玉屏风散与加味过敏煎干预PM2.5致肺泡巨噬细胞吞噬功能受损导致免疫失衡诱发肺部炎症反应的比较研究

The inhaling PM2.5 (particulate matter with a diameter of ≤2.5mm) may decrease the phagocytic activity of alveolar macrophages, which initiating the innate immune and adaptive immune response. PM2.5 exposure can cause the imbalance of Th1/Th2 and Th17/Treg responses induced by alveolar macrophages leading to pulmonary inflammation. Our previous study showed that Yupingfeng powder and the modified Guomin decoction could repair the imbalance of Th17/Treg responses and mucus hypersecretion induced by PM2.5. Therefore we put forward the scientific hypothesis that Yupingfeng powder and modified guomin decoction can repair alveolar macrophage phagocytic function, regulate innate immune and adaptive immune response and inhibit pulmonary inflammation. This study intends to use a real-life air pollution exposure protocol to simulate PM2.5 induced pulmonary injury in rat model. We will detective the alveolar macrophages phagocytosis and use whole rat genome microarray, immunohistochemical and molecular biology and flow cytometry method to detect the effect of Yupingfeng powder and the modified Guomin decoction on PM2.5 exposure rat. We will also detect the effect of Yupingfeng powder and the modified Guomin decoction in TLR2-My88-NF kappa B/MAPK pathway and Th1/Th2 and Th17/Treg responses, which lead to pulmonary inflammation. Besides, we will use whole rat genome microarray to screen differential expressed genes and possible intervention pathway of Yupingfeng powder and the modified Guomin decoction in rat model and validate through molecular biological methods.

PM2.5随呼吸进入体内后可抑制肺泡巨噬细胞吞噬功能，启动免疫应答，引起Th1/Th2和Th17/Treg免疫失衡，诱发肺部炎症反应。我们的前期研究表明玉屏风散、加味过敏煎可能通过干预免疫失衡对PM2.5致肺损伤起保护作用。据此，提出科学假说：玉屏风散与加味过敏煎可以修复PM2.5引起的肺泡巨噬细胞吞噬功能受损、调节免疫失衡，抑制肺部炎症反应。.本研究拟采用实际大气颗粒物下的控制暴露，复制PM2.5致肺损伤大鼠模型，分别给予玉屏风散和加味过敏煎干预，通过肺泡巨噬细胞吞噬功能检测、肺组织基因序列表达分析、运用免疫组化和分子生物学及流式细胞等方法检测TLR2-My88-NF-κB/MAPK通路、Th1/Th2和Th17/Treg失衡及相关的多种细胞因子；根据全基因组表达谱筛选表达差异基因，通过富集分析构建可能的干预途径，并用分子生物学方法进行验证。比较研究不同立法的方药作用机制。

本项目通过体内外实验，研究并比较玉屏风散与加味过敏煎干预PM2.5致肺损伤的作用机制。.体内实验.采用气道内滴注法建立PM2.5致肺损伤大鼠模型，分别给予玉屏风散和加味过敏煎，连续干预28天后采集肺组织，并进行肺泡灌洗，提取BALF和肺泡巨噬细胞。通过HE染色和AB-PAS染色观察肺组织病理形态；免疫组化法观察肺组织中炎症介质表达情况；ELISA、Western Blot和RT-PCR法检测BALF细胞因子含量、肺组织中炎症因子和TLR2-My88-NFκB，MAPKs，PI3K-AKT信号通路表达、和Th17/Treg平衡相关分子表达；同时通过基因组学、蛋白质组学推测药物的潜在靶点。结果表明：玉屏风散与加味过敏煎具有改善PM2.5致肺损伤的作用，通过抑制肺组织中炎症介质MCP-1和ICAM-1的表达和细胞因子IL-1β、IL-6、TNF-α、IgE、IL-4的合成分泌，干预PM2.5引起的肺组织炎症损伤，且玉屏风散的干预作用优于加味过敏煎；玉屏风散与加味过敏煎通过干预NFκB 和ERK、JNK发挥抑制炎症反应的作用；玉屏风散与加味过敏煎可以抑制杯状细胞增生和气道粘液分泌，其机制与下调PI3K-AKT信号通路有关，且玉屏风散的干预作用优于加味过敏煎；玉屏风散与加味过敏煎通过调节Th17/Treg平衡，减轻PM2.5导致的肺炎症损伤；转录组学和蛋白质组学研究表明，加味过敏煎可以抑制PM2.5模型大鼠肺组织炎症基因Alox12e、TLR12表达，抑制促癌蛋白，下调组蛋白和半乳糖凝集素，上调抑癌蛋白；玉屏风散可以抑制G蛋白偶联受体基因表达，上调抑癌基因Rasd1、Gas2，抑制促癌蛋白，下调组蛋白，上调水通道蛋白，推测这些分子是中药复方的潜在靶点。.体外实验.使用流式细胞术对大鼠肺泡巨噬细胞进行检测，分析中药复方对肺泡巨噬细胞凋亡的干预作用。结果表明：加味过敏煎可显著抑制PM2.5引起的肺泡巨噬细胞凋亡。.本项目发表核心期刊论文5篇,在投SCI论文1篇，进行全国学术报告3次，培养博士研究生4名。