Modulation of immune response, physical barrier and related signaling factors in the gills of juvenile grass carp (Ctenopharyngodon idella) fed supplemented diet with phospholipids

Fish & Shellfish Immunology 48 (2016) 79e93

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Modulation of immune response, physical barrier and related signaling factors in the gills of juvenile grass carp (Ctenopharyngodon idella) fed supplemented diet with phospholipids Lin Feng a, b, c, 1, Yong-Po Chen a, 1, Wei-Dan Jiang a, b, c, Yang Liu a, b, c, Jun Jiang a, b, c, Pei Wu a, b, c, Juan Zhao a, 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, Sichuan, Chengdu, 611130, China Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Sichuan, Chengdu, 611130, China c Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Sichuan Agricultural University, Sichuan, 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 8 September 2015 Received in revised form 27 October 2015 Accepted 10 November 2015 Available online 14 November 2015

This study was conducted to investigate the effects of dietary phospholipids (PL) on the gill immune response and physical barrier of juvenile grass carp (Ctenopharyngodon idella). A total of 1080 juvenile grass carp with an average initial weight of 9.34 ± 0.03 g were fed six semi-purified diets containing 0.40% (unsupplemented control group), 1.43%, 2.38%, 3.29%, 4.37% and 5.42% PL for 2 months. Compared with the control group, optimal PL supplementation increased (P < 0.05): (1) the lysozyme activity, acid phosphatase activity, complement component 3 (C3) content, liver expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 mRNA expression; (2) the relative mRNA expression of interleukin 10, transforming growth factor b1, inhibitor factor kBa (IkBa) and target of rapamycin (TOR); (3) the activities of antisuperoxide anion (ASA), anti-hydroxyl radical (AHR), copper/zinc superoxide dismutase (SOD1), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR), glutathione content and mRNA levels of SOD1, CAT, GPx, GR and NF-E2-related factor 2 (Nrf2) genes; (4) the transcription abundance of occludin, claudin b, claudin c, claudin 12 and zonula occludens 1 genes. At the same time, appropriate PL supplementation decreased (P < 0.05): (1) tumor necrosis factor a, interleukin 1b, nuclear factor kB p65 (NF-kB p65), IkB kinase b (IKKb) and IkB kinase g (IKKg) mRNA expression; (2) malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) content and the relative mRNA expression of Kelch-like-ECH-associated protein 1a (Keap1a) and Keap1b; (3) the transcription abundance of myosin light chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) genes. In conclusion, the positive effect of PL on gill health is associated with the improvement of the immunity, antioxidant status and tight junction barrier of fish gills. Finally, based on ACP activity, C3 content, PC content and ASA activity in the gills, the optimal dietary PL level for juvenile grass carp (9.34e87.50 g) was estimated to be 3.62%, 4.30%, 3.91% and 3.86%, respectively. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Phospholipids Gill Grass carp (Ctenopharyngodon idella) Immunity Antioxidant status Tight junction

1. Introduction The gills have the largest organ-specific surface interacting with the external milieu and provide an initial barrier to the entry of

* Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, 611130, 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.2015.11.020 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

pathogens in fish [1]. The gill-associated lymphoid tissues play important roles in governing immune repertoire in teleost mucosal immunity [2]. It is reported that gill dysfunction led to the seriously impaired growth performance [3], implying the importance of gill health status. Phospholipids (PL) serve as the integral part of the structure of the biological membranes in fish [4], may participate in maintaining fish gill health; however, to date, no studies have addressed about this topic. Study reported that phospholipids had antioxidant activity by means of its major functional groups

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(choline, ethanolamine and side-chain moieties) [5]. And Jiang et al. [6] found that the important component of PL, inositol, could attenuate oxidative damage in the gills of Jian carp (Cyprinus carpio var. Jian). It appears that there may be a close relationship between PL and the health status of fish gills, which needs to be investigated. The gill health status in fish mainly depends on the gill mucosal immune function [7], which is governed by gill-associated lymphoid tissue that consists of variably sized immune cells, such as lymphocytes, macrophages and granulocytes [8]. The immune cells could secrete humoral components, such as lysozyme (LZ), acid phosphatase (ACP), complement component 3 (C3) and antimicrobial peptides, which play crucial roles in the gill innate immune response of fish [7]. However, no studies have addressed the effects of dietary PL on the humoral components in the gills of fish. The available study showed that dietary PL improved plasma LZ activity in juvenile channel catfish, Ictalurus punctatus [9]. These data indicated a possible correlation between PL and the humoral components in fish gills, which is valuable for investigation. Furthermore, inflammation is a key element in the response of the innate immune system and is mediated by cytokines, such as IL-1b, TNF-a and IL-10 [10]. Meanwhile, the production of cytokines could be regulated by the signaling pathways of NF-kB [11] and TOR [12]. To date, no study has investigated the effect of PL on the cytokines through NF-kB and TOR signaling pathways in fish gills. Studies have shown that the important component of PL, choline, could inhibit NF-kB activation in endotoxin stimulated mouse macrophage-like cells [13] and modulate cytokines and TOR expression in fish [14]. These appear that PL may be related to the NF-kB and TOR signaling pathways of fish gills to influence cytokines production. This possibility is worth investigating. In addition to mucosal immunity, the physical barrier function is also critical in maintaining gill health status [15]. Structural integrity of the gills is the foundation of its physical barrier function in fish, and fish have developed an antioxidant defense system to protect the structural integrity of the gills [16]. Antioxidant defense systems in fish include antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and non-enzymatic compounds, such as glutathione (GSH) [17]. Moreover, the expression of antioxidant enzyme is mainly regulated by nuclear factor-E2-related factor 2 (Nrf2) in zebrafish [18]. However, there is no information about the effect of PL on antioxidant enzymes and Nrf2 in the gills of fish. The available study showed that dietary PL could increase SOD and GPx activities in the liver of blunt snout bream fingerlings [19]. The above data indicates that PL may affect the antioxidant capacity of fish gills. Moreover, the tight junction complex is also an important part of the physical barrier for the fish gills [20]. The tight junction (TJ) complex was mainly made up of both integral TJ proteins and TJ plaque proteins [21]: the integral TJ proteins, such as occludin and claudins, bridge the apical intercellular space and form a regulated permeability barrier; the TJ plaque proteins, such as zonula occludens 1 (ZO-1), serve as links between the integral TJ proteins and the actin cytoskeleton and as adapters for the recruitment of cytosolic molecules implicated in cell signaling. The myosin light chain kinase (MLCK) has emerged as key regulators of the tight junctions in terrestrial animals [22]. However, to date, studies to investigate the effect of dietary PL on TJ proteins and MLCK in fish gills have not been carried out. In terrestrial animal, dietary PL could inhibit intestinal cholesterol absorption [23]. Zhu et al. [24] reported that high cholesterol level could increase MLCK expression and activity in the aortas of rabbits. These data indicated PL might affect the tight junction via influencing the signaling molecules of MLCK in fish gills, however, these warrant investigation. This study is a part of a larger study aimed at determining the effects of PL on fish growth using the same growth trial as the

