Methionine hydroxy analogue improves intestinal immunological and physical barrier function in young grass carp (Ctenopharyngodon idella)

Fish & Shellfish Immunology 64 (2017) 122e136

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Methionine hydroxy analogue improves intestinal immunological and physical barrier function in young grass carp (Ctenopharyngodon idella) Fei-Yu Pan a, 1, Pei Wu a, b, c, 1, Lin Feng a, b, c, Wei-Dan 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, *, Yang Liu 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 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 c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 August 2016 Received in revised form 27 February 2017 Accepted 4 March 2017 Available online 6 March 2017

This study was conducted to test the hypothesis that methionine hydroxy analogue (MHA) enhances the defense against enteritis occurrence via improving intestinal barrier function in fish. After 630 young grass carp (Ctenopharyngodon idella) (259.70 ± 0.47 g) fed six graded levels of MHA (0, 2.4, 4.4, 6.4, 8.5 and 10.5 g/kg diet) and one DL-methionine group (6.4 g/kg diet) for 8 weeks. At the end of feeding trial, 15 fish from each treatment were challenged with Aeromonas hydrophila for 14 days. The results indicated that optimal MHA enhanced the capacity of fish against enteritis emergence, which might be related to the positive effects of MHA on intestinal immunological and physical barrier function in fish. Dietary MHA supplementation enhanced intestinal immunological barrier function via (1) lysozyme (LZM) and acid phosphatase (ACP) activities, complement 3 (C3), C4 and immunoglobulin M (IgM) contents and upregulated mRNA levels of liver-expressed antimicrobial peptide 2, hepcidin (head kidney), b-defensin-1; (2) repressing p38MAPK/IKKb/IkBa/NF-kB signaling pathway to down-regulate pro-inflammatory cytokines mRNA levels except IL-8 mRNA level only in mid and distal intestine; (3) potentiating TOR-signal cascades to up-regulate anti-inflammatory cytokines. Meanwhile, dietary MHA supplementation improved intestinal physical barrier via (1) down-regulating c-Jun N-terminal kinase mRNA levels to inhibit death receptor and mitochondria pathways induced apoptosis; (2) modulating Keap1a/Nrf2 system to elevate antioxidant enzymes genes isoforms mRNA levels and corresponding enzymes activities, subsequently alleviate oxidative damage; (3) down-regulating MCLK gene expression to upregulating occludin, zonula occluden 1 and claudins mRNA levels except claudin-7a and claudin-7b only in the proximal intestine. In conclusion, bases on the capacity defense against enteritis, proximal intestinal malondialdehyde content and lysozyme activity, the optimal MHA supplementation levels were 5.83, 5.59 and 6.07 g/kg diet (4.01 g/kg methionine basal), respectively. This study indicates that MHA exerts a positive effect on fish intestinal health status and a superior efficacy to DL-methionine based on the positive effects. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Methionine hydroxy analogue Enteritis Immunological barrier function Physical barrier Ctenopharyngodon idella

1. Introduction * Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan, China. ** Corresponding author. Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, Sichuan, China. E-mail addresses: [email protected], [email protected] (X.-Q. Zhou), [email protected] (Y. Liu). 1 These two authors contributed to this work equally. http://dx.doi.org/10.1016/j.fsi.2017.03.012 1050-4648/© 2017 Elsevier Ltd. All rights reserved.

Under intensive and pathogen rich aquatic conditions, fish intestine is susceptible to enteritis emergence which induces fish growth retardation and economic losses [1]. To defense against pathogen invasion, fish intestine develops its immunological and physical barrier [2]. It is now widely accepted that nutritional approaches are essential to protect intestine from damage via

F.-Y. Pan et al. / Fish & Shellfish Immunology 64 (2017) 122e136

improving immunological and physical barrier [3,4]. As we know, methionine is an indispensable amino acid for fish nutrition [5]. Nowadays, methionine hydroxy analogue (MHA) is widely used as a common synthetic source of dietary methionine in animal nutrition due to its lower price than crystalline methionine [6]. Fang et al. [7] reported that MHA firstly metabolized in the intestine and 29% proportion was utilized in the first pass by intestine in piglet. Currently, limit studies only focused on the effects of MHA on intestinal physical barrier in fish. Our previous study demonstrated that MHA could protect intestine from oxidative damage via increasing antioxidant enzymes activities in Jian carp [8]. However, the study of MHA on the intestinal immunological barrier is scarce and the effects of MHA on the intestinal physical barrier is lack of systematic and in-depth investigation in fish, which requires furthermore research. In fish, intestine executes immunological barrier function via producing humoral immune factors like lysozyme (LZM), acid phosphatase (ACP), complements, antimicrobial peptides and cytokines [2,9]. Cytokines could be regulated by nuclear factor kB (NFkB) and target of rapamycin (TOR) in human [10,11]. Our previous study only investigated that MHA could increase serum LZM and ACP activities, C3 and C4 contents in Jian carp [12]. However, there is no study to address the relationship of MHA with these humoral immune factors and related signaling molecules in fish intestine. It was reported that MHA could up-regulate insulin-like growth factor 1 (IGF-1) gene expression in broiler [13]. In human, IGF-1 could result in up-regulation of b-defensin gene expression [14]. In addition, methionine is the ingredient of forming S-adenosyl-Lmethionine (SAMe) which always participates in phospholipids synthesis [15]. Study in our lab indicated that phospholipids could down-regulate NF-kB gene expression in the intestine of grass carp (Ctenopharyngodon idella) [16]. Meanwhile, methionine involves in an important catabolic pathway to produce choline in vertebrates [17]. In Jian carp, choline could up-regulate intestinal TOR gene expression [18]. According to these data, MHA may exert a positive influence on intestinal immunological barrier function referred to NF-kB and TOR pathways in fish, which is worthy of investigation. The intestinal barrier function also relies on its physical barrier which includes the defense against apoptosis and oxidative damage, as well as tight junctions [19,20]. It was reviewed that apoptosis is mediated by the signaling molecule c-jun N-terminal kinase (JNK) in human [21,22]. Oxidative damage could be attenuated by antioxidant enzymes whose genes transcription were regulated by NF-E2-related factor 2 (Nrf2) in mammals [23]. Besides, tight junctions could be disordered by myosin light chain kinase (MLCK) in bovine cerebral cortices [24]. Studies demonstrated that MHA could increase intestinal antioxidant enzymes activities in Jian carp [8] and protect tight junction structure from damage in Caco-2 cells [20]. However, information about effects of MHA on apoptosis, antioxidant enzymes molecular mechanism and tight junctions in fish intestine is scarce. It was found that MHA could elevate intestinal taurine level in chicken [25]. In mice, taurine acted as a repressor for JNK [26]. Study revealed that methionine could increase insulin and thyroid hormone levels in mice [27]. In mice, insulin was found to promote Nrf2 nuclear translocation and thyroid hormone actually acted a repressor for MLCK [28,29]. Based on these findings, it is essential to investigate the effect of MHA on intestinal physical barrier and relevant signaling pathways in fish. MHA, as a methionine resource for animals' diets, is different from DL-methionine (DLM) in metabolism [30]. In addition, the metabolism of MHA in intestine was preferentially diverted to the transsulfuration pathway, leading to higher taurine and cysteine production in chicken intestine [25]. Study indicated that taurine could potentiate the immune response and inhibit apoptosis in

