Effect Of Threonine On Intestinal Structure Integrity Of Juvenile Grass Carp (Ctenopharyngodon Idella) And Involved Mechanism

Threonine（Thr）is an essential amino acid for aquatic animals.Studies have reported that dietary threonine deficiency can decrease the growth performance of aquatic animals.The growth of aquatic animals is closely related to the digestion and absorption of nutrients.Intestine is the main part for the digestion and absorption of nutrients.The structural integrity of the intestine is the basis for the intestinal normal function.Firstly,this study aimed to investigate the effect of dietary Thr on the growth performance of juvenile grass carp（Ctenopharyngodon idella）,the development index of digestive organs,the activities of digestive enzymes and brush border enzymes,and amino acid metabolism related enzymes.Secondly,Aeromonas hydrophila was employed for the challenge test.On the one hand,we systematically explored the effects of dietary Thr on intestinal structural integrity of juvenile grass carp by investigating the effects of dietary Thr on the anti-oxidative damage ability and the Nrf2 related signal pathways;On the other hand,we explored the effects of dietary Thr on the intestinal intercellular structural integrity of juvenile grass carp by investigating the effects of dietary Thr on tight junctions（TJs）and PKC related signal regulation.Thirdly,we established the oxidative damage model of primary intestinal epithelial cells（IECs）of grass carp to investigate the effect of Thr on the oxidative damage and antioxidant capacity in the grass carp IECs and explore the Nrf2signaling involved mechanisms;Furthermore,by silencing the Thr metabolism related genes such as Muc2,Muc3,TDH and Gal NT2 to investigate the possible ways by which Thr regulates the structural integrity in the grass carp IECs.Fourth,by establishing the TJs destruction model of grass carp IECs to investigate the effects of Thr on the transmembrane resistance,transmembrane permeability and TJs in the grass carp IECs and explore the PKC signaling involved mechanisms;Furthermore,by silencing the Thr metabolism related genes Muc2,Muc3,TDH and Gal NT2 to explore the possible ways by which Thr regulates the intercellular TJs integrity in the grass carp IECs.The main contents and results were displayed as follows:1.Effect of dietary Thr on the growth performance,digestion and absorption and amino acid metabolism of juvenile grass carp（Ctenopharyngodon idella）Method of Experiment1:1080 healthy juvenile grass carp（9.53±0.02 g）were selected and randomly divided into six treatments and with three replicates for each treatment.Each treatment group fed with different Thr diets（3.99,7.70,10.72,14.10,17.96 and 21.66 g/kg respectively）,and the breeding period was 8 weeks.Main results are displayed as follows:The results of the study showed that,compared with the dietary Thr deficiency group（3.99g/kg）,the optimal dietary Thr level increased the final body weight,percentage of weight gain,specific growth rate,food intake and feed efficiency of juvenile grass carp significantly（P<0.05）.Meanwhile,the optimal dietary Thr level significantly increased the intestinal weight（IW）,intestinal weight index（ISI）,intestinal length（IL）and intestinal length index（ILI）of juvenile grass carp（P<0.05）,and improved the integrity of intestinal morphology structure;increased the moisture,crude protein,crude fat and calcium contents of the whole body,as well as the protein deposition rate,fat deposition rate and ash deposition rate,and decreased the crude ash content in the fish body significantly（P<0.05）.Based on the specific growth rate（SGR）,using the quadratic regression model,dietary Thr requirement of juvenile grass carp（9.53-53.43g）was determined to be14.53g/kg diet（4.48 g Thr/100 g protein）.Meanwhile,the optimal dietary Thr level increased the activities of trypsin,lipase and amylase in the hepatopancreas and intestines,the activities of Na⁺-K⁺-ATPase（NKA）and alkaline phosphatase（AKP）,γ-glutamyl transpeptidase（γ-GT）and creatine kinase（CK）in the proximal intestine（PI）,middle intestine（MI）and distal intestine（DI）of juvenile grass carp significantly（P<0.05）;increased the glutamate-oxaloacetate