多黏菌素B对内毒素诱导的RAW264.7细胞分泌一氧化氮的影响

Effects of polymyxin B on nitric oxide production of RAW264.7 cells stimulated by lipopolysaccharide

目的以单核巨噬细胞株RAW264.7分泌一氧化氮(NO)为指标,观察多黏菌素B(PmB)对内毒素(LPS)作用的阻断能力,为运用PmB消除LPS生物学效应提供实验基础。方法实验分3类:①10μg·L^(-1)LPS预先与RAW264.7细胞作用一定时间后,加入各浓度PmB;②各浓度PmB与RAW264.7细胞作用一定时间后,加入10μg·L^(-1)LPS刺激;③将10μg·L^(-1)LPS与各浓度PmB混合,经不同时间预处理后用于刺激RAW264.7细胞。检测细胞释放NO水平,评价LPS的生物学效应。结果①LPS刺激RAW264.7细胞10min后加入各浓度PmB,NO水平显著降低(P<0.05),但最大抑制率仅为23%;LPS刺激20min后,PmB不能显著降低NO水平。②PmB先作用于RAW264.7细胞10、20 min,再加入LPS,NO水平均显著降低(P<0.05),最大抑制率均为39%;③PmB与LPS经过预处理,RAW264.7细胞分泌的NO显著降低(P<0.001),最大抑制率达80%,并具有量效和时效关系。结论预处理24h,PmB能最大程度地抑制10μg·L^(-1)LPS在RAW264.7细胞上的生物学效应。

AIM To investigate the effects of polymyxin B （PmB） on lipopolysaccharide-induced nitric oxide （NO） production of RAW264.7 cells and to provide an experimental basis for the correct use of PmB to eliminate biologi-cal activities of lipopolysaccharide （LPS）. METHODS Experiments were divided into three types：① RAW264.7 cells were stimulated with 10μg·L^-1 LPS for a period of time, and subsequently PmB was added; ② RAW264.7 cells were treated with PmB for a period of time before stimulation with 10μg·L^-1 LPS; ③ LPS was pre-incubated with PmB for a period of time, then the mixture was used to stimulate RAW264.7 cells. NO levels in cell culture supematant were mea-sured using Griess reagent. RESULTS ① After the cells were stimulated by LPS for 10 min, PmB could significantly inhibit NO release（ P 〈 0.05）. The maximum inhibition rate was 23%. But when PmB was added at 20 rain after LPS stimulation, this effect was not significant. ②When PmB was added to cells 10 or 20 min before LPS stimulation, NO production was significantly reduced（ P 〈 0. 05）. The maximal inhibition rate was 39%. ③ When LPS was pre-incubat-ed with PmB, NO secretion of RAW264.7 cells was significantly reduced（ P 〈 0.001）. The maximum inhibition rate was up to 80%. This inhibition effects were time-and dose- dependent. CONCLUSION If it pre-treated LPS for 24 h, PmB could effectively counteract LPS biological effects.