姜黄素对过氧化氢诱导小鼠巨噬细胞内一氧化氮及活性氧生成的影响

Inhibitory Effect of Curcumin on H_2O_2-Induced Production of Nitric Oxide and Reactive Oxygen Species in Mouse Embryo-Fibroblast

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2×10^8L-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 curcumin on H2O2-induced the production of nitric oxide（NO） and reactive oxygen species（ROS） in mouse embryo-fibroblast.Methods Macrophage were collected in abdominal cavity of 6-8 weeks Kunming mouse,cultured macrophage（2×10^8 L^-1） were divided into control group and curcumin groups randomly.Macrophage in H2O2 group were added into a single bolus of H2O2（1 mmol·L^-1） for 30 min,macrophage in curcumin group were preincubated with different concentration of curcumin for 2 h followed by a 30 min incubation with 1 mmol·L^-1 H2O2 and macrophage in control group were added into the same volume（0.1 mL） of 9 g·L^-1 so-dium chloride.Immunocytochemistry was used to measure the contents of inducible nitric-oxide synthase（iNOS）,NO production was determined by Griess reactive,ROS was determined by DCFH-DA Fluorescence proe.Results The production of NO in control group was little.The production of NO in H2O2 group markly highter than that in control group（P〈0.01）.When pretreated with different concentrations of curcumin for 2 h,the production of NO in isolation supernatants decreased gradually in a dose-dependent（P〈0.05）.The statistical analysis change trend of iNOS located in the cytoplasm was almost accord with production of NO.Conclusions The production of NO in mouse embryo-fibroblast was significantly inhibited by curcumin,and this effect is mediated through the inhibition of expression of iNOS,otherwise,the production of ROS in mouse embryo-fibroblast is significantly inhibited by curcumin,so it can decrease oxidative damage of histiocyte.