Protective Effects And Its Mechanism Of Helix B-surface Peptide Against Cardiac Microvascular Endothelial Cell Injury Induced By Ischemia/reperfusion

Background: Acute myocardial infarction(AMI) is the leading cause of death and morbidity worldwide. For patients presenting with acute myocardial infarction, timely myocardial reperfusion using either thrombolytic therapy or primary percutaneous coronary intervention(PCI) is the most effective therapy for limiting myocardial infarct(MI) size, preserving left-ventricular contractile function and reducing the onset of heart failure. However, reperfusion itself could initiate additional injury,i.e.,myocardial ischemia/ reperfusion injury(MI/RI). It could paradoxically reduce the beneficial effects of myocardial reperfusion and cause systolic dysfunction and cellular damage,lacking of effective strategies of prevention cure. As a critical regulator of hematopoiesis,Erythropoietin(EPO) has been widely used in the treatment of anemia. Over the past decade,many nonhematopoietic actions of EPOhave been identified,including suppression of atherosclerosis. Accumulating evidence has implicated that erythropoietin(EPO) could reduce ischemia/reperfusion associated tissue injury(for example, stroke, liver ischemia, myocardial infarction, hemorrhagic shock and acute kidney injury). However, prothrombotic adverse effects of EPO hinder its clinical utilization in nonanemic patients.A helix-B surface peptide(HBSP),which is composed of 11 amino acids derived from the aqueous face of helix B of EPO,recently was developed and retained tissue-protective but avoiding erythropoietic property of EPO.Our previous experiment results have demonstrated that HBSP could reduce myocardial ischemia/ reperfusion(MI/R) injury of Sprague-Dawley rats and increase the post ischemic myocardial functions via activating the phosphatidylinositol 3-kinase(PI3-K)-Akt cascade.However,it is still unclear whether HBSP can protect the cardiac microvascular endothelial cells(CMECs) when subjected to ischemia/reperfusion injury and the downstream effectors of Akt. Therefore,the aims of the this study were to investigate the protective effect of HBSP against cardiac microvascular endothelial cells(CMECs) injury induced by ischemia/ reperfusion and further explored the underlying mechanisms involved.Methods: CMECs isolated from the adult hearts of Sprague-Dawley rats were exposed to hypoxia(94% N2, 5% CO2,1% O2) and ischemia buffer for 2h followed by 4h reoxygenation(95% air, 5% CO2).Then CMECs were randomized to receive different concentrations of EPO-derived peptides HBSP,EPO,HBSP plus LY294002(specific inhibitor of PI3K),HBSP plus rapamycin(specific inhibitor of m TOR) at the start of reperfusion.The cell viability of CEMCs was measured by MTT colorimetric assay and the apoptosis of CEMCs was detected by Tunel method.The wound scratch assay and transwell method were performed to detect the migration of CMECs.The expression of p-Akt,p-m TOR and p-p70S6 K were analyzed by western blot analysis.Results: Both cell viability and migration ability of CMECs were impaired after SI/R(P<0.01vs.control), and the apoptotic index increased in comparison with control group(P<0.01).While administration of rh EPO and HBSP during reperfusion dramatically attenuated the dysfunction of CMECs.Compared with the SI/R group, HBSP treatment in CMECs exerted protective effects as evidenced by the increase of cell viability(P<0.05),inhibited CMECs apoptosis(P<0.01) and improved the migration ability of CMECs(P<0.05).Moreover,HBSP caused over Akt phosphorylation in the reperfusion CMECs,which was abrogated by the treatment of LY294002(P<0.05), but not by rapamycin. Furthermore, m TOR phosphorylation following HBSP treatment was prevented by either LY294002 or rapamycin(P<0.05).Similarly,the phosphorylation of the m TOR downstream molecule p70S6 K were up-regulated by HBSP treatment(P<0.05). While treating with LY294002 or rapamycin prevented HBSP-induced phosphorylation of p70S6K(P<0.05).Compared with the HBSP group,the apoptotic index incresed while treating with LY294002 or rapamycin(P<0.05).Conclusion: HBSP might has protective effect of CMECs against ischemia/ reperfusion injury,which may be related to the activation of PI3K/Akt/m TOR signaling pathway.