previous study [25]. The objective of this study was to further investigate the effects of PL on the immunity and the physical barrier function of fish gills, which could be used to preliminarily determine the PL-dependent mechanism of improving disease resistance. The optimum dietary PL levels for gill health related parameters in juvenile grass carp was also evaluated. 2. Materials and methods 2.1. Experimental diets The diet formulation and composition is shown in Table 1. 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. Linseed oil (Hunan Yama biotechnology Co., Ltd., Hunan, China), safflower oil (Shanghai Yuan Tian Edible Agricultural Products Ltd., Shanghai, China), coconut oil (Lvyuan natural flavor oil refinery, Jiangxi, China) and soybean lecithin (Shaanxi Huicheng biotechnology Co., Ltd., Shaanxi, China) were used as lipid sources. Six experimental diets were obtained by supplementing the control diet with soybean lecithin at concentrations of 0.00% (un-supplemented control), 1.00%, 2.00%, 3.00%, 4.00% and 5.00% diet, while adjusting coconut oil to maintain the diets iso-lipidic, the method according to Niu et al. [26]. The analyzed PL levels were 0.40% (unsupplemented control), 1.43%, 2.38%, 3.29%, 4.37% and 5.42% of the diets according to the method described by Juaneda and Rocquelin [27] and Li et al. [28]. After being prepared completely, the pellets were stored at
20  C until feeding according to Zhao et al. [29]. 2.2. Feeding trial All protocols were approved by the Institutional Animal Care

Table 1 Diet formulation and composition.a Ingredient

% (diet)

Fishmeal Casein Gelatin DL-Met (99%) Corn starch Alpha-starch Cellulose Vitamin premixb Mineral premixc Ca(H2PO4)2 (22%) Choline chloride (60%) Ethoxyquin (30%) Oil premixd Soybean lecithin premixe

3.00 28.00 7.50 0.14 11.55 24.00 5.00 1.00 2.00 2.26 0.50 0.05 5.00 10.00

a Crude protein and total lipids were measured to be 30.16% and 9.06% respectively. b Per kilogram of vitamin premix: retinyl acetate (500 000 IU g
1), 2.40 g; cholecalciferol (500 000 IU g
1), 0.40 g; D,L-a-tocopherol acetate (50%), 12.55 g; menadione (23%), 0.80 g; thiamine nitrate (98%), 0.05 g; riboflavin (80%), 0.55 g; pyridoxine hydrochloride (98%), 0.59 g; cyanocobalamin (1%), 0.83 g; niacin (99%), 2.24 g; D-biotin (2%), 4.91 g; mesoinositol (99%), 19.39 g; folic acid (96%), 0.40 g; ascorhyl acetate (93%), 7.16 g. All ingredients were diluted with corn starch to 1 kg. c Per kilogram of mineral premix: MgSO4$H2O, 60.530 g; FeSO4$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. All ingredients were diluted with corn starch to 1 kg. d Per kilogram of oil premix: linseed oil 185.80 g, safflower oil 237.60 g, corn starch 576.60 g. e Soybean lecithin premix: premix was added to obtain graded level of phospholipids.

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and Use Committee (IACUC) of Sichuan Agricultural University. Grass carp were acclimated to experimental conditions for 4 weeks according to Ji et al. [30] after obtainment from fisheries (Sichuan, China). Subsequently, a total of 1080 grass carp, with an average initial weight of 9.34 ± 0.03 g, were randomly distributed into 18 experimental cages (1.4  1.4  1.4 m), resulting in 60 fish in each cage. The cages were equipped with a disc of 100-cm diameter made of 1-mm gauze at the bottom to collect the uneaten feed as described by Tang et al. [31]. During the experimental period, fish were fed four times daily for 2 months. Thirty minutes after the feeding, the uneaten feed were collected, and then dried and weighted to calculate the feed intake as described by Anh et al. [32]. During the experimental period, the feeding trial was completed under natural light, the dissolved oxygen content was not less than 6.0 mg L
1 according to the experimental conditions described by Tang et al. [31]. The water temperature was 26 ± 2  C, and the water pH was at 7.0 ± 0.5.

from 10-fold serial dilutions according to Luo et al. [46] and Bustin et al. [47]. After verification that the primers amplified with an efficiency of approximately 100%, the results were analyzed using the 2
DDCT method according to Livak and Schmittgen [48].

2.3. Sample collection and biochemical analysis

3.1. Humoral components in fish gills

The procedures of sample collection were similar to those previously described in our another study [25]. According to the method of Martins et al. [33], 15 fish from each cage were anaesthetized in benzocaine bath (50 mg L
1), and then the gills were quickly removed, frozen in liquid nitrogen, then stored at
80  C for later analysis as described by Hu et al. [34]. The samples were homogenized in 10 volumes (w v
1) of ice-cold physiological saline and centrifuged at 6000  g for 20 min at 4  C according to Kotorman et al. [35], and the supernatant was collected to analyze the gill immune and antioxidant parameters. The protein concentrations in the gill samples were determined according to the method of Bradford [36]. The LZ activity, ACP activity and C3 content were assayed according to Zhou et al. [37], Molina et al. [38] and Wang et al. [39], respectively. The contents of ROS, malondialdehyde (MDA), protein carbonyl (PC) and GSH were assayed according to the procedures described by Felty [40], Yonar et al. [41], Armenteros et al. [42] and Beutler et al. [43], respectively. The anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) activities were measured according to Zhao et al. [44]. The SOD, CAT, GPx and GR activities were assayed as described by Mourente et al. [45].

As shown in Table 3, the LZ activity in the gills were the lowest in fish fed the control diet (P < 0.05), and no significant differences were found among other groups (P > 0.05). The ACP activity in the gills of fish fed PL-supplemented diets were found to be significantly higher than those fed control diet (P < 0.05), except for the group receiving dietary PL at 1.43%. The C3 content in the gills gradually increased with increasing PL levels up to 4.37%, and then significantly decreased (P < 0.05). As shown in Fig. 1, the relative expression of LEAP-1 in the gills of fish fed PL-supplemented diets were found to be significantly higher than those fed control diet (P < 0.05), except for the group receiving dietary PL at 1.43%; the relative expression of LEAP-1 in the gills of fish fed 4.37% PL diet were found to be significantly higher than other groups (P < 0.05), except for the group receiving dietary PL at 2.38%. The LEAP-2 mRNA level in the gills was higher for fish fed 4.37% and 5.42% PL diet than control group (P < 0.05), and no significant differences were found among other groups (P > 0.05).

