The Mechanism Of Porcine Small Intestinal Epithelial Cells Damage Induced By Soybean Antigen Protein

In order to further explore the mechanism of intestinal sensitization induced by soybean antigen protein in piglets,small intestinal epithelial cells of piglets were cultured in vitro.After cells were stimulated with different concentrations(0,1,5,10mg/mL)of glycinin(11S)and ?-conglycinin(7S)for 24 h,changes in cellular growth and morphology were observed by an inverted microscopy and a transmission electron microscopy.Cellular viability were determined by cell counting kit-8.The related protein expression and the related gene mRNA expression levels of MAPKs and NF-?B signal transduction pathways were determined by western blotting(WB)and Real-time PCR(qRT-PCR).The contents of serotonin(5-HT),NO,IL-6,IL-10 and diamine oxidase(DAO)in the cell supernatant were determined by ELISA kit.The results were as follows:Effect of 11 S or 7S on IPEC-J2 morphology:(i)In control group,IPEC-J2 presented irregular polygons with clear outlines and adherent growth.The cells began to swell with the concentration of 11 S or 7S increased and the cells with unclear outlines gradually increased.The total number of cells gradually decreased and the number of exfoliated cells gradually increased as concentrations of 11 S or 7S increased.Finally,the apoptotic bodies appeared.The nuclear of IPEC-J2 with the concentration of 11 S or 7S increased tended to shrink and nucleoli gradually disappeared.Chromatin in the nuclear gradually gathered to the nuclear membrane and disappeared.(ii)Compared with IPEC-J2 in 5 mg/mL 11 S or 7S group,IPEC-J2 presented irregular polygons with more clear outlines,more adherent growth,more complete nuclear and the chromatin distribution was more uniform after PDTC,L-NAME,SP600125 and SB202190 inhibitors were added.IPEC-J2 viability:(i)The cell viability decreases gradually with the concentration of 11 S or 7S increased.Compared with the control group,the survival rate of cell in 1mg/mL 11 S or 7S group had no significant difference(P> 0.05).However,the survival rate of cell was extremely significantly lower than the control group(P< 0.01)when the concentration of 11 S or 7S reached 5mg/mL or more.(ii)Compared with the 5 mg/mL 11 S or 7S group,cell viabilities were increased in all four inhibitor groups.Changes of 5-HT,diamine oxidase,IL-6,IL-10 and NO levels:(i)Compared with the control group,contents of 5-HT,DAO,IL-6,IL-10 and NO in IPEC-J2 in 1 mg/mL 11 S or 7S group were not significantly different(P> 0.05).Contents of 5-HT,DAO,IL-6 and NO were significantly increased(P <0.01)when the concentrations of 11 S and 7S reached more than 5mg/mL and the content of IL-10 was significantly decreased(P < 0.01).(ii)Compared with those in 5 mg/mL 11 S or 7S group,the contents of 5-HT,DAO,IL-6 and NO were increased in all four inhibitors groups,however,IL-10 content was significantly decreased(P<0.01).Related protein and gene expression levels of NF-?B signaling pathway:(i)p-NF-?B p65,iNOS,COX-2 protein expression and NF-?B p65,iNOS,COX-2,IKK? and IKK? mRNA relative expression levels gradually increased with 11 S concentration increased.Compared with those in 5mg/mL 11 S group,p-NF-?B p65,iNOS,COX-2 protein expression and NF-?B p65,IKK?,IKK? and iNOS mRNA relative expression levels in PDTC + 11S(5mg/mL)group and L-NAME + 11S(5mg/mL)group were decreased.(ii)p-NF-?B p65,iNOS protein expression and NF-?B p65,iNOS,IKK?,IKK?,COX-2 mRNA relative expression levels gradually increased with 7S concentration increased.Compared with those in 5mg/mL 7S group,p-NF-?B p65 protein expression and NF-?B p65,IKK?,IKK? mRNA related expression levels were decreased in PDTC + 7S(5mg/mL)group,however,iNOS protein expression and iNOS,COX-2 mRNA related expression levels had no significant difference(P>0.05).Compared with those in 5mg/mL 7S group,p-NF-?B p65,iNOS protein expression and NF-?B p65,iNOS,IKK?,IKK?,COX-2 mRNA related expression levels were decreased in L-NAME+ 7S(5mg/mL)group.Related protein and gene expression levels of MAPKs signaling pathway:(i)p-JNK,p-p38 protein expression and Bad,P53,Caspase-3,Bax mRNA relative expression levels gradually increased with 11 S concentration increased,however,Bcl-2 protein expression and Bcl-xl,Bcl-2 mRNA relative expression levels gradually decreased with 11 S concentration increased.Compared with those in 5mg/mL 11 S group,p-JNK,p-p38 protein expression and P53,Caspase-3,Bax mRNA relative expression levels decreased,however,Bcl-2 protein expression and Bcl-xl,Bcl-2 mRNA relative expression levels increased.(ii)p-JNK,p-p38 protein expression and Bad,P53,Caspase-3,Bax mRNA relative expression levels gradually increased with 7S concentration increased,however,Bcl-2 protein expression and Bcl-2,Bcl-xl mRNA relative expression levels gradually decreased with 11 S concentration increased.(iii)Compared with those in 5mg/mL 7S group,p-JNK,p-p38 protein expression significantly decreased(P<0.01)and Bcl-2 protein expression increased in SP600125+7S(5mg/mL)group and SB202190+7S(5mg/mL)group.Bad,Caspase-3,Bax mRNA relative expression levels decreased in SP600125+7S(5mg/mL)group and SB202190+7S(5mg/mL)compared with those in 5mg/mL 7S group.However,Bcl-xl,Bcl-2 mRNA relative expression levels increased(P<0.05)and P53 mRNA relative expression levels in SP600125+7S(5mg/mL)group compared with those in 5mg/ml 7S group.Bcl-xl,Bcl-2,P53 mRNA relative expression levels in SB202190+7S(5mg/mL)group had no significant difference(P>0.05).In conclusion,11 S and 7S can induce cellular apoptosis through MAPKs signaling pathway and induce cellular inflammatory damage through NF-?B signaling pathway.11 S and 7S can cause allergic damage in intestinal epithelial cells of piglets.