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
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摘要
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(End MT)的影响。方法:采用t-AUCB(50μmol/L)预处理HUVEC 40 min后,用TGF-β1(10μg/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(collagenⅠ、collagenⅢ、vimentin)及End MT中重要的下游转录因子(snail1、twist1、twist2、ZEB1)mRNA的表达。同时设TGF-β1和tAUCB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,collagenⅠ、collagenⅢ、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End MT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 endothelialto-mesenchymal transition( End MT) 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-8kit 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,collagenⅢ,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 EndM T.
Aim: To observe the effect of soluble epoxide hydrolase inhibitor t-AUCB on TGF-β1 induced endothelialto-mesenchymal transition( End MT) 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-8kit 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,collagenⅢ,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 EndM T.
引文
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[11]MARKWALD RR,FITZHARRIS TP,MANASEK FJ.Structural development of endocardial cushions[J].Am J Anat,1977,148(1):85
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[13]XU D,LI N,HE Y,et al.Prevention and reversal of cardiac hypertrophy by soluble epoxide hydrolase inhibitors[J].Proc Natl Acad Sci USA,2006,103(49):18733
[2]KRENNING G,ZEISBERG EM,KALLURI R.The origin of fibroblasts and mechanism of cardiac fibrosis[J].J Cell Physiol,2010,225(3):631
[3]BUJAK M,REN G,KWEON HJ,et al.Essential role of Smad3 in infarct healing and in the pathogenesis of cardiac remodeling[J].Circulation,2007,116(19):2127
[4]WIDYANTORO B,EMOTO N,NAKAYAMA K,et al.Endothelial cell-derived endothelin-1 promotes cardiac fibrosis in diabetic hearts through stimulation of endothelial-to-mesenchymal transition[J].Circulation,2010,121(22):2407
[5]FENG XH,DERYNCK R.Specificity and versatility in tgfbeta signaling through Smads[J].Annu Rev Cell Dev Biol,2005,21(21):659
[6]DAVIS BB,THOMPSON DA,HOWARD LL,et al.Inhibitors of soluble epoxide hydrolase attenuate vascular smooth muscle cell proliferation[J].Proc Natl Acad Sci USA,2002,99(4):2222
[7]QIN J,SUN D,JIANG H,et al.Inhibition of soluble epoxide hydrolase increases coronary perfusion in mice[J].Physiol Rep,2015,3(6):12427
[8]SHEN L,PENG H,ZHAO S,et al.A potent soluble epoxide hydrolase inhibitor,t-AUCB,modulates cholesterol balance and oxidized low density lipoprotein metabolism in adipocytes in vitro[J].Biol Chem,2014,395(4):443
[9]PORTER KE,TURNER NA.Cardiac fibroblasts:at the heart of myocardial remodeling[J].Pharmacol Ther,2009,123(2):255
[10]SIRISH P,LI N,LIU JY,et al.Unique mechanistic insights into the beneficial effects of soluble epoxide hydrolase inhibitors in the prevention of cardiac fibrosis[J].Proc Natl Acad Sci U S A,2013,110(14):5618
[11]MARKWALD RR,FITZHARRIS TP,MANASEK FJ.Structural development of endocardial cushions[J].Am J Anat,1977,148(1):85
[12]VALCOURT U,KOWANETZ M,NIIMI H,et al.TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition[J].Mol Biol Cell,2005,16(4):1987
[13]XU D,LI N,HE Y,et al.Prevention and reversal of cardiac hypertrophy by soluble epoxide hydrolase inhibitors[J].Proc Natl Acad Sci USA,2006,103(49):18733