Taurine Attenuates Arsenic-induced Pyroptosis And Nonalcoholic Steatohepatitis By Inhibiting Autophagic-inflammasomal Pathway

Objective:To investigate the damage of liver caused by As₂O₃ and the molecular mechanism of taurine protective effectMethods:The C57BL/6J mice were used as in vivo model and the HepG2 cells as the in vitro model.After treated with As₂O₃ and taurine,H&E staining and Oil Red O were performance for access the steatosis and accumulation of lipid.the inflammatory,NLRP3-inflammasome and autophgic proteins were detected with Western blot.identified the NASH by NAS scores.Transmission electron microscopy was used to detect the changes of autophagy in mouse liver tissue after As₂O₃ exposure.The activation of caspase-1 in liver tissue of mice was detected by immunofluorescence.MTT assay was used to detect the effect of As₂O₃ on HepG2 cell proliferation;NLRP3 inflammasome inhibitor MCC950 was used to determine the relationship between NLRP3inflammasome and cell apoptosis and lipid accumulation induced by As₂O₃;CA-074 Me was used to observe the role of cathepsin B in the activation of NLRP3 inflammasome and cell focal apoptosis induced by As₂O₃.Autophagy inhibitor 3-MA was used to determine As₂O₃-induced autophagy and NLRP3 inflammasome activation and pyroptosis.After taurine pretreatment,explore the taurine on As₂O₃-induced non-alcoholic steatohepatitis protective effect of the molecular mechanism.Flow cytometry was used to detect the change of pyroptosis in HepG2 cells after As₂O₃ exposure.The expression of inflammasome and autophagy was observed by Western blotting.The changes of HepG2 cells lipid accumulation of the situation were detected by Oil Red O staining.Results:In vivo experiments showed that:H&E and Oil Red O staining results showed that steatosis and accumulation of lipid in mice liver after exposure of As₂O₃.Immunofluorescent staining of liver macrophages and western blot results showed that inflammation related indicators increased with the increase of exposure dose.NAS score showed that non-alcoholic steatohepatitis occurred in the livers of 4 mg/L group mice.The expression of inflammasome-related proteins NLRP3,ASC and caspase-1 and the expression of autophagy-related proteins LAMP-1,LC3 and CTSB were increased,while the expression of p62 decreased.The autophagy bodies and autophagy lysosomes were found by transmission electron microscopy,indicating that the autophagy level of mouse liver tissue increased after exposure to As₂O₃.The results of active caspase-1 staining showed that focal necrosis of hepatocytes occurred in mice.The level of autophagy,the level of inflammation,the activation of inflammasome and pyroptotic cell death in the taurine intervention group were all improved,and the NAS score was decreased.However,the steatosis and lipid accumulation did not improve.In vivo,we found that the expression of NLRP3 inflammasome related proteins?NLRP3,caspase-1 p20 and IL-1?p17?in HepG2 cells increased after treated with As₂O₃.The results of flow cytometry showed that the percentages of pyroptotic cell increased significantly.After intervention with MCC950 and CTSB inhibitor CA-074 Me,the activation of inflammasome was inhibited and the percentage of pyroptotic cells was significantly decreased.The expression of CTSB,inflammasome related proteins,and the percentage of pyroptotic cells significantly decreased after treated with autophagy inhibitor 3-MA.After taurine intervention,the expression of autophagy-related proteins and inflammasome-related proteins in HepG2 cells were inhibited and the percentage of pyroptotic cells was significantly decreased.Consistent with in vivo experiments,lipid accumulation did not change after intervention with either MCC950,CA074 Me,3-MA or taurine.Conclusion:In conclusion,this study indicated for the first time that NLRP3-inflammasome was involved in arsenic-induced NASH,and the molecular mechanism of NLRP3 inflammasome activation underlies the autophagy and degradation of autolysosome by induction of arsenic,and the protective effect of taurine on As₂O₃-induced NASH...