < 0.05).
1178
3
Values are means ± SD (n = 18), and different superscripts in the same row are significantly different (P < 0.05).
1179
4
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05).
M AN U
Values are means ± SD for three replicate groups, with 30 fish in each group, and different superscripts in the same row
TE D
1180
IBW: Initial body weight (g/fish); FBW: final body weight (g/fish); PWG: percent weight gain (%), SGR: specific growth
Table 4 Immune parameters in the head kidney and spleen of young grass carp (Ctenopharyngodon idella)
1182
fed diets containing different vitamin C levels after infection with A. hydrophila for 14 days 1.
1183
AC C
EP
1181
1184
1
1185
Lysozyme activity (U/mg protein); ACP: acid phosphatase (U/mg protein); C3: complement component 3 (mg/g protein);
1186
C4: complement component 4 (mg/g protein).
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05). LA:
1187
1188
Table 5 Antioxidant status related parameters in the head kidney and spleen of young grass carp 52
1189
(Ctenopharyngodon idella) fed diets ACCEPTED containing different levels of vitamin C levels after infection with A. MANUSCRIPT
1190
hydrophila for 14 days 1.
1191
1192
1
1193
reactive oxygen species (% DCF florescence); MDA: malondialdehyde (nmol/mg protein); PC: protein carbonyl (nmol/mg
1194
protein); ASA: anti-superoxide anion (U/mg protein); AHR: anti-hydroxyl radical (U/mg protein); T-SOD: total superoxide
1195
dismutase (U/mg protein); SOD1: copper/zinc superoxide dismutase (U/mg protein); SOD2: manganese superoxide
1196
dismutase (U/mg protein); CAT: catalase (U/mg protein); GPx: glutathione peroxidase (U/mg protein); GR, glutathione
1197
reductase (U/mg protein); GSH, glutathione (mg/g protein).
M AN U
SC
RI PT
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05). ROS:
1198
1199
Table 6 Immune parameters in the skin of young grass carp (Ctenopharyngodon idella) fed diets containing
1200
different vitamin C levels after infection with A. hydrophila for 14 days 1.
1202
1
TE D
1201
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05).
1203
Table 7 Antioxidant status related parameters in the skin of young grass carp (Ctenopharyngodon idella) fed
1205
diets containing different levels of vitamin C levels after infection with A. hydrophila for 14 days 1.
1206
1207
1208
1
AC C
EP
1204
Values are means ± SD (n = 6), and different superscripts in the same row are significantly different (P < 0.05).
1209
Table 8 Correlation coefficients of NF-κB p65 with IL-4/13A, IL-6, IL-8, IL-10, IL-11, IL-12 P40, IL-15,
1210
IL-17D and TGF-β1, IκBα with NF-κB p65, IKKα, IKKβ and IKKγ, TOR with IFN-γ2, TNF-α and IL-1β in
1211
the head kidney and spleen. 53
ACCEPTED MANUSCRIPT
1213
Table 9 Correlation coefficients of CuZnSOD, MnSOD, CAT and GR activities with their mRNA levels in
1214
the head kidney and spleen, GPx activity with GPx1a, GPx1b, GPx4a and GPx4b mRNA levels in the head
1215
kidney and GPx1b and GPx4b mRNA levels in the spleen, GST activity with GSTR, GSTP1 and GSTP2
1216
mRNA levels in the head kidney and spleen, Nrf2 with CuZnSOD, MnSOD, CAT, GPx1a, GPx1b, GPx4a,
1217
GPx4b, GSTR, GSTP1, GSTP2, GR, Keap1a and Keap1b mRNA levels in the head kidney and spleen.
RI PT
1212
1218
Table 10 Correlation coefficients of FasL with caspase-8 and p38 MAPK, MAPKK6 with p38 MAPK,
1220
Apaf-1 with caspase-3,-7,-9, Bcl-2 and Bax, IAP with caspase-3,-7,-9 in the head kidney and spleen.
M AN U
SC
1219
1221
Table 11 Correlation coefficients of MLCK with ZO-1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, -12, -15a and
1223
