Effects Of Lysine On The Immune And Structural Barrier Functions And The Related Mechanisms Of Intestines In Fish

Fish growth mainly depends on food utilization,which is strongly related to the digestive and absorptive capacity.Thus,this study firstly investigated the effects of lysine on the growth,the growth and development of the digestive organs,activities and gene expressions of digestive and brush border enzymes in young grass carp(Ctenopharyngodon idellus).Second,we examined the effects of lysine on the antimicrobial peptides,cytokines and TLR/NF?B signaling molecules expression of intestine in grass carp to explore the effects of lysine on theimmune barrier of intestine in fish.Meanwhile,the effects of lysine on the gene expressions of cytokinesand the related signaling molecules of TLR/NF?B pathway in fish enterocytes were further investigated to explore the possible mechanisms by which lysine affects theintestinal immune in fish.Third,we determined the effects of lysine on intestinal tight junction proteins,MLCK and PKC signaling molecules expressionin grass carp in order to explore the effects of lysine on the physical barrier of intestine in fish.Fouth,we examined the effects of lysine on the gene expressions of antioxidant enzymes and the related signaling molecules of Nrf2 pathway in grass carp to explore the effects of lysine on thestructural integrity of intestine in fish.Based on the results of the study in vivo,we used grass carp IECs model to investigate the hypothesis that lysine protects enterocytes against oxidant stress via protection,prevent and repair pathways,which reveals the act pathways of lysine as an antioxidant.The main contents and results are presented as follow:1.The effects of lysine on growth,digestive and absorptive ability of young grass carpThe present research studied the effects of dietary lysine on growth performance and the digestion and absorption capacity of young grass carp(Ctenopharyn.godon idellus).A total of 720 young grass carp(254.9±1.1 g)were randomly distributed into six groups with four replicates each,fed semi-purified isonitrogenous diets containing graded levels of lysine(7.1,9.6,12.2,14.6,17.0 and 19.6 g/kg diets)for 56 days.The results showed that optimum dietary lysine increased feed intake(FI)and specific growth rate(SGR)(P<0.05),decreased the feed conversion ratio(P<0.05).Additionally,optimum dietary lysine improved the weight of intestine and hepatopancreas,activities of trypsin,lipase and chymotrypsin in intestine,alkaline phosphatase activity in proximal intestine(PI)and mid intestine(MI),activity of creatine kinase(CK)in MI and distal intestine(DI),Na+/K+-ATPase and y-glutamyl transpeptidase in PI,MI and DI(P<0.05).Furthermore,optimum dietary lysine up-regulated relative mRNA expressions of chymotrypsin and trypsin in intestine,Na+/K+-ATPase(PI,MI and DI)and CK(PI and MI)(P<0.05).Meanwhile,lysine supplementation increased relative mRNA expressions of target of rapamycin(TOR)in all intestine segment,and decreased eIF4E-binding protein2(4E-BP)in DI(P<0.05).Collectively,this study indicated that dietary lysine may improve fish growth,promotes the digestive and absorptive ability and regulates gene expression of the digestive and brush border enzymes via TOR and 4E-BP.Based on the quadratic regression analysis of PWG,the optimal lysine levels of young grass carp(255-489 g)was estimated to be 14.2 g/kg diet,corresponding to 47.3 g/kg of dietary protein.2.The effects of lysine on the immune barrier function of young grass carp intestine2.1 The effects of lysine on the gene expressions of cytokines,antimicrobial peptidesand the related signaling moleculesThe experiment design of the present study is the same as the experiment 1.This study investigated the effects of lysine on the immune factors in the intestine of fish to explore the effects of lysine on the immune barrier of intestine in fish.The results showed that optimal lysine supplementation increased the relative mRNA expressions of LEAP-2,Hepcidin,interleukin 10(IL-10)and transforming growth factor ?