transaminase（GOT）and glutamate-pyruvate transaminase transaminase（GPT）in the hepatopancreas and muscle,and reduced the plasma ammonia content（PAC）significantly（P<0.05）;up-regulated the threonine dehydrogenase（tdh）and N-acetylgalactosamine transferase 2（galnt2）m RNA levels in the hepatopancreas and of juvenile grass carp significantly（P<0.05）,only upregulated the threonine dehydratase（thal）in the hepatopancreas,but no effect on the m RNA levels of threonine aldolase（tha）（P>0.05）;up-regulated the m RNA levels of mucin 2（Muc2）and mucin 3（Muc3）in the PI,MI and DI of juvenile grass carp significantly（P<0.05）.Above results indicated that the optimal dietary Thr level could improve the growth,digestion and absorption of juvenile grass carp and amino acid metabolism.2.Effect of Thr on the intestinal structural integrity of juvenile grass carp（Ctenopharyngodon idella）Intestinal structure is the basis of intestinal function.Experiment 2 was conducted to explore the effect of dietary Thr on the intestinal structure integrity of aquatic animals systematically.After the growth trial,the juveniles were challenged with Aeromonas hydrophila for 14 days to investigate the effect of dietary Thr on the intestinal cellular structure integrity and intercellular structure integrity of juvenile grass carp.Results showed that the optimal dietary Thr level decreased the contents of reactive oxygen species（ROS）,malondialdehyde（MDA）and protein carbonyl（PC）,activities of anti-superoxide anion free radicals（ASA）,anti-hydroxyl free radicals（AHR）,copper and zinc superoxide dismutase（Cu/Zn-SOD）,glutathione peroxidase（GPX）,catalase（CAT）,glutathione sulfur transferase（GST）and the reduced content of glutathione（GSH）in the PI,MI and DI of juvenile grass carp significantly（P<0.05）,but had no effect on the manganese superoxide dismutase（Mn-SOD）（P>0.05）in the PI,MI and DI of juvenile grass carp.Meanwhile,the optimal dietary Thr could up-regulate the m RNA levels of Cu/Zn-SOD（not Mn-SOD）,CAT,GPX1a,GPX4a,GPX4b,GSTR,and GR in the PI,MI and DI of juvenile grass carp,and the GSTO1 and GSTO2 in the MI and DI（not PI）of juvenile grass carp significantly（P<0.05）.Furthermore,the optimal dietary Thr level increased the nuclear Nrf2 protein level and down regulated the Keap1a（not Keap1b）m RNA levels in the intestine of juvenile grass carp significantly（P<0.05）.Above results indicated that the optimal dietary Thr level could increase the antioxidant enzymes genes expression partly relate to the Keap1a/Nrf2 signaling to enhance the corresponding activities and antioxidant substance GSH content,thereby maintaining the intestinal structure integrity of juvenile grass carp.Results also showed that the optimal dietary Thr increased the Occludin,scaffold protein（zonula occludens 1/2,ZO-1/2）,and ion channel proteins Claudin-b,Claudin-c,Claudin-3,Claudin-7a,Claudin-7b and Claudin-12 m RNA levels in the PI,MI and DI of juvenile grass carp significantly（P<0.05）,but did not affect the Claudin-15b（P>0.05）;down-regulated the Claudin-15a m RNA levels in the PI（not MI and DI）significantly（P<0.05）.Furthermore,the optimal dietary Thr level enhanced the membranes protein levels of PKC（α,β2,ε,δ）in the PI,MI and DI of juvenile grass carp significantly（P<0.05）,but didn’t influence the PKC（β1,γ,θ,η,ζ,μ,ι）protein levels（P>0.05）.Above results indicated that the optimal dietary Thr level could regulate the tight junction protein genes transcripts partly by activating PKC（α,β2,ε,δ）subtypes signaling selectively,thereby maintaining the intestinal structure integrity of juvenile grass carp.3.Mechanism of Thr regulating the IECs cellular structural integrity of grass carpThe results of the experiment 2 in vivo showed that the optimal dietary Thr level could maintain the integrity of the intestinal cell structure in juvenile grass carp.However,it is still unclear whether it is regulated by related signal pathways and how to regulate it by Thr.Therefore,based on the oxidative damage model of IECs established by H₂O₂induction,the experiment 3 was carried out to investigate the effect of Thr on antioxidation in the IECs of grass carp.The experiment 3 consists of three parts.3.1 Effect of Thr on the IECs cellular structural integrity of grass