| Background and objectives:Coronary heart disease is the leading cause of death worldwide,and 3.8 million men and 3.4 million women die of the disease each year.After an acute myocardial infarction,early and successful myocardial reperfusion with the use of thrombolytic therapy or primary percutaneous coronary intervention(PCI) or coronary artery bypass graft(CABG) is the most effective strategy for reducing the size of a myocardial infarct and improving the clinical outcome.But according to the studies in animal and clinical observations,we can find that,in some animals or patients, the cell dysbolism and structural damage became more serious after ischemia-reperfusion.The phenomenon was called ischemia-reperfusion injury(IRI). The process of restoring blood flow to the ischemic myocardium also can induce injury.This phenomenon,termed myocardial ischemia-reperfusion injury,can paradoxically reduce the beneficial effects of myocardial reperfusion.Studies in animal models of acute myocardial infarction suggest mat lethal reperfusion injury accounts for up to 50%of the final size of a myocardial infarct.Therefore,to prevent or cure myocardial ischemia-reperfusion injury may improve the clinical outcome of acute myocardial infarction which was treated by reperfusion therapy.It has been a hot spot of medical science study,but,there is no drug for clinical use yet.The injury to the heart during myocardial reperfusion causes four types of cardiac dysfunction.The first type is myocardial stunning,a term denoting the"mechanical dysfunction that persists after reperfusion despite the absence of irreversible damage and despite restoration of normal or near-normal coronary flow."The myocardium usually recovers from this reversible form of injury after several days or weeks.The second type of cardiac dysfunction is no-reflow phenomenon, which was originally defined as the"inability to reperfuse a previously ischemic region."It refers to the impedance of microvascular blood flow encountered during opening of the infarct-related coronary artery.The third type of cardiac dysfunction is reperfusion arrhythmias.The last type of cardiac dysfunction is lethal reperfusion injury.Not only cellular necrosis but also cardiomyocyte apoptosis were found in myocardium after ischemia-reperfusion in studies,and cardiomyocyte apoptosis contributed to myocardial ischemia-reperfusion injury.As we know,the mechanism of myocardial ischemia-reperfusion injury maybe relate to oxygen paradox,calcium paradox,pH paradox and inflammation,and the cardiomyocyte apoptosis is an important kind of pathomechanism.Cardiomyocyte apoptosis is very important for the body to maintain stableness of tissue structure and function during myocardial ischemia-reperfusion injury.But abnormally increase of apoptosis would severely destroy the myocardium.The decrease of myocardium cells and the cardiac function disorder could be induced by cellular necrosis and cardiomyocyte apoptosis during reperfusion,they contributed to the outcome of myocardial ischemia-reperfusion injury all togeter.The apoptotic cells which had not been phagocytosed could turn to necrosis after being attacked by reactive oxygen species(ROS).There are two types of cell death which are necrosis and apoptosis during myocardial ischemia-reperfusion,but they are not inflexible,it has been proved that apoptosis could turn to necrosis while ATP was run-down or there was an inflammation.To comprehend the effect of apoptosis to myocardial ischemiareperfusion injury,apoptosis was inhibited by all kinds of methods by researchers, and the change of the structure and function of the heart was observed,the results proved that inhibition of apoptosis could diminish the myocardial infarct size almost to 50%-70%.As we known,oxygen paradox,calcium paradox and secondary mitochodria damage can induce cell apoptosis,they work all together or by oneself, they are connected with each other,everyone is cause and effect of each other.But these factors can not induce cell apoptosis directly,they activate survival or death gene by signal transmission,then the signal transmit to the incision enzyme intranuclear to perform the function of death.To block the signal transduction of cardiomyocyte