Effects Of β-conglycinin On Apoptosis In Grass Carp Enterocytes And The Involved Mechanism

This study first investigated the effects of dietaryβ-conglycinin on growth,intestinal development and intestinal apoptosis in juvenile grass carp（Ctenopharyngodon idella）,and explored the potential mechanisms ofβ-conglycinin-induced apoptosis among different intestinal segments.Second,the in vitro digestion trial was executed to digestβ-conglycinin,and determined theβ-conglycinin digestive products in triggering grass carp enterocyte apoptosis.Third,to explore the relationship betweenβ-conglycinin digestive products caused-apoptosis and the cytoskeleton,this study found out a new cytoskeleton F-actin binding protein from the Saccharomyces,and explored the“upstream”mechanism ofβ-conglycinin digestive products-caused apoptosis in grass carp enterocyte.This study further explored the“downstream”mechanism ofβ-conglycinin digestive products-caused apoptosis in grass carp enterocyte by adding the p38MAPK phosphorylation inhibitor.Fourth,this study investigated the protective effect and mechanism of glutamine（Gln）inβ-conglycinin digestive products-caused apoptosis in the grass carp enterocyte.The main contents and results are presented as follow:1.The effect of dietaryβ-conglycinin on growth performance,intestinal development and apoptosis of juvenile grass carpA total of 240 grass carp,with an average initial weight of 13.77±0.10 g,were randomly distributed into two groups with three replicates resulting in 40 fish per replicate.Fish were fed with the control diet（devoid of soybean protein）orβ-conglycinin diet（containing 80 gβ-conglycinin per kg diet）for 7 weeks,respectively.The results are presented as follow:1.1 The effect of dietaryβ-conglycinin on growth performance and intestinal development index of juvenile grass carpCompared with control,dietaryβ-conglycinin decreased the percent weight gain（PWG）,specific growth ratio（SGR）,feed intake（FI）and feed efficiency（FE）,intestinal weight（IW）,intestinal length（IL）and intestinal weight index（ISI）,the folds height in the proximal intestine（PI）,mid intestine（MI）and distal intestine（DI）（P<0.05）in the juvenile grass carp.These results suggest that dietaryβ-conglycinin suppression fish growth might be partly related to depress intestinal development.1.2 The effect and possible mechanisms of dietaryβ-conglycinin on apoptosis among different intestinal segments of juvenile grass carpIn the PI,in comparison to control,dietaryβ-conglycinin did not cause DNA fragmentation,had no significant effect on caspase-3,caspase-8 and caspase-9 activities,fas ligand（FasL）,apoptotic protease-activating factor-1（Apaf1）,Bcl-2 associated X protein（BAX）,B cell lymphoma 2（BCL-2）,myeloid cell leukaemia-1（MCL-1）,inhibitors of apoptosis（IAP）,caspase-2,-3,-7,-8,-9,Jun N-terminal kinase（JNK）,and p38mitogen-activated protein kinase（p38 MAPK）mRNA levels,total-and phospho-p38 MAPK protein expression levels（P>0.05）,tended to decrease ROS content（P=0.09）,and decreased NADPH oxidase（NOX）activity,NADPH oxidase 1（NOX1）,NADPH oxidase 2（NOX2）,dual oxidase（DUOX）,p22^PHOX（PHOX=phagocytic oxidase）（P22）,p47^PHOX（PHOX=phagocytic oxidase）（P47）,p67^PHOX（PHOX=phagocytic oxidase）（P67）,NADPH oxidase organizer 1a（NOXO1a）,NADPH oxidase organizer 1b（NOXO1b）and dual oxidase maturation factor（DUOXA）mRNA relative expression levels（P<0.05）.These data suggested that dietaryβ-conglycinin did not cause apoptosis in the PI of grass carp,which may be related to its’none effect on ROS content and thus could not activate the apoptotic signal pathways.In the MI,compared with control,dietaryβ-conglycinin caused DNA fragmentation,and increased the 180bp DNA band content,elevated the caspase-3,caspase-8 and caspase-9activities（P<0.05）,upregulated Apaf1,BAX,MCL-1,caspase-3,caspase-9,JNK and p38MAPK mRNA expression levels（P<0.05）,tended to increase IAP mRNA expression level（P=0.09）,did not