Claudin-15b in the head kidney, and TNF-α with ZO-1, ZO-2, Claudin-b, -c, -3c, -7a, -7b, -12, -15a and
1224
Claudin-15b in the spleen.
1227
1228
1229
1230
1231
EP
1226
AC C
1225
TE D
1222
1232
1233
1234
1235
54
1236
ACCEPTED MANUSCRIPT
Table 1
1237
1239
Nutrients content
g/kg
Fish meal
85.80
Crude protein 4
298.57
Casein
131.60
Crude lipid 4
45.40
Soybean protein concentrate
200.00
n-3 5
4.60
n-6 5
240.00
Corn starch
187.30
10.00
Available phosphorus 5
M AN U
α-starch
5.00
SC
DL-Met 99%
RI PT
g/kg
5.30
Soybean oil
19.30
Cellulose
50.00
Ca(H2PO4)2
30.60
Mineral premix 2 Vitamin C premix 3 Choline chloride (60%)
10.00 20.00
EP
Vitamin premix 1
TE D
Fish oil
AC C
1238
Ingredients
Ethoxyquin (30%)
10.00 5.00 0.50
1240
1241
1242 55
8.40
1243
ACCEPTED MANUSCRIPT
Table 2
1244 Primer sequence Forward (5’→3’)
Primer sequence Reverse (5’→3’)
Temperature (°C)
Accession number
Hepcidin
AGCAGGAGCAGGATGAGC
GCCAGGGGATTTGTTTGT
59.3
JQ246442.1
LEAP-2A
TGCCTACTGCCAGAACCA
AATCGGTTGGCTGTAGGA
59.3
FJ390414
LEAP2-B
TGTGCCATTAGCGACTTCTGAG
ATGATTCGCCACAAAGGGG
59.3
KT625603
β-defensin
TTGCTTGTCCTTGCCGTCT
AATCCTTTGCCACAGCCTAA
58.4
KT445868
IFN-γ2
TGTTTGATGACTTTGGGATG
TCAGGACCCGCAGGAAGAC
60.4
JX657682
TNF-α
CGCTGCTGTCTGCTTCAC
CCTGGTCCTGGTTCACTC
58.4
HQ696609
IL-1β
AGAGTTTGGTGAAGAAGAGG
TTATTGTGGTTACGCTGGA
57.1
JQ692172
IL-6
CAGCAGAATGGGGGAGTTATC
CTCGCAGAGTCTTGACATCCTT
62.3
KC535507.1
IL-8
ATGAGTCTTAGAGGTCTGGGT
ACAGTGAGGGCTAGGAGGG
60.3
JN663841
IL-15
CCTTCCAACAATCTCGCTTC
AACACATCTTCCAGTTCTCCTT
61.4
KT445872
IL-17D
GTGTCCAGGAGAGCACCAAG
GCGAGAGGCTGAGGAAGTTT
62.3
KF245426.1
IL-4/13A
CTACTGCTCGCTTTCGCTGT
CCCAGTTTTCAGTTCTCTCAGG
55.9
KT445871
IL-4/13B
TGTGAACCAGACCCTACATAACC
TTCAGGACCTTTGCTGCTTG
55.9
KT625600
IL-10
AATCCCTTTGATTTTGCC
GTGCCTTATCCTACAGTATGTG
61.4
HQ388294
IL-11
GGTTCAAGTCTCTTCCAGCGAT
TGCGTGTTATTTTGTTCAGCCA
57.0
KT445870
IL-12 P35
TGGAAAAGGAGGGGAAGATG
AGACGGACGCTGTGTGAGTGTA
55.4
KF944667.1
IL-12 P40
ACAAAGATGAAAAACTGGAGGC
GTGTGTGGTTTAGGTAGGAGCC
59.0
KF944668.1
TGF-β1
TTGGGACTTGTGCTCTAT
AGTTCTGCTGGGATGTTT
55.9
EU099588
TGF-β2
TACATTGACAGCAAGGTGGTG
TCTTGTTGGGGATGATGTAGTT
55.9
KM279716
NF-κB p52
TCAGTGTAACGACAACGGGAT
ATACTTCAGCCACACCTCTCTTAG
58.4
KM279720
NF-κB p65
GAAGAAGGATGTGGGAGATG
TGTTGTCGTAGATGGGCTGAG
62.3
KJ526214
IκBα
TCTTGCCATTATTCACGAGG
TGTTACCACAGTCATCCACCA
62.3
KJ125069
IKKα
GGCTACGCCAAAGACCTG
CGGACCTCGCCATTCATA
60.3
KM279718
IKKβ
GTGGCGGTGGATTATTGG
GCACGGGTTGCCAGTTTG
60.3
KP125491
IKKγ
AGAGGCTCGTCATAGTGG
CTGTGATTGGCTTGCTTT
58.4
KM079079
AC C
EP
TE D
M AN U
SC
RI PT
Target gene
56
TCCCACTTTCCACCAACT
ACACCTCCACCTTCTCCA
61.4
JX854449
S6K1
TGGAGGAGGTAATGGACG
ACATAAAGCAGCCTGACG
54.0
EF373673
4E-BP1
GCTGGCTGAGTTTGTGGTTG
CGAGTCGTGCTAAAAAGGGTC
60.3
KT757305
4E-BP2
CACTTTATTCTCCACCACCCC
TTCATTGAGGATGTTCTTGCC
60.3
KT757306
Occludin
TATCTGTATCACTACTGCGTCG
CATTCACCCAATCCTCCA
59.4
KF193855
ZO-1
CGGTGTCTTCGTAGTCGG
CAGTTGGTTTGGGTTTCAG
59.4
KJ000055
ZO-2
TACAGCGGGACTCTAAAATGG
TCACACGGTCGTTCTCAAAG
Claudin-b
GAGGGAATCTGGATGAGC
ATGGCAATGATGGTGAGA
Claudin-c
GAGGGAATCTGGATGAGC
CTGTTATGAAAGCGGCAC
Claudin-3c
ATCACTCGGGACTTCTA
CAGCAAACCCAATGTAG
Claudin-7a
ACTTACCAGGGACTGTGGATGT
Claudin-7b
ACCEPTED MANUSCRIPT
RI PT
TOR
KM112095
57.0
KF193860
59.4
KF193859
57.0
KF193858
CACTATCATCAAAGCACGGGT
59.3
KT625604
CTAACTGTGGTGGTGATGAC
AACAATGCTACAAAGGGCTG
59.3
KT445866
Claudin-12
CCCTGAAGTGCCCACAA
GCGTATGTCACGGGAGAA
55.4
KF998571
Claudin-15a
TGCTTTATTTCTTGGCTTTC
CTCGTACAGGGTTGAGGTG
59.0
KF193857
Claudin-15b
AGTGTTCTAAGATAGGAGGGGAG
AGCCCTTCTCCGATTTCAT
62.3
KT757304
MLCK
GAAGGTCAGGGCATCTCA
GGGTCGGGCTTATCTACT
53.0
KM279719
FasL
AGGAAATGCCCGCACAAATG
AACCGCTTTCATTGACCTGGAG
61.4
KT445873
MAPKK 6
GAGCATCTCCACAGCAACCT