2.4. Real-time PCR analysis The procedures of RNA isolation, reverse transcription and quantitative real-time PCR (qPCR) were similar to those previously described in our another study [25]. Total RNA was extracted from the gill samples using RNAiso Plus kit (TaKaRa Biotechnology (Dalian) Co., Ltd.) according to the manufacturer's instructions followed by DNase I treatment, and the RNA quality and quantity were assessed using agarose gel (1%) electrophoresis and spectrophotometric (A260: 280 nm ratio) analysis. Specific primers for the target genes and b-Actin were shown in Table 2. Real-time PCR was performed for these genes according to standard protocols and the published article in our laboratory [46]. The thermocycling conditions were initiated with a denaturation step of 95  C for 30 s followed by 40 cycles of denaturation at 95  C for 5 s, annealing for 30 s at their optimal anneal temperature, and elongation at 72  C for 30 s. A melting curve analysis was generated following the amplification to check and verify the specificity of PCR products. 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 that were generated

2.5. Data analysis Results were presented as means ± standard deviation (SD). All data were subjected to a one-way analysis of variance. Differences among the treatment means were determined using a Duncan's multiple-range test at a P < 0.05 level of significance. The optimal dietary PL levels based on physiological indices were calculated by the quadratic regression analysis according to Kuang et al. [49]. All statistical analyses were done using the SPSS 18.0 (SPSS, IL, USA) according to Rawling et al. [50]. 3. Results

3.2. Relative mRNA expression of cytokines and signaling molecules of NF-kB and TOR pathways in fish gills As shown in Fig. 2, the IL-1b and TNF-a mRNA levels in the gills significantly decreased with increasing dietary PL levels up to the 3.29% (P < 0.05), and plateaued thereafter (P > 0.05). Fish fed the diets containing 3.29%, 4.37% and 5.42% PL were found to have significantly lower IFN-g2 mRNA levels in the gills than those fed control diet (P < 0.05). As shown in Fig. 3, the relative expression of IL-10 significantly increased with increasing dietary PL levels up to the 3.29% (P < 0.05), and plateaued thereafter (P > 0.05). Fish fed the diets containing 3.29%, 4.37% and 5.42% PL were found to have significantly higher TGF-b1 mRNA levels in the gills than those fed control diet (P < 0.05). As shown in Fig. 4, fish fed PL-supplemented diets were found to have significantly lower NF-kB p65 mRNA levels in the gills than those fed control diet (P < 0.05); the relative expression of NF-kB p65 in the gills of fish fed 3.29% PL diet were found to be significantly lower than those fed the diet contenting 1.43% PL (P < 0.05). Fish fed PL-supplemented diets were found to have significantly higher IkBa mRNA levels in the gills than those fed control diet (P < 0.05). As shown in Fig. 5, no significant difference was found in IKKa mRNA levels in the gills of fish fed graded levels of PL (P > 0.05). The mRNA levels of IKKb in the gills of fish fed PLsupplemented diets were significantly lower than those fed the control diet (P < 0.05). Fish fed the diets containing 2.38%, 3.29% and 4.37% PL were found to have significantly lower IKKg mRNA

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Table 2 Real-time PCR primer sequences.a Gene LEAP-1 Forward Reverse LEAP-2 Forward Reverse IFN-g2 Forward Reverse TNF-a Forward Reverse IL-1b Forward Reverse TGF-b1 Forward Reverse IL-10 Forward Reverse NF-kB p65 Forward Reverse IkBa Forward Reverse IKKa Forward Reverse IKKb Forward Reverse IKKg Forward Reverse TOR Forward Reverse S6K1 Forward Reverse CK2 Forward Reverse SOD1 Forward Reverse CAT Forward Reverse GPx Forward Reverse GR Forward Reverse Nrf2 Forward Reverse Keap1a Forward Reverse Keap1b Forward Reverse Claudin b Forward Reverse Claudin c Forward Reverse

Sequences of primers

Annealing temperature ( C)

Amplification products (bp)

Amplification efficiency (%)