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mice [26,31]. Meanwhile, cysteine is proved as the ingredient of antioxidant molecule glutathione (GSH) [32]. Hence, there might be some differences between MHA and DLM affecting the intestinal immunological and physical barrier function in fish, which is essential to investigate. This study utilized the same growth trial as our previous study, which determined that 5.21 g/kg of MHA supplementation could improve fish growth [33]. This study was a further investigation with the principal aim to test the hypothesis that dietary MHA enhances the capacity of fish to defense against enteritis occurrence via improving intestinal immunological and physical barrier function. To test the hypothesis, this study was for the first time to investigate the influence of MHA on humoral immune factors, caspases, antioxidant enzymes and tight junction proteins molecular mechanism in the intestine of fish. In addition, we further investigated the influence of MHA on the relevant signaling molecules NF-kB, TOR, JNK, Nrf2 and MLCK, which might imply the partial modulation mechanism. 2. Materials and methods 2.1. Experimental diets and procedure The experimental diets were the same as our previous study [33]. Fish meal, soy protein concentrate, soybean meal, rapeseed meal and cottonseed meal were used as the main protein sources and were found to be limiting in methionine. The dietary amino acids except methionine were according to the grass carp wholebody profile [34]. Diets 1e6 were formulated according to MHA supplementation: 0 (basal), 2.4, 4.4, 6.4, 8.5 and 10.5 g MHA/kg diet, respectively. In addition, diet 7 was formulated by supplementing 6.4 g DLM/kg diet (positive control), which satisfied the young grass carp methionine requirement according to our lab investigation [35]. The methionine concentration in the basal diet was 4.01 g/kg diet, which was determined by the method according to Spindler et al. [36]. All the seven diets contained equal nitrogen, in addition, the 6.4 g MHA/kg diet was even equal sulfur to the 6.4 g DLM/kg diet. The prepared diets were store at
20  C until used. The procedures used in this study were approved by the University of Sichuan Agricultural Animal Care Advisory Committee. Grass carp were obtained from fisheries (Sichuan, China). Ahead of initiating the experiment, fish were acclimatized to the experiment conditions for 4 weeks. Then, 630 fish (mean weight 259.70 ± 0.47 g) were randomly assigned to 21 experimental aquaria (1.4 L  1.4 W  1.4 H m). Each cage was equipped with an aerator and a 100 cm diameter disc in the bottom to collect the uneaten feed. Each cage was randomly assigned to triplicate of the seven dietary treatments, and fish were fed with the respective diet four times daily for 8 weeks. During the experiment, water temperature, pH and dissolved oxygen were 28 ± 2  C, 7.0 ± 0.2 and not less than 6.0 mg/L, respectively. The experimental units were maintained under natural light cycle. 2.2. Challenge experiment After the 8 weeks feeding trial, 15 fish were obtained from each treatment, and were moved to the respective label new cage and acclimatized to the experimental condition for 5 days. Aeromonas hydrophila was friendly supplied by Veterinary Medicine College, Sichuan Agricultural University in China. At the end of acclimatize period, fish were injected by intraperitoneal injection with 1 mL A. hydrophila (2.5  108 cfu/mL), which was enough to activate the immune system and consequently enable the investigation of effluent on reactivity against a threatening disease according to our preliminary study (data not shown). And then, the challenge trial

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was conducted for 14 days, as described by Basha et al. [37]. The challenge trial conditions were the same as the feeding trial. At the end of challenge trial, the records of enteritis morbidity were evaluated according to our created rating system which is similar to the method of Song et al. [1]. 2.3. Sample collection and analysis At the end of the challenge trial, all survival fish from each cage were anaesthetized in a benzocaine bath (50 mg/L). After sacrificed, the intestines of fish were removed and immediately frozen in liquid nitrogen followed by storage at
80  C for total RNA isolation and histological examination. Proximal-intestine (PI), midintestine (MI) and distal intestine (DI) were quickly classified according to the position of the turns of the intestine (the proximal intestine was anterior to the first turn, the mid-intestine was the region located between the first turn and the last turn, and the distal intestine was posterior to the last turn) on ice, subsequently homogenized in 10 vol (w/v) of ice-cold physiological saline and centrifuged at 6000 g for 20 min at 4  C, then the supernatant was stored for antioxidant and immune related parameters analysis. The malondialdehyde (MDA), protein carbonyl (PC) and GSH contents and the superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) activities in the PI, MI and DI were assayed according to Feng et al. [8]. The LZM activity was assayed according to Nya and Austin [38]; ACP activity according to Classics et al. [39]; C3 and C4 contents according to according to Welker et al. [40], respectively. PI, MI and DI were determined by the method described by Chen et al. [16]. 2.4. Analysis of antimicrobial peptides, cytokines, caspases, antioxidant enzymes, tight junction proteins and relevant signaling molecules genes expression in intestine Total RNA of PI, MI and DI was extracted using RNAiso Plus (Takara, China) according to the manufacturer's instructions followed by DNase I treatment. Total RNA was quantified by spectrophotometry at 260 nm and 280 nm, and the quality was checked by agarose gel (1%) electrophoresis. cDNA synthesis was performed according to the manufacturer's instructions using the PrimeScript™RT reagent Kit (Takara, China). For quantitative real-time PCR, specific primers were designed according to the sequences cloned in our lab and the published sequences of grass carp in NCBI. All real-time PCR reactions were performed on a CFX96TM RealTime PCR Detection System. 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. The target and housekeeping gene amplification efficiency were calculated according to the specific gene standard curves generated from 10-fold serial dilutions. After verification that the primers amplified with an efficiency of approximately 100%, all test genes were calculated using the 2
DDCT method as described by Livak and Schmittgen [41].

Fig. 1. Enteritis morbidity of young grass carp (Ctenopharyngodon idella) fed diets supplemented with graded levels of MHA and 6.4 g/kg DLM. Values having different letters are significantly different (P < 0.05), and values in the 6.4 g/kg DLM group with no superscript are not significantly different from the 6.4 g/kg MHA group (P > 0.05).

3. Results 3.1. Effects of MHA on enteritis morbidity As shown in Fig. 1, enteritis morbidity significantly decreased with dietary MHA up to 4.4 g/kg diet (P < 0.05), and increased thereafter. According to the regression analysis of enteritis morbidity (Y ¼ 1.852x2 - 21.607x þ 74.995, R2 ¼ 0.739, P ¼ 0.134), the optimal MHA levels for young grass carp was 5.83 g/kg diet. Meanwhile, the morbidity of fish fed 6.4 g MHA/kg diet had no significant difference from that in fish fed 6.4 g DLM/kg diet (P > 0.05). As shown in Fig. 2, the enteritis symptom in fish fed diet with 6.4 g/kg MHA was much slighter than that in fish fed basal diet. 3.2. Effects of MHA on the immune function-related parameters in intestine of fish The LZM and ACP activities and C3 and C4 contents in intestine are shown in Table 1. The activities of LZM in PI and MI and ACP in DI, as well as the C3 content in PI, gradually increased with dietary MHA up to 6.4 g/kg diet, and significantly decreased thereafter (P < 0.05). The LZM activity in DI, and the contents of C3 in MI and DI and C4 in MI gradually increased with dietary MHA up to 6.4 g/kg diet, and gradually decreased. Meanwhile, the ACP activities in PI and MI and C4 content in PI significantly increased with dietary

2.5. Statistical analysis The results were presented as the mean ± standard deviation (SD). The data of the groups fed basal diet and graded levels of MHA were subjected to a one-way analysis of variance (ANOVA) with SPSS software, and followed by the Duncan's multiple-range test to determine significant differences among treatments at P < 0.05. In addition, the data of the group fed 6.4 g/kg DLM with the group fed t 6.4 g/kg MHA were subjected to unpaired student's t-test to determine significant differences at P < 0.05.

Fig. 2. Histological analysis of intestine in young grass carp (Ctenopharyngodon idella) fed basal diet (A) and 6.4 g/kg MHA diet (B) after challenged with A. hydrophila.