(TGF-P)and the activities of acid phosphatase andlysozyme as well as complement 3 content in PI,MI and DI(P<0.05).In contrast,optimal lysine supplementation decreased intestinal IFN-y2,IL-8,TNF-? and IL-1 mRNA levels in PI and DI(P<0.05).Moreover,results indicated that optimal lysine supplementation decreased TLR3,TLR4 and NF-?B gene expression in PI,MI and DI(P<0.05),andincreased I-?B gene expression in PI,MI and DI(P<0.05).In conclusion,dietary lysine regulates the immune barrier of intestine via regulating of antibacterial compounds and cytokines expression.Moreover,the regulation of cytokines gene expressions by lysine may be partially related to the regulation of TLR and NF-?B signaling molecules.2.2 The effects of lysine on the cytokine and TLR/NF-?B signaling expression in fish intestinal epithelial cellsThe experiment in vivo showed thatlysine improved the intestinal immune barrier of young grass carp.In order to reveal the detail mechanisms,we used in vitro model to further investigate the effects of lysine on cytokine expression via TLR/NF-?B signaling pathway in the presence or absence of inhibitor.Enterocytes were cultured in media containing lysine at concentration of 0,180,240 and 300 mg/L for 24 h,and exposed to 10?g/ml LPS for 1 h thereafter.For inhibition experiments,cells were pretreated with BAY 11-7082(NF-?B inhibitor)at 10 ?mol/L for 2 h,and then exposed to 240 mg/L lysine for 24 h or 10 ?g/ml LPSfor 1 h according to the experiments design.Our studies demonstrated that the Trif,IRF-3,TLR4,TLR3,NF-?B,MyD88,IL-8 and IL-1 mRNA levels increased and decreased I?BomRNA level in fish epithelial cells after LPS stimulation(P<0.05),and optimal lysine supplementation decreased TLR3,NF-?B,Trif,IRF-3,IL-1 and IL-8 and increased I?B mRNA levels compared to LPS only exposed control group in fish enterocytes(P<0.05).Morover,TLR3,TLR4,Trif and MyD88gene expression were significantly increased in LPS only exposed group,while I?B? mRNA abundant was depressed in comparison of the control group(P<0.001).Fish enterocytes exposed to 240 mg L-1 lysine(lysine+ LPS+)exhibited decreased TLR3 and Trif mRNA level compared to cells exposed to LPS alone(P<0.01 and P<0.001)and increased I?B?mRNA level compared to cells exposed to LPS alone(P<0.01).However,MyD88 and TLR4 gene expression have not change in LPS+Lys group compared to LPS group(P>0.05).In the LPS+Lys group,pretreated with BAY 11-7082 decreased the mRNA level of TLR3,I?B? and Trif and increased MyD88 mRNA levels compared to BAY 11-7082 absent group(P<0.05).In conclusion,lysine may inhibit cytokines expression via regulating TLR3-mediated pathway but not TLR4-mediated pathway in fish enterocytes.3.The effects of lysine on the physical barrier function of young grass carp intestine3.1 The effects of lysine on the gene expressions of tight junction proteinand the related signaling moleculesThe experiment design of this study is the same as the experiment 1.The effects of lysine on the gene expressions of tight junction proteins and signaling molecules of MAPKpathway in the intestine of young grass carp in order to explore the effect of lysine on the intestinal structural integrity in fish.The date showed that lysine up-regulated the relative mRNA expressions of Claudin 12,Claudin c,ZO-land Occludin in PI,MI and DI and Claudin b in PI and MI(P<0.05),down-regulated Claudin 15 in all intestinal segment and Claudin 3 in PI and MI(P<0.05).Conversely,the relative mRNA expression of PKC in the MI and DI showed an upward trend(P<0.05),while MLCK mRNA expression in the MI and DI showed an adverse trend(P<0.05).In conclusion,lysine could regulate gene transcriptions of intestinal tight junction proteins in fish.Moreover,the regulation of intestinal tight junction gene expressions by lysine may be partially related to the regulation of PKC and MLCK signaling molecules.3.2 The effects of lysine on the tight junction protein and PKC/MLCK signaling expression in fish intestinal enterocytesThe experiment in vivo showed thatlysine improved the intestinal physical barrier of young grass carp.In order to reveal the detail mechanisms,we used in vitro model to further investigate the effects of lysine on tight junction protein gene expression via PKC-MLCK signaling pathway.Studies demonstrated that the lactic acid dehydrogenase(LDH)activity and inllin fluxincreased and transepithelial electrical resistance(TEER)decreased in the supernatant of fish epithelial cells after LPS stimulation(P<0.05),and optimal lysine supplementation decreased LDH and inllin fluxand increased TEER compared to LPS only exposed control group in the supernatant of fish enterocytes(P<0.05),which indicated that lysine could inhibit LPS-induced increase of intestinal permeability.Morover,ZO-1?Claudin c?Claudin 3,Ocludin and PKC gene expression were elevated and MLCK gene expression decreased in LPS-exposed control group(P<0.05),and