Accession number

5ʹ-AGCAGGAGCAGGATGAGC-3ʹ 5ʹ-GCCAGGGGATTTGTTTGT-3ʹ

59.3

100

100.2

JQ246442

5ʹ-TGCCTACTGCCAGAACCA-3ʹ 5ʹ-AATCGGTTGGCTGTAGGA-3ʹ

59.3

76

99.9

FJ390414

5ʹ-TGTTTGATGACTTTGGGATG-3ʹ 5ʹ-TCAGGACCCGCAGGAAGAC-3ʹ

60.4

145

100.3

JX657682

5ʹ-CGCTGCTGTCTGCTTCAC-3ʹ 5ʹ-CCTGGTCCTGGTTCACTC-3ʹ

58.4

188

99.7

HQ696609

5ʹ-AGAGTTTGGTGAAGAAGAGG-3ʹ 5ʹ-TTATTGTGGTTACGCTGGA-3ʹ

57.1

234

100.1

JQ692172

5ʹ-TTGGGACTTGTGCTCTAT-3ʹ 5ʹ-AGTTCTGCTGGGATGTTT-3ʹ

55.9

173

99.5

EU099588

5ʹ-AATCCCTTTGATTTTGCC-3ʹ 5ʹ-GTGCCTTATCCTACAGTATGTG-3ʹ

61.4

256

100.2

HQ388294

5ʹ-GAAGAAGGATGTGGGAGATG-3ʹ 5ʹ-TGTTGTCGTAGATGGGCTGAG-3ʹ

62.3

197

100.3

KJ526214

5ʹ-TCTTGCCATTATTCACGAGG-3ʹ 5ʹ-TGTTACCACAGTCATCCACCA-3ʹ

62.3

197

100.3

KJ125069

5ʹ-GGCTACGCCAAAGACCTG-3ʹ 5ʹ-CGGACCTCGCCATTCATA-3ʹ

60.3

260

100.0

KM279718

5ʹ- GTGGCGGTGGATTATTGG-3ʹ 5ʹ- GCACGGGTTGCCAGTTTG-3ʹ

60.3

88

100.1

KP125491

5ʹ-AGAGGCTCGTCATAGTGG-3ʹ 5ʹ-CTGTGATTGGCTTGCTTT-3ʹ

58.4

115

99.9

KM079079

5/-TCCCACTTTCCACCAACT-3/ 5/-ACACCTCCACCTTCTCCA-3/

61.4

177

99.8

JX854449

5/-TGGAGGAGGTAATGGACG-3/ 5/-ACATAAAGCAGCCTGACG-3/

59.4

111

100.4

5ʹ-CCCCAACCACAGTGACCT-3ʹ 5ʹ-TCCCTGCTGATACTTCTCC-3ʹ

57.9

118

99.3

KF914143

5ʹ-CGCACTTCAACCCTTACA-3ʹ 5ʹ-ACTTTCCTCATTGCCTCC-3ʹ

61.5

218

100.5

GU901214

5ʹ-GAAGTTCTACACCGATGAGG-3ʹ 5ʹ-CCAGAAATCCCAAACCAT-3ʹ

58.7

158

99.1

FJ560431

5ʹ-GGGCTGGTTATTCTGGGC-3ʹ 5ʹ-AGGCGATGTCATTCCTGTTC-3ʹ

61.5

278

100.1

EU828796

5ʹ-GTGTCCAACTTCTCCTGTG-3ʹ 5ʹ-ACTCTGGGGTCCAAAACG-3ʹ

59.4

222

100.2

JX854448

5ʹ-CTGGACGAGGAGACTGGA-3ʹ 5ʹ-ATCTGTGGTAGGTGGAAC-3ʹ

62.5

234

99.8

KF733814

5ʹ-TTCCACGCCCTCCTCAA-3ʹ 5ʹ-TGTACCCTCCCGCTATG-3ʹ

63.0

205

100.0

KF811013

5ʹ-TCTGCTGTATGCGGTGGGC-3ʹ 5ʹ-CTCCTCCATTCATCTTTCTCG-3ʹ

57.9

87

99.7

KJ729125

5ʹ-GAGGGAATCTGGATGAGC-3ʹ 5ʹ-ATGGCAATGATGGTGAGA-3ʹ

57.0

122

100.9

KF193860

5ʹ-GAGGGAATCTGGATGAGC-3ʹ 5ʹ-CTGTTATGAAAGCGGCAC-3ʹ

59.4

241

100.6

KF193859

EF373673.1

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Table 2 (continued ) Gene Claudin 3 Forward Reverse Claudin 12 Forward Reverse Claudin 15 Forward Reverse ZO-1 Forward Reverse Occludin Forward Reverse MLCK Forward Reverse p38 MAPK Forward Reverse b-Actin Forward Reverse

Sequences of primers

Annealing temperature ( C)

5ʹ-ATCACTCGGGACTTCTA-3ʹ 5ʹ-CAGCAAACCCAATGTAG-3ʹ

57.0

82

99.9

KF193858

5ʹ-CCCTGAAGTGCCCACAA-3ʹ 5ʹ-GCGTATGTCACGGGAGAA-3ʹ

55.4

81

99.8

KF998571

5ʹ-TGCTTTATTTCTTGGCTTTC-3ʹ 5ʹ-CTCGTACAGGGTTGAGGTG-3ʹ

59.0

115

99.6

KF193857

5ʹ-CGGTGTCTTCGTAGTCGG-3ʹ 5ʹ-CAGTTGGTTTGGGTTTCAG-3ʹ

59.4

154

100.4

KJ000055

5ʹ-TATCTGTATCACTACTGCGTCG-3ʹ 5ʹ-CATTCACCCAATCCTCCA-3ʹ

59.4

208

99.7

KF193855

5ʹ-GAAGGTCAGGGCATCTCA-3ʹ 5ʹ-GGGTCGGGCTTATCTACT-3ʹ

53.0

161

99.0

KM279719

5ʹ- GGGAGCAGACCTCAACAA -3ʹ 5ʹ- CCATCGGGTGGCAACATA -3ʹ

61.0

235

99.7

KM112098

5ʹ-GGCTGTGCTGTCCCTGTA-3ʹ 5ʹ-GGGCATAACCCTCGTAGAT-3ʹ

61.4

101

99.9

M25013

Amplification products (bp)

Amplification efficiency (%)

Accession number

a LEAP-1, liver expressed antimicrobial peptide 1; LEAP-2, liver expressed antimicrobial peptide 2; IFN-g2, interferon g2; TNF-a, tumor necrosis factor a; IL-1b, interleukin 1b; TGF-b1, transforming growth factor b1; IL-10, interleukin 10; NF-kB p65, nuclear factor kappa B p65; IkBa, inhibitor protein kBa; IKK, IkB kinase; TOR, target of rapamycin; S6K1, ribosomal protein S6 kinase 1; ZO-1, zonula occludens 1; MLCK, myosin light chain kinase; p38MAPK, p38 mitogen-activated protein kinase; SOD1, copper, zinc superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase; Nrf2, NF-E2-related factor 2; Keap1, Kelch-like-ECH-associated protein 1.

Table 3 Immune parameters in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months.a Dietary phospholipids levels (% diet)

LZ ACP C3

Control

1.43

2.38

3.29

4.37

5.42

20.12 ± 1.71a 54.53 ± 3.23a 3.99 ± 0.28a

36.81 ± 2.47b 59.02 ± 1.47a 5.05 ± 0.32b

35.60 ± 3.65b 69.16 ± 2.65b 7.04 ± 0.61c

34.30 ± 1.66b 84.37 ± 3.98c 7.90 ± 0.47d

34.35 ± 1.85b 85.19 ± 6.22c 8.52 ± 0.44e

34.55 ± 2.10b 84.66 ± 5.28c 7.44 ± 0.42cd

a Values are mean ± SD (n ¼ 6). Mean values with the different superscripts in the same row are significantly different (P < 0.05). LZ: Lysozyme (U mg
1 protein); ACP: acid phosphatase (U mg
1 protein); C3: complement component 3 (mg g
1 protein).

Fig. 1. Relative expression of liver expressed antimicrobial peptide 1 (LEAP-1) and LEAP-2 genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

levels in the gills than those fed control diet (P < 0.05). As shown in Fig. 6, the relative expression of TOR in the gills of fish fed PLsupplemented diets were found to be significantly higher than those fed control diet (P < 0.05), except for the group receiving dietary PL at 1.43%; the relative expression of TOR in the gills of fish fed 3.29% PL diet were found to be significantly higher than those fed the diet contenting 1.43% PL (P < 0.05). The S6K1 mRNA levels in

the gills of fish increased significantly with dietary PL levels higher than 1.43% (P < 0.05). As shown in Fig. 7, the relative expression of CK2 in the gills of fish fed PL-supplemented diets were found to be significantly higher than those fed control diet (P < 0.05), except for the group receiving dietary PL at 1.43%; the relative expression of CK2 in the gills of fish fed 4.37% PL diet were found to be significantly higher than those fed the diet contenting 1.43% PL (P < 0.05).

