丫蕊花甾体皂苷YB16促进A549细胞凋亡及其机制研究

Study on promotion of A549 cells apoptosis by steroid saponin YB16 of Ypsilandra thibetica and its mechanism

目的观察丫蕊花甾体皂苷YB16对人肺癌细胞A549凋亡的影响,并探讨其促凋亡的作用机制。方法体外培养A549细胞,给予不同浓度的YB16干预,采用MTT比色法检测A549细胞增殖;荧光显微镜观察吖啶橙(AO)、5,5′,6,6′-四氯-1,1′,3,3′-四甲基咪唑并羰花青碘化物(JC-1)染色的细胞荧光变化;流式细胞术检测碘化丙啶(PI)单染、Annexin V-FITC/PI双染、细胞线粒体膜电位(JC-1染色)的变化;采用2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)法检测细胞活性氧(ROS)量的变化,免疫印迹法(Western blotting)检测YB16对细胞凋亡相关蛋白表达的影响。结果丫蕊花甾体皂苷YB16显著抑制A549细胞的生长(P<0.05),呈量效关系;随着药物浓度的增加,与对照组相比,细胞形态有显著变化;不同浓度YB16作用A549细胞后,细胞凋亡率上升(P<0.05);细胞线粒体膜电位下降明显;细胞内ROS水平上升,具有显著性差异(P<0.05);YB16作用A549细胞24 h后,抗细胞凋亡蛋白Bcl-2表达下降,促细胞凋亡蛋白Bax的表达增加,激活型半胱氨酸蛋白酶-3(cleaved-Caspase-3)表达增加(P<0.05)。结论丫蕊花甾体皂苷YB16能抑制细胞增殖,降低细胞线粒体的膜电位,提升细胞ROS水平,促进细胞凋亡,具有良好的抗肿瘤活性。

Objective To observe the effects of steroidal saponin YB16 of Ypsilandra thibetica on cell apoptosis of A549 human lung cancer cells,and explore the mechanism of apoptosis.Methods Cells were cultured in vitro and treated with different concentration of YB16;The proliferation of A549 cells was examined by MTT method;Cells fluorescence changes of A549 were examined by acridine orange （AO） and JC-1 staining;PI,Annexin V-FITC/PI double staining,and JC-1 staining were examined by flow cytometry.Intracellular level of reactive oxygen （ROS） was determined by DCFH-DA assay,the expression of YB16 on apoptosis-related proteins was examined by Western blotting.Results Steroidal saponin YB16 of Y.thibetica could inhibit A549 cells growth significantly with dose-effect relationship （P〈0.05）;With the increasing of drug concentration,cell morphology changed significantly compared with the control group;Cell apoptosis rate increased with the different concentration of YB16（P〈0.05）;Mitochondrial membrane potential decreased significantly;ROS level of cells was increased with a significant difference （P〈0.05）.Apoptotic protein,such as Bcl-2 expression of anti-apoptotic protein,was decreased,and the expression of pro-apoptotic protein Bax and cleaved caspase-3 was increased treated by YB16 after 24 h （P〈0.05）.Conclusion Steroidal saponin YB16 of Y.thibetica could inhibit the cell proliferation,reduce the mitochondrial membrane potential,enhance ROS level of A549 cells,and promote apoptosis.Therefore,it has a good anti-tumor activity.