肥胖哮喘Muc5ac高分泌和Munc18b上调的关系及机制

Airway mucus hypersecretion is hallmark of obese asthma, it is great important to clarify the mechanisms of mucus hypersecretion of obese asthma for optimizing the treatment and improving the outcome of patients. Our preliminary study found Munc18b, the most important scaffold protein in mucin exocytosis, in the lungs of obese asthma mice was up-regulated along with increasing serum leptin compared with non-obese asthma. Since leptin always takes effect by activating JAK2/Stat3 pathway, we presented our hypothesis that excessive leptin-leptin receptor(lepR) increase the transcription of Munc18b via activating Stat3 to bind the Stat3 binding sites in promoter of Munc18b gene, and then the Muc5ac secretion was enhanced. In this study, firstly obese asthma mice induced by high fat diet and ovalbumin (OVA) were compared with non-obese asthma by mucin secretion, expression of SNARE protein, SM protein, as well as leptin and lepR in lungs. And then Munc18b and Munc18c CCSP-conditional knockout mice were used to construct Munc18 mutant obese asthma and clarify the role of Munc18b and Munc18c in mucus hypersecretion. At last, Clara cells were isolated and cultured in different condition medium with or without S31-201(Stat3 inhibitor) for 1, 4 and 24h. Muc5ac were evaluated by vacuum blot, Munc18b/c by PCR, and Stat3-DNA binding activity by CHIP, to explore the mechanism of munc18b steering mucus hypersecretion. The founding in study will uncover the mechanisms of mucus hypersecretion in obese asthma and provide new potential molecular therapeutic targets for future treatment.

气道粘液高分泌是肥胖哮喘的重要特征，前期研究发现，肥胖哮喘小鼠体内粘蛋白囊泡运输蛋白Munc18b表达增加。据此我们提出——肥胖哮喘通过Leptin-leptin受体（LepR）激活STAT3，上调Munc18b，提高muc5ac的分泌。本项目通过动物实验和体外研究，在高脂饮食和OVA致敏诱导的肥胖哮喘小鼠上，运用RT-PCR、western blot、Vacuum blot方法检测Muc5ac分泌和SNARE、SM蛋白的变化；采用Munc18b/c条件敲除小鼠建立肥胖哮喘模型，揭示Munc18在气道粘液高分泌中的重要作用；并在气道粘液分泌细胞（Clara 细胞）培养基中加入IL-13和leptin模拟体内环境，运用信号通路阻断和免疫共沉淀等技术阐明Munc18b参与肥胖哮喘的粘液高分泌的分子途径，为揭示肥胖哮喘粘液高分泌的机制和开发针对性治疗奠定理论依据。

气道粘液高分泌是肥胖哮喘的重要特征，前期研究发现，肥胖哮喘小鼠体内粘蛋白囊泡运输蛋白Munc18b表达增加。据此我们提出——肥胖哮喘通过Leptin-leptin受体（LepR）激活STAT3，上调Munc18b，提高muc5ac的分泌。本项目通过动物实验和体外研究，在高脂饮食和OVA致敏诱导的肥胖哮喘小鼠上，运用RT-PCR、western blot、Vacuum blot方法检测Muc5ac分泌和SNARE、SM蛋白的变化；并在气道粘液分泌细胞培养基中加入IL-13和leptin模拟体内环境，采用基因沉默技术抑制上皮细胞Munc18b/c通路，运用信号通路阻断和免疫共沉淀等技术阐明Munc18b参与肥胖哮喘的粘液高分泌的分子途径，揭示Munc18在气道粘液高分泌中的重要作用；为揭示肥胖哮喘粘液高分泌的机制和开发针对性治疗奠定理论依据。由于美国合作单位转基因动物出现问题，未能进行转基因动物的研究。研究者通过体外转染实验完成了相关目标，得到了预期结果。为了进一步粘液分泌的调控机制，我们在4种非小细胞肺癌细胞株 (A549, NCI-H292, NCI-H460, NCI-H1703) 和人支气管上皮细胞株(16HBE)上构建质粒互补DNA SNHG16 cDNA（pcdna-snhg16）并进一步研究粘液分泌的靶向调节方式。.通过研究，确立了肥胖哮喘气道粘液高分泌的机制，并确立了Munc18b在肥胖哮喘粘液分泌中的重要枢纽作用。相关结果和结论为肥胖哮喘慢性气道炎症性疾病的治疗提供了新的诊治方向和理论依据，对慢性起到炎症性疾病的管理和科研工作提供了科学依据和科学基础，同时研究方法对于提高临床科研水平也发挥了积极作用。根据这一理论，我们通过调整Muncb/Munc18c数量，适当提高粘蛋白的基础分泌减少细胞内粘蛋白蓄积，减少刺激后哮喘发作时的粘液分泌，改善哮喘症状，减少致死性哮喘，减轻经济负担，具有切实的社会意义。.发表了论文4篇，其中SCI收录2篇，并出版专著1部，获得青岛市科技进步奖1项。多次参加学术会议并发言，组织三次学术活动，进行研究成果交流。培养了两名研究生和科室粘液分泌研究队伍。