Molecular Mechanism Of LPS-induced IPEC-J? Cells Expressing Reg??

Reg??,a novel type of intestinal antibacterial protein,plays critical roles in antimicrobe,anti-inflammation,wound healing,and promoting the proliferation of cells in different tissues of the body.It is very important for maintaining the homeostasis of the intestinal microbiota and protecting the integrity of intestinal mucosal barrier.Reg??expression is mediated by the signaling pathway of Myeloid differentiation primary response protein 88(MyD88),and the linker molecule downstream of MyD88 is currently unclear.In order to further study the regulation mechanism of Reg?? expression,the inactivated Escherichia(E.coli)and Lipopolysaccharide(LPS)were used to induce Reg??expression in IPEC-J? cells from piglets.The Reg?? expression level and the phosphorylation levels of signaling molecules downstream of MyD88,including p65,p38,JNK,and ERK were analyzed respectively to explore the molecular mechanism of Reg??expression.The methods and results are as follows:1.IPEC-J? cells were treated with different CFU of inactivate E.coli and different concentration of LPS for 24 h to induce the expression of Reg??.The morphology of IPEC-J? cells were observed and the cell viability was determined by MTT.The results showed that the effect of inactivation of E.coli and LPS on cells is similar.The cell morphology of the low-concentration group did not change significantly,the high-concentration group appeared elongated and translucent,and the cell viability decreased significantly as the concentration increased.2.The mRNA and protein levels of Reg?? were analyzed by Q-PCR and Western blot.The results showed both inactivation of E.coli and LPS increased the expression of Reg??.3.Signaling molecules downstream MyD88,including p38 MAPK kinase(also known as stress-activated protein kinase 2)in the nuclear factor kappa B(NF-?B)and mitogen-activated protein kinase(MAPK)signaling pathway,C-Jun N-terminal kinase(JNK)and Extracellular regulated protein kinases(ERK)were detected using anti-phosphorylated proteins as primary antibodies.The results showed that compared with the control group,the phosphorylation level of ERK protein did not change significantly,and the phosphorylation level of p65,p38 and JNK protein increased significantly.The results show that during the expression of Reg??,the protein phosphorylation levels of the signal molecules p65,p38 and JNK downstream of MyD88 increased significantly,indicating that the NF-?B and MAPK signaling pathways downstream of MyD88 are involved in the regulation of Reg?? protein expression.