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Fig. 2. Relative expression of interleukin 1b (IL-1b), tumor necrosis factor a (TNF-a) and interferon g2 (IFN-g2) genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

Fig. 3. Relative expression of interleukin 10 (IL-10) and transforming growth factor b1 (TGF-b1) genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

3.3. Antioxidant-related parameters in fish gills As shown in Fig. 8, the ROS content in the gills significantly decreased with increasing dietary PL levels up to the 2.38%

(P < 0.05), and increased thereafter (P < 0.05). As shown in Table 4, the MDA content in the gills of fish fed PL-supplemented diets were found to be significantly lower than those fed control diet (P < 0.05); the MDA content in the gills of fish fed 2.38% and 3.29%

Fig. 4. Relative expression of nuclear factor kappa B p65 (NF-kB p65) and inhibitor protein kBa (IkBa) genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

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Fig. 5. Relative expression of IkB kinase a (IKKa), IkB kinase b (IKKb) and IkB kinase g (IKKg) genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

Fig. 6. Relative expression of target of rapamycin (TOR) and ribosomal protein S6 kinase 1 (S6K1) genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (%) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

Fig. 7. Relative expression of casein kinase 2 (CK2) gene in the gills of juvenile grass carp fed diets with graded levels of phospholipids (%) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

PL diets were found to be significantly lower than those fed the diet contenting 5.42% PL (P < 0.05). The PC content in the gills significantly decreased with increasing dietary PL levels up to the 3.29% (P < 0.05), and increased above 4.37% (P < 0.05). The activities of

AHR, SOD1 and CAT in the gills significantly increased with increasing dietary PL levels up to the 3.29% (P < 0.05), and decreased thereafter (P < 0.05). The ASA activity in the gills significantly increased with increasing dietary PL levels up to the

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Fig. 8. Reactive oxygen species (ROS) production in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

Table 4 Antioxidant status related parameters in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months.a Dietary phospholipids levels (% diet) Control MDA PC ASA AHR SOD1 CAT GPx GR GSH

3.52 8.87 148.70 193.05 3.57 1.67 112.03 24.42 9.78

1.43 ± ± ± ± ± ± ± ± ±

0.18c 0.43d 9.35a 10.60a 0.22a 0.06a 10.64a 1.53a 0.47a

3.14 7.55 169.20 240.98 4.94 1.79 125.17 26.41 9.81

2.38 ± ± ± ± ± ± ± ± ±

0.20ab 0.50c 6.99b 16.83b 0.44b 0.09b 10.83ab 1.75b 0.85a

2.99 6.70 192.24 261.20 6.10 2.01 150.48 27.80 9.91

3.29 ± ± ± ± ± ± ± ± ±

0.18a 0.31b 3.65c 16.99c 0.57cd 0.11c 9.42c 2.31bc 0.98ab

3.03 5.79 201.61 288.29 6.17 2.26 145.16 26.86 10.01

4.37 ± ± ± ± ± ± ± ± ±

0.09a 0.31a 6.93d 16.38d 0.29d 0.07d 17.55c 1.43bc 0.34ab

3.13 5.37 208.00 273.55 5.72 1.95 136.53 28.73 10.71

5.42 ± ± ± ± ± ± ± ± ±

0.19ab 0.26a 8.49d 15.72cd 0.41cd 0.08c 12.14bc 0.79c 0.36b

3.25 6.63 188.10 267.72 5.61 1.96 130.32 27.02 9.71

± ± ± ± ± ± ± ± ±

0.07b 0.30b 5.93c 19.36c 0.44c 0.12c 9.02b 0.86bc 0.60a

a Values are mean ± SD (n ¼ 6). Mean values with the different superscripts in the same row are significantly different (P < 0.05). MDA: malondialdehyde (nmol mg
1 protein); PC: protein carbonyl (nmol mg
1 protein); ASA: anti-superoxide anion (U g
1 protein); AHR: anti-hydroxyl radical (U mg
1 protein); SOD1: copper/zinc superoxide dismutase (U mg
1 protein); CAT: catalase (U mg
1 protein); GPx: glutathione peroxidase (U mg
1 protein); GR, glutathione reductase(U g
1 protein); GSH, glutathione (mg g
1 protein).

3.29% (P < 0.05), and decreased above 4.37% (P < 0.05). Fish fed PLsupplemented diets had significantly higher GPx activity in the gills than those fed control diet (P < 0.05), except for the group receiving dietary PL at 1.43%. The GR activity in the gills of fish fed PLsupplemented diets were found to be significantly higher than those fed control diet (P < 0.05); fish fed 4.37% PL diet were found to have significantly higher GR activity in the gills than those fed the diet contenting 1.43% PL (P < 0.05). Fish fed the diet containing 4.37% PL were found to have significantly higher GSH content in the gills than those fed control diet (P < 0.05), and no significant differences were found among other groups (P > 0.05).

As shown in Fig. 10, the relative expression of Nrf2 in the gills of fish fed PL-supplemented diets were found to be significantly higher than those fed control diet (P < 0.05); fish fed 3.29% PL diets had significantly higher Nrf2 mRNA levels in the gills than those fed the diet contenting 1.43% PL (P < 0.05). The relative expression of Keap1a and Keap1b significantly decreased with increasing dietary PL levels up to the 3.29% (P < 0.05), and plateaued thereafter (P > 0.05).

3.4. Relative mRNA expression of antioxidant enzymes, Nrf2, Keap1a and Keap1b in fish gills

As shown in Fig. 11, the relative expression of occludin in the gills of fish fed PL-supplemented diets were found to be significantly higher than those fed control diet (P < 0.05), except for the group receiving dietary PL at 1.43%. Fish fed PL-supplemented diets had significantly higher ZO-1 mRNA levels in the gills than those fed control diet (P < 0.05), except for the group receiving dietary PL at 5.42%. As shown in Fig. 12, no significant difference was found in claudin 3 and claudin 15 mRNA levels in the gills of fish fed graded levels of PL (P > 0.05). The relative expression of claudin b in the gills of fish fed PL-supplemented diets were found to be significantly higher than those fed control diet (P < 0.05). Fish fed the diets containing 2.38%, 3.29% and 4.37% PL were found to have significantly higher claudin c mRNA levels in the gills than those fed control diet (P < 0.05). The relative expression of claudin 12 in the

As shown in Fig. 9, the relative expression of SOD1 in the gills of fish fed PL-supplemented diets were found to be significantly higher than those fed control diet (P < 0.05). The relative expression of CAT significantly increased with increasing dietary PL levels up to the 3.29% (P < 0.05), and plateaued thereafter (P > 0.05). The relative expression of GPx in the gills of fish fed PL-supplemented diets were found to be significantly higher than those fed control diet (P < 0.05); fish fed 4.37% PL diets had significantly higher GPx mRNA levels in the gills than those fed the diet contenting 1.43% PL (P < 0.05). The GR mRNA levels in the gills of fish increased significantly with dietary PL levels higher than 1.43% (P < 0.05).