apoptosis induced by myocardial ischemia-reperfusion may inhibit cardiomyocyte apoptosis,prevent or cure myocardial ischemia-reperfusion injury, and renew the function of cardiomyocyte.Erythropoietin(EPO) is a kind of sialoglycoprotein hormone,which can stimulate the marrow to produce red cell.EPO is chief made by the juxtaglomerular cells which located at the juncture between the cortex and medulla of kidney. Recombinant human Epo(rHuEpo) which had been manufactured to therapy all kinds of anemia had been synthetized artificially in 1985.Endogenous EPO has a molecular weight of 34 kDa,and the molecular weight of rHuEPO is 30.4 kDa.They have the same physico-chemical property and biologic activity.rHuEPO has a demiperiod of 4-12 hours while it was used by intravenous injection,the bioavailability of rHuEPO is 23%-42%while it was used by hypodermic injection.To produce a marked effect, the binding of EPO and EPO receptors on the membrane of target cells is necessary. EPO receptors have been discovered in numerous non-hematopoietic tissues including liver,cerebrum,womb,endothelial cell,smooth muscle cell, myocardium,and so on.Studies had found that EPO could inhibit cell apoptosis, attenuate oxidative stress,exhibit angiogenic potential,attenuate inflammation, promote stem cell migration,and protect the myocardium from ischemia-reperfusion injury in anoxia/reoxygenation models,Langendorff-perfused rat models or when it was administrated before ischemia in rats.The mechanism how EPO inhibited cell apoptosis has been studied widely and deeply.The binding of EPO and EPO receptors which were widely expressed in cardiovascular system could activate some signal transduction pathways that mediated the inhibition of apoptosis.The phosphatidylinositol-3-kinase(PI3K)/Akt pathway was one of the pathways which had been studied widely,and it was well known that the pathway mediated the inhibition of apoptosis.The earlier studies on the cardioprotection of EPO usually used anoxia/reoxygenation models or Langendorff-perfused models,and EPO was administrated before ischemia in animal studies,it maybe relate to that it need some time for EPO to be absorbed and to produce a marked effect.Obviously,the results of these researchs can not be applied to the patients who suffer from acute myocardial infarction.Therefore,it is necessary to prove the cardioprotection of EPO when it is administrated between ischemia and reperfusion,similar study is rare.In this study,we try to set up myocardial ischemia-reperfusion models in rats to simulate the pathophysiologic changes in patients which suffer from acute myocardial infarction and were treated with reperfusion therapy,and EPO was administrated by intravenous injection between ischemia and reperfusion,the whole procedure was similar to clinical treatment.The aim of this study is to demonstrate if EPO can inhibit apoptosis and protect myocardium from ischemia-reperfusion injury,discuss the mechanism involved,chiefly find out the relation between the protection and PI3K pathway,and find new evidence to protect myocardium from ischemiareperfusion injury.Our study includes three parts:The first part is to find out if EPO can protect myocardium from ischemia-reperfusion injury when it was administrated by intravenous injection between ischemia and reperfusion,especially to explore the effect of EPO on the change of cardiac ultrastructure,the marker of myocardial damage and the occurrence of arrhythmia after reperfusion.The second part is to demonstrate if EPO can inhibit apoptosis in both gene and protein,discuss the mechanism involved.The last part is to demonstrate if EPO could antioxidation.Methods:The first part of this study:The left anterior descending branch of coronary artery(LAD) of rats was ligated for 30 minutes and then loosed for 3 hours to establishischemia/reperfusion heart model.45 SD rats were randomly divided into 5 groups:The sham operation group(group SHAM),and 4 experimental groups:The ischemia/reperfusion group(group IR),the highly selected blocker of the PI3K/Akt pathway LY294002 group(group LY),the EPO group(group EPO) and the EPO plus LY294002 group(group EPO+LY).