Protective Effects And Its Mechanisms Of Cardiomypeptidin For Injection On The Immature Myocardium In Young Rats

Part I: The establishment of Langendorff isolated heart perfusion model and the effect of Cardiomyopeptidin for injection on the cardiac function of the isolated hearts of young ratsObjective: 1. This experiment was designed to establish a stable Langendorff isolated heart perfusion model in the hearts of young rats; 2. to investigate the effect of cardiomyopeptidin for injection on the cardiac function of the different states isolated hearts of young rats.Methods: 50 young healthy SD rats(aged 20±3 day and weighing 50-70g)were randomly divided into five groups, Normal control group (NC group ): the isolated heart were stable 20min, and then 150 min continuous perfusion ;Normal + cardiomyopeptidin group (NCMP group ): the same as normal control group, but K-H buffer solution were added with 50 mg/L cardiomyopeptidin for injection;Control group: the isolated rat hearts were perfused with K-H buffer and then arrested with cardioplegic solution; CMP1 group: the above ST.Thomas'II cardioplegic solution was added with 100 mg/L cardiomyopeptidin for injection; CMP2 group: the above K-H buffer and ST.Thomas'II cardioplegic solution were added with 50 mg/L and 100 mg/L cardiomyopeptidin for injection respectively. The isolated rat hearts were initially perfused with K-H buffer in 37℃Langendorff system for 20 min, following a procedure of perfusion with ST.Thomas'II cardioplegic solution at 4℃for 3min, and then arrest for 27min, with the Langendorff system kept in 32℃. This procedure was repeated three times. After arrested for 90min (undergone ischemia), the rat hearts were reperfused for 60min in normal temperature. The cardiac functional indexes were monitored, including heart rate, myocardial contractility and diastolic function, peak systolic and diastole myocardial velocities and coronary flow.Results: In NC group, after prolonged perfusion, the cardiac function of isolated hearts had been no significant change; cardiomyopeptidin for injection had been no significant effect on normal isolated hearts; Compared with the control group, the decreased degree of the cardiac function indexes and coronary flow in the groups treated with cardiomyopeptidin for injection were much less, Administration of cardiomyopeptidin in both K-H buffer and ST.Thomas'II cardioplegic solution is better than adding cardiomyopeptidin for injection in cardioplegic solution only.Conclusion: The Langendorff perfusion model of the rat's isolated hearts was stable, can be used for next experiments. Cardiomyopeptidin for injection had no significant effect on the cardiac function of normal isolated hearts. Cardiomyopeptidin for injection may improve coronary flow and cardiac function after reperfusion, thus protected immature myocardial against ischemia-reperfusion injury in young rats.Part II: Protective effects and its mechanisms of Cardiomypeptidin for injection on the isolated hearts of young rats from ischemia-reperfusion injuryObjective: This experiment was designed to investigate the effect of cardiomyopeptidin for injection on immature myocardium ischemia-reperfusion injury in the isolated hearts of young rats and its mechanism.Methods: 30 young healthy SD rats(aged 20±3 day and weighing 50-70g)were randomly divided into three groups, with 10 rats in each group. Control group: the isolated rat hearts were perfused with K-H buffer and then arrested with ST.Thomas'II cardioplegic solution; CMP1 group: the above ST.Thomas'II cardioplegic solution was added with 100 mg/L cardiomyopeptidin for injection; CMP2 group: the above K-H buffer and ST.Thomas'II cardioplegic solution were added with 50 mg/L and 100 mg/L cardiomyopeptidin for injection respectively. The isolated rat hearts were initially perfused with K-H buffer in 37℃Langendorff system for 20 min, following a procedure of perfusion with ST.Thomas'II cardioplegic solution at 4℃for 3min, and then arrest for 27min, with the Langendorff system kept in 32℃. This procedure was repeated three times. After arrested for 90min (undergone ischemia), the rat hearts were reperfused for 60min in normal temperature. The cardiac functional indexes was monitored, including heart rate, myocardial contractility and diastolic function, peak systolic and diastole myocardial velocities and coronary flow; myocardial structure was observed through optical microscopy and Transmission Electron Microscopy; the content of Na+-K+ ATPase, Ca2+-Mg2+ ATPase, total ATPase, superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide(NO), total nitric