Hepatoprotective Effect And Possible Mechanism Of Phytoestrogen Calycosin On Carbon Tetrachloride-induced Liver Fibrosis In Mice

Background:Liver fibrosis is a common pathophysiological process,an abnormal proliferation of connective tissue in the liver caused by various pathogenic causes and a key step in the development of liver cancer and cirrhosis.Clinical studies have found that women have a lower risk of liver fibrosis and cirrhosis than men,indicating that estrogen has a protective effect on the liver.However,estrogen treatment of liver fibrosis can bring many side effects,and phytoestrogens have potential estrogen-like effect,and are mostly safer than estrogen.Phytoestrogen calycosin has been shown to have effects on anti-oxidative stress,anti-intestinal fibrosis,anti-liver damage,inhibiting non-alcoholic steatohepatitis and other activities.Our previous studies also found that calycosin has an inhibitory effect on the activation,proliferation and migration of hepatic stellate cells?HSCs?.Therefore,calycosin has important value for the treatment of liver fibrosis.This study will further explore the role and mechanism of calycosin in liver fibrosis models through in vivo experiments.Objective:The study was to explore the hepatoprotective effect and possible mechanism of calycosin on CCl₄-induced liver fibrosis in mice.Methods:Male C57BL/6 mice were randomly divided into 6 experimental groups treated as follows:the control group?n=9?,the model group?n=12?,calycosin groups?20,40and 80 mg/kg,n=12 in each group?and the colchicine group?0.1 mg/kg,n=12?.Liver fibrosis was induced in male mice twice a week by intraperitoneal injection of1 m L/kg CCl₄diluted 1/10 in olive oil for 8 weeks.Calycosin and colchicine were administered once a day by gavage,starting from the first intraperitoneal injection of CCl₄.Liver histopathology and collagen fibrogenesis were assessed by HE and Masson trichrome staining.Liver indexes were calculated by the ratio of liver to body weight ratio.Serum ALT and AST activities,liver SOD activity,liver Hyp and MDA levels were determined by biochemistry assay.The m RNA expressions of?-SMA,Col-I,JAK2 and STAT3 were determined using q RT-PCR,whereas the protein levels of?-SMA,Collagen I,ER?,ER?,TIMP-1,MMP-1,JAK2,p-JAK2,STAT3 and p-STAT3 were detected by western blotting.The levels of?-SMA and ER?were measured by immunohistochemistry.Results:1. The HE and Masson staining results showed that there were a large amount of steatosis,cell necrosis,inflammatory infiltration,obvious damage to the lobular structure and excessive collagen deposition in the liver tissue of mice in the model group,suggesting that the liver fibrosis model was successfully established;Compared with the model group,the calycosin?40 mg/kg,80 mg/kg?group and the colchicine group had relatively less liver damage and collagen deposition,and liver histology were closer to normal histology.2. Calycosin markedly reduced liver index,serum ALT and AST activities,liver MDA and Hyp levels,and increased liver SOD activity.The m RNA and protein expressions of?-SMA,Collagen I were significantly lower in the calycosin group than that in the model group.3. Calycosin increased MMP-1 and inhibited TIMP-1 expression resulting in the improvement of MMP-1/TIMP-1 ratio.Importantly,calycosin significantly increased ER??but not ER??protein expression,JAK2 and STAT3 m RNA expression,p-JAK2and p-STAT3 protein expression,and p-JAK2/JAK2 and p-STAT3/STAT3 relative protein expression.However,total JAK2 and total STAT3 protein expressions remained relatively unchanged.Conclusion:Calycosin significantly inhibits CCl₄-induced liver fibrosis in mice,and its mechanism may involve:?1?Calycosin inhibits oxidative stress;?2?Calycosin balances MMP-1/TIMP-1 system and thereby inhibits collagen synthesis;?3?Calycosin increases liver ER?expression and activates JAK2-STAT3 pathway.