RhoA/ROCK信号通路调控肺泡巨噬细胞微囊泡形成介导VILI的分子机制研究

It have been convincingly shown that alveolar macrophage is the initial determinant in the VILI development. The activation and proliferation states of alveolar macrophage greatly augmented inflammation, and ultimately lead to waterfall inflammatory reaction that is the striking feature of acute lung injury. Our previous study demonstrated that alveolar macrophage initiate the inflammatory reaction mainly via NLRP3/IL-1β and mtDNA/TLR9/MyD88 signaling pathway. But how the inflammatory signal transmit from alveolar macrophage to other target cell that eventually lead to systemic inflammatory response syndrome? Its mechanism have not yet been fully elucidated. Our previous study have shown that microvesicles increased significantly with serious lung inflammation, whereas autophagosome and mtDNA expression shew down-regulation. Whether autophagosome and mtDNA were parceled by microvesicles? So we proposed that autophagosome, mtDNA and NLRP3 were parceled by microvesicles during inflammation, microvesicles package with inflammatory cargo that shed from cell via activation of signaling pathway, and the microvesicles transmit inflammatory cargo to other target recipient cells, thus mediate production of inflammatory cytokines that greatly augmented inflammation causing waterfall inflammatory reaction, and ultimately lead to VILI. To address the hypothesis, gene knockout technology, immunofluorescence microscopy, fluorescence RT-PCR and Western blot techniques et.al were adopt to elucidated the nature of signaling pathway responsible for the formation of microvesicles, and to explore the mechanism of triggering alveolar macrophages generates microvesicles that greatly augmented inflammation ultimately lead to VILI. This study will provide a new idea for the prevention and treatment of VILI.

我们前期研究证实：机械刺激主要通过NLRP3/IL-1β及mtDNA/TLR9/MyD88通路启动炎症反应诱发VILI，同时发现，当机械刺激越大时，炎性介质如自噬小体及逃逸mtDNA反而呈下调表达，而细胞内微囊泡则呈上调表达。炎症介质是否被微囊泡吞噬呈不表达？尚未见报道。因此，我们提出假说：当炎症发生到一定程度时，细胞内形成能吞噬炎症介质的MVs，MVs被激活后介导出胞释放并在细胞间传递炎症因子致瀑布式炎症级联反应，最终导致VILI发生。为验证假说，采用荧光标记、流式细胞仪探讨MVs形成机制，采用荧光RT-PCR、Western blot等技术分析MVs成分及功能；通过基因转染、基因敲除等技术，探讨调控肺泡巨噬细胞内微囊泡形成的分子机制；通过MVs与靶细胞共培养，明确MVs的致炎作用，探讨微囊泡在细胞间传递炎症信息导致VILI的分子机制，为VILI的防治提供新靶点与新思路。

项目背景：微囊泡作为一种细胞间通讯介质，近年来备受学者们的关注。我们前期研究证实，机械刺激主要通过NLRP3/IL-1β及mtDNA/TLR9/MyD88通路启动炎症反应诱发VILI，同时发现，当机械刺激越大时，炎性介质如自噬小体及逃逸mtDNA反而呈下调表达，而细胞内微囊泡则呈上调表达。炎症介质是否被微囊泡吞噬呈不表达？尚未见报道。为此，本项目通过科学实验方法对RhoA/ROCK信号通路调控肺泡巨噬细胞微囊泡形成介导VILI的分子机制研究进行探讨。.主要研究内容：（1）明确在呼吸机相关性肺损伤中微囊泡形成与炎症反应相关性；（2）探讨 RhoA/ROCK/LIM/ Cofilin 信号通路调控肺泡巨噬细胞微囊泡形成的作用机制；（3）明确含炎症介质的 MVs 在细胞间的致炎作用，并阐明 VILI 的发病机制。. 重要结果及数据：20ml/kg潮气量机械通气可诱发肺部微囊泡释放增加，且与机械通气时间正相关；在VILI模型小鼠中，微囊泡富含IL-1β和TNF-α；将VILI源性微囊泡打入正常小鼠后，可诱发小鼠肺部炎症，提示，微囊泡通过包裹IL-1β和TNF-α参与VILI的发生、发展。中细粒细胞来源的微囊泡在肺组织中有较高表达，但各组表达无差异，对VILI中炎性微囊泡贡献较小；肺泡上皮细胞源性微囊泡表达较低；肺泡巨噬细胞来源的微囊泡在VILI的发生进展过程中占主导地位。肺泡巨噬细胞通过20%的细胞牵张后可发生明显的炎症改变，同时可诱导RhoA、Rock1、Rock2、磷酸化Limk表达上调，微囊泡生成增多；应用RhoA及Rock抑制剂后，Rock1、Rock2、磷酸化Limk表达被抑制，微囊泡生成明显减少，培养上清中IL-1β、IL-6和TNF-α浓度下降，炎症改善；在VILI中，RhoA/Rock信号通路对肺泡巨噬细胞微囊泡形成具有调控作用。. 科学意义：明确 RhoA/ROCK/LIM/Cofilin 信号通路在 VILI 中形成微囊泡的重要作用；明确肺泡巨噬细胞微囊泡的致炎作用，阐明 MVs 作为细胞间传递信息分子导致炎症级联反应的机制。