大黄素通过线粒体通路诱导HepG2细胞凋亡

Emodin Induces Apoptosis in HepG2 Cells via Mitochondrial Pathway

目的：研究大黄素对Hep G2细胞的毒性和可能的作用机制。方法：通过细胞增殖与活性检测试剂（Am-blue）测定大黄素对Hep G2细胞的毒性;使用2＇,7＇-二氯荧光黄双乙酸盐（DCFH-DA）和花青染料（JC-1）探针检测细胞内活性氧与线粒体膜电位水平;使用流式细胞仪,通过磷脂结合蛋白V/碘化丙啶（Annexin V/PI）双染色试剂盒检测大黄素对Hep G2细胞凋亡的影响;通过蛋白免疫印迹法（Western blot）检测成熟型半胱氨酸天冬氨酸蛋白酶-3,8,9（cleaved Caspase-3,8,9）以及聚腺苷二磷酸-核糖聚合酶（cleaved-PARP）等凋亡相关蛋白表达。结果：与空白组比较,大黄素（15,30μmol·L^-1）对Hep G2细胞具有增殖抑制作用（P〈0.01）,并且具有浓度依赖性;大黄素（15,30μmol·L^-1）能够升高细胞内活性氧水平,并且使细胞线粒体膜电位降低（P〈0.01）;大黄素（15,30μmol·L^-1）能够使Hep G2细胞早期和晚期凋亡增加（P〈0.01）;大黄素（15,30μmol·L^-1）能够激活cleaved Caspases-8,9,3和PARP蛋白的表达（P〈0.01）。结论：大黄素对Hep G2细胞有增殖抑制作用,说明大黄素具有潜在的肝脏毒性,其毒性作用机制是通过线粒体凋亡途径实现的。

Objective： The aim of the study was to determine the potential cytotoxicity and the underlying mechanism of emodin on HepG2 cells. Method： Am-blue assay were used to detect the toxicity of emodin on HepG2 cells. The level of intracellular reactive oxygen species（ROS） was detected by the DCFH-DA fluorescent dye,and the mitochondrial membrance potential was measured by the mitochondrial-specific lipophilic cationic fluorescent dye JC-1. The apoptotic cells were quantified by the Annexin V/PI double staining kit and analyzed by flow cytometry,and Western blot was used to detect the protein expression levels of cleaved Caspase-3,8,9,cleaved-PARP,and other apoptosis-related proteins. Result： As compared with blank control group,emodin（15,30 μmol·L^-1） had significant toxicity on HepG2 cells in a dose-and time-dependent manner（P〈0. 01）,promoted the production of intracellular ROS and reduced mitochondrial membrane potential（P〈0. 01）. After treatment with emodin（15,30 μmol·L^-1） for 24 h,the early apoptotic and late apoptotic cells were increased obviously（P〈0. 01）. Further studies by Western blot indicated that emodin（15,30 μmol·L^-1） exposure to HepG2 cells up-regulated the levels of cleaved Caspases-8,9,3 and PARP in a dose-dependent manner（P〈0. 01）. Conclusion： Emodin has a toxic effect on HepG2 cells, indicating that emodin has potential hepatotoxicity,and its toxic mechanism is via mitochondrial apoptotic pathway.