桔梗皂苷D对肿瘤坏死因子相关凋亡诱导配体抗肺癌细胞活性的影响

摘要

目的：探讨中药活性成分桔梗皂苷D对肿瘤坏死因子相关凋亡诱导配体（TRAIL）抗肺癌细胞活性的影响及其机制。方法将人肺癌细胞系A549分为对照组、桔梗皂苷D组、TRAIL组及桔梗皂苷D联合TRAIL组，MTT法检测A549细胞活力，Annexin V/PI染色检测A549细胞凋亡，免疫共沉淀法检测A549细胞RIP1-FADD-caspase-8复合物的形成，Western blot法检测A549细胞caspase-8的活化。结果对照组、桔梗皂苷D组、TRAIL组、桔梗皂苷D+TRAIL组对A549细胞活力的抑制率分别为0、（7.6±0.7）%、（13.4±1.0）%、（60.3±4.2）%，桔梗皂苷D组、TRAIL组、桔梗皂苷D+TRAIL组的细胞活力抑制率与对照组比较差异均有统计学意义（P<0.05）。对照组、桔梗皂苷D组、TRAIL组、桔梗皂苷D+TRAIL组对A549细胞凋亡的诱导率分别为（1.6±0.2）%、（4.5±0.3）%、（7.2±0.5）%、（40.2±2.8）%，TRAIL组、桔梗皂苷D+TRAIL组的细胞凋亡率较对照组有明显提高（P<0.05）。桔梗皂苷D+TRAIL组A549细胞内的RIP1-FADD-caspase-8复合物水平及caspase-8的活化程度显著提高。RIP1 siRNA及z-IETD-fmk能显著抑制桔梗皂苷D对TRAIL的协同作用。对照组、桔梗皂苷D+TRAIL组、桔梗皂苷D+TRAIL+RIP1 siRNA组、桔梗皂苷D+TRAIL+z-IETD-fmk组对A549细胞活力的抑制率分别为0、（61.2±5.0）%、（24.5±1.9）%、（17.5±1.7）%，与桔梗皂苷D+TRAIL组比较，桔梗皂苷D+TRAIL+RIP1 siRNA组、桔梗皂苷D+TRAIL+z-IETD-fmk组的细胞活力抑制率明显降低（P<0.05）。对照组、桔梗皂苷D+TRAIL组、桔梗皂苷D+TRAIL+RIP1 siRNA组、桔梗皂苷D+TRAIL+z-IETD-fmk组对A549细胞的凋亡诱导率分别为（1.8±0.2）%、（39.5±2.6）%、（12.3±1.1）%、（9.4±0.8）%，与桔梗皂苷D+TRAIL组比较，桔梗皂苷D+TRAIL+RIP1 siRNA组、桔梗皂苷D+TRAIL+z-IETD-fmk组的细胞凋亡率明显降低（P<0.05）。结论桔梗皂苷D可能通过促进死亡受体复合物的形成，增强TRAIL对肺癌细胞的凋亡诱导效应。%Objective To investigate the role of platycodin-Din TNF-related apoptosis-inducing ligand(TRAIL)-induced cell death in lung cancer. Methods The A549 cells were divided into control group, platycodin-D group, TRAIL group, and platycodin-D+TRAIL group, then the cell death, cell apoptosis, and the formation of RIP1-FADD-caspase-8 complex were detected by MTT assay, Annexin V/PI staining, and co-immunoprecipitation, re-spectively. The activation of caspase-8 in A549 cells was evaluated by western blot assay. Results The cell via-bility inhibitory rate of A549 cells in control group, platycodin-D group, TRAIL group, and platycodin-D+TRAIL group was as follows: 0, (7.6±0.7)%, (13.4±1.0)%, and (60.3±4.2)%; significant differences in the cell viability inhibitory ratewas found between control group and platycodin-D group, TRAIL group, and platycodin-D+TRAIL group (all P<0.05). The apoptotic ratio of A549 cells in control group, platycodin-D group, TRAIL group, and platycodin-D+TRAIL group was as follows:(1.6±0.2)%, (4.5±0.3)%, (7.2±0.5)%, and (40.2±2.8)%, with signifi-cant differences between control group and TRAIL group and platycodin-D+TRAIL group (all P<0.05). The forma-tion of RIP1-FADD-caspase-8 complex and the activation of caspase-8 in TRAIL-treated A549 cells were signifi-cantly increased in platycodin-D+TRAIL group. Treatment of RIP1 siRNA and z-IETD-fmk significantly impaired the synergistic effect of platycodin-D on TRAIL-induced cell death. The cell viability inhibitory rate of A549 cells in control group, platycodin-D+TRAIL group, platycodin-D+TRAIL+RIP1 siRNA group, and platycodin-D+TRAIL+z-IETD-fmk group was as follows: 0, (61.2±5.0)%, (24.5±1.9)%, (17.5±1.7)%; significant differences in the cell vi-ability inhibitory ratewas noted between platycodin-D+TRAIL groupandplatycodin-D+TRAIL+RIP1 siRNA groupand platycodin-D+TRAIL+z-IETD-fmk group (all P<0.05). The apoptotic rate of A549 cells in control group, platy-codin-D+TRAIL group, platycodin-D+TRAIL+RIP1 siRNA group, and platycodin-D+TRAIL+z-IETD-fmk group was as follows:(1.8±0.2)%, (39.5±2.6)%, (12.3±1.1)%, and (9.4±0.8)%, with significant differences betweenplaty-codin-D+TRAIL groupand platycodin-D+TRAIL+RIP1 siRNA groupand platycodin-D+TRAIL+z-IETD-fmk group(all P<0.05). Conclusion platycodin-D promotes TRAIL-induced apoptosis by the formation of RIP1-FADD-caspase-8 complex in lung cancer.