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A Study of Oxidative Stress and the Protection in Rabbit Primary Left Atrial Caridomyocytes

Author: TianMiao
Tutor: DanZhaoLiang
School: PLA Postgraduate Medical School
Course: Cardiovascular within science
Keywords: Atrial fibrillation Oxidative Stress Peroxide Primary atrial cells Piperine Apocynin
CLC: R541.75
Type: Master's thesis
Year: 2011
Downloads: 51
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Abstract

Background and Purpose of oxidative stress in the past has proven to be an important factor involved in the development of many human diseases. A large number of studies have shown that the product of oxidative stress, such as reactive oxygen species can influence atrial electrical remodeling and structural remodeling in atrial fibrillation; atrial fibrillation, atrial myocytes increased oxidative stress products, as well as mitochondrial DNA oxidative damage exists that atrial fibrillation When atrial oxidative stress. However, the current study does not explicitly oxidative stress is initiating mechanisms of atrial fibrillation or a secondary reaction, and the lack of drugs targeted antioxidant effects. This study uses hydrogen peroxide Construction of primary cultured rabbit atrial muscle cells from oxidative injury model, and play a key role in the oxidation-reduction reaction of nicotinamide adenine dinucleotide phosphate (nicotinamide adenine dinucleotide phosphate, NADPH) oxidase inhibitor a The apocynin, and Huixin grass monomer ingredients Piperine intervention. Through the analysis of oxidative damage to key factors such as intracellular peroxide, oxidoreductases content on to explore the relationship and the mechanism of oxidative stress and atrial fibrillation. Research methods, the use of low concentrations of hydrogen peroxide (H202) to build primary cultured rabbit atrial myocytes, production of oxidative stress damage model and with of apocynin and Huixin grass monomer ingredients Piperine intervention. Observe atrial myocytes morphological changes in primary cultured under the above conditions, and MTT assay for cell viability, flow cytometry atrial rate of apoptosis;, xanthine oxidase test of superoxide dismutase ( superoxide dismutase, SOD). thiobarbituric acid colorimetric assay malondialdehyde content (malondialdehyd, MDA) and the colorimetric detection glutathione (glutathione hormone, GSH); calcium fluorescent probe to detect intracellular calcium ion concentration; 4, Real time-PCR for quantitative analysis of mitochondrial mRNA analysis detects NADPH oxidase subunit p22phox expression; Western Blotting Protein gel electrophoresis and bar with. Results, compared with the control group, the the primary atrial muscle cell viability H202 group was significantly lower than the control group (P lt; 0.05), the H202 group of SOD and GSH content decreased (P lt; 0.05), while MDA content and calcium concentration increased (P lt; 0.05); NADPH oxidase subunit p22phox mRNA expression was significantly higher (P lt; 0.05). Primary atrial myocytes apoptosis rate was significantly higher (P lt; 0.05). 2, the cell viability of piperine group was significantly higher than the H2O2 group (P lt; 0.05); the H2O2 groups and piperine group SOD and GSH content decreased (P lt; 0.05), but significantly higher than piperine group H202 group (P lt; 0.05). the H2O2 groups and piperine group MDA and calcium ion concentration increased (P lt; 0.05); piperine group above indicators are lower than the H202 group (P lt; 0.05) H2O2 group Compared with the control group, SOD and GSH decreased significantly (P lt; 0.05) of MDA increased (P lt; 0.05); compared with H202 group, of Piperine group cell apoptosis rate lower (P lt; 0.05). The Mitochondrial mRNA expression in atrial myocytes piperine group was significantly lower than the H202 group (P lt; 0.05). 3, apocynin cell viability was significantly higher than the H2O2 group (P lt; 0.05). Compared with H202, apocynin group SOD and GSH increased (P lt; 0.05), MDA significantly decreased (P lt; 0.05). Significantly increased intracellular calcium ion concentration in the the H202 group of apocynin group than in the control group (P lt; 0.05), the apocynin group was significantly less than the H202 group (P lt; 0.05). Compared with the control group, apocynin group and the H202 group of NADPH oxidase subunit p22phox expression were significantly increased (P lt; 0.05), but the apocynin group than in the group H202 decreased expression (P lt; 0.05 ). Compared with H202 group, the apocynin group of cells in cell apoptosis rate (P lt; 0.05), and at the same time the expression of mitochondrial nRNA apocynin group was significantly lower than H202 group (P lt; 0.05). Research Conclusions This study shows that H202 oxidative damage of primary atrial myocytes generation of oxygen free radicals, antioxidant enzyme activity, and more unsaturated fatty acid lipid peroxidation, and mitochondrial oxidative phosphorylation obvious impact . Found increased expression of oxidative stress injury of the atrial muscle NADPH oxidase subunit p22phox protective effect of SOD and GSH activity decreased oxidative stress, an increase in the content of MDA reflect cell damage, and cause mitochondrial damage, intracellular calcium content increases. Huixin grass piperine extract from the generation of oxygen free radicals clear links to reduce the accumulation of reactive oxygen radicals in cells, reducing lipid peroxidation of cell damage, protects mitochondrial oxidative phosphorylation function, reduce mitochondrial oxidative damage. Piperine can reduce intracellular calcium overload, reduce oxidative damage to cell viability, and its role link is likely to be through the inhibition of NADPH oxidase. In addition, piperine thereby reducing apoptosis due to oxidative damage caused by reducing the accumulation of oxygen free radicals. Piperine play a protective role in oxidative damage to the primary atrial myocytes. 3 apocynin, a specific NADPH oxidase inhibitor, by inhibiting the expression of the NADPH oxidase subunit p22phox alleviate oxidative stress injury. Apocynin oxidative damage from oxygen free radicals clear links to reduce the generation of reactive oxygen species, regulating the activity of antioxidant enzymes, reduce lipid peroxidation of cell damage, protection of mitochondrial oxidative phosphorylation function, reduce the oxidative damage of mitochondria. Apocynin also able to reduce oxidative stress-induced apoptosis, and its mechanism is likely to be through the inhibition of NADPH oxidase activation and ultimately effective in reducing ROS-mediated cell apoptosis.

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