optimal lysine can diminish LPS-induced ZO-1,Claudin c,Claudin 3,Occludin and PKC gene expression down-regulated and MLCK gene expressionup-regulated in fish enterocytes.In conclusion,lysine may promote tight junction protein expression via regulating PKC/MLCK-mediated pathway in fish enterocytes.4.The effects of lysine on the intestinal antioxidant ability and the possible action pathway 4.1 The effects of lysine on the intestinal antioxidant ability of young grass carpThe experiment design of this study is the same as the experiment 1.The effects of lysine on the intestinal glutathione(GSH)content,antioxidant enzyme activities and gene expressions of the signaling molecules of Nrf2 pathway in the intestine grass carp in order to explore the effect of lysine on the structural integrity of intestine in fish and the possible mechanisms.The date showed that optimal lysine decreased the protein carbonyl(PC)and malondialdehyde(MDA)contents in the intestine(P<0.05),and increased activities of catalase(CAT),superoxide dismutase(SOD),glutathione reductase(GR),glutathione peroxidase(GPx)and glutathione S-transferases(GST)and glutathione content(GSH)in the intestine(P<0.05).Furthermore,optimal lysine supplementation up-regulated relative mRNA expressions of CuZnSOD,CAT,GPx and GST in the intestine(P<0.05),down-regulated relative mRNA expressions of GR in the intestine(P<0.05).Furthermore,optimal lysine supplementation also up-regulated NF-E2-related factor 2(Nrf2)gene exprrssion in the intestine(P<0.05),down-regulated relative mRNA expressions of Keaplb in intestine(P<0.05).Moreover,there is no significant effect on Keapla mRNA expressions in the intestine(P>0.05).Collectively,this study indicated that dietary lysine improved non-enzymatic and enzymatic antioxidant capacity in the intestine,and depressed lipid peroxidation and protein oxidative damage,and then improvedthe structural integrity of intestine in fish.Moreover,lysineimproved enzymes activities in fish intestine may be partially related to the regulation of gene transcription of antioxidant enzymes,which may be regulated by Nrf2-Keap1 signaling pathway.4.2 The possible action pathway of lysine on intestinal antioxidant ability in fish enterocytesThe experiment in vivo showed thatlysine improved the intestinal antioxidant capacity of young grass carp.To explore the the possible action pathway of lysine on intestinal antioxidant ability,this parts of experiment include twotrials in vitro to investigate the prevention,interception and repair effects of lysine on grass carp enterocytes against oxidative damage,respectively.Firtstly,in order to investigate theoptimal concentration lysine on carp IECs against oxidative stress,the first experiment has seven treatments with four replicates each.IECs were pre-incubated with graded levels of lysine(0 mg/L,60 mg/L,120 mg/L,180 mg/L,240 mg/L and 300 mg/L)for 72 h,pre-,co-treatment and post-treatment exposed to 6 mg Cu/L of medium for 24 h(trial 1).The results indicated that compared with the basal control group(Lys:0 mg/L,Cu:0 mg/L),exposure to Cu alone(Lys:0 mg/L,Cu:6 mg/L)increased LDH activity,MDA and PC contents,and decreased MTT content and the activity of ALP(P<0.05).Compared with Cu alone group(Lys:0 mg/L,Cu:6 mg Cu/L),co-treatment and post-treatment with 180 mg/L lysine significantly inhibited the increase of LDH activity,MDA and PC contents induced by Cu(P<0.05)and restored the decrease of ALP activity and MTT content induced by Cu(P<0.05).To sum up,180 mg/L lysine is optimal concentration that protect IECs against oxidative stress.Prevention experiment has three treatments with four replicates each.IECs were pre-incubated with 180mg/L lysine for 72 h,and then exposed to 6 mg Cu/L of medium for 24 h(trial 2).The results showed that compared with the basal control group(Lys:0 mg/L,Cu:0 mg/L),exposure to Cu alone(Lys:0 mg/L,Cu:6 mg/L)increased LDH activity,PC and MDA contents as well as 4-Hydroxy-2-nonenal(4HNE),(P<0.05)and decreased the activity of alkaline phosphatase(ALP)and MTT(P<0.05).Compared with Cu alone group(Lys:0 mg/L,Cu:6 mg Cu/L),pre-treatment with lysine significantly inhibited the increase of LDH activity and PC contents induced by the Cu(P<0.05).Compared with the basal control group(Lys:0 mg/L,Cu:0 mg/L),treatment with Cu(Lys:0 mg/L,Cu:6 mg/L)decreased GSH content(P<0.05)and increased the activities of GPx,GST,SODand GR(P<0.05),and have no effect on CAT activity and GPx,SOD,GR and GST mRNA levels(P>0.05).Compared