3.5. Relative mRNA expression of tight junction proteins, MLCK and p38 MAPK in fish gills

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Fig. 9. Relative expression of antioxidant enzymes genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05). SOD1, copper/zinc superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase.

Fig. 10. Relative expression of Nrf2, Keap1a and Keap1b genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05). Nrf2, NF-E2-related factor 2; Keap1, Kelch-like-ECH-associated protein 1.

Fig. 11. Relative expression of occludin and zonula occludens 1 (ZO-1) genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

gills of fish fed PL-supplemented diets were found to be significantly higher than those fed control diet (P < 0.05); the relative expression of claudin 12 in the gills of fish fed 3.29% PL diet were

found to be significantly higher than those fed the diet contenting 1.43% PL (P < 0.05). As shown in Fig. 13, the relative expression of MLCK significantly

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Fig. 12. Relative expression of claudin 3, claudin b, claudin c, claudin 12 and claudin 15 genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

decreased with increasing dietary PL levels up to the 2.38% (P < 0.05), and plateaued thereafter (P > 0.05). The relative expression of p38 MAPK in the gills of fish fed PL-supplemented diets were found to be significantly lower than those fed control diet (P < 0.05); the relative expression of p38 MAPK in the gills of fish fed 3.29% and 4.37% PL diets were found to be significantly lower than those fed the diet contenting 1.43% PL (P < 0.05). 3.6. Optimal dietary PL level for juvenile grass carp The present study evaluated the optimal dietary PL level for the different physiological indices of juvenile grass carp. As shown in Fig. 14, based on ACP activity, C3 content, PC content and ASA activity in the gills, the optimal dietary PL level for juvenile grass carp (9.34e87.50 g) was estimated to be 3.62%, 4.30%, 3.91% and 3.86%, respectively. 4. Discussion 4.1. PL enhanced the gill immunity of fish 4.1.1. PL modulated gill innate immune and immune response of fish The innate immune of the immune system are the first line of defense against pathogens in teleost [8]. The immune cells in gill-

associated lymphoid tissue could secrete humoral components, such as lysozyme (LZ), acid phosphatase (ACP), complement component 3 (C3) and antimicrobial peptides, which play crucial roles in the gill innate immune response of fish [7]. The present study showed that optimal PL supplementation significantly improved the LZ, ACP activities, C3 contents and the expression of LEAP-1 and LEAP-2 in the gills of juvenile grass carp, suggesting that PL was contributed to enhance the innate immunity in the gills of juvenile fish. In juvenile channel catfish (I. punctatus), dietary PL improved plasma LZ activity [9]. The beneficial effect of PL on innate immunity in the gills of juvenile fish may be partly related to leucine. In't Veld et al. [51] reported that PL could affect leucine transport activity in Lactococcus lactis. Studies from our laboratory showed that leucine could increase LZ and ACP activities and C3 levels in the intestine of grass carp [52]. The inflammatory response is a key ingredient in the response of the innate immune system to a variety of challenges, and it is primarily mediated by cytokines [10]. TNF-a, IL-1b and IFN-g are pro-inflammatory cytokines, which initiate inflammatory processes and accelerate additional inflammatory processes by inducing other inflammatory molecules; and the anti-inflammatory cytokines IL-10 and TGF-b are able to suppress the production of pro-inflammatory cytokine thereby inhibit the excessive activation of the inflammatory response in teleost [53]. In the current study, the relative mRNA expression of TNF-a,

Fig. 13. Relative expression of myosin light chain kinase (MLCK) and p38 mitogen-activated protein kinase (p38 MAPK) genes in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. Values are mean ± SD (n ¼ 6), and different letters above a bar denote the significant difference (P < 0.05).

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Fig. 14. Quadratic regression analysis of ACP activity (A), C3 content (B), PC content (C) and ASA activity (D) in the gills of juvenile grass carp fed diets with graded levels of phospholipids (% diet) for 2 months. ACP, acid phosphatase; C3, complement 3; PC, protein carbonyl; ASA, anti-superoxide anion.

IL-1b and IFN-g2 significantly decreased as the dietary PL levels increased up to a certain level, whereas optimum supplementation of PL significantly up-regulated the IL-10 and TGF-b1 mRNA levels in the gills of juvenile grass carp. These results imply that an optimum dietary PL content could attenuate the inflammatory response in the gills of juvenile fish. However, the underlying mechanism by which PL influences the cytokine expression in fish is still unknown. As we know, production of cytokines could be regulated by the signaling pathways of NF-kB [11] and TOR [12]. Therefore, we next investigated the relationship between PL and signaling molecules in the NF-kB and TOR signaling pathways in the gills of juvenile grass carp. 4.1.2. PL regulated gene expression of inflammation-related signaling molecules involved in NF-kB and TOR signaling pathways in the gills of fish Study in mice monocyte demonstrated that inhibition of NF-kB p65 expression decreased the mRNA expression of proinflammatory cytokines (IL-1b and IL-8) [54]. In the present study, optimal PL supplementation significantly down-regulated NF-kB p65 mRNA expression in the gills of juvenile grass carp. Correlation analysis indicated that the mRNA expression of TNF-a, IL-1b and IFN-g2 in the gills of juvenile grass carp was positively related to NF-kB p65 expression (Table 5). The results suggested that dietary PL decreased the expression of pro-inflammatory cytokines (TNF-a, IL-1b and IFN-g2) may be partly attributed to decrease NF-kB p65 expression in juvenile fish. Additionally, the nuclear translocation of NF-kB p65 is the prerequisite of its regulation of cytokines expression [55]. It has been reported that IkBa, an NF-kB-binding protein, is well known to sequester NF-kB in the cytoplasm and thereby prevent its nuclear translocation in zebra fish [55]. Study reported that up-regulated IkBa expression could down-regulate the expression of pro-inflammatory cytokines, such

