Therapeutic Effects And Mechanism Of Total Flavones Of Bidens Bipinnata L (TFB) On Experimental Liver Fibrosis

Total flavones of Bipinnata L(TFB) were extracted from the leaves of Bidens pilosa L, which has been preliminarily assessed to be of protection against acute liver injury given via intragastric administration(ig) route.To further explore the effect of total flavones of Bidens Bipinnata L(TFB) on experimental liver fibrosis,the experimental model was induced by CCl₄(50%,0.1 mL/100 g body weight) twice a week in Sprague-Dawley rats lasting for 18 weeks.The mechanisms of action of TFB on liver fibrosis by using the methods in vivo and in vitro at body,cellular and molecular levels were studied.1.Therapeutic effects of TFB on CCl₄-indueed liver fibrosisTFB(160,80,40mg/kg,ig) significantly inhibited the elevation of index of liver and spleen in CCl₄-induced liver fibrosis rats.Furthermore,TFB markedly reduced the elevated levels of serum transaminases,HA,PCâ…¢,Câ…£and Hyp in liver tissue.Liver histopathological and electron microscopy examination showed TFB could also ameliorate fibrogenesis and pseudolobule formation.Further study showed that TFB treatment markedly reduced serum TNF-a,IL-1βcontents and MDA in liver tissue,increased GSH-px activity.Immunohistochemistry and RT-PCR analysis showed that TFB treatment decreased the expression of TGF-β1,NF-κβand TGF-βmRNA expression in liver.In addition TFB treatment for 10 weeks decreased the number of activated HSC and increased the quantity of apoptosis HSC.2.Effect of TFB on HSC TGF-β1/Smads pathwayThe rat hepatic stellate cell(HSC) was obtained with collagenaseâ…£infusion and commonly cultured.The cell growth and proliferation were assessed via MTT assay, smads2,smads7 mRNA,α2(â… )collagen mRNA and smads2 protein were measured by semi-quantitative RT-PCR and Western-blot methods respectively.The results showed that TFB could significantly inhibite the proliferation of HSC dose-dependently compared with the control group.Moreover the proliferation of HSC stimulated by TGF-β1 was also markedly inhibited dose-dependently with TFB treatment.In addition TFB treatment could significantly down-regulated Smads2 and a2(â… ) collagen mRNA expression and up-regulated Smads7 mRNA expression of HSC.Smads2 protein expression of HSC stimulated by TGF-β1 was also down-regulated by TFB.3.Effect of TFB on the apoptosis of activated HSCElectron microscopy examination and flow cytometry were used to analyze the apoptosis and apoptosis rate of HSC.The expression of p⁵³,bax of HSC was measured by immunohistochemistry.Fas/FasL mRNA expression of HSC was analyzed by semi-quantitative RT-PCR.The result showed that cell shrinkage,chromatin condensation and(or) ranked along inside of nuclear membrane could be found in HSC after TFB treatment for 48h and the apoptosis rate of HSC with TFB(0.1mg/ml) treatment was 16.05%±0.93%compared with 1.85%±0.31%in the control group(P＜0.05).Moreover the expression of p⁵³,bax and Fas/FasL mRNA was up-regulated after TFB treatment for 48h. Conclusion:TFB significantly had therapeutical effect on CCl₄-induced liver fibrosis in rats probably by its free-radical scavenging ability,inhibition of cytokines,suppressive effects on HSC proliferation and collagen production and stimulation of activated HSC apoptosis.