| Objective:To study the protective effects and mechanisms of Erythropoietin preconditioning on myocardial ischemia-reperfusion injury (MIRI) in rats.Methods:84 male SD rats were randomly divided into 3 groups:Normal group,ischemia-reperfusion group (group IR)and erythropoietin preconditioning group(group EP)。The hearts of normal group were perfused without cardioplegia and heart storage.Rats of EP group were injected with EPO(5000U/Kg) in abdominal cavity 24 h prior to operation respectively,the rats of IR group were injected with nomal sodium.We made rat MIRI model through create a animal model using a living rat take place of Langendorff modle. The hearts were arrested with St.ThomasII and stored in the St.ThomasII solution.All hearts were stored for 4h,6h and 8h with 4℃hypothermia,and underwent 60 minutes of reperfusion.The parameters of hemodynamics (HR,CF,LVDP,LVEDP,±dp/dtmax),the myocardial enzymes(LDH,CK,CK-MB)were measured before and during reperfusion. Detecting the apoptosis index(AI) of myocardial cells at the end of experiment by TUNEL. The expression of Caspase-3 protein in cardiac myocytes were assayed by the methods of Western blot. Optical microscopy and transmission electron microscopy were used to observe the pathological changes of myocardial tissue and cells respectively.Results: (1)After the hearts were stored for 4h,6h,8h,the recovery of hemodynamics and cardiac function of the EP group were significantly better than the IR group during reperfusion (P<0.05 or P<0.01 ).(2)The myocardial enzymes leakage was appeared in every group,With the time past , the levels of the myocardial enzymes(LDH,CK,CK-MB) in serum is gradually increased ,the levels of the myocardial enzymes of the EP group was less than it the IR group.(P<0.05 or P<0.01()3)All the hearts of EP group could beat again during reperfusion, but the heart function decreased.But 3 hearts stored for 8 hours of IR group could not beat again.(4) The results of TUNEL showed that cardiomyocyte apoptosis index(AI) in the Control group and EP group were significantly less than IR group(P<0.05 or P<0.01()5)The results of Westem blot showed that the expression of Caspase-3 protein can be found in all time points.In the EP group,protein expression of Caspase-3 became obviously lower than IR group at all time point(P<0.05 or P<0.01). (6)By the optical microscopy, in normal group, myocardial fibre arranged in order,the structural integrity of the nucleus. The myocardial tissue of IR group showed extensive necrosis and severe muscle fiber fracture, myocardial cells dissolution and disappearance, neutrophils exudation. EP group displayed a reduced degree of myocardial neutrophilic infiltrate,necrosis, hemorrhage, and spindle-shaped interstitial cells .After ischemia and reperfusion myocardial samples were qualitatively assessed by transmission electron microscopy for structural changes. Under the TEM, IR group showed myofibrils disarrangement, large areas of myofibrils fracture, disappearance, sarcomere structure unclear, myocardial nuclear swelling, the nuclear membrane rupture, nuclear chromatin asymmetry, condensation, margination, mitochondrial morphological abnormalities and swelling, the rige disarrangement, fracture, disappearance, forming cavity, some mitochondria parceled by double membrane, and completely desquamated from the mother cells, forming apoptotic bodies in IR group. In EP groups, myocardial ultrastructural changes were discriminating. The structural damage in EP group was relatively less than IR group.We can see more unclear sarcomeres,no significant dissolution,no fracture,almost normal mitochondria and unclear nucleolus.Conclusion: (1)Erythropoietin preconditioning in rats can promote the preserved heart's recovery of function, enhanced myocardial contractibility significantly,improved myocardial compliance.(2) Erythropoietin preconditioning can protect cardiomyocyte from ischemia reperfusion injure. The mechanism may be associated with that the Erythropoietin can anti-inflammatory, anti-oxidation, promote angiogenesis and reduce expression of caspase-3 gene and the occurrence of cardiomyocyte apoptosis. |
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