as TNF-a, IL-1b and IL-6 in mouse muscle fibers [56]. The current study showed that significantly increase in mRNA levels of IkBa was observed in the grass carp gills with optimal PL supplementation. Correlation analysis indicated that the mRNA expression of TNF-a, IL-1b and IFN-g2 was negatively related to IkBa expression in the gills of juvenile grass carp (Table 5). These data suggested that PLdecreased the relative mRNA expression of pro-inflammation cytokines may be partly attributed to inhibit NF-kB nuclear translocation by up-regulating IkBa mRNA expression. Moreover, in eukaryotes, the degradation of IkB promotes NF-kB p65 nuclear translocation and the signaling event is induced by activated IKK complex (IKKa, IKKb and IKKg) [57]. The results of our current study demonstrated that optimal PL supplementation significantly down-regulated IKKb and IKKg mRNA expression in the gills of juvenile grass carp, while the mRNA expression of IKKa did not vary among treatments. These results suggested that dietary PL may act through down-regulating the expression of IKKb and IKKg (not IKKa) to attenuate the degradation of IkB, thereby inhibit NF-kB p65 nucleus translocation to down-regulate the gene expression of pro-inflammatory cytokines in juvenile fish. However, this hypothesis and the underlying mechanism of this effect requires further investigation. Furthermore, the up-regulation of antiinflammatory cytokines expression by PL may be related to TOR. It was reported that promoted mTOR expression increased the IL-10 concentration of human monocytes [12]. S6K1 is one of the major downstream targets of TOR, ensuring the function integrity of TOR signaling pathway [58]. In the current study, the relative mRNA expression of TOR significantly increased in the gills of juvenile grass carp with optimal PL supplementation, S6K1 showed a similar trend with TOR. Correlation analysis indicated that the mRNA expression of anti-inflammatory cytokines IL-10 and TGF-b1 was positively related to TOR expression in the gills of juvenile grass carp (Table 5). This finding supports that PL elevated anti-

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L. Feng et al. / Fish & Shellfish Immunology 48 (2016) 79e93 Table 5 Correlation coefficient of NF-kB p65 and IkBa with IL-1b, TNF-a, IFN-g2; TOR and S6K1 with IL-10 and TGF-b1; CK2 with TOR; MLCK with tight junction proteins; Nrf2, Keap1a and Keap1b with antioxidant enzymes in the gills. Independent parameters

Dependent parameters

Correlation coefficients

P

NF-kB p65

IL-1b TNF-a IFN-g2 IL-1b TNF-a IFN-g2 IL-10 TGF-b1 IL-10 TGF-b1 TOR occludin ZO-1 claudin b claudin c claudin 12 SOD1 CAT GPx GR SOD1 CAT GPx GR SOD1 CAT GPx GR

þ0.984 þ0.940 þ0.917
0.908
0.921
0.817 þ0.955 þ0.879 þ0.876 þ0.752 þ0.954
0.766
0.909
0.822
0.665
0.960 þ0.977 þ0.925 þ0.874 þ0.915
0.931
0.966
0.977
0.918
0.948
0.917
0.916
0.883

<0.01 <0.01 <0.05 <0.05 <0.01 <0.05 <0.01 <0.05 <0.05 0.085 <0.01 0.076 <0.05 <0.05 0.150 <0.01 <0.01 <0.01 <0.05 <0.05 <0.01 <0.01 <0.01 <0.05 <0.01 <0.05 <0.05 <0.05

IkBa

TOR S6K1 CK2 MLCK

Nrf2

Keap1a

Keap1b

inflammatory cytokines (IL-10 and TGF-b1) mRNA levels partly due to the up-regulation of TOR gene expression. Additionally, TOR expression is regulated by its upstream signaling molecules, such as CK2. In vitro, the up-regulation of CK2 caused the up-regulation of the expression of mTOR in human glioblastoma cells [59]. The current study showed that optimal PL supplementation the significantly enhanced CK2 mRNA expression in the gills of juvenile grass carp. The correlation analysis indicated that juvenile grass carp TOR mRNA expression positively correlated with corresponding CK2 expression in the gills of juvenile grass carp (Table 5), suggesting benefits of PL on TOR mRNA expression may be partly explained by PL up-regulated CK2 mRNA expression in juvenile fish. However, the detailed mechanism by which PL regulates cytokines and these inflammation-related signaling molecules awaits further characterization. In addition to gill immunity, the physical barrier function is also critical in maintaining gill health status [15]. Thus, we next investigated the relationship between PL and physical barrier in the gills of juvenile grass carp. 4.2. PL improved the gill physical barrier of fish 4.2.1. PL elevated antioxidant status and gene expression of signaling molecules Nrf2 Keap1a and Keap1b in the gills of fish MDA and PC are commonly used as biomarkers for lipid peroxidation and protein oxidation, respectively [60]. In the present study, the MDA and PC contents in the gills of juvenile grass carp significantly decreased with optimal PL supplementation, suggesting that oxidative damage in fish gills was depressed by PL. To our knowledge, the oxidative damage mainly caused by excess ROS [61]. The current study found that optimal PL supplementation significantly decreased ROS content in the gills of juvenile grass carp. Superoxide radical ðO2 $
Þ and hydroxyl radical ð· OHÞ are two important ROS continually generated as byproducts of normal aerobic metabolism and strongly involved in oxidative damage

within the cell [61]. Thus, we then detected the influence of PL on O2 $
-scavenging ability (ASA) and · OH-scavenging ability (AHR) in the gills of fish. The results showed that the ASA and AHR activities were significantly enhanced by optimal PL supplementation. Correlation analysis showed that the ROS content were negatively related to ASA and AHR activities in the gills of juvenile grass carp (rASA ¼
0.818, P < 0.05; rAHR ¼
0.887, P < 0.05), suggesting that dietary PL could enhance the ROS scavenging ability in juvenile fish gills. ROS scavenging ability has been correlated with the enzymatic and non-enzymatic antioxidant defense systems [17]. The current study showed that SOD1, CAT, GPx and GR activities and GSH content in the gills increased with optimal PL supplementation, suggesting that PL could improve the antioxidant capacity of gills in juvenile fish. The antioxidant enzyme activities are closely related to their mRNA levels in fish [62]. In this study we found that optimal PL supplementation significantly up-regulated the mRNA expression of SOD1, CAT, GPx and GR in the gills of juvenile grass carp. Correlation analysis showed that SOD1, CAT, GPx and GR activities were positively correlated with their mRNA expression (rSOD1 ¼ þ0.946, P < 0.01; rCAT ¼ þ0.833, P < 0.05; rGPx ¼ þ0.729, P ¼ 0.100; rGR ¼ þ0.868, P < 0.05), indicating that PL might improve antioxidant enzyme activities partly through promoting their gene transcription in juvenile fish gills. Interestingly, this study showed that CAT expression and activity in the gills increased with PL supplementation, while the CAT expression and activity in the intestine were not affected by PL supplementation in our previous study. The different effects of PL on CAT in different tissue/organs may be partly explained by the different inducible expression of CAT in different tissues. Study in disk abalone (Haliotis discus discus) indicated that CAT expression was more sensitive to its inducer H2O2 in the gills than that in digestive tract [63]. And the gills is continuously in contact with high oxygen for hemolymph circulation and oxygen consumption proportionally induces the production of H2O2. However, more researches are needed to confirm this hypothesis.

