linoleic acid ratios modulate immune response, physical barrier and related signaling molecules mRNA expression in the gills of juvenile grass carp (Ctenopharyngodon idella)

Fish & Shellfish Immunology 62 (2017) 1e12

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Dietary alpha-linolenic acid/linoleic acid ratios modulate immune response, physical barrier and related signaling molecules mRNA expression in the gills of juvenile grass carp (Ctenopharyngodon idella) Yun-Yun Zeng a, 1, Lin Feng a, b, c, 1, Wei-Dan Jiang a, b, c, Yang Liu a, b, c, Pei Wu a, b, c, Jun Jiang a, b, c, Sheng-Yao Kuang d, Ling Tang d, Wu-Neng Tang d, Yong-An Zhang e, Xiao-Qiu Zhou a, b, c, * a

Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China c Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China d Animal Nutrition Institute, Sichuan Academy of Animal Science, Chengdu 610066, China e Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 July 2016 Received in revised form 29 December 2016 Accepted 3 January 2017 Available online 4 January 2017

This study was conducted to explore the possible effects of dietary ALA/LNA ratios on the gill immunity, tight junction and antioxidant capacity, and the related signaling factor mRNA levels of juvenile grass carp (Ctenopharyngodon idella). Fish were fed diets with different ALA/LNA ratios (0.01, 0.34, 0.68, 1.03, 1.41, 1.76 and 2.15) for 60 days. The present results showed that ALA/LNA ratio of 1.03 significantly enhanced lysozyme and acid phosphatase activities, complement 3 contents, promoted mRNA levels of antimicrobial peptides (Hepcidin and liver expression antimicrobial peptide-2), anti-inflammatory cytokines (interleukin 10 and transforming growth factor b1) and inhibitor protein kBa, whereas suppressed pro-inflammatory cytokines (interleukin 1b, interleukin 8, tumor necrosis factor a and interferon g2), and signal molecules (IkB kinase b, IkB kines g and nuclear factor kB p65) mRNA levels in the gill, indicating that optimal dietary ALA/LNA ratio improve gill immunity of juvenile fish. Besides, ALA/LNA ratio of 1.03 increased mRNA levels of the barrier functional proteins (occludin, zonula occludens-1, claudin-b, -c and
3), and reduced the pore-formation proteins (claudin-15a) and myosin light-chain kinase mRNA abundance in the gill of juvenile grass carp, indicating optimum ALA/LNA ratio strengthen gill tight junction of juvenile fish. Additionally, ALA/LNA ratio of 1.03 increased glutathione contents, copper/zinc superoxide dismutase, glutathione peroxidase, glutathione S-transferase and glutathione reductase activities and mRNA abundance, and nuclear factor erythoid 2-related factor 2 mRNA levels in the gill of fish, suggesting that optimal ALA/LNA ratio ameliorate gill antioxidant status of juvenile fish. Interestingly, dietary ALA/LNA ratios had no effect on IkB kinase a and catalase activities in fish gills. Collectively, optimal dietary ALA/LNA ratio could improve gill immunity and strengthen physical barrier of juvenile fish. Based on the quadratic regression analysis of complement 3 content in the gill, optimal dietary ALA/LNA ratio for maximum growth of juvenile grass carp was estimated to be 1.12. © 2017 Published by Elsevier Ltd.

Keywords: Alpha-linolenic acid Linoleic acid Grass carp Gill Immunity Physical barrier

1. Introduction

* Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan, China. E-mail addresses: [email protected], [email protected] (X.-Q. Zhou). 1 These two authors contributed to this work equally. http://dx.doi.org/10.1016/j.fsi.2017.01.003 1050-4648/© 2017 Published by Elsevier Ltd.

The fish gills serve several purposes as they not only participate in respiration, osmoregulation, but also represent a crucial organ for immune response [1,2]. It is reported that fish gill dysfunction often result in growth retardation and even high mortality [3]. Thus, maintaining gill health is of the utmost importance in fish.

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Y.-Y. Zeng et al. / Fish & Shellfish Immunology 62 (2017) 1e12

Hess et al. [4] reported that gill health status is mainly associated with immunity and physical barrier function in clownfish larvae (Amphiprion percula). Moreover, fish gill immunity and physical barrier function have close relation to nutrients [5,6]. Alphalinolenic acid (ALA, 18:3n-3) and linoleic acid (LNA, 18:2n-6) are considered as essential fatty acids (EFA) in fish [7]. Previous study demonstrated that optimal level of ALA and LNA could improve the growth performance and intestinal health status of juvenile grass carp [8]. Additionally, Bell et al. [9] noted that dietary ALA and LNA could significantly affect ALA and LNA concentration in the gill of juvenile turbot (Scophthalmus maximus L.), which suggested that ALA and LNA could be retained in fish gill. Thus, above data implies a potential effect of dietary ALA and LNA on the gill health of fish, which is an interesting topic to investigate. In Atlantic halibut (Hippoglossus hippoglossus), the gill health status is strongly dependent upon the mucosal immune response [10], which is correlated with humoral components (lysozyme, complement and antimicrobial peptides) [11], and cytokines [interleukin 1b (IL-1b), tumor necrosis factor a (TNF-a) and transforming growth factor b (TGF-b)] [12]. Moreover, cytokines have been shown to be transcriptionally regulated by intracellular signaling molecules nuclear factor-kB (NF-kB) and inhibitor factorkB (IkB) in the gill of catfish [13]. However, little information is available regarding on the effects of dietary ALA and LNA on the cytokines production via NF-kB signaling pathway in the gill of fish. Feldman et al. [14] reported that ALA and LNA stimulated Sirtuin 6 (SIRT6) activity in Escherichia coli strains. SIRT6 could suppress forkhead transcription factor (FOXO3a) expression in breast cancer cells [15], while FOXO3a could inhibit NF-kB activity in 239T cells [16]. These findings lead to the idea that dietary ALA and LNA may affect cytokines production via NF-kB signaling pathway in the gill of fish, which needs further investigation. Depart from the mucosal immunity, physical barrier function also play a prominent role in maintaining gill health of fish. It was reported that physical barrier was mainly relied on the tight junction (TJ) complex, such as occludin, claudins and zonular occludens 1 (ZO-1) [17]. Study in the gill of grass carp exhibited that TJ protein expression could be modulated by myosin light-chain kinase (MLCK) [18]. However, information about the effects of dietary ALA and LNA on the MLCK expression in fish gill is scarce. Pervious study has indicated that optimal level of dietary ALA and LNA could inhibit TNF-a expression in the intestine of grass carp [8]. Meanwhile, Thomas et al. [19] pointed out that TNF-a increased myosin light chain kinase (MLCK) expression in Caco-2 cells. These observations indicate that dietary ALA and LNA might have effects on TJ protein mRNA levels via a MLCK-dependent way in the gill of fish, and this topic is worthy of further investigation. Additionally, fish gill barrier function was also relied on the cell structural integrity [20]. It was reported that fish have developed nonenzymatic and enzymatic antioxidant defense system to maintain gill structural integrity [21]. Antioxidant enzyme activities were partly associated with antioxidant enzyme gene transcriptions, which were regulated by the nuclear factor erythoid 2-related factor 2 (Nrf2) in Jian carp (Cyprinus carpio var. Jian) [22]. However, limited study is available regarding the effects of dietary ALA and LNA on the gill antioxidant enzyme activities through modulating their gene transcriptions link to Nrf2 in juvenile grass carp. Linolenic acid metabolite, DHA, could induce Nrf2 expression in mice [23]. Linoleic acid metabolite, ARA, could activate Nrf2 in HepG2 cells [24]. These observations indicate that dietary ALA and LNA might have effects on the gill antioxidant capacity of fish, which requires further investigation. This study is a part of a large research aimed at determining the effects of dietary ALA/LNA ratios on fish growth and intestinal health of juvenile grass carp [8]. The objective of this study was to

