| ObjectiveMyocardial ischemia-reperfusion (ischemic reperfusion, I/R) injury is a common clinical practice of organs and tissue injury, its precise mechanism is not yet very clear. Studies have shown that a variety of growth factors have a protective effect on the heart to ischemia and other damage from the damage caused, Although these myocardial protection factor transduction pathway is very complex, but in many cases, anti-apoptosis is a common feature of their. PI3-K (phosphatidylinositol 3-kinase)-AKT(v-akt murine thymoma viral oncogene homolog,PKB)signal transduction pathway is an important intracellular signal transduction pathway, playing an important biological function in cell apoptosis, survival, proliferation and cytoskeletal changes and so on, and the particular importance is its regulation of the apoptosis and survival.In recent years, on the I/R injury mechanism of the increasing depth of understanding of the protection of immature myocardium to further deepen and put forward some new protection strategy. Of these, erythropoietin (EPO) for myocardial protection attracted attention. Our study had proved that EPO pretreatment can significantly reduce the myocardial ischemia-reperfusion injury, has a certain role in myocardial protection on the immature myocardium, and the amount to the most significant effect is 5000U/kg. In this study, application of Langendorff isolated heart working model of immature rabbits to explore the relationship between the protection effect of EPO injection of immature myocardial ischemia-reperfusion injury and PI3-K/AKT signal transduction pathway.Materials and Methods14～21d-born Japanese long ear rabbits 32, weighing250±50g, male and female informal, offered by China Medical University animal hospital room Shengjing.30 immature rabbits were randomly divided into control group and EPO pretreatment group.15 rabbits for each group.24 hours before the model building, EPO pretreatment group by intraperitoneal injection of EPO,5000U/Kg, Control group, intraperitoneal injection of normal saline. Experiments with intraperitoneal injection of 10% immature 10ml/Kg urethane anesthesia, by intraperitoneal injection of 500IU heparin anticoagulation. The middle thoracic chest incision and the heart quickly removed and immediately immersed in 4℃K-H NS, after aortic cannulation was made after the connection langendorff isolated heart perfusion device to 37℃KH retrograde perfusion fluid, perfusion pressure constant as 60mmHg. After 15min perfusion of the natural balance, the control group and EPO group with 4℃St.ThomasⅡcardiologic for cardiologic. Four groups of heart cardiologic after 30min to 37℃K-H solution to the resumption of reperfusion after the jump 120min. At the end of the experiment, take specimens from part of the left ventricular myocardium and stored in frozen -70℃refrigerator. Until after the end of all experimental, remove the myocardial tissue, thawed, made into paraffin blocks and slice. Immunohisto chemical method to observe the myocardial reperfusion 120min expression of PI3-K, AKT, and caspase9. And Detection of apoptosis by TUNNEL method. SPSS 13.0 statistical software used for data processing, measurement data to mean±standard deviation (s) that the comparison group using repeated measures analysis of variance of data, compared with 22 multiple comparisons t test, P<0.05 for difference statistical significance.Results1. Myocardial PI3-K and AKT expression changes:The control group after ischemia-reperfusion PI3K, AKT almost no expression, EPO group after ischemia-reperfusion significantly increased expression of PI3-K and AKT.2. Myocardial caspase9 expression changes:EPO group after ischemia-reperfusion of the caspase9 almost no expression, the control group after ischemia-reperfusion significantly increased expression of caspase9.3. The changes of Cell Apoptosis:The control group shows a large number of apoptotic cells, EPO group significantly less apoptotic cells, by T test comparison, the two groups was statistically significant difference in Apoptotic index. Conclusion1. EPO pretreatment can significantly reduce the myocardial apoptosis after ischemia-reperfusion.2. EPO pretreatment significantly increased the expression of the PI3k and AKT in myocardial tissue, further phosphorylation of caspase-9 to make it lose its proteolytic enzyme activity to achieve its anti-apoptosis, thus play its role in myocardial protection of the myocardium... |
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