Tanshinone ⅡA And Berberine Induce Apoptosis Of Rat Hepatic Stellate Cells In Vitro

Objective Activation of hepatic stellate cell (HSC) is the key event in liver fibrosis.The therapeutic goal in liver fibrosis is the reversal of fibrosis and clearance of activated HSC which has a key role in extracellular matrix accumulation. Thus the induction of HSC apoptosis has been proposed as an antifibrotic treatment strategy. This study was designed to investigate the effects of Tanshinone II A and Berberine on apoptosis and the expressions of MMP-1,TIMP-1 levels in HSC.Methods Rat HSC line (HSC-T6) was treated with Tanshinone II A or Berberine, the optimal concentrations were assessed by MTT. a-SMA and collagen I were detected by immunecytoche-mical stain.ELISA was used to analyze the TGF-β1 levels.The morphology of HSC-T6 cells was observed with HE staining.Cell apoptosis was detected with PI staing followed by flow cytometry cytometric analysis. The expressions of MMP-1, TIMP-1 mRNA were evaluated with RT-PCR.Results 1. MTT tests showed the cell survival rate with the concentration of Tanshinone II A 1.5mg/L was 93.9%, and the cell survival rate with the concentration of Berberine 20μmol/L was 96.8%,therefore,we chose Tanshinone II 1.5mg/L, Berberine 20μmol/L as the experimental concentrations of the drugs.2. HSC-T6 cells is the activated phenotype of HSC which can express a-SMA and collagenⅠ,after the effect of Tanshinone II A or Berberine,both of them decreased.3.When treated with Tanshinone II A or Berberine in the experimental concentration for 24 hours, TGF-β1 levels in the supernatant of the culture became lower compared to the control group (P<0.05).4. HE staining showed cell shrinkage,and also found apoptotic bodies in Tanshinone II A or Berberine group.5.Tanshinone II A and Berberine were associated with increases in the apoptosis index of 9.880±0.064%,17.756±1.184% respectively, compared to the control 3.051±0.061%, P< 0.01,and the ratios of HSC-T6 cells in G0/G1 phase increased.6. HSC-T6 cells can express MMP-1, TIMP-1 mRNA,after adding Tanshinone II A or Berberine for 24 hours, the expression of MMP-1 mRNA increased but TIMP-1 mRNA decreased.Conclusions Tanshinone II A and Berberine can caused the apoptosis of activated HSC-T6 cells with the concomitant decrease in the a-SMA,collagen I and TGF-β1 levels obviously,which may resulted by inhibiting the activity of TIMP-1 in order to increase the expression of MMP-1, so suggested that TanshinoneⅡA and Berberine as effective antifibrotic agents of hepatic fibrosis.