MiR-199a-3p通过mTOR-Atg13/Rab8a通路调控肺泡巨噬细胞分泌自噬小体介导ARDS炎症反应的机制

Alveolar macrophage (AM) activation is an important trigger in inflammatory cascade of ARDS, However, the exact mechanism is still unclear. We have found that LPS-induced AM could secrete large numbers of autophagosomes, which contains lots of inflammatory mediators and may play an important role in triggering excessive inflammation during ARDS. It has been demonstrated that mTOR-Atg13 signaling pathway plays a key role in the induction of autophagy, and its downstream gene Rab8a is an important gene which mediates autophagosome secretion to the extracellular domain. What’s more, we found that miR-199a-3p, which could specifically bind to 3’-mTOR, were significantly elevated in patients with ARDS. Therefore, it is hypothesized that miR-199a-3p regulates autophagosome-mediated secretion through mTOR-Atg13/Rab8a pathway to induce inflammation during ARDS. Firstly, LPS is used to stimulate miR-199a-3p high/low expression of AM, to clarify the effect of miR-199a-3p on the autophagosome secretion of macrophages; Secondly, secondly, we plan to activate or inhibit mTOR pathway in vitro, to clarify the molecular mechanism of autophagosome secretion by macrophage regulated by mTOR-Atg13/Raba8a pathway; Finally, animal experiments were performed to verify the lung protective effect of miR-199a-3p genetic knock-out on ARDS mice, providing a novel idea for the treatment of ARDS.

肺泡巨噬细胞(AM)激活是ARDS过度炎症的重要始动环节，但具体机制不明。我们发现LPS刺激AM导致富含炎症介质的自噬小体分泌增加，可能是导致炎症加重的始动因素。mTOR-Atg13在自噬中起关键作用，其下游Rab8a是介导自噬小体分泌至胞外的关键基因。我们发现ARDS患者血清miR-199a-3p表达显著升高，并可特异结合mTOR'3端。推测miR-199a-3p通过mTOR-Atg13/Rab8a调控AM分泌自噬小体介导ARDS炎症。本课题先采用LPS刺激miR-199a-3p高/低表达的AM，明确miR-199a-3p对巨噬细胞自噬小体分泌的影响；其次体外激活和抑制mTOR通路，明确miR-199a-3p通过mTOR-Atg13/Rab8a调控巨噬细胞分泌自噬小体的分子机制；最后通过动物实验验证miR-199a-3p基因敲除对ARDS小鼠的肺保护作用，为治疗ARDS提供了全新的思路。

项目背景：急性呼吸窘迫综合征（ARDS）是ICU常见临床危重症，死亡率达40%。过度的炎症反应是 ARDS 发生的根本机制。肺泡巨噬细胞(AM)作为炎症始动的主要效应细胞，AM源性自噬小体可能是介导ARDS失控炎症的重要途径，减少 AMs 通过自噬小体分泌介导的炎症介质释放，可能是抑制 ARDS炎症级联放大的新靶点。.主要研究内容：体外培养AM收集分泌的自噬小体，破膜后检测其内含炎症因子表达水平，在细胞和动物层面明确巨噬细胞来源的自噬小体对ARDS失控炎症反应的作用；通过质粒转染构建高表达及低表达 miR-199a-3p 的小鼠巨噬细胞系，明确 miR-199a-3p 对巨噬细胞自噬小体分泌的影响；在此基础上通过PAK4信号通路的抑制剂以及siRNA干扰Rab8a基因的表达，明 确 miR-199a-3p 通过PAK4-Rab8a 调控巨噬细胞分泌自噬小体的分子机制；进一步构建 miR-199a-3p 高/低表达的 C57BL/6 小鼠，证实 miR-199a-3p 低表达可以减缓 ARDS 小鼠肺部炎症损伤，改善 ARDS 小鼠生存。.重要结果、关键数据.本研究发现：⑴ LPS诱导小鼠肺泡巨噬细胞分泌自噬小体增加，且AM自噬小体诱导小鼠发生肺损伤。⑵AM源性自噬小体可释放炎症因子IL-1β，而IL-1RA减轻巨噬细胞来源自噬小体诱导的小鼠肺损伤。⑶LPS可上调巨噬细胞miR-199a-3p的表达，高表达miR-199a-3p的巨噬细胞自噬小体分泌增多。⑷miR-199a-3p低表达可显著改善ARDS小鼠肺损伤，减轻肺部炎症反应，提高生存率。⑸Rab8a参与调节巨噬细胞分泌自噬小体，干扰Rab8a表达后巨噬细胞自噬小体分泌明显减少，Rab8a基因敲除可改善ARDS小鼠肺损伤。⑹细胞实验发现，高表达miR-199a-3p的巨噬细胞Rab8a表达增多，而抑制PAK4通路可上调低表达miR-199a-3p巨噬细胞Rab8a的表达并促进自噬小体的分泌，提示miR-199a-3p通过PAK4信号通路调节Rab8a表达调控巨噬细胞分泌自噬小体。.科学意义.本研究证实了ARDS时，miR-199a-3p通过下游PAK4/Rab8a通路，调节肺泡巨噬细胞释放内含炎症因子IL-1β的分泌型自噬小体，调控ARDS炎症反应，为探讨ADRS治疗新策略提供了重要的理论基础和关键靶点。