基于Akt/mTOR通路研究地榆皂苷II诱导肝癌细胞凋亡和自噬作用机制

目的 探讨地榆皂苷II抑制肝癌细胞增殖、诱导肝癌细胞凋亡和自噬的作用机制。方法 采用不同浓度（0、10、20、40 μmol/L）地榆皂苷II处理人肝癌HepG2细胞和小鼠肝癌Hepa1-6细胞，通过CCK-8和EdU实验观察地榆皂苷II对HepG2和Hepa1-6细胞增殖的影响；运用流式细胞术检测地榆皂苷II对细胞凋亡的影响；通过Western blotting检测地榆皂苷II对细胞凋亡和自噬相关蛋白半胱氨酸天冬氨酸蛋白酶-3（cysteine-aspartic acid protease-3，Caspase-3）、Caspase-8、Caspase-9、B细胞淋巴瘤2（B-cell lymphoma-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2-associated X protein，Bax）、cleaved Caspase-3、微管相关蛋白1A/1B-轻链3-II（microtubule-associated protein light chain3-II，LC3II）、微管相关蛋白1A/1B-轻链3-Ⅰ（microtubule-associated protein light chain3-Ⅰ，LC3Ⅰ）、自噬相关蛋白p62、Beclin1及蛋白激酶B（protein kinase B，Akt）/哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）通路关键蛋白表达的影响。结果 地榆皂苷II显著抑制HepG2和Hepa1-6细胞活力，且呈现一定的剂量相关性；流式细胞检测发现地榆皂苷II给药后细胞凋亡率显著增加（P＜0.01、0.001）；Western blotting结果显示，与对照组比较，地榆皂苷II给药后Caspase-3、Caspase-8、Caspase-9、Bcl-2表达显著降低（P＜0.05、0.01、0.001），cleaved Caspase-3、Bax蛋白表达显著升高（P＜0.05、0.01），LC3Ⅱ/LC3I值显著升高（P＜0.05、0.01、0.001），Beclin1蛋白表达量显著升高（P＜0.05、0.01），p62蛋白表达量显著下降（P＜0.05、0.01），p-Akt/Akt、p-mTOR/mTOR值显著降低（P＜0.05、0.01、0.001）。结论 地榆皂苷II通过Akt/mTOR通路诱导HepG2和Hepa1-6细胞自噬和凋亡实现对肝癌细胞增殖的抑制，为抗肝细胞癌新药发现提供药理学证据。

Mechanism of ziyuglycoside II on apoptosis and autophagy in hepatocellular carcinoma cells based on Akt/mTOR pathway

Objective To investigate the mechanism of ziyuglycoside II on inhibiting liver cancer cell proliferation, inducing liver cancer cell apoptosis and autophagy. Methods HepG2 and Hepa1-6 cells were treated with different concentrations (0, 10, 20, 40 μmol/L) of ziyuglycoside Ⅱ, and the effect of ziyuglycoside Ⅱ on proliferation of HepG2 and Hepa1-6 cells were observed by CCK-8 and EdU experiments; Flow cytometry was used to detect the effect of ziyuglycoside Ⅱ on cell apoptosis; Western blotting was used to detect the effect of ziyuglycoside II on expressions of apoptosis and autophagy-related proteins, including cysteine-aspartic acid protease-3 (Caspase-3), Caspase-8, Caspase-9, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved Caspase-3, microtubule-associated protein light chain3-Ⅱ (LC3-II), microtubule-associated protein light chain3-I (LC3-I), p62, Beclin1 and key proteins in protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway. Results Ziyuglycoside Ⅱ significantly inhibited the viability of HepG2 and Hepa1-6 cells in a dose-dependent manner; Flow cytometry showed that the apoptosis rate was significantly increased after administration of ziyuglycoside Ⅱ (P < 0.01, 0.001); Western blotting results showed that comparison with control group, the expressions of Caspase-3, Caspase-8, Caspase-9 and Bcl-2 were significantly decreased after treatment with ziyuglycoside II (P < 0.05, 0.01, 0.001), cleaved Caspase-3 and Bax protein expressions were significantly increased (P < 0.05, 0.01), LC3Ⅱ/LC3I was significantly increased (P < 0.05, 0.01, 0.001), Beclin1 protein expression was significantly increased (P < 0.05, 0.01), p62 protein expression was significantly decreased (P < 0.05, 0.01), and p-Akt/Akt and p-mTOR/mTOR were significantly decreased (P < 0.05, 0.01, 0.001). Conclusion Ziyuglycoside Ⅱ can induce autophagy and apoptosis in HepG2 and Hepa1-6 cells through Akt/mTOR pathway to inhibit the proliferation of hepatoma cells, which provides pharmacological evidence for the development of novel therapies for hepatocellular carcinoma.