CTTCGCCACTGAATCCACAA
57.1
KT445869
p38 MAPK
TGGGAGCAGACCTCAACAAT
TACCATCGGGTGGCAACATA
60.4
KM112098
JNK
ACAGCGTAGATGTGGGTGATT
GCTCAAGGTTGTGGTCATACG
62.3
KT757312
Bcl-2
AGGAAAATGGAGGTTGGGAT
CTGAGCAAAAAAGGCGATG
60.3
JQ713862.1
Mcl-1
TGGAAAGTCTCGTGGTAAAGCA
ATCGCTGAAGATTTCTGTTGCC
58.4
KT757307
Bax
CATCTATGAGCGGGTTCGTC
TTTATGGCTGGGGTCACACA
60.3
JQ793788.1
Apaf-1
AAGTTCTGGAGCCTGGACAC
AACTCAAGACCCCACAGCAC
61.4
KM279717
IAP
CACAATCCTGGTATGCGTCG
GGGTAATGCCTCTGGTGCTC
58.4
FJ593503.1
Caspase-2
CGCTGTTGTGTGTTTACTGTCTCA
ACGCCATTATCCATCTCCTCTC
60.3
KT757313
Caspase-3
GCTGTGCTTCATTTGTTTG
TCTGAGATGTTATGGCTGTC
55.9
JQ793789
Caspase-7
GCCATTACAGGATTGTTTCACC
CCTTATCTGTGCCATTGCGT
57.1
KT625601
AC C
EP
TE D
M AN U
SC
60.3
57
1247
1248
TCCATCTGATGCCCATACAC
59.0
KM016991
CTGTGGCGGAGGTGAGAA
GTGCTGGAGGACATGGGAAT
59.0
JQ793787
CuZnSOD
CGCACTTCAACCCTTACA
ACTTTCCTCATTGCCTCC
61.5
GU901214
MnSOD
ACGACCCAAGTCTCCCTA
ACCCTGTGGTTCTCCTCC
60.4
GU218534
CAT
GAAGTTCTACACCGATGAGG
CCAGAAATCCCAAACCAT
58.7
FJ560431
GPx1a
GGGCTGGTTATTCTGGGC
AGGCGATGTCATTCCTGTTC
61.5
EU828796
GPx1b
TTTTGTCCTTGAAGTATGTCCGTC
GGGTCGTTCATAAAGGGCATT
GPx4a
TACGCTGAGAGAGGTTTACACAT
CTTTTCCATTGGGTTGTTCC
GPx4b
CTGGAGAAATACAGGGGTTACG
CTCCTGCTTTCCGAACTGGT
GSTR
TCTCAAGGAACCCGTCTG
CCAAGTATCCGTCCCACA
GSTP1
ACAGTTGCCCAAGTTCCAG
GSTP2
RI PT
Caspase-9
ACCEPTED MANUSCRIPT
KT757315
60.4
KU255598
60.3
KU255599
58.4
EU107283
CCTCACAGTCGTTTTTTCCA
59.3
KM112099
TGCCTTGAAGATTATGCTGG
GCTGGCTTTTATTTCACCCT
59.3
KP125490
GSTO1
GGTGCTCAATGCCAAGGGAA
CTCAAACGGGTCGGATGGAA
58.4
KT757314
GSTO2
CTGCTCCCATCAGACCCATTT
TCTCCCCTTTTCTTGCCCATA
61.4
KU245630
GR
GTGTCCAACTTCTCCTGTG
ACTCTGGGGTCCAAAACG
59.4
JX854448
Nrf2
CTGGACGAGGAGACTGGA
ATCTGTGGTAGGTGGAAC
62.5
KF733814
Keap1a
TTCCACGCCCTCCTCAA
TGTACCCTCCCGCTATG
63.0
KF811013
Keap1b
TCTGCTGTATGCGGTGGGC
CTCCTCCATTCATCTTTCTCG
57.9
KJ729125
β-actin
GGCTGTGCTGTCCCTGTA
GGGCATAACCCTCGTAGAT
61.4
M25013
TE D
M AN U
SC
60.3
EP
1246
ATCTGGTTGAAATCCGTGAA
AC C
1245
Caspase-8
1249
1250
1251
1252 58
1253
ACCEPTED MANUSCRIPT
Table 3
1254 Dietary vitamin C levels (mg/kg diet)
44.2
89.1
133.8
179.4
224.5
IBW2
264.56±0.84
264.67±0.67
264.11±0.38
264.00±0.33
264.33±1.20
264.56±0.51
FBW2
784.67±8.50a
874.00±19.67b
1024.00±18.03c
1031.33±6.35c
1025.33±14.29c
1025.67±29.30c
PWG2
196.60±3.29a
230.22±6.97b
287.71±6.29c
290.66±2.84c
287.88±3.64c
287.69±10.61c
SGR2
1.55±0.02a
1.71±0.03b
1.94±0.02c
1.95±0.01c
1.94±0.01c
1.94±0.04c
FI2
728.95±0.43a
811.95±0.42b
943.98±0.71c
946.36±0.23c
FE2
71.35±1.16a
75.04±2.36b
Weight3
1.412±0.104a
1.864±0.137b
Index3
0.181±0.014a
0.213±0.018b
VC concentration4
7.25±0.61a
14.53±0.58b
Weight3
1.107±0.080a
Index3
0.141±0.007a
Skin
SC
M AN U
80.43±1.42c
80.42±3.04c
2.200±0.137c
2.212±0.248c
2.211±0.152c
2.209±0.152c
0.214±0.015b
0.214±0.019b
0.215±0.014b
0.215±0.012b
18.16±0.84c
19.16±1.54c
23.04±1.38d
23.44±1.37d
1.340±0.091b
1.573±0.122c
1.593±0.110c
1.587±0.083c
1.587±0.092c
0.153±0.008b
0.153±0.006b
0.154±0.007b
0.155±0.007b
0.155±0.007b
TE D
EP
VC concentration4
946.44±0.24c
81.08±0.70c
AC C
VC concentration4
946.22±0.46c
80.50±1.92c
Head kidney
Spleen
RI PT
2.9
13.81±1.05a
28.79±1.05b
42.20±3.35c
42.75±2.83c
42.10±2.49c
42.96±2.36c
3.47±0.25a
5.09±0.39b
8.37±0.47c
9.24±0.52d
8.97±0.27d
9.50±0.63d
Regression
YFI = 2.5010X + 714.7500
Ymax = 945.75
R2 = 0.989
P = 0.07
YFE = 0.1064X + 70.8010
Ymax = 80.61
R2 = 0.992
P = 0.06
59
Ymax = 1.94 ACCEPTED MANUSCRIPT
YSGR = 0.0045X + 1.5275
R2 = 0.988
P = 0.07
YVC concentration in head kidney = 0.0814X + 9.1096
Ymax = 23.24
R2 = 0.915
P
0.05
Yspleen weight = 0.0054X + 1.0947
Ymax = 1.585
R2 = 0.999
P
0.05
YVC concentration in spleen = 0.3289X + 13.3340
