消退素D1促进ARDS炎症消退新机制：NF-κB/COX-2功能研究

The uncontrolled inflammation caused by disregulated resolution of inflammation is the most important mechanism in the development of ARDS. Resolvin D1(RvD1) is one of the important endogenous anti-inflammatory and pro-resolution mediators. Our previous study demonstrated that lipopolysaccharide (LPS) induces COX-2 bimodal expression, RvD1 promotes the expression of the second peak of COX-2 and its production prostaglandin D2 (PGD2). Moreover, the new function of COX-2 in promoting inflammation resolution is mediated by NF-κB p50/p50. Therefore, we hypothesize that RvD1 could act on the NF-κB/COX-2/PGD2 axis to promote the resolution of inflammation. We will use EMSA-supershift, laser confocal, dual-luciferase report gene, bioinformatics, ChIP, DNA pull down to measure the different expression of COX-2, PGD2 and PGE2 synthetase and related derivatives. To discuss the specific mechanism of NF-κB p50/p50 mediated the expression of COX-2. And to confirm the underlying mechanism of RvD1 regulating NF-κB/COX-2/PGD2 pathway. In addition, the methods for overexpression of related gene, siRNA interference and knockout technology will be used to verify the hypothesis. The main aim of this grant is to reveal a novel mechanism of the resolution of inflammation and provide experimental data for the prevention and treatment of ARDS.

炎症消退障碍所致的失控性炎症为ARDS重要发病机制。消退素D1是新近发现的重要内源性抗炎促消退介质。在我们发现LPS诱导COX-2双峰表达，消退素D1促进COX-2第二峰及其产物PGD2表达，NF-κB p50/p50介导COX-2促消退作用基础上。我们推测：消退素D1作用于NF-κB/COX-2/PGD2轴促进炎症消退。为此，拟采用原代肺成纤维细胞、内毒素性小鼠ARDS模型，应用EMSA-supershift、激光共聚焦、双荧光素酶报告基因、生物信息学、ChIP、DNA pull down等技术，观察炎症发生期和消退期COX-2、PGD2和PGE2合成酶及衍生物的差异性；探讨NF-κB p50/p50介导COX-2第二峰表达的具体机制；研究消退素D1调控NF-κB/COX-2/PGD2轴的具体通路；并应用基因过表达、敲低和基因敲除技术验证假说；为防治ARDS新策略提供实验依据。

炎症消退障碍所致的失控性炎症为ARDS重要发病机制。消退素D1是新近发现的重要内源性抗炎促消退介质，被称为炎症反应的“刹车信号”。我们在前期研究证实消退素D1能促进肺水的清除，减轻肺损伤的基础上，结合新近发现：LPS诱导COX-2和L-PGDS双峰表达，消退素D1促进COX-2第二峰及其产物PGD2表达，NF-κB p50/p50介导COX-2促消退作用等线索。我们推测：消退素D1作用于NF-κB/COX-2/PGD2轴促进炎症消退。为验证这一假说，我们采用内毒素性大小鼠ARDS模型，探究炎症发生期和消退期COX-2、PGD2和PGE2合成酶及衍生物的差异性；探讨NF-κB p50/p50介导COX-2第二峰表达的具体机制；进一步应用消退素受体及转导信号通路抑制剂、激动剂以及基因敲除技术验证假说。我们的研究结果表明LPS诱导ARDS大鼠肺中COX-2和L-PGDS呈双峰表达，第二峰的COX-2可以通过促进下游L-PGDS-PGD2表达促进炎症消退的作用，也提示我们在临床应用非甾体类等抗炎药物治疗炎性疾病的时间窗和治疗时间，为我们临床治疗和干预提供一定的指导；另外我们进一步证实NF-κB p50/p50介导COX-2第二峰表达促进炎症消退，RvD1可通过调控NF-κB p50/p50活性促进COX-2及PGD2表达，从而促进急性肺损伤的炎症消退。从强调促进炎症消退的角度，遵循机体自身的内源性修复规律，将失控性炎症逐渐转向对机体有益的方向调控，RvD1的抗炎促消退作用为防治ARDS新策略提供实验依据。

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