Inhibition Effect Of Phytoestrogen Calycosin On TGF-?₁-Induced Hepatic Stellate Cells Activation,Proliferation And Migration Via Estrogen Receptor ?

Liver fibrosis is a kind of abnormal proliferation of connective tissue in the liver caused by various pathogenic factors.Inhibition of liver fibrosis can effectively prevent liver cirrhosis and liver cancer.Activated hepatic stellate cells?HSC?play an important role in the progression of liver fibrosis and are a major source of extracellular matrix?ECM?in liver fibrosis.Liver fibrosis can be effectively prevented and treated by inhibiting HSC activation,proliferation,and collagen synthesis.In the epidemiological statistics,the incidence of liver fibrosis in premenopausal women was significantly lower than that in men.It was confirmed that estrogen in women has a protective effect on the liver,but estrogen such as estradiol is directly used to treat liver fibrosis due to its target.The body is widely distributed in the body,and the strong estrogen effect and poor selectivity to the target will bring many serious side effects.Therefore,it is very promising to choose phytoestrogens with relatively weak estrogen effect as an alternative treatment.The phytoestrogens,calycosin,are partial agonists of estrogen receptors,which have anti-oxidative free radicals,protect liver cells,inhibit hepatocellular carcinoma and other pharmacological activities,and can significantly reduce the pathological state of liver fibrosis.This study will further investigate the effects of calycosin on HSC function and explore the role of estrogen receptors in it.Objective:To investigate the effects of calycosin on hepatic stellate cell function and to explore whether the drug exerts its effect through the estrogen receptor.Methods:In order to screen the appropriate concentration of the drug,the cells were treated directly with calycosin?0,50,100,200,400,800?mol/L?,and the appropriate concentration was determined by MTT assay;the appropriate concentration of TGF-?₁was screened and grouped into normal groups.TGF-?₁ groups?2.5,5,10,15 ng/ml?were analyzed by MTT method and the optimal concentration was selected.After the above concentration was determined,cells were divided into normal group,TGF-?₁group,calycosin groups?50,100,200?mol/L?,and ICI182,780 group?calycosin 50,100,200?mol/L+ICI182,780 1?mol/L?.The cells were preincubated with ICI182,780 for half an hour before adding calycosin,and the cells were incubated with TGF-?₁?10 ng/ml?for 12 h after 12 hours of the addition of calycosin.The cytotoxicity and safe concentration of calycosin on HSC were determined by LDH method.The proliferation and migration of HSC were determined by MTT assay and Transwell chamber assay,respectively.The mRNA and protein expression of?-SMA,Col-I and ER?were detected by real-time quantitative PCR and Western blotting.Immunofluorescence was used to detect the colocalization and expression of?-SMA and ER?proteins.Results:The safe concentration of calycosin on HSC-T6 is 25-200?M,which has no obvious effect on the proliferation of HSC-T6 induced by TGF-?₁.Calycosin?200?M?significantly inhibited the proliferation and migration of HSC-T6 induced by TGF-?₁?P<0.05?,and significantly decreased the protein expression of?-SMA and Col-I in activated HSC-T6?P<0.05?.The results suggest that calycosin has an inhibitory effect on the proliferation,activation and migration of HSC-T6 induced by TGF-?₁.2.Calycosin significantly down-regulated the expression of ER?protein in HSC-T6?P<0.05?,and found that the ER?subtype in HSC-T6 was ER?5 with a molecular weight of 47 kDa.The expression of ER?decreased with the down-regulation of?-SMA expression,suggesting that ER?changes its expression level with the degree of HSC activation.3.In the co-treatment group of the complete estrogen receptor antagonist ICI 182,780,the above-mentioned effects of calycosin on HSC-T6 were significantly reversed.In view of the fact that only ER?was expressed on HSC without ER?expression,suggesting that ER?could mediate the effect of calycosin on the function of HSC-T6.Conclusion:Calycosin inhibits proliferation,activation,and migration of TGF-??induced HSCs.The effect may be related to binding and downregulation of ER?₅.