Ymax = 42.50
R2 = 0.997
P
0.05
YVC concentration in skin = 0.0470X + 3.3667
Ymax = 9.24
SC
1258
1259
1260
1267
1268
EP AC C
1266
TE D
1261
1265
R2 = 0.957
M AN U
1257
1264
RI PT
Ymax = 2.208
1256
1263
P = 0.05
Yhead kidney weight = 0.0091X + 1.4114
1255
1262
R2 = 0.994
1269
1270
1271
60
P
0.05
1272
ACCEPTED MANUSCRIPT
Table 4
1273 Dietary vitamin C levels (mg/kg diet)
2.9
44.2
89.1
133.8
LA
133.83±13.01a
182.07±12.49b
200.17±5.46c
251.36±11.43d
ACP
545.42±16.32a
567.81±24.21a
607.23±20.80b
637.00±20.72c
C3
53.18±3.23a
62.41±4.83b
83.24±6.76c
95.92±7.67d
C4
3.05±0.22a
3.84±0.29b
4.57±0.42c
5.05±0.48d
LA
92.68±6.38a
239.99±17.38b
241.21±16.56b
ACP
575.51±25.83a
624.99±14.44b
C3
101.91±5.96a
119.54±9.15b
C4
7.81±0.56a
9.37±0.78b
179.4
224.5
RI PT
Head kidney 240.99±13.94d
651.99±21.35c
650.51±23.08c
97.81±8.88d
100.77±7.31d
SC
244.72±13.07d
5.22±0.21d
238.30±17.34b
248.78±15.71b
254.75±19.48b
611.33±31.06b
624.96±20.14b
612.67±26.39b
621.23±30.32b
132.28±4.12c
133.02±11.27c
130.63±9.45c
130.64±4.53c
11.00±0.52cd
11.46±0.56d
11.43±0.54d
YACP in head kidney = 0.7188X + 540.8400
Ymax = 651.25
R2 = 0.993
P
0.01
YC3 in head kidney = 0.3412X + 50.6580
Ymax = 98.17
R2 = 0.984
P
0.01
YC3 in spleen = 0.3513X + 101.9600
Ymax = 131.64
R2 = 0.986
P = 0.07
YC4 in head kidney = 0.0153X + 3.0914
Ymax = 5.17
R2 = 0.985
P
TE D
Spleen
M AN U
5.23±0.42d
AC C
EP
Regression
10.41±0.60c
1274
1275
1276
1277 61
0.01
1278
ACCEPTED MANUSCRIPT
Table 5
1279 Dietary vitamin C levels (mg/kg diet)
2.9
44.2
89.1
133.8
179.4
ROS
100.00±5.26c
81.21±7.83b
64.18±5.65a
61.02±3.60a
MDA
5.01±0.41d
4.53±0.30c
4.17±0.12b
3.63±0.19a
PC
5.88±0.18b
4.30±0.22a
4.12±0.31a
4.19±0.18a
AHR
151.23±12.51a
160.76±13.09a
175.50±14.41b
180.50±11.24b
ASA
50.98±4.09a
59.82±2.96b
67.28±4.53c
T-SOD
4.74±0.13a
6.82±0.33b
CuZnSOD
3.00±0.06a
MnSOD
224.5
RI PT
Head kidney 59.84±4.87a
3.66±0.23a
3.61±0.21a
4.21±0.14a
4.38±0.13a
SC
60.36±5.14a
177.46±9.17b
77.20±4.46d
76.61±5.30d
78.71±4.82d
7.62±0.33c
7.63±0.30c
7.44±0.40c
7.40±0.30c
3.84±0.22b
4.32±0.14c
4.40±0.14c
4.27±0.18c
4.33±0.19c
1.74±0.13a
2.97±0.28b
3.29±0.23b
3.23±0.30b
3.17±0.31b
3.07±0.29b
CAT
0.86±0.06a
0.91±0.04ab
0.96±0.09b
1.09±0.10c
1.07±0.02c
1.09±0.04c
GPx
187.76±14.80a
225.94±16.45b
223.97±17.21b
234.01±15.18b
227.09±19.73b
231.79±20.80b
GST
35.92±3.16a
36.27±2.00a
40.66±3.59b
41.98±3.78b
42.63±3.23b
42.40±1.59b
GR
13.85±0.71a
15.52±0.41b
17.24±0.42c
17.81±0.77c
18.19±1.62c
17.91±0.77c
AC C
EP
TE D
M AN U
176.97±4.86b
0.98±0.08a
0.99±0.09a
1.25±0.12b
1.24±0.11b
1.40±0.09c
1.39±0.09c
100.00±7.43d
84.91±7.10c
74.86±6.92b
67.90±6.38ab
66.39±6.48a
66.20±3.28a
MDA
5.97±0.27c
4.85±0.92b
4.30±0.31a
4.37±0.23a
4.39±0.38a
4.41±0.10a
PC
4.01±0.19b
3.62±0.26a
3.64±0.09a
3.59±0.17a
3.53±0.24a
3.68±0.41a
AHR
165.53±13.32a
171.05±13.37a
167.56±12.96a
166.03±8.85a
193.13±19.29b
193.89±9.65b
GSH
Spleen
ROS
62
86.53±3.79 85.15±2.84 ACCEPTED MANUSCRIPT
82.07±5.87b
b
T-SOD
4.38±0.15a
5.15±0.11b
6.45±0.25c
CuZnSOD
2.63±0.08a
2.91±0.10b
MnSOD
1.74±0.11a
CAT
b
84.42±7.09b
86.75±1.56b
7.79±0.25d
7.97±0.32d
7.90±0.13d
3.52±0.24c
4.06±0.10d
4.32±0.11e
4.34±0.20e
2.23±0.18b
2.92±0.20c
3.73±0.25d
3.66±0.24d
3.56±0.20d
0.48±0.03a
0.50±0.04a
0.67±0.05b
0.65±0.05b
0.67±0.03b
0.68±0.04b
GPx
182.37±11.82a
199.20±13.73b
203.37±14.46b
211.67±13.24b
240.97±18.51c
241.23±4.07c
GST
26.70±2.11a
29.96±2.86b
30.99±2.90b
31.14±1.50b
31.34±2.56b
31.06±2.55b
GR
18.32±1.04a
19.39±1.14b
20.27±0.74b
20.18±0.59b
20.14±0.71b
20.18±0.82b
GSH
1.31±0.10a
1.34±0.05ab
1.45±0.13ab
1.46±0.12b
RI PT
64.61±5.80a
SC
ASA
1.98±0.15c
Ymin = 61.35
R2 = 0.997
P
Ymin = 4.37
R2 = 0.953
