Merlin信号通路在LPS诱导微血管内皮细胞损伤中的机制研究

Acute lung injury (ALI) is a common respiratory critical illness with higher incidence and mortality, of which the most common risk factor is sepsis. The pathogenesis of acute lung injury is not well understood, thus specific remedies to treat the disease remain limited. Our previous studies confirmed that RhoA activation played an important role in LPS-induced endothelial cell injury, whereas Rac1 inactivation and RhoA activation are both important for LPS-induced endothelial cell barrier compromise. In the present study, Merlin encoded by NF2 gene can regulate the activity of small molecule GTPase and negatively regulates the activity of Rac-1. Moreover, LPS-mediated upregulated expression of Merlin significantly reduced the expression of tight junction proteins( ZO-1 and Claudin5) on cytomembrane, resulting in shuttling of Vinculin to the focal adhesion and instability of Vinculin-α-Catenin-VE-cadherin complexes, eventually leading to integrity compromise of microvascular endothelial. Furthermore, knockout of Merlin could alleviate the inhibitory effect of LPS on cell cycle progression. Therefore, we hypothesize that the increased expression of Merlin under LPS stimulation inactivates Rac-1, inhibits cell cycle progression and promotes cell apoptosis, leads to endothelial barrier compromise and injury. The purpose of study is to fully illustrate the cellular and molecular biological mechanisms of septic ALI, and to provide strategic methods for clinical treatments.

急性肺损伤(ALI)是发病率和死亡率较高的常见呼吸系统危重症，以脓毒症ALI最为常见，其发病机制尚未完全明确。我们前期研究结果证实RhoA活化在LPS诱导内皮细胞损伤中具有重要作用，而Rac1失活和RhoA活化对于LPS所致内皮细胞屏障损伤均尤为重要。既往研究表明Merlin蛋白可负性调节Rac-1的活性。本项目的预实验结果提示Merlin在LPS刺激下表达增加，而内皮细胞过表达Merlin后紧密连接蛋白ZO-1以及Claudin5表达明显减少、Vinculin移位，内皮通透性增高。此外，抑制Merlin表达后，LPS所致的细胞周期阻滞效应被减弱。因此本研究推测LPS刺激下Merlin蛋白的表达增加可能通过抑制Rac-1的活性导致连接蛋白屏障受损，同时通过抑制细胞周期、促凋亡的效应加剧内皮细胞损伤，破坏内皮完整性，从而更加全面地理解脓毒症ALI的机制，为临床防治提供新的思路。

本课题主要研究Merlin蛋白在急性肺损伤中对肺微血管内皮通透性及肺损伤严重程度中的作用， 探讨Merlin蛋白对人肺微血管内皮细胞凋亡的影响及其机制研究，以及Merlin蛋白及其信号通路对人肺微血管内皮通透性调控的机制。结果表明，1. 敲低肺血管内皮细胞Merlin蛋白表达，可以明显缓解急性肺损伤严重程度，抑制肺血管通透性增加，减轻急性肺水肿；2. NF2基因敲低可缓解LPS诱导的单层融合肺微血管内皮细胞通透性，减轻内皮细胞间连接结构的破坏；3. Merlin蛋白通过活化Mst1/YAP/Caspase 3信号通路来促进肺微血管内皮细胞凋亡。4. LPS诱导肺微血管内皮细胞Merlin表达增加，从而使Merlin竞争性结合Angiomotin，使游离Rich1增加，导致Rac1和Cdc42活性下降，最终增加肺微血管内皮细胞通透性。本研究结果提示， Merlin蛋白通过激活Mst1/YAP/Caspase 3信号通路来诱导肺微血管内皮细胞凋亡； LPS增加Merlin表达从而通过Merlin/Amot/Rich1信号通路抑制Rac1和Cdc42活性，导致肺微血管内皮通透性增加，而靶向抑制Merlin/Amot/Rich1 Rac1/Cdc42信号通路可以改善肺微血管内皮通透性。