The Effect Of Hydrogen On Hypoxia/reoxygenation Injury In Cultured Neonatal Rat Cardiomyocytes

BackgroundMyocardial ischemia/reperufsion injury (MIRI) is a clinical common pathophysical phenomenon. It is found in the Coronary reopening (thrombolysis, coronary angioplasty and coronary artery bypass grafting et.al), open heart surgery. MIRI can affect great harm but it is difficult to aviod. Transitory coronary artery obstruction does not lead to irreversible damage, while coronary artery reopening induces irreversible configuration and function damage, accelete injury cells death. Therefore, prevention and treatment to MIRI has been one of the hotspots in the clinic and basic study of cardiology. According to recently studies, free radical induce cardiomyocytes apoptosis may be one of the machnisms of MIRI.Ohsawa provided evidence that inhaled hydrogen gas (H₂) has antioxidant and antiapoptotic activities that protect the brain against ischemia-reperfusion injury and stroke by selectively reducing cytotoxic oxygen radicals in Nature Medicine in July 2007. Until recently, Hayashida reported inhalation of hydrogen gas reduces infarct size in the rat model of myocardial ischemia/reperfusion injury. But there are no reports so far about whether the hydrgeon has protective effect in cultured neonatal rat cardiomyoeytes. ObjectiveThe aim of the study is to investigate the effects and the possible mechanism of the hydrgeon induced by myocardial ischemia/reperfusion in cultured neonatal rat cardio-myoeytes. Then observe the survival ability,cell apoptosis rate, lipid peroxide, explore the possible mechanism from cell apoptosis.MethodsThe model of hypoxia-roxygenation injury in cultureded cardiomyoeytes was set up by making the primary cullutred neonaatl rat cardiomyocytes by lack of oxygen of 60min and then afford oxygen 30min. The nomral culltured 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 four groups: control group; hyoxia/ reoxygenation (H/R)goup; group of low concentration of hydrogen:H/R+2% hydrgeon precondition group; group of high concentration of hydrogen:H/R+4% hydrgeon precondition group. The photos of all groups were taken. The cell vialibity was detected by MTT assay. The cell apoptosis rate was detected by TUNEL way, To investigate the change of mitochondrial membrane potential (△ψm), the Rhodaminel23(Rhl23)fluorescence intensity of cardiomyocytes with Rhl23 stain was observed by fluorescence microscope, the activity of MDA in culutre was measured.Stastical analysisAll data are expressed as mean value±S.D. and analyzed by statistical software SPSS 10.0.The level of statistical significant was set at 0.05.Results1. Morphological changes Observed under inverted microscope, normal cardiomyocyte after 72h culture, cell clusters and extend pseudopodia growth, high refractive index, and pulsatility significantly. Diversified morphology,such as round, and spindle-shaped vertebra. Being injuerd by hypoxia and reoxygenation, refracting power of the cardiomyocytes declined, pseudo-podium becmae shortened or disppeared, cardiomyoeytes beat became weak or stopped.2. The cell purity was 95% identified by immunocytochemical method. We got high purity cardiomyocyte.3. In pretreatment hydrgeon group, the cell viability was significantly increased when compared with H/R group (P<0.05). The viability of high concentration hydrgeon group was significantly increased when compared with low concentration hydrgeon group, suggesting that hydrogen can enhance cell viability and the higher concentration is better.4. In pretreated hydrgeon group, the apoptosis rate of cardiomyocyte was significantly decreased when compared with H/R group(P<0.05). The apoptosis rate of high concentration hydrgeon group was significantly decreased when compared with low concentration hydrgeon group, suggesting that hydrogen can inhibit apoptosis and the higher concentration is better.5. The Rhl23 fluorescence treated were significantly decreased. However, the decrease in Rh123 fluorescence and MFI induced by was prevented by,suggesting that hydrgeon can inhibit the decrease of△ψm induced by H/R, suggesting that hydrgeon can inhibit the decrease of△ψm induced by H/R.6. The contents of MDA in hydrgeon group was significantly decreased when compared with H/R respectively (P<0.05). The contents of MDA in high concentration hydrgeon group was significatly decreased when compared with low concentration hydrgeon group (P<0.05), suggesting that hydrgeon can inhibit lipid peroxidation and the higher concentration is better.ConclusionsHydrgeon has a protective effect on hypoxia/ reoxygenation injury in cultured rat cardiomyocytes in vitro. The effect maybe involved in protecting the integrity of the mitochondrial membrane, inhibition mitochondrial membrane potential decline,inhibit lipid peroxidation, thereby reducing cell apoptosis and enchancing the cell viability. The higher concentration hydrgeon has better protection of cardiomyocyte.