The Regulation And Mechanism Of Autophagy On Chemokines Expression In Intestinal Epithelial Cells In Inflammatory Bowel Disease

ObjectiveTo investigate the regulation and mechanism of autophagy-related protein p62 and ATG16L1 on chemokines expression in intestinal epithelial cells(IECs)under inflammatory condition.MethodsIntestinal epithelial model in vitro was established by Caco-2 cells.Inflammatory bowel disease(IBD)model in vitro was induced by interleukin-1?(IL-1?).Western blot was used for analysis of protein expression of p62,p65,ATG16L1,microtubule-associated proteins 1 light chain 3(LC3),nuclear factor-?B(NF-?B)and mitogen-activated protein kinase(MAPK).Real-time PCR was used for analysis mRNA expression of p62,interleukin-8(IL-8)and monocyte chemoattractant protein-1(MCP-1).ELISA was used for analysis of IL-8 and MCP-1 secreted by cells.Small interfering RNA(siRNA)of p62,p65 and ATG16L1 were transfected into cells to knockdown target genes respectively.Results 1.The mechanism of IL-1? regulating p62 expression in IECs.IL-1? increased p62 protein without changing expression of LC3B-II indicating that p62 elevation is not caused by inhibition of autophagy.IL-1? increased mRNA level of p62 suggesting that p62 elevation is related to transcription activation.Protein synthesis inhibitor cycloheximide decreased expression of p62 and autophagy inhibitor bafilomycin significantly accumulated p62 in cells after IL-1? stimulation.These results show that p62 elevation is due to increased transcription and protein synthesis.Knockdown of p65,a subunit of NF-?B,inhibited expression of p62 induced by IL-1? indicating that NF-?B regulates p62 expression 2.The mechanism of p62 regulating chemokines production in IECs.NF-?B and MAPKs were activated by IL-1?.The expression of p62 increased with enhanced activation of NF-?B indicating p62 may regulate NF-?B activation.Knockdown of p62 resulted in inhibition of NF-?B activation confirming that p62 regulates NF-?B activation induced by IL-1?.Knockdown of p65 by siRNA decreased expression of IL-8 and MCP-1 induced by IL-1? suggesting that NF-?B regulates chemokines production induced by IL-1?.Knockdown of p62 by siRNA inhibited expression of IL-8 and MCP-1,while accumulation of p62 by bafilomycin A1 promoted expression of IL-8 and MCP-1.These data demonstrate that p62 positively regulates expression of IL-8 and MCP-1 induced by IL-1? through NF-?B.3.ATG16L1 regulates chemokines expression in IECs.Knockdown of ATG16L1 by siRNA promoted expression of IL-8 and MCP-1 induced by IL-1? indicating ATG16L1 negatively regulates IL-8 and MCP-1 expression induced by IL-1?.ConclusionIn our study,using the IBD model in vitro,we demonstrate that IL-1? increased p62 expression through NF-?B and p62 in turn enhances activation of NF-?B and expression of chemokines after IL-1? stimulation.ATG16L1 inhibits chemokines expression induced by IL-1?,the mechanism of which needs further investigation.