| Erucic acid(EA),an anti-nutritional factor naturally present in rapeseed meal,could inhibit animal growth.While most studies about the effect of EA have been restricted to terrestrial animal,and the information on aquatic animal is lacking.First of all,this study investigated the growth performance,apparent nutrient digestibility,intestinal structure,serum diamine oxidase activity,serum D-lactic acid content,intestinal tight junction,intestinal adherins junction,intestinal oxidative damage,intestinal apoptosis and related signaling molecules of on-growing grass carp(Ctenopharyngodon idellus)and evaluated its maximum tolerance levels of EA.Second,we investigated the effect of EA on the activity of signaling molecule RhoA and the expression of tight junction and adherins junction-related genes and proteins of on-growing grass carp intestine by an in vitro experiment.Third,combined in vivo with in vitro experiment,we investigated the effect of EA on the enterocyte ultrastructure and the expression of ER stressrelated genes and proteins of grass carp intestine.Fourth,we investigated the effect of ER stressmediated unfolded protein response(UPR)signaling pathways(PERK/eIF2α,IRE1/XBP1 and ATF6)on EA-disrupted intercellular structural integrity of grass carp intestine by in vivo experiments.The main contents and results are presented as follow:1.The effect of EA on the growth performance and intestinal structural integrity of ongrowing grass carp540 healthy fish(average initial weight 129.17 ± 0.19 g)were randomly divided into 18 experimental cages(length,width and height were all measured to be 1.4 m)with 3 cages being assigned to each treatment(n=6 treatments)and 30 fish per cage.The actual EA contents of six different diets were 0.00%,0.29%,0.60%,0.88%,1.21% and 1.50% diet,respectively.During the 60 days-growth experiment,fish obtained their respective EA diets 4 times a day to visual satiation.The main results are presented as follow:1.1 The effect of EA on the growth performance and intestinal histology of on-growing grass carp1)Incremental EA level in the diet linearly(P < 0.01)and quadratically(P < 0.05)decreased FBW,PWG,SGR,FI,FE,IW and ISI,linearly(P < 0.01)decreased IL,ILI and the apparent digestibility of dry matter,crude protein and crude lipid of on-growing grass carp,linearly(P < 0.01)and quadratically(P < 0.05)increased the residue of EA in PI and serum Dlactic acid content,and linearly(P < 0.01)increased the residues of EA in MI and DI and the activity of serum diamine oxidase;2)intestinal hyperemia of fish fed diet with EA level up to0.88% or higher diet was detected,and fish fed diets with EA levels up to 0.88% or higher diet had hyperplasia of intestine villi;3)Broken-line regression analysis demonstrated that the maximum tolerance levels of EA for on-growing grass carp(129.17–471.18 g)for 60 days based on PWG,MDA in PI,ROS in MI and PC in DI were estimated to be 0.64%,0.48%,0.48%and 0.53%.These results showed that EA(≥ 0.64%)could inhibit the growth performance and disrupt intestinal structure of on-growing grass carp.1.2 The effect of EA on the cellular structural integrity of on-growing grass carp intestine1)A linear(P < 0.01)and quadratic(P < 0.05)increase in ROS,MDA and PC was verified with incremental EA level in on-growing grass carp intestine.Incremental EA level in the diet linearly(P < 0.01)and quadratically(P < 0.05)decreased the activities of AHR,Mn SOD,GPX and GST in the PI,decreased the activities of AHR,ASA,Mn SOD,CAT,GST and GR in the MI,and decreased the activities of Mn SOD,GPX,GST and GR,and the content of GSH in the DI of on-growing grass carp.The activities of ASA,CAT and GR in PI,and the activity of GPX in MI,and the activities of AHR,ASA and CAT in DI,and the contents of GSH in PI and MI of on-growing grass carp decreased in a linear fashion with incremental EA levels(P < 0.01);2)A linear reduction in