change caspase-2,caspase-7,FasL,TNF-αand BCL-2 mRNA levels（P>0.05）,down-regulate caspase-8 mRNA level（P<0.05）,increased total-and phospho-p38MAPK protein expression levels（P<0.05）,had a tendency to increase ROS content（P=0.07）,raised NOX2,P47,NOXa1 and NOXO1b mRNA relative expression levels（P<0.05）,while did not change NOX1,NOX4,DUOX,P67,NOXO1a and DUOXA mRNA levels（P>0.05）.All the above data indicated that dietaryβ-conglycinin caused apoptosis in the MI of grass carp,which might be associated with the activation of p38MAPK and then increased the apoptotic effector caspase activity.In the DI,compared with control,dietaryβ-conglycinin caused DNA fragmentation,and increased the 180bp DNA band content,elevated the activities of caspase-3,caspase-8 and caspase-9（P<0.05）,decreased the mRNA levels of Apaf1,BAX,MCL-1,FasL,BCL-2,IAP and JNK（P<0.05）,tended to decrease caspase-2 mRNA level（P=0.08）,increased the mRNA levels of TNF-α,caspase-3,-7,-8 and p38 MAPK（P<0.05）,significantly increased total-and phospho-p38 MAPK protein expression levels,ROS content,NOX activity,NOX2,P22,P47,NOXa1 and NOXO1b mRNA levels（P<0.05）,tended to increase DUOX mRNA level（P=0.06）,did not change NOX1,NOX4,NOXO1a and DUOXA mRNA levels（P>0.05）.All these results suggested that dietaryβ-conglycinin might through increase TNF-αand caspase-8;increasing NOX-related molecules mRNA levels and enzyme activities to increase ROS content and thus activate p38MAPK signalling pathway,resulting in the activation of caspase which caused the apoptosis in the DI of grass carp.2.Effects ofβ-conglycinin digestion products on the apoptosis in grass carp enterocyteThe results in the animal trial showed thatβ-conglycinin triggered apoptosis in the grass carp intestine,but there are some differences among different intestinal segments.This might be associated with the difference ofβ-conglycinin digestive products existed in different intestinal segments.In order to determine theβ-conglycinin digestive products in apoptosis,researches need to be conducted:The in vitro digestion ofβ-conglycinin trial was conducted to simulate the grass carp in vivo digestion in the trial one including:extracting the digestive enzyme from the intestine,determining the ratio of enzyme and substrate.To compareβ-conglycinin digestion difference between the control group fish andβ-conglycinin group fish in the trial one,the in vitro digestion trial was designed two groups,the control group fish digestive enzyme digestβ-conglycinin（CON-BC）and theβ-conglycinin group fish digestive enzyme digestβ-conglycinin（BC-BC）.Theβ-conglycinin was digested by the two enzymes for 0.5h,1.5h,2.5h,3.5h,5.5h and 7.5h,then the sample were collected to SDS-PAGE analysis.Theβ-conglycinin digestive products from BC-BC group were purified by ultrafiltration according the molecule size,and then were used to treat the grass carp intestine enterocyte to find theβ-conglycinin digestive products in apoptosis.Seven products（includingβ-conglycinin and its 6 digestive products:0.5h,1.5h,2.5h,3.5h,5.5h and 7.5h digestion）were used to evaluate their effect in apoptosis of grass carp enterocyte.For each product,five group were designed,and three replicates for each group.Group 1 was control group,from group 2 to 5 added 1,2,4 and 8 mg/mL of the seven products to cell culture,respectively.Enterocyte were cultured in the media for 48h then treated with the seven products for another 24h,respectively.Cells were collected to analyse the DNA fragmentation.The results showed that theβ-conglycinin cannot be completely digested in both CON-BC and BC-BC group.In the CON-BC groups,theα’-andα-subunit ofβ-conglycinin were hydrolysed to about 60kDa and 50kDa polypeptides need about 0.5 and 1.5 hours,respectively.However,in the BC-BC group,the two subunits need 1.5 and 5.5 hours to be hydrolysed to the same products as CON-BC group.Theβ-subunit ofβ-conglycinin cannot be hydrolysed