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Table 1 LZM and ACP activities (U/mg protein), IgM contents (g/g protein), C3 and C4 contents (mg/g protein) in the PI, MI and DI of young grass carp (Ctenopharyngodon idella) fed diets with graded levels of MHA and 6.4 g/kg DLM.1 Dietary MHA levels (g/kg diet)

PI LZ ACP IgM C3 C4 MI LZ ACP IgM C3 C4 DI LZ ACP IgM C3 C4

Basal

2.4

4.4

6.4

8.5

10.5

DLM (6.4)2

134.14 ± 11.61a 78.61 ± 5.97a 75.31 ± 6.86a 12.39 ± 1.16a 1.26 ± 0.07a

156.83 ± 11.17b 112.12 ± 6.92c 87.85 ± 6.32b 15.30 ± 0.57bc 1.50 ± 0.05b

194.34 ± 13.55cd 148.78 ± 7.71e 90.62 ± 2.83b 17.99 ± 0.46d 1.74 ± 0.12c

206.48 ± 15.06d 155.67 ± 3.42f 91.63 ± 4.88b 19.04 ± 1.04d 1.94 ± 0.15d

186.87 ± 16.98c 127.02 ± 3.51d 88.77 ± 4.93b 16.22 ± 1.42c 1.66 ± 0.10c

157.17 ± 13.70b 102.20 ± 5.75b 85.03 ± 7.50b 14.60 ± 1.00b 1.23 ± 0.07a

210.13 ± 20.22 149.10 ± 7.73 91.27 ± 3.84 18.33 ± 1.47 1.78 ± 0.11

73.80 ± 5.75a 116.47 ± 3.70a 95.62 ± 4.79a 9.63 ± 0.54a 1.33 ± 0.11a

129.94 ± 10.18b 148.79 ± 4.60c 109.26 ± 8.63bc 11.97 ± 1.12b 1.55 ± 0.05b

187.31 ± 13.97d 174.19 ± 4.03e 113.71 ± 9.27c 16.11 ± 0.82d 1.65 ± 0.13bc

198.67 ± 14.03d 181.58 ± 5.21f 115.39 ± 5.34c 16.64 ± 1.55d 1.69 ± 0.09c

166.61 ± 12.25c 159.38 ± 5.43d 102.82 ± 8.99ab 15.55 ± 0.87d 1.58 ± 0.13bc

117.18 ± 7.83b 137.87 ± 2.63b 98.38 ± 6.65a 13.84 ± 0.81c 1.31 ± 0.10a

194.95 ± 17.89 180.76 ± 15.92 111.43 ± 7.83 15.80 ± 1.20 1.68 ± 0.10

84.21 ± 6.36a 154.69 ± 11.69a 105.82 ± 3.51a 11.70 ± 0.82a 1.23 ± 0.06a

152.43 ± 10.18b 210.58 ± 10.91c 111.27 ± 3.99a 13.47 ± 1.12b 1.64 ± 0.07c

212.88 ± 17.81c 251.22 ± 13.39d 122.38 ± 10.23bc 16.46 ± 1.21c 1.98 ± 0.14e

222.10 ± 10.88c 261.89 ± 8.39d 127.70 ± 6.25c 16.69 ± 1.43c 1.86 ± 0.10de

155.41 ± 12.89b 221.36 ± 7.55c 114.86 ± 10.61ab 15.35 ± 1.45c 1.76 ± 0.15cd

162.12 ± 14.43b 189.61 ± 6.09b 107.61 ± 9.56b 12.72 ± 0.71ab 1.50 ± 0.06b

239.21 ± 24.16 248.15 ± 14.52 122.01 ± 4.32 16.41 ± 1.45 1.88 ± 0.11

YLZ in PI ¼
1.912x2 þ 23.228x þ 127.19 YACP in PI ¼
2.1847x2 þ 25.477x þ 74.52 YIgM in PI ¼
0.4181x2 þ 5.1597x þ 76.168 YC3 in PI ¼
0.1769x2 þ 2.0776x þ 12.1 YC4 in PI ¼
0.0213x2 þ 0.2336x þ 1.1845 YLZ in MI ¼
3.4886x2 þ 41.613x þ 66.796 YACP in MI ¼
1.8214x2 þ 21.313x þ 114.13 YIgM in MI ¼
0.6417x2 þ 6.7551x þ 96.198 YC3 in MI ¼
0.1598x2 þ 2.1361x þ 9.1145 YC4 in MI ¼
0.0129x2 þ 0.1376x þ 1.3176 YLZ in DI ¼
3.2531x2 þ 40.087x þ 84.499 YACP in DI ¼
3.0329x2 þ 35.14x þ 151.96 YIgM in DI ¼
0.6257x2 þ 6.9653x þ 103.48 YC3 in DI ¼
0.1541x2 þ 1.7822x þ 11.217 YC4 in DI ¼
0.0203x2 þ 0.2372x þ 1.2284

R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

0.902 0.940 0.969 0.926 0.878 0.962 0.967 0.912 0.925 0.976 0.841 0.964 0.814 0.913 0.955

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

< 0.05 < 0.05 < 0.01 < 0.05 < 0.05 < 0.01 < 0.01 < 0.05 < 0.05 < 0.01 ¼ 0.064 < 0.01

P < 0.05 P < 0.01

1 Values are means ± SD (n ¼ 6), and superscripted different letters in the same row are significantly different (P < 0.05). LZM, lysozyme; ACP, acid phosphatase; IgM, immunoglobulin M; C3, complement 3. 2 Values in the 6.4 g/kg DLM group with no subscript are not significantly different from the 6.4 g/kg MHA group (P > 0.05).

MHA up to 6.4 g/kg diet (P < 0.05), and significantly decreased thereafter (P < 0.05). The C4 content in DI significantly increased with dietary MHA up to 4.4 g/kg diet (P < 0.05), and then gradually decreased. Based on the regression analysis of PI lysozyme activity (Y ¼
1.912x2 þ 23.228x þ127.192, R2 ¼ 0.902, P < 0.05), the optimal MHA levels for young grass carp was 6.07 g/kg diet. As shown in Table 1, these parameters in the intestines of fish fed 6.4 g MHA/kg diet had no significant difference from that in fish fed 6.4 g DLM/kg diet (P > 0.05). 3.3. Effects of MHA on the mRNA levels of antimicrobial peptides and cytokines in intestine of fish As shown in Fig. 3, the mRNA levels of liver expressed antimicrobial peptide 2 (LEAP2), hepcidin and b-defensin in PI, MI and DI gradually increased with dietary MHA up to 6.4 g/kg diet, and then gradually decreased. The mRNA levels of cytokines in intestine of fish are presented in Fig. 4. Dietary MHA had no significant effect on the mRNA levels of interleukin 4/13B (IL-4/13B) and transforming growth factor b2 (TGF-b2) in intestine, as well as IL-8 in PI (P > 0.05). In PI, the mRNA levels of tumor necrosis factor a (TNF-a), interferon g2 (IFN-g2) and IL-1b gradually decreased with dietary MHA up to 6.4 g/kg diet, and then increased. The IL-12p35 mRNA level significantly decreased with dietary MHA up to 4.4 g/kg diet (P < 0.05), and gradually increased thereafter. Meanwhile, the mRNA levels of IL-12p40, IL10, IL-4/13A and TGF-b1 gradually increased with dietary MHA up

to 6.4 g/kg diet, and gradually decreased thereafter. In MI, the tendency of TNF-a, IFN-g2, IL-1b, IL-12p40, IL-10 and TGF-b1 mRNA levels were as the same as that in PI. The Il-8 and IL12p35 mRNA levels gradually decreased with dietary MHA up to 6.4 g/kg diet, and then increased. Meanwhile, the IL-4/13A mRNA level significantly increased with dietary MHA up to 2.4 g/kg diet (P < 0.05), and then plateaued. In DI, the tendency of TNF-a, IFN-g2, IL-1b, IL-12p40, IL-10, IL-4/ 13A and TGF-b1 mRNA levels were as the same as that in PI, and the tendency of IL-12p35 mRNA level was as the same as that in MI. Meanwhile, the IL-8 mRNA level gradually decreased with dietary MHA up to 6.4 g/kg diet, and then significantly increased (P < 0.05). As shown in Fig. 4, TNF-a and IL-1b mRNA levels in the PI of fish fed 6.4 g MHA/kg diet were significantly lower, and IL-10 mRNA level in the PI was significantly higher than that in fish fed 6.4 g DLM/kg diet (P < 0.05), and the others in the intestines were not significantly different between the two groups (P > 0.05). 3.4. Effects of MHA on the mRNA levels of immune-related signaling molecules in intestine of fish As shown in Fig. 5, dietary MHA had no significant effect on IkB kinase a (IKKa) and IKKg mRNA levels in PI, MI and DI of fish (P > 0.05). In PI, the NF-kBp65, c-Rel, IKKb and 4E-BP1 mRNA levels gradually decreased with dietary MHA up to 6.4 g/kg diet, and gradually decreased thereafter. The mRNA level of p38 mitogen activated

