microRNA 200家族调控线粒体DAMPs释放介导香烟诱导气道炎症与黏液高分泌的机制研究

Cigarette smoke-induced airway inflammation and mucus hypersecretion is one the most important features of COPD, while its mechanism remains unclear. Our previous study found that miR-200 family, which target genes related with mitochondrion, is involved in cigarette smoke -induced airway inflammation and mucus hypersecretion, and miR-200 family may play a role in the regulation of MTDs release, which mediated subsequent airway epithelium inflammatory injury and mucus hypersecretion through TLR-9 and FPR signal pathways. To validate the above academic hypothesis, in this study, we will perform studies involving clinical research, gene knockout and transgene, RNA interference, specific inhibitor/ agonist to explore: 1) the effects on cigarette smoke exposure on miR-200, MTDS and MTDs related signal pathways, and investigate the possibility of miR-200 and MTDs as biomarker of COPD; 2) the characteristics of different MTDs induced airway inflammatory response, and the molecular mechanism of miR-200 family mediated MTDs release and its role in cigarette smoke-induced airway inflammation and mucus hypersecretion; 3) the effects of interventions based on miR-200 family, MTDs related signal pathway (TLR-9/FPR-1/FPR-2) on cigarette smoke-induced airway inflammation and mucus hypersecretion, and try to find a novel therapeutic target for cigarette smoke induced inflammation and mucus hypersecretion.

香烟诱导的气道炎症与黏液高分泌是慢阻肺重要的病理生理学特征，其具体机制尚未明确。我们前期研究发现靶基因与线粒体调控相关的miR-200家族参与了香烟诱导气道上皮炎症损伤与黏液高分泌,其机制可能与调控线粒体损伤相关分子模式（MTDs）释放，介导TLR-9与FPR信号通路有关。为验证上述学术假说，本研究将通过临床研究、基因敲除/转基因、RNA干扰、特异性抑制剂/激动剂等研究手段，探明：1）吸烟对miR-200家族、MTDs及其相关信号通路的影响，明确其慢阻肺标记物功能；2）不同类型MTDs诱导气道上皮炎症损伤的机制与特点，miR-200家族调控MTDs释放参与香烟诱导炎症与黏液高分泌的具体分子机制；3）开展基于miR-200家族及MTDs相关信号通路（TLR-9/FPR-1/FPR-2）干预性研究，为香烟诱导的气道炎症与黏液高分泌寻找到新的干预靶点。

慢性阻塞性肺疾病（慢阻肺）的发病机制复杂，其中香烟诱导的气道炎症与黏液高分泌是慢阻肺重要的病理生理学特征。本项目探索了microRNA-200家族与线粒体损伤相关分子模式（MTDs）参与香烟诱导气道炎症损伤与黏液高分泌的机制。本项目首先通过测序与生物信息数据挖掘初步揭示了microRNA在慢阻肺中的潜在作用与机制，临床研究发现与对照组相比，慢阻肺患者血清中microRNA-200家族成员存在差异性表达 其中血清microRNA-200b与肺功能呈正相关，与C反应蛋白呈负相关；慢阻肺患者血清MTDs（线粒体DNA）的表达水平显著高于对照组，并且与C反应蛋白呈正相关，提示microRNA-200家族和MTDs对慢阻肺具备一定的标志物功能。然后本项目通过体外试验证实microRNA-200b/429可能通过β-catenin/PPARδ信号网络缓解香烟提取物（CSE）诱导的气道上皮炎症反应，借助人原代小气道上皮细胞研究了microRNA-141/429通过调控上皮间质转化参与CSE诱导气道上皮炎症反应、气道重塑进而参与慢阻肺发病的相关机制。microRNA-141/429能够降低CSE刺激气道上皮细胞线粒体DNA的释放，提示microRNA-200家族可能通过调控MTDs释放进而参与调节香烟诱导气道炎症。本项目在已证实MTDs诱导气道上皮炎症损伤、肺部炎症的基础上，构建了MTDs主要受体TLR9和FPR1基因敲除小鼠，研究发现，TLR9和FPR1敲除能显著降低香烟诱导的小鼠气道炎症损伤与气道重塑；线粒体靶向抗氧化药物Mitoquinone和SS-31能显著减轻香烟暴露导致的小鼠气道炎症损伤，炎症因子释放，氧化应激反应、气道黏液高分泌、部分MTDs释放，其作用机制与维持线粒体功能、调控NF-κB、MAPK信号通路有关。综上，本项目基本明确了microRNA-200家族、MTDs对慢阻肺标记物功能，阐释了microRNA-200家族调控香烟诱导气道炎症、气道重塑相关分子机制，评估了基于MTDs干预香烟诱导的气道炎症与黏液高分泌的可行性，为慢阻肺的干预提供了进一步的研究证据。