青蒿提取物对镉暴露致人肺上皮细胞恶性转化及对Wnt、β-catenin表达的影响

Effect of Artemisia annua extract on malignant transformation of human lung epithelial cells induced by cadmium exposure and expression of Wnt and β-catenin

目的 研究青蒿提取物对镉暴露致人肺上皮细胞恶性转化及Wnt、β-catenin表达的影响。方法 设人肺上皮细胞A549组、镉暴露组(100μmol/L CdCl_(2))及青蒿提取物低、中、高剂量人肺上皮细胞A549组(100μmol/L CdCl_(2)+15、30、60μmol/L的青蒿提取物);各组每孔设6个平行样,培养72 h。细胞计数试剂盒-8测定细胞增殖水平,Transwell室、划痕试验测定细胞侵袭水平,流式细胞仪测定细胞凋亡水平,实时荧光逆转录法及蛋白印法测定细胞Wnt、β-catenin水平。结果 与人肺上皮细胞A549组比较,镉暴露组OD值(0.81±0.07 vs 0.92±0.05)、存活率[(80.62±6.69)%vs(89.63±9.57)%]、穿膜数[(198.54±82.65)vs(980.92±89.05)个]、迁移距离[(35.69±9.63)vs(105.69±8.05)μm]、Wnt mRNA(1.12±0.32 vs 3.28±0.79)、β-catenin mRNA(1.15±0.26 vs 3.56±0.23)、Wnt蛋白(0.26±0.05 vs 1.24±0.06)、β-catenin蛋白(0.34±0.07 vs 1.32±0.06)表达水平均升高(均P<0.05),凋亡率降低[(2.17±0.45)%vs(0.90±0.52)%](P<0.05);与镉暴露组比较,青蒿提取物各剂量组OD值、存活率、穿膜数、迁移距离、Wnt、β-catenin mRNA蛋白表达水平降低(均P<0.05),凋亡率升高[(0.90±0.52)%vs(1.22±0.39)%、(2.39±0.48)%、(4.12±0.51)%](P<0.05),且随着青蒿提取物干预剂量的增加,青蒿提取物各剂量组OD值、存活率、穿膜数、迁移距离、Wnt、β-catenin mRNA蛋白表达水平逐渐降低,凋亡率逐渐升高,具有剂量-效应关系(P<0.05)。结论 青蒿提取物能抑制镉暴露下人肺上皮细胞恶性转化,其机制可能与青蒿提取物抑制Wnt、β-catenint的表达进而抑制Wnt/β-catenin通路的激活有关。

Objective To study the effect of Artemisia annua extract on the malignant transformation of human lung epithelial cells induced by cadmium exposure and the expression of Wnt andβ-catenin.Methods There were human lung epithelial cell A549group,cadmium exposure group(100μmol/L CdCl_(2)),low,medium and high dose of Artemisia annua extract human lung epithelial cell A549 group(100μmol/L CdCl_(2)+15,30,60μmol/L Artemisia annua extract),and parallel samples were set up for each well in each group,cultured for 72 h.The cell counting kit-8 was used to measure cell proliferation level.Transwell chamber and scratch test were used to measure cell invasion levels.The flow cytometry was used to measure cell apoptosis levels.The real-time fluorescence reverse transcription and western blotting were used to measure cell Wnt andβ-catenin levels.Results Compared with the human lung epithelial cell A549 group,the OD value(0.81±0.07 vs 0.92±0.05),survival rate[(80.62±6.69)%vs(89.63±9.57)%],transmembrane number[(198.54±82.65)vs(980.92±89.05)cells],migration distance[(35.69±9.63)vs(105.69±8.05)μm],and levels of Wnt mRNA(1.12±0.32 vs 3.28±0.79),β-catenin mRNA(1.15±0.26 vs 3.56±0.23),Wnt protein(0.26±0.05 vs1.24±0.06),β-catenin protein(0.34±0.07 vs 1.32±0.06)in the cadmium exposure group were increased(all P<0.05),and apoptosis rate were decreased[(2.17±0.45)%vs(0.90±0.52)%](P<0.05).Compared with the cadmium exposure group,the OD value,survival rate,transmembrane number,migration distance,Wnt,β-catenin mRNA protein expression levels in Artemisia annua extract groups of were decreased(all P<0.05),while the apoptosis rate were increased[(0.90±0.52)%vs(1.22±0.39)%,(2.39±0.48)%,(4.12±0.51)%](P<0.05).With the increase of the intervention dose of Artemisia annua,the OD value,survival rate,transmembrane number,migration distance,Wnt,β-catenin mRNA protein expression levels in each dose group of Artemisia annua extract gradually decreased,and the apoptosis rate gradually increased,with a dose-effect relationship(P<0.05).Conclusion Artemisia annua extract can inhibit the malignant transformation of human lung epithelial cells under cadmium exposure,and its mechanism may be related to the inhibition of the expression of Wnt andβ-catenint by the extract of Artemisia annua,so as to inhibit the activation of Wnt/β-catenin pathway.