| Background and objectivesIn the past studies, it is shown that oxidative stress has been an important factor to be involved in the development of many diseases. Recent studies pointed out that the product of oxidative stress such as active oxygen can affect electrical remodeling and structural remodeling in atrial fibrillation. In atrial fibrillation the produce of oxidative stress increased, and mitochondria damaged. However, it is undefined that oxidative stress is a primary pathogenetic or a consequence of atrial fibrillation. The aim of this study was to build oxidative stress model of rabbit primary atrial cardiomyocytes with low-consistence H2O2, and to study the protective effect of apocynin aganist oxidative stress. The relationship between oxidative stress and atrial fibrillation was probed by means of analysis key factors of oxidative stress such as the content of intracellular peroxides and oxidoreductases.Methods1. Establish a model of oxidative stress by low concentration H2O2 for rabbit primary arterial cells.2. The morphological change of primary arterial cell was observed. Cell viability was determined by MTT assay, cell apoptosis rate were analyzed by flow cytometer.3. Superoxide dismutase was measured by xanthine oxidase; and the content of malondialdehyd and reduced glutathione hormone were both tested by colorimetric methods.4. Fluorescence probe technique was used to measure intracellular free calcium concentration.5. RealTime-PCR analysis suggested the level of mitochondria mRNA. And Two-dimensional protein gel electrophoresis were used to detect the expression level of nicotinamide adenine dinucleotide phosphate(NADPH) p22phox.Results1. Compared with NC group, the cell viability decreased in H2O2 group (P<0.05).But the cell viability in piperine group was higher than that in in H2O2 group (P<0.05). The activity of superoxide dismutase (SOD) and the content of glutathione hormone (GSH) decreased (P<0.05) in H2O2 group. But the content of Malondialdehyd (MDA), the intracellular free calcium concentration and the expression of mitochondrial mRNA increased obviously (P<0.05).Meanwhile, gene expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22phox in H2O2 group is more than that in NC group (P<0.05). In H2O2 group the rate of apoptosis was significantly higher in H2O2 group (P<0.05)2. Compared with H2O2 group, the cell viability, the activity of superoxide dismutase (SOD) and the content of glutathione hormone (GSH) decreased (P<0.05) in piperine group. The content of Malondialdehyd (MDA), the intracellular free calcium concentration, the expression of mitochondrial mRNA and the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits p22phox increased obviously (P<0.05). Compared with H2O2 group, the rate of apoptosis decreased in piperine group.3. Compared with NC group, the activity of SOD and the content of GSH decreased (P<0.05) in apocynin group, whereas the content of MDA, the intracellular free calcium concentration and gene expression of NADPH oxidase subunits p22phox increased (P<0.05). In apocynin group, the activity of SOD and the content of GSH is higher (P0.05), the content of MDA, the intracellular free calcium concentration, gene expression of NADPH oxidase subunits p22phox and the expression of mitochondrial mRNA declined evidently compared with the H2O2 group. Conclusions1. The research shows that oxidative stress by H2O2 had a notable influence on the produce of oxygen free radical, the activity of antioxidase. the lipid peroxidation and the mitochondria oxidative phosphorylation in primary culture cell. The demage of oxidative stress caused the activity of antioxidant enzyme decreased, the mitochondria damage, and calcium overload.2. Piperine can protect the oxidative stress induced by H2O2 in rabbit primary left atrial cardiomyocytes. In process of oxidative stress, piperine play a role by means of reducing the generation of active oxygen free radical, restrainting NADPH oxidase, and increasing activity of antioxidant enzyme. Piperine also can ease intra-cellular calcium overload, weaken harmful role of oxidative stress. Moreover, piperine can lighten cell apoptosis by reducing the produce of oxygen free radical. So piperine can protects the oxidative stress induced by H2O2 in rabbit primary left atrial cells.3. Our results suggest that apocynin can play an important protective role in rabbit primary atrial cardiomyocytes against oxidative stress in the production and elimination processes of reactive oxygen species(ROS). Apocynin may function by reducing the generation of ROS, restrainting NADPH oxidase, and increasing activity of antioxidant enzyme.
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