The Protective Effects of Erythropoietin on Myocardial Ischemia-reperfusion Injury
|School||China Medical University|
|Keywords||erythropoietin (EPO) ischemia reperfusion injury (IRI) TNF-α IL-6 NF-κB HIF-1α Akt|
ObjectivesPercutaneous coronary intervention(PCI)has already been the main mechanism in the therapy of myocardial infarction,since then ischemia reperfusion injury(IRI) associated with percutaneous coronary intervention(PCI)also became the hot issue in research region.With the in-depth understanding of erythropoietin(EPO),EPO is administered in sufficient amount can protect cardiac myocytes from ischemic reperfusion injury.Currently,only animal can be used in the study and ligation of coronary artery is the base of the IRI model establishment.The first part of this study is going to establish IRI model by coronary occlusion through saccule intervention.This model is more similar to the clinical therapy of PCI in myocardial infarction.Based on this to detect ventricular arrhythmia,left ventricular function,myocardial cells death markers and myocardial infarct area after IRI and observe the protective role of EPO from IRI.With the in-depth research in cardiac myocyte ischemia reperfusion injury(IRI), more and more evidences showed that the inflammation mainly caused by neutrophils may play prominent roles in the pathogenesis of ischemia-reperfusion injury(IRI). TNF-αand IL-6 are important mediators of inflammation.The past researches showed that the nuclear factor-kappa B(NF-κB)can regulate the previous two genes in the inflammation.In the course of myocardial ischemia/reperfusion injury,angiogenesis and and promotion of branch circulation as to increase the coronary reserve are also important to protect myocardium from injury.Hypoxia-inducible factor-1(HIF-1)plays key roles in angiogenesis under hypoxia condition by regulating expressions of other growth factors.Under the hypoxia situation,the expression of HIF-1αincreased significantly.Protein kinase B(Akt)can also up-regulate HIF-1αexpression.Many studies have comfirmed that EPO can promote the proliferation of endothelial cells under endocardium.The other part of this study is to establish IRI model in rats to observe the effects of EPO on inflammation factors and HIF-1αafter myocardium IRI and to discuss the possible mechanism.MethodsThe first part1、A total of 40 pigs were divided into four groups:sham-operation group,IRI group,EPO-treated group after ischemia(EPO-1)and EPO-treated group after reperfusion(EPO-2).IRI model was established by occluding the anterior descending branch of coronary artery.The pigs in EPO-1 group received erythropoietin(EPO) 5000 units/kg injected subcutaneously through belly at the time of occlusion,the pigs in EPO-2 group received the same dose injection subcutaneously through belly at the time of occlusion released.2、And then make a record of the kinetic parameter of blood flowing,LVSP, LVEDP,dp/dtmax and dp/dtmin for 120 minutes reperfusion.3、Make a record of ventricular arrhythmia.4、And detect the CK and LDH levels respectively for 120 minutes reperfusion.5、Take out the heart and calculate the ratio of left ventricular infarct area to the quality of left ventricular.The second and third part1、A total of 40 male Wistar rats were randomly divided into sham-operation group, IRI group,EPO-treated group after ischemia(EPO-1)and EPO-treated group after reperfusion(EPO-2).Establishment of the model is through ligating the anterior descending branch of coronary artery.The rats in EPO-1 received erythropoietin(EPO) 5000 units/kg injected subcutaneously on belly at the time of ligation,the rats in EPO-2 received the same injection subcutaneously on belly at the time of ligation released.2、Collected vein blood for 120 minutes Reperfusion and detected TNF-α、IL-6 by ELISA assay.3、Killed the rats and took out the hearts,detected the expression of NF-κB、HIF-1αand Akt protein in myocardium by Western blots and the expression of HIF-1αmRNA in myocardium tissue by RT-PCR assay.ResultsThe first part1、Compared to the sham-operation group,all the indexes of IRI group significantly changed(P＜0.01).2、Compared to IRI group,blood engorging speed(p＜0.01),left ventricular contractive and diastoling function(p＜0.05)showed significant increase after reperfusion pretreated with Epo;however,both myocardial cells necrosis markers (p＜0.01)levels and myocardial infarct area(p＜0.05)significantly reduced.3、Compared to EPO-2,EPO-1 showed much lower myocardial cells necrosis levels and myocardial infarction areas,the difference is significant(P＜0.05).The second part1、Compared to the sham-operation group,IRI group showed increased expression of TNF-α,IL-6 and NF-κB significantly(P＜0.01).2、Compared to IRI group,groups pretreated with Epo demonstrated that expression of TNF-α、IL-6 and NF-κB decreased significantly(P＜0.01).3、Expression of TNF-α、IL-6 and NF-κB was lower in EPO-1 than EPO-2(P＜0.05).4、The expression of NF-κB and TNF-α,IL-6 showed direct correlation.The third part1、Compared to the sham-operation group,the expression of HIF-1αin IRI group significantly increased(P＜0.01),but Akt expression had no significant change(P＞0.05).2、Compared to IRI group,the expression of HIF-1αand Akt increased significantly in Epo pretreated groups(P＜0.01).3、Compared to EPO-2,the expression of HIF-1αand Akt had more significant increase in EPO-1(P＜0.05).4、HIF-1αand Akt are positive correlated.Conclusion1、EPO-treatment is effective in reducing ventricular arrhythmia,improvement of left ventricular function,decreasing myocardial infarction marker levels and myocardial infarction area.2、In aspects of decreasing myocardial infarction marker levels and myocardial infarction area,the effectiveness of EPO-treatment at the time of ischemia is much better than at the time of reperfusion,which suggests that Epo treatment in early ischemia phase may be more effective in protection of myocardial cells.3、EPO treatment can reduce the serum level of TNF-αand IL-6 to play a protective role from IRI by reducing expreesion of NF-κB pathway.4、The effectiveness of EPO-treatment administrated at the time of ischemia is much better than at the time of reperfusion.5、EPO treatment can play protective role by increasing HIF-1αand Akt expression after IRI.6、The effectiveness of EPO-treatment administrated at the time of ischemia is much better than at the time of reperfusion.