Protective Effect Of The Ethanolic Extract Of Radix Morindae Officinalls On Hypoxia/reoxygenation Injury In Cultured Neonatal Rat Cardiomyocytes

Myocardial ischemia/reperfusion injury (MIRI) is one of the most important complications in reopening therapy of coronary artery.But the real mechanisms of the phenomenon have been subjected to debate. Therefore, prevention and treatment of MIRI has been one of the hotspots in the clinic and basic study of cardiology. Recently, there has been great development in Chinese medicine to protect myocardium against ischemia reperfusion injury. Some of them have been used in clinical treatment of myocardial ischemic disease. The extract of Radix moridae officinalls (RMO) has been reported to increase antioxidants in brain and liver. Previous study has demonstrated that the aqueous extract of RMO has evidently protective effects on MIRI induced by ligation of coronary artery, but there are no reports so far about whether the ethanolic extract of RMO has protective effect in cultured neonatal rat cardiomyocytes. The purpose of this study is to investigate the effects of the ethanolic extract of RMO on hypoxia/reoxygenation injury in cultured neonatal rat cardiomyocytes. MethodsThree different concentrations of water fraction and butanol fraction from the ethanolic extract of RMO: 0.1g·L^-1, 0.3g·L^-1 and 0.9g·L^-1 were prepared. The heartswere isolated from l³-day-old Wistar rats and the ventricles were minced in phosphate buffer solution. The myocardial cells were dissociated with 0.125% pancreatin. After dispersed, the cells were collected, purified and incubated in 25ml culture bottles(the cells density was adjusted to 5xl05cells-mr1 with Dulbecco's modified eagle culture medium consisting of 15% fetal calf serum). The models of hypoxia/reoxygenation were made with the first generation of cultured cardiomyocytes. The normal cultured medium was replaced by hypoxia solution to induce hypoxia, then the hypoxia solution was replaced by reoxygenation solution to induce reperfusion. Cultured cardiomyocytes of neonatal rats were divided into eight groups: control group; hypoxia/reoxygenation(H/R) group; H/R+low dose of butanol fractionCO.lg-L"1) group; H/R+medium dose of butanol fractionCO.Sg-L'1) group; H/R+high dose of butanol fraction(0.9gL'') group; H/R +low dose of water fractionCO.lg-L"1) group; H/R+medium dose of water fraction^.Sg-L"1) group; H/R+high dose of water fraction (O^g-L"1) group. The photos of all groups were taken. The activities of cardiomyocytes SOD were measured by the method of xanthine oxidase and the contents of cardiomyocytes MDA by the method of thiobarbituric acid after 60min of hypoxia and 30min of reoxgynation. The activities of LDH in culture were evaluated after 30min of hypoxia and 40min of reoxgynation. The contents of cardiomyocytes NO were measured by the method of nitrifying ferment and the activities of cardiomyocytes NOS by the method of chemic colorimetry after 60min of hypoxia and 30min of reoxgynation. ResultsBeing injured by hypoxia and reoxygenation, refracting power of the cardiomyocytes declined, pseudo-podium became shortened or disappeared, cardiomyocytes beat became weak or stopped. The activities of SOD were obviously decreased in H/R group ( PO.01, compared with the control group). The activities of iNOS were significantly decreased in H/R group ( P<0.05,compared with the control group ). The activities of tNOS were significantly decreased in H/R group ( P<0.01,compared with the control group ). The contents of MDA in H/R group werehigher than those in any other groups ( P<0.01). The activities of LDH in H/R group were higher than those in any other groups ( P<0.01). The contents of NO in H/R group were lower than those in any other groups ( P<0.01). Water fraction and butanol fraction of the ethanolic extract of RMO ( 0.9 g*L'\ 0.3 g-lA 0.1 g-L"1) relieved the injury: the morphologic appearance of cells comparatively approached normal, the activities of SOD, iNOS and tNOS were significantly higher, the contents of LDH and MDA were significantly lower compared with H/R group, while the contents of NO were significantly higher compared with H/R group. Conclusions1. Water fraction and butanol fraction from the ethanolic extract of RMO increase antioxidants and inhibit lipid peroxidate in cultured neonatal rat cardiomyocytes injured by hypoxia/reoxygenation.2. Water fraction and butanol fraction from the ethanolic extract of RMO increase the level of NO, iNOS in cultured neonatal rat cardiomyocytes injured by hypoxia/reoxygenation, that may be concerned with its cardioprotective effects.3. The experiment shows that v/ater fraction and butanol fraction from the ethanolic extract of RMO have obviously protective effects on the cultured neonatal rat cardiomyocyte injured by hypoxia/reoxygenation. Some effects of RMO were dose-dependant.