further investigate the relationship between dietary ALA/LNA ratios and gill health by determining the effects of dietary ALA/LNA ratios on immune response, tight junctions, antioxidant capacities and signaling molecule expression (e.g., NF-kB, MLCK and Nrf2) in the gills of juvenile grass carp.

2. Materials and methods 2.1. Experimental diets and design The formulation of the experimental diets is shown in Table 1. Seven iso-nitrogenous and iso-lipidic experimental diets varying only in the dietary lipid source were formulated. Meanwhile, the experiment diets contained 5% of lipid and 35% of protein, respectively according to Ji et al. [25] and NRC [26]. Fish meal (Pesquera Lota Protein Ltd., Lota, Chile), casein (Hulunbeier Sanyuan Milk Co., Ltd., Inner Mongolia, China) and gelatin (Rousselot Gelatin Co., Ltd., Guangdong, China) were used as the protein sources. According to Li et al. [27], three different lipid sources: linseed oil (Hunan Yama biotechnology Co., Ltd., Hunan, China), safflower oil (Shanghai Yuan Tian Edible Agricultural Products Ltd., Shanghai, China) and coconut oil (Lvyuan natural flavor oil refinery, Jiangxi, China) were utilized to formulate the experiment diet containing varying ratios of ALA/LNA (0.00, 0.35, 0.70, 1.05, 1.40, 1.75 and 2.10), with a constant total C18 PUFA (ALA þ LNA) content. Ethoxyquin was added as the antioxidant. According to the method described by Otsuka et al. [28], final ratios of dietary ALA/LNA of the seven experimental diets were measured to be 0.01, 0.34, 0.68, 1.03, 1.41, 1.76 and 2.15. All ingredients were mixed, pelleted, and stored at
20  C until use as described by our previous study [29].

Table 1 Diet formulation and composition .a Ingredients

g kg
1

Fish meal Casein Gelatin DL-Methionine (99%) Vegetable oil premix b Alpha-starch Corn starch Vitamin premix c Mineral premix d Ca(H2PO4)2 (220 g kg
1) Choline chloride (600 g kg
1) Cellulose Ethoxyquin (300 g kg
1)

30.0 280.0 75.0 1.4 50.0 240.0 215.5 10.0 20.0 22.6 5.0 50.0 0.5

a Crude protein and total lipids were measured to be 347.4 g kg
1 and 46.9 g kg
1. b Linseed oil and safflower oil were added to achieve different ALA/LNA ratios (0.00, 0.35, 0.70, 1.05, 1.40, 1.75 and 2.10). Each mixture was made isolipidic with the addition of coconut oil. c Per kilogram of vitamin premix (g kg
1): retinyl acetate (500 000 IU g
1), 2.40 g; cholecalciferol (500 000 IU g
1), 0.40 g; D, L-atocopherol acetate (500 g kg
1), 12.55 g; menadione (230 g kg
1), 0.80 g; cyanocobalamin (10 g kg
1), 0.83 g; D-biotin (20 g kg
1), 4.91 g; folic acid (960 g kg
1), 0.40 g; thiamin nitrate (980 g kg
1), 0.05 g; ascorhyl acetate (930 g kg
1), 7.16 g; niacin (990 g kg
1), 2.24 g; meso-inositol (990 g kg
1), 19.39 g; calcium-D-pantothenate (980 g kg
1) 2.89 g; riboflavine (800 g kg
1), 0.55 g; pyridoxine hydrochloride (980 g kg
1), 0.59 g. All ingredients were diluted with corn starch to 1 kg. d Per kilogram of mineral premix (g kg
1): FeSO4$H2O, 23.110 g; CuSO4$5H2O, 0.010 g; ZnSO4$H2O, 0.620 g; MnSO4$H2O, 1.640 g; KI, 0.070 g; NaSeO3, 0.005 g; MgSO4$H2O, 60.530 g. All ingredients were diluted with corn starch to 1 kg.

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2.2. Feeding trial The procedures used in this study were approved by the University of Sichuan Agricultural Animal Care Advisory Committee. Juvenile grass carp were obtained from fisheries (Sichuan, China). The feeding schedule was the same as our previous study [29]. Before starting the experiment, juvenile grass carp were acclimated to the experimental conditions for 4 weeks [30]. Subsequently, a total of 1260 juvenile grass carp, with an average initial weight of 8.78 ± 0.03 g, were randomly distributed into 21 experimental cages (1.4  1.4  1.4 m), each of which was equipped with a 100 cm diameter disc of 1-mm gauze in the bottom to collect the uneaten food, as described by Tang et al. [31]. The treatments were randomly assigned to 3 cages, and the fish were fed to apparent satiation 4 times daily for 60 days, according to Hu et al. [32]. Thirty minutes after feeding, uneaten feed was collected, dried, and weighed to calculate the feed intake, as described by Lim and Lee [33]. During the experiment, water temperature was 26 ± 2  C. The dissolved oxygen was not less than 6.0 mg L
1, the pH was maintained at 7.0 ± 0.5 and the experimental units were under natural light and dark cycle as described by Tang et al. [31]. 2.3. Sample collection and analysis The procedures of sample collection were similar to those previously described in another study conducted in our laboratory [34]. Fish feed intake has been reported in our previous study and the specific value was ‘64.40 g, 72.37 g, 81.32 g, 82.25 g, 81.76 g, 78.40 g and 72.91 g’ [29]. Additionally, based on the measured ALA/ LNA ratios, the estimated dietary ALA intake was ‘0.01 g, 0.38 g, 0.67 g, 0.84 g, 0.96 g, 1.00 g, 1.02 g’ and ALA intake was ‘1.33 g, 1.11 g, 0.98 g, 0.81 g, 0.68 g, 0.56 g and 0.47 g’, respectively. At the end of the feeding trial, fish from each treatment were anaesthetized in a benzocaine bath (50 mg L
1). According to the method of Shi et al. [35], the blood of each fish was drawn from the caudal vain. After sacrificed, the gills of fish were quickly removed, frozen in liquid nitrogen, and stored at
80  C for subsequent analysis, according to Rizzello et al. [36]. According to the method of Chen et al. [34], the samples were homogenized on ice in 10 vol (w v
1) of ice-cold physiological saline and centrifuged at 6000 g for 20 min at 4  C, and then the supernatant was collected to analyze the gill immune and antioxidant parameters. Gill protein content was determined by the method of Bradford [37]. The lysozyme (LZ) and acid phosphatase (ACP) activities, and complement 3 (C3) content in the gill were determined as described by Shiu et al. [38], Wei et al. [39] and Wang et al. [40], respectively. The gill reactive oxygen species (ROS) was measured by the method according to Rosa et al. [41]. The protein carbonyl (PC) and malonaldehyde (MDA) contents were determined according to the procedures described by Tokur and Korkmaz [42]. The anti-superoxide anion (ASA) (O
2 -scavenging ability) and antihydroxy radical (AHR) (OH-scavenging ability) were measured as described by Jiang et al. [43]. The total superoxide dismutase (TSOD) and copper/zinc superoxide dismutase (Cu/ZnSOD) activities were assayed as described by Richard et al. [44], glutathione (GSH) content and glutathione peroxidase (GPx), catalase (CAT) activities were assayed as described by Vardi et al. [45]. The glutathione-Stransferase (GST) and glutathione reductase (GR) activities were measured as the method according to Lushchak et al. [46]. 2.4. Real-time polymerase chain reaction (PCR) analysis The procedures of total RNA isolation, reverse transcription and quantitative real-time PCR were similar to our previous study [8]. Total RNA of gills was isolated using RNAiso Plus (TaKaRa, Japan)

