他莫昔芬缓解高糖诱导大鼠腹膜上皮-间质转分化的机制研究

The mechanism by which tamoxifen attenuates high glucose-induced epithelial-to-mesenchymal transition of rat peritoneal mesothelial cells

目的观察他莫昔芬(TAM)对高糖诱导大鼠腹膜上皮-间质转分化(EMT)的作用及其机制。方法2015年1月至2016年6月取正常SD雄性大鼠腹膜,原代培养腹膜间皮细胞,分为空白对照组、高糖刺激组和药物干预组。高糖刺激组:60 mmol/L高浓度葡萄糖刺激诱导大鼠腹膜间皮细胞(RPMCs)发生EMT。药物干预部分:(1)予高糖、不同浓度梯度的他莫昔芬(0.5μmol/L,2μmol/L)干预刺激RPMCs 72 h提取蛋白。(2)予高糖、2μmol/L他莫昔芬或同时予2μmol/L ER-α阻断剂干预RPMCs 1 h提取蛋白和6 h提取RNA。(3)予高糖、2μmol/L他莫昔芬或同时予1μmol/L蛋白酶体抑制剂干预RPMCs 1 h提取蛋白。Western blot分析E-cadherin、α-SMA、Smad2、p-Smad2、Smad3、p-Smad3、Smad4蛋白含量的变化,实时荧光定量聚合链式反应(PCR)检测Smad2、Smad3、结缔组织生长因子(CTGF)、纤溶酶原激活物抑制剂-1(PAI-1)的mRNA表达变化。结果他莫昔芬能够改善EMT表现,可使高糖刺激诱导下的RPMCs表达E-cadherin回升,α-SMA下降,且随着药物浓度的增加,效果更为显著(tE-cadherin=2.31、tα-SMA=-2.53,均P<0.05)。高糖刺激TGF-β1/R-Smad信号通路的Smad2/3蛋白磷酸化水平及CTGF、PAI-1的mRNA表达增加,他莫昔芬干预可显著下调上述蛋白及相关靶基因的mRNA表达(t_(p-Smad2)=-3.38、tCTGF=-3.81,均P<0.05),引入ER-α阻断剂可阻断此抑制作用。蛋白酶体抑制剂可减弱他莫昔芬对p-Smad2的抑制作用,提升p-Smad2/3蛋白水平(t_(p-Smad2)=3.94,P<0.05)。结论他莫昔芬激活腹膜间皮细胞上的ER-α,通过减少p-Smad2蛋白水平而减弱TGF-β1/R-Smad信号通路活性,有效缓解高糖刺激诱导的EMT进程,此抑制作用可能是通过蛋白酶体途径降解p-Smad2蛋白而产生。他莫昔芬在EMT中的作用可为腹膜纤维化的研究和防治提供可能的方向。

Objective To investigate the effects of tamoxifen on high glucose-induced epithelial-to-mesenchymal transition of rat peritoneal mesothelial cells and the underlying mechanism.Methods The peritoneal mesothelial cells of normal male SD rats were selected between January 2015 and June 2016 and then cultured and divided into blank control,high-glucose stimulation and drug intervention groups.High-glucose stimulation group:primary cultured rat peritoneal mesothelial cells(RPMCs)were treated with 60 mmol/L high-concentration glucose to induce epithelial-to-mesenchymal transition.Drug intervention group:(1)RPMCs were treated with 60 mmol/L high-concentration glucose and different concentrations(0.5μmol/L,2μmol/L)of tamoxifen.After 72 hours of stimulation,protein was extracted.(2)RPMCs were treated with 60 mmol/L high-concentration glucose and 2μmol/L tamoxifen with or without 2μmol/L ER-αantagonist for 1 hour to extract protein and for 6 hours to extract RNA.(3)RPMCs were treated with high-concentration glucose and 2μmol/L tamoxifen with or without 1μmol/L 1μM proteasome inhibitor for 1 hour to extract protein.Western blot analysis was performed to analyze change in E-cadherin,α-SMA,Smad2,p-Smad2,Smad3,p-Smad3 and Smad4 protein.Real-time fluorescence quantitative PCR was performed to detect the change in mRNA expression of Smad2,Smad3,connective tissue growth factor and plasminogen activator inhibitor 1.Results Tamoxifen attenuated epithelial-to-mesenchymal transition on RPMCs induced by high-level glucose,showing increased expression of epithelial cell marker E-cadherin and decreased expression ofα-SMA in a concentration-dependent manner(tE-cadherin=2.31,tα-SMA=-2.53,both P<0.05).TGF-β1/R-Smad signal pathway was activated by high-concentration glucose.Phosphorylation of Smad2/3 and mRNA expression of CTGF and PAI-1 were increased.Tamoxifen remarkably reduced protein and mRNA level of above mentioned protein and related target genes(t_(p-Smad2)=-3.38,tCTGF=-3.81,P<0.05),which could be blocked by ER-αantagonist.Finally,proteasome inhibitor could weaken the inhibitory effects of tamoxifen on p-Smad2/3 and increase p-Smad2/3 protein level(t_(p-Smad2)=3.94,P<0.05).Conclusion Tamoxifen activates ER-αon RPMCs,weakens the activation of TGF-β1/R-Smad signal pathway through decreasing p-Smad2 protein level,and effectively inhibits the progression of high-concentration glucose-induced epithelial-to-mesenchymal transition possibly through degrading p-Smad2 protein through proteasome.The role of tamoxifen in epithelial-to-mesenchymal transition may provide a possible guide for research,prevention and treatment of peritoneal fibrosis.