IRF-1调控肺泡巨噬细胞焦亡在急性肺损伤中的作用及信号机制

The pathogenesis of ALI/ARDS remains unclear. Basing on our previous study on the role of IRF-1 in sepsis, this study focus on the role of alveolar macrophages pyroptosis in the pathogenesis of the ALI/ARDS, aimed to find new therapeutic targets for ALI/ARDS. Our previous study found that IRF-1 gene knockout relieved sepsis-induced lung injury and mortality in mice, accompanied by decreased expression of active caspase-1 in lung tissue and alveolar macrophages. But it is unclear whether IRF-1 involves in the pathogenesis of ALI / ARDS mainly by modulating alveolar macrophages pyroptosis. With wild type, IRF-1 knockout mice as the object,this project uses pulmonary and extrapulmonary pathogenic factors-induced ALI/ ARDS model, prepared by intratracheal instillation or intraperitoneal injection of LPS,to study the role of alveolar macrophages pyroptosis regulated by IRF-1 in the pathogenesis of the ALI/ARDS in vivo.Furthermore, in vitro experiments are performed to clarify the role of IRF-1 in regulation of macrophage pyroptosis and lung injury，and the upstream and downstream signal regulatory mechanism of IRF-1 regulation of alveolar macrophage pyroptosis,by primary culturing alveolar macrophages from wild-type and IRF-1 knockout mice and macrophage cell line RAW264.7,which will lay a solid theoretical basis for new therapeutic approach for ALI/ARDS.

ALI/ARDS的发病机制还未清楚。本研究在我们以前对IRF-1调控脓毒症发病机制的基础上，研究肺泡巨噬细胞细胞焦亡在ALI/ARDS的作用,以期发现新的治疗靶点。我们前期研究发现IRF-1基因敲除减轻了脓毒症相关性肺损伤和死亡率，并伴有肺组织和肺泡巨噬细胞caspase-1活化下降。但尚不清楚IRF-1是否主要通过调控肺泡巨噬细胞焦亡参与ALI/ARDS的发生。本项目以野生型、IRF-1 基因敲除小鼠为研究对象，分别以LPS气管内滴入和腹腔注射制备肺内和肺外致病因素ALI/ARDS模型，在体阐明IRF-1调控肺泡巨噬细胞焦亡在ALI/ARDS中的作用；并进一步通过原代培养野生型和IRF-1基因敲除小鼠肺泡巨噬细胞和巨噬细胞株RAW264.7，体外证实IRF-1调控肺泡巨噬细胞焦亡致肺损伤的细胞分子机制及其上下游信号调控机制，为ALI/ARDS寻找新的治疗途径打下坚实的理论基础。

本课题在我们以前研究发现巨噬细胞焦亡（pyroptosis）与急性呼吸窘迫综合征（ARDS）局部炎症风暴相关的研究基础上，进一步研究发现IRF-1通过促进肺泡巨噬细胞（AMs）焦亡从而导致细胞内炎症内容物的大量释放，并且发现这一过程受LPS介导激活的Toll样受体4激活的密切调控。通过检测LPS诱导的脓毒症急性肺损伤模型中肺泡巨噬细胞和向模型中腹腔注射焦亡重要调节分子caspase-1抑制剂（Ac-YVAD-cmk）的方法，发现能够抑制脓毒症相关性急性肺损伤喘模型中肺组织AMs的焦亡，从而减轻小鼠肺内和全身炎症反应。同期的研究发现IRF-1能促进ALI小鼠肺组织的焦亡水平，加重气道炎症。体外实验发现敲除IRF-1基因能够抑制小鼠巨噬细胞caspase-1的激活。在LPS+ATP的刺激下，IRF-1蛋白水平上调能够促进肺泡巨噬细胞的焦亡和细胞因子的释放。进一步研究发现IRF-1的作用依赖于LPS刺激巨噬细胞表面的TLR-4受体，NLRP3经典焦亡小体的激活与这一过程密切相关。提示拮抗AMs表面的TLR4受体和细胞内IRF-1基因表达可减轻LPS导致的脓毒症相关肺损伤，而巨噬细胞内IRF-1可能是预防ALI发生的靶点。