the mRNA levels of Mn SOD,CAT,GPX1 a,GPX1b,GPX4 a,GPX4b,GSTP1,GSTO1,GSTO2,GR,Nrf2,IAP and Mcl-1,and the protein levels of cytosolic and nuclear Nrf2(P < 0.01)and a linear increase in the mRNA levels of keap1 a,caspase-2,-7,-8,-9,Apaf-1,Bax,Fas L and p38MAPK(P < 0.01)in on-growing grass carp intestine with incremental EA level;3)DNA fragmentation of on-growing grass carp intestine was detected when EA level up to 0.88% or higher diet.Interestingly,dietary EA had no impact on Cu Zn SOD activity,and the mRNA levels of Cu Zn SOD,GSTP2,keap1 b and JNK of on-growing grass carp intestine(P > 0.05).Meanwhile,incremental EA level had a linear increase response on caspase-3 mRNA levels in PI and MI(Rather than DI)(P < 0.01),a linear decrease response X on Bcl-2 mRNA level in MI(rather than PI and DI)(P < 0.01)of on-growing grass carp.These results showed that EA might inhibit the Nrf2 signaling pathway and activate the p38 MAPK signaling pathway to disrupt the cellular structural integrity of on-growing grass carp intestine.1.3 The effect of EA on the intercellular structural integrity of on-growing grass carp intestine1)Incremental EA level linearly(P < 0.01)decreased the mRNA levels of tight junction protein ZO-1,ZO-2,occludin,claudin-b,-c,-f,-3c,-7a,-7b,-11 and adherins junction α-catenin,β-catenin,E-cadherin,nectin,afadin,and the protein levels of E-cadherin,β-catenin,ZO-1 and linearly(P < 0.01)increased the mRNA levels of claudin-15 a and signaling molecules NMII,ROCK and MLCK;2)Incremental EA level linearly(P < 0.01)increase the ratio of GTP-RhoA protein level to Total-RhoA protein level in on-growing grass carp intestine.Interestingly,claudin-12 and claudin-15 b mRNA levels in on-growing grass carp intestine were not affected by dietary EA(P > 0.05).These results showed that EA might activate RhoA signaling pathway to disrupt the intercellular structural integrity of on-growing grass carp intestine.2.EA activated the RhoA signaling pathway to disrupt intercellular structural integrity of grass carp enterocyteThe results of trial 1 showed that EA might activate RhoA signaling pathway to disrupt the intercellular structural integrity of on-growing grass carp intestine.In order to further verify whether EA activates this signaling pathway to disrupt the intercellular structural integrity of grass carp intestine,we investigated the effect of EA on the intercellular structural integrity of grass carp by an in vitro experiment.The in vitro experiment included two parts: Part 1 designed6 EA levels(0,1,2,3,4 and 5 mmol/L)with 6 replicates per EA level.The enterocytes were treated by EA for 24 h to achieve an appropriate EA level.The results showed that 3 mmol/L or higher EA levels could increase the activity of lactate dehydrogenase(LDH)of supernatant of grass carp enterocytes and the MDA content of grass carp enterocyte,increase the mRNA level of ion channel protein-related gene claudin-15 a and decrease the mRNA levels of tightjunction-related genes ZO-1,occludin,claudin-b,JAM,E-cadherin,α-catenin and β-catenin(P< 0.05),indicating that 3 mmol/L or higher EA levels could efficiently disrupt the intercellular structural integrity of grass carp enterocyte.Part 2 designed 4 treatments: control group,RhoA inhibitor group(Rhosin hydrochloride+)、EA group(EA+)and EA+RhoA inhibitor group(EA+Rhosin hydrochloride+),with 6 replicates per treatment,to verify whether EA could activate the RhoA signaling pathway to disrupt the intercellular structural integrity of grass carp intestine.The main results are presented as follow: 1)compared to control group,EA decreased the mRNA levels of tight junction-related genes ZO-1,occludin,claudin-7a and claudin-7b(P< 0.05),the mRNA levels of adherins junction-related genes nectin,E-cadherin and β-catenin(P < 0.05),and the protein levels of ZO-1,E-cadherin and β-catenin(P < 0.05)of grass carp enterocyte.Meanwhile,EA increased