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Fig. 3. Relative mRNA levels of LEAP2, hepcidin and b-defensin in the PI (A), MI (B) and DI (C) of young grass carp (Ctenopharyngodon idella) fed diets supplemented with graded levels of MHA and 6.4 g/kg DLM. Data represent means of six fish in each group, error bars indicate S.D. Values having different letters are significantly different (P < 0.05), and values in the 6.4 g/kg DLM group with no superscript are not significantly different from the 6.4 g/kg MHA group (P > 0.05).

protein kinase (p38MAPK) gradually decreased with dietary MHA up to 6.4 g/kg diet, and then significantly increased (P < 0.05). The 4E-BP2 mRNA level significantly decreased with dietary MHA up to 4.4 g/kg diet (P < 0.05), and then gradually increased. Meanwhile, the mRNA levels of inhibitor of kBa (IkBa), TOR and ribosomal protein S6 kinase 1 (S6K1) gradually increased with dietary MHA up to 6.4 g/kg diet, and gradually decreased thereafter. In MI, the tendency of c-Rel, IKKb, IkBa, TOR, S6K1 and 4E-BP1 mRNA levels were as the same as that in PI. The mRNA levels of p38MAPK and 4E-BP2 gradually decreased with dietary MHA up to 6.4 g/kg diet, and gradually decreased thereafter. In DI, the tendency of NF-kBp65, c-Rel, IKKb, IkBa, TOR, S6K1 and 4E-BP1 mRNA levels were as the same as that in PI, and the tendency of p38MAPK and 4E-BP2 mRNA levels were as the same as that in MI. Meanwhile, these mRNA levels of signaling molecules in the intestines of fish 6.4 g MHA/kg diet had no significant difference from that in fish fed 6.4 g DLM/kg diet (P > 0.05).

3.5. Effects of MHA on the mRNA levels of caspases and related signaling molecules in intestine of fish The mRNA levels of caspases and related signaling molecules in intestine of fish are displayed in Fig. 6. In PI, the mRNA levels of caspase-3, capase-8, caspase-9, Fas ligand (FasL) and JNK gradually decreased with dietary MHA up to 6.4 g/kg diet, and gradually increased thereafter. The caspase-7 and apoptosis proteaseactivating factor 1 (Apaf-1) mRNA levels gradually decreased with dietary MHA up to 6.4 g/kg diet, and then significantly increased (P < 0.05). The Bcl-2 associated X protein (Bax) mRNA level significantly decreased with dietary MHA up to 4.4 g/kg diet (P < 0.05), and then gradually increased. Meanwhile, the B-cell lymphoma 2 (Bcl-2) mRNA level in PI significantly increased with dietary MHA up to 2.4 g/kg diet (P < 0.05), and plateaued thereafter. In MI, the tendency of caspase-3, capase-8, caspase-9, Fas ligand (FasL) and JNK mRNA levels were as the same as that in PI. The mRNA levels of caspase-7, Apaf-1 and Bax gradually decreased with dietary MHA up to 6.4 g/kg diet, and gradually increased thereafter. Meanwhile, the Bcl-2 mRNA level gradually increased with dietary MHA up to 6.4 g/kg diet, and gradually decreased thereafter. In DI, the tendency of caspase-3, caspase-7, capase-8, FasL, Apaf1, Bax, Bcl-2 and JNK mRNA levels were as the same as that in MI. Meanwhile, capsase-9 mRNA level gradually decreased with

dietary MHA up to 6.4 g/kg diet, and then significantly increased (P < 0.05). As shown in Fig. 6, the mRNA levels of caspase-3 in the three intestine segments and caspase-7 in the PI of fish fed 6.4 g MHA/kg diet were significantly lower than that in fish fed 6.4 g DLM/kg diet (P < 0.05), and the others in the intestines were not significantly different between the two groups (P > 0.05). 3.6. Effects of MHA on the oxidative status and antioxidant-related parameters in the intestine of fish The oxidative status and antioxidant enzymes activities in the intestine of fish are presented in Table 2. The MDA contents in PI and MI gradually decreased with dietary MHA up to 6.4 g/kg diet, and significantly increased thereafter (P < 0.05). The contents of PC in PI, MI and DI, as well as MDA in DI of fish with graded levels of dietary MHA were significantly lower than that of fish fed basal diet (P < 0.05). Based on the regression analysis of PI MDA content (Y ¼ 0.032x2 - 0.358x þ 3.013, R2 ¼ 0.878, P < 0.05), the optimal MHA levels for young grass carp was 5.59 g/kg diet. In addition, the CAT activities in PI, MI and DI, GST and GR activities in DI, and GSH contents in PI, MI and DI gradually increased with dietary MHA up to 6.4 g/kg diet, and then gradually decreased. The activities of SOD in PI and GPx in DI significantly increased with dietary MHA up to 4.4, 6.4 and 6.4 g/kg diet respectively (P < 0.05), and then gradually decreased. The activities of SOD in MI and DI and GST in MI significantly increased with dietary MHA up to 4.4 g/ kg diet (P < 0.05), and gradually decreased thereafter. The activities of GPx in PI and MI, GST in PI and GR in PI and MI gradually increased with dietary MHA up to 6.4 g/kg diet, and then significantly decreased (P < 0.05). As shown in Table 2, MDA contents in the PI and DI, and PC content in the MI of fish fed 6.4 g MHA/kg diet were significantly lower, and GSH content in the PI was significantly higher than that in fish fed 6.4 g DLM/kg diet (P < 0.05), and the others in the intestines were not significantly different between the two groups (P > 0.05). 3.7. Effects of MHA on the mRNA levels of antioxidant enzymes and related signaling molecules in intestine of fish The mRNA levels of antioxidant enzymes and related signaling molecules in intestine of fish are shown in Fig. 7. Dietary MHA had

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Fig. 4. Relative mRNA levels of TNF-a, IFN-g2, IL-1b, IL-8, IL-12p35, IL-12p40, IL-10, IL-4/13A, IL-4/13B, TGF-b1 and TGF-b2 in the PI (A), MI (B) and DI (C) of young grass carp (Ctenopharyngodon idella) fed diets supplemented with graded levels of MHA and 6.4 g/kg DLM. Data represent means of six fish in each group, error bars indicate S.D. Values having different letters are significantly different (P < 0.05), and values in the 6.4 g/kg DLM group superscripted asterisk are significantly different from the 6.4 g/kg MHA group (P > 0.05).

no significant effect on CuZnSOD, GSTP1 and Kelch-like-ECHassociated protein 1b (Keap1b) mRNA levels in intestine of fish (P > 0.05). In PI, the mRNA levels of MnSOD, CAT, GPx1, GPx4, GSTP2, GR and Nrf2 gradually increased with dietary MHA up to 6.4 g/kg diet, and gradually decreased thereafter. Meanwhile, the Keap1a mRNA levels gradually decreased with dietary MHA up to 6.4 g/kg diet, and then gradually increased. In MI, the tendency of MnSOD, CAT, GPx1, GPx4, GSTP2, GR, Nrf2 and Keap1a were as the same as that in PI. In DI, the tendency of MnSOD, CAT, GPx4, GSTP2, GR, Nrf2 and Keap1a mRNA levels were as the same as that in PI. Meanwhile, the

GPx1 mRNA level significantly increased with dietary MHA up to 4.4 g/kg diet (P < 0.05), and then gradually decreased. As shown in Fig. 7, the mRNA levels of these antioxidant enzymes isoforms, Nrf2, Keap1a and Keap1b in the intestines of fish fed 6.4 g MHA/kg diet had no significant difference from that in fish fed 6.4 g DLM/kg diet (P > 0.05). 3.8. Effects of MHA on the mRNA levels of tight junction proteins and MLCK in intestine of fish As shown in Fig. 8, dietary MHA had no significant effect on claudin-7a and claudin-7b mRNA levels in MI and DI (P > 0.05).