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according to the manufacturer's instructions followed by DNAse I treatment, and agarose gel (1%) electrophoresis and spectrophotometric analysis (A260: 280 nm ratio) were used to assess the RNA quality and quantity. Subsequently, cDNA was synthesized with 2ul of total RNA using the PrimeScript™ RT reagent Kit (TaKaRa Biotechnology (Dalian) Co., Ltd.). For quantitative real-time PCR, specific primers were designed according to the sequences cloned in our laboratory and the published sequences of grass carp (Table 2). According to the results of our preliminary experiment concerning the evaluation of internal control genes (data not shown), b-actin was used as a reference gene to normalize cDNA loading. Target and housekeeping gene amplification efficiency were calculated according to the specific gene standard curves generated from 10-fold serial dilutions according to Luo et al. [47]. After verification that the primers amplified with an efficiency of approximately 100%, the results were analyzed using the 2
DDCT method as described by Livak et al. [48]. 2.5. Western blotting The processes for gill protein extract preparation, antibodies and western blotting are similar to those previously described in another study conducted in our laboratory [49]. Briefly, gill nuclear and cytosolic proteins were extracted and the concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 2 h at room temperature and then incubated with primary antibody overnight at 4  C. We used the same anti- Nrf2, b-actin and lamin B1 antibodies as those in another study conducted in our laboratory [49], which were checked and also successfully cross-reacted with grass carp proteins of interest. b-Actin and lamin B1 were used as control proteins for cytosolic and nuclear protein, respectively. The blots were washed three times, followed by 2 h incubation with horseradish peroxidase-conjugated secondary antibody in TBST. Immune complexes were visualized, using an ECL kit (Millipore). The signals of Western blot were quantitatively measured using the image analysis software Quantity One, v4.62 (BioRad, Hercules, CA, USA). This experiment was repeated at least three times, and similar results were obtained each time. 2.6. Statistical analysis The results were presented as the mean ± standard deviation (SD). All data were subjected to a one-way analysis of variance (ANOVA) followed by the Duncan's multiple-range test to determine significant differences among treatments at P < 0.05 with SPSS 20.0 (SPSS Inc., IL, USA). A quadratic regression model was used to determine the optimal dietary ALA/LNA ratio, according to Zeitoun et al. [50]. 3. Results 3.1. Gill immune parameters As shown in Table 3, LZ activity was significantly improved with increasing dietary ALA/LNA ratios up to 1.03, and decreased thereafter (P < 0.05). ACP activity in the gill was the highest for fish fed with ALA/LNA ratio of 1.03, and the lowest for fish fed with ALA/ LNA ratio of 0.01 (P < 0.05). The C3 content of gill showed a similar trend with acid phosphatase activity. As shown in Fig. 1, based on the quadratic regression analysis of gill C3 content of juvenile grass carp, optimal dietary ALA/LNA ratio was found to be 1.12.

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Table 2 Real-time primer sequences, thermocycling conditions and gene bank numbers.a Gene Hepcidin Forward Reverse LEAP-2 Forward Reverse IgM Forward Reverse IgD Forward Reverse IgZ Forward Reverse IL-1b Forward Reverse IL-8 Forward Reverse TNF-a Forward Reverse IFN-g2 Forward Reverse IL-10 Forward Reverse TGF-b1 Forward Reverse IkBa Forward Reverse NF-kB P65 Forward Reverse IKKa Forward Reverse IKKb Forward Reverse IKKg Forward Reverse Claudin 3 Forward Reverse Claudin b Forward Reverse Claudin c Forward Reverse Claudin 12 Forward Reverse Claudin 15a Forward Reverse Occludin Forward Reverse ZO-1 Forward Reverse MLCK Forward Reverse CuZnSOD Forward

Sequences of primers

Thermocycling conditions

Accession number

5/-AGCAGGAGCAGGATGAGC-3/ 5/-GCCAGGGGATTTGTTTGT-3/

95  C 30 s, 40 cycles of 95  C 5 s, 59.3  C 30 s and 72  C 30 s

JQ246442

5/-TGCCTACTGCCAGAACCA-3/ 5/-AATCGGTTGGCTGTAGGA-3/

95  C 30 s, 40 cycles of 95  C 5 s, 59.3  C 30 s and 72  C 30 s

FJ390415

5/-CGATGCTTTTGACTACTGGGGA-3/ 5/-AGAAGAACACTGAGACAGGGCG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 55  C 30 s and 72  C 30 s

DQ417927

5/-GCAACTAAATGGGACGAAACTC-3/ 5/-ATCACCGAGGCATACAAGTTCT-3/

95  C 30 s, 40 cycles of 95  C 5 s, 55.5  C 30 s and 72  C 30 s

GQ429174

5/-TGAACCATCTCCGCCGAAGT-3/ 5/-TCACTCCCGAACGCTGGATACT-3/

95  C 30 s, 40 cycles of 95  C 5 s, 55.5  C 30 s and 72  C 30 s

GQ201421

5/-AGAGTTTGGTGAAGAAGAGG-3/ 5/-TTATTGTGGTTACGCTGGA-3/

95  C 30 s, 40 cycles of 95  C 5 s, 57.1  C 30 s and 72  C 30 s

JQ692172

5/-ATGAGTCTTAGAGGTCTGGGT-3/ 5/-ACAGTGAGGGCTAGGAGGG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 60.3  C 30 s and 72  C 30 s

