清肺口服液调节呼吸道微生物群介导Th17/Treg免疫应答改善哮喘气道炎症的机制研究

The imbalance of respiratory microbes and metabolites play an important role in airway inflammation and remodeling of asthma. The immune response of Th17/Treg was a key of regulation the respiratory microbial and metabolic disorder via HIF-1ɑ signaling pathway. Previous studies has found that Qingfei Oral Liquid could be anti-respiratory pathogens and play anti-inflammatory, immune regulation, reduce the role of respiratory resistance. But Qingfei Oral Liquid can or not reduce airway inflammation and slow down airway remodeling effects by regulating respiratory microbes imbalance is not clear. Our researchs intends to carry on the three aspects: clinical,animal model of asthma and in vitro cell. 1. High throughput sequencing and GC-MS techniques were used to study the characteristics of respiratory microbial and metabolites in children of asthma and the role of Qingfei Oral Liquid on regulate respiratory microbial and their metabolites. we will elucidate that Qingfei Oral Liquid adjust Th17/Treg immune response imbalance by regulating respiratory microbial and their metabolites in animal models of asthma. On this basis,we will use the cell model, we will clear that the mechanism of Qingfei Oral Liquid regulated respiratory microbes and metabolic imbalance lead to Th17/Treg immune response balance via HIF-1ɑ signaling pathway. And to provide theoretical basis and new ideas for the treatment of airway inflammation in Asthma with traditional Chinese Medicine.

呼吸道微生物群及代谢物失衡在哮喘气道炎症损伤与气道重塑中发挥重要作用，呼吸道微生物群及代谢物失衡通过HIF-1ɑ信号通路调控Th17/Treg免疫应答是其发挥作用的关键环节。前期研究发现清肺口服液可以通过抗呼吸道致病菌而起到抗炎、调节免疫、减轻呼吸道阻力的作用，但清肺口服液是否可通过调节呼吸道微生物群失衡而减轻气道炎症尚未明确。本课题拟从临床、实验动物、细胞三个层面开展研究：1.通过高通量测序及GC-MS技术对哮喘患儿呼吸道微生物群及代谢物失衡进行研究；并明确清肺口服液对其调节作用。2.建立微生物群干预体内哮喘动物模型，研究清肺口服液通过对呼吸道微生物群及代谢物失衡的调控对Th17/Treg免疫应答的影响。3.构建体外细胞模型明确清肺口服液是否通过调节呼吸道微生物群失衡调控HIF-1ɑ信号通路促使Th17/Treg免疫应答平衡而发挥作用。以期为中药治疗哮喘气道炎症及气道重塑提供理论依据。

目的：研究清肺口服液对哮喘呼吸道微生物群及代谢物失衡的调控作用及对HIF-1α信号通路的作用机制。方法：1.临床水平：成功完成111例哮喘儿童呼吸道菌群16S rRNA测序工作，从哮喘儿童病原体和呼吸道微生物特征谱方面进行研究。2.动物水平：（1）气道炎症：成功利用OVA诱导建立哮喘小鼠模型，肺组织病理（HE、PAS、Masson）切片观察气道炎症、ELISA法检测气道IgE及炎症因子（IL-4、IL-6、IL-10、及IL-17A）变化，流式检测小鼠脾脏中Th1、Th2、及Th17细胞数量，免疫组化和免疫荧光检测肺组织中HIF-1α相关信号通路分子HIF-1α、MMP-9和VEGF-A蛋白表达和定位；（2）呼吸道/肠道菌群多样性：利用16S rRNA测序对清肺口服液干预前后的哮喘小鼠的鼻腔灌洗液（NLF）、肺泡灌洗液（BALF）、及粪便的V3-4区进行测序，借助QIIME2、LEfSe、PICRUSt2等软件分析菌群结构、丰度、多样性以及特征菌属。3.细胞水平：将IL-13刺激人支气管上皮细胞株16-HBE体外模拟呼吸道上皮损伤，ELISA法检测加入清肺口服液前后各组培养上清中炎性因子（IL-6、IL-8）的变化，Western Blot检测HIF-1α信号通路相关蛋白表达。结果：1.哮喘患儿变形菌门和嗜热菌门丰度增高与哮喘发作次数呈正相关。2.清肺口服液明显降低OVA诱导的哮喘小鼠BLAF中IgE、OVA-IgE、炎症因子（IL-4、IL-6、IL-10、及IL-17A）、及HIF-1α水平，且脾细胞中IL-17A表达阳性的细胞比例下降，与CON组相比有显著差异（P<0.05）；呼吸道菌群分析表明，变形杆菌门水平和假单胞菌属水平的丰度显著增加，同时肠道菌群分析显示，OVA组乳杆菌属、普氏菌属、及臭杆菌属等丰度明显下降，经QFL治疗后均可改善。3.清肺口服液能够抑制IL-13激活的16-HBE中HIF-1α和NF-kB的表达。.结论：清肺口服液通过调节呼吸道/肠道菌群紊乱抑制HIF-1α信号通路促使Th17/Treg平衡能显著缓解哮喘小鼠气道炎症，这可能是清肺口服液治疗哮喘气道炎症的机制之一。