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Fig. 5. Relative mRNA levels of NF-kBp65, c-Rel, IkBa, IKKa, IKKb, IKKg, p38MAPK, TOR, S6K1, 4E-BP1 and 4E-BP2 in the PI (A), MI (B) and DI (C) of young grass carp (Ctenopharyngodon idella) fed diets supplemented with graded levels of MHA and 6.4 g/kg DLM. Data represent means of six fish in each group, error bars indicate S.D. Values having different letters are significantly different (P < 0.05), and values in the 6.4 g/kg DLM group with no superscript are not significantly different from the 6.4 g/kg MHA group (P > 0.05).

In PI, the mRNA levels of occludin, zonula occluden 1 (ZO-1), claudin-c, claudin-7a, claudin-7b and claudin-11 gradually increased with dietary MHA up to 6.4 g/kg diet, and gradually decreased thereafter. The claudin-b, claudin-c and claudin-15 mRNA levels significantly increased with dietary MHA up to 2.4 g/kg diet (P < 0.05), and then plateaued. Meanwhile, the MLCK mRNA level gradually decreased with dietary MHA up to 6.4 g/kg diet, and then gradually increased. In MI, the tendency of occludin, ZO-1, claudin-c, claudin-11 and MLCK mRNA levels were as the same as that in PI. The mRNA levels of claudin-3, claudin-11 and claudin-15 gradually increased with dietary MHA up to 6.4 g/kg diet, and gradually decreased thereafter. The claudin-b mRNA level gradually increased with dietary MHA up

to 6.4 g/kg diet, and significantly decreased thereafter (P < 0.05). In DI, the tendency of occludin, ZO-1, claudin-c, claudin-3 and claudin-15 mRNA levels were as the same as that in MI. The mRNA level of claudin-b gradually increased with dietary MHA up to 6.4 g/ kg diet, and gradually decreased thereafter. The claudin-11 mRNA level significantly increased with dietary MHA up to 6.4 g/kg diet (P < 0.05), and gradually decreased thereafter. Meanwhile, the MLCK mRNA level gradually decreased with dietary MHA up to 6.4 g/kg diet, and significantly increased thereafter (P < 0.05). Meanwhile, the mRNA level of occludin in the PI of fish fed 6.4 g MHA/kg diet were significantly higher than that in fish fed 6.4 g DLM/kg diet (P < 0.05), and the others in the intestines were not significantly different between the two groups (P > 0.05).

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Fig. 6. Relative mRNA levels of caspase-3, 7, 8, 9, FasL, Apaf-1, Bax, Bcl-2 and JNK in the PI (A), MI (B) and DI (C) of young grass carp (Ctenopharyngodon idella) fed diets supplemented with graded levels of MHA and 6.4 g/kg DLM. Data represent means of six fish in each group, error bars indicate S.D. Values having different letters are significantly different (P < 0.05), and values in the 6.4 g/kg DLM group superscripted asterisk are significantly different from the 6.4 g/kg MHA group (P > 0.05).

4. Discussion This study was a part of a larger research that MHA improved fish growth and immunity [33], which indicated that intestinal

immune response was in accordance with the systemic response after A. hydrophila infection. The intestine is an important immune organ in teleost, which constitutes the immune system with other immune organs (such as head kidney, spleen and skin) and

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Table 2 MDA (nmol/g tissue) and PC contents (nmol/mg protein), capacities of AHR (U/mg protein) and ASA (U/g protein), and activities of SOD (U/mg protein), CAT (U/mg protein), GPx (U/mg protein), GR (U/g protein) and GST (U/mg protein), and reduced GSH contents (mg/g protein) in the PI, MI and DI of young grass carp (Ctenopharyngodon idella) fed diets with graded levels of MHA and 6.4 g/kg DLM.1 Dietary MHA levels (g/kg diet)

PI MDA PC SOD CAT GPx GST GR GSH MI MDA PC SOD CAT GPx GST GR GSH DI MDA PC SOD CAT GPx GST GR GSH

Basal

2.4

4.4

6.4

8.5

10.5

DLM (6.4)2

2.97 ± 0.24c 8.65 ± 0.44d 6.48 ± 0.16a 1.82 ± 0.12a 70.24 ± 3.05a 43.69 ± 3.31a 27.30 ± 1.84a 4.75 ± 0.34a

2.37 ± 0.17b 6.98 ± 0.46b 7.63 ± 0.33b 2.34 ± 0.21bc 91.02 ± 7.69b 50.01 ± 3.61b 30.49 ± 1.68b 5.27 ± 0.47b

2.25 ± 0.21b 6.27 ± 0.48a 9.81 ± 0.24d 2.58 ± 0.24d 118.65 ± 10.02d 53.32 ± 2.93bc 34.62 ± 1.41c 5.87 ± 0.46cd

1.79 ± 0.11a 6.22 ± 0.28a 11.14 ± 0.27e 2.60 ± 0.09d 122.04 ± 11.97d 55.55 ± 3.59c 35.52 ± 3.42c 6.24 ± 0.54d

2.39 ± 0.19b 6.52 ± 0.28a 9.49 ± 0.24c 2.50 ± 0.06cd 102.88 ± 2.80c 51.71 ± 1.04b 33.54 ± 1.24c 5.96 ± 0.30d

2.84 ± 0.15c 7.49 ± 0.28c 6.33 ± 0.30a 2.29 ± 0.08b 82.22 ± 7.54b 45.87 ± 3.02a 29.98 ± 2.59b 5.44 ± 0.16bc

2.14 ± 0.20* 6.32 ± 0.20 10.68 ± 0.50 2.60 ± 0.20 114.34 ± 8.01 55.57 ± 2.83 33.97 ± 1.99 5.65 ± 0.12*

6.67 ± 0.61c 10.71 ± 0.58f 11.85 ± 0.65a 1.94 ± 0.15a 98.47 ± 4.21a 43.81 ± 1.89a 28.43 ± 2.16a 4.54 ± 0.25a

5.12 ± 0.26b 7.49 ± 0.37d 16.22 ± 0.45b 2.75 ± 0.20bc 113.35 ± 7.71b 52.32 ± 2.99bc 32.64 ± 1.36b 5.11 ± 0.24b

4.07 ± 0.35a 5.82 ± 0.30b 17.16 ± 0.34c 2.95 ± 0.27cd 136.25 ± 9.70d 59.48 ± 3.62d 37.97 ± 3.50c 5.57 ± 0.39cd

3.72 ± 0.22a 4.82 ± 0.31a 16.84 ± 0.68c 3.05 ± 0.20d 143.95 ± 6.10d 58.83 ± 4.41d 38.18 ± 2.99c 5.74 ± 0.41d

4.70 ± 0.21b 6.94 ± 0.16c 15.97 ± 0.45b 2.85 ± 0.17bcd 123.43 ± 7.63c 55.73 ± 3.29cd 35.03 ± 1.38b 5.16 ± 0.41bc

6.34 ± 0.55c 8.14 ± 0.28e 12.30 ± 0.49a 2.62 ± 0.10b 103.25 ± 7.85a 48.48 ± 4.35b 29.37 ± 1.53a 4.92 ± 0.43ab

