Chen Xiao, Cai Jiejie, Zhou Xi, Chen Lingzhi, Gong Yongsheng, Gao Zhan, Zhang Huaiqin, Huang Weijian, Zhou Hao
Department of Cardiology, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
Cell Physiol Biochem. 2015;36(1):191-200. doi: 10.1159/000374063. Epub 2015 Apr 30.
Fibrosis results in excessive buildup of extracellular matrix proteins along with abnormalities in structure and is partly derived by a process involving transforming growth factor β (TGF-β) called endothelial-to-mesenchymal transition (EndMT). We investigated whether the aldosterone receptor-blocker spironolactone could abrogate TGF-β-induced fibrosis in EndMT and the underlying mechanism.
Human umbilical vein endothelial cells (HUVECs) were divided into 5 groups for treatment: blank; vehicle control; TGF-β (10 ng/ml); spironolactone (1 μM)+TGF-β; and spironolactone+TGF-β+DAPT (10 μM). Cell chemotaxis was assayed by transwell assay. The expression of CD31 and vimentin was determined by Immunofluorescence staining and western blot analysis. Notch1 protein level was detected by western blot analysis.
Spironolactone significantly prevented TGF-β-stimulated EndMT by down-regulate vimentin and up-regulate CD31 in HUVECs (p<0.01).It inhibited cell migration during EndMT (p<0.01). The protective effect of spironolactone against EndMT could be attenuated by blocking the Notch signal pathway with DAPT (p<0.01). Notch signaling was activated and cross-interacted with TGF-β and spironolactone in regulating EndMT in HUVECs and reversed the spironolactone-related signaling by abrogating the antifibrotic actions with decreased Notch1 protein expression (p<0.01).
Spironolactone may have a protective role in TGF-β-induced EndMT in HUVECs mediated by the Notch signal pathway.
纤维化导致细胞外基质蛋白过度积聚以及结构异常，部分是由一种涉及转化生长因子β（TGF-β）的称为内皮-间充质转化（EndMT）的过程引起的。我们研究了醛固酮受体阻滞剂螺内酯是否可以消除TGF-β诱导的EndMT中的纤维化及其潜在机制。
将人脐静脉内皮细胞（HUVECs）分为5组进行处理：空白组；溶剂对照组；TGF-β（10 ng/ml）组；螺内酯（1 μM）+TGF-β组；以及螺内酯+TGF-β+DAPT（10 μM）组。通过Transwell实验检测细胞趋化性。通过免疫荧光染色和蛋白质印迹分析确定CD31和波形蛋白的表达。通过蛋白质印迹分析检测Notch1蛋白水平。
螺内酯通过下调HUVECs中的波形蛋白并上调CD31，显著预防了TGF-β刺激的EndMT（p<0.01）。它抑制了EndMT过程中的细胞迁移（p<0.01）。用DAPT阻断Notch信号通路可减弱螺内酯对EndMT的保护作用（p<0.01）。在调节HUVECs的EndMT过程中，Notch信号被激活并与TGF-β和螺内酯相互作用，并通过降低Notch1蛋白表达消除抗纤维化作用，从而逆转了螺内酯相关信号（p<0.01）。
螺内酯可能在Notch信号通路介导的HUVECs中TGF-β诱导的EndMT中起保护作用。
