土茯苓活性分子落新妇苷联合顺铂对人肺癌A549细胞凋亡的影响

Effect of Astilbin from Smilax glabra Combining Cisplatin on the Apoptosis of Human Lung Cancer A549 Cell

目的:研究土茯苓活性分子落新妇苷联合顺铂对人肺癌A549细胞凋亡的影响。方法:培养人肺癌A549细胞至对数生长期,采用MTT法观察不同浓度落新妇苷作用不同时间对A549细胞活力的影响;将A549细胞分为对照组、顺铂(100μmol/L)组、顺铂(100μmol/L)+落新妇苷(300μmol/L)组及落新妇苷(300μmol/L)组。药物作用48 h后,倒置显微镜下观察各组细胞形态学变化,Annexin V-FITC/PI流式细胞法检测细胞凋亡情况,Western blot法检测细胞Bax、Bcl-2、cleaved-Caspase-3蛋白的表达情况。结果:落新妇苷能抑制A549细胞活力,其作用随着其浓度和作用时间的增加而增强;倒置显微镜下观察显示,对照组细胞正常生长,各给药组细胞出现体积缩小、变形与凋亡,部分细胞悬浮于培养基中,尤其以顺铂+落新妇苷组明显;Annexin V-FITC/PI流式细胞凋亡检测结果显示,各给药组A549细胞凋亡率较对照组显著升高(P<0.01);与顺铂组比较,顺铂+落新妇苷组凋亡率显著升高(P<0.05);Western blot结果显示,各给药组Bax、cleaved-Caspase-3蛋白表达显著上调,Bcl-2蛋白表达显著下调(P<0.01),且顺铂+落新妇苷作用更显著(P<0.01)。结论:落新妇苷能增强顺铂对人肺癌A549细胞的促凋亡作用,其机制可能与调控Bax/Bcl-2/Caspase-3凋亡通路有关。

Objective： To study the effect of astilbin from Smilax glabra combining cisplatin on the apoptosis of human lung cancer A549 cell. Methods： The proliferation effect of astilbin with different concentrations and action time on A549 cell was detected by MTT assay,when A549 cell were cultured to logarithmic growth phase. The A549 cell were divided into control group,cisplatin group（ 100μmol/L）,cisplatin（ 100 μmol/L） ＋astilbin（ 300 μmol/L） group and astilbin group（ 300 μmol/L）. The morphological changes of the cells were observed under inverted microscope after 48 h of drug action. The apoptosis was detected by Annexin V-FITC/PI Flow cytometry,and the protein expression of Bax,Bcl-2 and cleaved-Caspase-3 were detected by Western blot. Results： Astilbin had an effect of inhibiting the activity of A549 cell,and with its concentrations and action time increasing to increase the inhibitory effect. The inverted microscope showed that the growth of A549 cell in control group was normal,while the cells of each administration group were reduced,deformed and apoptosis,some cells were suspended in the medium,especially in cisplatin＋astilbin group. The Annexin V-FITC/PI flow cytometry showed that the apoptosis rate of A549 cell in each administration group increased significantly compared with control group（ P0. 01）,and the apoptosis rate of cisplatin＋astilbin group compared with cisplatin group（ P0. 05）. The results of Western blot showed that cisplatin,astilbin and cisplatin ＋ astilbin could significantly up-regulate Bax,cleaved-Caspase-3 protein expression and down-regulate Bcl-2 protein expression,especially in cisplatin＋astilbin group（ P0. 01）. Conclusion： Astilbin can enhance the proapoptotic effect of cisplatin on human lung cancer A549 cell which mechanism may be related to the regulation of Bax,Bcl-2 and Caspase-3 expression.