Gasdermin D基因敲除对慢加急性肝衰竭小鼠肝组织坏死性凋亡通路RIPK1、RIPK3、M LKL、p-MLKL表达的影响OA

Objective:To investigate the effect of Gasdermin D(GSDMD)gene knockout on the expression of necroptosis pathway RIPK1,RIPK3,MLKL and p-MLKL in liver tissue of mice with acute-on-chronic liver failure(ACLF).Methods:Bioin-formatics was used to analyze the expression of pyroptosis and necroptosis pathway and related genes in different chronic liver diseas-es.The protein-protein interaction(PPI)network between the execution protein GSDMD of pyroptosis and necroptosis key protein MLKL was contructed.And the linear correlation between GSDMD and necroptosis pathway and its effector protein MLKL was an-alyzed.Carbon tetrachloride(CCl4)combined with Klebsiella pneumoniae(K.P.)was used to establish ACLF mouse model.Wild type(wild type,WT)blank control group,4 w and 8 w CCl4 liver injury and ACLF group,GSDMD gene knockout(knockout,KO)blank control group and ACLF group were set up.The levels of ALT/AST and IL-33/IL-1α were detected by ELISA.The histopathology of liver tissue was observed by H&E staining.The expressions of pyroptosis related gene and proteins Caspase-1/11,GSDMD-FL/N,IL-18/1β and necroptosis key proteins RIPK1/3,MLKL,p-MLKL in liver tissue were detected by real-time fluorescence quantitative PCR and Western blot.And the fluorescence intensity of GSDMD and MLKL in liver tissue were detected by immunofluorescence.Results:Bioinformatics analysis showed that the expression of genes related to pyroptosis and necroptosis pathway was significantly up-regulated in ACLF,and GSDMD was positively correlated with necroptosis pathway and MLKL in ACLF.The in vivo experiment showed that compared to the blank control,the expression of pyroptosis and nrcroptosis related pro-teins in liver tissue of ACLF mice was up-regulated(P<0.01),and immunofluorescence showed that the fluorescence intensity of GSDMD was significantly increased(P<0.001).Compared to the wild type ACLF group,the survival time of the GSDMD knock-out ACLF group was prolonged,the levels of ALT and IL-33 were decreased(P<0.05),the H&E staining showed that the hyper-plastic fibrous tissue was less than the wild type ACLF group,the hepatocytes were necrotic,some of them had inflammatory cell infiltration,the expressions of RIPK1/3,MLKL and p-MLKL were significantly down-regulated(P<0.05),and the immunofluo-rescence intensity of hepatocyte MLKL was significantly decreased(P<0.001).Conclusion:Hepatocyte pyroptosis and necropto-sis are two important ways of cell death in ACLF.Knockout executive protein GSDMD can inhibits the expression of RIPK1/3,MLKL and p-MLKL in necroptosis pathway,reduce the release of inflammatory cytokines of IL-33 and IL-1α,reduce hepatocyte death and reduce liver inflammation,so as to improve the degree of liver injury and improve the survival rate of ACLF mice.