the mRNA levels of ion channel protein-related gene claudin-15 a and the signaling molecule MLCK,NMII and ROCK(P < 0.05)of grass carp enterocyte.While RhoA inhibitor(Rhosin hydrochloride)could increase the mRNA levels of ZO-1,occludin,claudin-7a,claudin-7b,nectin and E-cadherin(P < 0.05)decreased by EA,and increased the protein levels of ZO-1,E-cadherin and β-catenin(P < 0.05)decreased by EA.Meanwhile,RhoA inhibitor(Rhosin hydrochloride)decreased the mRNA levels of claudin-15 a,MLCK,NMII and ROCK(P < 0.05)increased by EA.RhoA inhibitor(Rhosin hydrochloride)has the trend to increase the mRNA level of β-catenin(P = 0.072)decreased by EA;2)compared to control group,EA increased the ratio of GTP-RhoA protein level to Total-RhoA protein level of enterocyte(P < 0.05),while RhoA inhibitor(Rhosin hydrochloride)decreased the ratio of GTP-RhoA protein level to Total-RhoA protein level(P < 0.05)increased by EA.These results showed that EA could activate the RhoA signaling pathway to disrupt the intercellular structural integrity of grass carp enterocyte.3.The effect of ER stress on EA-disrupted intercellular structural integrity of grass carp intestine3.1 EA induced ER stress in on-growing grass carp intestineThe present study explored whether EA could induce ER stress in on-growing grass carp intestine by investigating the effect of EA on the ultrastructure of enterocyte and the expression of ER stress-related indicators.The main results are presented as follow: 1)EA induced the occurrence of swelling of endoplasmic reticulum and mitochondria in the intestine of ongrowing grass carp;2)Incremental EA level linearly(P < 0.01)increased the ER-stress related marker GRP78,CHOP,eIF2α and XBP1 mRNA levels in on-growing grass carp intestine;3)Incremental EA level linearly(P < 0.01)increased the ER-stress related proteins GRP78,p- PERK,p-IRE1 and ATF6 protein levels in on-growing grass carp intestine.These results showed that EA could induce ER stress in the intestine of on-growing grass carp.3.2 The effect of ER stress on EA-disrupted intercellular structural integrity of grass carp enterocyteThe results of trial 3.1 showed that EA could induce ER stress in the intestine of ongrowing grass carp.In order to explore whether the occurrence of ER stress induced by EA was participated in EA-disrupted intercellular structural integrity of grass carp intestine,we investigated the effect of ER stress on EA-disrupted intercellular structural integrity of grass carp enterocyte.The experiment designed 4 groups: control group,ER stress inhibitor group(4-PBA+),EA group(EA+)and EA+ER stress inhibitor group(EA+4-PBA+),with 6 replicates per treatment.The main results are presented as follow: 1)compared to control group,EA induced the occurrence of swelling of endoplasmic reticulum and mitochondria in grass carp enterocyte,while ER stress inhibitor(4-PBA)could relieve the pathological symptoms in grass carp enterocyte caused by EA;2)compared to control group,EA increased the mRNA levels of GRP78,CHOP,eIF2α and XBP1 of grass carp enterocyte(P < 0.05),while ER stress inhibitor(4-PBA)could decrease the mRNA levels of GRP78,CHOP,eIF2α and XBP1(P <0.05)increased by EA;3)compared to control group,EA increased the protein levels of GRP78,p-PERK,p-IRE1 and ATF6 of grass carp enterocyte(P < 0.05),while ER stress inhibitor(4-PBA)decreased the protein levels of GRP78,p-PERK,p-IRE1 and ATF6(P < 0.05)increased by EA;4)compared to control group,EA decreased the mRNA levels of tight junction-related genes ZO-1,occludin,claudin-7a and claudin-7b(P < 0.05),the mRNA levels of adherins junction-related genes nectin,E-cadherin and β-catenin(P < 0.05),and the protein levels of ZO-1,E-cadherin and β-catenin of grass carp enterocyte(P < 0.05).Meanwhile,EA increased the mRNA levels of ion channel protein-related gene claudin-15 a and the signaling moleculesrelated genes MLCK,NMII and ROCK(P < 