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目的:研究大黄素对HepG2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HepG2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(Armexin V/P1)双染色试剂盒检测大黄素对HepG2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HepG2细胞具有显著毒性(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HepG2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HepG2细胞有毒性作用,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: To study the toxicity and possible mechanism of emodin on HepG2 cells. Methods: The toxicity of emodin to HepG2 cells was assayed by Am-blue. DCFH-DA and JC-1 were used in this study. The levels of reactive oxygen species (ROS) and mitochondrial membrane potential were measured by using a probe. Flow cytometry was used to detect the effect of emodin on the apoptosis of HepG2 cells by double staining with Armexin V / PI. The expressions of cleaved Caspase-3, 8, 9 and cleaved-3, 8, 9 were detected by Western blot. PARP) and other apoptosis-related protein expression. RESULTS: Emodin (15,30 μmol·L -1) had significant toxicity (P <0.01) to HepG2 cells in a dose-dependent manner. Emodin (15 μmol·L -1) 1)) increased the intracellular reactive oxygen species (ROS) and decreased the mitochondrial membrane potential (P <0.01). Emodin (15,30 μmol·L -1) increased the early and late apoptosis of HepG2 cells P <0.01). Emodin (15,30 μmol·L -1) activated cleaved Caspases-8,9,3 and PARP protein expression (P <0.01). Conclusion: Emodin has a toxic effect on HepG2 cells, indicating that emodin has potential liver toxicity and its toxicity mechanism is through the mitochondrial apoptotic pathway.