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

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

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、40μmol/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。

Objective To investigate the mechanism of ziyuglycosideⅡon inhibiting liver cancer cell proliferation,inducing liver cancer cell apoptosis and autophagy.Methods HepG2 and Hepa1-6 cells were treated with different concentrations(0,10,20,40μmol/L)of ziyuglycosideⅡ,and the effect of ziyuglycosideⅡon proliferation of HepG2 and Hepa1-6 cells were observed by CCK-8 and EdU experiments;Flow cytometry was used to detect the effect of ziyuglycosideⅡon cell apoptosis;Western blotting was used to detect the effect of ziyuglycosideⅡon 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 ZiyuglycosideⅡsignificantly 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 ziyuglycosideⅡ(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 ziyuglycosideⅡ(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 ZiyuglycosideⅡcan 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.