P = 0.14
Ymax = 77.51
R2 = 0.997
P
0.01
Ymax = 7.89
R2 = 0.990
P
0.01
Ymax = 4.33
R2 = 0.968
P=0.12
Ymax = 4.33
R2 = 0.982
P
0.01
Ymax = 3.65
R2 = 0.992
P
0.01
YCAT in head kidney = 0.0017X + 0.8405
Ymax = 1.08
R2 = 0.940
P
0.05
YGPx in spleen = 0.2933X + 181.1500
Ymax = 241.10
R2 = 0.909
P
0.05
YGR in head kidney = 0.0393X + 13.7520
Ymax = 17.79
R2 = 0.999
P
0.05
YGSH in head kidney = 0.0025X + 0.9502
Ymax = 1.40
R2 = 0.899
P
0.05
YGSH in spleen = 0.0031X + 1.2179
Ymax = 1.96
R2 = 0.744
P = 0.06
M AN U
1.94±0.13c
Regression
YROS in head kidney = -0.415X + 100.6400 YMDA in spleen = -0.0193X + 5.9149
YT-SOD in spleen = 0.0264X + 4.1606 YCuZnSOD in head kidney = 0.0152X + 3.0278
AC C
YMnSOD in spleen = 0.0152X + 1.6261
EP
YCuZnSOD in spleen = 0.0102X + 2.5679
TE D
YASA in head kidney = 0.1967X + 50.5400
1280
63
0.05
1281
ACCEPTED MANUSCRIPT
Table 6
1282 Dietary vitamin C levels (mg/kg diet)
44.2
89.1
133.8
179.4
224.5
LA
91.94±6.28a
91.58±5.47a
148.59±5.59b
150.32±8.00b
143.53±7.29b
144.31±8.19b
ACP
248.65±12.62a
249.43±12.80a
259.48±6.91a
262.66±18.03a
C3
12.18±0.90a
15.44±0.87b
16.35±1.22b
16.07±0.99b
C4
2.74±0.27a
2.95±0.23a
3.36±0.29b
3.39±0.30b
1284
1285
1286
EP AC C
1290
TE D
1287
1289
267.41±17.13a
262.24±15.84a
15.20±1.23b
16.11±0.73b
3.35±0.20b
3.38±0.14b
SC M AN U
1283
1288
RI PT
2.9
64
1291
ACCEPTED MANUSCRIPT
Table 7
1292
1294
44.2
89.1
133.8
179.4
224.5
ROS
100.00±8.30d
67.19±4.85c
39.22±2.60b
33.55±2.83a
32.16±2.12a
32.55±3.14a
MDA
11.98±0.69c
11.69±0.88c
7.11±0.51b
4.61±0.38a
PC
4.48±0.12d
4.44±0.38d
3.66±0.21c
2.93±0.22b
AHR
106.33±4.33a
116.43±9.41b
147.15±9.48c
145.31±5.26c
ASA
59.49±4.39a
65.64±4.14b
80.66±5.51c
84.47±7.04c
T-SOD
16.85±0.15a
17.04±0.18ab
17.24±0.36b
CuZnSOD
9.26±0.38a
9.33±0.30a
MnSOD
7.58±0.29a
CAT
RI PT
2.9
4.70±0.37a
2.37±0.20a
2.60±0.19a
150.94±8.77c
146.27±5.06c
81.16±5.57c
80.30±3.63c
18.36±0.16c
18.17±0.21c
18.28±0.21c
9.39±0.36a
10.59±0.55b
10.75±0.25b
10.89±0.67b
7.71±0.36a
7.85±0.37a
7.77±0.56a
7.41±0.36a
7.39±0.64a
1.70±0.11a
1.82±0.17ab
1.88±0.10b
1.90±0.11b
1.97±0.12b
1.95±0.08b
GPx
88.76±7.82a
106.13±6.49b
119.08±9.33c
130.48±10.79cd
133.78±11.96d
136.64±11.52d
GST
44.91±2.89a
51.00±3.05b
51.10±4.24b
52.58±4.94b
53.79±4.65b
53.72±2.68b
GR
31.94±2.39a
35.85±3.23b
38.64±3.37b
38.11±1.51b
39.00±2.01b
39.08±2.74b
GSH
6.97±0.35a
7.12±0.69a
10.12±0.44b
10.43±0.87b
10.38±0.54b
10.15±0.40b
M AN U
TE D
EP
SC
4.75±0.43a
AC C
1293
Dietary vitamin C levels (mg/kg diet)
1295
1296
1297
1298 65
1299
ACCEPTED MANUSCRIPT
Table 8
1300 Correlation coefficients
NF-κB p65
IL-4/13A
-0.973
0.01
IL-10
-0.905
0.05
IL-11
-0.991
0.01
TGF-β1
-0.963
0.01
TGF-β2
-0.976
0.01
RI PT
Independent parameters
SC
Head kidney
Dependent parameters
IL-8
0.990
M AN U
IL-6
0.01
IL-12 P40
0.947
0.01
IL-15
0.904
0.05
0.992
0.01
NF-κB p65
-0.957
0.01
IKKα
-0.980
0.01
IKKβ
-0.989
0.01
IKKγ
-0.911
0.05
IL-1β
0.938
0.01
TNF-α
0.972
0.01
IFN-γ2
0.884
0.05
IL-4/13A
-0.968
0.01
IL-10
-0.994
0.01
IL-11
-0.843
0.05
TE D EP
AC C TOR
Spleen
NF-κB p65
0.01
0.967
IL-17D IκBα
P
66
1303
1304
1305
1306
TGF-β2
-0.935
0.01
IL-6
0.983
0.01
IL-8
0.927
0.01
IL-12 P40
0.971
0.01
IL-15
0.981
0.01
IL-17D
0.955
0.01
NF-κB p65
-0.948
0.01
SC
RI PT
0.01
IKKβ IKKγ TOR
IL-1β
TE D
TNF-α
-0.919
M AN U
IKKα
IFN-γ2
EP
1302
IκBα
-0.960
AC C
1301
TGF-β1 ACCEPTED MANUSCRIPT
1307
1308
1309
67
0.01
-0.845
0.05
-0.909
0.05
0.977
0.01
0.989
0.01
0.994
0.01
1310
ACCEPTED MANUSCRIPT
Table 9
1311 Correlation coefficients
CuZnSOD activity
CuZnSOD mRNA level
0.944
0.01
MnSOD activity
MnSOD mRNA level
0.852
0.05
CAT activity
CAT mRNA level
0.863
0.05
GPx activity
GPx1a mRNA level
