秦皮乙素调节AMPK/NOTCH1通路对TGF-β1诱导的肺癌A549细胞上皮间质转化的影响研究

Aesculetin Inhibits the Epithelial-Mesenchymal Transition of Lung Cancer A549 Cells Induced by TGF-β1 through Modulating AMPK/Notch1 Pathway

目的:研究秦皮乙素对转化生长因子(TGF)-β1诱导的肺癌A549细胞上皮间质转化的影响及对腺苷酸激活蛋白激酶(AMPK)/缺刻基因(NOTCH)1通路的调节作用。方法:A549细胞加入终浓度为5 ng/mL的TGF-β1诱导建立A549细胞上皮间质转化,设为模型对照组、秦皮乙素200、400、800μg/mL组,另设空白对照组,分别加入相应浓度药物或培养液,四甲基偶氮唑盐(MTT)法测定各组A549细胞增殖率,测定A549细胞迁移抑制率,测定A549细胞侵袭率,流式细胞术测定A549细胞凋亡率,实时定量聚合酶链式反应(RT-qPCR)测定A549细胞上皮型钙黏蛋白(E-cadherin)、纤维粘连蛋白(Fibronectin)、波形纤维蛋白(Vimentin)、蜗牛同源蛋白(Snail)、Ampk、Notch1 mRNA表达,免疫印迹法(Western blot)测定A549细胞E-cadherin、Fibronectin、Vimentin、Snail、AMPK、p-AMPK、NOTCH1蛋白表达。结果:与空白对照组比较,模型对照组A549细胞迁移抑制率、凋亡率、E-cadherin mRNA及蛋白表达明显降低(P<0.05),Fibronectin、Vimentin、Snail、Notch1 mRNA及蛋白表达明显升高(P<0.05);与模型对照组比较,秦皮乙素200、400、800μg/mL组A549细胞24、48、72 h细胞增殖率、细胞侵袭率、细胞Fibronectin、Vimentin、Snail、Notch1 mRNA及蛋白表达明显降低(P<0.05),细胞迁移抑制率及凋亡率、细胞E-cadherin mRNA及蛋白表达明显升高(P<0.05),且上述指标变化与秦皮乙素的剂量呈依赖性。结论:秦皮乙素能够抑制TGF-β1诱导的A549细胞上皮间质化,并抑制A549细胞增殖、迁移和侵袭,促进细胞凋亡,其机制可能与调节AMPK/NOTCH1通路有关。

Objective:To study the effect of aesculetin on the epithelial-mesenchymal transition of lung cancer A549 cells induced by transforming growth factor(TGF)-β1 and its regulation of AMPK/Notch1 pathway.Methods:A549 cells were mixed with TGF-β1(final concentration:5 ng/mL)to induce the epithelial-mesenchymal transition of the cells.Then cells were classified into model control group and aesculetin groups(200,400,800μg/mL),and a blank control group was designed.Then corresponding drugs or culture were added.Methyl thiazolyl tetrazolium(MTT)assay was used to detect the proliferation rate.The migration inhibition rate and the invasion rate of A549 cells were detected,and flow cytometry was used to detect the apoptosis rate of A549 cells.Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)was employed to determine the mRNA levels of epithelial cadherin(E-cadherin),fibronectin,vimentin,Snail,AMPK,and Notch1 in A549 cells,and Western blotting to determine the protein levels of E-cadherin,fibronectin,vimentin,Snail,AMPK,p-AMPK,and Notch1 in A549 cells.Results:The migration inhibition rate,apoptosis rate,and mRNA and protein levels of E-cadherin of A549 cells were lower(P<0.05)and the mRNA and protein levels of fibronectin,vimentin,Snail,and Notch1 of A549 cells were higher(P<0.05)in the model group than in the blank control group.The proliferation rate(at 24 h,48 h,and 72 h),invasion rate,and the mRNA and protein levels of fibronectin,vimentin,Snail,and Notch1 of A549 cells were lower(P<0.05)and the migration inhibition rate,apoptosis rate,and the mRNA and protein levels of E-cadherin of the A549 cells were higher(P<0.05)in the aesculetin groups(200,400,and 800μg/mL)than in the model group.Moreover,the changes of the above indexes were dose-dependent.Conclusion:Aesculetin inhibits the epithelial-mesenchymal transition of A549 cells induced by TGF-β1 and it also suppresses the proliferation,migration,and invasion of A549 cells and promotes the apoptosis of the cells,possibly by regulating the AMPK/Notch1 pathway.