Study On Molecular Mechanism Of NOD2 Activated By MDP In Colorectal Epithelial Cells To Induce Tolerant CXC Chemokines

Objective:The role and function of chemokines are closely related to chronic inflammation.However,the immunoregulatory mechanism of NOD2 plays an important role in chronic inflammatory diseases such as Crohn’s disease,but the regulatory mechanism of NOD2-induced inflammatory response remains unclear in colorectal epithelial cells.This study aims to investigate the molecular mechanism of NOD2-induced chemokine expression in colorectal epithelial cells activated by muramyl dipeptide(MDP).To clarify whether NOD2-induced chemokine production in colorectal epithelial cells is a phenomenon of tolerance,and whether A20,a negative regulator of inflammation,regulates NOD2-induced immune responses.By elucidating the molecular mechanism of MDP-activated NOD2-induced chemokine expression in colorectal epithelial cells,this paper can provide a theoretical basis for the clinical treatment of intestinal chronic inflammatory diseases.Methods:1.RT-PCR is used to detect the mRNA expression levels of various cytokines in monocytes and colorectal epithelial cells treated by MDP.2.qRT-PCR is used to detect the quantitative mRNA expression levels of cytolines in monocytes and colorectal epithelial cells treated by MDP.3.Western blotting is used to detect the protein expression levels of p-ERK,p-JNK,p-P38 and IκB-α,NOD2,A20,and RIP2 in colorectal epithelial cells treated by MDP.4.Lipofactamine is used to overexpress A20 by transfection of A20 plasmid,and Lipofactamine is used to downexpress NOD2 by transfection of NOD2 si RNA.5.ELISA assay is used to detect the cytokine expression levels of IL-8 in the supernatant of colorectal epithelial cells treated by MDP.Results:1.MDP induces the expression of chemokines CXCL1,CXCL2,CXCL3 and CXCL8 in colorectal epithelial cells.2.MDP induces the expression of chemokines and inflammatory factors in monocytes,while MDP only induces chemokines in colorectal epithelial cells.3.MDP induces chemokines expressed by colorectal epithelial cells in a time-and dose-dependent manner.4.MDP induces chemokine production in colorectal cells by activating NOD2.5.The chemokine produced by MDP-induced NOD2 is resistant.6.The ubiquitin editing enzyme A20 does not regulate NOD2-induced chemokine expression.7.IL-1β-induced A20 does not down-regulate MDP-induced chemokines.8.MDP activates NOD2 to produce chemokine by activating MAPK and NF-κB signaling pathways.9.MDP-activated NOD2 induces chemokine expression related to the signaling linker molecule RIP2.Conclusion:1.MDP-induced inflammatory response in colorectal epithelial cells is different from that in monocytes.2.MDP-induced inflammatory response is tolerant,but it is not regulated by A20.3.MDP-induced inflammatory response is related to ERK,P38 and NF-κB signaling pathways4.The negative regulation mechanism of MDP-induced inflammatory response may be related to the down-regulation of RIP2.