MicroRNA-21 在脂氧素促进 ARDS 肺水清除中的作用及机制研究

Acute respiratory distress syndrome (ARDS)consisting of acute hypoxemic respiratory failure,occur with a high incidence and mortality. The pathological changes seen in ARDS comprise diffuse alveolar damage,alveolar capillary damage,disruption of the alveolar epithelium and devastating lung fibrosis. Edema fluid resorption is critical for gas exchange and both alveolar epithelial sodium channel (ENaC) and Na,K-ATPase are accreditedwith key roles in the resolution of pulmonary edema. Lipoxins (LXs) are unique structures derived from arachidonic acid. Lipoxins were the first mediators recognized to have dual anti-inflammatory and pro-resolution activities, which have been described as the endogenous “braking signal”for inflammation. We previous research shows that the lipoxin concentration and lipoxin receptor expression decrease in ARDS patients. Lipoxin can promote alveolar fluid clearance in ARDS, microRNA-21 can affect alveolar epithelial sodium channel. Base on the finding, our hypothesis is that microRNA-21 may play a role in the development of edema. The main aim of this grant is to find the mechanism of microRNA-21 on lipoxin promoting alveolar fluid clearance in ARDS. Lipoxin may provide new opportunities to design “reabsorption targeted” therapies with high degree of precision in controlling ARDS.

炎症消退调控障碍导致失控性炎症是ARDS发病重要机制。脂氧素是机体重要内源性抗炎促消退介质,为炎症反应刹车信号。在前期证实ARDS病人脂氧素浓度和受体表达明显下调，脂氧素促进肺水清除的基础上；结合我们发现内毒素上调miRNA-21，而miRNA-21负性调控钠通道蛋白；脂氧素通过降低miRNA-21进而上调钠通道表达等线索。我们推测:miRNA-21在ARDS肺水肿发生中起重要作用并介导脂氧素促进肺水清除作用。为此，应用PCR、流式细胞仪、膜片钳、高效液相-质谱、激光共聚焦、western等技术,检测ARDS病人miRNA-21动态变化；研究miRNA-21对钠通道功能、Na,K-ATP酶活性及肺水清除影响；siRNA干扰和报告基因技术探讨miRNA-21介导脂氧素促进肺水清除机制；应用miRNA-21过表达和抑制剂进一步验证。旨在揭示揭示ARDS发病新机制并为探寻防治新靶标提供实验依据。

炎症消退机制出现障碍导致炎症失控是ARDS发生重要因素。脂氧素（Lipoxin）是炎症消退过程产生的最重要内源性抗炎促消退介质，通过调节促炎/抗炎因子的平衡，限制炎症损伤，被喻为炎症反应的“刹车信号”。内毒素刺激模型上调miRNA-21，且miRNA-21负性调控钠通道蛋白。为探索miRNA-21在ARDS肺水肿发生中起重要作用并介导脂氧素促进肺水清除作用。我们课题组建立内毒素性ARDS动物模型，应用PCR、流式细胞仪、膜片钳、高效液相-质谱、激光共聚焦、western等技术，研究miRNA-21对钠通道，Na,K-ATP 酶表达和活性及肺水清除的影响；采用siRNA干扰和报告基因技术探讨miRNA-21介导脂氧素促进肺水清除机制；应用miRNA-21过表达和抑制剂进一步验证并且检测ARDS病人血液和肺泡灌洗液miRNA-21的动态变化。研究表明内毒素组miRNA-21表达量明显增高且miRNA-21通过负性调控肺泡二型上皮细胞钠通道，Na,K-ATP酶的表达和活性，使肺水清除下调，发生肺水肿。而脂氧素能逆转内毒素的作用miRNA-21表达量明显下调，钠通道蛋白表达增加，减轻肺部炎症反应，促进肺水清有显著作用。在转染miRNA-21 inhibitor基因沉默后，钠通道蛋白表达量则进一步增加。转染miRNA-21 mimic过表达后，脂氧素上调钠通道蛋白表达作用受到明显抑制，表明脂氧素通过降低miRNA-21水平进而上调钠通道的功能。基于ARDS治疗侧重抑制炎症发生之现状，本项目首先提出促进炎症消退的治疗新策略，强调适度炎症反应对机体保护作用，倡导通过增强机体内在防御能力和促进肺泡水肿液的主动重吸收，达到维持机体自身的平衡。这是脂氧素促进炎症消退的新机制，也是miRNA-21一个全新的生理功能，是对现有ARDS发病机制和治疗策略的创新。