摘要
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.
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2018年第3期104-108,共5页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81473149)