until 7.5 hours in both CON-BC and BC-BC groups.Among these products,only the 5.5-and 7.5-hoursβ-conglycinin hydrolysis products can induce DNA fragmentation in the grass carp enterocyte.The above results indicated thatβ-conglycinin is indigestible in the grass carp intestine enzyme,especially theβ-subunit.The grass carp fedβ-conglycinin for 7weeks was more incompetent to digestβ-conglycinin than that of the control group.Finally,the 5.5-and 7.5-hoursβ-conglycinin hydrolysis products are the effector in the apoptosis of grass carp enterocyte.3.The effect ofβ-conglycinin hydrolysis products on cytoskeleton-mediated apoptosis in in grass carp enterocyteThe results of trial one found that dietaryβ-conglycinin could activation mitochondrial apoptotic pathway and regulation p38MAPK phosphorylation which are the downstream of apoptosis signaling.The in vitro digestion trial detected theβ-conglycinin digestive products in apoptosis of grass carp enterocyte.Study in eukaryotes found that the cytoskeleton could as the upstream siginaling to trigger apoptosis.To further explore the relationship between β-conglycinin digestive products and cytoskeleton and p38MAPK in apoptosis,the cytoskeleton detecting probe for grass carp enterocyte should be constructed,and the p38MAPK phosphorylation inhibitor should be added to determine it effect.Thus,we conducted two parts researches:3.1 The construction of cytoskeleton F-actin detecting probeA new cytoskeleton F-actin binding protein was found from the Saccharomyces Genomic Database（SGD）.After the localization pattern examination and in vitro proteins binding assay,the F-actin binding protein was tagged with green fluorescent protein（GFP）as a probe to detect the cytoskeleton F-actin.Finally,this probe was transfected into the grass carp enterocyte by lipofection to detect its cytoskeleton.The results showed that the cable domain from Ecm25 C-terminus is a potential protein binding with cytoskeleton F-actin.After the localization pattern and living cell imaging assays in budding yease,as well as the F-actin directly binding assay in vitro,the Ecm25 cable domain was determined to be a cytoskeleton F-actin detecting probe.This probe can be transfected and expressed in grass carp enterocyte,and its localization is same as the cytoskeleton F-actin.This result indicating that the cytoskeleton F-actin detecting probe for grass carp enterocyte was conscructed successfully,which provided the technology support for the further study.3.2.The mechanism ofβ-conglycinin digestive products in grass carp enterocyte apoptosisFour groups were designed in this trial:control group,control+p38MAPK phosphorylation inhibitor group,treatment group（add 8mg/mLβ-conglycinin 7.5h digestive product[7S-7.5hD]）,treatment+p38MAPK phosphorylation inhibitor group（add 8mg/mL7S-7.5hD and p38MAPK phosphorylation inhibitor）,three replicates per group.Enterocyte were cultured in the media for 24h then transfected by F-actin detection probe,24h culture latter added the p38MAPK inhibitor,incubate 2h,then add 8mg/mL 7S-7.5hD for 24h.The results showed that the localization of grass carp enterocyte cytoskeleton F-actin is polarized in the control group;after 7S-7.5hD stress,the cytoskeleton F-actinpolymerization and localized depolarized（distributed randomly around the cytomembrane）;add the p38MAPK phosphorylation inhibitor alone did not affect the localization pattern of cytoskeleton F-actin;after adding both p38MAPK phosphorylation inhibitor and7S-7.5hD,the cell phenotype is normal,the localization and distribution of cytoskeleton F-actin is similar as control group.In addition,compared with control group,adding 7S-7.5hD alone increased the fragment DNA（180bp）and ROS contents,T-p38 MAPK and P-p38 MAPK proteins levels;caspase-3,-8,-9 activities（P<0.05）.Compared with treatment group（added 7S-7.5hD alone）,adding both p38MAPK phosphorylation inhibitor and 7S-7.5hD