大柴胡汤含药血清通过Sirt3线粒体途径诱导人肝癌HepG2细胞凋亡的研究

Apoptosis of human hepatocellular carcinoma cell line Hep G2 induced by Dachaihu Decotion containing drug serum via Sirt3 mitochondrial pathway

目的:探讨大柴胡汤含药血清抑制人肝癌Hep G2细胞增殖的作用机制。方法:采用血清药理学方法制备大柴胡汤含药血清。以Hep G2人肝癌细胞为对象,采用噻唑蓝法测定其对生长抑制的影响,倒置相差显微镜观察含药血清对Hep G2细胞作用后形态改变,罗丹明123荧光染色观察细胞线粒体膜电位的变化,免疫印迹实验探讨其作用机制。结果:与空白血清组比较,大柴胡汤组(8.1 g/kg)5%、10%和20%含药血清能够抑制Hep G2人肝癌细胞增殖并呈时间-浓度依赖性特点。5%、10%和20%大柴胡汤含药血清作用24 h后,能够显著降低细胞线粒体膜电位并下调Sirt3、PI3K、Akt、NF-κB和Bcl-2蛋白表达,上调Bax和Caspase3蛋白表达。结论:大柴胡汤含药血清能够显著抑制人肝癌Hep G2细胞增殖并通过线粒体依赖的Sirt3/PI3K途径发挥作用。

Objective： To investigate the effects of Dachaihu Decotion（ DCHD） containing drug serum on the cell proliferation of hepatocarcinoma Hep G2 cell. Methods： DCHD containing drug serum was prepared. The cells were separated into 10% control serum group,5% DCHD containing drug serum,10% DCHD containing drug serum and 20% DCHD containing drug serum. The Hep G2 cell morphologic changes were observed by light microscope. The changes of mitochondrial transmembrane potential（ Δ Ψ m） in the Hep G2 cells were analyzed with rhodamine 123 staining. Western blot analysis was used to evaluate the levels of Sirt3,PI3 K,Akt,NF-κB,Bcl-2,Bax and Caspase3 expressions. Results： The results showed that DCHD containing drug serum（ 5% ~ 20%） induced apoptosis in Hep G2 cells in a dose-dependent manner,as determined by MTT assay,and demonstrated that the proper time and dose is 24 h and 10% DCHD containing drug serum,respectively. DCHD containing drug serum（ 5% ~ 10%） treatment with 24 h significantly reduced Δ Ψ m. Further analysis by western blotting showed that the expression of Sirt3,PI3 K,Akt,NF-κB and Bcl-2 were decreased,while the expression of Bax and Caspase3 were increased. Conclusion： DCHD containing drug serum could inhibit the proliferation of hepatocarcinoma Hep G2 cells and inducing apoptosis via mitochondrial-dependent Sirt3 / PI3 K pathway.