旱莲苷A对HepG-2细胞凋亡作用的研究

Effect of Ecliptasaponin A on the apoptosis of HepG-2 cells

目的研究旱莲苷A(Ecliptasaponin A)对肝癌细胞(HepG-2)的抑制增殖作用,探讨其诱导HepG-2细胞凋亡的机制。方法旱莲苷A处理HepG-2细胞后,MTT法检测细胞增殖,AO/EB染色法观察细胞凋亡,流式细胞仪检测细胞凋亡和周期变化,DCFH-DA荧光探针法检测细胞内ROS,JC-1荧光探针法检测细胞线粒体膜电位的变化。Western blot法检测凋亡相关蛋白的表达情况。结果旱莲苷A处理细胞48 h后,旱莲苷A能明显抑制HepG-2细胞的生长,且呈浓度依赖性增长趋势,其IC50为(29.8±1.6)μmol/L。与对照组相比,浓度为12.5、25μmol/L的旱莲苷A组AO/EB染色可看到凋亡细胞明显增加。流式细胞仪检测显示12.5、25μmol/L旱莲苷A组细胞周期被阻滞在S期的比例增加,分别为24.03%、43.19%,其细胞早期凋亡比例分别增加了4.06%和11.82%。浓度为12.5μmol/L、25μmol/L的旱莲苷A组细胞内ROS水平明显增加,细胞线粒体膜电位显著下降,且随着旱莲苷A浓度的增加,线粒体膜电位下降更为明显。Western blot结果显示,旱莲苷A组凋亡相关蛋白Bcl-2表达减少,Bak表达增加。结论旱莲苷A可有效抑制HepG-2细胞增殖,其作用机制可能是通过线粒体途径抑制Bcl-2表达,促进Bak和Caspase-3的表达,诱导细胞凋亡。

Objective To investigate the effect of Ecliptasaponin A on the growth and apoptosis of HepG-2cells.Methods HepG-2cells were treated with Ecliptasaponin A,and the proliferation of HepG-2cells was measured by MTT assay.AO/EB staining was used to detect the morphological changes of apoptotic cells.The cell cycle and apoptosis of HepG-2cells were determined by flow cytometry.Reactive oxygen species(ROS)was detected by DCFH-DA assay.The potential of mitochondrial membrane of cells was detected by JC-1assay.The proteins of Bcl-2and Bak were detected by western blot.Results The proliferation of HepG-2cells was inhibited by Ecliptasaponin A.IC50was29.8±1.6μmol/L.After incubated with12.5μmol/L and25μmol/L of Ecliptasaponin A for48h,the cell apoptosis was induced and the cell cycle was arrested at S phase,in which the ratios of S phase were increased by24.03%and43.19%and the ratios of apoptosis were increased by4.06%and11.82%.Meantime,the level of cell ROS was increased and mitochondrial membrane potential was decreased.The expression of Bcl-2was decreased and Bak was increased in HepG-2cells.Conclusion The growth of HepG-2cells can be inhibited after incubated with Ecliptasaponin A,which induces cell apoptosis in mitochondrial dysfunction pathway.