基于内质网应激（ERS）下蛋白质未折叠反应（UPR）抑制内源性炎症探讨固本防哮饮防治哮喘的机制

The study found that children with asthma remission had state of lung spleen kidney deficiency, GubenFangxiao Decoction(GBFXD) is a compound prescription of TCM. It demonstrated the exact efficacy in clinical application of reducing asthma attacks in frequency and degree, enhancing the children's physical fitness, improving the quality of life in our previous study. Experimental studies have established a stable and reliable animal models of asthma remission and model assessment system, confirmed that the compound can reduce airway hyperresponsiveness, improve inflammatory cells infiltration around the bronchi. High domestic and foreign research shows that higher expression of asthma susceptibility genes ORMDL3 can further activate the endoplasmic reticulum stress under UPR signaling pathway,which is closely related to continuous endogenous inflammation and airway remodeling in the process of asthma,which reflects the children's physical fitness to some extent. This project proposed by in vivo and in vitro experiments, using gene cloning and signaling pathway block technology, laser confocal scanning microscopy techniques, gene transcription in vitro, immunofluorescence technique, Real - time PCR, Western Blot and ELISA experiment technology, from the point of gene protein and cell and tissue morphology to study the effect of GBFXD on higher expression of asthma susceptibility ORMDL3 gene's activation of endoplasmic reticulum stress under UPR signaling pathways IRE1α、PERK、ATF6 and their related regulatory proteins after channel blocking, Finding the mechanism of TCM in asthma prevention and treatment in children with asthma.

固本防哮饮是名老中医经验方，前期临床研究已证实该复方能改善哮喘患儿体质，防治哮喘。实验研究已建立稳定可靠的哮喘缓解期动物模型及模型评估体系，证实该复方能降低气道高反应性、改善支气管周围炎性细胞浸润。国内外研究表明，哮喘易感基因ORMDL3的高表达可进一步激活内质网应激下UPR相关信号通路，与哮喘发病中持续存在的内源性炎症及气道重塑密切相关，是哮喘反复发作和加剧的重要机制。本项目拟通过体内和体外实验，采用基因克隆和信号通路阻滞技术、基因体外转录、激光共聚焦扫描显微技术、PCR、Western Blot等技术，研究固本防哮饮对哮喘易感基因ORMDL3激活的内质网应激下UPR下游信号通路IRE1α、PERK和ATF6三条信号通路相关调控蛋白表达的影响，从RNA、基因蛋白水平及细胞、组织形态学等不同层次探讨固本防哮饮防治哮喘的作用机理，多层次、多靶点揭示中药复方防治哮喘的作用机制。

哮喘是一种多基因遗传病，2007 年发表在《Nature》上的研究发现血清类黏蛋白1样蛋白3 (orosomucoid 1-like protein 3, ORMDL3) 基因是儿童时期起病的哮喘的易感基因。近年来的研究证实，其主要参与细胞应激及鞘脂类代谢，介导慢性炎症。课题组前期研究发现固本防哮饮可抑制哮喘缓解期小鼠模型中ORMDL3的表达。本项目进一步发现哮喘模型小鼠内质网应激下蛋白未折叠反应通路激活，且伴随上皮完整性损伤，体外实验揭示ORMDL3过表达可激活IRE1α-XBP1通路，并抑制上皮紧密连接蛋白表达，部分揭示了ORMDL3作为哮喘易感基因与哮喘发作的相关性。复方固本防哮饮及其有效成分黄芪甲苷可改善上皮屏障功能，纠正内质网应激。从细胞、分子层面丰富中医学“益气固表”等哮喘治法的科学内涵，为研究中医药防治哮喘提供思路，亦为临床应用提供依据。