半边旗二萜类成分PAG通过ROS诱导肝癌细胞凋亡的作用研究

Induction of Apoptosis by PAG from Pteris semipinna in Hepatocarcinoma Cells through ROS

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。

This study was aimed to investigate the effects and mechanisms of PAG from Pteris semipinna on proliferation and apoptosis of human hepatocarcinoma HepG2 cells.Cell growth inhibition of PAG was evaluated by MTT assay after treated with different concentrations of PAG HepG2 cells.Cell-cycle distribution and apoptosis were analyzed by flow cytometry after treatment of PI and Annexin V-FITC/PI,respectively.The expression of apoptosis-related proteins and mRNA were measured by Western bolt and RT-PCR,respectively.ROS generation was also measured by flow cytometry after treatment of DCFH-DA.The ROS dependence of the cytotoxicity of PAG was evaluated by pre-treatment of NAC.The results showed that at 24 h,48 h and 72 h,PAG was dose dependent to inhibit the proliferation of HepG2 cells（p〈0.05）,and IC_（50） was 64.8 μmol/L,38.5 μmol/L,24.8 μmol/L,respectively.When taking drugs for 24 h,PAG was dose dependent to make the HepG2 cells block to G2/M,and the apoptosis rate was increased（p〈0.05）.PAG could significantly reduce the protein expression of caspase 3,PARP and Bcl- 2,and the mRNA expression of Bcl- 2（p〈0.05）,while increase the protein expression of actived-caspase 3,cleaved-PARP and Bax,and the mRNA expression of Bax（p〈0.05）.When using 1 mmol/L ROS inhibitors NAC pretreatment HepG2 cells,cell proliferation inhibition of PAG to HepG2 was blocking significantly.These results indicated that PAG from Pteris semipinna could induce apoptosis in hepatocarcinoma cells by upregulated the ratio of Bax/Bcl- 2 both in gene and protein level,which might be related with its intracellular ROS generation effect.