黄芪-莪术协同作用调节SIRT3/HIF-1α通路对人肺腺癌A549细胞侵袭迁移的影响

Effects of milkvetch root and zedoray rhizome combined on the invasion and migration of A549 cells through regulating the SIRT3/HIF-1αpathway

目的观察黄芪-莪术药对通过调节SIRT3/HIF-1α信号通路对人肺腺癌A549细胞增殖、凋亡及侵袭和迁移能力的影响。方法复制C57BL/6小鼠Lewis肺癌移植瘤模型,观察黄芪-莪术不同配比(1:1、2:1、3:1)干预14 d后对移植瘤重和肺转移的影响,计算抑瘤率,以抑瘤率优选黄芪-莪术配比;体外培养人肺腺癌A549细胞,采用CCK-8法检测不同浓度黄芪-莪术提取物对A549细胞增殖的抑制作用,筛选其最佳给药浓度和作用时间,流式细胞仪检测A549细胞凋亡率,细胞划痕实验和Transwell实验检测A549细胞移动性及侵袭能力变化,Western-blot检测侵袭相关蛋白(COX-2、CD44v6、MMP-9)的表达水平,Western-blot及RT-PCR检测SIRT3/HIF-1α通路蛋白和mRNA表达。结果黄芪-莪术不同配比干预后,小鼠瘤重和肺转移灶数明显减少(P<0.05),其中3:1配比抑瘤作用最显著(P<0.05);黄芪-莪术提取物0.4~0.8 mg/mL干预48 h对A549细胞增殖的抑制作用明显,应用0.4、0.6、0.8 mg/mL浓度处理48 h后,A549细胞凋亡率较空白对照组明显提升,迁移能力减弱,创面愈合率明显降低,侵袭细胞数明显减少(P<0.05),细胞COX-2、CD44v6、MMP-9的蛋白表达显著降低(P<0.05),同时明显下调了HIF-1α的蛋白和m RNA表达,上调了SIRT3的蛋白和m RNA表达(P<0.05)。结论黄芪-莪术药对可有效抑制肺癌肿瘤的生长和转移,并能通过调节SIRT3/HIF-1α信号通路发挥对A549细胞的促凋亡和抗侵袭、迁移作用。

Objective To observe the effects of couplet medicines of milkvetch root and zedoray rhizome on the proliferation,apoptosis,invasion and migration of human lung adenocarcinoma A549 cells through regulating the SIRT3/HIF-la signal pathway.Methods A transplanted tumor model of Lewis lung cancer in C57BL/6 mice was replicated.The effects of different ratios of milkvetch root and zedoray rhizome(1:1,2:1,3:1)on the weight of the transplanted tumor and its lung metastasis after 14 days of intervention were observed,and their tumor inhibition rates were calculated.The ratio of milkvetch root and zedoray rhizome was optimized according to the tumor inhibition rate.Human lung adenocarcinoma A549 cells were cultured in vitro.The inhibitory efiects of different concentrations of milkvetch root and zedoray rhizome extracts on the proliferation of A549 cells were detected by the CCK-8 method,and the optimal administration concentration and action time were selected.The apoptosis rate of A549 cells was detected by the flow cytometry,while the mobility and invasion ability ofA549 cells were detected by the cell scratch test and the Transwell test.The expression levels of invasion related proteins(COX-2,CD44v6,MMP-9)were detected by the Western-blot.The protein and mRNA expression levels of the SIRT3/HIF-1αpathway were detected by the Western-blot and RT-PCR.Results After the intervention of different ratios of milkvetch root and zedoray rhizome,the tumor weight and the number of lung metastases in mice decreased significantly(P<0.05),and the 3:1 ratio had the most significant antitumor effect(P<0.05).The milkvetch root and zedoray rhizome extract(0.4-0.8 mg/mL)intervention for 48 hours significantly inhibited the proliferation ofA549 cells.Compared with the blank control group,after a treatment with the concentrations of 0.4,0.6 and 0.8 mg/mL for 48 hours,the apoptosis rate of A549 cells increased significantly,the migration ability decreased,the wound healing rate decreased significantly,and the number of invasive cells decreased significantly(P<0.05).The protein expression levels of COX-2,CD44v6 and MMP-9 decreased significantly(P<0.05).The protein and mRNA expression levels of HIF-la were down-regulated significantly while the protein and mRNA expression levels of SIRT3 were up-regulated(P<0.05).Conclusion The couplet medicines of milkvetch root and zedoray rhizome can effectively inhibit the growth and metastasis of lung cancer tumors,promoting the apoptosis and resisting the invasion and migration ofA549 cells through regulating the SIRT3/HIF-1αsignal pathway.