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The promoted gene transcription of antioxidant enzymes by PL may be partly related to Nrf2. Nrf2 is well known as a key nuclear transcription factor that could bind the antioxidant response element (ARE) to regulate the transcription of antioxidant genes in fish [18]. Studies reported that up-regulation of Nrf2 expression could promote the SOD1 and GPx mRNA expression in the intestine of grass carp [64]. Data in this study displayed that optimal PL supplementation significantly up-regulated the mRNA expression of Nrf2 in the gills of juvenile grass carp, and showed that the mRNA expression of SOD1, CAT, GPx and GR were positively related to the Nrf2 expression, suggesting that the benefits of PL on antioxidant enzyme gene expression may be partly due to up-regulate Nrf2 gene expression in juvenile fish gills. As we know, the Nrf2 nuclear translocation is a critical event for provoking gene transcription of antioxidant enzymes in HT29 human colon carcinoma cell [65]. Keap1 is identified as an Nrf2-binding protein that prevents Nrf2 translocation to the nucleus [66]. It has been reported that the down-regulation of Keap1 gene in mice could increase the Nrf2 nuclear translocation, resulting in transcriptional induction of antioxidant genes [67]. In this study, the mRNA expression of Keap1a and Keap1b significantly decreased with optimal PL supplementation. Correlation analysis indicated that the mRNA expression of SOD1, CAT, GPx and GR were negatively related to the expression of Keap1a and Keap1b (Table 5). These results indicated that PL increased the expression of antioxidant enzymes may be partly attributed to promote the nuclear translocation of Nrf2, which is related to the decreased Keap1 expression in juvenile fish, but an exact mechanism needs to be further investigated. Interestingly, in this study, both Keap1a and Keap1b expression in the gills of juvenile grass carp decreased with dietary PL supplementation. Whereas, our previous study showed that dietary PL supplementation did not affect Keap1b expression in the intestine of this fish [25]. However, to date, no study has been conducted to investigate the effects of PL on Keap1b in different tissue/organs, and thus the reasons are still unknown; here, we hypothesize that different effects of PL on Keap1b in different tissue/organs in fish could be partly related to the tissue distribution of Keap1b. Li et al. [68] reported that both Keap1a and Keap1b were able to facilitate the degradation of Nrf2 protein and repress Nrf2-mediated target gene activation, while the expression of keap1b was relatively abundant in the gills and scarce in the intestine of zebrafish. These data suggest that the gills instead of intestine are one of the functional area of Keap1b. Nevertheless, the hypothesis and the underlying mechanisms require more investigation. 4.2.2. PL promoted the gene expression of tight junction proteins and signaling molecules MLCK and p38 MAPK in the gills of fish The gill epithelial tight junction (TJ), which was mainly made up of TJ complex (occludin, claudins and zonula occludens (ZOs)), also plays an important role in physical barrier of fish gills [20]. It is reported that up-regulation of occludin, claudin 12 and ZO-1 were indicated to lead to attenuate the gill epithelial TJ barrier function damage in grass carp [69]. The current study showed that optimal PL supplementation significantly increased the mRNA expression of occludin, claudin b, claudin c, claudin 12 and ZO-1 in the gills of juvenile grass carp, suggesting that optimal PL supplementation had the ability to maintain tight junction barrier in fish gills. The positive effects of PL on the gene expression TJ proteins could be ascribed to MLCK. It is well known that MLCK has emerged as a key regulator of tight junction barrier [22]. Study reported that downregulation of MLCK expression attenuates impairment of TJ barrier of intestinal epithelium in mice [70]. Meanwhile, the suppressed TJ barrier impairment mediated by the decrease in MLCK expression was accompanied by up-regulating expression of ZO-1 in Caco-2 cells [71]. In the present study, the mRNA expression of

91

MLCK significantly decreased with optimal PL supplementation. Further correlation analysis found that the mRNA expression of occludin, claudin b, claudin c, claudin 12 and ZO-1 was negatively related to MLCK expression in the gills of juvenile grass carp (Table 5). The results indicated that the up-regulation of TJ proteins in the gills by dietary PL could partly be related to the MLCK expression of juvenile fish. Moreover, MLCK expression is regulated by its upstream signaling molecule p38 MAPK [72]. Study in burninduced mice found that inhibition of p38 MAPK decreased expression of intestinal MLCK [73]. The current study showed that the mRNA expression of p38 MAPK in the gills of juvenile grass carp significantly decreased with PL supplementation. Further correlation analysis showed that the mRNA expression of MLCK was positively related to p38 MAPK expression the gills of juvenile grass carp (Table 5), suggesting that down-regulation of MLCK expression in the gills by PL may be partly related to the down-regulation of p38 MAPK expression in juvenile fish. However, more studies are required to thoroughly explore the mechanism by which PL regulates the gill tight junction barrier in juvenile fish. It is worth noting that the relative mRNA expression of claudin 3 in the gills of juvenile grass carp was not affected by dietary PL in this study. Our previous studies in the intestine of this fish found that optimal dietary PL increased the relative mRNA expression of claudin 3 [25]. Until now, there is no information regarding the effect of PL on claudin 3 gene expression in different tissue/organs, and thus the reasons for the discrepancies in the data remain unclear. Existing researches indicated that claudin 3 acts as a general barrierforming protein, while its special function is unresolved as yet [74]. Further investigation should be conducted to explain the different effect of PL on claudin 3 gene expression in different tissue/organs.

5. Conclusion In summary, the results presented here demonstrate that dietary PL enhances gill health status in grass carp by improving the immune response and physical barrier. Furthermore, results of the oxidative damage, antioxidant status, tight junction and TNF-a, IL1b, IFN-g2, IL-10 and TGF-b1 gene expression go further to support these positive effects. Moreover, dietary PL regulates NF-kB, TOR, Nrf2 MLCK and p38 MAPK gene expression in the gills and could be used to determine a preliminary model of PL's influence the gill health status. Finally, based on ACP activity, C3 content, PC content and ASA activity in the gills, the optimal dietary PL level for juvenile grass carp (9.34e87.50 g) was estimated to be 3.62%, 4.30%, 3.91% and 3.86%, respectively.

Acknowledgments This research was financially supported by the National Basic Research Program of China (973 Program) (2014CB138600), 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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