with Cu alone group(Lys:0 mg/L,Cu:6 mg Cu/L),pretreatment with optimum lysine decreased CAT activity.To sum up,lysine could ont improve the antioxidant capacity of fish IECs,reduce lipid peroxidation and protein oxidation of enterocytes induced by Cu.The third objective of this study was to investigate whether lysine can intercept Cu-induced oxidative stress.IECs were treated with 180 mg/L lysine in the presence of 6 mg Cu/L of medium for 24 h(trail 3).Compared with the basal control(Lys:0 mg/L,Cu:0 mg/L),exposure to Cu alone increased MTT,4HNE,8-hydroxydeoxyguanosine(80HdG),LDH activiy,MDA and PC contents(P<0.05),decreased ALP activity(P<0.05).Compared with Cu alone group(Lys:0 mg/L,Cu:6 mg Cu/L),co-treatment with lysine significantly inhibited MTT,4HNE,80HdG,LDH activity,MDA and PC contents(P<0.05).Compared with the basal control(Lys:0 mg/L,Cu:0 mg/L),Cu alone group significantly increased GPx,SOD,GR and GST activities and gene expression as well as Nrf2 mRNA level(P<0.05),decreased GSH content and Keapl mRNA expression(P<0.05),and have no significant effect on CAT activity and gene expression(P>0.05).Compared with Cu alone group(Lys:0 mg/L,Cu:6 mg Cu/L),co-treatment with lysine significantly decreased GR,SOD,GST and GPx activities and gene expression as well as Nrf2 mRNA level(P<0.05),increased GSH content and Keapl mRNA expression(P<0.05)and have no significant effect on CAT activity and gene expression in enterocytes(P>0.05).Meanwhile,compared with Cu alone group(Lys:0 mg/L,Cu:6 mg Cu/L),co-treatment with lysine significantly decreased free Cu2+ion concentrations in culture medium.To sum up,co-treatment with lysine may chelate Cu2+ to intercept the damage of enzymatic antioxidant system of enterocytes,reduce lipid peroxidation and protein oxidation of enterocytes in fish.In the repair trail,enterocytes were pre-treated with 6 mg/L Cu for 24 hours,and then treated with 180 mg/L lysine for 72 h to study the reparative effects of lysine.The data showed that compared with the basal control(Lys:0 mg/L,Cu:0 mg/L),exposure to Cu alone(Lys:0 mg/L,Cu:6 mg Cu/L)increased LDH activity,MTT,80HdG,PC and MDA contents(P<0.05),and decreased ALP and PSM activities(P<0.05).Compared with Cu alone group(Lys:0 mg/L,Cu:6 mg Cu/L),post-treatment with lysine significantly inhibited LDH activity,MDA and PC contents(P<0.05).Compared with the basal control group(Lys:0 mg/L,Cu:0 mg/L),treatment with Cu alone(Lys:0 mg/L,Cu:6 mg Cu/L)induced decreases in GR activity and GR as well as Keapl mRNA expression and GSH content(P<0.05),increases CAT,GPx,SOD,GST activities and GPx,GST and CAT mRNA levels(P<0.05),but have no effect on SOD and Nrf2 gene expression(P>0.05).Compared with Cu alone group(Lys:0 mg/L,Cu:6 mg Cu/L),subsequent treatment with lysine increased the activity and mRNA level of GR and GSH content(P<0.05),decreased CAT and GSTactivities as well as GST mRNA level(P<0.05),but have no impact on SOD and GPx activities as well as SOD,CAT,GPx,Nrf2 and Keapl mRNA levels(P>0.05).In a word,subsequent treatment with lysine played a reparative effect on the damage of enzymatic antioxidant system of enterocytes caused by copper,reduced oxidative damage of enterocytes in fish.Collectively,lysine could improve fish growth and the digestive and absorptive function in fish.The improved digestive ability and absorptive function was correlated with the improved growth and development of intestine,the increased activites of digestive and brush border enzymes as well as the regulation of gene expressions of digestive enzymes and brush border enzymes.Lysine couldregulate immune barrier in the intestine of fish.Lysine could improve non-enzymatic and enzymatic antioxidant abitliy,and decrease lipids prooxidation and protein oxidation,and then improve the structural integrity of intestine in fish.Meanwhile,lysine improved the antioxidant ability of intestine is related to the regulation of gene transcriptions of antioxidant enzymes and Nrf2 signaling-related molecues in fish intestine.Moreover,lysine could regulate gene transcriptions of intestinal tight junction proteins in fish,which may be partially related to the regulation of PKC and MLCK signaling molecules.Furthermore,lysine could improve the antioxidant ability in IECs of fish via interception and repair function.Based on the quadratic regression analysis of PWG,the optimal lysine levels of young grass carp(255-489 g)was estimated to be a 14.2 g/kg diet,corresponding to 47.3 g/kg of dietary protein.