The Role And Mechanism Of NLRP3 Inflammasome Activation And Inflammatory Bowel Disease Induction Triggered By High Level Colonic Deoxycholic Acid

Objective A high-fat diet(HFD)is closely associated with the development of inflammatory bowel disease.High level fecal deoxycholic acid(DCA)caused by HFD contributes to the colonic inflammatory injury of IBD,however,the mechanism concerning the initiation of inflammatory response by DCA remains unclear.Increasing evidences suggest the pivotal role of NLRP3 inflammasome in the development of intestinal inflammation.In our study,we mainly investigated the role and mechanism of DCA in the induction of gut inflammation via promoting NLRP3inflammasome activation.MethodsPart Ⅰ Different concentrations of DCA was used to stimulate LPS-primed murine macrophages(cell line and bone marrow derived macrophages),DCA-untreated group was regarded as control.Western blot,ELISA were adopted to determine the expression and secretion of caspase-1 and mature IL-1β,which play key roles in the NLRP3 inflammasome activation.To confirm the activation of NLRP3inflammasome induced by DCA,the expression of NLRP3 was knocked down by transfecting macrophages with si RNA specific for NLRP3 or caspase-1 inhibitor was used to block NLRP3 inflammasome activation,and then ELISA was applied to detect DCA-induced mature IL-1βsecretion.Part Ⅱ Reactive oxygen species(ROS)production,lysosomal cathepsin B leakage and potassium efflux are the three common mechanisms of NLRP3 inflammasome activation.Therefore,LPS-primed murine macrophages was stimulated by DCA,and the production of intracellular ROS,integrity of lysosomes and release of cathepsin B were measured by fluorescence staining,the changes of intracellular K~+level were examined by inductively coupled plasma optical emission spectrometry(ICP-OES);meanwhile,ROS production,cathepsin B release or K~+efflux were blocked by corresponding inhibitor pre-treatment,then the changes of mature IL-1βproduction in DCA-stimulated macrophages were tested by ELISA.To clarify the molecular mechanism of DCA-induced NLRP3 activation,the major bile acid receptors were selectively inhibited by inhibitors or si RNA technology,then DCA was added to stimulate the cells.IL-1βsecretion,intracellular ROS production,cathepsin B release were tested to identify the model and molecular mechanism underlying the induction of NLRP3 inflammasome activation by DCA.Part Ⅲ To evaluate the role of NLRP3 inflammasome activation in the DCA-exacerbated DSS-induced colonic inflammation,acute colitis was induced in mice with 2.5%dextran sodium sulfate(DSS)for 7 days.Mice were divided into four groups:water intake control group,DSS group,DSS plus colorectal instillation of DCA group,DSS plus colorectal instillation of DCA and intraperitoneal injection of caspase-1 inhibitor group.Mental state,body weights changes and fecal properties of mice were recorded daily and disease activity index was evaluated.On day 8,mice were sacrificed,HE staining and immunohistochemical staining of colon sections were performed to detect inflammatory cells infiltration and intestinal mucosa injury;colonic tissue homogenate was used to determine the mature IL-1βlevel and myeloperoxidase(MPO)activity.To investigate the role of macrophages in the DCA-exacerbated DSS-induced colonic inflammation,acute colitis was induced in mice with 2.5%DSS and macrophages depletion was performed by intraperitoneal injection of clodronate liposomes.Part Ⅳ To investigate the effect of long-term exposure of high level DCA on colonic inflammation,wild-type C57BL/6 mice were fed with routine diet supplemented with0.2%DCA,routine diet-fed mice were as control.The development of colonic inflammation was evaluated.HE staining was used to examine intestinal mucosa injury,western blot was adopted to detect the expression of mature IL-1β,realtime PCR was performed to test the expression of proinflammatory cytokine IL-6 and MCP-1,and colonic tissue homogenate was prepared to determine MPO activity.ResultsPart Ⅰ DCA could time-and dose-dependently induce NLRP3 inflammasome activation and proinflammatory cytokine IL-1βproduction.Part Ⅱ DCA triggered NLRP3 inflammasome activation and mature IL-1βsecretion through promoting lysosomal cathepsin B release.This effect was mainly mediated by the bile acid membrane receptor sphingosine-1-phosphate receptor 2(S1PR2)on macrophages.Blockage of S1PR2 or cathepsin B release could significantly reduce DCA-induced mature IL-1βsecretion.Part Ⅲ Colorectal instillation of DCA significantly exacerbated colonic inflammation and injury induced by 2.5%DSS.Compared with DSS alone group,the addition of DCA enema caused much more severe colitis,as evidenced by significant decrease of body weight and bloody,diarrhea,strongly increased mature IL-1βlevel in colonic tissue and more severe intestinal mucosa injury.Blockage of NLRP3inflammasome activation by caspase-1 inhibitor or macrophage depletion dramatically ameliorated the aggravated inflammatory injury imposed by DCA.Part Ⅳ Wild type C57BL/6 mice were fed with diet supplemented with 0.2%DCA for 3 months,HE staining exhibited colonic inflammatory injury,the m RNA level of IL-6,MCP-1 elevated obviously and mature IL-1βproduction increased significantly.Conclusion Our findings show that high level fecal DCA may serve as an endogenous danger signal to activate NLRP3 inflammasome and induce proinflammatory cytokine secretion,which could be one of the important factors in HFD-related colonic inflammation.NLRP3 inflammasome may represent a new potential therapeutical target for treatment of IBD.