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目的:利用低浓度过氧化氢(H2O2)建立兔原代左心房肌细胞氧化损伤模型,探讨胡椒碱减轻氧化应激损伤的保护作用。方法:将18只新西兰白兔随机分为3组,每组6只:即正常对照(NC)组、H2O2组和胡椒碱组,均分别进行左心房肌细胞的原代培养。NC组对培养的心房细胞直接进行检测,H2O2组直接在培养的原代心房肌细胞中加入终浓度为100μmol/L的H2O2培养2 h,胡椒碱组以7×10-6mol/L浓度的胡椒碱处理细胞1 h之后,加入终浓度为100μmol/L的H2O2共同培养2 h。检测氧化和抗氧化指标的变化。MTT法检测三组原代细胞活力,黄嘌呤氧化酶法检测超氧化物歧化酶(SOD)活力、比色法检测丙二醛(MDA)含量及还原型谷胱甘肽(GSH)含量,Fura-2AM检测细胞内钙离子浓度,RT-PCR对线粒体mRNA进行定量分析。结果:与NC组相比较,H2O2组细胞的活力、SOD的活力及GSH的含量明显下降(P<0.05);MDA的含量、钙离子浓度和线粒体mRNA的表达均明显增加(P<0.05)。胡椒碱组和H2O2组比较,上述指标均有显著改善(P<0.05)。结论:胡椒碱能够在氧自由基的产生清除等环节,减轻兔原代左心房肌细胞的氧化应激损伤。
OBJECTIVE: To establish a model of primary oxidative damage in rabbit atrial myocytes by using low concentration of hydrogen peroxide (H2O2), and to explore the protective effect of piperine against oxidative stress injury. Methods: Eighteen New Zealand white rabbits were randomly divided into 3 groups, 6 rats in each group. They were normal control (NC) group, H2O2 group and piperine group, and were respectively primary culture of left atrial myocytes. The cultured atrial cells were directly detected by NC group in the H2O2 group. The primary atrial myocytes were incubated with H2O2 at a final concentration of 100 μmol / L for 2 h. The piperine group was treated with 7 × 10 -6 mol / L pepper Alkali-treated cells were incubated with H2O2 at a final concentration of 100 μmol / L for 1 h. Detection of changes in oxidation and antioxidant indicators. The activity of superoxide dismutase (SOD) was measured by xanthine oxidase method, the content of malondialdehyde (MDA) and the content of reduced glutathione (GSH) were detected by MTT assay. Fura -2AM detection of intracellular calcium concentration, quantitative analysis of mitochondrial mRNA by RT-PCR. Results: Compared with NC group, the viability, the activity of SOD and the content of GSH in H2O2 group were significantly decreased (P <0.05). The contents of MDA, calcium ion and mitochondrial mRNA were significantly increased (P <0.05). Piperine group and H2O2 group, the above indicators were significantly improved (P <0.05). Conclusion: Piperine can reduce the oxidative stress injury of rabbit primary atrial myocytes in the process of oxygen free radical scavenging.