carpPart 1was designed with seven groups as:Ctrl（Thr:0m M,H₂O₂:0μM）,hydrogen peroxide induction group:H₂O₂（Thr:0m M,H₂O₂:100μM）and graded level of Thr treatment for 48h after H₂O₂induction for 12h:T_0.5,T_1.0,T_1.5,T_2.0and T_2.5（Thr:0.5,1.0,1.5,2.0 and 2.5m M）.Each treatment was designed with six replicates.Result showed that,compared with the Ctrl group,the H₂O₂group increased the MDA and PC content,the activities of CAT and GPX,and decreased the activities of T-SOD and GR,the GSH content significantly（P<0.05）;up-regulated the m RNA levels of Cu/Zn-SOD,Mn-SOD,GPX1a,GPX1b,GPX4a,GPX4b and GR（P<0.05）.Compared with the H₂O₂group,2.0m M Thr decreased the MDA and PC contents,increased the activities of T-SOD,CAT,GPX and GR,GSH content,and up-regulated the Cu/Zn-SOD,CAT,GPX1a,GPX4a,GPX4b and GR m RNA levels significantly（P<0.05）,but did not alter the Mn-SOD and GPX1b（P>0.05）.Above results indicated that Thr could enhance the antioxidant capacity of grass carp IECs to decrease the lipid peroxidation and protein oxidation caused by hydrogen peroxide,thus maintaining the structure integrity of grass carp IECs.3.2 The signal mechanism of Thr on the regulation of cellular structural integrity in the IECs of grass carpPart 2 aimed to explore the signal regulation mechanism of Thr on the structural integrity of grass carp IECs.Five treatment groups were designed as:the blank control group:Ctrl（Thr:0m M,H₂O₂:0μM）,hydrogen peroxide induction group:H₂O₂（Thr:0m M,H₂O₂:100μM）,optimal Thr level supplementation for 48h after H₂O₂induction for 12h:Thr（Thr:2.0m M,H₂O₂:100μM）,Nrf2 inhibitor（ML385）treated medium without Thr for48h after H₂O₂induction for 12h:ML385（Thr:0m M,ML385:10μM）and Nrf2 inhibitor treated medium with Thr for 48h after H₂O₂induction for 12h:ML385+Thr（Thr:2.0m M,ML385:10μM）.Each treatment was designed with six replicates.The Nrf2 inhibitor（ML385）could inhibit the decreased MDA and PC,increased GSH contents,T-SOD,CAT,GPX and GR activities,and up-regulated m RNA levels of Cu/Zn-SOD,GPX1a,GPX4a,GPX4b and GR by Thr significantly（P<0.05）.Above results indicated that Thr could enhance the genes transcription of antioxidant enzymes and corresponding enzymatic activities by Nrf2 signaling activation,and the non-enzymatic antioxidant GSH content to improve the antioxidation,thereby maintaining the integrity of grass carp IECs.3.3 The modulated ways by which Thr regulated the signaling of cellular structural integrity in the IECs of grass carpPart 3 aimed to further explore the signal action mode of Thr on regulating the cell structural integrity of grass carp IECs.Total four of the in vitro trials were included in this part,and each was designed with five treatment groups as:blank control group:Ctrl（Thr:0m M,H₂O₂:0μM）,hydrogen peroxide induction group:H₂O₂（Thr:0m M,H₂O₂:100μM）,optimal Thr level supplementation for 48h after H₂O₂induction for 12h:Thr（Thr:2.0m M,H₂O₂:100μM）,si RNA transfection in medium without Thr after H₂O₂induction for 12h:si RNA（Thr:0m M,si RNA:10n M）and si RNA transfection in medium with Thr after H₂O₂induction for 12h:si RNA+Thr（Thr:2.0m M,si RNA:10n M）.Each treatment was designed with six replicates.Result showed that:Firstly,si Muc2/3 transfection could inhibit the Thr decreased MDA and PC contents,increased GSH content,activities of T-SOD,CAT,GPX and GR,and up-regulated m RNA levels of Cu/Zn-SOD,CAT,GPX1a,GPX4a,GPX4b and GR significantly（P<0.05）.Moreover,si Muc3（not Muc2）transfection could inhibit the nucleus Nrf2 protein levels enhanced by Thr（P>0.05）.Combined with the results of part 2,Nrf2 inhibitor（ML385）could not inhibit the m RNA upregulation of Muc2/3 by Thr（P>0.05）.Above results indicated that Muc3（not Muc2）mediated the improvement of antioxidation by Thr in the Nrf2-dependent way in the grass carp IECs.Secondly,si Gal NT2/TDH transfection inhibited the decreased MDA and PC and increased GSH contents by Thr significantly（P<0.05）.si Gal NT2 transfection could inhibit the increased activities of T-SOD,CAT,GPX and GR,GSH content,and the up-regulated m RNA levels of Cu/Zn-SOD,CAT,GPX1a,GPX4a,GPX4b and GR by Thr significantly（P<0.05）.However,si TDH just inhibited the increased GR activity and GSH content,the up-regulated GPX1a and GR m RNA