Ethyl Acetate Fraction of Dicliptera chinensis(L.)Juss.Ameliorates Liver Fibrosis by Inducing Autophagy via PI3K/AKT/m TOR/p70S6K Signaling Pathways

摘要

Objective:To investigate the molecular mechanism underlying the anti-hepatic fibrosis activity of ethyl acetate fraction Dicliptera chinensis(L.)Juss.(EDC)in human hepatic stellate cells(HSCs)in vitro and in a carbon tetrachloride(CCl4)-induced hepatic fibrosis mouse model in vivo.Methods:For in vitro study,HSCs were pre-treated with platelet-derived growth factor(10 ng/mL)for 2 h to ensure activation and treated with EDC for 24 h and 48 h,respectively.The effect of EDC on HSCs was assessed using cell counting kit-8 assay,EdU staining,transmission electron microscopy,immunofluorescence staining,and Western blot,respectively.For in vivo experiments,mice were intraperitoneally injected with CCl4(2μL/g,adjusted to a 25%concentration in olive oil),3 times per week for 6 weeks,to develop a hepatic fibrosis model.Forty 8-week-old male C57 BL/6 mice were divided into 4 groups using a random number table(n=10),including control,model,positive control and EDC treatment groups.Mice in the EDC and colchicine groups were intragastrically administered EDC(0.5 g/kg)or colchicine(0.2 mg/kg)once per day for 6 weeks.Mice in the control and model groups received an equal volume of saline.Biochemical assays and histological examinations were used to assess liver damage.Protein expression levels ofα-smooth muscle actin(α-SMA)and microtubule-associated protein light chain 3 B(LC3 B)were measured by Western blot.Results:EDC reduced pathological damage associated with liver fibrosis,downregulated the expression ofα-SMA and upregulated the expression of LC3 B(P<0.05),both in HSCs and the CCl4-induced liver fibrosis mouse model.The intervention of bafilomycin A1 and rapamycin in HSCs strongly supported the notion that inhibition of autophagy enhancedα-SMA protein expression levels(P<0.01).The results also found that the levels of phosphoinositide(PI3 K),p-PI3 K,AKT,p-AKT,mammalian target of rapamycin(mTOR),p-mTOR,and p-p70 S6 K all decreased after EDC treatment(P<0.05).Conclusion:EDC has anti-hepatic fibrosis activity by inducing autophagy and might be a potential drug to be further developed for human liver fibrosis therapy.