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目的探讨非对称二甲基精氨酸(ADMA)预处理心肌细胞后诱导氧化应激发生,观察其活性氧类(ROS)产生和自噬分子beclin-1表达的机制。方法将培养的大鼠心肌细胞随机分为对照、ADMA-3、ADMA-5、ADM A-10、ADM A-15、ADM A-20和ADM A-30μmol/L等7组,干预48 h后以流式细胞术检测胞内活性氧水平,采用免疫印迹法(WB)测定心肌细胞自噬分子Beclin-1和PI3K/AKT蛋白表达水平,并检测以上分组所对应的细胞存活率。结果 ADMA干预心肌细胞48 h后,活性氧水平依ADMA浓度变化明显上调(9.86±0.86、15.91±0.97、19.15±3.13、28.76±1.69、29.57±1.99和35.09±2.22;P<0.05)且降低心肌细胞存活率(%)(90.3±6.5、76.2±8.7、76.2±6.4、71.4±5.6、67.8±5.0、63.5±4.2和61.2±6.9;P<0.05),均存在剂量依赖性关系;同时可以上调beclin-1蛋白的表达(P<0.05),抑制PI3K-AKT信号通路。结论 ADMA可以诱导心肌细胞活性氧产生从而抑制PI3K-AKT通路,该效应促进心肌细胞自噬分子beclin-1蛋白表达并导致其存活率明显降低。
Objective To investigate the oxidative stress induced by asymmetric dimethylarginine (ADMA) preconditioning in cardiomyocytes and to investigate the mechanism of its production of reactive oxygen species (ROS) and the expression of beclin-1. Methods The cultured rat cardiomyocytes were randomly divided into 7 groups: ADMA-3, ADMA-5, ADM A-10, ADM A-15, ADM A-20 and ADM A-30μmol / The level of intracellular reactive oxygen species (ROS) was detected by flow cytometry. The expression of Beclin-1 and PI3K / AKT protein in cardiomyocytes was detected by Western blotting (WB). The cell viability was also detected. Results After ADMA intervention for 48 h, the level of reactive oxygen species (ROS) was significantly up-regulated by ADMA (9.86 ± 0.86,15.91 ± 0.97,19.15 ± 3.13,28.76 ± 1.69,29.57 ± 1.99 and 35.09 ± 2.22; P <0.05) Cell survival rates (%) (90.3 ± 6.5, 76.2 ± 8.7, 76.2 ± 6.4, 71.4 ± 5.6, 67.8 ± 5.0, 63.5 ± 4.2, and 61.2 ± 6.9, P <0.05) were all dose- Beclin-1 protein expression (P <0.05), inhibit the PI3K-AKT signaling pathway. Conclusion ADMA can induce the production of reactive oxygen species (ROS) in cardiomyocytes and thus inhibit the PI3K-AKT pathway. This effect promotes the expression of beclin-1 protein in cardiomyocytes and significantly reduces the survival rate.