大气颗粒物急性暴露对C57BL/6小鼠肺脏病理改变和气道上皮细胞IL-6和IL-8表达的影响

Effect of acute exposure to atmospheric particulate matter on lung patho⁃logical changes and expression of IL-6 and IL-8 in bronchial epithelial cells of C57BL/6 mice

目的:探讨大气颗粒物(PM)急性暴露下C57BL/6小鼠肺脏的病理改变和气道上皮细胞炎症介质白细胞介素6(IL-6)和白细胞介素8(IL-8)分泌的分子机制。方法:选取雄性C57BL/6小鼠随机分为空白对照组和PM实验组,每组10只:PM实验组连续2 d经气管滴注PM悬浮液,建立PM短期暴露的小鼠模型;空白对照组连续2 d经气管滴注PBS。获取肺组织进行HE染色和PAS染色以评估组织病理变化;获取支气管肺泡灌洗液(BALF),检测IL-6和IL-8的表达。建立PM刺激人气道上皮细胞的体外细胞模型:200 mg/L的PM刺激BEAS-2B细胞,于3、6、12和24 h时点检测IL-6和IL-8的mRNA表达;25、50、100、200和400 mg/L的PM刺激BEAS-2B细胞24 h,检测IL-6和IL-8蛋白的分泌;200 mg/L的PM刺激BESA-2B细胞5、10和15 min,检测PI3K信号通路相关蛋白的变化;预先给予5μmol/L PI3K抑制剂LY294002干预0.5 h,再用200 mg/L的PM刺激BESA-2B细胞15 min,检测PI3K信号通路相关蛋白的变化。结果:在体内动物模型中,与空白对照组相比,PM组小鼠气道周围可见炎症细胞浸润,气道上皮细胞变厚,BALF中IL-6和IL-8显著升高(P<0.01)。在体外细胞模型中,与空白对照组相比,PM刺激可引起BEAS-2B细胞中IL-6和IL-8的mRNA表达和蛋白分泌(P<0.01),同时能显著提高PI3K信号通路的磷酸化水平(P<0.01),PI3K抑制剂LY294002能部分逆转PM诱导的PI3K信号通路的磷酸化(P<0.05),同时显著逆转PM诱导的IL-6和IL-8蛋白分泌(P<0.01)。结论:本研究的动物实验揭示了PM急性暴露下C57BL/6小鼠肺脏以气道炎症为主的病理改变,体外细胞实验揭示PI3K信号通路介导了PM诱导的气道上皮细胞IL-6和IL-8分泌。

AIM:To investigate the pathological changes of lung tissues in C57BL/6 mice exposed to atmospheric particulate matter(PM)and the molecular mechanisms of secretion of inflammatory mediators interleukin-6(IL-6)and interleukin-8(IL-8)from bronchial epithelial cells.METHODS:For in vivo study,the C57BL/6 mice were randomly divided into vehicle group and PM group.The mice in PM group were given intratracheal instillation of PM for 2 consecutive days.The harvested lung tissues were stained with HE staining and PAS staining,and the inflammatory mediators in bronchoalveolar lavage fluid(BALF)were quantified.For in vitro study,human bronchial epithelial cells were treated with PM at various doses for indicated time.The mRNA and protein expression levels of IL-6 and IL-8 were quantified.The changes of PI3K signaling pathway-related proteins were detected.RESULTS:In the C57BL/6 mice,PM exposure resulted in infiltration of inflammatory cells around the pulmonary airways,thickening of airway epithelial cells,and upregulated secretion of IL-6 and IL-8 in BALF.In human bronchial epithelial cells,PM stimulated the mRNA transcription and protein synthesis of IL-6 and IL-8.Exposure to PM promoted phosphorylation of PI3K signaling pathway,and inhibition of PI3K signaling pathway partially reversed the secretion of IL-6 and IL-8.CONCLUSION:The pulmonary pathological changes in mice exposed to PM were mainly airway inflammation.The PI3K signaling pathway mediates PM-induced IL-6 and IL-8 secretion from bronchial epithelial cells.