川芎嗪诱导肺癌细胞凋亡及其机制研究

Investigation of effect of ligustrazine on induction of apoptosis in lung cancer A549 cell

目的探究川芎嗪对肺癌A549细胞凋亡的影响及其机制。方法体外培养肺癌A549细胞,将细胞分为空白对照组(NC组)以及用不同浓度(200μg/ml、400μg/ml、800μg/ml)的川芎嗪处理人肺癌A549细胞实验组,用吖啶橙/嗅化乙锭(AO/EB)双荧光染色观察细胞凋亡形态;流式细胞仪检测细胞凋亡率;Western blot检测A549细胞凋亡相关蛋白表达情况;实时荧光定量PCR(real-time PCR)和Western Blot检测Fox M1的表达水平。转染过表达Fox M1质粒,用800μg/ml川芎嗪干预,分别用AO/EB双荧光染色和流式细胞仪检测细胞凋亡情况。结果相对于NC组,AO/EB双荧光染色结果显示不同浓度的川芎嗪干预后,可见橘红色荧光的凋亡细胞,并且随着川芎嗪的浓度增加而增加,呈浓度依赖性。流式细胞仪检测结果显示川芎嗪干预的A549细胞凋亡数显著增加。WB结果显示不同浓度的川芎嗪干预后,Bcl-2蛋白的表达水平下调,Bax蛋白的表达水平上调,呈浓度依赖。qPCR结果显示Fox M1 mRNA显著下降,WB结果同样显示Fox M1蛋白表达水平降低,并随着川芎嗪的干预浓度增加而下降,具有浓度依赖性。过表达Fox M1蛋白后,相对于阴性对照组,AO/EB双荧光染色结果显示肺癌A549细胞橘红色荧光减少,流式细胞仪检测结果显示,细胞凋亡率降低。结论川芎嗪能有效地诱导肺癌A549细胞凋亡,其作用机制可能与下调Fox M1蛋白表达有关。

Objective To investigate the effect of ligustrazine on apoptosis of lung cancer A549 cells and its mechanism. Methods The lung cancer A549 cells were cultured in vitro,and were divided into blank control group（normal control group,NC group） and A549 cells were treated with different concentrations of ligustrazine respectively. The morphology of apoptotic cells were observed by acridine orange/ethidium bromide（AO/EB） double fluorescence staining; The apoptosis rate was detected by flow cytometry. The expression of apoptosis related proteins in A549 cells were detected by western blot. The expression of FoxM1 was detected by real-time PCR and Western Blot. The overexpression of FoxM1 plasmid was transfected and then treatment with 800 μg/ml ligustrazine,the apoptosis was detected by AO/EB double fluorescence staining and flow cytometry. Results Compared with NC group,AO/EB double fluorescent staining showed that apoptosis cells of orange fluorescence could be seen after different concentrations of ligustrazine,and in a dose-dependent manner. The results of flow cytometry showed that the apoptosis of A549 cells was significantly increased by ligustrazine. WB results showed that after different concentrations of ligustrazine intervention,the expression level of Bcl-2 was down-regulated,and the expression level of Bax was up-regulated,and in a dose-dependent manner. q PCR showed that FoxM1 mRNA was decreased significantly and WB also showed that the expression level of FoxM1 was decreased,and in a dose-dependent manner. Overexpression FoxM1 protein,compared with negative control group,AO/EB double fluorescent staining showed that the apoptosis of lung cancer A549 cells were decreased,and the flow cytometry showed that the apoptosis rate decreased. Conclusion Ligustrazine can induce apoptosis of A549 cells and up-regulate the expression of FoxM1.