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目的研究姜黄素(Cur)对过氧化氢(H2O2)诱导小鼠巨噬细胞内一氧化氮(NO)及活性氧(ROS)生成的影响。方法健康6~8周龄昆明种小鼠腹腔提取巨噬细胞,培养成活后调整细胞水平为2×108L-1,H2O2组加入1 mmol.L-1H2O2刺激30 min,Cur干预组加入不同水平Cur,作用细胞2 h后加入1 mmol.L-1H2O2刺激30 min,对照组加入等量9 g.L-1盐水。免疫细胞化学法观察诱导型一氧化氮合酶(iNOS)的表达,Griess法测定上清液NO的生成量,荧光探针DCFH-DA法测定巨噬细胞内ROS生成。结果对照组上清液NO2-水平(相当于NO累积生成量)很低,H2O2组显著高于对照组(P<0.01),不同水平Cur干预组NO2-水平降低,即NO表达减少,并且呈水平依赖性降低(P<0.05)。各组胞质内iNOS表达与上清液NO的生成量呈相应的变化趋势。H2O2组小鼠巨噬细胞内ROS显著升高,与对照组比较差异有统计学意义(P<0.01)。不同水平Cur预先干预2 h后,ROS生成量呈水平依赖性降低(P<0.01)。结论Cur抑制H2O2诱导的小鼠巨噬细胞内NO生成,可能是通过抑制iNOS表达抑制NO生成,Cur也可抑制小鼠巨噬细胞内ROS生成,从而减轻组织细胞的氧化损伤。
Objective To investigate the effects of Cur on the production of nitric oxide (NO) and reactive oxygen species (ROS) in macrophages induced by hydrogen peroxide (H2O2). Methods Healthy macrophages were obtained from peritoneal cavity of 6-8 week old Kunming mice. The viable cells were cultured and adjusted to 2 × 108 L-1. The H2O2 group was stimulated with 1 mmol.L-1H2O2 for 30 min, and the Cur intervention group was added with different levels of Cur The cells were stimulated with 1 mmol.L-1H2O2 for 2 h after stimulated for 30 min, while the control group received the same amount of 9 gL-1 saline. The expression of inducible nitric oxide synthase (iNOS) was observed by immunocytochemical method. The amount of NO produced in supernatant was determined by Griess method. The ROS production in macrophages was determined by fluorescent probe DCFH-DA. Results Compared with the control group, NO2-level (equivalent to NO accumulation) in the control group was significantly lower than that in the control group (P <0.01), while the levels of NO2- The level of dependence decreased (P <0.05). The expression of iNOS in cytoplasm and NO production in supernatant showed a corresponding trend. ROS in H2O2 group increased significantly compared with that in control group (P <0.01). The levels of ROS were decreased in a level-dependent manner (P <0.01) after pretreatment with different levels of Cur for 2 h. CONCLUSION: Cur inhibits H2O2-induced NO production in mouse macrophages by inhibiting the expression of iNOS and inhibiting the production of NO. Cur also inhibits the ROS production in mouse macrophages and thus reduces the oxidative damage of the cells.