NLRP3 Inflammasome Formation And Activation In Non-alcoholic Steatohepatitis

Background:Non-alcoholic fatty liver disease(NAFLD)has become the most common liver disease in the world,and about 20%of patients may develop into non-alcoholic steatohepatitis(NASH)which is high-risk continue to liver fibrosis,liver cirrhosis and even to liver cancer.Therefore,NAFLD has become a social health problem.The relevant mechanisms of NAFLD include the classic "two-hits" theory.That is the combination of fat deposition and inflammatory infiltration further leads to the progression of NASH.In addition,the "gut-liver axis" theory,namely the intestinal dysbiosis may lead to the steatosis aggravation in liver,has become a hot topic recently.However,the pathogenesis of either theory is still unclear,especially inflammatory pathogenesis and its potential as a therapeutic target in the early stage of NASH.NLRP3 inflammasome mainly consists of NLRP3,ASC and caspase-1,and plays an important role in the early inflammatory pathogenesis and progression of NAFLD.In the present study,we characterize the roles of NLRP3 inflammasomes play in liver and gut at the early stage of NASH,and also examined whether FTZ(an herbal medicine compound)could prevents NASH development by targeting the effects of NLRP3 inflammasome activation on both inflammatory response and steatosis in the liver.METHODS:Part Ⅰ:HSCs cells in the liver were stimulated by palmitic acid(PA)in cell experiments.The formation and activation of NLRP3 inflammasomes were detected by immunofluorescence.The expressions of activated caspase-1 and IL-lbeta were detected by WB and ELISA.ESR was used to detect the release of O2·-to understand the upstream ROS pathway of NLRP3 inflammasomes.Part Ⅱ:C57BL/6J(WT)mice were treated with high-fat diet(HFD)and normal diet(ND),and part of them were treated with FTZ gavaged,so as to further understand the role of NLRP3 inflammasomes in early stage of NASH and the inhibitory effect of FTZ in vivo used the methods as mentioned before.Part Ⅲ:Damage of intestinal mucosa of inflammasomes deficient mice(ASC-/-)with HFD was detected by IHC and its dysbiosis detected by 16S rDNA sequencing.The expression of HMGB1 in intestinal mucosa was understood by IHC and immunofluorescence methods.After that,the intestinal exosomes were observed by NanoSight,and the contents of HMGB1 were determined by WB method.Results:part I:The formation and activation of NLRP3 inflammasomes were increased in hepatic HSCs cells,and after that the expression of activated caspase-1 and IL-lbeta were increased.These effect can be reversed by Nlrp3 siRNA gene silencing.The high-fat status can lead to the formation of lipid raft-redox signal platform in the HSCs cell membrane,and then generate a large amount of O2·-,which subsequently promote the formation and activation of NLRP3 inflammasomes.Furthermore,the exogenous HMGB1 led to lipid deposition in HSCs.Part Ⅱ:HFD led to the formation and activation of NLRP3 inflammasomes in mice liver,and then the expression of activated caspase-1,IL-lbeta and IL-18 were increased.It finally generated inflammations and lipid depositions in liver.However,the activation of NLRP3 inflammasomes were surpressed when treated with FTZ.Co-location of liver fibrosis markers(alpha-SMA and vimentin)with activated caspase-1 were increased,and it suggested that NLRP3 inflammasomes were involved in fibrogenic phenotype changed in the early formation of liver fibrosis,which can be improved by FTZ.Part Ⅲ:ASC-/-mice fed with HFD showed that intestinal mucosal injury and hepatic steatosis were aggravated.The gut dysbiosis,Firmicutes:Bacteroidetes ratio and Streptomyces were abnormal increased.Moreover,HMGB1 increased significantly in the intestinal tissue and was co-localized with an exosomal marker.We revealed that HMGB1 was significantly elevated in the exosomes of the ASC-/--HFD group.Conclusion:In summary,the present study demonstrated that increased NLRP3 inflammasome formation and activation are an important pathogenic mechanism initiating and promoting the development of NASH.NLRP3 inflammasome activation was due to formation of MR redox signaling platform and subsequent O2·-production.NLRP3 inflammasome activation was not only associated with liver inflammation and lipid deposition,but also leading to fibrogenesis changed.FTZ suppressed this NLRP3 inflammasome activation to prevent steatosis,hepatic inflammation and fibrogenic phenotype changed.Our current study suggests that exosomal HMGB1 released from the gut may aggravate hepatic steatosis,but further examination will be needed to confirm the cross-talk action of HMGB1 between the gut and liver.