0.866
0.05
GPx1b mRNA level
0.934
0.01
0.838
0.05
0.889
0.05
GSTR mRNA level
0.923
0.01
GSTP1 mRNA level
0.960
0.01
GSTP2 mRNA level
0.941
0.01
GR mRNA level
0.957
0.01
CuZnSOD
0.973
0.01
MnSOD
0.975
0.01
CAT
0.976
0.01
GPx1a
0.984
0.01
GPx1b
0.996
0.01
GPx4a
0.936
0.01
GPx4b
0.982
0.01
GSTR
0.977
0.01
GSTP1
0.987
0.01
EP
Nrf2
P
GPx4b mRNA level
TE D
GST activity
GR activity
SC
M AN U
GPx4a mRNA level
RI PT
Independent parameters
AC C
Head kidney
Dependent parameters
68
GSTP2 ACCEPTED MANUSCRIPT GR
0.932
0.01
Keap1a
-0.941
0.01
Keap1b
-0.994
0.01
CuZnSOD activity
CuZnSOD mRNA level
0.958
0.01
MnSOD activity
MnSOD mRNA level
0.923
0.01
CAT activity
CAT mRNA level
0.941
0.01
GPx activity
GPx1a mRNA level
0.754
SC
0.05
0.861
0.05
GSTR mRNA level
0.943
0.01
GSTP1 mRNA level
0.998
0.01
GSTP2 mRNA level
0.951
0.01
GR mRNA level
0.993
0.01
CuZnSOD
0.981
0.01
MnSOD
0.949
0.01
CAT
0.969
0.01
GPx1a
0.959
0.01
GPx1b
0.983
0.01
GPx4a
0.900
0.05
GPx4b
0.989
0.01
GSTR
0.968
0.01
GSTP1
0.964
0.01
EP
Nrf2
0.08
GPx4b mRNA level
TE D
GST activity
GR activity
0.813
M AN U
GPx1b mRNA level
RI PT
0.01
AC C
Spleen
0.957
69
GSTP2 ACCEPTED MANUSCRIPT
0.966
0.01
GR
0.950
0.01
Keap1a
-0.988
0.01
Keap1b
-0.944
0.01
RI PT
1312
1313
1314
SC
1315
M AN U
1316
1317
1318
1319
1323
1324
1325
1326
1327
EP
1322
AC C
1321
TE D
1320
1328
1329
1330
70
1331
ACCEPTED MANUSCRIPT
Table 10
1332 Correlation coefficients
FasL
caspase-8
0.950
0.01
p38 MAPK
0.977
0.01
MAPKK 6
p38 MAPK
0.968
0.01
Apaf-1
caspase-3
0.959
0.01
caspase-7
0.859
0.05
0.849
0.05
RI PT
Independent parameters
SC
Head kidney
Dependent parameters
Bcl-2
-0.987
0.01
0.941
0.01
caspase-3
-0.991
0.01
caspase-7
-0.979
0.01
caspase-9
-0.974
0.01
caspase-8
0.961
0.01
p38 MAPK
0.984
0.01
p38 MAPK
0.956
0.01
caspase-3
0.995
0.01
caspase-7
0.966
0.05
caspase-9
0.944
0.01
Bcl-2
-0.966
0.01
Bax
0.996
0.01
caspase-3
-0.986
0.01
Bax
EP
FasL
MAPKK 6
AC C
Spleen
TE D
IAP
M AN U
caspase-9
Apaf-1
IAP
P
71
caspase-7 ACCEPTED MANUSCRIPT caspase-9
-0.981
0.01
-0.946
0.01
1333
1334
RI PT
1335
1336
1337
SC
1338
M AN U
1339
1340
1341
1342
1346
1347
1348
1349
1350
EP
1345
AC C
1344
TE D
1343
1351
1352
1353
72
1354
ACCEPTED MANUSCRIPT
Table 11
1355 Correlation coefficients
MLCK
ZO-1
-0.983
0.01
ZO-2
-0.986
0.01
Claudin-b
-0.924
0.05
Claudin-c
-0.951
0.01
Claudin-3c
-0.853
0.05
RI PT
Independent parameters
SC
Head kidney
Dependent parameters
-0.929
M AN U
Claudin-7a
0.01
-0.943
0.01
Claudin-12
0.976
0.01
Claudin-15a
0.980
0.01
Claudin-15b
0.947
0.01
ZO-1
-0.993
0.01
ZO-2
-0.993
0.01
Claudin-b
-0.937
0.01
Claudin-c
-0.998
0.01
Claudin-3c
-0.991
0.01
Claudin-7a
-0.891
0.05
Claudin-7b
-0.999
0.01
Claudin-12
0.927
0.01
Claudin-15a
0.997
0.01
Claudin-15b
0.957
0.01
TE D
Claudin-7b
EP
TNF-α
AC C
Spleen
P
73
1356
Fig.1. Effects of dietary vitamin C ACCEPTED levels on survival rate (A) and skin lesion mrobidity (B) of young MANUSCRIPT
1357
grass carp (Ctenopharyngodon idella) after infection with A. hydrophila for 14 days.
1358
Fig.2. Deficiency of vitamin C led to obviously skin lesion, compared with optimal vitamin C
1360
supplementation, after infection with A. hydrophila in young grass carp.
RI PT
1359
1361
Fig.3. Relative expression of LEAP-2A, LEAP-2B, Hepcidin and β-defensin in the head kidney (A)
1363
and spleen (B) of young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of
1364
vitamin C after infection with A. hydrophila for 14 days. Data represent means of six fish in each group,
1365
error bars indicate S.D. Values having different letters are significantly different (P < 0.05). LEAP-2: liver
1366
expressed antimicrobial peptide 2.
