大气细颗粒物经p66Shc蛋白/MAPK通路上调支气管和肺动脉受体的作用

Airborne fine particulate matter (PM2.5) is a risk factor for respiratory system diseases. The receptor upregulation is the basis of airway hyperresponsiveness. The p66Shc/MAPK pathway is associated with PM2.5 and receptor upregulation. Therefore, we assume that PM2.5 upregulates the contractile receptors in smooth muscles via a p66Shc/MAPK pathway, which enhances the contraction of bronchi to induce respiratory diseases. On the basis of our previous in vitro study that PM2.5 upregulated receptors in cultured rat bronchi and arteries, the present project aims to confirm that PM2.5 induced over-expression of contractile receptor in rat bronchi and pulmonary arteries in PM2.5 inhalation models (the model of directly inhaling polluted air and the model of aerosol inhaling collected PM2.5). Next, the mechanisms of how p66Shc/MAPK pathway is involved PM2.5-induced upregulation of contractile receptors are studied by p66Shc knockout mouse model with PM2.5 inhalation, p66Shc RNAi technique in bronchus and pulmonary artery smooth muscle cells, and MAPK inhibitors. Further, the contractile responses of isolated bronchi and pulmonary arteries are studied by wire myograph. The mRNA and protein expressions for the receptors and p66Shc are examined by qRT-PCR, Western blotting and immunofluorescence, respectively. The effects of PM2.5 on bronchus function are evaluated by an airway resistance determining system. The findings will unveil the mechanisms of PM2.5-induced respiratory diseases, which may provide an experimental basis for prevention and treatment of the diseases.

大气细颗粒物(PM2.5)是呼吸系统疾病的危险因素，受体上调是平滑肌高反应性的基础， p66Shc蛋白/MAPK通路与PM2.5和受体上调相关。我们假设PM2.5经p66Shc蛋白/MAPK通路，上调收缩性受体，增强支气管收缩，诱发呼吸系统疾病。本项目拟在以往离体培养显示PM2.5上调受体的基础上，用中重度污染大气直接吸入和收集的PM2.5雾化吸入整体暴露模型，确证PM2.5上调大鼠支气管和肺动脉收缩性受体的作用；用p66Shc基因敲除小鼠、人支气管和肺动脉平滑肌细胞siRNA及MAPK通路抑制剂研究p66Shc蛋白及其相关MAPK通路对PM2.5上调受体的机理，反向证实假说。用肌动描记技术检测支气管和肺动脉环的收缩；用基因/蛋白定量、免疫荧光技术研究受体和p66Shc蛋白表达，用气道阻力测定系统测气道阻力变化。本项目的完成将揭示PM2.5诱发呼吸系统疾病的机理，为其防治策略提供实验依据。

大气细颗粒物是呼吸系统和血管性疾病的重要危险因子，我们前期研究已证实PM2.5通过上调支气管和动脉收缩性受体，增加平滑肌的收缩反应性，诱发呼吸和血管系统疾病。在本项目中，我们发现: ① PM2.5与离体支气管环或肠系膜动脉环共培养后，能激活MAPK通路，上调支气管平滑肌乙酰胆碱和内皮素A（ETAR）和内皮素B受体（ETBR），上调肠系膜动脉平滑肌5羟色胺受体、ETAR和ETBR，诱发平滑肌的高反应性。② 慢性污染大气颗粒物暴露能引起大鼠肺部炎性因子升高，激活MEK/ERK1/2信号通路，从而诱导肺小动脉平滑肌收缩性ETBR上调，可能参与大鼠肺小动脉高反应性和重构。③ 长期实时大气颗粒物暴露能导致大量颗粒物在肺部沉积，升高肺部炎性因子的水平，引起肺部病变，从而致使大鼠肺功能减弱，并且表现出年龄相关的差异。大气颗粒物暴露对老年鼠呼吸系统的损害作用最大，幼年鼠和成年鼠次之。本项目的完成将揭示大气细颗粒物诱发呼吸系统疾病的机理，为其防治提供依据和可能的靶点。