布洛芬对人离体中性白细胞MyD88-p38MAPK通路的影响

Effect of Ibuprofen on LPS-induced MyD88-p38MAPK Pathway in Human Neutrophils in Vitro

目的：研究布洛芬对脂多糖（LPS）介导人中性白细胞MyD88-p38MAPK通路的影响。方法取正常健康人外周静脉血20 mL。用密度梯度法分离中性白细胞和用免疫磁珠纯化纯化中性白细胞。用Nucleofection转染系统转染MyD88 siRNA基因。用5μmol/L、10μmol/L、50μmol/L和100μmol/L布洛芬和LPS 1μg/mL刺激上述细胞。用Western Blot检测 MyD88和p38MAPK磷酸化水平。结果布洛芬呈剂量依赖性抑制LPS介导的正常人中性白细胞和MyD88基因沉默中性白细胞产生氧自由基。正常人中性白细胞和MyD88基因沉默中性白细胞，浓度为50μmol/L和100μmol/L时，可明显抑制ROS的产生达到50.0%±2.4%和42.5%±4.7%，40.0%±3.7%和30.0%±4.6%，与单纯LPS刺激相比P＜0.01。同时在正常和MyD88基因沉默细胞，50μmol/L布洛芬可明显抑制LPS介导的MyD88和p38MAPK的表达。结论布洛芬通过下调MyD88-p38MAPK通路抑制人中性白细胞细胞内氧自由基的产生。

Objective To study the effect of ibuprofen on LPS induced MyD88-p38MAPK pathway in hu-man neutrophils in vitro. Methods Neutrophils were isolated from the peripheral blood 20 mL and subjected to Ficoll-Hypaque density gradient centrifugation and the puriifcation using the Easysep Neutrophil Enrichment Kit. Isolated and puriifed neutrophils were transiently transfected using the Nucleofection technology. The cells were inbucated with ibuprofen （5μmol/L, 10μmol/L, 50μmol/L and 100μmol/L） and LPS 1μg/mL. Then, the level of ROS and the phosphorylation of MyD88 and NK-κB were determined by lfow cytometry and Western Blot, respectively. Results LPS-induced ROS production were inhibited by ibuprofen in dose dependent in normal neu-trophil and MyD88-knockdown human neutrophils, and the inhibitory ability of 50μmol/L and 100μmol/L ibu-profen on LPS-ROS production is 50.0%±2.4%and 42.5%±4.7%;40.0%±3.7%and 30.0%±4.6%in normal neutrophil and MyD88-knockdown human neutrophils, respetively. Ibuprofen （50μmol/L） inhibited the phosphor-ylation of MyD88 and p38MAPK in normal and MyD88-knockdown human neutrophils. Conclusion Ibuprofen inhibits LPS-ROS production via the down-regulation of MyD88 and p38MAPK in human neutrophils.