核因子-κB信号通路在尼古丁影响哮喘大鼠树突状细胞表达白细胞介素-12的作用研究

Effect of NF-κB signaling pathway on IL-12 expression of dendritic cells in rats with nicotine-induced asthma

目的研究核因子-κB信号通路在尼古丁影响支气管哮喘大鼠树突状细胞(DCs)表达(IL)-12中的作用,探讨吸烟加重哮喘的病理学机制。方法建立支气管哮喘大鼠模型,体外培养骨髓来源的DCs,采用不同实验条件尼古丁进行干预,酶联免疫吸附法(ELISA)测定细胞上清液中IL-12蛋白的浓度,观察尼古丁作用的最佳浓度和时间。进一步用NF-κB抑制剂二硫基氨基甲酸吡咯烷(PDTC)干预DCs,免疫细胞化学法测定各组细胞NF-κB的表达,ELISA法测定各组细胞上清液中IL-12蛋白的表达水平,同时进行混合淋巴细胞反应,四甲基偶氮唑盐(MTT)法检测淋巴细胞的增殖活性。结果 (1)与对照组相比,200μg/ml尼古丁组DCs IL-12蛋白表达增加,400μg/ml尼古丁组DCs IL-12蛋白表达减少,差异均具有统计学意义(均P<0.01);(2)以200μg/ml尼古丁分别与DCs孵育0、4、8、12、24及72 h,IL-12蛋白表达在12 h达到高峰,72 h降低,差异均具有统计学意义(均P<0.01);(3)与对照组相比,尼古丁组DCs IL-12蛋白表达增加,PDTC组DCs IL-12蛋白表达减少,差异均具有统计学意义(均P<0.01);与尼古丁组相比,尼古丁+PDTC组DCs IL-12蛋白表达减少,差异具有统计学意义(P<0.05);(4)与对照组相比,尼古丁组DCs刺激同种异体淋巴细胞增殖能力升高,PDTC组DCs刺激同种异体淋巴细胞增殖能力降低,差异均具有统计学意义(均P<0.01);与尼古丁组相比,尼古丁+PDTC组DCs刺激同种异体淋巴细胞增殖能力降低,差异具有统计学意义(P<0.05)。结论 NF-κB信号通路在尼古丁诱导DCs表达IL-12中发挥一定作用,这可能是烟雾暴露恶化哮喘气道免疫平衡的机制之一。

Objective To determine the effect of nuclear factor（NF）-κB signaling pathway in the interleukin（IL）-12 expression of dendritic cells（DCs） in rats with nicotine-induced bronchial asthma, and to investigate the pathological mechanism of asthma exacerbated by smoking. Methods The rat models of bronchial asthma were established, and the bone marrow-derived DCs were cultured in vitro. Nicotine intervention was given, under different experimental conditions. The concentration of IL-12 protein in the supernatant was measured by ELISA, and the optimum concentration and time of nicotine effect were determined. Furthermore, DCs were intervened with NF-κB inhibitor, pyrrolidine dithiocarbamate（PDTC）. The expression of NF-κB was determined by immunocytochemistry. The levels of IL-12 protein in the supernatant of each group were determined by ELISA, and the mixed lymphocyte reac-tion was performed. The proliferation activity of lymphocytes was determined by MTT assay. Results（1）Compared with the control group, the expression of IL-12 protein in DCs increased in the 200 μg/ml nicotine group and decreased in the 400 μg/ml nicotine group, and the difference was statistically significant（all P〈0.01）.（2）After the DCs were incubated with 200 μg/ml nicotine for 0, 4, 8, 12, 24 and 72 h, the expression of IL-12 protein reached a peak at12 h and decreased at 72 h, respectively, and the differences were statistically significant（all P〈0.01）.（3）Compared with the control group, the DCs IL-12 protein expression increased in the nicotine group and decreased in the PDTC group, and the difference was statistically significant（all P〈0.01）. Compared with the nicotine group, the DCs IL-12 protein expression decreased in the nicotine＋PDTC group, and the difference was statistically significant（ P〈0.05）.（4）Compared with the control group, the proliferation of allogeneic lymphocytes stimulated by DCs increased in the nico-tine group, and decreased in the PDTC group, and the difference was statistically significant（all P〈0.01）. Compared with the nicotine group, the proliferation of allogeneic lymphocytes stimulated by DCs decreased in the nicotine＋PDTC group, and the difference was statistically significant（P〈0.05）. Conclusion NF-κB signaling pathway may play a certain role in the IL-12 expression in DCs induced by nicotine, which may be one of the mechanisms of airway immune balance of asthma exacerbated by smoking exposure.