JN663841

5/-CGCTGCTGTCTGCTTCAC-3/ 5/-CCTGGTCCTGGTTCACTC-3/

95  C 30 s, 40 cycles of 95  C 5 s, 58.4  C 30 s and 72  C 30 s

HQ696609

5/-TGTTTGATGACTTTGGGATG-3/ 5/-TCAGGACCCGCAGGAAGAC-3/

95  C 30 s, 40 cycles of 95  C 5 s, 60.4  C 30 s and 72  C 30 s

FJ766439

5/-AATCCCTTTGATTTTGCC-3/ 5/-GTGCCTTATCCTACAGTATGTG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 61.4  C 30 s and 72  C 30 s

HQ388294

5/-TTGGGACTTGTGCTCTAT-3/ 5/-AGTTCTGCTGGGATGTTT-3/

95  C 30 s, 40 cycles of 95  C 5 s, 55.9  C 30 s and 72  C 30 s

EU099588

5/-TCTTGCCATTATTCACGAGG-3/ 5/-TGTTACCACAGTCATCCACCA-3/

95  C 30 s, 40 cycles of 95  C 5 s, 62.3  C 30 s and 72  C 30 s

KJ125069

5/-GAAGAAGGATGTGGGAGATG-3/ 5/-TGTTGTCGTAGATGGGCTGAG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 62.3  C 30 s and 72  C 30 s

KJ526214

5/-GGCTACGCCAAAGACCTG-3/ 5/-CGGACCTCGCCATTCATA-3/

95  C 30 s, 40 cycles of 95  C 5 s, 60.3  C 30 s and 72  C 30 s

KM279718

5/- GTGGCGGTGGATTATTGG-3/ 5/- GCACGGGTTGCCAGTTTG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 60.3  C 30 s and 72  C 30 s

KP125491

5/-AGAGGCTCGTCATAGTGG-3/ 5/-CTGTGATTGGCTTGCTTT-3/

95  C 30 s, 40 cycles of 95  C 5 s, 58.4  C 30 s and 72  C 30 s

KM079079

5/-ATCACTCGGGACTTCTA-3/ 5/-CAGCAAACCCAATGTAG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 57  C 30 s and 72  C 30 s

KF193858

5/-GAGGGAATCTGGATGAGC-3/ 5/-ATGGCAATGATGGTGAGA-3/

95  C 30 s, 40 cycles of 95  C 5 s, 57.0  C 30 s and 72  C 30 s

KF193860

5/-GAGGGAATCTGGATGAGC-3/ 5/-CTGTTATGAAAGCGGCAC-3/

95  C 30 s, 40 cycles of 95  C 5 s, 59.4  C 30 s and 72  C 30 s

KF193859

5/-CCCTGAAGTGCCCACAA-3/ 5/-GCGTATGTCACGGGAGAA-3/

95  C 30 s, 40 cycles of 95  C 5 s, 55.4  C 30 s and 72  C 30 s

KF998571

5/-TGCTTTATTTCTTGGCTTTC-3/ 5/-CTCGTACAGGGTTGAGGTG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 59.0  C 30 s and 72  C 30 s

KF193857

5/-TATCTGTATCACTACTGCGTCG-3/ 5/-CATTCACCCAATCCTCCA-3/

95  C 30 s, 40 cycles of 95  C 5 s, 59.4  C 30 s and 72  C 30 s

KF193855

5/-CGGTGTCTTCGTAGTCGG-3/ 5/-CAGTTGGTTTGGGTTTCAG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 59.4  C 30 s and 72  C 30 s

KJ000055

5/-GAAGGTCAGGGCATCTCA-3/ 5/-GGGTCGGGCTTATCTACT-3/

95  C 30 s, 40 cycles of 95  C 5 s, 53.0  C 30 s and 72  C 30 s

KM279719

5/-CGCACTTCAACCCTTACA-3/

95  C 30 s, 40 cycles of 95  C 5 s,

GU901214

Y.-Y. Zeng et al. / Fish & Shellfish Immunology 62 (2017) 1e12

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Table 2 (continued ) Gene

Sequences of primers

Thermocycling conditions

Reverse CAT Forward Reverse GPx Forward Reverse GST Forward Reverse GR Forward Reverse Nrf2 Forward Reverse Keap1a Forward Reverse Keap1b Forward Reverse b-Actin Forward Reverse

5/-ACTTTCCTCATTGCCTCC-3/

61.5  C 30 s and 72  C 30 s

Accession number

5/-GAAGTTCTACACCGATGAGG-3/ 5/-CCAGAAATCCCAAACCAT-3/

95  C 30 s, 40 cycles of 95  C 5 s, 58.7  C 30 s and 72  C 30 s

FJ560431

5/-GGGCTGGTTATTCTGGGC-3/ 5/-AGGCGATGTCATTCCTGTTC-3/

95  C 30 s, 40 cycles of 95  C 5 s, 61.5  C 30 s and 72  C 30 s

EU828796

5/-TCTCAAGGAACCCGTCTG-3/ 5/-CCAAGTATCCGTCCCACA-3/

95  C 30 s, 40 cycles of 95  C 5 s, 58.4  C 30 s and 72  C 30 s

EU107283

5/-GTGTCCAACTTCTCCTGTG-3/ 5/-ACTCTGGGGTCCAAAACG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 59.4  C 30 s and 72  C 30 s

JX854448

5/-CTGGACGAGGAGACTGGA-3/ 5/-ATCTGTGGTAGGTGGAAC-3/

95  C 30 s, 40 cycles of 95  C 5 s, 62.5  C 30 s and 72  C 30 s

KF733814

5/-TTCCACGCCCTCCTCAA-3/ 5/-TGTACCCTCCCGCTATG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 63.0  C 30 s and 72  C 30 s

KF811013

5/-TCTGCTGTATGCGGTGGGC-3/ 5/-CTCCTCCATTCATCTTTCTCG-3/

95  C 30 s, 40 cycles of 95  C 5 s, 57.9  C 30 s and 72  C 30 s

KJ729125

5/-GGCTGTGCTGTCCCTGTA-3/ 5/-GGGCATAACCCTCGTAGAT-3/

95  C 30 s, 40 cycles of 95  C 5 s, 61.4  C 30 s and 72  C 30 s

M25013

a LEAP-2, liver expressed antimicrobial peptide 2; IgM, immunoglobulin M; IgD, immunoglobulin D; IgZ, immunoglobulin Z; IL-1b, interleukin 1b; IL-8, interleukin 8; TNF-a, tumor necrosis factor a; IFN-g2, interferon g2; IL-10, interleukin 10; TGF-b1, transforming growth factor b1; IkBa, inhibitor protein kBa; NF-kB p65, nuclear factor kappa B P65; IKK, IkB kinase; ZO-1, zonula occludens 1; MLCK, myosin light chain kinase; CuZnSOD, copper/zinc superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione-S-transferase; GR, glutathione reductase; Nrf2, NF-E2-related factor 2; Keap1, Kelch-like-ECH-associated protein 1.

