Pharmacodynamic Evaluation Of SM934 In A Mouse Model Of Nonalcoholic Steatohepatitis

Non-alcoholic steatohepatitis（NASH）is an advanced form of non-alcoholic fatty liver disease（NAFLD）which pathological changes were hepatocyte vesicular steatosis,lobular inflammation with punctate necrosis,hepatocyte balloon degeneration and so on.The pathology of NASH involves a complex network of mechanisms.Up to date,there are no anti-NASH drugs have been approved.With the huge market demand,the research and development of the first marketed NASH drug has become a hot spot pursued by pharmaceutical companies all over the world.There is growing evidence that immune cells and inflammatory cytokines play a crucial role in promoting liver inflammation.Our previous work had found that SM934,a water-soluble artemisinin derivative,could significantly improve the inflammation and pathological changes of tissues and organs caused by various causative factors.Treatment of passive Hyman glomerulonephritis rats with SM934 attenuated the progression of glomerulonephritis and renal fibrosis by inhibiting TGF-β/SMAD signal pathway.SM934 had therapeutic effects on spontaneous lupus-prone mice by inhibiting the pathogenic helper T cell development and enhancing anti-inflammatory cytokine IL-10 production.Moreover,In the model of dry eye disease,topical SM93can directly inhibit the expression of Toll-like receptor 4 in macrophages and reduce the production of inflammatory factors in conjunctiva.This research focuses on the intervention effect of SM934 on nonalcoholic steatohepatitis,exploring the application of SM934 to non-alcoholic steatohepatitis and determining the regulatory effect of SM934 on the immune microenvironment related to the progression of NASH,to find potential new therapeutic drugs for NASH.First of all,we established a free fatty acid-induced hepatic steatosis system in Huh7 cells in vitro to investigate the effects of SM934 on liver fat accumulation and adipogenesis-related genes.The content of triglyceride in cells was detected by GPO-PAP method.The results showed that SM934 reduced the accumulation of triglycerides in a concentration-dependent manner.Subsequently,we evaluated the activity of SM934 in inhibiting liver fibrosis in vitro based on the ability of SM934 to inhibit hepatic stellate cell activation and the expression of fibrosis-related genes.The results showed that SM934 inhibited the migration of HSC cells in a concentration-dependent manner.In addition,we confirmed that SM934 inhibits the production of fat-induced inflammatory factors in macrophages treated with free fatty acids These results suggest that SM934 may change the inflammatory environment of the liver in which NASH occurs and control the progression of liver fibrosis.In this study,we used High-fat diet（HFD）with CCl₄micro-dose induced NASH animal models to verify the efficacy of SM934 in vivo.The results showed that SM934 significantly attenuated the level of serum ALT,AST,TBIL and TG,improved liver lobular inflammation and reversed liver fibrosis in NASH mice,suggesting that SM934 had therapeutic effects on NASH in vivo.For studying the mechanism of therapeutic effect,we used flow cytometry to systematically analyze the hepatic leucocytes of NASH mice.The results showed that the SM934 group significantly reduced the proportion of inflammatory macrophages(F4/80^lowLy6C⁺)infiltrated in the liver and up-regulated the proportion of restorative macrophages(F4/80⁺Ly6C^low).It is suggested that oral administration of SM934 can reduce liver inflammation caused by fat accumulation and chemical injury,promote phenotype switch of inflammatory to a restorative phenotype and accelerates the fibrosis regression.Due to the enhanced phagocytosis and matrix metalloproteinase secretion of reparative macrophages,it can accelerate the clearance of apoptotic hepatocytes and fiber degradation in the liver.SM934 promotes the transformation of this subgroup in vivo,which may be the key factor for its therapeutic effect on NASH.In summary,this study systematically evaluated the therapeutic effect of water-soluble artemisinin derivative SM934 on non-alcoholic steatohepatitis.Through the liver parenchyma and immune cell system in vitro,it was proved that SM934could interfere with cell degeneration and inflammatory response induced by free fatty acid.In the animal model,it was found that SM934 participated in liver restorative immune regulation,which provided strong experimental support for the development of new indications of SM934 for NASH.In addition,based on liver parenchyma cells and immune cells,we have screened a series of natural-derived compounds for anti-fat accumulation and immunosuppressive activities,among which many active compounds are expected to be further studied and developed.