基于内病外治法揭示喘康贴对哮喘大鼠肺组织miRNA网络调控机制的研究

Bronchial asthma, characterized by chronic airway inflammation and airway hyper-responsiveness, is a chronic airway disease which seriously endangers children's health. Pure Chinese medicine Chuna-Kang Plaster was set on the basis of theory of “external treatment for internal illness”, which is effective in clinical treatment . In previous studies, the research team found that Chuan-Kang Plaster had significantly inhibiting effect on bronchial inflammation and modulatory effects on immune system in asthmatic rats. Establishing asthma models, the present study will compare the miRNAs expression profiles of asthmatic rats treated with those untreated by Chuan-Kang Plaster through high-throughput miRNA chip techonology. The miRNAs with differential expression will be analyzed by bioinformatics analytic systerm, including gene-set enrichment analysis, target genes calculation and prediction, cluster analysis, pathway analysis, and establishing interrelation of target proteins. On that basis, using methods of RT-qPCR to verify the predicted target genes and Western Blot to test involved proteins’ expression in the lung tissue will further be implemented, so as to explore potential pathogenesis of asthma in both gene level and molecular level in rats, also build regulatory network involved in the effected miRNAs of Chuan-Kang Plaster. The study will provide experimental basis and evidence to future clinical research on external therapy to asthma by Chuan-Kang Plaster.

支气管哮喘以慢性气道炎症和气道高反应性为临床特征，是一种严重危害儿童健康的慢性气道疾病。运用中医经典理论内病外治的中药穴位贴敷喘康贴治疗哮喘，临床疗效显著。课题组前期研究表明，喘康贴对哮喘大鼠的慢性气道炎症有显著抑制作用和免疫调节作用。本次研究拟通过建立哮喘大鼠模型，利用miRNA高通量芯片技术观察喘康贴干预后的哮喘大鼠肺组织miRNA表达谱变化，将芯片检测结果和差异表达的miRNAs进行生物信息学分析，对其靶基因进行计算预测和功能注释、富集分析，构建其所涉及的信号通路及相关蛋白相互作用关系；并运用RT-qPCR法对差异表达的miRNAs预测靶基因进行验证，Western blot法对相应的蛋白表达进行检测，从而探寻支气管哮喘大鼠从相关miRNA到分子信号的可能致病机理，构建喘康贴作用的miRNA调控网络，为内病外治喘康贴治疗支气管哮喘的临床应用提供实验基础和理论依据。

运用中药复方网络药理学和分子生物学的方法及相关数据库进行检索，筛选出喘康贴治疗哮喘的主要活性成分、作用靶点及相关通路；通过全转录组测序、ceRNA网络分析与构建、基因功能与通路富集分析等方法，筛选喘康贴治疗哮喘相关的分子靶标，鉴定出哮喘相关的ceRNA网络以及潜在的分子标志物，进一步探讨喘康贴对哮喘大鼠治疗作用的分子机制，结果表明：①喘康贴具有73个活性成分，槲皮素、山柰酚、豆甾醇、甲基丁香酚等4种物质可能是发挥治疗作用的关键疗效物质；②筛选出喘康贴发挥治疗作用的关键靶点为TNF、IL-1B、IL-6、CXCL8、JUN等；③对潜在靶点进行通路富集分析，相关性较强的IL-17信号通路、Th17分化调控通路、MAPK信号通路、NF-κB信号通路、TNF信号通路等是与免疫和炎症关联密切的信号通路；④ceRNA调控机制在哮喘的发生、发展中有重要作用，为哮喘后续的靶向治疗提供新的理论基础；⑤IL-1B、IRF-1是哮喘相关的关键基因，在哮喘的发生、发展中有重要作用，可能成为治疗哮喘的潜在靶点；⑥MAPK信号通路、TNF信号通路、IL-17信号通路是与哮喘发生、发展相关的重要信号通路；⑦分子生物学研究表明在差异表达基因（DEG）方面，与空白对照组相比，模型组上调DEG（炎症反应）数目为434个，下调基因（免疫反应）数目为174个；与模型组组相比，给药组上调基因为137个，下调基因为264个。模型组较对照组相比差异表达基因（DEG）数目明显增多，说明哮喘造模成功，另差异表达基因（DEG）与免疫和炎症因素相关，也表示模型组致敏后的病理状态与临床实际相符。另给药组与模型组相比，上调基因下降，而下调基因上升，阐述喘康贴防治哮喘的调节免疫和炎症机制。.综上所述喘康贴治疗哮喘具有多成分、多靶点、多通路的特点。通过对喘康贴治疗哮喘的复杂调控网络进行系统的分析，构建ceRNA调控网络，所筛选出的多种信号通路、分子靶标和关键基因多与免疫因素和炎症因素有关，揭示了喘康贴治疗哮喘调节免疫和炎症的治疗机制，为后续的研究及临床应用奠定了良好的基础。