Table 3 LA (U mg
1 protein), ACP activity (U mg
1 protein) and C3 (mg g
1 protein) content in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Dietary ALA/LNA ratios 0.01 LA ACP C3

0.34 b

17.62 ± 0.50 49.35 ± 3.22a 3.04 ± 0.12a

0.68 ab

17.43 ± 0.74 56.96 ± 3.64c 3.73 ± 0.21b

1.03 c

28.37 ± 1.40 62.52 ± 2.85d 4.40 ± 0.27d

1.41 d

39.19 ± 1.58 67.47 ± 4.66e 4.92 ± 0.23e

1.76 c

29.14 ± 1.23 63.11 ± 4.47de 4.41 ± 0.32d

2.15 a

16.19 ± 0.67 54.19 ± 4.75bc 4.05 ± 0.21c

16.94 ± 0.79ab 50.19 ± 2.72ab 3.26 ± 0.21a

Values are means ± SD (n ¼ 6). Values within the same row with different superscripts are significantly different (P < 0.05).

Fig. 1. Quadratic regression analysis of C3 content in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days.

3.2. Antibacterial peptide, immunoglobulin, cytokines and related signaling molecules mRNA levels in the gill of fish As shown in Fig. 2, antibacterial peptide Hepcidin and LEAP-2 mRNA levels were significantly elevated with increasing ALA/LNA ratios up to 1.03, and reduced subsequently (P < 0.05). As shown in Fig. 3, IgM mRNA levels were the highest at the ratio of 0.68, 1.03

Fig. 2. Hepcidin and LEAP-2 mRNA levels in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 6). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

and 1.41; IgD mRNA levels were the highest at the ratios of 0.68 and 1.03; IgZ mRNA levels were the highest at the ratios of 1.03 and 1.41. As shown in Fig. 4a, IL-1b mRNA level was the lowest at the ratio of 1.03, and the highest at the ratio of 0.01, 1.76 and 2.15, respectively. IL-8 and IFN-g2 mRNA levels were the lowest for fish fed with ALA/

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Fig. 3. IgM, IgD and IgZ mRNA levels in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 6). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

Fig. 5. IkBa and NF-kB p65 (a), IKKa, IKKb and IKKg (b) mRNA levels in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 6). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

lowest for fish fed with ratio of 1.03. However, IKKa mRNA levels showed no significant difference among all groups (P > 0.05). 3.3. Relative mRNA levels of tight junction proteins and MLCK in the gill of fish

Fig. 4. IL-1b, IL-8, TNF-a and IFN-g2 (a), IL-10 and TGF-b 1 (b) mRNA levels in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 6). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

LNA ratio of 1.03, and the highest for fish fed with ratios of 0.01 and 2.15, respectively. TNF-a mRNA level was the lowest at the ratio of 1.03 and highest at the ratio of 2.15. As shown in Fig. 4b, IL-10 and TGF-b2 mRNA levels were the highest at the ratio of 1.03, and the lowest at the ratio of 0.01 (P < 0.05). As shown in Fig. 5a, IkBa mRNA level was significantly enhanced with increasing dietary ALA/LNA ratios up to 1.03, and then decreased (P < 0.05). Conversely, NF-kB p65 mRNA level was the lowest at the ratio of 1.03 and highest at the ratio of 2.15 (P < 0.05). As shown in Fig. 5b, IKK b and IKKg transcript levels were the

As shown in Fig. 6a, claudin-3, -b, -c and
12 mRNA levels were significantly improved with increasing dietary ALA/LNA ratios up to 1.03, and depressed subsequently (P < 0.05). However, claudin-15a mRNA level was the lowest for fish fed with ratio of 1.03, and the highest for fish fed with ratios of 0.01 and 2.15, respectively (P < 0.05). As shown in Fig. 6b, the maximum mRNA levels of occludin and ZO-1 were obtained at the ratio of 1.03, while the minimum mRNA levels were observed at the ratios of 0.01 and 2.15 (P < 0.05). As shown in Fig. 7, MLCK mRNA level in the gill was significantly reduced with increasing dietary ALA/LNA ratios up to 1.03, and enhanced thereafter (P < 0.05). Nevertheless, there was no significant difference between 0.68 and 1.03 groups (P > 0.05). 3.4. Antioxidant-related parameters, Nrf2, Keap1a and Keap1b mRNA levels in the gill of fish As shown in Fig. 8, ROS content was the lowest for fish fed with ALA/LNA ratio of 1.03, and the highest at the ratio of 0.01 (P < 0.05). As shown in Table 4, the maximum MDA and PC contents were observed at the ratio of 0.01, and the minimum contents were obtained at the ratio of 1.03 (P < 0.05). The gill ASA and AHR activities were markedly elevated with increasing ALA/LNA ratios up to 1.03, and reduced thereafter (P < 0.05). Cu/ZnSOD activity in the gill was the highest for fish fed with ratio of 1.03, and the lowest for

Y.-Y. Zeng et al. / Fish & Shellfish Immunology 62 (2017) 1e12

7

Fig. 8. ROS production in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 6). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

mRNA levels showed no significant difference among all groups (P > 0.05). Relative mRNA levels of GPx, GST and GR were markedly enhanced with increasing dietary ALA/LNA ratios up to 1.03, and depressed thereafter (P < 0.05). As shown in Fig. 10, signaling molecule Nrf2 mRNA level in the gill was sharply enhanced with improving ALA/LNA ratios up to 1.03, and then depressed (P < 0.05). The minimum Keap1a and Keap1b mRNA levels were observed at the ratio of 1.03 and the maximum values were obtained at the ratio of 2.15 (P < 0.05). Fig. 6. Claudin-3, claudin-b, claudin-c, claudin-12 and claudin-15a (a), occludin and ZO-1 (b) mRNA levels in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 6). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

3.5. Nrf2 protein levels in the gill of fish As shown in Fig. 11a, the cytosolic Nrf2 protein levels were the highest at the ratio of 0.68, 1.03 and 1.41, and the lowest at the ratio of 0.01, 0.34, 1.76 and 2.15. As shown in Fig. 11b, the nuclear localization of Nrf2 was significantly depressed at the ratio of 1.03 and 1.41. However, the nuclear Nrf2 protein levels showed no significant difference from dietary ALA/LNA ratio of 0.34e1.41. 4. Discussion 4.1. Optimal dietary ALA/LNA ratio enhanced gill immunity of juvenile fish