tended to decrease fragment DNA（180bp）content and T-p38 MAPK protein level（0.05<P<0.1）;decreased P-p38 MAPK protein level,caspase-3,-8,-9 activities（P<0.05）.All the above results indicated that7S-7.5hD might through affect cytoskeleton F-actin to increase ROS content and then activated p38 MAPK signal,increased the caspase activities,finally caused the apoptosis in grass carp enterocyte.4.The effect of Gln inβ-conglycinin digestive products induced apoptosis of grass carp enterocyteFor study the protective effect of glutamine（Gln）againstβ-conglycinin digestive products induced apoptosis,two researches need to be conducted.First,checking whether Gln could protect grass carp enterocyte againstβ-conglycinin digestive products induced apoptosis,and determining its concentration.Second,after the Gln concentration determined,invesgitated the possible mechanism of Gln protecting grass carp enterocyte againstβ-conglycinin digestive products induced apoptosis.4.1 Determining the effect and concentration of Gln inβ-conglycinin digestive products induced apoptosis in grass carp enterocyteThe present trial was designed 6 groups:control group,7S-7.5hD treatment group（add8mg/mLβ-conglycinin 7.5h digestive product）,and four groups 7S-7.5hD plus graded levels of Gln（0.7,1.4,2.8 and 5.6 mM）,three replicates per group.Enterocytes were cultured in the media for 24h,then add different contents of Gln to culture another 24 hours,add 8mg/mL7S-7.5hD for 24h culture.Collecting cells for DNA fragmentation analysis.The results showed that the DNA fragments gradually decreased with the increased Gln concentration compared with 7S-7.5hD treatment group.The DNA fragments are no difference when the Gln concentration is 2.8mM.When the Gln concentration is 5.6mM,the DNA fragments are same with control.These data indicated that Gln can protect grass carp enterocyte against 7S-7.5hD induced apoptosis,and the optimum concentration is 5.6 mM.4.2 The effect of Gln inβ-conglycinin digestive products induced apoptosis in grass carp enterocyteThree groups were designed:control group,7S-7.5hD treatment group（add 8mg/mLβ-conglycinin 7.5h digestive product）,7S-7.5hD treatment+Gln group（add 8mg/mL7S-7.5hD and 5.6mM Gln）,three replicates per group.Enterocytes were cultured in the media for 24h,then transfected by F-actin detection probe,add 5.6mM Gln culture another 24h,add8mg/mL 7S-7.5hD for 24h culture.The data showed that after 7S-7.5hD stress,the cytoskeleton F-actin localized depolarized and distributed randomly around the cytomembrane.Preculture grass carp enterocytes with Gln 24h,the 7S-7.5hD partly affect the localization of cytoskeleton F-actin.These results suggest that Gln might through protecting cytoskeleton to protect the grass carp enterocyte against 7S-7.5hD induced apoptosis.Collectively,dietaryβ-conglycinin could suppress fish growth,which might be partly related to its role in inducing intestine apoptosis.Dietaryβ-conglycinin triggered apoptosis in the MI and DI rather than in the PI,but the mechanism of apoptosis is different between MI and DI.Dietaryβ-conglycinin mainly through mitochondrial apoptotic pathway to trigger apoptosis in the MI,and through both mitochondrial apoptotic pathway and death receptor apoptotic pathway to cause apoptosis in the DI,both pathways are regulated by the p38MAPK signaling in the MI and DI.The in vitro digestion trial and the cell culture trial found that the effector in triggering apoptosis areβ-conglycinin 5.5h and 7.5h digestive products.The further study found that theβ-conglycinin 7.5h digestive product might through changing cytoskeleton F-actin to increase the ROS content and then activate p38MAPK signal to increase caspase activity and finally caused the apoptosis in the grass carp enterocyte.Besides,Gln might through protecting cytoskeleton to protect the grass carp enterocyte againstβ-conglycinin digestive product trggered apoptosis.