0.05).While ER stress inhibitor(4-PBA)increased the mRNA levels of ZO-1,occludin,nectin and E-cadherin(P < 0.05)decreased by EA,and increased the protein levels of ZO-1,E-cadherin and β-catenin(P < 0.05)decreased by EA.ER stress inhibitor(4-PBA)also decreased the mRNA levels of claudin-15 a,MLCK,NMII and ROCK(P < 0.05)increased by EA.Meanwhile,ER stress inhibitor(4-PBA)has the trend to increase the mRNA levels of claudin-7a(P = 0.053),claudin-7b(P = 0.055)and β-catenin(P= 0.087)decreased by EA;5)compared to control group,EA increased the ratio of GTP-RhoA protein level to Total-RhoA protein level of grass carp enterocyte(P < 0.05),while ER stress inhibitor(4-PBA)decreased the ratio of GTP-RhoA protein level to Total-RhoA protein level(P < 0.05)increased by EA.These results showed that ER stress induced by EA is involved in the process of EA-disrupted intercellular structural integrity of grass carp intestine.4.The effect of ER stress-mediated UPR on EA-disrupted intercellular structural integrity of grass carp intestine4.1 The effect of PERK/ eIF2α signaling pathway on EA-disrupted intercellular structural integrity of grass carp enterocyteIn order to explore the effect of PERK/eIF2α signaling pathway on EA-disrupted structural integrity of grass carp intestine,we investigated the effect of EA on the expression of tight junction and adherins junction-related protein,p-PERK and eIF2α.The main results are presented as follow: 1)compared to control group,EA decreased the mRNA levels of tight junction-related genes ZO-1,occludin,claudin-7a and claudin-7b(P < 0.05),and the mRNA levels of adherins junction-related genes nectin,E-cadherin and β-catenin(P < 0.05),and the protein levels of ZO-1,E-cadherin and β-catenin(P < 0.05)of grass carp enterocyte.Meanwhile,EA increased the mRNA levels of claudin-15 a,MLCK,NMII and ROCK in grass carp enterocyte(P < 0.05).While PERK inhibitor(GSK2656157)increased the mRNA levels of ZO-1,claudin-7a and claudin-7b(P < 0.05)decreased by EA,increased the protein levels of ZO-1,E-cadherin and β-catenin(P < 0.05)decreased by EA,and decreased the mRNA levels of claudin-15 a,MLCK,NMII and ROCK(P < 0.05)increased by EA.Meanwhile,PERK inhibitor(GSK2656157)had the trend to increase the mRNA level of nectin(P = 0.067)decreased by EA;2)compared to control group,EA increased the ratio of GTP-RhoA protein level to Total-RhoA protein level of grass carp enterocyte(P < 0.05),while PERK inhibitor(GSK2656157)decreased the ratio of GTP-RhoA protein level to Total-RhoA protein level(P< 0.05)increased by EA;3)compared to control group,EA increased the protein level of pPERK and mRNA level of eIF2α of grass carp enterocyte(P < 0.05),while PERK inhibitor(GSK2656157)decreased the protein level of p-PERK and mRNA level of eIF2α(P < 0.05)increased by EA.These results showed that PERK/ eIF2α signaling pathway was participated in the process of EA-disrupted intercellular structural integrity of grass carp intestine.4.2 The effect of IRE1/ XBP1 signaling pathway on EA-disrupted intercellular structural integrity of grass carp intestineIn order to explore the effect of IRE1/XBP1 signaling pathway on EA-disrupted structural integrity of grass carp intestine,we investigated the effect of EA on the expression of tight junction and adherins junction-related proteins,p-IRE1 and XBP1.The main results are presented as follow: 1)compared to control group,EA decreased the mRNA levels of tight junction-related genes ZO-1,occludin,claudin-7a and claudin-7b(P < 0.05),the mRNA levels of adherins junction-related genes nectin,E-cadherin and β-catenin(P < 0.05),and the protein levels of ZO-1,E-cadherin and β-catenin(P < 0.05)of grass carp enterocyte.Meanwhile,EA increased the mRNA levels of ion channel protein-related gene claudin-15 a and the signaling molecules MLCK,NMII and ROCK of grass carp enterocyte(P < 0.05).While,IRE1 inhibitor(STF-083010)increased