猫爪草总皂苷对A549细胞增殖的抑制及诱导凋亡作用的研究

Inhibitory Effect of Radix Ranunculus Ternati Saponins on Proliferation of A549 Cells and Its Induction Apoptosis

目的:观察猫爪草总皂苷(RRTS)对人非小细胞肺癌A549细胞增殖抑制及诱导凋亡的作用。方法:采用系统溶剂法提取RRTS,RRTS(400、200、100、50、25、12.5 mg/L)作用于A549细胞,实时细胞分析系统(RTCA)和CCK-8法观察RRTS对细胞增殖的抑制作用,及磷脂酰肌醇3-激酶(PI3K)抑制剂、表皮生长因子受体(EGFR)抑制剂、丝裂原活化蛋白激酶(MAPK)抑制剂、半胱氨酸天冬氨酸蛋白酶3(Caspase-3)抑制剂对上述作用的影响;JC-1染色法观察线粒体膜电位的改变;qRT-PCR法检测凋亡相关Caspase-3、Caspase-9、Bax、Bcl-2的mRNA表达情况;比色法检测Caspase-3、Caspase-9蛋白的表达。结果:400~50 mg/LRRTS能明显降低细胞存活率;400~25 mg/L浓度能明显降低细胞指数(CI)值,50μmol/LCaspase-3抑制剂能阻断这一作用;200~50 mg/LRRTS能降低线粒体膜电位;100 mg/LRRTS能显著升高Bax、Caspase-3、Caspase-9的mRNA表达量,使Bcl-2 mRNA表达略有降低;200~50 mg/L RRTS能显著升高Caspase-3、Caspase-9的蛋白表达。结论:RRTS能抑制A549细胞增殖并诱导其凋亡,可能通过上调Bax表达,激活Caspase-9、Caspase-3级联反应,从而激活线粒体通路诱导A549细胞凋亡。

Objective:To observe the effects of Radix Ranunculus Ternati Saponins(RRTS)on proliferation and apoptosis of human non-small cell lung cancer A549 cells.Methods:RRTS was extracted by the technique of solvent-refining.Real-time cell analysis system(RTCA)and CCK-8 method were used to observe the inhibitory effect of RRTS(400,200,100,50,25,12.5 mg/L)on cell proliferation,and the effects of phosphatidylinositol 3-kinase(PI3K)inhibitor,epidermal growth factor receptor(EGFR)inhibitor,mitogen-activated protein kinase(MAPK)inhibitor and caspase-3 inhibitor on the above effects.The changes of mitochondrial membrane potential were observed by JC-1 staining.The mRNA expression of Caspase-3,Caspase-9,Bax and Bcl-2 related to apoptosis was detected by qRT-PCR.The expressions of Caspase-3 and Caspase-9 protein were detected by colorimetry.Results:400~50 mg/L RRTS could significantly reduce the cell survival rate,400~25 mg/L RRTS could obviously reduce the cell index(CI),and 50μmol/LCaspase-3 inhibitor could block this effect.Mitochondrial membrane potential were reduced by 200~50 mg/L RRTS,100 mg/L RRTS could significantly increase the mRNA expression of Bax,Caspase-3 and Caspase-9,and slightly decrease the mRNA expression of Bcl-2.The protein expressions of Caspase-3 and Caspase-9 were significantly increased by 200~50 mg/L RRTS.Conclusion:RRTS can inhibit proliferation and induce apoptosis of A549 cells,and may activate caspase-9 and caspase-3 cascade by up-regulating Bax expression,thus activating mitochondrial pathway to induce apoptosis of A549 cells.