白藜芦醇阻断TGF-β1/Smad信号通路抑制脑胶质瘤细胞迁移侵袭及上皮间质转化

Resveratrol inhibits the migration and invasion of glioma cells and epithelial-mesenchymal transition by blocking TGF-β1/Smad signaling pathway

目的探究白藜芦醇对人脑胶质瘤细胞黏附、迁移、侵袭、上皮间质转化(EMT)及转化生长因子-β1(TGF-β1)/Smads信号通路的调控作用。方法体外培养人脑胶质瘤T98G细胞,分为对照组(不做干预)、实验组(12.5μmol·L^(-1)白藜芦醇组、25μmol·L^(-1)白藜芦醇组、50μmol·L^(-1)白藜芦醇组)、5-氟尿嘧啶组(50 mg·L^(-1)的5-氟尿嘧啶)、抑制剂组(50μmol·L^(-1)白藜芦醇+10μmol·L^(-1) TGF-β1/Smad通路抑制剂LY2109761)和激活剂组(50μmol·L^(-1)白藜芦醇+10μmol·L^(-1)TGF-β1/Smad通路激活剂SRI-011381),干预24 h。用细胞计数试剂盒比色(CCK-8)、倒置显微镜、黏附实验、划痕实验、Transwell小室及蛋白印迹(Wb)法对细胞活力、形态、黏附、迁移、侵袭能力及EMT和TGF-β1/Smad信号通路相关蛋白表达水平进行分析。结果与对照组比较,12.5、25μmol·L^(-1)白藜芦醇组对细胞活力没有显著变化(P>0.05),50μmol·L^(-1)白藜芦醇组和5-氟尿嘧啶组的细胞活力显著降低(P<0.05),12.5、25μmol·L^(-1)白藜芦醇组较5-氟尿嘧啶组细胞活力显著升高(P<0.05)。其中50μmol·L^(-1)白藜芦醇组细胞活力接近50%且与5-氟尿嘧啶组差异无统计学意义(P>0.05),因此,选择在该浓度基础上分别加入TGF-β1/Smad通路抑制剂LY2109761和激活剂SRI-011381进行TGF-β1/Smad通路验证,且实验组中不再选取12.5和25μmol·L^(-1)白藜芦醇组进行后续实验,其他组别不变。与对照组相比,50μmol·L^(-1)白藜芦醇组和5-氟尿嘧啶组T98G细胞生长受到抑制、细胞黏附、迁移、侵袭能力及N-钙粘蛋白(N-cadherin)、波形蛋白(Vimentin)、纤维粘连蛋白(FN)、TGF-β1、p-Smad3蛋白表达水平显著降低,而E-钙粘蛋白(E-cadherin)、Smad7蛋白表达水平显著增加(P<0.05);与50μmol·L^(-1)白藜芦醇组相比,抑制剂组T98G细胞生长进一步受到抑制、细胞黏附、迁移、侵袭能力、N-cadherin、Vimentin、FN、TGF-β1、p-Smad3蛋白表达水平显著降低(P<0.05),而E-cadherin、Smad7蛋白表达水平显著增加(P<0.05);激活剂组较50μmol·L^(-1)白藜芦醇组T98G细胞生长状态较好,细胞黏附、迁移、侵袭能力、N-cadherin、Vimentin、FN及TGF-β1、p-Smad3蛋白表达水平显著增加(P<0.05),而E-cadherin、Smad7蛋白表达水平显著降低(P<0.05)。结论白藜芦醇可显著抑制人脑胶质瘤T98G细胞的生长、黏附、迁移及侵袭,其作用机制可能与通过抑制TGF-β1/Smad通路的信号转导抑制EMT进程相关。

Objective To investigate the regulatory effects of resveratrol on the adhesion,migration invasion,epithelial-mesenchymal transition(EMT)and transforming growth factor-β1(TGF-β1)/Smads signaling pathway.Methods Human glioma T98G cells were cultured in vitro,they were divided into control group(without intervention),experimental group(12.5μmol·L^(-1)resveratrol group,25μmol·L^(-1)resveratrol group,50μmol·L^(-1)resveratrol group),positive drug group(50 mg·L^(-1)5-fluorouracil),inhibitor group(50μmol·L^(-1)resveratrol+10μmol·L^(-1)TGF-β1/Smad signaling inhibitor LY2109761)and activator group(50μmol·L^(-1)resveratrol+10μmol·L^(-1)TGF-β1/Smad signaling activator SRI-011381),intervention in 24 h.Cell viability,morphology,adhesion,migration,invasion ability,EMT and TGF-β1/Smad signaling pathway related protein expression levels were analyzed by cell count(CCK-8),inverted microscope,adhesion assay,scratch assay,transwell and western blotting.Results Compared with the control group,the proliferation activity of 12.5 and 25μmol·L^(-1)resveratrol group had no significant change(P>0.05),the cell viability of 50μmol·L^(-1)resveratrol group and positive drug group was significantly decreased(P<0.05),the cell viability of 12.5 and 25μmol·L^(-1)resveratrol groups was significantly higher than that of positive drug group(P>0.05).The cell viability of 50μmol·L^(-1)resveratrol group was close to 50%and had no significant difference from that of positive drug group(P>0.05).Therefore,TGF-β1/Smad pathway inhibitor LY2109761 and activator SRI-011381 were added to verify TGF-β1/Smad pathway on the basis of this concentration.Resveratrol groups 12.5 and 25μmol·L^(-1)were not selected for subsequent experiments in the experimental group,and the other groups were unchanged.Compared with the control group,growth of T98G cells was inhibited,the cell adhesion,migration,invasion ability and the protein expression levels of N-cadherin,Vimentin,fibronectin(FN),TGF-β1,p-Smad3 in 50μmol·L^(-1)resveratrol group and positive drug group were significantly decreased.The protein expression of E-cadherin,Smad7 were significantly increased(P<0.05).Compared with the 50μmol·L^(-1)resveratrol group,the growth of T98G cells in the inhibitor group was further inhibited,cell adhesion,migration,invasion ability,N-cadherin,Vimentin,FN,TGF-β1,p-Smad3 protein expression levels were significantly decreased(P<0.05),while the protein expression level of E-cadherin,Smad7 were significantly increased(P<0.05);Compared with 50μmol·L^(-1)resveratrol group,the growth status of T98G cells in activator group was better,the cell adhesion,migration,invasion ability,N-cadherin,Vimentin,FN,TGF-β1,p-Smad3 protein expression levels were significantly increased(P<0.05),while the protein expression level of E-cadherin,Smad7 were significantly decreased(P<0.05).Conclusion Resveratrol can significantly inhibit the growth,adhesion,migration and invasion of human glioma T98G cells,and its mechanism may be related to the inhibition of EMT process by inhibiting TGF-β1/Smad pathway signal transduction.