3.94 ± 0.13 5.49 ± 0.22* 16.28 ± 0.44 2.85 ± 0.24 132.24 ± 13.19 58.45 ± 3.77 37.77 ± 3.98 5.72 ± 0.22

6.70 ± 0.57f 5.33 ± 0.40d 12.77 ± 0.29b 1.76 ± 0.11a 105.37 ± 8.50a 42.32 ± 2.90a 21.80 ± 1.59a 4.59 ± 0.26a

3.28 ± 0.31c 4.40 ± 0.29b 15.63 ± 0.27c 2.52 ± 0.19bc 123.60 ± 8.16b 52.28 ± 3.50bc 26.70 ± 1.50bc 5.05 ± 0.21b

2.47 ± 0.24b 3.59 ± 0.23a 17.63 ± 0.59d 2.66 ± 0.10cd 143.99 ± 13.92c 55.66 ± 2.92cd 28.27 ± 2.22cd 5.66 ± 0.27c

2.00 ± 0.12a 3.36 ± 0.14a 17.47 ± 0.18d 2.73 ± 0.13d 157.50 ± 8.84d 57.91 ± 4.69d 29.69 ± 2.95d 5.75 ± 0.27c

4.60 ± 0.41d 4.15 ± 0.22b 15.19 ± 0.43c 2.51 ± 0.10bc 128.99 ± 7.88b 53.03 ± 1.77bc 26.57 ± 0.86bc 5.11 ± 0.19b

5.98 ± 0.29e 4.84 ± 0.17c 11.79 ± 0.46a 2.43 ± 0.15b 110.99 ± 3.85a 49.44 ± 2.87b 24.91 ± 1.46b 4.92 ± 0.44ab

2.51 ± 0.12* 3.52 ± 0.11 17.05 ± 0.51 2.67 ± 0.08 145.29 ± 11.40 55.88 ± 4.60 29.12 ± 1.13 5.61 ± 0.07

YMDA in PI ¼ 0.0324x2 - 0.3573x þ 3.0097 YPC in PI ¼ 0.0687x2 - 0.828x þ 8.6181 YSOD in PI ¼
0.1438x2 þ 1.6069x þ 5.8142 YCAT in PI ¼
0.02x2 þ 0.2515x þ 1.8343 YGPx in PI ¼
1.5554x2 þ 17.834x þ 66.568 YGST in PI ¼
0.3549x2 þ 4.0132x þ 43.192 YGR in PI ¼
0.2282x2 þ 2.7398x þ 26.659 YGSH in PI ¼
0.034x2 þ 0.4398x þ 4.6359 YMDA in MI ¼ 0.0958x2 - 1.0602x þ 6.8247 YPC in MI ¼ 0.1483x2 - 1.7849x þ 10.757 YSOD in MI ¼
0.1911x2 þ 2.0452x þ 11.9990 YCAT in MI ¼
0.0273x2 þ 0.3421x þ 1.9875 YGPx in MI ¼
1.3728x2 þ 15.385x þ 94.092 YGST in MI ¼
0.4755x2 þ 5.4946x þ 43.322 YGR in MI ¼
0.3288x2 þ 3.6493x þ 27.643 YGSH in MI ¼
0.0327x2 þ 0.3772x þ 4.5049 YMDA in DI ¼ 0.1522x2 - 1.6162x þ 6.5397 YPC in DI ¼ 0.0575x2 - 0.6559x þ 5.4162 YSOD in DI ¼
0.1939x2 þ 1.9677x þ 12.5521 YCAT in DI ¼
0.0226x2 þ 0.2866x þ 1.8339 YGPx in DI ¼
1.4877x2 þ 16.546x þ 101.24 YGST in DI ¼
0.4031x2 þ 4.8284x þ 42.563 YGR in DI ¼
0.2066x2 þ 2.4188x þ 21.901 YGSH in DI ¼
0.0326x2 þ 0.3716x þ 4.5373

R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2 R2

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

0.882 0.997 0.830 0.992 0.928 0.972 0.938 0.926 0.975 0.960 0.980 0.972 0.890 0.980 0.942 0.915 0.941 0.952 0.985 0.927 0.870 0.975 0.953 0.858

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

< 0.05 < 0.01 ¼ 0.071 < 0.01 < 0.05 < 0.01 < 0.05 < 0.05 < 0.01 < 0.01 < 0.01 < 0.01 < 0.05 < 0.01 < 0.05 < 0.05 < 0.05 < 0.01 ¼ 0.071 < 0.05 < 0.05 < 0.01 < 0.01 ¼ 0.054

1 Values are means ± SD (n ¼ 6), and superscripted different letters in the same row are significantly different (P < 0.05). MDA, malondialdehyde; PC, protein carbonyl; AHR, anti-hydroxy radical; ASA, anti-superoxide anion; and GSH, glutathione; SOD, superoxide dismutase; GST, glutathione-S-transferase; CAT, catalase; GPx, glutathione peroxidase; GR, glutathione reductase. 2 Values in the 6.4 g/kg DLM group subscripted asterisk are significantly different from the 6.4 g/kg MHA group (P < 0.05), and no superscript are not significantly different (P > 0.05).

circulating blood. It was reported that enteritis is also a common consequence of A. hydrophila infection to fish [42]. Accordingly, enteritis morbidity is used as an index to evaluate the ability of fish defense against this disease [1]. In this study, optimal MHA supplementation reduced the enteritis morbidity of young grass carp, suggesting that MHA could improve intestinal health status in fish. Generally, the intestinal health status depends on its immunological and physical barrier function.

4.1. MHA enhanced intestinal immunological barrier function In fish, LZM, ACP, complements and antimicrobial peptides exert positive effect on intestinal defense against pathogen invasion [2,9]. This study demonstrated that optimal MHA supplementation elevated LZM and ACP activities, C3 and C4 contents, and antimicrobial peptides hepcidin, LEAP2 and b-defensin mRNA levels in the intestine of young grass carp. Our previous study on MHA showed similar results in Jian carp serum [12].

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Fig. 7. Relative mRNA levels of CuZnSOD, MnSOD, CAT, GPx1, GPx4, GSTP1, GSTP2, GR, Nrf2, Keap1a and Keap1b in the PI (A), MI (B) and DI (C) of young grass carp (Ctenopharyngodon idella) fed diets supplemented with graded levels of MHA and 6.4 g/kg DLM. Data represent means of six fish in each group, error bars indicate S.D. Values having different letters are significantly different (P < 0.05), and values in the 6.4 g/kg DLM group with no superscript are not significantly different from the 6.4 g/kg MHA group (P > 0.05).

In addition to these humoral immune factors, cytokines also perform important roles in fish intestinal immune response [2]. However, inflammation occurrence in intestine can cause excessive tissue damage and dysfunction [43]. It was reported that downregulation of pro-inflammatory cytokines such as TNF-a, IL-1b, IL8, IFN-g2 and IL-12p35, leads to suppression on inflammation in fish [44e46]. This study showed that optimal MHA supplementation down-regulated the mRNA levels of TNF-a, IFN-g2, IL-1b and IL-12p35 in the intestine, as well as IL-8 in the MI and DI of young

grass carp. The interesting result that the down-regulation of IL-8 only in the MI and DI (not in PI) might be attributed to MHA absorption. Rombout et al. [2] reviewed that nutrients absorption appears to take place in PI. Meanwhile, MHA cross the apical membrane of Caco-2 cells requires the participation of a transport mechanism with many of the properties of system monocarboxylate transporter 1 (MCT1), which is consistent with the cooperative Naþ/Hþ exchanger [47]. Nemeth et al. [48] reported that the Naþ/Hþ exchange process could facilitate the up-regulation