①SHAM group(5 rats):chest was opened,a suture was passed under the LAD,but the LAD was not ligated,0.5ml dimethyl sulfoxide(DMSO) was injected via vena caudalis before opening chest,0.5ml NS was injected an hour later,the rat was executed 3.5 h after opening chest;②IR group(10 rats):chest was opened,the LAD was ligated by a suture,0.5ml DMSO was injected via vena caudalis 30 min before opening chest,and 0.5ml NS was injected before reperfusion,the rat was executed after the LAD was ligated for 30 min with subsequent 3h reperfusion.③LY group(10 rats):the same surgical procedure like that of IR group,0.3mg/kg LY294002 which was resolved into 0.5 ml DMSO was injected 30 min before opening chest,and 0.5ml NS was injected before reperfusion.④EPO group(10 rats):the same surgical procedure like that of IR group,0.5ml DMSO was injected 30 min before opening chest,and 1000u/kg EPO which was resolved into 0.5 ml NS was injected before reperfusion.⑤EPO+LY group(10 rats):the same surgical procedure like that of IR group,0.3mg/kg LY294002 was injected 30 min before opening chest,and 1000u/kg EPO was injected before reperfusion.After the experiment had been done,we killed the rats by decapitation,mutilated the great vessels and tissues from the cardiac base,took out of the heart,removed the atriums,cut the heart to take the damaged cardiac muscles according to the need of detection,divided the cardiac muscles in three parts,the first part was fixed with 3%glutaral,the second part was kept in liquid nitrogen,the third part was kept in icebox at 40℃below zero.We observed the occurrence of arrhythmia in electrocardiogram of leadⅡ,and scored the ventricular arrhythmias (VA) according to the method of Ravingerova T.The changes of cardiac ultrastructure were examed by transmission electron microscope.The levels of serum CK-MB and cTnI were detected just before DMSO injection and after reperfusion.The procedure was just as below:To anesthetize rat→to collect blood sample,DMSO or LY+DMSO was injected via vena caudalis→to open chest 30minutes later,and ligate LAD→NS or EPO+NS was injected via vena caudalis 30 minutes after ligating→reperfusion began with ligation released→to collect blood sample 3 hours after reperfusion and decapitation,to collect myocardium sample.The second part of this study:The models and samples were the same as the first part,but in the first part,it had been proved that LY294002 could obviously aggravate the myocardial ischemia-reperfusion injury,so we deleted the LY group in second part.There were 35 SD rats which were divided into four groups in the second part: group SHAM,group IR,group EPO and group EPO+LY.Myocardial cells apoptosis were estimated by in situ nick end labeling(TUNEL) method,to calculate the apoptotic index(AI).The cardiac ultrastructure were examed by transmission electron microscope in order to observe the apoptotic bodies.bcl-2,box and caspase-3 mRNA transcription were detected by RT-PCR,to calculate the Ct values and relative copy numbers of samples.The third part of this study:The models,the groups were the same as the second part,the damaged cardiac muscles were collected in the second part to detect the content of malondialdehyde(MDA) in myocardium tissues by thiobarbituric acid (TBA) method,and the content of superoxide dismutase(SOD) in myocardium tissues was detected by xanthine oxidase method.Results:The first part of this study:1,The ultrastructural changes of ischemia-reperfused myocardium:The ultrastructural changes of ischemia-reperfused myocardium were observeded by transmission electron microscopy.Except the SHAM group,diffuse swelling of myocardial cells,fragmentation and depletion of myofilament,contraction of muscle rod,swelling of mitochondria,and expansion of sarcoplasmic reticulum were observed in IR,LY,EPO and EPO+LY groups,but these changes were significantly lightened in EPO group,and obviously aggravated in LY group.2,The levels of serum CK-MB and cTnI:The levels after operation were increased in all groups significantly(P＜0.05) comparing to the preoperative levels.The results reflected the necrosis of myocardium,and the coronary artery was ligated successfully.The muscles were damaged during the operation in the SHAM group. The levels in EPO group were significantlylower than those in IR,LY and EPO+LY groups(P＜0.001);The