基于IL-13/STAT6与纤毛自噬介导气道粘液增多探讨小青龙汤温肺蠲饮治疗哮喘发作机制研究

Asthma is a chronic airway inflammatory disease that threatens the public health. According to the Global Initiative for Asthma (GINA) 2012, China ranked first in terms of rate of fatality caused by asthma around the world. The excessive airway mucus and mucus plug contribute to mortality in many patients. The IL-13/STAT6 signaling pathways, as key events for mucus production and ciliophagy, as the impairment of mucous clearance mechanism, may both have the greatest potential for inhibition of excessive mucus production and become the new pharmacotherapy. From perspective of Chinese medicine, phlegm is the primary pathogenesis of asthma and thus treating asthma from phlegm plays the key role in the treatment of asthma. Xiaoqinglong decoction is a famous prescription to treat differentiation of Cold Fluid, cough and asthma, and Ganjiang-xixin-wuweizi is classical herb pairs for Zhongjing treating retention of phlegm, but their roles on the excessive airway mucus, IL-13/STAT6 signaling pathways and ciliophagy are unknown. The aim of this study is to explore effects of Xiaoqinglong decoction and Ganjiang-xixin-wuweizi on the excessive airway mucus in OVA-induced asthma using AB-PAS and immunohistochemistry, and to explore its effects and mechanisms on the IL-13/STAT6 signaling pathways and ciliophagy using RT-PCR, Western blot, and transmission electron microscope. This research will be helpful for deep understanding of the relationship between the excessive mucus production and phlegm.

哮喘是威胁全球公共健康重要的慢性肺部疾病。根据2012全球哮喘防治创议,中国已成为全球哮喘死亡率最高的国家。患者气道粘液增多和粘液栓引起气道梗阻和窒息，提高哮喘病死率。IL-13/STAT6 信号途径在气道粘液高分泌中发挥重要作用，而纤毛自噬可致粘液清除受损，两者成为抑制气道粘液增多的重要靶点。中医认为痰饮是哮喘发作的重要病机，从痰治喘是治疗哮喘发作关键环节。小青龙汤可解表散寒、温肺蠲饮，是治疗支气管哮喘经典名方，方中的干姜-细辛-五味子是仲景治疗痰饮的经典药对，但其对气道粘液增多、IL-13/STAT6信号途径与纤毛自噬的影响未见报道。本研究通过建立OVA诱导小鼠哮喘模型，运用AB-PAS、免疫组化法、RT-PCR、Western blot及透射电镜研究小青龙汤及干姜-细辛-五味子药对对气道粘液、IL-13/STAT6信号途径及纤毛自噬的影响及机制，有助于理解气道粘液增多与痰饮的关系。

目的：小青龙汤可解表散寒、温肺蠲饮，是治疗支气管哮喘经典名方。本研究通过建立OVA诱导小鼠哮喘模型，研究小青龙汤对气道粘液高分泌和纤毛自噬的影响。.内容及方法： 建立OVA 诱导的小鼠哮喘模型：于试验第0天和第14天分别腹腔注射OVA 20μg和氢氧化铝 4mg（溶于0.1ml生理盐水）；于试验第18-22天，以1% OVA雾化攻击20分钟。 动物随机分为正常、模型、羧甲司坦组和 XQL组。在第二次致敏的同时给予正常对照组、哮喘模型组生理盐水0.4ml 灌胃、羧甲司坦组（2mg/kg）、小青龙汤组(10 g/kg)水煎液灌胃。每次雾化攻击前1h 给予小青龙汤、羧甲司坦灌胃，而正常对照组、哮喘模型组则给予生理盐水。运用AB-PAS染色、血清IL-13、肺泡灌洗液分析、Western blot、透射电镜以及荧光免疫法观察纤毛自噬(Light Chain 3 ,LC3) 和乙酰化a-tubulin共表达情况，以评价小青龙汤对气气道粘液、IL-13水平及纤毛自噬的影响及机制，有助于理解气道粘液增多与痰饮的关系。.结果： 小青龙汤 (10 g/kg) 减轻气道粘液分泌、减少肺组织病理AB-PAS染色阳性区域的面积 (P <0.05)，血清IL-13 的表达(P <0.01)，OVA诱导炎症改变，以及肺泡灌洗液中白细胞数目也明显减少(P <0.01) 。小青龙汤组还能够改善纤毛长度、降低气管上皮LC3 的表达和纤毛自噬，对粘液分泌的影响较与羧甲司坦组作用明显。.结论：小青龙汤能够减轻纤毛缩短，增加纤毛上皮细胞的清除粘液的能力，减少OVA诱导哮喘小鼠模型气道粘液，同时抑制粘液高分泌，对其深入研究有助于新靶点的发现和药物开发。