清肺口服液调节IL-17激活的ERK1/2信号通路抑制呼吸道合胞病毒加重哮喘发作的机制研究

Respiratory viral infection is the major causes of asthma exacerbation, in infancy, infections with respiratory viruses, particularly Respiratory Syncytial Virus (RSV), cause wheezing and bronchiolitis, which frequently induce asthma exacerbation. Many studies have suggested that RSV infection were associated with epithelial cells injury and subsequent airway remodeling. However, the precise mechanisms are still unclear. Recent studies indicated that early activation of ERK1/2 MAPK plays a critical role in regulating virus-induced asthma exacerbations. Our previous studies have provided evidence that Qi Fei oral liquid can inhibit RSV proliferation directly, and regulate TGF-β FAS signaling, which associated with the lung fibroblast proliferation. The aims of the present study plan are to evaluate the effection of Qi Fei Oral Liquid on the IL-17 activated ERK1/2 signaling pathway, and probe into the mechanisms of using Qi Fei oral liquid to reduce RSV induced asthma exacerbation. To reach these aims, in vitro models of differentiated tracheobronchial epithelium have being built. In this research plan, gene cloning, gene expressing regulation, signal pathways block, and laser cofocus light microscopy technology etc. would be used in vivo and vitro, and the cellular and molecular mechanism would be analyzed. Through analysis the associations between RSV infection and epithelial basal cells differentiation, chemokine expression, airway hyperresponsiveness and airwayremolding, probes into the mechanism of RSV induced asthma exacerbation. This research is expected to provide experimental basis for preventing and/or treatment RSV induced asthma exacerbations in Chinese Traditional Medicine pediatrics.

病毒感染是导致儿童哮喘急性发作的重要诱因，呼吸道合胞病毒是引起呼吸道感染最常见的病原体，其致病机制与合胞病毒感染后上皮修复异常所致的气道重塑有关，而IL-17激活的ERK1/2信号通路与合胞病毒感染所致气道上皮损伤与修复密切相关。前期研究显示清肺口服液对呼吸道合胞病毒增殖具有直接抑制作用，且可通过调控TGF-β1、 Fas等影响肺成纤维细胞分裂、增殖。本项目拟通过构建具有复合立体结构的气道模型，应用激光共聚焦技术研究清肺口服液对合胞病毒感染致哮喘加重时气道上皮细胞损伤修复的影响；应用基因克隆表达和信号通路阻滞技术，从分子、细胞水平研究合胞病毒感染后ERK1/2信号通路与气道上皮细胞增殖、分化的关系以及清肺口服液的调控作用。探讨清肺口服液抗合胞病毒感染致哮喘气道炎症损伤的可能机制，为运用中医药治疗呼吸道合胞病毒感染加重哮喘发作提供实验基础和新思路。

目的：研究清肺口服液对哮喘合并RSV感染小鼠模型气道炎症损伤的保护和促进损伤修复的作用及对IL-17激活的ERK1/2 信号通路的作用机制。方法：1.动物实验：用HEp－2细胞孵育RSV A2 株（ATCC）病毒，复制了哮喘+RSV感染模型，检测肺泡灌洗液中EOS的变化；使用先进的Buxco RC 系统测定了小鼠的肺功能；采用HE、PAS、VG三种方法的病理切片观察气道炎症。Western Blot 检测ERK1/2 MAPK信号通路相关蛋白表达。 2.细胞实验：(1)炎症因子及蛋白表达：IL-17A作用于人呼吸道上皮细胞株16-HBE，NCI-H292组成的体外模拟气道模型。ELISA 法检测炎症因子（IL-6、IL-8、IL-4、IFN-γ）变化，Western Blot 检测ERK1/2 MAPK信号通路相关蛋白表达的影响。 (2)细胞信号通路阻滞实验：采用MTT法评估清肺口服液、抑制剂、激活剂对两组细胞的毒性；Western Blot 检测加入通路抑制剂、激活剂p-ERK，ERK、p38，phospho-p38，JNK，phospho-JNK 等信号通路相关蛋白表达；ELISA 法：检测加入阻滞剂及激活剂后各组培养上清中细胞因子、趋化因子（IL-6、IL-8、IL-4、IFN-γ）。结果：1.清肺口服液可以明显降低BALF中嗜酸粒细胞比例，与正常组相比差异有极显著性(P<0.01)；清肺口服液可显著改善气道高反应性(P<0.05)，其中中剂量清肺口服液效果尤为显著(P<0.05)。2.RSV病毒对MAPK信号通路p-p38蛋白表达无影响，对p-ERK，p-JNK有明显的浓度及时间依赖关系，随着时间延长及浓度增大，RSV可以明显升高p-ERK，p-JNK蛋白的表达。3.信号通路阻滞实验提示:清肺口服液可以明显提高加入激活剂后（p-ERK，p-JNK）的表达，可减轻气道炎症因子IL-6、IL-8的表达。.结论：1.呼吸道合胞病毒感染能明显加重哮喘模型小鼠肺部组织炎症反应,同时气道阻力明显增高。2.清肺口服液能明显减轻RSV感染所致哮喘小鼠急性发作肺组织炎症反应，可以明显改善哮喘小鼠气道高反应性。3.清肺口服液能明显降低IL-17A激活的ERK1/2，JNK信号通路ERK1/2、JNK磷酸化蛋白的表达，这可能是清肺口服液调节RSV感染哮喘气道炎症反应的机制之一。