Study On Mechanisms Of Anti-hepatofibrotic Effects Of Compound Astragalus Extract

Chronic hepatitis is very common in our country that threatens the health of many people. Most of the patients can develop into hepatic fibrosis or hepatic cirrhosis. Hepatic fibrosis is very common in many kinds of chronic liver disease. Excess production of fibre in liver is one of their characters. Recently years much research has approved activated hepatic stellate cell transdifferentiating into Myofibroblast,which results in synthesis of large quantities of extracellular matrix components,on the other hand,the imbalance of production and assimilation of liver extracellular matrix,is the key pathological evidence. TGF-β₁ plays an important role in liver fibrogenesis. At the present study,TGF-β₁ signals are transduced directly from the cell membrane to the nucleus and regulate transcription by smad pathway,consequently exert diverse biology effects.Compound Astragalus Extract (CAE) is the combination of active compound extract from the Astragalus. Previous study from our laboratory showed that CAE had significantly activating blood to resolve stagnation, anti-inflammatory, anti-oxidative, anti-hepatic injury,anti-hepatofibrotic and immuno-regulated effects.Moreover,it suppressed the proliferation of MFBs, collagen production, NF-κB effects, and induced apoptosis.Based on the previous study in our laboratory, Immunoprecipitation and Western blotting molecular biology methods were used, this article was designed to explore the mechanisms of anti-hepatic fibrosis effects of CAE at cellular and molecular levels. The main contents are divided into the following sections: 1. Inhibitory effects of CAE on MFBsVitro models for proliferation of MFBs stimulated with serum and TGF-β₁ respectively were established,which were measured by MTT.The results of concentration and time-inhibition of CAE on proliferation in vitro showed as the followings:(1)CAE(10,20,40,80,160mg?L-1)inhibited the proliferation of MFBs stimulated by 10% NBS in a dose and time dependent manner. (2) CAE(10,20,40,80,160mg?L-1)inhibited the proliferation of MFBs stimulated by TGF-β₁(9 pmol/L)in a dose dependent manner. The results suggested that CAE may suppress MFBs proliferation in vitro.2. Effect of CAE on migration of MFBs induced by TGF-β₁To investigate the effects of CAE on the migration of MFBs induced by TGF-β₁, vitro models were employed to partially mimic invivo microenvironment of Disse space of normal and liver fibrosis. The effects were observed via cell migration experiments. The results showed that TGF-β₁ could induce the invasive capacity of MFBs ,and CAE could decrease invasive capacity of MFBs induced by TGF-β₁. Research suggested the change of Disse space microenvironment accelerate the migratory of MFBs in hepatic fibrosis. The inhibitory effects of CAE on migration of MFBs may be related to its mechanism of anti-hepatic fibrotic effect.3. The effects of CAE on Smad signal transductionThe effects of CAE on the expression levels of pSmad2C, pSmad2L, pSmad3L and Smad2/3 in MFBs induced by TGF-β₁ were investigated by immunoprecipitation and Western blotting methods.The results shows that Smad2C, Smad2L,Smad3L phosphorylation was not only in a concentration dependent manner in MFBs induced by TGF-β₁(0.33,1,3,9pmol/L)but also in a time dependent manner in MFBs co-incubated with TGF-β₁(9 pmol/L)for 5³0min. (2) CAE (10,20,40,80 mg?L-1) inhibited Smad2C, Smad2L phosphorylation in MFBs induced by TGF-β₁ ( 9 pmol/L,30 min) in a dose dependent manner. The results suggested that CAE could interfere in the intracellular signal transduction of TGF-β₁ in MFBs by inhibiting Smad2/3 phosphorylation, which may be one of the mechanisms of anti-hepatic fibrotic effects of CAE.