Study On Preparation And Anti-hepatic Fibrosis Effect Of Ginsenoside Rg₂

Liver fibrosis is caused by abnormal wound healing response caused by long-term chronic inflammation,including alcohol drinking,non-alcoholic fatty liver disease（NAFLD）,viral hepatitis,cholestasis and autoimmune hepatitis.If liver fibrosis is not treated in time,it may further develop into liver cirrhosis and even lead to liver cancer.At the same time,according to epidemiological investigations,about 2 million people die of liver cirrhosis complications and liver cancer every year.However,it is regrettable that liver cirrhosis is almost irreversible,so it is of great clinical significance to give effective treatment in the stage of liver fibrosis.Ginsenoside Rg₂,as one of the protopanaxatriol saponins,is one of the active components in the roots,stems and leaves of ginseng（Panax ginseng C.A.Mey.）.Ginsenoside Rg₂ has many pharmacological effects,such as antioxidation,anti-inflammation,anti-apoptosis and antidepressant.However,the content of ginsenoside Rg₂ in ginseng is not high,so it is difficult to separate a large number of ginsenoside Re directly from ginseng,while ginsenoside Re is high in ginseng and easy to obtain,so we use glutamic acid to hydrolyze ginsenoside Re to prepare rare ginsenoside Rg₂,and explore the anti-liver fibrosis effect of ginsenoside Rg₂ and its possible mechanism.Methods:（1）Rare ginsenoside Re was prepared by hydrolysis of ginsenoside Rg₂with glutamic acid.The hydrolysate was purified and identified by thin layer chromatography,semi-preparative liquid chromatography and high performance liquid chromatography.（2）The mouse model of hepatic fibrosis was established by feeding CDAHFD for 10 weeks.The anti-fibrosis effect of ginsenoside Rg₂ in vivo and its possible mechanism were evaluated by histological analysis,immunohistochemistry,QPCR and Western blotting.（3）The primary hepatocyte injury model was induced by oleic acid（OA）.The toxicity of ginsenoside Rg₂ was detected by CCK8,and the expression of TGF-β1 and autophagy related protein was detected by Western blotting.（4）Lipopolysaccharide（LPS）was used to establish the activation model of HSC-T6cells in vitro,and the expression ofα-SMA and COL1A1 protein was traced by Western blotting and immunofluorescence.（5）AKT/m TOR signal pathway and autophagy related protein expression were further detected by Western blotting and immunofluorescence,and m TOR inhibitor rapamycin and AKT inhibitor MK2206were given to verify the possible mechanism of ginsenoside Rg₂ inhibiting the activation of HSC-T6 cells.Results:（1）Ginsenoside Re was hydrolyzed by glutamic acid to ginsenoside Rg₂,and the conversion rate was 59.9%.After further purification by semi-preparative liquid chromatography,the purity of ginsenoside Rg₂ was 98.89%,and the yield was 21.6%.（2）Ginsenoside Rg₂ significantly improved the pathological changes of liver tissue induced by CDAHFD,and inhibited the levels of serum transaminase,plasma LPS,liver HYP and the expression of TGF-β1,α-SMA and COL1A1.Significantly activate AKT/m TOR signal pathway and inhibit the expression of autophagy-related proteins in liver tissue.（3）Ginsenoside Rg₂ restored OA-induced autophagy flux impairment and inhibited the expression of TGF-β1 by promoting p62 degradation.（4）Ginsenoside Rg₂could decrease the expression ofα-SMA and COL1A1 induced by LPS in a dose-dependent manner without obvious toxic effect on HSC-T6 cells,and inhibit the activation of HSC-T6 cells.（5）Ginsenoside Rg₂ significantly increased the expression of p-AKT and p-m TOR proteins and inhibited the expression of autophagy-related proteins in HSC-T6 cells.Pretreatment with rapamycin and MK2206 reversed the AKT/m TOR signal pathway activated by ginsenoside Rg₂ and abolished the inhibitory effect of ginsenoside Rg₂ on autophagy andα-SMA in HSC-T6 cells.Conclusions:the preparation of ginsenoside Rg₂ by hydrolysis of ginsenoside Re by glutamic acid is simple and the product is relatively simple.Ginsenoside Rg₂ has obvious protective effects on liver injury and liver fibrosis induced by CDAHFD,and this effect may be mediated by activating AKT/m TOR pathway to inhibit autophagy.Ginsenoside Rg₂ protects hepatocytes from basic autophagy and inhibits the expression of TGF-β1.Ginsenoside Rg₂ inhibits the increase of autophagy flux induced by LPS in HSC-T6 cells by activating AKT/m TOR signal pathway,which in turn inhibits the activation of HSC-T6 cells.