Effect And Mechanism Of Sodium Butyrate On Apoptosis Of Intestinal Epithelial Cells Induced By Rotavirus Infection

Rotavirus(RV)is one of the main pathogenic factors of piglet viral diarrhea.It mainly infects mature intestinal epithelial cells of piglets,leading to cell apoptosis,shedding,intestinal villi atrophy,and thus disturbing malabsorption and severing diarrhea.Sodium butyrate(SB)is a kind of short chain fatty acid compounds.Recently,some reports have shown that SB played an pivotal role in antiviral.The aim of this study was to investigate whether SB could alleviate the intestinal epithelial cells apoptosis which was mediated by endoplasmic reticulum stress(ERS)with RV infection through short chain fatty acid receptor GPR109 A.Experiment 1 RV infection induced ERS-mediated apoptosis of IPEC-J2 cellsTo investigate the effects of RV(OSU strain)infection on apoptosis in piglets jejunal epithelial cells(IPEC-J2).The effects of RV infection on apoptosis in IPEC-J2 cells were investigated by measuring cell activity,DNA fragmentation,cell apoptosis rate,the ERS-related pathway gene and protein expression levels.The results showed that RV infection induced a dose-dependent decreasing IPEC-J2 cell activity compared with the control group(P < 0.05).Under 10 MOI RV infection,RV significantly increased DNA fragmentation and apoptosis rate of IPEC-J2 cells.The m RNA levels of PKR-like ER kinase(PERK),78-k Da glucose-regulated protein(GRP78),C/EBP homologous protein(CHOP),caspase9 and caspase3 were up-regulated in cells with RV infection(P < 0.05).In conclusion,these results indicated that RV could induced IPEC-J2 cells apoptosis.Meanwhile,in order to determine the apoptosis pathway in IPEC-J2 cells with RV infection,the expression of ERS-induced apoptosis-related genes and proteins were measured by adding PERK inhibitor GSK.The results shown that GSK could significantly:increased IPEC-J2 cell activity(P < 0.05);decreased m RNA levels of Bcl-2 by partial blocking of RV;increased caspase9 and caspase3 m RNA levels(P < 0.05);reduced PERK,EIF2α phosphorylation levels and caspase9,caspase3 protein levels(P < 0.05).Experiment 2 Effect of SB on apoptosis of IPEC-J2 cells induced by RV infectionTo investigate the protective effects of SB against RV-induced IPEC-J2 cell apoptosis.Firstly,the IPEC-J2 cells were pretreated with different concentrations of SB.We measured the concentration range of SB tolerance in IPEC-J2 cells for 24 h.The results indicated that SB at a concentration below 8 m M had no toxic effect on the cells.Meanwhile,cell activity,apoptosis rate,ERS-related pathway genes and protein levels were measured after SB preconditioning with RV infection.The results shown that 4 m M SB significantly increased the activity of IPEC-J2(P < 0.05);reduced the apoptosis rate with RV infection(P < 0.05).Compared with RV treatment alone,pretreatment of IPEC-J2 with 4 m M SB significantly reduced PERK,GRP78,CHOP,caspase9,and caspase3 m RNA levels(P < 0.05).These results suggest that SB inhibits the expression of caspase9 and caspase3 by decreasing the expressions of ERS-related molecules(PERK,GRP78,and CHOP)to protect IPEC-J2 cells from RV-induced apoptosis.Experiment 3 Mechanism of SB alleviating apoptosis of IPEC-J2 cells induced by RV infectionTo investigate whether SB inhibited ERS-induced apoptosis by GPR109 A with RV infection.In this study,si RNA interference was used to knock down the expression of GPR109 A.The apoptosis rate,ERS-related pathway genes and protein expression levels were detected.The results shown that 4 m M SB significantly inhibited the decrease of GPR109 A m RNA level(P < 0.05).The apoptosis rate,PERK,EIF2α,activation of transcription factors-4(ATF4),GRP78,CHOP,caspase9,and caspase3 m RNA levels in GPR109 A knockdown treatment up-regulated but down-regulated the expression of Bcl-2significantly compared with that in RV treatment.Furthermore,knockdown CPR109 A increased the phosphorylation levels of PERK and EIF2α proteins and the abundances of caspase9 and caspase3 fragments of proteins with RV infection.The results showed that afer silencing GPR109 A,SB was unable to played a protective role in IPEC-J2 against RV-induced apoptosis.These results further confirmed that SB protected IPEC-J2 against RV-induced ERS-mediated apoptosis.In summary,this study confirmed that IPEC-J2 cells infected with RV induced apoptosis via PERK/EIF2α signaling pathway.SB might alleviate the apoptosis of IPEC-J2 cells induced by RV to a certain extent,and its mechanism is to up-regulate the expression of short-chain fatty acid receptor GPR109 A,thus inhibiting the ERS-induced apoptosis signaling pathway.