Objective To investigate the effect and possible mechanism of the drug hirudin on hypoxia induced human microvascular endothelial cells(HCMECs)mesenchymal transition(EndMT).Methods HCMECs cells cultured con-ventionally were randomly divided into control group,Hypoxia group and Hirudin group.The control group was routinely cultured without any treatment.The hypoxic group was placed in a hypoxic incubator for 72h,and the Hirudin group was pre-added with Hirudin working solution,and then placed in a hypoxic incubator for 72h after 4h.The proliferation capacity of HCMECs was detected by MTS colorimetry.The morphology of HCMECs was observed by inverted microscope.Immu-nofluorescence identification of HCMECs interstitial trans differentiation,Western Blot detection of endothelial interstitial trans differentiation related proteins:Endothelial cells labeled platelet endothelial cell adhesion molecule(PECAM-1/CD31)and vascular endothelial cadherin(VE-cadherin),and stromal cells labeled α smooth muscle actin(α-SMA)and fibroblast specific protein 1(FSP-1).And the expression of hypoxia inducible factor-1α(HIF-1α),transforming growth factor β1(TGF-β1),Smad homology 2/3(Smad2/3),Zinc finger transcription factor(snail)related signaling pathways.Results MTS showed that hypoxia significantly inhibited cell activity(P＜0.01),and the cell activity was strongest when hirudin concentration was 100μg/ml(P＜0.01).After 72h of cell culture in each group,the cells in the control group showed a pave-like or pebble-like structure under an inverted microscope,while the cells in the hypoxic group changed from pebble-like structure to dispersed long spindle shape,close to the shape of fibroblasts.Western Blot and immunofluorescence results showed:Compared with the normal group,the protein levels of CD31 and VE cadherin in hypoxia group were decreased(P＜0.01),and the expres-sion of vWF was decreased.α-SMA and FSP-1 protein levels increased(P＜0.01)and vimentin expression was enhanced.Compared with hypoxia group,hirudin significantly increased the expression of CD31 and VE-cadherin(P＜0.01),enhanced the expression of vWF,and down-regulated α-SMA.The expression of FSP-1 protein(P＜0.01)decreased the expression of vimentin.Compared with the control group,hypoxic group of TGF-β1,HIF-1α,p smad2/3,snail protein expression(P＜0.01)were higher,than to the hypoxia group,Cut HIF-1α,hirudin TGF-β1,p smad2/3,snail protein expression(P＜0.01).Conclu-sion Hirudin can improve hypoxia induced EndMT in HCMECs cells,and the mechanism may be related to HIF-α/TGF-β1/smad/snail pathway.
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