[关键词]
[摘要]
目的 探究羽扇豆醇-3β-琥珀酸酯对HepG2细胞凋亡的作用及机制。方法 采用0、2.5、5、10、20、40、80 μmol/L的羽扇豆醇-3β-琥珀酸酯作用于HepG2细胞24、48、72 h,采用CCK-8法检测各组细胞的增殖活性。采用0、4、8、16 μmol/L的羽扇豆醇-3β-琥珀酸酯作用于HepG2细胞48 h后,AO/EB染色法和Annexin V-FITC/PI双染法检测各组细胞的凋亡率;JC-1染色法观察HepG2细胞的线粒体膜电位变化;DCFH-DA染色法观察细胞的活性氧(ROS)水平。Western blotting法检测各组细胞线粒体凋亡途径相关蛋白表达变化。结果 羽扇豆醇-3β-琥珀酸酯呈时间和浓度相关性地抑制HepG2细胞增殖(P<0.01)。与对照组相比,羽扇豆醇-3β-琥珀酸酯组细胞的ROS水平、总凋亡率、Bcl-2相关X蛋白(Bax)、细胞色素C、裂解半胱氨酸蛋白酶9(cleaved-Caspase-9)、cleaved Caspase-3蛋白水平呈浓度相关性升高(P<0.01、0.001);线粒体的膜电位、Bcl-2蛋白表达则显著降低(P<0.01)。结论 羽扇豆醇-3β-琥珀酸酯可能通过调控线粒体凋亡途径诱导人肝癌HepG2细胞凋亡,进而发挥抗肝癌作用。
[Key word]
[Abstract]
Objective To investigate the effect of lupinol-3β-succinate on apoptosis of HepG2 cells and mechanism. Methods HepG2 cells were treated with lupinol-3β-succinate (0, 2.5, 5, 10, 20, 40 and 80 μmol/L) for 24, 48, and 72 h, and the proliferation activity of the cells was detected by CCK-8 method. HepG2 cells were treated with lupinol-3β-succinate (0, 4, 8, and 16 μmol/L) for 48 h, and the apoptosis rate of each group was detected by AO/EB staining and Annexin V-FITC/PI double staining. Changes of mitochondrial membrane potential in HepG2 cells were observed by JC-1 staining. Levels of ROS were observed by DCFH-DA staining. Western blotting method was used to detect the changes of mitochondrial apoptosis-related protein expression in each group. Results Lupinol-3β-succinate showed significant time and dose dependent inhibition of HepG2 cell proliferation (P < 0.01). Compared with control group, ROS level, total apoptosis rate, Bax, Cyt-C, cleaved Caspase-9, and cleaved Caspase-3 protein levels were increased in a dose-dependent manner after lupinol-3β-succinate treatment (P < 0.01, 0.001). Mitochondrial membrane potential and Bcl-2 protein expression were significantly decreased (P < 0.01). Conclusion Lupinol-3β-succinate may induce apoptosis of human hepatoma HepG2 cells by regulating mitochondrial apoptosis pathway, and finally play an anti-hepatocellular carcinoma role.
[中图分类号]
R285
[基金项目]
中央支持地方高校改革发展资金人才培养项目(21B024);齐齐哈尔医学科学院科研项目(QMSI2022Z-01)