levels in LY group were higher than those in other groups (P＜0.001),and the levels in SHAM group were lower than those in other groups (P＜0.001).3,Effect to reperfusion arrhythmia:Arrhythmia was found in 40 rats,arrhythmia occurred mainly at about 5 min after the ligation of LAD and the beginning of reperfusion.The arrhythmia appeared in rats including ventricular premature beat,ventricular tachycardia(VT) and ventricular fibrillation(VF).The arrhythmia occurred after the ligation of LAD was transient,it did not last to the period of reperfusion.There were 3 rats appeared ventricular premature beat which less than 5 times,the arrhythmia occurred at an hour after the opening of chest while other rats were reperfused,so we also scored the VA in SHAM group in order to compare with other groups.There were 37 rats appeared reperfusion arrhythmia among 40 rats which had been reperfused,reperfusion arrhythmia were found in all the rats in IR,LY and EPO+LY group,and 7 rats in EPO group.There were 2 dead rats in LY group. The VA score in EPO group was more than that in SHAM group,but it was significantly less than those in IR,LY and EPO+LY groups(P＜0.05).The score in LY group was the highest(P＜0.05).The second part of this study:1,The apoptosis of myocytes:The apoptosis of myocytes was observeded by transmission electron microscopy.Disorganization of myocardium in IR,EPO and EPO+LY groups was observed.The apoptotic bodies in EPO group was more than that in SHAM group,but it was less than those in IR and EPO+LY groups.2,The apoptotic index of myocardium cell:The AI in EPO group was significantly higher than that in SHAM group(P＜0.001),but it was markedly lower than those in IR and EPO+LY groups(P＜0.001).3,The mRNA levels of bcl-2,bax and caspase-3:the mRNA level of bcl-2 in EPO group was significantlyhigher than those in SHAM,IR and EPO+LY groups (P＜0.001).The mRNA levels of box and caspase-3 in EPO group were higher than those in SHAM group(P＜0.001),but they were markedly lower than those in IR and EPO+LY groups(P＜0.001).The ratio of bcl-2/bax in EPO group was significantly higher than those in EPO,IR and EPO+LY groups(P＜0.001). The third part of this study:1,The content of SOD in myocardium tissues:the content of SOD in myocardium tissues in EPO group was less than that in SHAM group,but it was more than those in IR and EPO+LY groups,and the content in IR group was less than that in EPO+LY group,all differences between groups were significantly(P＜0.001).2,The content of MDA in myocardium tissues:the content of MDA in myocardium tissues in EPO group was more than that in SHAM group,but it was less than those in IR and EPO+LY groups,and the content in IR group was more than that in EPO+LY group,all differences between groups were significantly(P＜0.001).Conclusion:1,When EPO was administrated by intravenous injection between ischemia and reperfusion,it could significantly lighten the destruction of cardiac ultrastructure, decrease the leakage of CK-MB and cTnI which are the markers of myocardial damage,and lighten reperfusion arrhythmia.The results indicated that EPO could attenuate myocardial ischemia reperfusion injury.2,When EPO was administrated by intravenous injection between ischemia and reperfusion,it could inhibit the apoptosis of myocardium cells,but the effect could be attenuated by LY294002.In a word,EPO could:①down-regulate the mRNA expression of bax which can promote apoptosis,up-regulate the mRNA expression of bcl-2 which can inhibit apoptosis,and prevent apoptosis directly;②down-regulate the mRNA expression of caspase-3,prevent the execution of the apoptotic procedure;③activate the anti-apoptosis pathway of PI3K,inhibit the apoptosis of myocardium cells.3,Myocardial ischemia reperfusion could decrease the content of SOD which is antioxidase,increase the content of MDA which reflect the severity of the cells attacked by oxyradical.When EPO was administrated by intravenous injection between ischemia and reperfusion,it could increase the content of SOD and decrease the content of MDA in myocardium tissues,decrease the production of oxyradical, and attenuate oxidative stress.4,The cardioprotection by EPO required the PI3K pathway.
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