骨髓间充质干细胞外泌体通过miR-384-5p/Beclin-1通路调控肺泡巨噬细胞自噬在急性肺损伤中的作用

Alveolar macrophages (AMs) play a key role in modulation of inflammatory response in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Recent studies have found that bone marrow mesenchymal stem cell (BMSC)-derived exosomes are expected to treat ALI/ARDS. However, the role of BMSC derived-exosomes in modulation of AMs in response to inflammation and the underlying molecular mechanism have not been completely understood, and need to be further elucidated. Our previous study has shown that the level of microRNA-384-5p (miR-384-5p) is markedly increased, whereas the expression level of Beclin-1 is downregulated in the AMs after the BMSC derived-exosomes treatment. According to bioinformatic analysis, Beclin-1 may be direct targets of miR-384-5p. The 3’-untranslated region (3’-UTR) of Beclin-1 mRNA have binding sites for miR-384-5p. To date, the mechanism of miR-384-5p in regulation of AMs remains unclear. Therefore, we put forward the hypothesis that miR-384-5p from BMSC-derived exosomes may suppress the Beclin-1-mediated AMs autophagy, which has an effect on inflammatory response. We aim to investigate the role of miR-384-5p in the inflammatory response of ALI/ARDS, and confirm the molecular mechanism of AMs autophagy regulated by exosomes-mediated miR-384-5p/Beclin-1 signaling pathway, from molecular, cellular, tissue to whole animal level. The present study may lay a foundation for revealing the pathogenesis and progression of ALI/ARDS from the new viewpoint of AMs autophagy dysfunction, and may also provide a novel concept for the prevention and treatment of ALI/ARDS.

肺泡巨噬细胞（AMs）在急性肺损伤（ALI）的发病过程中有重要的调控作用，同时，BMSCs来源的外泌体参与了炎症反应的调控，并有望用于ALI的治疗。但外泌体与AMs的关系尚不清楚。我们的预实验发现：BMSCs外泌体作用于AMs的炎症模型，可以使AMs中的miR-384-5p水平升高，自噬基因Beclin-1表达下调。生物信息学预测Beclin-13’-UTR存在miR-384-5p的结合位点。据此我们提出假说：BMSCs通过外泌体传递miR-384-5p，负向调控Beclin-1介导的AMs自噬，进而影响对炎症反应的调节。本研究拟从分子、细胞、组织及动物整体水平等多层次探讨miR-384-5p在ALI炎症反应中的作用，明确外泌体通过miR-384-5p/Beclin-1通路调控AMs自噬的分子机制。本研究将从AMs自噬功能紊乱这个新视点探讨ALI发生、发展机制，为ALI的防治提供新的思路。

急性肺损伤（ALI）及其严重阶段的急性呼吸窘迫综合征（ARDS）是急危重症领域的常见疾病，死亡率高达40%。近年来国内外多项研究发现骨髓间充质干细胞（BMSC）释放的外泌体有望用于ALI/ARDS的治疗，但其具体机制不明。我们前期研究发现，Beclin-1介导的巨噬细胞自噬与炎症反应在ALI/ARDS过程中可能发挥了重要作用，用LPS刺激肺泡巨噬细胞后，Beclin-1的蛋白表达水平呈时间及剂量依赖性增加。本课题组以此为切入点，通过大鼠ALI模型，研究了BMSC来源的外泌体是否会对肺泡巨噬细胞自噬产生调节作用及其分子机制。我们使用全骨髓贴壁培养法成功分离出BMSC，并提取了外泌体。通过体内外研究证实外泌体可被肺泡巨噬细胞所摄取并富集到ALI大鼠的肺组织中。我们研究发现，炎症刺激时，BMSC来源的外泌体可以通过携带并释放miR-384-5p至肺泡巨噬细胞中，提高ALI大鼠肺组织中miR-384-5p的水平，荧光素酶报告基因的结果显示miR-384-5p通过直接结合Beclin-1 3’-UTR而发挥抑制作用，从而负向调控 Beclin-1介导的自噬，稳定自噬水平，控制炎症反应，缓解肺泡-毛细血管膜的高通透性，减轻肺损伤的程度，我们初步探索证实BMSC来源的外泌体有效提高大鼠的生存率，且气管内注射方式在调控自噬、控制炎症、缓解肺血管高通透性和减轻大鼠肺损伤等方面均要优于尾静脉注射方式。该研究为BMSC来源外泌体在急性肺损伤中的作用分子机制提供了新的认识，为ALI/ARDS的临床治疗提供了新的可能策略。