从TLR-NF-κB通路研究平喘方和拆方对哮喘小鼠气道炎症及气道重塑的作用及机制

Airway inflammation and remodeling play important roles in the development of asthma，it is conventionally believed that airway inflammation induces airway remodeling subsequently, which is the main cause of refractory asthma. "Sputum" and "stasis" in Chinese medicine correlate with airway inflammation and remodeling respectively, TLR-NF-κB signaling pathway is of great importance in the pathogenesis..Our previous studies showed that Pingchuan Formula and its stasis demolition supplement formula had better effect on airway inflammatory factors in acute asthmatic model mice than the sputum demolition supplement did. Therefore, we hypothesize that airway remodeling may occur and interact with airway inflammation in acute stage of asthma, application of blood-activating herbs in the acute phase can improve airway inflammation and remodeling by regulating the TLR-NF-κB pathway. .This project applies molecular biology techniques to observe the lung tissue pathology and to detect the proliferation, secretion and expression of related cells, proteins, factors and collagen fibers in alveolar lavage fluid, lung tissue and airway smooth muscle in acute asthmatic mice, to explore the effect and mechanism of Pingchuan Formula and it’s supplements on the TLR-NF-κB pathway to interfere with airway inflammation and remodeling, thus to provide experimental evidence for early application of stasis dissolving herbal medicine in treatment of asthma, especially refractory asthma clinically.

气道炎症与气道重塑是哮喘发病的核心。传统认为哮喘气道重塑由气道炎症导致，是难治性哮喘的主要原因。中医“痰”与气道炎症、“瘀”与气道重塑相关；TLR-NF-κB信号通路在难治性哮喘发病中具有重要作用。.前期研究发现平喘方及化瘀拆方对哮喘小鼠急性期气道炎症相关因子影响优于化痰拆方。因此我们假设：气道重塑也可能在哮喘早期出现，并与气道炎症相互影响；早期使用活血中药对气道炎症及重塑均有改善，其作用可能通过调控TLR-NF-κB通路实现。.本课题运用分子生物技术，通过观察哮喘小鼠急性期肺组织病理，检测肺泡灌洗液、肺组织及气道平滑肌相关细胞、蛋白、因子及胶原纤维增殖、分泌、表达等情况，从哮喘气道炎症、重塑和TLR-NF-κB通路角度，研究平喘方及拆方的作用及机制，为临床早期使用化瘀中药治疗哮喘特别是难治性哮喘提供实验依据。

背景：气道炎症和气道重塑是哮喘发生发展的核心。传统观念认为气道炎症先于气道重塑，近年来研究提示气道塑可能与气道炎症同时发生于哮喘早期。TLR2作为一种模式识别受体，在哮喘中可触发MyD88-NF-κB信号通路，介导气道炎症和气道重塑。.研究内容：本研究通过建立哮喘小鼠模型，探讨平喘方及拆方通过TLR-MyD88-NF-κB信号通路对哮喘气道炎症和气道重塑的影响，揭示平喘方及拆方治疗哮喘作用机制和环节，为临床上早期使用活血化瘀中药治疗哮喘提供有效依据。.结果：给药后，HE和Masson染色下观察各组小鼠病理变化，可见模型组小鼠肺组织出现气道炎性浸润及胶原沉积，提示气道炎症和气道重塑可能同时出现于急性期；用药组小鼠BALF中TNF-α、IL-6、IL-4、IL-17、TGF-β1、PDGF浓度含量较模型组明显下降（均P＜0.05），IL-10、IFN-γ上升；与模型组相比，给药组MyD88 、TAK1、p65、TLR2蛋白及mRNA水平显著下降，IκBαmRNA水平显著升高；地塞米松组小鼠IκBα蛋白显著升高（P＜0.01），中药组小鼠IκBα蛋白升高，但不具有统计学意义，提示平喘方及拆方可能通过TLR2-MyD88-NF-κB发挥作用；哮喘小鼠主气管中α-SMA mRNA水平，CollagenⅠ及E-cadherin蛋白表达水平显著升高（均P＜0.01），SM-MHC、Calpolin mRNA水平显著降低（均P＜0.01），与模型组相比，平喘方组及拆方组小鼠α-SMA mRNA水平，CollagenⅠ及E-cadherin蛋白表达水平显著降低（P＜0.05），SM-MHC、Calpolin mRNA水平显著升高（P＜0.05），提示祛瘀药物和化痰药物对于改善气道重塑均有改善作用。.科学意义：气道重塑和气道炎症可同时出现在哮喘早期；平喘方及拆方可能通过降低TLR2-My-D88-NF-κB通路及其相关因子的活性，抑制气道重塑，发挥哮喘治疗作用；早期应用化痰祛瘀法是治疗哮喘的关键。