加味四君子汤含药血清对肝癌Hep-G2细胞的影响

Effect of Jiawei Sijunzi Decoction Containing Serum on Human Hepatocellular Carcinoma Hep-G2 Cells

目的:探讨加味四君子汤含药血清对肝癌Hep-G2细胞增殖、凋亡、周期的影响及其作用机制。方法:不同浓度加味四君子汤含药血清处理Hep-G2细胞后,采用细胞活性检测试剂盒(CCK-8)检测细胞增殖;Annexin V/碘化丙啶(PI)流式细胞术检测细胞凋亡率和周期;hoechst33342荧光染色观察细胞凋亡形态;免疫印迹法(Western blot)检测细胞中磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/雷帕霉素靶蛋白(m TOR)信号通路相关蛋白表达水平。结果:加味四君子汤含药血清呈浓度依赖性抑制肝癌Hep-G2细胞的增殖,诱导Hep-G2细胞凋亡,阻滞细胞于G0/G1期。hoechst33342染色后,随着含药血清浓度的增加,Hep-G2细胞核显著呈碎块状致密浓染。同时,加味四君子汤含药血清能够抑制Hep-G2细胞Akt,m TOR,核糖体S6蛋白激酶(S6),真核翻译起始因子4E结合蛋白1(4EBP1)的磷酸化,从而上调Bcl-2相关X蛋白(Bax)和下调细胞周期蛋白(Cyclin D1),B淋巴细胞瘤-2(Bcl-2)的表达。加味四君子汤含药血清与300 nmol·L-1的PI3K/m TOR双重抑制剂VS-5584联合使用具有协同作用,含药血清能增强PI3K/m TOR双重抑制剂VS-5584对Hep-G2细胞中PI3K/Akt/m TOR信号通路靶点Akt和m TOR磷酸化的抑制作用。结论:加味四君子汤含药血清能抑制Hep-G2细胞的增殖,诱导其凋亡,阻滞其于G0/G1期,其机制可能通过阻断PI3K/Akt/m TOR信号通路而实现。

Objective： To investigate the effects of Jiawei Sijunzi decoction containing serum on proliferation,apoptosis,and cell cycle of human hepatocellular carcinoma Hep-G2 cells and explore its action mechanism. Method： After Hep-G2 cells were processed with different concentrations of Jiawei Sijunzi decoction containing serum,cell counting kit-8（ CCK-8） was used to measure the proliferation of Hep-G2 cells,apoptosis rate and cell cycle were detected by Annexin V / propidium iodide（ PI） flow cytometry. Apoptotic morphology was determined by hoechst33342 staining. The expression levels of related proteins of phosphatidylinositol 3-kinase（ PI3K） / protein kinase B（ Akt） / mammalian target of rapamycin（ m TOR） signaling pathway in Hep-G2 cells were evaluated by Western blot assay. Result： Jiawei Sijunzi decoction containing serum inhibited the proliferation and induced apoptosis of human hepatoma Hep-G2 cells in a dose-dependent manner. Cells were arrested in G0/ G1 phase. Hoechst33342 staining results showed prominent morphological changes with chromatin condensation,fragmentation and formation of apoptotic bodies in dose-dependent manner. Western blot analysis showed that Jiawei Sijunzi decoction containing serum could inhibit phosphorylation of Akt,m TOR,ribosomal S6 kinase（ S6）,and eukaryotic translation initiation factor 4E-binding protein 1（ 4EBP1）,up-regulate the expression levels of Bax,and down-regulate the expression levels of Cyclin D1,Bcl-2. Jiawei Sijunzi decoction containing serum and PI3K/m TOR dual inhibitor VS-5584 had a synergism effect,and Jiawei Sijunzi decoction containing serum enhanced the inhibitory effects on phosphorylation of Akt and m TOR. Conclusion： Jiawei Sijunzi decoction containing serum could inhibit the proliferation of Hep-G2 cells,induce apoptosis and arrest Hep-G2 cells in G1 phase. This inhibitory effect may be induced by blocking the PI3 K / Akt / m TOR pathway.