Fig. 7. Myosin light chain kinase (MLCK) mRNA levels in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 6). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

fish fed with ratio of 0.01 and 2.15 (P < 0.05). However, CAT activity showed no significant difference among all groups (P > 0.05). GPx and GST activities in the gill were significantly enhanced with increasing dietary ALA/LNA ratios up to 1.03, and reduced subsequently (P < 0.05). GR activity was the highest for fish fed with ALA/ LNA ratio of 1.03. Nevertheless, no significant difference existed between ratios of 0.68 and 1.03. GSH content in the gill showed a similar tendency with Cu/ZnSOD activity, and the maximum content was obtained at the ratio of 1.03 (P < 0.05). As shown in Fig. 9, Cu/ZnSOD mRNA level was the highest at the ratio of 1.03 and lowest at the ratio of 0.01 (P < 0.05). However, CAT

Fish gill innate immunity is governed by the gill-associated lymphoid tissue, which is comprised of variably sized immune cells [51]. The immune cells could secret humoral components, including lysozyme, acid phosphatase, complement and antimicrobial peptides, which play a vital role in the gill innate immune response of fish [34]. Current study showed that dietary ALA/ LNA ratio of 1.03 significantly increased LZ and ACP activities, C3 content, Hepcidin, LEAP-2 and immunoglobulin (IgM, IgD and IgZ) mRNA levels in the gill of juvenile grass carp, indicating that optimal dietary ALA/LNA ratio was contributed to improve immune function in the gill of juvenile fish. Fish immune function has close relation to inflammatory response, which is primarily mediated by cytokines [52]. Studies have demonstrated that up-regulating pro-inflammatory cytokines (IL-1b, IL-8 and IFN-g) in Atlantic cod (Gadus morhua) [53], and down-regulating anti-inflammatory cytokines (IL-10 and TGF-b) in common carp (Cyprinus carpio L.) [54], would trigger the inflammatory response. The present study demonstrated that dietary ALA/LNA ratio of 1.03 significantly reduced IL-1b, IL-8, TNF-a and IFN-g2 mRNA abundance, while promoted IL-10 and TGF-b1 gene transcription in the gill of juvenile grass carp. These results indicated that optimal dietary ALA/LNA ratio could impair

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Table 4 MDA (nmol mg
1 protein) and PC (nmol mg
1 protein) content, ASA (U g
1 protein) and AHR (U g
1 protein) capacities, Cu/ZnSOD (U mg
1 protein), CAT (U mg
1 protein), GPx (U mg
1 protein), GST (U mg
1 protein), GR (U g
1 protein) activities and GSH (mg g
1 protein) content in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Dietary ALA/LNA ratios

MDA PC ASA AHR Cu/ZnSOD CAT GPx GST GR GSH

0.01

0.34

0.68

1.03

1.41

1.76

2.15

5.94 ± 0.28f 7.17 ± 0.33d 102.99 ± 6.92a 189.49 ± 8.11a 3.92 ± 0.18a 1.36 ± 0.04a 59.30 ± 4.13a 71.60 ± 5.37a 36.47 ± 3.17a 6.57 ± 0.28a

5.15 ± 0.51e 6.75 ± 0.48cd 125.45 ± 8.27bc 203.73 ± 8.93b 4.43 ± 0.23b 1.38 ± 0.05a 70.48 ± 4.83b 76.00 ± 6.68ab 38.67 ± 2.10a 7.08 ± 0.35b

4.26 ± 0.28c 5.97 ± 0.51b 136.49 ± 6.07de 238.37 ± 13.82c 4.91 ± 0.24c 1.39 ± 0.07a 89.75 ± 7.50de 81.17 ± 7.52b 42.25 ± 2.34b 7.92 ± 0.36c

3.63 ± 0.20a 4.61 ± 0.25a 147.95 ± 6.35f 258.11 ± 10.97d 5.42 ± 0.29d 1.39 ± 0.07a 103.64 ± 7.70f 90.17 ± 4.52c 43.15 ± 2.87b 8.96 ± 0.39d

3.79 ± 0.29ab 5.57 ± 0.38b 138.46 ± 9.91e 235.48 ± 9.38c 4.97 ± 0.18c 1.37 ± 0.07a 90.67 ± 6.83e 81.22 ± 5.96b 39.31 ± 2.14a 7.41 ± 0.32b

4.11 ± 0.35bc 6.02 ± 0.54b 129.10 ± 5.95cd 205.23 ± 9.54b 4.21 ± 0.34ab 1.37 ± 0.07a 82.40 ± 7.44cd 76.15 ± 2.95ab 38.76 ± 2.17a 7.05 ± 0.46b

4.73 ± 0.26d 6.65 ± 0.37c 119.16 ± 7.77b 190.66 ± 8.04a 3.91 ± 0.27a 1.34 ± 0.07a 75.66 ± 6.53bc 71.23 ± 5.44a 36.16 ± 2.38a 6.15 ± 0.39a

Values are means ± SD (n ¼ 6). Values within the same row with different superscripts are significantly different (P < 0.05).

Fig. 9. Cu/ZnSOD, CAT, GPx, GST and GR mRNA levels in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 6). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

Fig. 10. Nrf2, Keap1a and Keap1b mRNA levels in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 6). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

inflammatory response in the gill of juvenile fish. A possible mechanism for dietary ALA/LNA ratio on cytokines gene expression has been proposed to be via NF-kB signaling pathway [55]. NF-kB is a critical regulator of inflammatory cytokines expression in bony fish [56]. Study has shown that NF-kB could promote IL-1b and TNF-a gene expression, and reduced IL-10 and TGF-b2 mRNA levels in the intestine of juvenile Jian carp [57]. Current

Fig. 11. Cytolosic Nrf2 protein levels (a) and nuclear Nrf2 protein levels in the gill of juvenile grass carp fed diets containing different ALA/LNA ratios for 60 days. Values are means ± SD (n ¼ 3). Different letters above a bar indicate statistically significant differences among treatments (P < 0.05).

