多黏菌素B抗炎作用机制的研究

Anti-inflammation Mechanism of Polymyxin B

目的观察多黏菌素B（PMB）和内毒素（LPS）共同处理大鼠肺泡巨噬细胞（PAM）后对核因子-κB（NF-κB）信号通路的变化。方法分离、培养大鼠PAM,分为正常对照组、LPS刺激组及PMB干预组。采用原位杂交（ISN）技术、凝胶电泳迁移率改变（EMSA）及ELISA法,检测刺激后0、15、30、60、120和240min各时相点PAM中IKK-βmRNA及IκB-α的表达、NF-κB的活性和TNF-α的含量。结果LPS刺激组IKK-βmRNA的水平在刺激后15min出现升高,30min达高峰,60min后逐渐下降;IκB-α的水平的变化趋势在各时相点与IKK-βmRNA刚好相反;NF-κB活性的峰值相出现在60min,15min出现升高,120min后逐渐下降;TNF-α的含量峰值相出现在60min,30min出现升高,120-240min后恢复至刺激前水平。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〈0.01）。IκB-α水平的最低值显著高于LPS刺激组（P〈0.01）;而IKK-βmRNA的峰值则显著低于LPS刺激组（P〈0.01）。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。而PMB则能抑制LPS诱导的IKK-β激活、IκB-α降解、NF-κB活化和TNF-α释放。

Objective To observe the pathway changes of nuclear factor kappa B （NF-κB） after rat pulmonary alveolar macrophages （PAM） were treated with LPS and polymyxin B （PMB）. Methods Wistar rat PAM were isolated and cultured, then treated with normal solution as normal control group, LPS as LPS stimulation group and LPS ＋ PMB as interference group. PAM were fixed respectively at 0, 15, 30, 60, 120 and 240 rain after stimulation. IKK-β mRNA expression in PAM by in situ hybridization, NF-κB activity in nucleoprotein extracted from PMB by electrophoretic mobility shift assay （EMSA）, κB-α and TNF-α content in culture supernatant by ELISA were detected. Results In LPS group, IKK-βmRNA level increased at 15 min, reached the peak at 30 min and decreased at 60 min, while Iκ-α level turned out contrary to IKK-βmRNA level; NF-κB activity peaked at 60 min, and started to decrease at 120 min, significantly higher than that before stimulation and of normal control group （P〈0. 01）; TNF-α level reached the peak at 60 min, started to increase at 30 rain and got back to the level before stimulation at 120-240 min. In 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 and 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 and accelerated TNF-α release, but PMB can counteract those effects.