丹参多酚酸盐通过线粒体途径诱导人肝癌SMMC-7721细胞的凋亡

Salvianolate induces apoptosis of human hepatoma SMMC-7721 cells through mitochondrial pathway

目的:研究丹参多酚酸盐(salvianolate)体外诱导人肝癌细胞SMMC-7721凋亡作用及其可能机制。方法:不同质量浓度丹参多酚酸盐(0.5、1、2mg/ml)与肝癌细胞共培养24h后,流式细胞仪检测肝癌细胞凋亡,线粒体膜电位试剂盒(JC-1)检测线粒体膜电位变化;比色法测定1.0mg/ml丹参多酚酸盐作用后肝癌细胞内caspase-8、caspase-9及caspase-3的活性,流式细胞仪检测培养体系内加入caspase-9抑制剂(z-LEHD-fmk)或caspase-3抑制剂(z-DEVD-fmk)后细胞凋亡率的变化,Western blotting检测肝癌细胞内线粒体凋亡途径相关蛋白Bax、Bcl-2表达水平。结果:丹参多酚酸盐显著诱导肝癌细胞SMMC-7721凋亡(P<0.05),同时线粒体膜电位随着药物浓度的升高而加剧下降(P<0.05)。1.0mg/ml丹参多酚酸盐处理肝癌细胞24h后caspase-9与caspase-3的活性明显升高(P<0.05),而caspase-8的活性无明显变化(P>0.05);当培养体系内加入caspase-9或caspase-3活性抑制剂后,丹参多酚酸盐诱导肿瘤细胞凋亡的作用明显降低(P<0.05)。Western blotting检测显示,丹参多酚酸盐处理组前凋亡蛋白Bax表达明显升高,抗凋亡蛋白Bcl-2表达降低。结论:丹参多酚酸盐(0.5～2.0mg/ml)剂量具有促进肝癌细胞凋亡的作用,且有剂量依赖的趋势,其机制与线粒体凋亡途径有关。

Objective：To explore the apoptosis-inducing effect of salvianolate on hepatoma SMMC-7721 cells and the underlying mechanism.Methods：SMMC-7721 cells were co-cultured in vitro with different concentrations （0.5,1,2 mg/ml） of salvianolate for 24 h.The apoptotic SMMC-7721 cells were examined by flow cytometry,and the changes of mitochondrial transmembrane potential were examined by mitochondrial transmembrane potential JC-1 kit.The activities of caspase-8,caspase-9,and caspase-3 were detected by spectrophotometry in the hepatoma SMMC-7721 cells after co-cultured with 1 mg/ml salvianolate.The changes of apoptotic SMMC-7721 cells induced by salvianolate in the presence or Absence of caspase-9 inhibitor or caspase-3 inhibitor were measured by flow cytometry.The expressions of pro-apoptotic protein Bax and anti-apoptotic protein Bcl-2 were detected by Western blotting analysis.Results：Salvianolate significantly induced apoptosis of hepatoma SMMC-7721 cells （P〈0.05）,and the decline of mitochondrial membrane potential increased with the increase of salvianolate concentration （P〈0.05）.The activities of caspase-9 and caspase-3,but not caspase-8,were increased in hepatoma cells after treatment with 1 mg/ml salvianolate for 24 h （P〈0.05）.The apoptosis-inducing effect of salvianolate was significantly decreased in the presence of caspase-9 or caspase-3 inhibitors （P〈0.05）.Western blotting results showed that salvianolate increased pro-apoptotic protein Bax expression and decreased anti-apoptotic protein Bcl-2 expression.Conclusion：Salvianolate can induce the apoptosis of human hepatoma SMMC-7721 cells in a dose-dependent manner,which is probably mediated by mitochondrial apoptosis pathway.