Y.-Y. Zeng et al. / Fish & Shellfish Immunology 62 (2017) 1e12

study exhibited that dietary ALA/LNA ratio of 1.03 significantly reduced NF-kB p65 mRNA levels in the gill of juvenile grass carp. Correlation analysis showed that IL-1b, IL-8, TNF-a and IFN-g2 were positively related to NF-kB p65, while IL-10 and TGF-b1 were negatively related to NF-kB p65 mRNA abundance (Table 5), suggesting that the suppressive pro-inflammatory cytokines and elevated anti-inflammatory cytokines mRNA levels by dietary ALA/ LNA ratio may be partly ascribed to the reduced NF-kB p65 transcript levels in the gill of juvenile grass carp. Besides, the inhibitor of kBa (IkBa) could trap the activated NF-kB in inactive cytoplasmic complexes, thereby preventing its nuclear translocation, and ultimately leading to the suppressed IFN expression in the gill of catfish [58]. Data reported herein showed that dietary ALA/LNA ratio of 1.03 significantly promoted IkBa mRNA abundance in the gill of juvenile grass carp. Correlation analysis exhibited that NF-kB p65 mRNA levels were negatively related to IkBa mRNA abundance (r ¼
0.891, P < 0.01), indicating that optimal dietary ALA/LNA ratio regulated cytokines gene transcription may be partly through upregulating IkBa expression, thereby depressing NF-kB p65 mRNA abundance in the gill of fish. Moreover, studies have noted that IkB kinase (IKK, including IKKa, IKKb and IKKg three subunits) could phosphorylate IkB molecules and promote their degradation, thereby release NF-kB in Helen cells [59,60]. Current results showed that dietary ALA/LNA ratio of 1.03 significantly reduced IKKb and IKKg mRNA levels in the gill of juvenile grass carp. However, dietary ALA/LNA ratios had no influence on IKKa mRNA abundance in the gill of juvenile grass carp. The unchanged mRNA levels of IKKa may be partly explained by protein kinase C q (PKC-q). ALA increased the synthesis of ceramides in C2C12 muscle cells [61]. CLA (LNA isomeride) was found to enhance ceramide content in the skeletal muscle of human [62]. Meanwhile, ceramide could reduce PKC-q activity, which induced IKKb but not IKKa activation in human T cells [63,64]. These data implied that optimal dietary ALA/LNA ratio attenuated inflammatory response may be partly through reducing IKKb and IKKg (rather than IKKa) mRNA levels, thereby inhibiting NF-kB p65 gene transcription in the gill of juvenile fish. Nevertheless, the underlying mechanism still awaits further investigation. 4.2. Optimal dietary ALA/LNA ratio regulated tight junction protein mRNA levels partly through MLCK pathway in the gill of juvenile fish In fish, the gill structural integrity has close relation to the physical barrier, which is mainly consisted with the tight junction

9

complex, such as claudins, occludin and ZO-1 [65]. It was reported that occludin, ZO-1, claudin-b and
3, are barrier-forming TJs, while claudin-15a is pore-forming TJ protein in fish [6]. Moreover, repressive gill claudin-3 and occludin mRNA levels of rainbow trout, and elevated gill claudin-12 mRNA abundance of goldfish [66], resulted in the disruption of tight junction. Current study exhibited that dietary ALA/LNA ratio of 1.03 significantly enhanced occludin, ZO-1, claudin-3, -b, ec and
12 mRNA levels, while inhibited claudin-15a mRNA abundance in the gill of juvenile grass carp. The enhanced claudin-12 mRNA levels in the gill of fish may be partly ascribed to the calcium. EPA (ALA metabolism) and GLA (LNA isomeride) increased calcium content in the bone of rat [67]. The increased calcium content could stimulate claudin-12 mRNA expression in the jejunum of mouse [68]. These findings indicated that optimal dietary optimal ALA/LNA ratio enhanced claudin-12 mRNA levels may be partly attributed to the increased content of calcium in juvenile fish. Nevertheless, this hypothesis awaits further investigation. The benefit effects of dietary ALA/LNA ratios on the TJ protein gene transcription may be partly attributed to myosin light chain kinase (MLCK). It is well known that MLCK has emerged as a key regulator of TJ protein expression, and upregulating MLCK could disturb TJs in fish [69]. Current study showed that dietary ALA/LNA ratio of 1.03 significantly decreased MLCK mRNA levels in the gill of juvenile grass carp. Correlation analysis exhibited that claudin-3, -b, -c,
12, occludin and ZO-1 were negatively related to the MLCK, while claudin-15a was positively related to MLCK mRNA abundance in the gill of juvenile carp (Table 5), suggesting that dietary ALA/LNA ratio strengthen tight junction may be partly ascribed to the decreased MLCK mRNA levels in the gill of juvenile fish. 4.3. Optimal dietary ALA/LNA ratio elevated antioxidant status and relevant signaling molecules mRNA levels in the gill of juvenile fish In fish, physical barrier of the gill is also associated with epithelial structural integrity [70]. Study has shown that the cell structure integrity is connected with antioxidant status in the gill of Channa punctata (Bloch) [21]. Jiang et al. [71] reported that the excessive ROS (mainly including superoxide radical and hydroxyl radical) overwhelmed the cellular antioxidant defense system would result in lipid peroxidation and protein oxidation in Jian carp. Generally, MDA and PC are used as biomarkers for lipid peroxidation and protein oxidation, while ASA and AHR activities are used to reflect radical-scavenging ability in fish [72]. The present

Table 5 Correlation coefficients of IL-1b, IL-8, TNF-a and IFN-g2 with NF-kB p65; tight junction protein mRNA levels with MLCK; antioxidant enzyme mRNA levels with Nrf2 in the gill of juvenile grass carp. Independent parameters

Dependent parameters

Correlation coefficients

P

NF-kB p65

IL-1b IL-8 TNF-a IFN-g2 IL-10 TGF-b1 Claudin-3 Claudin-b Claudin-c Claudin-12 Claudin-15a Occludin ZO-1 Cu/ZnSOD CAT GPx GST GR

þ0.918 þ0.980 þ0.970 þ0.987
0.940
0.858 - 0.854 - 0.964 - 0.853 - 0.797 þ0.943 - 0.969 - 0.959 þ0.978 þ0.844 þ0.938 þ0.988 þ0.921

P P P P P P P P P P P P P P P P P P

MLCK

Nrf2

< < < < < < < < < < < < < < < < < <

0.01 0.01 0.01 0.01 0.01 0.05 0.05 0.01 0.05 0.05 0.01 0.01 0.01 0.01 0.05 0.01 0.01 0.01

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Y.-Y. Zeng et al. / Fish & Shellfish Immunology 62 (2017) 1e12