the mRNA levels of ZO-1 and nectin(P < 0.05)decreased by EA,increased the protein levels of ZO-1,E-cadherin and β-catenin(P < 0.05)decreased by EA.Meanwhile,IRE1 inhibitor(STF-083010)decreased the mRNA levels of claudin-15 a,MLCK,NMII and ROCK(P < 0.05)increased by EA,and EA had the trend to increase the mRNA levels of occludin(P = 0.097),claudin-7a(P = 0.071),claudin-7b(P = 0.078)and E-cadherin(P =0.082)decreased by EA;2)compared to control group,EA increased the ratio of GTP-RhoA protein level to Total-RhoA protein level of grass carp enterocyte(P < 0.05),whileIRE1inhibitor(STF-083010)decreased the ratio of GTP-RhoA protein level to Total-RhoA protein level increased by EA;3)compared to control group,EA increased the protein level of p-IRE1 and mRNA level of XBP1 of grass carp enterocyte(P < 0.05),whileIRE1 inhibitor(STF-083010)decreased the protein level of p-IRE1 and mRNA level of XBP1(P < 0.05)increased by EA.These results showed that IRE1/ XBP1 signaling pathway was also participated in the process of EA-disrupted intercellular structural integrity of grass carp intestine.4.3 The effect of ATF6 signaling pathway on EA-disrupted intercellular structural integrity of grass carp intestineIn order to explore the effect of ATF6 signaling pathway on EA-disrupted structural integrity of grass carp intestine,we investigated the effect of EA on the expression of tight junction and adherins junction-related proteins and ATF6.The main results are presented as follow: 1)compared to control group,EA decreased the mRNA levels of tight junction-related genes ZO-1,occludin,claudin-7a and claudin-7b(P < 0.05),the mRNA levels of adherins junction-related genes nectin,E-cadherin and β-catenin(P < 0.05),and the protein levels of ZO-1,E-cadherin and β-catenin(P < 0.05)of grass carp enterocyte.Meanwhile,EA increased the mRNA levels of ion channel protein-related gene claudin-15 a and the signaling molecule MLCK,NMII and ROCK of grass carp enterocyte(P < 0.05).And ATF6 inhibitor(AEBSF)further decreased the mRNA levels of ZO-1,occludin,claudin-7a,claudin-7b,nectin,Ecadherin and β-catenin(P < 0.05)decreased by EA,and the protein levels of ZO-1,E-cadherin and β-catenin(P < 0.05)decreased by EA.Meanwhile,ATF6 inhibitor(AEBSF)further increased the mRNA levels of claudin-15 a,MLCK,NMII and ROCK(P < 0.05)increased by EA;2)compared to control group,EA increased the ratio of GTP-RhoA protein level to TotalRhoA protein level of grass carp enterocyte(P < 0.05),and ATF6 inhibitor(AEBSF)further increased the ratio of GTP-RhoA protein level to Total-RhoA protein level(P < 0.05)increased by EA;3)compared to control group,EA increased the protein level of ATF6 of grass carp enterocyte(P < 0.05),while ATF6 inhibitor(AEBSF)decreased the protein level of ATF6(P <0.05)increased by EA.These results showed that ATF6 signaling pathway was not participated in the process of EA-disrupted intercellular structural integrity of grass carp intestine,to a certain degree,this signaling pathway could attenuate the process of EA-disrupted intercellular structural integrity of grass carp enterocyte.In summary,EA decreased the growth performance of on-growing grass carp,which might be related to the alteration of intestinal structure by EA and the alteration of intestinal structure was related to the disrupted cellular structural integrity and intercellular structural integrity of on-growing grass carp intestine.EA could activate RhoA signaling pathway to disrupt the intercellular structural integrity of grass carp intestine.ER stress was participated in the process of EA-disrupted intercellular structural integrity of grass carp intestine.The ER stress-mediated UPR signalling pathways PERK/eIF2α and IRE1/XBP1,but not ATF6,were participated in the process of EA-disrupted intercellular structural integrity of grass carp intestine. |
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