可溶性环氧化物水解酶抑制剂在TGF-β1诱导的人脐静脉内皮细胞内皮-间质转化中的作用*
Effect of soluble epoxide hydrolase inhibitor on endothelial-to-mesenchymal transition of human umbilical vein endothelial cells induced by TGF-β1

目的:观察可溶性环氧化物水解酶抑制剂t-AUCB对TGF-β1诱导的人脐静脉内皮细胞(HUVEC)内皮-间质转化(EndMT)的影响。方法:采用t-AUCB(50 μmol/L)预处理HUVEC 40 min后,用TGF-β1(10 μg/L)诱导HUVEC 24 h,采用Western blot法检测Smad2/3的磷酸化水平; 诱导72 h后,光镜观察细胞形态的变化,采用CCK-8试剂盒检测细胞活性,采用实时荧光定量PCR检测内皮细胞标记物(CD31)、间质细胞标记物(collagen Ⅰ、 collagen Ⅲ、vimentin)及EndMT中重要的下游转录因子(snail1、twist1、twist2、ZEB1)mRNA的表达。同时设TGF-β1和t-AUCB单独作用组。结果:光镜下,TGF-β1组细胞转变为狭长形,细胞间隙疏松,TGF-β1+t-AUCB组、t-AUCB组细胞形态正常。TGF-β1组细胞CD31 mRNA表达下调,collagen Ⅰ、 collagen Ⅲ、vimentin mRNA表达上调,snail1、twist1、twist2、ZEB1 mRNA表达上调,Smad2及Smad3蛋白磷酸化水平升高(P＜0.05)。与TGF-β1组比较,TGF-β1+t-AUCB组、t-AUCB组上述指标的表达均有一定程度的逆转(P＜0.05)。结论:t-AUCB可通过抑制内皮细胞的EndMT而发挥抗纤维化作用。
Aim: To observe the effect of soluble epoxide hydrolase inhibitor t-AUCB on TGF-β1 induced endothelial-to-mesenchymal transition(EndMT)of human umbilical vein endothelial cell(HUVEC).Methods: The HUVEC were pretreated by t-AUCB(50 μmol/L)for 40 min,then were induced by TGF-β1(10 μg/L)for 24 h,and Western blot was used to detect the phosphorylation levels of Smad2/3; after 72 h, light microscope was used to observe cell morphology,CCK-8 kit was used to test cell viability, and Real-time PCR was used to determine the mRNA expressions of epithelial marker(CD31)and mesenchymal markers(collagen Ⅰ,collagen Ⅲ,vimentin), as well as the key downstream transcription factors(snail1,twist1,twist2,ZEB1). TGF-β1 group and t-AUCB group were set.Results: The cells in TGF-β1 group changed into a long and narrow shape with intercellular porosis, while the cells in TGF-β1+t-AUCB group and t-AUCB group had normal forms. In TGF-β1 group,CD31 mRNA expression was down-regulated, but those of collagen Ⅰ, collagen Ⅲ, vimentin,snail1,twist1,twist2,and ZEB1 were up-regulated,and Smad2 and Smad3 protein phosphorylation levels in creased(P＜0.05). Compared with those of TGF-β1 group, the indexes mentioned above in TGF-β1+t-AUCB group and t-AUCB group showed reversed effect to a certain extent(P＜0.05).Conclusion: t-AUCB has an anti-fibrosis effect on the HUVEC by inhibiting the progress of EndMT