Role Of Cardiolipin Activating NLRP3 Inflammasome In NAFLD Pathogenic Progression

Objective To establish NAFLD mouse model,explore the mechanism of cardiolipin activating NLRP3 inflammasome component and promoting the progress of NAFLD.Methods 1.Each group of 10 C57BL/6J mice were fed with high fat diet or low fat diet respectively.Liver tissues of each group were harvested after 16 weeks.Oil red O staining,HE staining,immunohistochemistry and serum analysis of liver function were used to assess the establishment of NAFLD model.2.The total protein of liver tissues from both NAFLD group and control group mice were extracted,and the level of NLRP3 inflammasome were determined by Western blot.3.The kupffer cells of mice were isolated and cultured,then they were stimulated with palmitic acid or saline,the expression level of NLRP3 inflammasome component was determined by Western blot,and the concentration of IL-1? in the culture supernatant were determined by ELISA.In addition,His-tagged NLRP3 overexpression plasmid was transfected into kupffer cells.After 48 hours' of coculturation,the total protein of these cell was extracted,then the protein-lipid overlay was used to detect the interaction between cardiolipin and NLRP3 protein.Results 1.Compared with low fat diet mice,the high fat diet mice showed swell liver,steatosis hepatocytes,proliferative kupffer cells and elevated serum ALT.2.NAFLD mice expressed significantly higher level of NLRP3 inflammasome component than that of control mice.3.The palmitic acid stimulated kupffer cells released more IL-1? than saline stimulated kupffer cells,the expression level of IL-1? from these two group kupffer cells were 169.3±6.8 pg/ml and134.6±2.4 pg/ml respectively(p <0.01).PA stimulated kupffer cells express 103.68% higher NLRP3 protein level and 38.78% higher c-IL-1?protein level than control group.PIP strip membrane was primed with kupffer cell total protein,followed by priming of anti His antibody.The well containing cardiolipin was immunblotted.Conclusion 1.NAFLD mouse model was successfully established.2.NLRP3 inflammasome took part in the pathogenesis of NAFLD and soared the process of NAFLD.3.Cardiolipin activated NLRP3 inflammasome in kupffer cells and exacerbated NAFLD.