血管紧张素Ⅱ2型受体激动剂对机械通气所致肺损伤大鼠模型中肺泡巨噬细胞异常极化、功能的调控作用

Angiotensin Ⅱpromoted ventilator-induced lung injury (VILI) via type 1 receptor (AT1R); blockade of AT1R had a protective effect against VILI. The abnormal polarization of alveolar macrophage (AMφ) played an important role in the pathogenesis of acute lung injury (ALI). AT1R promoted M1 macrophages polarization; whereas angiotensin Ⅱtype 2 receptor (AT2R) counteracted the AT1R function, and activation of AT2R stimulated the macrophages suppressive cytokines production, the role of AT2R and AMφ is still obscure in the development of VILI. We previously found AT2R expression increased in VILI patients, AT2R agonist alleviated monocytes/macrophages-mediated inflammatory injury. In present study, we will set up the rat model of VILI to get insights into the changes of the different stage of disease course, which contains the change of AT2R and AT1R level in AMφ and the polarization and function of AMφ after treating with AT2R agonist and AT1R blocker, using the laser confocal microscopy, co-immunoprecipitation, electrophoretic mobility shift assay, RT-PCR, and AT2R siRNA. The designed work will provide experimental evidence for the beneficial role of AT2R agonist in promoting M2 AMφ polarization and inhibiting M1 AMφ-mediated inflammatory injury within VILI.

血管紧张素Ⅱ通过1型受体（AT1R）促进机械通气所致肺损伤（VILI）；AT1R阻断剂缓解VILI进展。肺泡巨噬细胞（AMφ）异常极化、功能在急性肺损伤（ALI）中发挥重要作用。AT1R促进M1型巨噬细胞极化；而2型受体（AT2R）拮抗AT1R功能，活化AT2R刺激巨噬细胞产生抗炎介质，目前尚无AT2R与AMφ极化在VILI中的研究报道。课题组发现，VILI患者AT2R表达增高；AT2R激动剂缓解单核/巨噬细胞介导炎症免疫损伤。本课题拟建立VILI大鼠模型，利用AT2R、AT1R的药理学工具和流式细胞术、激光共聚焦、免疫共沉淀、电泳迁移实验、qPCR、Western blot、RNA干扰等技术，观察病程不同阶段AMφ的AT2R、AT1R分布、表达变化及其对AMφ极化、功能的调控作用，为揭示AT2R激动剂促进M2型AMφ极化，抑制M1型AMφ介导炎症免疫损伤，缓解VILI进展提供实验依据。

本课题目标拟发现机械通气肺损伤（VILI）小鼠模型的病程初期、进展期肺巨噬细胞（LMφ）的动态变化和血管紧张素2型受体（AT2R）分布、表达的关系；明确体内给予VILI小鼠AT2R激动剂（C21）调节肺组织中LMφ的变化，是否缓解VILI；进一步阐明AT2R对体外培养的Mφ的表型和功能及信号传导的作用。.. 选择成年雄性C57BL/6小鼠，建立VILI模型。机械通气频率80次／min，持续时间4 h，Vt=40 ml／kg，FiO2=21％，I：E=1：2，PEEP=0。机械通气前30min预先给予VILI小鼠模型C21，机械通气开始后动态观察小鼠血气分析、组织病理学和支气管肺泡灌洗液（BALF）中炎症介质水平、白细胞数目的变化。分离肺组织并观察VILI模型小鼠LMφ的表型变化，检测Mφ的生物学功能变化，观察肺组织Mφ的AT2R分布表达、基因转录变化以及与AT2R偶联的Giα蛋白、Giα-cAMP-PKA信号转导和下游MAPKs-NF-κB信号通路的变化。.. C21改善大潮气量机械通气组小鼠肺组织出现明显急性炎性损伤性改变（肺间隔明显增厚，炎性细胞浸润以及肺泡中出现渗出物），改善BALF中蛋白渗出，降低肺组织W/D比值，降低BALF中白细胞计数和中性粒细胞计数，减少炎性介质水平。流式细胞术检测发现，在大潮气量机械通气开始后早期，LMφ数量显著增多，而后随着机械通气时间的持续，LMφ数量逐渐降低。但是外周循环中的单核细胞不断的募集到小鼠肺组织，单核细胞逐渐分化CD11b(hi)Ly6C(low)细胞，细胞活化程度和抗原提呈能力相对增强，C21抑制这种细胞分化。AT2R活化后，Giα蛋白呈现增高趋势，AT2R发生内化。AT2R活化后下调cAMP-PKA途径，调节MKP-1。胞内p-ERK1/2和p-IκBα的水平出现下降趋势。NF-κB的p65、p50亚基胞浆分布出现升高趋势。.. 研究表明，AT2R通过下游信号通路调节LMφ的分化和表型，进而缓解VILI的临床表现，提示A2R药理学工具对急性肺损伤有保护作用。