多黏菌素B对内毒素诱导的肺泡巨噬细胞中NF-κB活化的影响

Effects of polymyxin B on LPS-induced activation of NF-κB in pulmonary alveolar macrophages

目的 :观察多黏菌素B(PMB)对内毒素 (LPS)刺激大鼠肺泡巨噬细胞 (PAM)中核因子 κB(NF κB)激活通路的影响 ,并探讨PMB可能的抗炎效应。方法 :分离、培养大鼠PAM ,分为正常对照组、LPS刺激组及PMB +LPS干预组。各组PAM在刺激后 0、15、30、6 0、12 0和 2 4 0min分别固定 ,提取PAM核蛋白并收集细胞培养上清 ,采用原位杂交 (ISN)技术、凝胶电泳迁移率改变 (EMSA)及ELISA法 ,观察PAM中IKK βmRNA及IκB α的表达 ,检测PMB核蛋白提取物中NF κB的活性和上清液中TNF α的含量。结果 :LPS刺激组IKK βmRNA的水平 ,显著高于刺激前和正常对照组 (P <0 .0 1) ;IκB α的水平的变化趋势与IKK βmRNA刚好相反。NF κB活性的峰值相对于刺激前和正常对照组有显著升高 (P <0 .0 1)。培养上清中TNF α的含量 ,亦显著高于刺激前和正常对照组(P <0 .0 1)。PMB干预组NF κB的活性与TNF α的含量虽较刺激前和正常对照组升高 ,但均显著低于LPS刺激组 (P <0 .0 1)。IκB α水平的最低值显著高于LPS刺激组 (P <0 .0 1) ;而IKK βmRNA的峰值则显著低于LPS刺激组 (P <0 .0 1)。结论 :LPS能诱导PAM中的IKK β激活、IκB α降解和NF κB活化 ,并促进TNF α释放。PMB则能抑制LPS诱导的IKK β激活、IκB α降解、NF κB活化和TNF α?

AIM: To observe effects of polymyxin B (PMB) on LPS induced activation of NF κB of pulmonary alveolar macrophages(PAMs) and explore the possible anti inflammation efficiency of PMB. METHODS: Rat PAMs were isolated and cultured. The PAMs were divided into 3 groups, namely, normal control group (PAMs+normal solution), LPS stimulation group (PAMs+10 mg/L LPS) and PMB interference group(after PMB treatment for 30 min, using LPS stimulation). The PAMs were fixed respectively at 0,15,30,60,120 and 240 min after stimulation. Nucleoprotein was extracted from cultured PAMs and culture supernatant of the PAMs was collected. The expression of IKK β mRNA in the PAMs, NF κB activity in PMB nucleoprotein extractive, TNF α content in culture supernatant of the PAMs were detected by in situ hybridization (ISN), electrophoretic mobility shift assay (EMSA) and ELISA, respectively. RESULTS: IKK β mRNA level in LPS group increased at 15 min, reached the peak at 30 min, while IκB α level turned out contrary to IKK β mRNA level. The peak of NF κB activity appeared at 60～120 min after stimulation was significantly higher than those of pre stimulation and normal control group ( P <0.01). In PMA interference group, NF κB activity and TNF α level were markedly lower than those in LPS stimulation group ( P <0.01). The lowest level of IκB α was notably higher than that in LPS stimulation group, while the peak of IKK β mRNA was obviously lower than that in LPS stimulation group ( P <0.01). CONCLUSION: LPS can induce IKK β and NF κB activation, IκB α degradation, accelerate TNF α release, but PMB can counteract those effects induced by LPS stimulation.