| OBJECTIVE ①To investigate the role of P38MAPK, iNOS, COX-2 and mitochondrial K+ATP channel, and explore the association among them in the late phase of ischemic preconditioning (IPC) in neonatal rat cardiomyocytes.②To study the late cardioprotection of nitroglycerine preconditioning and survey the role of P38MAPK, iNOS, COX-2 and mitochondrial K+ATP channel and the association among them in neonatal rat cardiomyocytes. ③To explore the cardioprotection of the cardiac fibroblasts and cardiomyocytes after ischemic (hypoxemic) preconditioning on normal cardiomyocytes .METHOD The protein expression of phospho-P38MAPK, iNOS and COX-2 was determined by Western blotting techniques and immunocytochemistry assay . The signal of COX-2 mRNA was determined by RT-PCR techniques. The injure of cardiomyocytes was assessed by LDH release and tyrpan blue exclusion in each group. Data were reported as mean ± SEM. Measurements were analyzed with ANOVA and Chi-square test.RESULTS ①The role of P38MAPK, iNOS, COX-2 and mitochondrial K+ATP channel and the association among them: The protein expression of phospho-P38MAPK culminated at 24h after IPC, decreased at 48 h and retured to control values by 96h. In cytochemical staining, the signal of phospho-P38MAPK was little in cytoplasm before IPC, occured aboundly innuclears at 48 after IPC. The protein expression of iNOS and COX-2 increased markedly at 24h after IPC, culminated at 48h, decreased at 72 h and retured to control values by 96h. The levels of COX-2 mRNA could be detected at 1h after IPC, culminated at 3h and retured to control values by 24h. The staining of iNOS and COX-2 was little in cytoplasm before IPC, occured aboundly in cytoplasm and was concentrated on the nuclear envelope at 48 after IPC. SB203580 (P38MAPK inhibitor), SMT (iNOS inhibitor) and NS398 (COX-2 inhibitor) could block the cardioprotection of IPC. SB203580 could inhibit the induction of iNOS and COX-2 following IPC. SMT decreased the protein expression of COX-2, but had no effect on the amount of phospho-P38MAPK following IPC. NS398 could not decrease the protein expression of phospho-P38MAPK and iNOS. Diazoxide (the mitochondrial K+ATP channel opener) mimicked the cardioprotection of late IPC. 5-HD (mitochondrial K+ATP channel inhibitor) could decrease the protein expression of phospho-P38MAPK, iNOS and COX-2 and block the cardioprotection administered before IPC. Administered after IPC, 5-HD had no effect on their expression but blocked the cardioprotection too. ② The cardioprotection and mechanism of nitroglycerine preconditioning: From the concentration of 1ng/ml to 80 ng/ml, nitroglycerine preconditioning could mimic the cardioprotection of the late IPC. SB203580 and SMT could block the cardioprotection of nitroglycerine preconditioning below 10ng/ml concentration, but had no effect on that above the concentration of 20ng /ml. NS398 could block the cardioprotection of nitroglycerine preconditioning from the concentration of 4ng/ml to 40 ng/ml. After 10ng/ml (low concentration) and 40ng/ml (high concentration) of nitroglycerine preconditioning, the protein expression and change of phospho-P38MAPK, iNOS COX-2 were similar with that of IPC at different time. The level of COX-2 mRNA could be detected at 2h after IPC, culminated at 3h and retured to control values by 24h. On the condition of low concentration nitroglycerine preconditioning, the effect of SB203580, SMT, NS398 and 5-HD on the expression of P38MAPK, iNOS, COX-2 and the cardioprotection was the same as that in IPC. It was different from low concentration nitroglycerine preconditioning that SB203580 and SMT could not decrease the expression of COX-2, 5-HD could not decrease the protein expression of COX-2 after high concentration nitroglycerine preconditioning, but could block the cardioprotection.③ Cardioprotection of cardiac fibroblasts and cardiomyocytes following IPC on normal cardiomyocytes: IPC did not induce the late protection on the cardiac fibroblast. The medium of cardiac fibroblasts... |
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