| The purpose of this study was to elucidate the mechanism of transmissible gastroenteritis virus(TGEV)on the inflammatory regulation of small intestinal epithelial cells,and to explore whether leucine(Leu)can alleviate the inflammatory response of small intestinal epithelial cells to TGEV infection,and to reveal the regulatory role of Leu in the inflammatory response of small intestinal epithelial cells to TGEV infection.The IPEC-J2 cells were used as the in vitro model and this study included three experiments.In experiment 1,we investigated the effect of TGEV infection on IPEC-J2 cell proliferation,and constructed a TGEV infected IPEC-J2 cell model.To investigate the effect of TGEV on cell proliferation,apoptosis and autophagy,TGEV(MOI=5)was co-cultured with IPEC-J2 cells at different time points(1h,3h,6h,12 h,24h,36h).The results showed:(1)TGEV replication increased with the prolongation of infection time,and the virus replication reached its peak at 24-36 h after TGEV infection.(2)At 36 h post-infection(p.i.),TGEV(MOI=5)significantly inhibited cell proliferation(P<0.01),promoted apoptosis and autophagy(P<0.01).(3)At 36 h post-infection(p.i.),TGEV(MOI=5)significantly activated NF-?B phosphorylation(P<0.05)and inhibited mTOR phosphorylation(P<0.05).In experiment 2,we investigated the effect of TGEV infection of IPEC-J2 cells on inflammatory response and the role of mTOR in inflammatory regulation.To investigate the role of mTOR in the regulation of inflammation,TGEV(MOI=5)co-acted on IPEC-J2 cells cultured for 36 h with 10 nM mTOR inhibitor(Rapamycin)or 10?M mTOR activator(MHY1485).Meanwhile,the TGEV encoding protein IPEC-J2 stable cell line was constructed to screen the TGEV encoding protein inhibiting mTOR,and the relationship between TGEV encoding protein and mTOR was investigated.The results showed:(1)At 36 h post-infection(p.i.),TGEV increased the mRNA expression levels of TLR2,TLR3,TLR9,RIG-I and MDA5(P<0.01),activated NF-?B(P<0.01),and induced the production of cytokines TNF-?,IL-1?,IL-6,IL-8,IFN-? and IFN-?(P<0.01),but significantly inhibited the mRNA expression of IFN-?(P<0.05).(2)The mRNA expression levels of TLR2,TLR3,RIG-I and MDA5 were down-regulated after mTOR inhibition in the early stage of TGEV infection(P<0.01),decreased the NF-?B activity,and then decreased the mRNA expression levels of TNF-?,IL-6 and IL-8(P<0.01).TGEV replication was inhibited in early infection(P<0.01).Upon mTOR activation,it inhibited TGEV replication(P<0.01),upregulated RIG-I and MDA5 expression(P<0.01),increased the mRNA expression level of IFN-?(P<0.01),and decreased the mRNA expression level of NF-?B and IL-8(P<0.01).(3)TGEV encoding proteins NSP4,NSP10,NSP12,ORF3 a,S,ORF7 significantly inhibited mTOR phosphorylation(P<0.05).(4)After NF-?B inhibition,mTOR mRNA expression was promoted(P<0.01)and TGEV mRNA expression was inhibited(P<0.01).In Experiment 3,we investigated the mechanism by which leucine alleviates TGEV-induced inflammatory responses.IPEC-J2 cells were infected with TGEV(MOI=5)and co-cultured for 36 h with different doses of leucine(5mM,10mM).The results showed:(1)10mM leucine could activate mTOR signaling pathway and reduce TGEV replication and autophagy(P<0.05).(2)Under the infection of TGEV,leucine down-regulated the expression of TLR2(P<0.01),inhibited the activity of NF-?B(P<0.01),further reduced the mRNA expression level of TNF-?(P<0.05)and increased the mRNA expression level of IL-10(P<0.05),thereby alleviated the inflammatory response caused by TGEV.In conclusion,TGEV replication could reach the peak at 24-36 h p.i.in IPEC-J2 cells.TGEV promoted NF-?B phosphorylation and up-regulated the expression of inflammatory factors through the TLRs(TLR2,TLR3,TLR9)and RLRs(RIG-I,MDA5)signaling pathways,a response dependent on mTOR.Adding leucine activated mTOR activity,inhibited NF-?B phosphorylation,surpressed TNF-? and IL-10 expression,and ultimately TGEV replication and inflammation could be inhibited. |
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