Thrormbopoietin Protects Against Chemical Hypotoxia-induced Damage In Endothelial Cells

Background:Thrombopoietin(TPO)is a major cytokine for megakaryopoiesis and platelet production.The function of TPO not only in the hematopoietic system,but also in the other system,for example,endothelial cells,nerve cells,myocardium cells and ovarian tissue.Related research found that TPO has a protective effect.So we speculate that TPO has a protective effect on hypoxic-ischemic injury of cells.TPO has an influence on cell proliferation,maturation and apoptosis through the receptor(c-Mpl)by several intracellular signal transduction pathways.Therefore,we speculate that TPO plays a protective role through its receptor by relevant signaling pathways.Therefore,the aim of this study is to investigate the effect of TPO on hypoxia-induced apoptosis of endothelial cells.And to explore its potential mechanism.Methods:Part I The effect of TPO on CoCl2-induced apoptosis of endothelial cells1.To investigate the best conditions of TPO to protect the hypoxia-damaged endothelial cells by CCK-8.2.The cell apoptosis and the expression of Caspase-3 and Mitochondrial Membrane Potential(MMT)are determined by flow cytometry.Part II The potential mechanism of TPO to protect the hypoxia-damaged endothelial cells1.Identification of TPO receptor expressed on endothelial cells by Q-PCR and Western blot.2.The effect of TPO in PI3K/AKT pathway was detected by using Western Blot.Results:Part ? The effect of TPO on CoCl2-induced apoptosis of endothelial cells.1.The cell viability of HUVEC cells was decreased gradually with CoCl2 at a gradient of chemical concentrations,then we used CoCl2 800?mol/L(lower than IC50)as a hypoxia injury model.Pre-treated HUVEC cells with TPO 100?g/L 48h markedly increased the cell viability,compared to CoCl2 treatment alone[(45.39 ±9.22)%versus(72.10 ± 5.11)%,P = 0.025],and as a cell protection model.2.CoCl2 dramatically increased apoptosis of HUVEC cells,whereas pre-treatment with TPO rescued cell apoptosis induced by CoCl2[(12.19 ± 0.84)%versus(19.54±1.40)%,P<0.001].3.Further investigation found that TPO protection group decreased the expression of Caspase-3 induced by CoCl2 group[(28.83±3.00)%versus(43.62±1.83)%,P = 0.039].4.TPO also inhibited the reduction of MMP induced by CoCl2[(14.51 ±2.46)%versus(41.09 ± 4.51)%,P = 0.038].TPO could increased the activation of PI3K/AKT pathway in HUVECs.Part II The potential mechanism of TPO to protect the hypoxia-damaged endothelial cells.1.TPO receptor were detected in the surface of HUVECs.2.TPO could increased the activation of PI3K/AKT pathway to protect the hypoxia-damaged endothelial cellsConclusion:1.TPO has a protective effect against CoCl2-induced apoptosis of HUVECs.2.The potential mechanism is to decrease the expression of apoptpsis protease Caspase-3 and inhibiting the reduction of MMP through the PI3K/AKT pathway.