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探讨半边旗二萜类成分Pteisolic acid G(PAG)对人肝癌细胞HepG2增殖和凋亡的影响及作用机制。用不同浓度的PAG处理HepG2细胞后,采用MTT法检测细胞存活率;采用PI单染法检测细胞周期分布;采用Annexin V-FITC/PI双染法检测细胞凋亡率;采用RT-PCR和Western Blotting检测细胞内mRNA和蛋白表达情况;采用DCFH-DA法检测细胞内ROS水平,采用ROS抑制剂乙酰半胱氨酸(NAC)评价PAG细胞增殖抑制作用对ROS的依赖性。结果表明,在24 h、48 h和72 h时,PAG可剂量依赖性地抑制HepG2细胞的增殖(p<0.05),IC_(50)分别为64.8μmol/L,38.5μmol/L和24.8μmol/L;用药24 h时PAG可剂量依赖性地使HepG2细胞阻滞在G_2/M期,同时增加HepG2细胞凋亡率(p<0.05);PAG可剂量依赖性地降低HepG2细胞内Bcl-2 mRNA和caspase 3、PARP、Bcl-2蛋白的表达(p<0.05),增加Bax mRNA和actived-caspase 3、cleaved-PARP、Bax蛋白的表达(p<0.05)。当使用1 mmol/L的ROS抑制剂NAC预处理HepG2细胞时,PAG对HepG2细胞增殖抑制作用被显著阻断。上述结果表明,半边旗二萜类成分PAG可提高Bax/Bcl-2的基因和蛋白表达比值,从而诱导肝癌细胞HepG2凋亡,该作用可能是通过升高细胞内ROS水平来实现的。
To investigate the effect and mechanism of Pteisolic acid G (PAG) on proliferation and apoptosis of HepG2 human hepatoma cell line HepG2. Cell viability was detected by MTT assay. Cell cycle distribution was detected by PI staining. Cell apoptosis rate was detected by Annexin V-FITC / PI double staining. The cell viability was detected by RT-PCR and Western blot Blotting was used to detect the expression of intracellular mRNA and protein. The intracellular ROS level was detected by DCFH-DA assay. ROS-dependent inhibition of PAG cell proliferation was evaluated by ROS inhibitor acetylcysteine (NAC). The results showed that PAG inhibited the proliferation of HepG2 cells in a dose-dependent manner at 24 h, 48 h and 72 h (IC50: 64.8 μmol / L, 38.5 μmol / L and 24.8 μmol / L, L; At 24 h, PAG dose-dependently blocked HepG2 cells in G 2 / M phase and increased the apoptosis rate of HepG2 cells (p <0.05); PAG dose-dependently reduced Bcl-2 mRNA in HepG2 cells (P <0.05), and increased the expressions of Bax mRNA and actived-caspase 3, cleaved-PARP and Bax protein (p <0.05). When HepG2 cells were pretreated with 1 mmol / L ROS inhibitor NAC, the inhibitory effect of PAG on HepG2 cell proliferation was significantly blocked. These results indicated that PAG could increase the ratio of Bax / Bcl-2 gene and protein, and induce apoptosis of HepG2 cells.