三七总皂苷对肺癌A549细胞上皮间质转化的抑制作用

Inhibitory Effect of Panax Notoginseng Saponins on Epithelial-mesenchymal Transition in Lung Cancer A549 Cells

目的观察三七总皂苷(PNS)对体外经转化生长因子-β_1(TGF-β_1)诱导肺腺癌A549细胞后发生上皮细胞间质化(EMT)的作用。方法首先进行PNS MTT实验,然后再进行PNS干预EMT实验,将A549细胞随机分为5组:空白组加入F-12培养基,模型组及低、中、高浓度中药组分别加入10 ng/mL TGF-β_1和62.5、125、250μg/mL PNS,继续培养72 h。采用光学显微镜观察各组细胞的形态变化,采用实时荧光定量PCR、Western Blot及免疫荧光技术检测纤维连接蛋白(FN)、波形蛋白(Vim)、E-钙黏素(E-Cad)mRNA及蛋白表达水平。结果 31.25～500μg/mL PNS对A549细胞无明显增殖作用。与空白组比较,模型组FN mRNA及蛋白表达升高(P<0.01,P<0.05),Vim蛋白表达升高(P<0.05),E-Cad mRNA及蛋白表达降低(P<0.05)。与模型组比较,高浓度中药组FN mRNA表达降低(P<0.05),E-Cad及蛋白mRNA表达升高(P<0.01,P<0.05);低浓度、高浓度中药组FN蛋白表达降低(P<0.05),低、中浓度中药组Vim蛋白表达降低(P<0.05)。结论 PNS体外干预可提高EMT过程中上皮标志物E-Cad并抑制间质标志物FN、Vim的表达,高浓度时结果更为显著,对EMT过程有一定的干预作用。

Objective To investigate the effect of Panax notoginseng saponins(PNS) on the epithelial-mesenchymal transition(EMT) induced by TGF-β1 in lung adenocarcinoma A549 cells. MethodsPNS MTT experiment was conducted, and then A549 cells were randomly divided into blank group, model group, low, medium and high concentration PNS intervention group. And the blank group was added F-12 medium, model group was added TGF-β1(10 ng/mL), the PNS groups was added low(62.5 μg/mL), middle(125 μg/mL) and high(250 μg/mL) concentration PNS respectively. The morphological changes of cells in each group were observed by optical microscopy, and the expression levels of fibronectin(FN), Vimentin(Vim), E-Cadherin(E-Cad) mRNA and protein were detected by Real-time quantitative PCR, Western Blot and immunofluorescence. Results PNS at 31.25-500 μg/mL had no obvious effect on the proliferation of A549 cells. Compared with the blank group, PNS intervention EMT showed increased FN mRNA and protein expression in the model group(P<0.01, P<0.05), Vim protein expression increased(P<0.05), E-Cad mRNA and protein expression decreased(P<0.05).Compared with the model group, FN mRNA expression decreased in the high concentration Chinese medicine group(P<0.05), E-Cad and protein mRNA expression were increased(P<0.01, P<0.05);FN protein expression decreased in the group with low concentration and high concentration of TCM(P<0.05), Vim protein expression decreased in the group with low and medium concentration(P<0.05). Conclusion PNS can improve the epithelial marker E-Cad and inhibit the expression of mesenchymal markers FN and Vim in the EMT process in vitro, and the results are more significant at high concentration, and thereby PNS showing certain intervention effect on the EMT process was concluded.