Chikusetsu Saponin Ⅳa Ameliorates Fatty Liver Disease Through Inhibiting CD36-NLRP3 Signaling Pathway

Objective There has been a rise in the prevalence of non-alcoholic fatty liver disease（NAFLD）in recent years in the world.NAFLD is a clinic pathologic syndrome which includes simple fatty liver disease,fatty hepatitis,liver fibrosis and liver cirrhosis.The fatty hepatitis is the most critical for the NAFLD progression.The limited understanding of the NAFLD molecular mechanism has hindered the development of effective therapeutic strategies.Therefore,this study was to investigate the effect of Chikusetsu Saponin Ⅳa（CS Ⅳa）on intervening fatty hepatitis and explore its possible molecular mechanism.Methods（1）In vivo:Male BALB/c mice were divided into normal group（control）,high fat diet group（HFD+CCl₄）,low and high dose CS Ⅳa group（CS Ⅳa-L,CS Ⅳa-H）.The mice were treated by high fat diet combined with CCl₄ intraperitoneal injection which induced fatty liver fibrosis.Different doses of CS Ⅳa were mixed in the high fat diet and quantitative feeding to the mouse every day.After 5 weeks of treatment,the mice were killed for following research.The liver was examined and weight was weighed.Serum was collected to detect the level of ALT,triglyceride and glucose by biochemical kits.The pathology of hepatic steatosis and hepatic fibrosis were detected by H&E and Masson’s staining.Lipid metabolism-related genes such as Lxr,Fasn,Chrebp,Acc1 and inflammatory-relative genes such as NLRP3,Caspase-1,Asc were detected by semi-quantitative RT-PCR in the mouse liver tissue.The expression of CD36,NLRP3,IL-1 beta andα-SMA was detected by real-time quantitative PCR,Western Blot and indirect immunofluorescence.（2）In vitro:The cellular model of inflammatory response was established by palmitic acid in the endothelial cell line MS-1 cells.The expression of CD36 and NLRP3 was detected by semi-quantitative RT-PCR and Western Blot in MS-1cells.Immunofluorescence assay was used to detect the expression of NLRP3 after transient transfection of CD36-siRNA.After the intervention of CS Ⅳa,the expression of CD36 and NLRP3 was detected by semi-quantitative RT-PCR and Western Blot.Results（1）Compared with that of normal mice,the serum level of ALT,triglyceride and glucose,as well as liver weight were increased in HFD+CCl₄ group（p<0.05）.H&E and Masson’s staining showed significant lipid deposition,inflammatory cell infiltration and increased fibrosis in liver tissue of HFD mice.And semi-quantitative RT-PCR assay detected that the genes of lipid metabolism-related（Lxr,Fasn,Chrebp（p<0.05）,Acc1）and inflammatory-relative genes（NLRP3（p<0.001）,Caspase-1（p<0.01）,Asc）were up-regulated.Immunofluorescence staining showed that CD36 and Nlrp3 were mainly co-localized in hepatic sinusoidal endothelial cells.Real-time quantitative PCR and Western Blot results showed that CS Ⅳa could significantly reduce the expression levels of CD36（p<0.0001）and NLRP3（p<0.01）and IL-1beta compared with HFD mice.（2）Palmitic acid treatment could induce inflammatory cell model in endothelial cell MS-1 which were increased expression of cytokines IL-6,Tnf-α.Semi-quantitative RT-PCR results showed that PA treatment could induce high expression of CD36,NLRP3 and inflammatory cytokines in MS-1 cells.Transfected CD36-siRNA could reduce the expression of NLRP3 protein in the MS-1 cells.Also,CS Ⅳa treatment significantly decreased the expression levels of CD36and NLRP3 by semi-quantitative RT-PCR and Western Blot.Conclusion（1）The expression of CD36 and NLRP3 were increased in HFD mice;（2）CS Ⅳa can reduce lipid deposition,and improve liver inflammation and fibrosis through CD36-NLRP3 signaling pathway,which provides a new idea for the treatment of NAFLD.