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[摘要]
目的 研究大黄素对人肝癌HepG2细胞线粒体凋亡的影响。方法 培养人肝癌HepG2细胞,与5、10、20、40、60、80、100 μmol/L的大黄素作用24、48 h,MTS法检测细胞增殖;40、80、160 μmol/L大黄素作用HepG2细胞24 h,AO/EB双荧光染色法观察细胞凋亡的形态学改变;Annexin V/PI染色经流式细胞仪检测细胞凋亡;分光光度法检测caspase 3活性;ATP试剂盒检测细胞ATP含量,不同荧光探针加载后流式细胞仪测定大黄素对HepG2细胞内活性氧(ROS)含量、Ca2+浓度、线粒体膜电位(MMP)变化的影响。结果 大黄素抑制HepG2细胞生长,且呈时间、浓度相关性,半数抑制浓度(IC50)为(77.42±1.25)μmol/L;随着大黄素浓度升高,AO/EB双染观察到细胞核浓缩、碎裂、凋亡小体等凋亡形态;与对照组比较,大黄素40、80、160 μmol/L作用于HepG2细胞24 h后细胞凋亡率显著增加,caspase 3活性显著增强,ROS水平、Ca2+浓度明显增加(P<0.05、0.01、0.001),80、160 μmol/L组线粒体膜电位明显降低,ATP含量显著下降(P<0.05、0.01、0.001)。结论 大黄素造成HepG2细胞内ROS堆积,ATP合成功能障碍,线粒体膜电位明显下降,进而诱导线粒体通透转运孔开放,导致钙离子和细胞色素C外流,活化caspase蛋白家族,导致细胞凋亡。
[Key word]
[Abstract]
Objective To study the effect of emodin on mitochondrial apoptosis in human hepatocellular carcinoma cell line (HepG2). Methods HepG2 cells were treated with 5, 10, 20, 40, 60, 80, and 100 μmol/L of emodin for 24 and 48 h, then the anti-proliferative effect was assessed by MTS assay. HepG2 cells were treated with 40, 80, 160 μmol/L of emodin for 24 h, and another control group was also established; The morphological changes of apoptosis were observed by AO/EB double fluorescence staining. Apoptosis was detected by flow cytometry (FCM) with Annexin V/PI staining. Caspase3 activity was detected by spectrophotometry. ATP assay kit was used to detect the content of ATP. Flow cytometry was used to determine the Ca2+ concentration, the content of ROS, and mitochondrial membrane potential (MMP) of emodin in HepG2 cells after loading with different fluorescence probes. Results Emodin could significantly inhibit the proliferation of HepG2 cells and showed a time and concentration correlation. The IC50 values were determined as (77.42±1.25) μmol/L. With the increase of emodin concentration, the apoptotic morphology of nucleus condensation, fragmentation and apoptotic bodies were observed by AO/EB double staining. Compared with the control group, after HepG2 cells treated with 40, 80, and 160 μmol/L emodin for 24 h, the apoptosis rate increased significantly, the activity of caspase3 increased significantly, and the level of ROS and the concentration of Ca2+ increased significantly (P<0.05, 0.01 and 0.001). Mitochondrial membrane potential decreased significantly in 80 and 160 μmol/L emodin group, and ATP content decreased significantly (P<0.05, 0.01, and 0.001). Conclusion Emodin causes the accumulation of ROS in HepG2 cells, the dysfunction of ATP synthesis, the decrease of mitochondrial membrane potential, which leads to the opening of the mitochondrial permeable transport pores, resulting in the efflux of calcium ions and cytochrome C, activating the caspase protein family and promoting cell apoptosis.
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[基金项目]
国家科技重大新药创制项目(2015ZX09501004);天津市科技计划项目(16PTGCCX00090)