芳香烃受体通过p38MAPK/p65NF-κB信号通路调节绿脓菌素诱导的巨噬细胞中炎症因子的表达

Aryl hydrocarbon receptors regulate pyocyanin-induced inflammatory factor expression in macrophages via p38MAPK/p65NF-κB signaling pathway

目的探讨芳香烃受体(aryl hydrocarbon receptor,AhR)对绿脓菌素(pyocyanin PCN)诱导的巨噬细胞RAW264.7中炎性因子表达的影响及其信号通路的调控机制。方法分别采用不同浓度的PCN处理RAW264.7细胞24 h,用CCK8法检测PCN对细胞活性的影响,确定最适PCN浓度制造感染模型。将细胞分为对照组(给予0.1%dimethyl sulfoxide,DMSO)、PCN组、PCN+AhR抑制剂(CH223191)组、PCN+AhR激动剂(FICZ)组,应用免疫荧光法检测AhR的表达情况;ELISA法检测炎症因子(IL-6、IL-1β和TNF-α)的表达水平;Western blot法检测AhR、p-p38MAPK和p-p65NF-κB的蛋白表达情况。结果PCN诱导的RAW264.7细胞中AhR的表达与PCN的浓度具有明显的量效关系;与对照组相比,CH223191使PCN诱导的炎症因子分泌增加,并增强p38MAPK和p65NF-κB的磷酸化能力;FICZ降低了PCN诱导的炎症因子的产生并降低了p38MAPK和p65NF-κB的磷酸化能力。结论AhR能够调节PCN诱导的RAW264.7细胞炎症因子的表达,且p38MAPK/p65NF-κB信号通路可能是AhR参与免疫调节的重要途径。

Aim To investigate the effect of the aryl hydrocarbon receptor(AhR)on the expression of inflammatory factors in macrophages RAW264.7 induced by pyocyanin(PCN)and the regulatory mechanism of its signaling pathway.Methods RAW264.7 cells were treated with different concentrations of PCN for 24 h,respectively,and the effect of PCN on cell activity was detected by CCK8 assay to determine the optimal PCN concentration for manufacturing infection models.The cells were divided into the control group(given 0.1%dimethyl sulfoxide DMSO),PCN group,PCN+AhR inhibitor(CH223191)group,and PCN+AhR agonist(FICZ)group,and the expression of AhR was detected by immunofluorescence.The expression levels of inflammatory factors(IL-6,IL-1β,and TNF-α)were detected by ELISA.The protein expression of AhR,p-p38 MAPK and p-p65NF-κB,was detected by Western blotting.Results PCN induced a significant quantitative effect on AhR expression in RAW264.7 cells.CH223191 increased PCN-induced inflammatory factor secretion and enhanced the phosphorylation of p38MAPK and p65NF-κB compared with the control group.FICZ decreased PCN-induced inflammatory factor production and reduced the phosphorylation of p38MAPK and p65NF-κB phosphorylation capacity.Conclusions AhR can regulate PCN-induced inflammatory factor expression in RAW264.7 cells,and the p38MAPK/p65NF-κB signaling pathway may be an essential pathway for the involvement of AhR in immune regulation.