FAK信号通路在严重烧伤早期肝素结合蛋白介导血管内皮细胞损伤中的作用机制

In the early severe burns, increasing level of Heparin-binding protein (HBP) in plasma is closely associated with expression of cytokines and dysfunction of systemic vascular permeability. Our previous studies confirmed that GEF-H1/RhoA signaling pathway mediated endotoxin-induced expression of inflammatory cytokines and permeability dysfunction in endothelial cells. However, changes of microvascular vascular permeability not only depend on intercellular adhesion, but also depend on changes of focal adhesion through which the cytoskeleton of endothelial cells connect to the extracellular matrix. Focal adhesion is regulated by focal adhesion kinase (FAK). In the present study, we found HBP could activate FAK within a short time in microvascular endothelial cells. HBP not only increased significantly activity of GEF-H1 and p-MYPT-1, but also upregulated significantly expression of p-p65, MCP-1, VCAM-1 in endothelial cells. Taken together, we assume that HBP regulates expression of inflammatory cytokines and vascular permeability compromise through activation of FAK signaling pathway in the early severe burns. The purpose of study is to fully illustrate the cellular and molecular biological mechanisms of burn shock during the early stage after severe burns, and to provide strategical methods for clinical treatments.

严重烧伤早期，血浆肝素结合蛋白（HBP）的水平已证实与炎症因子产生和毛细血管通透性的增加等密切相关。我们前期研究结果表明GEF-H1/RhoA信号通路的活化是血管内皮细胞炎症因子的产生与释放以及内皮细胞通透性增加的重要因素之一。然而微血管内皮屏障的变化不仅取决于细胞间紧密粘附连接，而且还取决于内皮细胞与周围基质之间的接触位点黏着斑的变化。黏着斑的调控却有赖于黏着斑激酶（FAK）的活性。本项目的预实验结果提示HBP可在短时间内激活内皮细胞中FAK，通过FAK的活化不仅细胞内GEF-H1和 p-MYPT-1活性显著增加，而且p65的磷酸化水平以及MCP-1和VCAM-1 的表达均明显升高。因此，我们推测严重烧伤早期，HBP通过激活内皮细胞FAK信号通路，介导炎症因子的表达和内皮细胞通透性的改变。从而更加全面地理解严重烧伤早期烧伤休克的细胞和分子生物学机制，为临床防治提供新的思路。

HBP可以激活GEF-H1并将刺激信号传导至RhoA/ROCK、磷酸化下游效应分子MLC和MYPT1，引起肌动肌球蛋白交联增加、细胞内肌动蛋白骨架重建和应力纤维生成，最终导致内皮细胞收缩，破坏内皮细胞屏障功能。此外，微血管内皮细胞通透性调节不仅取决于细胞间紧密粘附连接，而且还取决于内皮细胞与周围基质之间的接触位点黏着斑的变化。黏着斑的调控却有赖于黏着斑激酶（Focal adhesion kinase，FAK）的活性，细胞通过精确调控FAK活性与PI3K/Akt和GEF-H1/RhoA/ROCK信号通路共同调节细胞黏着斑、细胞骨架及炎症介质的变化，从而调控血管内皮通透性。 PMVECs中FAK 磷酸化对PI3K/Akt信号通路和 GEF-H1/RhoA信号通路的调控作用。活化内皮细胞中FAK后， PI3K/Akt信号通路和 GEF-H1/RhoA信号通路之间的关系，探索PI3K/Akt信号通路和p38 / NF-κB信号通路在FAK下游信号通路中的作用。激活PMVECs中FAK信号通路并通过其介导， 一方面通过介导PMVECs细胞骨架收缩，破坏内皮细胞屏障完整性，从而增加肺血管通透性；另一方面，激活下游的PI3K/Akt和p190RhoGAP / RhoA /ROCK/ NF-κB信号通路，促发内皮细胞的促炎反应，造成细胞损伤。在整体、细胞、分子水平上进一步分析FAK信号通路和 HBP作为新的干预手段，改善严重烧伤后烧伤休克和肺损伤。