小气道高分泌及NLRP3/IL-1β信号通路在臭氧诱导的哮喘急性加重模型中的作用

Small airways are important sites of airway inflammation, remodeling and hypersecretion in asthma with different types and severities. Dysfunction of the small airways is related to poor control and frequent exacerbations of asthma. Mucous cell proliferation and mucus hypersecretion are important pathological features of asthma. Mucus embolism contributes to asthmatic mortality. Our preliminary study demonstrated that ozone exposures induced acute exacerbation of asthma in mice, with mucus hypersecretion and increased mucus secretion cells which were more significantly in small airways. The mucus hypersecretion of small airways was associated with the increase of IL-1β, but not that of IL-13 or IL-4. Hypotheses of this study was that ozone could induce activation of NLRP3 inflammasome in the peripheral airway of asthmatic mice, and then regulate the activation of caspase-1 and the production of IL-1β, which will aggravate goblet cell metaplasia and mucus hypersecretion through downstream signaling pathways. The asthma exacerbation model will be established by OVA sensitization/challenge combined with ozone exposures in C57/BL6 mice. Main concern of this study is the difference between small airways and large airways. Airway secretion and activation of NLRP3/IL-1β signaling pathway will be detected. NLRP3 gene knockout mouse and NLRP3 inhibitors will be used to elucidate the role of NLRP3/IL-1β signaling pathway in airway hypersecretion. Our study will provide new theoretical evidence of molecular mechanism of small airway dysfunction in ozone-induced asthma exacerbation.

小气道是炎症、重构和高分泌发生的重要位点，参与哮喘的发病机制，并影响其急性发作和预后。黏液高分泌是导致哮喘急性发作的重要原因之一，黏液栓塞与哮喘死亡率相关。我们的前期研究表明臭氧暴露诱导哮喘小鼠急性加重，小气道出现黏液高分泌和黏液分泌细胞增多，比大气道更明显。小气道高分泌伴随白介素（IL）-1β升高，而非IL-13和IL-4。本研究的假设是臭氧诱导哮喘小鼠NLRP3炎症小体活化，激活caspase-1，参与调节IL-1β，并诱导下游信号通路，促使小气道为主区域的杯状细胞化生及黏液高分泌。拟采用上述哮喘急性加重模型，检测气道分泌和NLRP3/IL-1β信号通路活化情况，并给予NLRP3活性干预（基因敲除和抑制剂），揭示NLRP3/IL-1β活化在该模型中对气道高分泌的影响，并重点比较大小气道间差异，为小气道功能障碍在哮喘急性加重中的影响和分子学机制提供新的理论依据。

气道炎症，气道高分泌和气道高反应性是哮喘发病机制的核心要素，并影响其急性发作和预后。本课题，我们完成臭氧诱导的哮喘急性加重小鼠模型构建；完成不同炎症模型大小气道通气功能评估及相关性分析，并探索小鼠通气功能中小气道相关参数与炎症因子以及气道高分泌之间的相关性，证实在臭氧暴露+OVA致敏/激发模型中，大、小气道的功能障碍基本平行，可能并行出现和发展。小气道功能相关参数（MMEF﹑FEF75﹑FEF50）与IL-4﹑IL-5﹑IL-13以及IL-17A呈负相关（p均小于0.05），相关性比大气道相关参数更密切。MMEF﹑FEF75﹑FEF50与Muc5ac也呈显著负相关。muc5AC与IL-4﹑IL-5﹑IL-13以及IL-17A呈显著正相关（p均小于0.05）。FVC与上述炎症及高分泌相关参数之间均无相关性。FEV25与IL-5负相关。FEV50与IL-4﹑IL-5﹑IL-17A以及Muc5ac负相关。FEV75与IL-4﹑IL-5以及TNF-a负相关。小鼠哮喘模型中小气道相关参数与T2型炎症及气道高分泌的相关性比大气到更强，更值得临床和基础科研关注。肺组织p38MAPK浓度与大小气道代表性指标MMEF负相关。caspase-1mRNA的表达量与大小气道相关参数（FEV25和MMEF）均呈正相关，提示气道功能障碍小鼠可能存在caspase1的消耗，以及MAPK磷酸化的增多。IL-1β在OVA致敏/激发小鼠模型中明显升高，且伴随caspase-1表达量的下降。NLPR3炎症小体-caspase-1信号通路的异常参与调节IL-1β的产生与激活，而IL-1β的下游信号通路可能包括PI3K/AKT、MAPK和NF-κB信号通路，并同时参与大小气道的功能调节。NLRP3基因敲除可以改善气道阻塞，逆转气道高反应性。增加临床数据探索，证实胸闷症状及年龄增加提示单用吸入激素治疗效果不佳；嗜酸性粒细胞性气道炎症和小气道功能障碍均参与气道高反应性的形成，且发生机制可能相对独立。