Merlin蛋白负性调控CD40L介导肺微血管内皮炎症损伤的机制研究

There is a key challenge in the treatment and high mortality of post-burn patients with acute lung injury (ALI). The injury of pulmonary microvascular endothelial cells (PMVECs) is a central part of the pathophysiology of ALI, and our work has indicated that the proinflammatory factor CD40L plays a critical role in this process.We have previously demonstrated that CD40L could stimulate the expression of a growth inhibitor merlin in the endothelial cells. Merlin knockdown increased the inflammation responds intensifies, while merlin overexpression significantly inhibite the CD40L-mediated proinflammatory effects. The negative regulation of CD40L-mediated inflammation by Merlin was at least part through the modulation of Hippo signalling pathways and YAP/TAZ proteins .Based on previous experiments, in this study we attempt to confirm the role of merlin in the regulation of CD40L-mediated inflammatory injury of PMVECs and reveal the underlying mechanisms with the detection of the changes of both cell characteristic and histopathology and with a combined anlysis between the expression, subcellular localization and activation of the proteins involved in the Hippo pathways and YAP/TAZ proteins, thus identifying one or more targets for potential interventions to reduce the injury of PMVECs and providing foundations for the clinical treatment of ALI.

严重烧伤所致的急性肺损伤(ALI)死亡率高且治疗困难。肺微血管内皮细胞(PMVECs)受损是ALI发病核心环节，我们前期研究表明，促炎因子CD40L在这一核心环节中发挥关键作用。我们预实验发现，CD40L能刺激血管内皮细胞中一种生长抑制因子Merlin的反馈性表达；敲减Merlin表达能进一步加剧炎症反应；而过表达Merlin可明显抑制CD40L介导的促炎效应；且Merlin抑制CD40L介导的内皮炎症可能是通过调控Hippo信号通路和YAP/TAZ蛋白实现。本课题基于前期研究结果，拟在体外细胞实验和基因敲除小鼠模型中，通过细胞特性/组织病理变化这两个方面，结合关键信号的蛋白表达/定位/活性变化这三个角度，明确Merlin对CD40L介导的PMVECs炎症损伤的负性调控作用以及潜在分子机制，以期能揭示一个或若干个潜在干预靶点，旨在改善PMVECs炎症损伤，从而为ALI的防治提供新思路。

本研究通过体外实验、敲减及过表达NF2基因，已初步证实Merlin蛋白可以负性调控CD40L介导的内皮细胞炎症因子表达，其机制分为抑制YAP-TAZ蛋白信号通路；此外本研究还通过体内及体外实验证实Merlin 蛋白对于肺微血管内皮细胞间连接蛋白有着重要的调节功能，可以明显诱导细胞间连接的破坏，增加细胞应力纤维形成；可以明显缓解LPS诱导急性肺损伤严重程度， 减轻肺部炎症细胞渗出，缓解肺血管内皮通透性增加。内皮细胞促炎信号通路活化的同时，抗炎信号通路的“负性调节力量”可能决定内皮炎症的发展方向。我们首次探究 Merlin/Hippo 信号对 CD40L 介导血管内皮细胞炎症的关键环节 YAP/TAZ 表达和活性的影响，并且通过LPS诱导急性肺损伤小鼠模型中验证干预Merlin/Hippo 信号对于急性肺损伤发生发展的影响，为急性肺损伤中提供可能的干预靶点。