study exhibited that dietary ALA/LNA ratio of 1.03 significantly reduced ROS production, MDA and PC contents, and elevated ASA and AHR activities in the gill of juvenile grass carp. These findings indicated that optimal dietary ALA/LNA ratio prevented protein oxidation and lipid peroxidation may be partly attributed to the enhanced radical scavenging ability, thereby impairing fish gill oxidative damage. Additionally, fish has evolved antioxidant defense system, which is mainly constituted of antioxidant enzymes [SOD, CAT, GPx, GST and GR] and non-enzyme antioxidant like GSH [73,74]. Current study showed that dietary ALA/LNA ratio of 1.03 significantly enhanced Cu/ZnSOD, GPx, GST and GR activities, and GSH contents in the gill of juvenile grass carp, suggesting that optimal dietary ALA/LNA ratio could improve antioxidant capacity in the gill of fish. Interestingly, dietary ALA/LNA ratios have no effects on CAT activities in the gill of grass carp. The discrepancy might be partly ascribed to GPx, which is involved in a process similar to that of CAT and has the ability to neutralize hydrogen peroxide in the gill of mussel (Perna viridis) [75]. Meanwhile, fish gill has the ability to eliminate hydrogen peroxide into the aquatic environment [76]. These evidences led to a hypothesis that dietary ALA/LNA ratios scavenged hydrogen peroxide may be partly through GPx rather than CAT in the gill of juvenile grass carp. However, this hypothesis awaits further investigation. In fish, the antioxidant enzyme activities are closely related to their mRNA levels [77]. Current study exhibited that dietary ALA/ LNA ratio of 1.03 significantly enhanced Cu/ZnSOD, GPx, GST and GR mRNA levels in the gill of juvenile grass carp. Correlation analysis showed that Cu/ZnSOD, GPx, GST and GR activities were positively related to their mRNA abundance (rCu/ZnSOD ¼ þ0.980, P < 0.01; rGPx ¼ þ0.886, P < 0.01; rGST ¼ þ0.984, P < 0.01; rGR ¼ þ0.891, P < 0.01), suggesting that optimal dietary ALA/LNA ratio increased antioxidant enzyme activities may be partly attributed to their enhanced mRNA levels. Moreover, the promoted gene transcription of antioxidant enzymes by dietary ALA/LNA ratios may be partly ascribed to Nrf2. Nrf2 is a critical nuclear transcription factor that could bind the antioxidant response element (ARE) to regulate the transcription of antioxidant genes in fish [78]. Our results showed that dietary ALA/LNA ratio of 1.03 significantly up-regulated Nrf2 transcript levels in the gill of juvenile fish. Correlation analysis showed that Cu/ZnSOD, GPx, GST and GR mRNA levels were positively related to Nrf2 transcript levels (Table 5), suggesting that the benefits of dietary ALA/LNA ratios on antioxidant enzyme gene expression may be partly due to the elevated Nrf2 mRNA levels in the gill of fish. Additionally, further study was conducted to explore the effects of dietary ALA/LNA ratios on fish gill Nrf2 protein levels and showed that dietary ALA/LNA ratio of 1.03 significantly induced cytosolic Nrf2 protein levels whereas depressed the nuclear Nrf2 protein levels. A possible reason may be that the enhanced

antioxidant levels could negatively feedback regulate the nuclear translocation of Nrf2. However, this hypothesis needs further investigation. Besides, keap1 is a prominent Nrf2-binding protein, which could prevent Nrf2 translocation to the nucleus, thereby inhibiting antioxidant gene transcription in European eel (Anguilla anguilla) [79]. Current study showed that dietary ALA/LNA ratio of 1.03 significantly depressed Keap1a and Keap1b mRNA levels in the gill of juvenile grass carp. Correlation analysis exhibited that Nrf2 mRNA levels were negatively related to Keap1a and Keap1b mRNA abundance in the gill (rKeap1a ¼
0.920, P < 0.01; rKeap1b ¼
0.887, P < 0.01). In line of these data, we can suspect that optimal dietary ALA/LNA ratio enhanced antioxidant enzyme gene transcription may be partly link to Nrf2-Keap1 signaling pathway, but the underlying mechanism awaits further investigation. 4.4. Optimal dietary ALA/LNA ratio evaluated for juvenile fish The above data clearly demonstrated that optimal dietary ALA/ LNA ratio could ameliorate gill immunity, tight junction and antioxidant defense capacity of juvenile fish. Additionally, correlation analysis showed that immune and anti-oxidant index (AHR, ASA, Cu/ZnSOD, GPx, GST, GR and GSH) were positively related to feed intake; whereas anti-oxidant index (ROS, MDA and PC) were negatively related to the feed intake (Table 6). Based on the quadratic regression analysis of gill C3 content, optimal dietary ALA/LNA ratio for maximum growth of juvenile grass carp (8.78e72.00 g) was estimated to be 1.12 (10.71 g kg
1 ALA þ 9.59 g kg
1 LNA), which was higher than the previous study based on PWG [1.08 (10.55 g kg
1 ALA þ9.75 g kg
1 LNA)] [29]. The results implied for the first time that optimal dietary ALA/LNA ratio of fish for gill immunity was higher than that for growth. 5. Conclusion Summarily, the results presented here demonstrated the following findings for the first time: (1) Optimal dietary ALA/LNA ratio improved gill immunity in part by increasing antibacterial compounds, inhibiting pro-inflammatory cytokines mRNA levels and promoting anti-inflammatory cytokines mRNA abundances, which may be partly ascribed to the reduced IKKb and IKKg (rather than IKKa) mRNA levels, thereby suppressing NF-kB p65 gene expression in the gill of juvenile fish. (2) Optimal dietary ALA/LNA ratio strengthened gill tight junctions via increasing barrierforming TJ protein mRNA levels, which might be partly attributed to the reduced MLCK gene expression in the gill of fish. (3) Optimal dietary ALA/LNA ratio improved gill antioxidant status, accompanied by enhancing antioxidant enzyme activities and their respective mRNA levels, which may be partly associated with the

Table 6 Correlation coefficients of gill immune index (LA, ACP and C3) and anti-oxidant index (AHR, ASA, ROS, MDA, PC, Cu/ZnSOD, GPx, GST, GR and GSH) with feed intake of juvenile grass carp. Independent parameters

Dependent parameters

Correlation coefficients

P

Feed intake

LA ACP C3 AHR ASA ROS MDA PC Cu/ZnSOD GPx GST GR GSH

þ0.709 þ0.845 þ0.928 þ0.848 þ0.959 - 0.988 - 0.970 - 0.873 þ0.817 þ0.948 þ0.796 þ0.792 þ0.724

P P P P P P P P P P P P P

¼ 0.074 < 0.05 < 0.05 < 0.05 < 0.01 < 0.01 < 0.01 ¼ 0.01 < 0.05 ¼ 0.01 < 0.05 < 0.05 ¼ 0.066

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Nrf2-Keap1 signaling pathway in the gill of fish. (4) Based on the quadratic regression analysis of gill C3 content, optimal ALA/LNA ratio for maximum growth of juvenile grass carp (8.78e72.00 g) was estimated to be 1.12. [16]

Acknowledgments This research was financially supported by the National Basic Research Program of China (973 Program) (2014CB138600), Outstanding Talents and Innovative Team of Agricultural Scientific Research (Ministry of Agriculture), the National Department Public Benefit Research Foundation (Agriculture) of China (201003020), Science and Technology Support Program of Sichuan Province of China (2014NZ0003), Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China (2012NC0007; 2013NC0045), The Demonstration of Major Scientific and Technological Achievement Transformation Project of Sichuan Province of China (2015CC0011), Natural Science Foundation for Young Scientists of Sichuan Province (2014JQ0007) and Sichuan Province Research Foundation for Basic Research (2013JY0082). The authors would like to thank the personnel of these teams for their kind assistance.

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