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Fig. 8. Relative mRNA levels of occludin, ZO-1, claudin-b, c, 3, 7a, 7b, 11, 15 and MLCK in the PI (A), MI (B) and DI (C) of young grass carp (Ctenopharyngodon idella) fed diets supplemented with graded levels of MHA and 6.4 g/kg DLM. Data represent means of six fish in each group, error bars indicate S.D. Values having different letters are significantly different (P < 0.05), and values in the 6.4 g/kg DLM group superscripted asterisk are significantly different from the 6.4 g/kg MHA group (P > 0.05).

of IL-8 gene expression in Caco-2 cells. Thus, the process of MHA absorption might counteract the down-regulation of IL-8 in the PI of fish. However, the underlying mechanism requires further research. In addition, the down-regulation of TNF-a, IL-1b, IFN-g2

and IL-12p35 might be correlated with IKK/IkBa/NF-kB pathway. Dejardin et al. [49] demonstrated that activated IKK (composed of two catalytic subunits IKKa and IKKb, and a regulatory subunit IKKg) lead to IkBa degradation, subsequently induce NF-kB/Rel

F.-Y. Pan et al. / Fish & Shellfish Immunology 64 (2017) 122e136

family members (such as NF-kBp65 and c-Rel) nuclear translocation and activation. Studies indicated that NF-kBp65 was the pivotal role for TNF-a and IL-1b genes expression in human [11], and c-Rel was for IFN-g and IL-12p35 genes expression in mice [45,46]. This study demonstrated that optimal MHA supplementation down-regulated NF-kBp65, c-Rel and IKKb mRNA levels and up-regulated IkBa mRNA levels in the intestine of young grass carp. In addition, TNF-a and IL-1b mRNA levels were positively correlated with NF-kBp65 mRNA levels, and IFN-g2 and IL-12p35 mRNA levels were positively correlated with c-Rel mRNA levels in the intestine (Table 3). These results indicate that MHA might be via IKKb/IkBa/ (NF-kBp65 and c-Rel) pathway to down-regulate pro-inflammatory cytokines genes expression in fish intestine. Furthermore, MHA down-regulating IKKb (not IKKa or IKKg) might be attributed to p38MAPK. Study indicated that p38MAPK could up-regulate c-myc gene expression in mice [50]. In human, c-myc was found to repress N-myc downstream regulated gene 1 (Ndrg1) expression [51]. Hosoi et al. [52] reported that Ndrg1 could down-regulate IKKb (not IKKa or IKKg) gene expression in mice. This study showed that optimal MHA supplementation down-regulated p38MAPK mRNA levels in the intestine of fish, suggesting that MHA might be via down-regulating p38MAPK gene expression to suppress IKKb gene expression in fish intestine. In addition, this study demonstrated that optimal MHA supplementation up-regulated IL-12p40 mRNA levels in the intestine of young grass carp. The result might be related to TOR signaling pathway. Activation of TOR immediately leads to the downstream molecules S6K1 activation and 4E-BP degradation [53]. Attur et al. [54] reported that TOR could elevate prostaglandin E 2 (PGE2) production in mice. In human, PGE2 was found to be an inducer of IL-12p40 production [55]. In this study, optimal MHA supplementation up-regulated TOR and S6K1 mRNA levels, and down-regulated 4E-BP1 and 4E-BP2 mRNA levels in the intestine of young grass carp. Interestingly, IL-12p40, a subunit of IL-12p70 like IL-12p35, performed the inhibitor of colitis in contrast to IL-12p70 in mice [56]. Above results indicate that MHA might be via p38MAPK/IKKb/NF-kB and TOR pathways to suppress intestinal pro-inflammatory cytokines genes expression in fish intestine. The inflammatory process also could be suppressed by antiinflammatory cytokines, such as TGF-b, IL-10 and IL-4/13 [57,58]. In this study, optimal MHA supplementation up-regulated TGF-b1 (not TGF-b2), IL-10 and IL-4/13A (not IL-4/13B) mRNA levels in the intestine of young grass carp. MHA up-regulating TGF-b1 (rather than TGF-b2) gene expression might be related to insulin. Studies indicated that methionine could increase insulin which could potentiate (epidermal growth factor) EGF-signal in mice [27,59]. Cupp et al. reported that EGF could up-regulate TGF-b1 (rather than TGF-b2) gene expression in mice. These results indicate that MHA might be via insulin/EGF to up-regulate TGF-b1 (not TGF-b2) in fish intestine, which need further study. Meanwhile, the up-regulation of IL-10 and IL-4/13A (not IL-4/13B) might be attributed to TOR. Studies demonstrated that TOR could elevate IL-10 gene expression in human [10] and GATA-3 translation in mice [60]. Interestingly, it was reported that GATA-3 has a more important direct enhancing

133

function for IL-4/13A than IL-4/13B [61]. These results indicate that MHA might be via TOR signaling pathway to up-regulate IL-10 and IL-4/13A (not IL-4/13B) genes expression in the intestine of fish. Thus, MHA could up-regulate fish intestinal anti-inflammatory cytokines genes expression, which might be related to TOR-signal and potential influence on hormone. 4.2. MHA inhibited apoptosis in intestine of fish To our knowledge, the physical barrier of animal intestine is linked to the defense against apoptosis [62]. It was reported that caspases (such as caspase-3 and caspase-7) execute apoptosis in fish [21,63]. This study found that optimal MHA supplementation downregulated intestinal caspase-3 and caspase-7 mRNA levels in the intestine of young grass carp, indicating that MHA could inhibit apoptosis in fish intestine. Studies demonstrated that both caspase-3 and caspase-7 could be activated via death receptor pathway (FasL/ caspase-8) [64] and blockaded by the inhibition of Bcl-2 on mitochondria pathway (Bax/Apaf-1/caspase-9) [65]. In this study, optimal MHA supplementation down-regulated the mRNA levels of caspase8, caspase-9, FasL, Apaf-1 and Bax, and up-regulated Bcl-2 mRNA levels in the intestine of young grass carp, suggesting that MHA might be via suppression on death receptor pathway and mitochondria pathway to inhibit apoptosis in fish intestine. Furthermore, in HeGP2 cells, JNK was found to be the trigger for death receptor pathway and mitochondria pathway induced apoptosis [22]. This study demonstrated that optimal MHA supplementation downregulated JNK mRNA levels in the intestine of young grass carp, suggesting that MHA exerted suppression on apoptosis via downregulating JNK gene expression to inhibit the process of death receptor and mitochondria pathways in fish intestine. 4.3. MHA alleviated oxidative damage and improved antioxidant status in intestine of fish It was reported that apoptosis could be induced by oxidative damage [66]. ROS could cause oxidant damage (lipid peroxidation and protein oxidation) in tissues, which include hydroxyl and superoxide radicals [67]. Whereas, ROS could be scavenged by GSH and antioxidant enzymes in Carassius auratus [68]. In this study, optimal MHA supplementation decreased MDA and PC contents, and increased GSH contents and antioxidant enzymes SOD, CAT, GPx, GST and GR activities in the intestine of young grass carp. These results were similar to our previous study on Jian carp [8]. The antioxidant enzymes activities were found to be related to corresponding genes expression [69]. In this study, optimal MHA supplementation up-regulated MnSOD, CAT, GPx1, GPx4, GSTP2 and GR mRNA levels in the intestine of young grass carp, which might contribute to increase of their corresponding enzymes activities. Meanwhile, the interesting result that MHA up-regulating MnSOD but not CuZnSOD mRNA levels might be related to insulin. As the above-mentioned, dietary methionine could increase insulin level in mice [27]. In human, insulin could up-regulate

