Effect Of Taurine On TLR4 Pathway In LPS-induced IPEC-J2 Cells

Objective: Previous studies of our group found that taurine may inhibit inflammatory overactivation,protect intestinal mucosal epithelial cells and prevent diarrhea of piglets by acting on relevant targets of TLR4 signaling pathway.However,the specific target of taurine is not clear.In this study,taurine is used to pretreat piglet jejunum epithelial cell line IPEC-J2 cells,add LPS to the medium to induce the release of inflammatory factors,and detect the protein expression levels of key factors in TLR4 signal transduction pathway by Western-blot.Combined with immunofluorescence and pathway inhibitors,the post-action target of taurine on TLR4 receptor is identified.To reveal the inhibitory mechanism of taurine on inflammatory hyperactivation and provide theoretical basis for the use of taurine as a nutritional immune additive to prevent diarrhea in piglets.Methods: IPEC-J2 cells are cultured in vitro,and four groups are set up,including blank control group(C group,1640 medium),taurine control group(T group,20 m M taurine+1640medium),LPS treatment group(L group,10 (?)g/ m L LPS+1640 medium),LPS+ taurine group(LT group,10 (?)g/ m L LPS+20 m M taurine +1640 medium).After the addition of inhibitors,LPS+NF-κB inhibitor group(L1 group,L group +10μmol/ L NF-κB inhibitor)LPS+NF-κB inhibitor + taurine group(LT1 group,LT group +10μmol/ L NF-κB inhibitor).The effect of taurine on the levels of inflammatory cytokines in the supernatant of cell culture is detect by ELISA.The effect of taurine on MyD88 dependent NF-κB Is detect by Q-PCR,Western-blot and immunofluorescence.Influence and localization of MAPK and MyD88 signaling pathway related genes and proteins.Results: 1.Compared with group C,IFN-β levels in L group are significantly higher than those in blank control group(p<0.001),the IFN-β level in LT group is significantly lower than that in L group(p<0.001),IFN-β levels are significantly higher in the taurine control group than in the control group(p<0.01).2.The relative mRNA expressions of p65,ERK,JNK,p38 and IRF3 genes in L group are significantly higher than those in C group(p<0.001),compared with L group,the gene expression levels in LT group are reduce to varying degrees.3.Compared with group C,the average fluorescence intensity of p-p65,p-JNK,p-ERK1/2,p-p38 and p-IRF3 in group L is significantly increased(p<0.001),while the average fluorescence intensity in LT group is significantly lower than that in L group(p<0.001).p-ERK1/2 is significantly lower than that in L group(p<0.01),there is no significant difference between p-p65,p-JNK,p-ERK1/2,p-p38,p-IRF3.C group and T group(p(?)0.05).In addition,the fluorescence trace of L group is mainly distributed in the nucleus,indicating that LPS could induce nuclear translocation of p-p65,p-JNK,p-ERK1/2,p-p38 and p-IRF3.4.Compared with group C,the relative protein expression of p65 in group L is significantly increased(p<0.01),p-p65,p-JNK,p-ERK1/2,p-p38 and p-IRF3 in L group are significantly increased(p<0.001),ERK1/2,IRF3 increased(p<0.05),there is no significant difference in JNK and p38,while the relative protein expression of p65 in LT group is significantly lower than that in L group(p<0.01),p-p65,p-JNK,p-ERK1/2,p-p38,p-IRF3 are significantly lower than those of L group(p<0.001),ERK1/2 is lower than that of L group(p<0.05).The relative protein expression levels of JNK,p38 and IRF3 in LT group are not significantly different from those in L group.Conclusion: Taurine inhibited the activation of LPS/TLR4 pathway through MyD88 dependent NF-κB and MAPK signaling pathways and MyD88 non-dependent signaling pathways,and have a certain protective effect on LPS induced cell damage.