木犀草素、欧当归内酯A和黄芪皂苷Ⅰ单用及联用对人肝星状细胞株LX-2增殖、活化的影响

Effect of luteolin,levistilide A,and astragalosideⅠon the proliferation and activation of hepatic stellate cell line LX-2 by single and combined administration

目的 探讨木犀草素(Lut)、欧当归内酯A(Lev)和黄芪皂苷Ⅰ(Ast)单用及联用对人肝星状细胞株LX-2增殖、活化的影响。方法 取对数生长期的LX-2细胞,采用噻唑蓝(MTT)法考察Lut、Lev和Ast对转化生长因子-β1(TGF-β1)诱导的LX-2细胞增殖的影响、联用比例确定及Lut、Lev和Ast联用对TGF-β1诱导LX-2细胞增殖的影响;应用CalcuSyn软件计算Lut、Lev及Ast联用抑制LX-2细胞增殖的联合指数(CI),分析3者联用类型;通过蛋白免疫印迹法(Western blot)检测3者对LX-2细胞中α-平滑肌肌动蛋白(α-SMA)和TGF-β1蛋白表达的影响。结果 MTT结果显示,Lut、Lev和Ast联用摩尔比为1∶1∶10,3者单用及联用对TGF-β1诱导的LX-2细胞增殖均具有抑制作用(P<0.05),并呈浓度依赖性;CalcuSyn软件分析结果显示,3者联用对LX-2细胞的增殖抑制率>15%时CI<1,说明3者联用具有协同增效的作用;Western blot结果显示,Lut、Lev及Ast联用能够显著降低TGF-β1诱导的LX-2细胞中α-SMA蛋白的表达(P<0.01),降低LX-2细胞中TGF-β1蛋白的表达(P<0.05)。结论 Lut、Lev和Ast单用及联用均能抑制LX-2细胞的增殖、活化,3者联用作用更强、具有协同增效的作用,该作用可能与下调肝星状细胞TGF-β1蛋白表达有关。

Objective To investigate the synergistic effect of luteolin(Lut),levistilide A(Lev),and astragalosideⅠ(Ast)on the proliferation and activation of human hepatic stellate cell line LX-2 by single and combined administvation.Methods LX-2 cells in the logarithmic growth phase were evaluated by methyl thiazolyl tetrazolium(MTT)method for the effects of Lut,Lev,and Ast on the proliferation of LX-2 cells induced by transforming growth factor-β1(TGF-β1),determination of combination ratio,and combination of Lut,Lev,and Ast on the proliferation of LX-2 cells induced by TGF-β1.The combination indexes(CI)of Lut,Lev,and Ast to inhibit the proliferation of LX-2 cells were calculated by CalcuSyn software to determine the types of combined use of the three.The effects of them on the protein expression ofα-smooth actin(α-SMA)and TGF-β1 in LX-2 cells were evaluated by Western blot.Results The results of MTT experiments showed that the molar ratio of Lut,Lev,and Ast in combination was 1∶1∶10 and the single use of the and in combination of Lut,Lev,and Ast had inhibitory effects on the proliferation of LX-2 cells induced by TGF-β1(P<0.05)in a concentration-dependent manner;CalcuSyn software analysis results displayed CI<1 when the cell proliferation inhibition rate of LX-2 cells induced by TGF-β1 was more than 15%,indicating that combination of the three compounds was more synergistic and effective.Western blot results showed that the combined use of Lut,Lev,and Ast significantly reduced the expression ofα-SMA protein in LX-2 cells induced by TGF-β1(P<0.01)and the expression of TGF-β1 protein in LX-2 cells(P<0.05).Conclusion Although the single use of each of Lut,Lev,and Ast can inhibit the proliferation and activation of LX-2 cells,the combination use of the three has stronger and more synergistic clinical result.It may be related to the down-regulation of TGF-β1 protein expressions in hepatic stellate cells.