Table 3 Correlation analysis. Independent parameters

Dependent parameters

Correlation coefficients

NF-kBp65

TNF-a IL-1b INF-g2 IL-12p35 CAT GPx1 GPx4 GR

rPI rPI rPI rPI rPI rPI rPI rPI

c-Rel Nrf2

**Correlation is significant at P < 0.01; *Correlation is significant at P < 0.05.

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

þ0.973**; þ0.950**; þ0.959**; þ0.951**; þ0.963**; þ0.958**; þ0.935**; þ0.966**;

rMI rMI rMI rMI rMI rMI rMI rMI

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

þ0.977**; rDI ¼ þ0.997** þ0.996**; rDI ¼ þ0.979** þ0.960**; rDI ¼ þ0.938** þ0.988**; rDI ¼ þ0.913* þ0.905*; rDI ¼ þ0.974** þ0.876*; rDI ¼ þ0.980** þ0.955**; rDI ¼ þ0.970** þ0.935**; rDI ¼ þ0.990**

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apolipoprotein A-I (ApoA-I) gene expression [70]. Importantly, ApoA-I was found to up-regulate gene expression of MnSOD rather than CuZnSOD in mice [71]. Thus, MHA might be via insulin/ApoA-I to up-regulate MnSOD gene expression only in fish, but the mechanism is still need furthermore investigation. Meanwhile, the up-regulation of CAT, GPx1, GPx4 and GR mRNA levels might be owing to the effect of MHA on Keap1/Nrf2 system. It was reviewed that suppression on Keap1 leads to Nrf2 release and nuclear translocation, which promotes antioxidant enzymes transcription in mammals [72]. This study showed that optimal MHA supplementation up-regulated Nrf2 mRNA levels and down-regulated Keap1a mRNA levels in the intestine of fish. In addition, the mRNA levels of CAT, GPx1, GPx4 and GR were positively related to Nrf2 mRNA levels (Table 3), indicating that MHA was able to upregulate these antioxidant enzymes genes expression via Keap1a/ Nrf2 pathway. Meanwhile, the interesting result that MHA downregulating Keap1a rather than Keap1b mRNA levels might be attributed to the MHA's metabolite S-adenosyl-L-methionine which was found to be closely related to phospholipids synthesis [15]. Our previous study demonstrated that dietary phospholipids supplementation resulted in down-regulation of Keap1a rather than Keap1b mRNA levels in the intestine of juvenile grass carp. Thus, MHA down-regulating Keap1a (not Keap1b) gene expression might be via promoting synthesis of phospholipids in the intestine of fish, which need further investigation. In addition, the interesting result that MHA up-regulating GSTP2 rather than GSTP1 might be owing to Keap1a. It was reported that knockout mice Keap1 could upregulated gene expression of GSTP2 rather than GSTP1 [73]. This might partially explain that MHA only up-regulated GSTP2 gene expression in the intestines of fish. These results indicate that MHA might be via modulating Keap1a/Nrf2 pathway to improve antioxidant status and then alleviate oxidative damage in the intestine of fish. 4.4. MHA improved tight junctions in intestine of fish The physical barrier of animal intestine always depends on its tight junctions [4]. In this study, optimal MHA supplementation up-

regulated the mRNA levels of tight junction proteins occludin, ZO-1, claudin-b, c, 3, 11 and 15 in the intestine, as well as claudin-7a and 7b mRNA levels only in the PI of young grass carp. These results demonstrate that MHA could improve intestinal tight junctions in fish, which is similar to the study on MHA in Caco-2 cells [20]. Interesting, the up-regulation of claudin-7a and 7b mRNA levels were only found in the PI but not MI or DI, which might be related to MHA absorption. As above-mentioned, the MHA absorption might take place in the PI of fish [2], and the process is closely related to Naþ/Hþ exchange [47]. Noteworthy, Claudin-7 is recognized as a channel to Naþ [74]. Thus, the up-regulation of claudin7a and 7b mRNA levels only in the PI might be correlated with MHA absorption in this intestinal segment. However, the underlying mechanism needs to be further investigated. Meanwhile, the mRNA levels up-regulation of occludin, ZO-1, claudin-b, c, 3, 11 and 15 might be related to MLCK. It was reported that MLCK could cause tight junctions dysfunction in bovine brain cortices [24]. This study showed that MLCK mRNA levels in the intestine were downregulated with dietary MHA up to optimal levels. These results suggest that MHA might be via repressing MLCK gene expression to improve intestinal tight junctions in fish. 4.5. The comparison between MHA and DLM on the intestinal barrier function of fish In the present study, compared to the 6.4 g/kg DLM, equal sulfur MHA (6.4 g/kg) supplementation showed significantly lower TNF-a, IL-1b, caspase-3 and caspase-7 mRNA levels, and MDA and PC contents, as well as higher GSH content, IL-10 and occludin mRNA levels in the intestine of fish. These results suggesting that MHA was superior to DLM on improving the immunological and physical barrier function of intestine in fish. The mechanism might be correlated with different transport and metabolism between MHA and DLM. In chicken small intestine, MHA was preferentially diverted to the transsulfuration pathway, thus leading to higher contents of taurine and cysteine [25]. In mice, taurine could elevate serum IL-10 production [31]. Furthermore, taurine was found to be the inhibitor to JNK and p38MAPK activation in mice [26], which

Fig. 9. The potential action pathways involved in MHA improving intestinal immunological and physical barrier function in fish.

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might partly explain the lower mRNA levels of TNF-a and IL-1b. Besides, study indicated that L-Met transport was a concentration and energy dependent while MHA transport was only concentration dependent [75]. Thus, the transport of DLM might induce more ROS generation in the intestine. I addition, the antioxidant properties of taurine are based on its capacity to scavenge HOCl generating chlorotaurine which itself could scavenge NO and its oxygen-containing radical metabolites [76]. Meanwhile, cysteine is the ingredient for GSH synthesis [32]. Besides, in Pekin duck, study demonstrated that compared to DLM supplementation, dietary supplementation with MHA resulted in less plasma homocysteine production [77]. In mice brain, homocysteine could downregulated occludin mRNA expression [78]. 5. Conclusion In this study, we report the primary result that dietary MHA supplementation could enhance the capacity of young grass carp to defense against enteritis, which is found in further investigation that MHA improves fish intestinal barrier function (Summarized in Fig. 9). Dietary MHA supplementation-enhanced fish intestinal immunological barrier function might be via (1) increasing the activities or contents of antimicrobial substances to block pathogen invasion; (2) modulating p38MAPK/IKKb (not IKKa or IKKg)/IkBa/ (NF-kBp65 and c-Rel) pathway and TOR-signal cascades to downregulate pro-inflammatory cytokines and up-regulate anti-inflammatory cytokines mRNA levels, which repress inflammation. Simultaneously, dietary MHA supplementation-improved fish intestinal physical barrier might be via [1] down-regulating JNK gene expression to suppress death receptor pathway (FasL/caspase-8) and mitochondria pathway [Bcl-2/Bax/Apaf-1/caspase-9] induced apoptosis [2]; modulating Keap1a (rather than Keap1b)/Nrf2 signaling pathway to up-regulate MnSOD (not CuZnSOD), CAT, GPx1, GPx4, GSTP2 (not GSTP1) and GR mRNA levels, which contributes to alleviating oxidative damage [3]; suppressing MLCK gene expression to improve intercellular tight junctions. Besides, MHA was superior to equal sulfur DLM on improving the intestinal barrier function in fish. In summary, on the premise of 4.01 g/kg methionine basal, the optimal MHA supplementation levels for young grass carp were 5.83, 6.09 and 5.59 g/kg diet, which bases on the capacity defense against enteritis, lysozyme activity and MDA content in the PI, respectively. Acknowledgements This research was financially supported by the National Basic Research Program of China (973 Program) (2014CB138600), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20135103110001), 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. References [1] X.H. Song, J. Zhao, Y.X. Bo, Z.J. Liu, K. Wu, C.L. Gong, Aeromonas hydrophila induces intestinal inflammation in grass carp (Ctenopharyngodon idella): an experimental model, Aquaculture 434 (2014) 171e178. [2] J.H.W.M. Rombout, L. Abelli, S. Picchietti, G. Scapigliati, V. Kiron, Teleost intestinal immunology, Fish. Shellfish Immun. 31 (2011) 616e626.

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