levels by Thr significantly（P<0.05）,but not inhibited the enhanced activities of T-SOD,CAT and GPX,and the up-regulated m RNA levels of Cu/Zn-SOD,CAT,GPX4a and GPX4b by Thr（P>0.05）.Furthermore,si Gal NT2（not TDH）transfection could inhibit the enhanced nucleus Nrf2 level by Thr significantly（P<0.05）.Above results indicated that not only the Thr catabolism participated by TDH was involved in the improvement of the antioxidant status by Thr in the Nrf2-independent way,but also the Mucin anabolism participated by Gal NT2 was involved in the enhancement of the antioxidative ability by Thr in the Nrf2-dependent way,thus maintaining the cellular structure integrity in the grass carp IECs.4.Mechanism of Thr regulating the intercellular tight junctions（TJs）structural integrity in the IECs of grass carpThe results of the experiment 2 in vivo showed that the optimal dietary Thr could maintain the intestinal structural integrity of juvenile grass carp.However,it is still unclear whether it is regulated by related signals and how to regulate it by Thr.Therefore,based on the tight junction destruction model of grass carp IECs established by hydrogen peroxide,the experiment 4 aimed to investigate the effect of Thr on tight junction complex of grass carp IECs.The experiment 4 is divided into four parts.4.1 Effect of Thr on the intercellular TJs structural integrity of grass carp IECsPart 1 aimed to explore the effect of threonine on the TJs structural integrity of grass carp primary IECs.Seven groups were designed as:blank control group:Ctrl（Thr:0m M,H₂O₂:0μM）,hydrogen peroxide induction group:H₂O₂（Thr:0m M,H₂O₂:100μM）and graded level of Thr treated 6-48h after H₂O₂induction for 4h:T_0.5,T_1.0,T_1.5,T_2.0and T_2.5（Thr:0.5,1.0,1.5,2.0 and 2.5m M）.Each treatment was designed with six replicates.Result showed that,compared with the Ctrl group,the H₂O₂group reduced the transmembrane resistance（TER）significantly（P<0.05）and increased the influx rate of FITC-dextran（4KD）（P<0.05）,but did not cause oxidative damage（changes in MDA and PC content）and cell viability（MTT）alteration（P>0.05）,thus establishing the tight junction destruction model in grass carp IECs successfully.After H₂O₂induction for 4h,compared with the H₂O₂group,2.0 m M Thr could increase the TER and decrease the influx rate of FITC-dextran（4KD）significantly（P<0.05）at t≥36h.Meanwhile,compared with the Ctrl group,H₂O₂group could down-regulated the TJ proteins Occludin,scaffold protein ZO-1,ion channel protein Claudin-b,Claudin-c and Claudin-2,and up-regulated Claudin-12,-7a,-7b,-15a and-15b m RNA levels significantly（P<0.05）.Moreover,compared with the H₂O₂group,2.0 m M Thr up-regulated the Occludin,ZO-1,Claudin-b and Claudin-c,and down-regulated the Claudin-7a,-7b and-15a m RNA levels significantly（P<0.05）,but had no effect on Claudin-2,-12 and-15b（P>0.05）.Above results indicated that Thr could promote the TJs assembly by differentially modulating the of TJs protein genes expression,thereby maintaining the intercellular structural integrity of grass carp IECs.4.2 The signal mechanism of Thr on the regulation of intercellular TJs structural integrity of grass carp IECsPart 2 aimed to explore the signal mechanism of Thr on the intercellular TJs structural integrity of grass carp IECs.Five treatment groups was designed as:blank control group:Ctrl（Thr:0m M,H₂O₂:0μM）,hydrogen peroxide induction group:H₂O₂（Thr:0m M,H₂O₂:100μM）,optimal Thr level supplementation for 48h after H₂O₂induction for 4h:Thr（Thr:2.0m M,H₂O₂:100μM）,PKC inhibitor（LY333531）treated medium without Thr for 48h after H₂O₂induction for 4h:LY333531（Thr:0m M,LY333531:1μM）and PKC inhibitor（LY333531）treated medium with Thr for 48h after H₂O₂induction for 4h:LY333531+Thr（Thr:2.0m M,LY333531:1μM）.Each treatment was designed with six replicates.Result showed that LY333531 could inhibit the Thr up-regulated Occludin,ZO-1 and Claudin-c m RNA levels significantly（P<0.05）but did not inhibit the up-regulated Claudin-b,-7a,-7b and-15a（P>0.05）.Above results indicated that Thr could regulate TJ proteins genes expression by activating the PKC signaling,thereby maintaining the intercellular TJs structural integrity of grass carp IECs.Part 3 aimed to further explore by which