ApoE/LDLR信号通路抑制流感病毒H3N2诱发COPD急性加重气道炎症风暴分子机制研究

Viral infection is the earliest and most common trigger for acute exacerbation of COPD. After the alveolar macrophage recognizes the danger signal of infection, it quickly triggers a series of inflammatory reactions to cause acute exacerbation of COPD, however the mechanism is unknown. Pre-experimental results confirmed that in AECOPD patients with influenza virus positive, the levels of apolipoprotein E in serum and BALF were significantly reduced and the levels of IFN-γ, TNF-α, IL-6 and CCL-2 were significantly increased. Therefore, we hypothesized that ApoE plays an important role in the regulation of influenza virus-induced AECOPD airway inflammation storms by binding to high-affinity low-density lipoprotein receptor (LDLR) in airway epithelial cells. This experiment used: 1. Infecting COPD mouse models induced with ApoE or LDLR gene knockout with influenza virus H3N2. 2. On the basis of primary airway epithelial cells with CRISPR/Cas9 knockout or LDLR overexpressed transfected by lentivirus, detecting the expression of ApoE/LDLR and the levels of cytokines or inflammatory factors respectively. We aim to explore the molecular mechanism of ApoE/LDLR signaling pathway in the occurrence of airway inflammation storm in order to provide theoretical basis for the clinical search of new targets for blocking virus-induced AECOPD.

病毒感染是COPD急性发作最早最常见触发诱因，肺泡巨噬细胞识别感染危险信号后，快速启动一系列炎症反应致COPD急性发作，但发生机制不明。预实验结果证实：流感病毒检测阳性AECOPD患者，其血清和BALF中载脂蛋白ApoE水平显著降低,IFN-γ,TNF-α,IL-6,CCL-2等含量显著升高。因此，我们提出假设：ApoE通过与气道上皮细胞高亲和性低密度脂蛋白受体LDLR结合，在调控流感病毒诱发AECOPD气道炎症风暴中扮演重要角色。本实验采用：①流感病毒H3N2诱发ApoE和LDLR基因敲除的COPD小鼠模型；②原代气道上皮细胞CRISPR/Cas9敲除和慢病毒过表达LDLR，然后分别检测ApoE/LDLR表达和细胞/炎症因子含量的改变，以探讨ApoE/LDLR信号通路在气道炎症风暴发生中分子机制，为临床寻找阻断病毒诱导AECOPD的药物新靶点提供理论依据。

背景：流感病毒是COPD急性加重最早最常见的诱因，肺泡巨噬细胞识别感染危险信号后快速启动一系列炎症反应致COPD急性加重，但发生机制不明。预实验结果证实：流感病毒检测阳性AECOPD患者血清和BALF中IL-1β、IL-18和IFN-γ水平显著升高，而载脂蛋白ApoE水平显著降低。因此，我们提出假设：ApoE通过与气道上皮细胞高亲和性低密度脂蛋白受体LDLR结合，在调控流感病毒诱发AECOPD气道炎症风暴中扮演重要角色。.研究内容：（1）探讨流感病毒H3N2诱导COPD原代气道上皮细胞损伤和炎症风暴发生的分子机制；（2）研究ApoE/LDLR路径调控流感病毒诱发AECOPD气道炎症风暴和粘液高分泌的分子机制。.结果：（1）原代支气管上皮细胞和流感病毒H3N2共培养24h的转录组高通量测序结果表明DHBE细胞NLRP3炎性体信号通路在mRNA表达水平上显著高于NHBE；抑制NLRP3水平明显改善DHBE炎症反应。MCC950干预通过抑制NLRP3明显减轻H3N2感染诱导的COPD大鼠肺损伤。（2）在H3N2感染的野生型小鼠血清ApoE水平明显降低，ApoE敲除诱导小鼠肺组织出现更严重的肺损伤。在H3N2感染的小鼠骨髓来源巨噬细胞（BMDMs），re-ApoE3干预促进H3N2诱导的M1 BMDMs向M2极化，减轻细胞损伤。（3）在H3N2感染的COPD小鼠，出现明显肺损伤，Lm扩大，RGX104通过上调ApoE明显抑制AECOPD小鼠肺部炎症，减轻粘液高分泌。在CSE和H3N2联合刺激的Raw264.7细胞，ApoE表达明显降低，RGX104明显促进M1型Raw264.7细胞向M2型极化；同时，相比单纯CSE组和单纯H3N2组，CSE和H3N2联合刺激的16HBE细胞IL-1β、MUC5AC和GNE表达进一步升高，而RGX104和re-ApoE3干预明显抑制细胞焦亡和IL-1β介导的粘液高分泌。.结论：流感病毒激活 AECOPD 患者支气管上皮细胞NLRP3信号通路诱发更早、更强的气道炎性反应，而激活ApoE/LDLR通路有助于改善流感病毒H3N2诱发的巨噬细胞异常极化和焦亡，抑制肺损伤和COPD急性加重气道炎症风暴的发生，并减轻粘液高分泌。外源性ApoE活性肽有助于改善H3N2诱导的气道上皮炎症风暴和粘液高分泌，为流感病毒诱导COPD急性加重患者的治疗提供理论依据。