葛根素抑制非小细胞肺癌A549细胞增殖、侵袭和迁移的机制探讨

目的:探讨葛根素对非小细胞肺癌A549细胞增殖、侵袭和迁移的作用及其机制。方法:不同浓度葛根素处理非小细胞肺癌A549细胞后，采用CCK-8法检测葛根素对非小细胞肺癌A549细胞增殖抑制作用及半数抑制浓度（half-inhibitory concentration，IC50）；显微镜下观察葛根素对非小细胞肺癌A549细胞形态的影响；平板克隆实验检测葛根素对非小细胞肺癌A549细胞体外生长能力的影响；Annexin V/PI双染流式细胞术检测肺癌细胞凋亡率；Transwell实验和细胞划痕愈合实验检测葛根素对非小细胞肺癌A549细胞侵袭和迁移能力的影响；Western blot实验检测葛根素作用于非小细胞肺癌A549细胞后MMP-2、MMP-9、p-p65、p65蛋白表达的变化。结果:与对照组相比，葛根素呈剂量依赖性（0.8 mmol/L、1.6 mmol/L、3.2 mmol/L）抑制A549细胞的细胞活力（P＜0.05），葛根素作用于A549细胞48 h后的IC50为（1.917±0.119）mmol/L。葛根素能够明显改变A549细胞的形态，表现为细胞数目减少，细胞皱缩变圆，细胞贴壁能力减弱，细胞间距变大，细胞丝状伪足减少。与对照组相比，2 mmol/L和3 mmol/L的葛根素均能明显抑制A549细胞的集落形成能力（P＜0.05）。与对照组相比，葛根素呈剂量依赖性（1 mmol/L、2 mmol/L、3 mmol/L）诱导A549细胞凋亡（P＜0.05）。与对照组相比，2 mmol/L和3 mmol/L的葛根素均能明显抑制A549细胞侵袭能力（P＜0.05）。与对照组相比，葛根素呈剂量依赖性（1 mmol/L、2 mmol/L、3 mmol/L）抑制A549细胞迁移能力（P＜0.05）。与对照组相比，葛根素呈剂量依赖性（1 mmol/L、2 mmol/L、3 mmol/L）抑制A549细胞中MMP-2蛋白、MMP-9蛋白表达和降低p-p65/p65的比值（P＜0.05）。结论:葛根素可能通过NF-κB信号通路抑制肺癌A549细胞增殖、侵袭和迁移能力。

Study on the mechanism of puerarin in inhibiting the proliferation,invasion and migration of non-small cell lung cancer A549 cells

Objective:To investigate the effect of puerarin on the proliferation,invasion and migration of non-small cell lung cancer A549 cells and its mechanism.Methods:After treating non-small cell lung cancer A549 cells with different concentrations of puerarin,CCK-8 was used to detect the inhibitory effect of puerarin on the proliferation of non-small cell lung cancer A549 cells and the IC₅₀.Microscope was used to observe the effect of puerarin on the cell morphology of non-small cell lung cancer A549 cells.Plate cloning experiment was used to detect the effect of puerarin on the growth of non-small cell lung cancer A549 cells in vitro.Annexin V/PI double staining flow cytometry was used to detect lung cancer cell apoptosis rate.Transwell test and scratch test were used to detect the effect of puerarin on the invasion and migration ability of non-small cell lung cancer A549 cells.Western blot test was used to detect the effect of puerarin on MMP-2,MMP-9,p-p65,p65 protein expression of non-small cell lung cancer A549 cells.Results:Compared with the control group,puerarin inhibited the cell viability of A549 cells in a dose-dependent manner(0.8 mmol/L,1.6 mmol/L,3.2 mmol/L)(P<0.05),and the IC₅₀ of puerarin after acting on A549 cells for 48 hours was(1.917±0.119)mmol/L.Puerarin can significantly change the morphology of A549 cells,which is manifested as a decrease in cell number,shrinkage and rounding of cells,weakened cell attachment ability,increased cell spacing,and decreased filopodia.Compared with the control group,both 2 mmol/L and 3 mmol/L puerarin can significantly inhibit the colony forming ability of A549 cells(P<0.05).Compared with the control group,puerarin induced A549 cell apoptosis in a dose-dependent manner(1 mmol/L,2 mmol/L,3 mmol/L)(P<0.05).Compared with the control group,both 2 mmol/L and 3 mmol/L puerarin can significantly inhibit the invasion ability of A549 cells(P<0.05).Compared with the control group,puerarin inhibited the migration ability of A549 cells in a dose-dependent manner(1 mmol/L,2 mmol/L,3 mmol/L)(P<0.05).Compared with the control group,puerarin inhibited the expression of MMP-2 protein,MMP-9 protein and p-p65/p65 in A549 cells in a dose-dependent manner(1 mmol/L,2 mmol/L,3 mmol/L)(P<0.05).Conclusion:Puerarin may inhibit the proliferation,invasion and migration of lung cancer A549 cells through the NF-κB signaling pathway.