PKC isotype Thr regulating the TJs structural integrity of grass carp IECs.Part 3 included four in vitro trials.Each trial was designed with five treatment groups as:blank control group Ctrl:（Thr:0m M,H₂O₂:0μM）,the hydrogen peroxide induction group H₂O₂（Thr:0m M,H₂O₂:100μM）,optimal Thr level supplementation for 48h after H₂O₂induction for 4h:Thr（Thr:2.0m M,H₂O₂:100μM）.si RNA transfection in medium without Thr after H₂O₂induction for 4h:si RNA（Thr:0m M,si RNA:10n M）and si RNA transfection in medium with Thr after H₂O₂induction for 4h:si RNA+Thr（Thr:2.0m M,si RNA:10n M）.Each treatment was designed with six replicates.Result showed that,si PKCα（not PKCβ2）transfection could inhibit the upregulated Occludin and ZO-1 significantly（P<0.05）but not Claudin-c m RNA levels by Thr（P>0.05）.Meanwhile,si PKCε/δtransfection could inhibit the up-regulated m RNA levels of Occludin,ZO-1 and Claudin-c by Thr significantly（P<0.05）.Above results indicated that Thr could selectively regulate the TJ proteins expression by PKC（α,ε,δ）activation,thereby maintaining the intercellular TJs structural integrity of grass carp IECs.4.3 The modulated ways by which Thr regulated the signaling of intercellular TJs structural integrity of grass carp IECsTo explore the action mode by which Thr modulated the PKC signaling to regulate intercellular TJs structural integrity of grass carp IECs,the part 4 was designed with four in vitro trials.Each trial of the part 4 was designed with five treatment groups as:blank control groups:Ctrl（Thr:0m M,H₂O₂:0μM）,hydrogen peroxide induction group:H₂O₂（Thr:0m M）,H₂O₂:100μM),optimal Thr level supplementation for 48h after H₂O₂induction for 4h:Thr（Thr:2.0m M,H₂O₂:100μM）,si RNA transfection in medium without Thr after H₂O₂induction for 4h:si RNA（Thr:0m M,si RNA:10n M）and si RNA transfection in medium with Thr after H₂O₂induction for 4h:si RNA+Thr（Thr:2.0m M,si RNA:10n M）.Each treatment group was designed with 6 replicates.Results showed that:Firstly,si Muc2/3 transfection could inhibit the up-regulated Occludin,ZO-1 and Claudin-c m RNA levels by Thr significantly（P<0.05）.However,si Muc3（not Muc2）could inhibit the down-regulated Claudin-7a,-7b and-15a m RNA levels by Thr（P<0.05）.Meanwhile,LY333531 could inhibit the upregulated Muc2/3 m RNA levels significantly by Thr（P<0.05）.Above results indicated that Muc3（not Muc2）mediated the TJ proteins regulation by Thr in the PKC（α,ε,δ）-dependent way in the grass carp IECs.Secondly,si Gal NT2（not TDH）transfection inhibited the up-regulated Occludin,ZO-1,Claudin-b and Claudin-c and down-regulated Claudin-7a,-7b and-15a m RNA levels by Thr significantly（P<0.05）.Furthermore,si Gal NT2（not TDH）transfection could inhibit the enhanced PKCα,-εand-δprotein levels by Thr（P>0.05）.Above results indicated that,the Gal NT2 participated in the Mucin anabolism metabolism but not TDH participated in the Thr catabolism mediated the TJ proteins regulation by Thr in the PKC（α,ε,δ）-dependent way in the grass carp IECs.In summary,the optimal dietary Thr level could promote the growth of juvenile grass carp（Ctenopharyngodon idella）,which was related to its enhanced intestinal function including the digestion and absorption,amino acid metabolism and Mucin synthesis;The improved the intestinal function was related to the enhancement of the intestinal antioxidant system and the strengthening of the TJs,thereby maintaining the intestinal structural integrity of juvenile grass carp;Not only the Thr catabolism participated by TDH was involved in the improvement of the antioxidant status by Thr in the Nrf2-independent way,but also the Muc3（not Muc2）anabolism participated by Gal NT2 was involved in the enhancement of the antioxidative ability by Thr in the Nrf2-dependent way in grass carp IECs.The Gal NT2 participated in the Muc3（not Muc2）anabolism metabolism but not TDH participated in the Thr catabolism mediated the TJ proteins regulation by Thr in the PKC（α,ε,δ）-dependent way in grass carp IECs.Based on the GSH in the PI,the optimal Thr level of for enhancing the intestinal health of juvenile grass carp was determined to be15.41g/kg diet（4.76g Thr/100g protein）,which is little higher than that based on SGR（14.53g/kg diet,4.48g Thr/100g protein）.