大环内酯类激活苦味受体舒张气道平滑肌的机制研究

Bitter taste receptors (TAS2Rs) were newly found G-protein coupled receptors in airway smooth muscle, their agonists were certified as bronchodilators and erythromycin was agonist for TAS2R10. In this study we assumed that macrolides could activate the TAS2Rs to dilate the airway smooth muscle. We first used three macrolides, erythromycin, azithromycin and clarithromycin, to observe their relaxant effect in ex-vivo tracheal segments and the effects on Ca2+ concentration in human bronchial smooth muscle cells. Then the TAS2R10 plasmid was constructed and transfected to human embryo kidney(HEK293T) cells to validate whether the azithromycin and clarithromycin was agonists of TAS2R10. The TAS2R10 desensitization and TAS2R10 silencing was also performed to find the correlation between relaxant effect of microlides and TAS2Rs. Also the inhibitors of G protein subunits and their downstream molecules and ion channel were used to investigate the molecular targets of the bronchodilation; furthermore, microlides were administrated by inhalation to asthma model mice to observe the effect on airway hyperresponsiveness, thus to supply evidence for the clinical use of microlides in asthmatic patients.

苦味受体（TAS2R）是最近在气道中发现的具有舒张潜能的G蛋白偶联受体，红霉素为TAS2R10的激动剂。本研究假设大环内酯类药物通过激活苦味受体发挥舒张气道平滑肌作用。以3种大环内酯类药物（红霉素、阿奇霉素、克拉霉素）为研究对象，首先测定其对离体气管环肌张力和人支气管平滑肌细胞内钙离子浓度的影响，充分探讨其舒张效应。其次构建TAS2R10质粒并转染人胚肾（HEK293T）细胞，确认TAS2R10和3种大环内酯类药物的受体-配体关系；并通过TAS2R10受体脱敏和TAS2R10基因沉默的方法，观察其舒张作用与TAS2R10的关系。然后从气道上皮、G蛋白亚单位及其下游分子、离子通道等方面进一步研究大环内酯类激活苦味受体后舒张气道平滑肌的作用靶点。最后建立屋尘螨哮喘动物模型，雾化给予大环内酯类，验证其对哮喘气道痉挛的缓解作用和控制作用，为大环内酯类用于哮喘防治提供依据。

苦味受体（TAS2R）激动剂被证实具有舒张支气管作用。大环内酯类抗生素红霉素属于TAS2R10激动剂，但其对气道平滑肌的作用尚不清楚。阿奇霉素可舒张兔气管条，但其是否激动TAS2R发挥舒张作用未见报道。因此，大环内酯类抗生素对气道平滑肌的作用仍有待研究。本项目拟充分研究大环内酯类抗菌药物对气道平滑肌的舒张作用，并从气道上皮、TAS2R、G蛋白以及离子通道等角度探讨其对气道平滑肌的作用及机制。另外，本项目拓展了苦味受体在其他部位的研究，观察了苦味受体在动脉和鼻腔的表达及其作用。.大环内酯类抗生素红霉素和阿奇霉素可激动TAS2R10，舒张预收缩的气管环，可在β受体激动剂、M受体阻断剂基础上进一步发挥舒张作用。红霉素和阿奇霉素可通过G蛋白亚单位和TAS2R途径升高气道平滑肌细胞内钙离子浓度；同时，可逆转KCl升高的胞内钙离子浓度。红霉素和阿奇霉素的舒张作用与L-电压门控性钙通道有关。红霉素可降低哮喘小鼠的气道阻力；阿奇霉素可缓解哮喘小鼠气道炎症和气道高反应性。鼻腔的筛窦、中鼻甲、下鼻甲和鼻中隔表达苦味受体和孤立化学感觉细胞，以筛窦最高。大鼠肠系膜动脉、脑基底动脉和人大网膜动脉平滑肌和内皮细胞表达苦味受体，苦味受体介导动脉舒张。.本研究所证实了大环内酯类抗生素的舒张气道平滑肌作用，因此兼有抗炎和平喘作用的大环内酯类药物可能作为治疗气道阻塞性疾病的新药物。另外，对于苦味受体在鼻部和动脉的研究为慢性鼻窦炎和血管活性药物研发提供了新的靶点和思路。