1367
M AN U
SC
1362
Fig.4. Relative expression of IL-4/13A, IL-4/13B, IL-10, IL-11, TGF-β1 and TGF-β2 in the head
1369
kidney (A) and spleen (B) of young grass carp (Ctenopharyngodon idella) fed diets containing graded
1370
levels of vitamin C after infection with A. hydrophila for 14 days. Data represent means of six fish in
1371
each group, error bars indicate S.D. Values having different letters are significantly different (P < 0.05). IL:
1372
interleukin; TGF-β: transforming growth factor β.
EP
AC C
1373
TE D
1368
1374
Fig.5. Relative expression of IFN-γ2, TNF-α, IL-1β, IL-6, IL-8, IL-12 P35, IL-12 P40, IL-15 and
1375
IL-17D in the head kidney (A) and spleen (B) of young grass carp (Ctenopharyngodon idella) fed diets
1376
containing graded levels of vitamin C after infection with A. hydrophila for 14 days. Data represent
1377
means of six fish in each group, error bars indicate S.D. Values having different letters are significantly
1378
different (P < 0.05). IFN-γ2: interferon γ2; TNF-α: tumor necrosis factor α. 74
Fig.6. Relative expression of NF-κBACCEPTED p65, NF-κB p52, IκBα, IKKα, IKKβ, IKKγ, TOR, S6K1, 4E-BP1 MANUSCRIPT
1380
and 4E-BP2 in the head kidney (A) and spleen (B) of young grass carp (Ctenopharyngodon idella) fed
1381
diets containing graded levels of vitamin C after infection with A. hydrophila for 14 days. Data
1382
represent means of six fish in each group, error bars indicate S.D. Values having different letters are
1383
significantly different (P < 0.05). NF-κB: nuclear factor kappa B; IκBα: inhibitor of κBα; IKK: IκB kinase;
1384
TOR: target of rapamycin; S6K1: ribosomal protein S6 kinase 1; 4E-BP: eIF4E-binding protein.
RI PT
1379
1385
Fig.7. Relative expression of CuZnSOD, MnSOD, CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR,
1387
GSTP1, GSTP2, GSTO1, GSTO2 and GR in the head kidney (A) and spleen (B) of young grass carp
1388
(Ctenopharyngodon idella) fed diets containing graded levels of vitamin C after infection with A.
1389
hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate S.D. Values
1390
having different letters are significantly different (P < 0.05). CuZnSOD: copper, zinc superoxide dismutase;
1391
MnSOD: manganese superoxide dismutase; CAT: catalase; GPx: glutathione peroxidase; GST: glutathione
1392
S-transferase; GR: glutathione reductase.
M AN U
TE D
1393
SC
1386
Fig.8. Relative expression of Nrf2, Keap1a and Keap1b in the head kidney (A) and spleen (B) of young
1395
grass carp (Ctenopharyngodon idella) fed diets containing graded levels of vitamin C after infection
1396
with A. hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate S.D.
1397
Values having different letters are significantly different (P < 0.05). Nrf2: NF-E2-related factor 2; Keap1:
1398
Kelch-like-ECH-associated protein 1.
AC C
EP
1394
1399
1400
Fig.9. Relative expression of FasL, MAPKK 6, p38 MAPK, JNK, Bcl-2, Mcl-1, Bax, Apaf-1, IAP,
1401
caspase-2, caspase-3, caspase-7, caspase-8 and caspase-9 in the head kidney (A) and spleen (B) of 75
young grass carp (Ctenopharyngodon idella) fedMANUSCRIPT diets containing graded levels of vitamin C after ACCEPTED
1403
infection with A. hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate
1404
S.D. Values having different letters are significantly different (P < 0.05). FasL: Fas ligand; MAPKK 6:
1405
mitogen-activated protein kinase kinase 6; p38 MAPK: p38 mitogen-activated protein kinases; JNK: c-Jun
1406
N-terminal kinases; Bcl-2: B-cell lymphoma-2; Mcl-1: myeloid cell leukemia-1; Bax: B-cell lymphoma
1407
protein 2 associated X protein; Apaf-1: apoptotic protease activating factor-1; IAP, inhibitor of apoptosis
1408
protein.
RI PT
1402
SC
1409
Fig.10. Relative expression of Occludin, ZO-1, ZO-2, Claudin -b, -c, -3c, -7a, -7b, -12, -15a, 15b and
1411
MLCK in the head kidney (A) and spleen (B) of young grass carp (Ctenopharyngodon idella) fed diets
1412
containing graded levels of vitamin C after infection with A. hydrophila for 14 days. Data represent
1413
means of six fish in each group, error bars indicate S.D. Values having different letters are significantly
1414
different (P < 0.05). ZO: zonula occludens; MLCK: myosin light chain kinase.
TE D
M AN U
1410
1415
Fig.11. Relative expression of IL-10, TGF-β1, IFN-γ2, TNF-α, IL-1β, IL-8 and NF-κB p65, IκBα,
1417
IKKα, IKKβ, IKKγ, TOR and S6K1 in the skin of young grass carp (Ctenopharyngodon idella) fed
1418
diets containing graded levels of vitamin C after infection with A. hydrophila for 14 days. Data
1419
represent means of six fish in each group, error bars indicate S.D. Values having different letters are
1420
significantly different (P < 0.05).
AC C
1421
EP
1416
1422
Fig.12. Relative expression of CuZnSOD, MnSOD, CAT, GPx1a, GSTR, GR, Nrf2, Keap1a and
1423
Keap1b in the skin of young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of
1424
vitamin C after infection with A. hydrophila for 14 days. Data represent means of six fish in each group, 76
1425
error bars indicate S.D. Values havingACCEPTED different lettersMANUSCRIPT are significantly different (P < 0.05).
1426
Fig.13. Relative expression of caspase-3, -8 and caspase-9 in the skin of young grass carp
1428
(Ctenopharyngodon idella) fed diets containing graded levels of vitamin C after infection with A.
1429
hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate S.D. Values
1430
having different letters are significantly different (P < 0.05).
