Effects Of Leucine On TGEV Infection Of IPEC-J2 Cells And Intestinal Health Through Regulating MTOR Activity

Transmissible gastroenteritis(TGE)is an acute and highly contagious intestinal infectious disease caused by transmissible gastroenteritis virus(TGEV).In recent years,TGE has a severe epidemic form in China,seriously affecting the pig industry.Our previous study found that TGEV could significantly inhibit mTOR signaling pathway.Leucine(Leu),as one of the effective amino acids to activate mTOR signaling pathway,can alleviate the infection of TGEV.By investigating the effect of mTOR on TGEV infection and intestinal health,our study aims to reveal the interaction between mTOR and STAT1,explore the mechanisms of TGEV successfully infecting the host and Leu alleviating the virus infection,and provide a new theoretical basis for the prevention and treatment of transmissible gastroenteritis.This study consists of four experiments.Exp.1 Effects of mTOR inhibition on intestinal health and immune function in SD ratsWe used SD rats as the in vivo study model to investigate the effect of rapamycin(RAPA)on the growth performance,intestinal health,and immune function of SD rats.By investigate the effect of mTOR on the expression of STAT1 and interferon regulated genes(ISGS),the relationship between mTOR activity and immune regulation was preliminarily revealed.A total of 12 germ-free 5-week SD rats were randomly assigned to two treatments(n=6),which were intraperitoneally injected with 5% DMSO and 5 mg/kg BW rapamycin solution.Rats were fed with basic diet for 18 days.The results showed that RAPA significantly reduced the average body weight(P<0.01),small intestine weight(P<0.01),the height of jejunum villi and VCR(P<0.01),and significantly increased the content of serum free amino acids of SD rats(P<0.01).Among them,leucine and arginine increased the most(P<0.01).RAPA significantly reduced the expression of occludin,claudin-1,mucin1 and ZO-1 in jejunum(P<0.05).RAPA significantly reduced the gene expression of NF-κB in jejunum and inhibited the expression of IL-1β,IL-6,IL-10,TGF-β and TNF-α(P<0.01).RAPA significantly promoted the expression of STAT1,downstream ISG56,Mx A and OASL(P<0.01),but had no effect on PKR.Exp.2 Effect of L-leucine on TGEV infection and its molecular mechanism by regulating mTOR activityIn this study,IPEC-J2 cells were used as the in vitro study model,which was divided into three parts.By investigating the effect of mTOR activity on TGEV-infected IPEC-J2 cells,the mechanism of TGEV infection was elucidated.The ability of mTOR activity to regulate the resistance of IPEC-J2 cells to TGEV and the molecular mechanism of leucine alleviating TGEV infection were revealed.Firstly,1000 U/ml IFN-β was added into IPEC-J2 cells for 1 h as a positive control after IPEC-J2 cells were infected with TGEV for 48 h to investigate the effect of TGEV infection on the expression of genes related to mTOR and type I interferon signaling pathway.The results showed that TGEV could significantly inhibit the expression of mTOR,STAT1,downstream ISG15,ISG56,PKR and Mx A(P<0.01).Then we used a2×2 factor design.IPEC-J2 cells were pretreated with 10 n M mTOR-specific inhibitor Rapamycin or 10 μM activator MHY1485 for 30 min to detect TGEV replication,mTOR,STAT1 and ISGs expression.The results showed that The pretreatment of IPEC-J2 cells infected by TGEV with rapamycin can inhibit the expression of p-STAT1,downstream ISG15,ISG56,PKR and Mx A(P<0.01),thereby promoting the replication of virus.On the contrary,the pretreatment of MHY1485 promoted the expression of p-STAT1,downstream ISG15,ISG56,PKR and Mx A(P<0.01),thereby inhibiting the replication of virus.On this basis,a 2×2 factor design was used.Cells were starved for 3 h and then treated with 10 m M L-leucine for 30 min to detect the gene and protein expression of TGEV replication,mTOR,STAT1 and ISGs.The results showed that 10 m M leucine significantly promoted the expression of p-STAT1 and downstream ISG15,ISG56,PKR and Mx A(P<0.01),thereby inhibiting the replication of TGEV.In conclusion,mTOR plays an important role in promoting intestinal health and regulating antiviral innate immune function.The ability of IPEC-J2 cells to resist virus can be changed by regulating mTOR activity.Leu can promote the expression of STAT1 and ISGs by activating mTOR signaling pathway,to play a crucial part in alleviating TGEV infection.