| Objective: Ischemic Prencondition (IP) is a phenomenon in which brief repetitive periods of ischemic/reperfusion protect the cardiomyocytes against subsequent prolong ischemic and it is an endogenous cardioprotective mechanism. Exercise Prenconditioning (EP) is the especial way of IP, which induced by repeated brief strenuous exercise which induced relative ischemic/reperfusion. Study demonstrated that endogenous protectant plays an important role in the IP and EP. Adrenomedullin (ADM) is newfound and important endogenous protectant, which could be excreted by cardiocytes and blocked by its specificity receptor antagonist. ADM could induce cardioprotection by exogenous administering or endogenous releaseing in exercise. EP is an effective cardioprotection measure and ADM is an important endogenous protectant, which could release in exercise. Whether cardioprotection of EP were induced by ADM and What difference were long-term and short-term protective effect of EP and its mechanisms, there have not been reported by present studies. Therefore, we established short-term and long-term EP animal model and discussed the influence of EP on ADM and its mechanisms, which provided theoretical and experimental basis for the effect cardioprotection of EP.Methods: Male Sprague-Dawley rats (n=90) were randomly divided into six groups: control group (C), isoproterenol group (ISO), three days(short-term)group (3dEP), three weeks (long-term) group (3wEP), three days EP plus isoproterenol group (3dEP+ISO), three weeks EP plus isoproterenol group (3wEP+ISO). The rats of 3dEP group and 3dEP+ISO group performed 3 days intermittent treadmill exercise in order to estabelish short-term treadmill EP animal model. The rats of 3wEP group and 3wEP+ISO group performed 3 weeks intermittent treadmill exercise in order to estabelish long-term treadmill EP animal model. Thirty minutes after the last exercise session, the rats of 3dEP+ISO group and 3wEP+ISO group were injected intraperitioneally with ISO (4mg/kg). At the same time, the rats of ISO group were injected intraperitioneally with the same doses of ISO to reproduce the model of acute cardiomyocytes ischemia injury. The level of cTnI in serum was measured by immunchemiluminescent method. Histological structure of cardiocytes was observed by HE staining. Change of ischemia and anoxic was studied by HBFP staining. Change of cardiomyocytes ultrastructure was observed by transmission electron miscroscope (TEM). The expression of ADM mRNA in heart was showed by hybridization in situ and quantitatively analyzed by the system of computer image analysis.The expression of ADM in heart were showed by immunihistochemistry method and quantitatively analyzed by the system of computer image analysis. The content of ADM in heart and serum were measured by euzymelinked immunosorbent assay (ELISA).Results: (1) The cTnI of ISO group was higher than that of C group in serum (p<0.05). There was no significant difference in the cTnI level between 3dEP, 3wEP and C group in serum.The cTnI level of 3dEP+ISO group was higher than that of C group in serum (p<0.05), but the cTnI level of 3dEP+ISO group was significant lower than that of ISO group in serum (p<0.05). Compared with C group, the cTnI level of 3wEP+ISO in serum had no difference, but was significant lower than that of ISO group in serum (p<0.05). (2) Cardiomyocytes structures of C group, 3dEP group and 3wEP group were normal, there were no red change of ischemia and anoxic. Cardiomyocytes of ISO group had a great red change of ischemia and anoxic.Cardiomyocytes of 3dEP+ISO group and 3wEP+ISO group had a little red change of ischemia and anoxic.(3) Compared with C group,there was no significant difference in the ADM level in 3dEP and 3wEP group in serum. (4) The cardiomyocytes ultrastructure of 3dEP group, 3wEP and C group were nomal.The damage of cardiomyocytes ultrastructure of ISO was serious.However, the damage of cardiomyocytes ultrastructure of 3dEP and 3wEP group were lighter than ISO group. (5) There were ADM mRNA positive hybridization signal in intracytoplasm of cardiocytes of C group, whose colour was brown. There was no significant difference in hybridization signal between 3dEP and C group, but 3wEP was significant higher than that of C group. The computer image analysis showed that immunoreation area and mean optinal density of ADM mRNA of 3wEP were higher than those of C group in cardiocytes (p<0.05). There were ADM mRNA positive hybridization signal in vessel wall of coronary vessels of C group, whose colour was brown-black. There was no significant difference in hybridization signal between 3dEP and C group in coronary vessels, but 3wEP was significant lighter than that of C group. The computer image analysis show that immunoreation area and optinal density of ADM mRNA in cardiocytes of 3wEP were lighter than those of C group (p<0.05).(6)There were ADM positive signal of cardiocytes of C group, which colour was brown and was lower intensity in intracytoplasm. There were high intensity in vessel wall of coronary vessels. The computer image analysis show that immunoreation area and mean optinal density of ADM of 3wEP were higher than those of C group in cardiocytes(p<0.05), and the immunoreation area and mean optinal density of ADM of 3wEP were lower than those of C group in coronary vessels (p<0.05).(7) Compared with C group, the contents of ADM in cardiocytes of 3dEP had no difference,but ADM of 3wEP group were higher than those of C group (p<0.05).Conclusion: (1)3days and 3 weeks consecutive exercise protocol could establish short-term and long-term animal EP model successfully. The changes of cTnI level in serum, ischemis/anoxic and ultastructure of cardiomyocytes could evaluate the injury of cardiomyocytes in animal EP model. (2)After long-term EP, the expression of ADM mRNA in cardiocytes increased, which indicated exercises induced ADM synthesis, mainly contributing for enhanced transcription in gene.(3)After long-term EP,the totel expression of ADM in heart increased, the expression of ADM in cardiocytes increased, while the expression of ADM in endothelium and smooth muscle decreased, and there is no change in serum, which indicated hypoxia and contractility in exercises can induce ADM synthesis in cardiocytes ,and ADM releasing from cardiocytes ,endothelium and smooth muscle effected by autocrine and paracrine in cardioprotection.(4)After long-term EP, the totel expression of ADM in heart increased, whose hemodynamics stablization and antioxidation may reduce the damage of ISO.(5)After short-term EP, the totel expression of ADM in heart had no change, the relationship between ADM and short-time EP needed a profound research. |
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