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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 the effect of polymyxin B (PMB) on the activation of nuclear factor κB (NF-κB) in rat alveolar macrophages (PAM) stimulated by endotoxin (LPS) and to explore the possible anti-inflammatory effects of PMB. Methods: The rat PAM was isolated and cultured and divided into normal control group, LPS stimulation group and PMB + LPS intervention group. The PAMs of each group were fixed at 0, 15, 30, 60, 120 and 240 min after stimulation, PAM nuclear protein was extracted and the cell culture supernatants were collected. The mobility of PAM was changed by in situ hybridization (ISN) (EMSA) and ELISA were used to observe the expression of IKKβmRNA and IκBα in PAM. The activity of NF κB in PMB nuclear extract and the content of TNFα in supernatant were detected. Results: The level of IKKβ mRNA in LPS stimulation group was significantly higher than that in stimulation group and normal control group (P <0.01). The trend of IκBα level was opposite to that of IKK β mRNA. The peak of NF-kappaB activity was significantly increased relative to pre-stimulation and normal controls (P <0.01). The content of TNFα in the culture supernatant was also significantly higher than that before stimulation and normal control (P <0.01). The activity of NF κB and the level of TNFα in PMB intervention group were significantly higher than those in the control group before stimulation and in the normal control group, but both were significantly lower than those in the LPS stimulation group (P <0.01). The lowest level of IκB α was significantly higher than that of LPS stimulation group (P <0.01), while the peak of IKK β mRNA expression was significantly lower than that of LPS stimulation group (P <0.01). CONCLUSION: LPS can induce IKK β activation, IκB α degradation and NF κB activation in PAM, and promote TNF α release. PMB inhibited LPS-induced IKK β activation, IκB α degradation, NF κB activation, and TNFα?