Study On The Effect And Mechanism Of Enterococcus Faecium HDRsEf1 On LPS-Induced Down-Regulation Of Tight Junction Protein ZO-1 Expression

The intestinal epithelial barrier is the main defense mechanism against bacterial infections and inflammation,and is essential for the health of the gastrointestinal tract of humans and animals.Tight junctions are the important part of the intestinal barrier structure and play a decisive role in maintaining the integrity of the barrier.The probiotic Enterococcus faecium can maintain intestinal stability and improve the host's intestinal health.Although the protective effect of Enterococcus faecium on the intestinal barrier has been reported,its molecular mechanism is extremely complex,especially the mechanism of regulating tight junction proteins is currently unclear.In this study,IPEC-J2 cells with tight junction injury induced by LPS were used as a model to explore the regulation and mechanism of the probiotic Enterococcus faecium HDRs Ef1 on the expression of tight junction protein ZO-1,and provide a theory for probiotics to regulate the expression of tight junction protein in the intestine Foundation,to provide technical support for probiotics to prevent and treat animal gastrointestinal diseases.The main contents and results are as follows:1.The effect of LPS and HDRs Ef1 on the expression of ZO-1 in IPEC-J2 cells(1)The effect of LPS on the expression of ZO-1 in IPEC-J2 cells.A model of LPS-induced intestinal epithelial cell injury was established,and the effect of LPS on the expression of ZO-1 m RNA was detected by q RT-PCR to determine the optimal concentration and time of LPS.The results showed that LPS significantly down-regulated the expression of ZO-1,with a certain concentration-dependent.(2)The effect of HDRs Ef1 on the expression of ZO-1 in IPEC-J2 cells.The CCK8method was used to detect whether HDRs Ef1 has cytotoxic effect on IPEC-J2 cells.The results showed that 10~7CFU/m L HDRs Ef1 live bacteria and IPEC-J2 cells did not produce cytotoxicity within 8 hours.The effect of HDRs Ef1 on the expression level of ZO-1 was analyzed by q RT-PCR and Western blot.The results showed that HDRs Ef1could alleviate the down-regulation of ZO-1 expression by LPS.Cellular immunofluorescence test was used to detect the effect of HDRs Ef1 and LPS on the structure and morphology of ZO-1.The results showed that HDRs Ef1 could inhibit the damage of LPS to the structure of ZO-1.Therefore,HDRs Ef1 could inhibit the destructive effect of LPS on the expression and structure of ZO-1.2.Study on the mechanism of HDRs Ef1 on LPS-induced down-regulation of ZO-1(1)The effect of transcription factor AP-1 on the regulation of ZO-1 expression.The q RT-PCR method was used to detect the effects of HDRs Ef1 and LPS on transcription factors,and to determine the transcription factors involved in regulation.The results showed that AP-1 might be involved in the regulation of ZO-1 expression.The effect of AP-1 on the regulation of ZO-1 was analyzed by gene overexpression test and RNA interference test.The results showed that AP-1 overexpression could inhibit the expression of ZO-1,and AP-1 gene interference could increase the expression of ZO-1.Therefore,AP-1 could participate in the regulation of ZO-1 expression.(2)The mechanism of JNK/MKK7/ASK1 pathway on HDRs Ef1 alleviating LPS down-regulation of ZO-1 expression.Western blot was used to detect the upstream JNK activation of AP-1.The results showed that HDRs Ef1 could inhibit LPS-induced JNK phosphorylation.By adding JNK inhibitor(SP600125)and gene interference test to detect whether HDRs Ef1 regulated ZO-1 expression through this pathway,the results showed that after JNK pathway was blocked,ZO-1 expression increased,and alleviated LPS down-regulates the expression of ZO-1.Therefore,HDRs Ef1 stabilizeed the expression of ZO-1 by inhibiting the JNK pathway,and HDRs Ef1 alleviated the upregulation of AP-1 expression by LPS,which also depended on the JNK pathway.Western blot and gene interference tests were used to detect whether JNK upstream factors are involved in the regulation of HDRs Ef1 on ZO-1.The results showed that HDRs Ef1 could inhibit LPS-induced phosphorylation of MKK7 and ASK1.After the interference of MKK7 and ASK1 genes,the expression of ZO-1 was up-regulated,and could significantly inhibit LPS from down-regulating the expression of ZO-1.Therefore,HDRs Ef1 inhibited LPS-induced down-regulation of ZO-1 by inhibiting the JNK/MKK7/ASK1 pathway.(3)Study on the mechanism of TLRs on HDRs Ef1 alleviating LPS down-regulation of ZO-1 expression.The q RT-PCR method was used to detect whether TLRs play a role in the regulation of ZO-1 expression by HDRs Ef1.The results showed that after LPS treatment,TLR2 expression was significantly down-regulated,while TLR4 expression was significantly up-regulated;after HDRs Ef1 treatment,TLR2 and TLR4 expression were both significant up-regulation,and can alleviated the effect of LPS down-regulating TLR2.The effect of TLR2/4 was analyzed by RNA interference test,and the results showed that after TLR2 and TLR4 gene interference,it could inhibit HDRs Ef1 and alleviated the effect of LPS down-regulating ZO-1.Therefore,HDRs Ef1 inhibited LPS-induced down-regulation of ZO-1 through the TLR2/4 pathway.(4)Study on the mechanism of TNF-?on HDRs Ef1 alleviating LPS down-regulation of ZO-1 expression.The q RT-PCR method was used to detect the effect of HDRs Ef1 on inflammatory factors,and the results showed that HDRs Ef1 could inhibit the up-regulation of inflammatory factors induced by LPS.The influence of inflammatory factors stimulated by IPEC-J2 cells on the expression of ZO-1 was detected by q RT-PCR and Western blot.The results showed that IL-6 and TNF-?could down-regulate the expression of ZO-1,but HDRs Ef1 only relieveed the down-regulation of TNF-?.RNA interference test was conducted to detect whether HDRs Ef1 inhibited LPS-induced TNF-?expression through TLR4.The results showed that HDRs Ef1 could alleviate LPS-induced TNF-?production by inhibiting TLR4.Therefore,HDRs Ef1 inhibited TNF-?-induced down-regulation of ZO-1 through TLR4.Conclusion:HDRs Ef1 could inhibit LPS down-regulating the expression of ZO-1and stabilize the ZO-1 grid structure.HDRs Ef1 signaled through TLR2/4,thereby inhibiting the LPS-induced ASK1/MKK7/JNK/AP-1 cascade to stabilize the expression of ZO-1.In addition,HDRs Ef1 could inhibit the down-regulation of ZO-1 induced by TNF-?,and might alleviate the production of TNF-?induced by LPS by inhibiting TLR4.