小檗碱通过ROS及caspase通路对肝癌HepG2细胞凋亡的作用

目的 探究小檗碱(BBR)对肝癌HepG2细胞的增殖抑制及作用机制.方法 采用噻唑蓝(MTT)法检测不同浓度(0、10、20、40、80 μmol/L) BBR对肝癌HepG2细胞的抑制作用;采用Hoechst 33342染色观察处理后HepG2细胞形态学变化;用流式细胞仪检测HepG2细胞凋亡率;间接荧光标记法检测BBR对细胞内活性氧簇(ROS)水平的影响;JC-1染色法检测细胞内线粒体膜电位的变化;应用Western blot法检测凋亡相关蛋白Bax、caspase-3、Cleaved-caspase-3的表达变化.结果 MTT法显示BBR可呈剂量、时间依赖性抑制肝癌HepG2细胞活力(P＜0.05);Hoechst 33342染色发现细胞形态发生明显变化,0μmol/L BBR处理的细胞呈淡蓝色,处理组细胞核固缩,形成凋亡小体,细胞凋亡率与药物浓度呈正相关(P＜0.05).流式细胞术发现BBR能使细胞内ROS升高,线粒体膜电位水平下降.Western blot法显示随着药物浓度升高,Bax、Cleaved-caspase-3、cleaved-聚腺苷二磷酸核糖聚合酶表达增强,Bcl-2蛋白表达降低,凋亡相关蛋白caspase-3表达下降.结论 BBR在体外可能通过增加ROS、提高促凋亡蛋白Bax并激活caspase-3表达抑制肝癌HepG2细胞增殖并诱导细胞凋亡.

Mechanism of liver cancer cell apoptosis induced by berberine: the roles of ROS and caspase pathway

Objective To investigate the effects of berberine on inhibiting the proliferation and inducing the apoptosis of liver cancer HepG2 cells.Methods The inhibitory effect of berberine on the proliteration of liver cancer HepG2 cells was detected by MTT.The effect of inducing apoptosis was detected by contrast phase microscope and flow cytometry.The level of ROS and mitochondrial membrane potential were detected by flow cytometry.The expression of bax,caspase-3 and cleaved caspase-3 was determined by Western blot.Results Compared with the blank control group,berberine significantly inhibited the proliferation of HepG2 cells.The cell of HepG2 cells was significantly altered in morphology,presenting with ruptured cellular membrane,weaken brightness.Annexin V/7-AAD double staining showed that cell apoptosis was positively correlated with drug concentration.Berberine increased the level of ROS,and reduced mitochondrial membrane potential.Western blot showed that berberine exposure resulted in up-regulation of Bax protein expression and promoting cleavage of caspase-3 and PARP.Conclusion Berberine exerts anti-proliferative and pro-apoptotic effects through the activation of caspase signal pathway and increasing the level of ROS.