RI PT
1427
1431
Fig.14. Relative expression of Occludin, ZO-1, Claudin -b, -c, -3c, -12 and Ccaudin-15a in the skin of
1433
young grass carp (Ctenopharyngodon idella) fed diets containing graded levels of vitamin C after
1434
infection with A. hydrophila for 14 days. Data represent means of six fish in each group, error bars indicate
1435
S.D. Values having different letters are significantly different (P < 0.05).
M AN U
SC
1432
1436
Fig.15. Broken-line analysis of PWG (A), LA activity (B) and MDA content (C) in head kidney and C4
1438
content (D) and ROS production (E) in spleen for young grass carp (Ctenopharyngodon idella) fed
1439
diets containing graded levels of vitamin C.
EP
1440
TE D
1437
Fig.16. The potential action pathways of vitamin C in the head kidney and spleen immunity and
1442
structural integrity of fish.
1443
1444
AC C
1441
1445
1446
1447
77
ACCEPTED MANUSCRIPT
Fig.1
RI PT
1448
SC
1449
1453
1454
1455
1456
TE D EP
1452
AC C
1451
M AN U
1450
1457
1458
1459
78
ACCEPTED MANUSCRIPT
SC
RI PT
Fig. 2
M AN U
1460
1464
1465
1466
1467
1468
EP
1463
AC C
1462
TE D
1461
1469
1470
1471
79
ACCEPTED MANUSCRIPT
RI PT
Fig. 3
SC
1472
M AN U
1473
1476
EP AC C
1475
TE D
1474
1477
1478
1479
80
ACCEPTED MANUSCRIPT
RI PT
Fig. 4
SC
1480
M AN U
1481
1484
EP AC C
1483
TE D
1482
1485
1486
1487
81
ACCEPTED MANUSCRIPT
RI PT
Fig. 5
SC
1488
M AN U
1489
1492
EP AC C
1491
TE D
1490
1493
1494
1495
82
ACCEPTED MANUSCRIPT
RI PT
Fig. 6
SC
1496
M AN U
1497
1500
EP AC C
1499
TE D
1498
1501
1502
1503
83
ACCEPTED MANUSCRIPT
RI PT
Fig. 7
SC
1504
M AN U
1505
1508
EP AC C
1507
TE D
1506
1509
1510
1511
84
ACCEPTED MANUSCRIPT
RI PT
Fig. 8
SC
1512
M AN U
1513
1516
EP AC C
1515
TE D
1514
1517
1518
1519
85
ACCEPTED MANUSCRIPT
RI PT
Fig. 9
SC
1520
M AN U
1521
1524
EP AC C
1523
TE D
1522
1525
1526
1527
86
ACCEPTED MANUSCRIPT
RI PT
Fig. 10
SC
1528
M AN U
1529
1532
EP AC C
1531
TE D
1530
1533
1534
1535
87
ACCEPTED MANUSCRIPT
RI PT
Fig. 11
SC
1536
M AN U
1537
1538
1539
1543
1544
1545
1546
1547
EP
1542
AC C
1541
TE D
1540
1548
1549
1550
1551 88
ACCEPTED MANUSCRIPT
RI PT
Fig. 12
SC
1552
M AN U
1553
1554
1555
1559
1560
1561
1562
1563
EP
1558
AC C
1557
TE D
1556
1564
1565
1566
1567 89
ACCEPTED MANUSCRIPT
RI PT
Fig. 13
SC
1568
M AN U
1569
1570
1571
1575
1576
1577
1578
1579
EP
1574
AC C
1573
TE D
1572
1580
1581
1582
1583 90
ACCEPTED MANUSCRIPT
RI PT
Fig. 14
SC
1584
M AN U
1585
1586
1587
1591
1592
1593
1594
1595
EP
1590
AC C
1589
TE D
1588
1596
1597
1598
1599 91
ACCEPTED MANUSCRIPT
Fig. 15
RI PT
1600
M AN U
SC
1601
1604
1605
EP AC C
1603
TE D
1602
1606
1607
1608
1609 92
1614
1615
1616
Vitamin C deficiency
Optimal vitamin C
Inhibition signaling
LA and
receptor pathway:
signaling
than NF-κB P52) and TOR
FasL
molecule:
ACP
Mitochondria
MLCK (only in
activities
pathway: activation
head kidney)
, C3 and
Pro-inflammatory cytokines:
Bcl2 (rather than
C4
TNT-α, IFN-γ2, IL-1β, IL-6,
Mcl-1), and
contents
IL-8, IL-12 P35 (only in spleen),
inhibition Bax
IL 12-P40, IL-15, IL-17D
junctional
Anti-inflammatory cytokines:
complexes
SC
Tight
caspase-2 (only in spleen),
IL-4/13A, IL-4/13B (only in
caspase-3, caspase-7,
M AN U
head kidney), IL-10, IL-11,
caspase-8, caspase-9
TGF-β1, TGF-β2
Antioxidant enzymes mRNA
RI PT
molecules: NF-κB P65 (rather
Activation signaling molecule: Nrf2
Inhibition death
Inhibition
expression: CuZnSOD,
MnSOD, CAT, GPx1a, GPx1b, GPx4a, GPx4b, GSTR, GSTP (rather than GSTO), GR
Antioxidant enzymes activities: CuZnSOD, MnSOD, CAT, GPx, GST and GR
Head kidney and spleen structural integrity
Head kidney and spleen immunity
Fish disease resistance
TE D
1613
Fish
Fish growth performance
EP
1611 1612
ACCEPTED MANUSCRIPT
Fig. 16
AC C
1610
93
ACCEPTED MANUSCRIPT Highlights Compared with optimal vitamin C supplementation:
Vitamin C deficiency decreased fish head kidney, spleen and skin immunity under injection of A. hydrophila. Vitamin C deficiency aggravated inflammation in the head kidney, spleen and skin of fish under injection of A. hydrophila.
RI PT
Vitamin C deficiency induced oxidative injury via disruption of antioxidant system in the head kidney, spleen and skin of fish under injection of A.
SC
hydrophila.
Vitamin C deficiency disturbed tight junctional complexes in the head kidney,
M AN U
spleen and skin of fish under injection of A. hydrophila.
AC C
EP
TE D
Vitamin C regulated NF-κB, TOR, Nrf2, apoptosis and MLCK signaling.