| Background Heart transplantation has been one of the most effective treatments by saving the lives of patients with end-stage heart disease. Heart transplantation in brain death donor is currently the only source of organs. Research has shown that: brain death itself before organ transplantation has previously led to a series of pathophysiological changes of graft function and morphology, which may experience systemic hemodynamic disorders, apoptosis, severe acute stress reactions, and the release of inflammatory mediators and related cytokines and so on. For now, improving the quality of donor heart is an important way to reduce short-term and long-term complications of heart transplant patients. Cell apoptosis or called programmed cell death is an active process, which is of the integrated results of cell’s outside environment factors and cell itself; as the cell mitosis, the starting of this progress has a precise gene regulation with cell death due to the interaction of those gene products. In recent years, studies have shown cell apoptosis after myocardial ischemia-reperfusion injury is the most major form of cell death, in which attention is put to the role of mitochondria in apoptosis. Mitochondrial apoptotic signaling pathway is the endogenous signalingpathways to induce apoptosis.c-Jun N-terminal kinase as one of the important members of the MAPK family, with biological functions of activating of JNK signaling pathway which causes the open of mitochondrial permeability transition pore, resulting in the decrease of mitochondrial transmembrane potential, and leading to releasing of cytochrome-c, eventually activating Caspase-3, the key apoptosis performers through a series of reactions. SP600125 is a commonly highly selective inhibitor of JNK.Objective In this study, we have built a SD rat model for the study of brain death to observe the myocardial cell apoptosis, Cyt-C, Caspase-3 and its m RNA expression levels changes, trying to explore whether brain death status trigger mitochondrial apoptotic pathway in myocardial cells of rats, resulting in myocardial cell injury and apoptosis. And to observe furtherly the protective effect of SP600125 on myocardial cell apoptosis in rats under brain death status and its possible molecular mechanism.Methods Twenty Sprague-Dawley(SD) rats which are purchased from the laboratory animal Center of Zhengzhou University were randomly divided into four groups: SHAM group(n=5), which were only subjected to intracranial insertion of Fogarty tube after anesthesia; Brain-death group(n=5), which were subjected to brain death for 6h; SP600125 group(n=5), which were subjected to brain death for 6h, and 1 hour before achieving brain death, peritoneal injection of SP600125(10mg/kg) was given immediately; and DMSO group(n=5), which were subjected to brain death for 6h, and 1 hour before achieving brain death, peritoneal injection of placebo solution DMSO(10mg/kg).To establish improved brain death models of rat, then the apoptosis in cardiac muscle sections were determined by terminal deoxynucleotidyl transferase d UTP nick end labelling(TUNEL) assay and then Western blot analysis and Real-timePCR were performed to detect the expression of Cyt-C and Caspase-3 in protein and m RNA level respectively.Results(1)15 SD-rats of the models of brain death were all successfully established, which can maintain a state of brain death for 6hours and have a MAP over 80 mm Hg.( 2) The results of TUNEL figured out that the apoptosis rate of cardiomyocytes in BD group was(26.39?4.99)% with significant difference to that in SHAM group(1.41?0.35)%(p<0.05),the apoptosis rate in BD group was similar with that in DMSO group(26.28?4.92)%(p>0.05)and the apoptosis rate in SP600125 group(7.63?3.09)% made a significant decreasing with that in BD group(p<0.05).(3)Western blot revealed that the expressions of Caspase-3 and Cyt-C in BD group(145.63?7.21、73.67?7.35)increased significantly with that in SHAM group(38.86?8.95、18.74?8.77)(p<0.05),the expressions of Caspase-3 and Cyt-C in BD group were similar with that in DMSO group(146.56?6.87、72.45?6.84)(p>0.05)and the expressions of Caspase-3 and Cyt-C in SP600125 group(39.45?5.73、20.34?5.69)decreased significantly with that in BD group(p<0.05).(4)The results of Real-time PCR showed that the expressions of Caspase-3 m RNA and Cyt-C m RNA in BD group were(2.37?0.12、2.03?0.08)times more than that in SHAM group(p<0.05),the expressions of Caspase-3 m RNA and Cyt-C m RNA in BD group were similar with that in DMSO group(2.38?0.11、2.06?0.09)(p>0.05) and the expressions of Caspase-3 m RNA and Cyt-C m RNA in SP600125 group(1.21?0.05 、 1.23?0.24) were significantly less than that in BD group(2.38?0.11、2.06?0.09)(p<0.05).1. Using the improved slow intermittent intracranial pressure method for establishing brain death models of rat is simple and reproducible, easy to be standardized, and with high success rate. The model has the similar pathophysiology to clinical brain death and has advantages for studying the influence of brain death on human organs, providing a stable and reliable platform. 2. Caspase-3 and Cyt-C expression in myocardial cells of brain death rats significantly increased suggesting that mitochondrial apoptosis pathway activation can cause myocardial damage; 3.SP600125 has a protective effect on myocardial cell injury mediated by mitochondrial apoptotic pathway and provides a new way of targets and ideas about how to protect the quality of isolated heart well. Conclusion... |
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