Soluble Epoxide Hydrolase Inhibitor Regulates The TGF-?1 Induced Transition From Human Umbilical Vein Endothelial Cells To Mesenchymal Cells

BackgroundsMyocardial fibrosis refers to the excessive proliferation of myocardial fibroblasts,over-deposition,imbalance and abnormal distribution of collagens,which can lead to cardiac interstitial remodeling under multiple pathological factors.MF is an adaptive response of the heart to a variety of stimulis,and it can also be the common pathological changes of the myocardial ischemia,cardiomyopathy,heart failure,hypertension,diabetes,pulmonary hypertension and a variety of other heart diseases to a certain stage.The occurrence of myocardial fibrosis is closely related to renin-angiotensin-aldosterone system,endothelin,nuclear factor ?B,nitric oxide,TGF-? and so on.Reducing myocardial remodeling can improve the prognosis of patients.Thus finding specific drugs to reverse myocardial fibrosis and to reduce myocardial remodeling has become a hot topic in current research.In recent years,with the gradually deepening understanding of the mechanism of myocardial fibrosis,the role of endothelial-mesenchymal transition in myocardial fibrosis has been attracting more and more researchers attention.Endothelialmesenchymal transition is a special type of epithelial-mesenchymal transition.It is a process in which endothelial cells are gradually lost its morphology and function under the stimulation of various factors,and eventually transformed into mesenchymal cells and then they can proliferate and synthesis collagens.TGF-? signaling,Notch signaling,Wnt signaling,microRNAs and some inflammatory factors may be involved in the regulation of EndMT,in which TGF-?1 / Smads signaling pathway is the most studied one.Epoxide hydrolase is commonly found in mammals and it has influences on multiple aspects of cardiovascular diseases,for examples,it can diastoles blood vessels,reverses inflammatory,stimulates the formation of blood vessels and other effects.Soluble epoxide hydrolase inhibitor(sEHI)is an inhibitor of EH.It is involved in the inhibition of polycyclic aromatic hydrocarbons epoxides,epoxidecatrienoic(EET)acid and other epoxide metabolisms.It has the role of reversing myocardial fibrosis,regulating blood lipids,lowering blood pressure and reducing myocardial ischemia and reperfusion injury and so on.However,whether sEHI has the role of anti-myocardial fibrosis by inhibiting EndMT and whether its anti-fibrosis effect is achieved by inhibiting TGF-?1 / Smads signaling pathway have not been reported.ObjectiveTo observe the effects of soluble epoxide hydrolase inhibitor(t-AUCB)on human umbilical vein endothelial cells(HUVEC-12)undergoing TGF-?1 induced Endothelial-to-mesenchymal transition and investigate the potential molecular mechanism.MethodsTGF-?1(10?g/L)was used to induce EndMT.CCK-8 kit was used to test cell growth after stimulated by TGF-?1(10ng/ml)and/or t-AUCB(50?mol/L)for 72 h.Real-Time PCR(RT-PCR)was used to determine the expressions of epithelial and mesenchymal markers as well as the expressions of the key regulatory factors.Microscope was used to test changes of the cell morphology;Western blot(WB)was used to detect changes of signaling pathways.Results(1)The TGF-?1(10?g/L)and/or t-AUCB(50?mol/L)had similar effect on cell viability(all P>0.05).(2)The results of RT-PCR indicated that the expression of endothelial cell marker CD31 was significantly down-regulated,while gene expressions of mesenchymal markers Collagen I,Collagen III and vimentin were significantly up-regulated after stimulated by TGF-?1(10?g/L)for 72 h.This phenomenon could be significantly reversed after stimulated by TGF-?1 and t-AUCB(50?mol/L)for 72h(all P<0.05).(3)The control group presented cobblestone-appearance under the light microscope and they connected tightly.Cells of TGF-?1(10?g/L)group were converted to elongated cells,and they connected loosely.After the joint intervention of TGF-?1 and t-AUCB(50?mol/L)for 72 h,cell morphology was similar to the control group(all P<0.05).(4)The results of WB indicated that TGF-?1(10?g/L)can significantly increase the phosphorylation of Smad2/3,and t-AUCB(50?mol/L)can significantly reverse the above changes(all P<0.05).(5)Gene expressions of snail1,twist1,twist2,ZEB1 were significantly higher than the control group;The joint intervention of t-AUCB(50?mol/L)and TGF-?1(10?g/L)can significantly inhibit their expressions(all P<0.05).Conclusionst-AUCB can inhibit the TGF-?1 induced transition from human umbilical vein endothelial cells to mesenchymal cells,and it provides the experimental evidence for clinical prospects of anti-myocardial fibrosisi medicine.