满天星异荭草苷对人肝癌HepG2细胞增殖和凋亡的调控作用机制

摘要

目的：研究满天星异荭草苷对人肝癌HepG2细胞增殖和凋亡的影响及其作用机制。方法满天星异荭草苷5，10，20，40，80和160μmol·L-1作用于HepG2细胞24，48和72h后，用MTT法检测细胞存活率。满天星异荭草苷5，10和20μmol·L-1作用细胞48 h，检测细胞内乳酸脱氢酶（LDH）的释放量；作用24 h，荧光探针H2DCF-DA测定细胞内活性氧（ROS）含量，流式细胞仪检测HepG2细胞凋亡和线粒体膜电位，比色法检测胱天蛋白酶3，8和9的活性，RT-PCR检测Bcl-2和Bax mRNA的表达；Western蛋白印迹法检测Bcl-2，Bax和细胞色素c蛋白的表达。结果满天星异荭草苷浓度依赖性地（5~160μmol∙L-1）抑制HepG2细胞存活，作用24，48和72 h时IC50分别为62.7±9.1，47.2±11.4和（18.2±7.5）μmol∙L-1。与细胞对照组相比，满天星异荭草苷5，10和20μmol∙L-1能明显提高LDH水平和细胞凋亡率（P<0.05），10和20μmol∙L-1可显著促进ROS的产生，降低线粒体膜电位，并增强胱天蛋白酶3和9的活性；RT-PCR和Western蛋白印迹结果表明，满天星异荭草苷能明显降低Bcl-2 mRNA和蛋白的表达，但升高Bax mRNA和蛋白表达（P<0.05），降低细胞色素c蛋白表达（P<0.05）。结论满天星异荭草苷抑制HepG2细胞增殖，促进其凋亡，该作用与线粒体凋亡途径密切相关。%OBJECTIVE To investigate the effect and underlying mechanism of Gypsophila elegans isoorientin on the proliferation and apoptosis of human HepG2 cells. METHODS HepG2 cells were treated with isoorientin 5,10,20,40,80 and 160μmol∙L-1 for 24,48 and 72 h,respectively. Cell survival was analyzed by MTT assay. HepG2 cells were treated with isoorientin 5,10 and 20μmol∙L-1 for 48 h before the lactate dehydrogenase(LDH)level was detected. After treatment with isoorientin for 24 h, the variation of reactive oxygen species (ROS) was monitored by a fluorescence probe H2DCF-DA. HepG2 apoptosis and mitochondria membrane potential(MMP)were evaluated by flow cytometry. The activi⁃ties of caspase 3,8 and 9 were determined by colorimetry. The mRNA expression of Bcl-2 and Bax was determined by RT-PCR,and the protein expression of Bcl-2, Bax and cytochrome c was detected by Western blotting. RESULTS Isoorientin(5-160μmol∙L-1)inhibited HepG2 cell survial in a concen⁃tration-dependent manner,the 50%inhibitory concentration(IC50)was 62.7±9.1,47.2±11.4 and(18.2± 7.5)μmol∙L-1 after treatment with isoorientin for 24,48 and 72 h,respectively. Compared with the cell control group,treatment with isoorientin 5,10 and 20μmol∙L-1 significantly increased the LDH level and cell apoptosis rate(P<0.05). Moreover,isoorientin 10 and 20μmol∙L-1 notably increased the production of ROS,decreased the MMP(P<0.05),and increased the activities of caspase 3 and 9. RT-PCR analysis and Western blotting showed that isoorientin significantly decreased the mRNA and protein expressions of Bcl-2,decreased the mRNA and protein expressions of Bax(P<0.05),and inhibited the protein expression of cytochrome c(P<0.05). CONCLUSION Isoorientin inhibits HepG2 cell prolif⁃eration,but promotes cell apoptosis,which is closely related to the regulation of the mitochondrial apoptosis pathway.