整合素β5-MMP9信号轴调控巨噬细胞极化平衡在脓毒症肺损伤中的作用及机制研究

Acute lung injury is the leading cause of death in sepsis patients, which is hard to defense. The imbalance of macrophage polarization plays an indispensable role in acute lung injury, which can be served as a promising target. Integrin β5 and MMP9 can be expressed in macrophage, and have pivotal roles in secreting inflammatory cytokines. Our preliminary studies found that integrin β5 and MMP9 protein levels were significantly enhanced in lung macrophages from septic mice; MMP9 promoted marker gene transcriptions of M1 macrophages and can bind integrin β5 to activate the downstream signals; MMP9 expression was markedly decreased in the lung from integrin β5 gene knockout mice subjected to sepsis; MMP9 gene knockout significantly decreased lung permeability in the mice suffered from ALI. Thus, we proposed the hypothesis “Integrin β5-MMP9 axis involves in macrophage polarization, and affects the outcome of acute lung injury”. Based on our preliminary results, we will take some methods, including gene knockout mice and plasmid transfection, to determine whether integrin β5-MMP9 axis could involve in acute lung injury, and then explore whether integrin β5-MMP9 axis could regulate macrophage polarization. Last, we will uncover whether protease inhibitor could prevent the imbalance of macrophage polarization and attenuate acute lung injury via targeting MMP9. The conclusion of this study might contribute to provide new perspectives for the protease inhibitor therapy and underlying mechanism of acute lung injury.

急性肺损伤（ALI）是脓毒症患者围术期常见并发症，死亡率高。巨噬细胞极化失衡是ALI重要的病理特征之一，以此为靶点的防治具有重要临床意义。整合素β5和基质金属蛋白酶9(MMP9)均可表达于巨噬细胞，在介导巨噬细胞释放炎症因子过程中发挥重要作用。本项目组预实验发现：①ALI小鼠肺巨噬细胞的整合素β5与MMP9蛋白表达水平明显上调；②MMP9促进M1型巨噬细胞标志基因的转录，且可结合整合素β5激活下游信号；③整合素β5基因敲除可显著下调ALI肺组织MMP9的表达，MMP9基因敲除显著改善ALI肺组织渗透性。结合文献报道，我们提出：“整合素β5-MMP9信号轴参与调控巨噬细胞极化，最终影响ALI转归”。本项目拟通过体内外实验明确此信号轴在ALI中的作用与机制；探讨抑制此信号轴关键分子表达的药物能否改善巨噬细胞极化失衡，减轻ALI；研究结果为急性肺损伤的防御与治疗提供新的理论基础及临床防治依据。

急性肺损伤（ALI）是一种严重的呼吸系统疾病，发病率和死亡率都很高。脓毒症期间巨噬细胞极化失衡是ALI重要的病理特征之一，整合素β5和基质金属蛋白酶9(MMP9)均可表达于巨噬细胞。在脓毒症致急性肺损伤过程中整合素β5与MMP9能否以及如何参与调节巨噬细胞极化尚不清楚。本研究通过小鼠脓毒症在体模型以及LPS刺激巨噬细胞的离体模型，从细胞、动物表型到细胞内在的下游信号通路水平研究整合素β5与MMP9在巨噬细胞以及脓毒症小鼠中的作用与机制。研究发现，在细胞模型中，单独MMP9刺激无明显影响巨噬细胞极化，外源性MMP9刺激或者过表达MMP9可显著促进LPS刺激的巨噬细胞向M1型巨噬细胞极化，敲减MMP9则可抑制LPS刺激的巨噬细胞向M1型极化；内源性免疫共沉淀显示MMP9可与整合素β5结合，且敲减整合素β5可显著下调MMP9的表达水平；进一步发现，整合素β5-MMP9信号轴可显著影响NF-κB信号通路的激活。我们还发现，显著抑制MMP9表达与活性的药物沙奎那韦（SQV），可显著抑制LPS刺激的巨噬细胞向M1型巨噬细胞极化，且显著抑制促炎因子的分泌。在动物模型中，我们提取小鼠肺组织单个细胞，利用流式细胞术检测，发现SQV可显著抑制脓毒症小鼠肺组织内的巨噬细胞向M1型极化，还发现SQV后处理可显著减轻脓毒症小鼠肺组织的损伤程度，且显著延长了脓毒症小鼠的七天生存率。本研究首次提出，“整合素β5-MMP9 信号轴参与调控巨噬细胞极化，影响急性肺损伤的转归”,阐明了一条可以调控巨噬细胞极化的新机制，有助于为探索ALI的病理机制和干预研究提供新的思路。另外，沙奎那韦是已经被FDA批准的应用于临床抗击HIV病毒的药物，通过本项目的研究，以期拓展沙奎那韦的药理作用范围，安全用于临床脓毒症患者，可能有助于预防脓毒症患者的肺损伤甚至提高脓毒症患者围术期的生存率、降低死亡率。