流感病毒激活COPD患者气道上皮细胞炎症小体NLRP3/caspase-1信号转导机制研究

Respiratory viral infection is recognized as an important trigger for acute exacerbations of COPD. After rapid recognization of viral infection as a dangerous message, NLRP3 inflammasome in epithelial cell cytoplasm is activated and triggers downstream inflammatory response inducing a large number of synthesis and release of inflammatory mediators which ultimately resulted in exacerbations of COPD. However, its possible mechanism remains unclear. Our previous research has confirmed that influenza virus was the most frequently isolated virus in AECOPD, and the expression of NLRP3 and caspase-1increased significantly in BAL in those patients with AECOPD. Furthermore, after co-culture of influenza virus and primary cultured human bronchial epithelial cells from patients of COPD for 24 hours, there was significantly elevated expression of NLRP3 and caspase-1 as well as its downstream cytokins IL-1β and IL-18. After the interference of siRNANLRP3 or transfection of lentivirus changing the expression of NLRP3 inflammasome in bronchial epithelial cells, the secretion of IL-1β and IL-18 were obviously regulated. Therefore, we make a hypothesis that viruses activated NLRP3/caspase-1 signaling pathway plays an important role in inflammatory response in patients of AECOPD. Based on the culture of primary airway epithelial cells, we want to explore the mechanism of influenza virus inducing inflammatory response of AECOPD at molecular biological level, which may provide evidence for exploring therapeutic targets in patients with COPD.

病毒感染是COPD急性加重最常见诱因，而气道上皮细胞炎症小体NLRP3可快速识别病毒感染“危险”信号，从而触发系列炎症反应致COPD急性加重，但其发生机制不甚明了。本项目组研究证实：流感病毒是AECOPD最常见感染病毒，其患者肺泡灌洗液中NLRP3/caspase-1水平显著增高；流感病毒和COPD患者原代气道上皮细胞共培养24h，NLRP3和caspase-1表达显著上调，下游细胞因子IL-1β 和IL-18含量升高。采用慢病毒转染或siRNANLRP3干涉抑制气道上皮细胞改变NLRP3表达量，则其IL-1β 和IL-18表达量呈相应的正、负调控。因此，我们提出假设：病毒激发NLRP3/caspase-1信号通道在AECOPD炎症反应中扮演重要角色。本项目旨在研究流感病毒感染与炎症小体NLRP3在气道炎症信号转导中作用及分子机制，为临床寻找阻断病毒诱导AECOPD药物新靶点提供理论依据。

慢性阻塞性肺疾病（COPD）是呼吸系统最常见慢性气道炎症性疾病，在中国40 岁以上人群中的患病率为13.7%，同时致残率和致死率高，造成了严重的社会危害和经济负担。慢阻肺急性发作（AECOPD）影响患者病情整体严重程度，导致肺功能下降，甚至呼吸衰竭。流感病毒感染是COPD急性加重最早最常见诱因，而气道上皮细胞炎症小体NLRP3可快速识别病毒感染“危险”信号，从而触发系列炎症反应致COPD急性加重，临床症状急剧恶化，甚至死亡，但AECOPD的诱因与气道炎性发生的分子生物学机制研究甚少。本项目研究从分子生物学水平上深入地探讨病毒感染导致COPD急性加重的发病机制，探讨临床上早期诊治的有效途径。研究证实：（1）流感病毒是AECOPD最早、最常见的感染病毒，其患者肺泡灌洗液中NLRP3/caspase-1水平显著增高；（2）成功进行了COPD患者气道上皮细胞的分离、鉴定和体外培养；（3）流感病毒和COPD患者原代气道上皮细胞共培养24h后，NLRP3和caspase-1表达显著上调，下游细胞因子IL-1β和IL-18含量升高；（4）采用慢病毒转染或siRNANLRP3干涉抑制气道上皮细胞改变NLRP3表达量，则其IL-1β和IL-18表达量呈相应的正、负调控；（5）首次证实了中西药联合（疏风解毒胶囊）在抗流感病毒中作用。. 流感病毒激发NLRP3/caspase-1信号通道在AECOPD炎症反应中扮演重要角色，为临床AECOPD的治疗提供分子生物学依据，为临床寻找阻断病毒诱导AECOPD药物新靶点提供理论依据。本课题还解决了有关人原代支气管上皮细胞基础研究中长期存在分离纯化和培养困难的问题，为今后其他实验室进行类似研究提供技术支持和借鉴，首次验证了中药疏风解毒胶囊联合西药奥司他韦在抗流感病毒中的作用，促进传统医学与现代医学的交融。本项目培养和训练了3名博士研究生和7名硕士研究生，使他们受到了严格的实验技能训练，也提高了科研思维能力，提升了对实验结果的分析、整理、论文的撰写及投稿等一系列研究生综合素质。在本课题的资助下，已发表相关SCI论文5篇(另有在投2篇)，北大中文核心期刊4篇(另有已接收1篇)，其他期刊会议论文2篇。