脂氧素调控巨噬细胞表型转换促进炎症消退的机制研究

Acute respiratory distress syndrome (ARDS) consisting of acute hypoxemic respiratory failure, occur with a high incidence and mortality. 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. We also find lipoxin not only promotes neutrophils apoptosis and efferocytosis, but also increases M1 transfer into M2 macrophages. Base on these findings, our hypothesis is that lipoxin may mediate M1 and M2 transformation to promote inflammation resolution, which is the important mechanism of acute lung injury. Therefore, low dose LPS induced lung injury was established, primary monocytes and macrophages were also isolated and cultured, blood and BALF from ARDS patients were harvest. Real-time PCR, flow cytometry, laser confocal microscope and western blot et al were used to measure the dynamic of lipoxin, lipoxin receptor and macrophage phenotype changes, discuss the effect of lipoxin on M1 and M2 phenotype. Finally, to gain a better understanding of the mechanisms, we also investigated the effect of BML-111 (ALX agonist), butoxycarbonyl-Phe-Leu-Phe-Leu-Ph (BOC-2 [ALX antagonist]) and siRNA on macrophages phenotype in ARDS. The main aim of this grant is to reveal a novel mechanism of pro-resolution and provide experimental data explore the prevention and treatment of ARDS.

炎症消退调控障碍导致炎症失控是急性呼吸窘迫综合征发病的重要机制。脂氧素是机体最重要内源性抗炎促消退介质,为炎症反应的“刹车信号”。在前期证实脂氧素促进肺泡液体清除率，保护肺损伤作用的基础上，结合我们新近发现脂氧素可促进巨噬细胞吞噬凋亡的中性粒细胞以及脂氧素促进M2型肺泡巨噬细胞增多，抑制M1型肺泡巨噬细胞表达等线索。我们推测：脂氧素调控巨噬细胞表型转换可能是促进炎症消退的重要机制。为此，我们拟采用静脉注入内毒素型大鼠ARDS炎症损伤模型, 培养人原代单核细胞，巨噬细胞，应用实时定量PCR、流式细胞仪、激光共聚焦显微镜、western blot等技术,动态观察脂氧素及其受体，巨噬细胞表型变化规律；探讨脂氧素对巨噬细胞M1型和M2型的影响及相互间转化的机制；进一步应用脂氧素受体激动剂, 抑制剂和siRNA技术验证我们假说。旨在揭示促炎症消退新机制并为防治ARDS提供实验资料。

炎症消退调控障碍导致炎症失控是急性呼吸窘迫综合征发病的重要机制。脂氧素是机体最重要内源性抗炎促消退介质,为炎症反应的“刹车信号”。巨噬细胞是机体免疫系统的重要组成部分。骨髓髓系前体细胞在外周血循环系统中分化为单核细胞, 继而进入外周组织分化成熟为巨噬细胞。作为机体的“清道夫”细胞，巨噬细胞表面不仅存在大量能够识别，吞噬和破坏病原菌的表面受体和细胞内介质，而且对凋亡细胞抗原呈递免疫耐受信号，从而吞噬凋亡的中性粒细胞。本课题主要是探讨脂氧素对巨噬细胞表型转化的影响及机制。首先我们发现脂氧素不仅受体依赖性促进中性粒细胞凋亡，而且促进巨噬细胞吞噬凋亡的中性粒细胞，这一过程也依赖于脂氧素受体。第二，通过分离肺组织巨噬细胞和人单核细胞，我们发现脂氧素能够下调M1型巨噬细胞表达，上调M2型巨噬细胞表达，促进巨噬细胞由促炎症发生的M1型向促炎症消退的M2型转化，进而促进炎症消退。 第三，为进一步研究M2型巨噬细胞的来源及脂氧素调控M2巨噬细胞的具体机制，我们分离LPS刺激的小鼠肺组织并匀浆后流式细胞术检测M2来源。结果显示，相较于正常对照组，LPS组F4/80+Ly6c-常驻巨噬细胞中M2含量减少（P<0.05），F4/80+Ly6c+炎症巨噬细胞中M2型巨噬细胞增多（P<0.05）；相较于LPS组，脂氧素治疗组F4/80+Ly6c-常驻巨噬细胞中M2含量增多（P<0.05），F4/80+Ly6c+炎症巨噬细胞中M2型巨噬细胞减少（P<0.05）。M2型巨噬细胞主要来源于肺组织炎症巨噬细胞，脂氧素通过抑制炎症巨噬细胞M2型进入到肺组织，同时增强肺组织常驻巨噬细胞向M2型转化，进而促进炎症消退。此外，脂氧素上调炎症消退模型中M2型巨噬细胞表达促进炎症消退。总之，本研究提示脂氧素可通过上调巨噬细胞吞噬功能，调控M1/M2型巨噬细胞转化，进而促进炎症消退，为临床炎症治疗提供新的思路和策略。