oxide synthase (TNOS), inducible nitric oxide synthase (iNOS) and aldosereductase were measured in myocardium tissue; the relative expression levels of iNOS mRNA, eNOS mRNA and Akr1b4 mRNA in myocardial tissue were detected by relative real-time fluorescence quantitative PCR.Results: Compared with the control group, the decreased degree of the cardiac function indexes and coronary flow in the groups treated with cardiomyopeptidin for injection were much less. Cardiac myofibrillar fragmentation and mitochondrial swelling were observed in the control group, while in the CMP groups, the myocardial structure was near-complete, and only mild mitochondria swelling and degeneration could be seen. There was a less decline in the contents of Na+-K+ ATPase, Ca2+-Mg2+ ATPase and Total ATPase in the treatment groups, and a increase in the activity of SOD (P<0.01 or P<0.05). The concentration of NO and MDA produced after ischemia-reperfusion injury were much less in two CMP groups; moreover, the activity of iNOS and aldosereductase were inhibited, the expression levels of iNOSmRNA and Akr1b4mRNA were significantly down-regulated in two CMP groups. These changes were more prominent in the CMP 2 group (P<0.01 or P<0.05). Besides, the eNOS mRNA levels in CMP2 group was up-regulated (P<0.05).Conclusion: Cardiomyopeptidin for injection may improve coronary blood flow and cardiac function, thus protect immature myocardial against ischemia-reperfusion injury in young rats. The mechanism may be associated with inhibited the expression of iNOS mRNA and Akr1b4 mRNA in cardiomyocytes, the inhibited activity of iNOS and aldosereductase, and the reduced NO production. Administration of cardiomyopeptidin for injection in both ST.Thomas'II cardioplegic solution and K-H buffer is better than adding cardiomyopeptidin for injection in cardioplegic solution only.PartⅢCardiomypeptidin for injectionon preconditioning attenuates immature myocardial ischemia - reperfusion injury in young ratsObjective: This experiment was designed to investigate the Cardioprotective of cardiomyopeptidin for injectionon the immature hearts of young rats from ischemia-reperfusion injury and its mechanism.Methods 30 young healthy SD rats(aged 20±3 day and weighing 50-70g)were randomly divided into three groups, with 10 rats in each group. Control group, sham-operated group and cardiomyopeptidin for injection group (CMP group). The serum Concentration of LDH, CK-MB were detected by CX7-type automatic biochemical analyzer, the serum Concentration of troponin Tn-T were detected by Elisa; the content of superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide(NO), total nitric oxide synthase (TNOS), inducible induse nitric oxide synthase (iNOS)and eNOS were measured in myocardium tissue; the expression level of NOS2 and NOS3 were detected by Immunohistochemical; the relative expression levels of iNOS, eNOS and Caspase-3mRNA in myocardial tissue were also detected by real-time fluorescence quantitative PCR; myocardial structure were observed through optical microscopy and Transmission Electron Microscopy; the expression level of active Caspase-3 protein was detected by Immunohistochemical; each groups Myocardial apoptosis was observed by TUNEL. Results: Compared with the control group, the concentration of LDH, CK-MB, Tn-T was much lesser in CMP group; Compared with the sham-operated group, the content of MDA, SOD, TNOS, and iNOS were increased and the expression levels of iNOS mRNA and Caspase-3 mRNA were significantly down-regulated in CMP groups, but less than the control group,(P<0.01 or P<0.05. Several pieces of myocardial hemorrhage and inflammatory cell infiltration were observed in the control group, severe degeneration and necrosis of myocardial cells could be seen, apoptotic myocardial cells were significant increased; while in the CMP group, myocardial infarct size was reduced, myocardial pathological injuries were mitigated, only mild degeneration and necrosis of myocardial cells could be seen, the vascular structure was near-complete, the number of apoptotic myocardial cells was less than the control group; In the sham operation group, the structure of myocardial tissue were basically normal, there was a small amount of myocardial cell apoptosis.Conclusion: Cardiomyopeptidin for injection preconditioning protected immature myocardium against ischemia-reperfusion injury in young rats. The mechanism may be associated with reduced Oxygen free radical (OFR) and NO production, and the inhibited expression of iNOS mRNA, Caspase-3mRNA and Caspase-3 protein in cardiomyocytes, thereby reduced apoptosis.