PLD2在ARDS血管内皮细胞多糖包被损伤中的作用及其机制研究

Acute respiratory distress syndrome (ARDS) is a complex issue in intensive care medicine. An increase in pulmonary vasopermeability is the basic pathological feature of ARDS, whereas damage to the glycocalyx of vascular endothelial cells is a critical factor that increases pulmonary vasopermeability. Heparanase (HPA) is a key enzyme that can damage the glycocalyx. HPA can specifically cut the side chain of heparan sulfate (HS), which is an important component of the glycocalyx. Phospholipase D2 (PLD2) is an important signal transduction-related enzyme. The prophase study revealed that a decrease in HPA activity and reduction in HS of the glycocalyx resulted from the intervention of PLD2 inhibitor on the injury model of human umbilical vein endothelial cells induced by LPS. This finding indicated that PLD2 was an important factor for regulating damage to the glycocalyx in ARDS. This study aimed to further analyze: effects of changes in PLD2 expression (over-expression, siRNA intervention / gene deletion) on damage to the glycocalyx of vascular endothelial cells in ARDS; expression of PLD2 and changes in PA in ARDS; and effects of PLD2/PA on HPA activity of vascular endothelial cells and its activation/release in lysosomes in ARDS, as well as clarify the contributions of PLD2 in damage to the glycocalyx of vascular endothelial cells in ARDS and its mechanism. The results of this study will elucidate novel mechanisms of glycocalyx injury in ARDS, and provide a new theoretical basis for ARDS therapy.

急性呼吸窘迫综合征（ARDS）是呼吸与重症医学的热点和难点。肺血管通透性增加是ARDS基本病理特点，而血管内皮细胞多糖包被损伤是引发通透性增加的关键因素。乙酰肝素酶（HPA）是导致多糖包被损伤的关键酶，可特异性切割多糖包被重要组分-硫酸乙酰肝素（HS）侧链。磷脂酶D2 (PLD2)是机体重要信号转导酶，前期研究发现，PLD2抑制剂干预LPS诱导的人脐静脉内皮细胞损伤模型，可导致HPA活性降低、多糖包被HS降解减少，提示PLD2是调控ARDS多糖包被损伤的重要因子。本研究拟进一步解析: PLD2表达改变（过表达、siRNA干扰/基因缺失）对ARDS血管内皮细胞多糖包被损伤的影响；ARDS中PLD2表达及其催化产物磷脂酸（PA）的变化；ARDS中PLD2/PA对血管内皮细胞HPA活性及其在溶酶体中活化与释放的影响。揭示ARDS血管内皮细胞多糖包被损伤调控新机制，为ARDS治疗提供新的理论基础。

肺血管通透性增加是急性呼吸窘迫综合征（ARDS）的基本病理特点。血管内皮细胞多糖包被(GCX)作为血管的第一道屏障，其损伤是引发通透性增加的关键因素。乙酰肝素酶（HPA）是导致GCX损伤的关键酶。磷脂酶D2（PLD2）是机体重要的信号转导酶。前期研究发现，PLD2抑制剂干预可降低LPS诱导人脐静脉内皮细胞GCX损伤及抑制HPA活性升高，提示PLD2是调控ARDS多糖包被损伤的重要因子。在此基础上，以PLD2/GCX为切入点，通过体内/体外实验解析PLD2在ARDS血管内皮细胞GCX损伤中的作用及其机制。研究表明：利用CRISPR/Cas9技术构建PLD2基因缺陷（PLD2-/-）小鼠。在LPS诱导ARDS模型下，与野生型小鼠相比，PLD2-/-小鼠肺组织中内皮细胞多糖包被损伤、HPA表达以及肺血管通透性显著降低，同时肺组织病理改变、炎症反应、肺水肿明显减轻；巨噬细胞是机体产生HPA的重要来源，在LPS刺激条件下，与野生型小鼠骨髓来源的巨噬细胞（BMDM）相比，PLD2-/- BMDM中HPA表达显著降低；外源添加PLD2的催化产物磷脂酸（PA）可诱发血管内皮细胞GCX损伤和胞内HPA活性升高。此外，PA生成抑制剂1-丁醇干预可显著抑制LPS诱发血管内皮细胞GCX损伤；溶酶体组织蛋白酶L(Cathepsin L)是使HPA酶原活化的主要水解酶，PLD2抑制剂CAY10594干预可降低 LPS 刺激血管内皮细胞中Cathepsin L的表达；PLD2基因缺失可显著抑制LPS刺激条件下小鼠肺组织和小鼠BMDM中Cathepsin L的表达。以上结果揭示了ARDS中血管内皮细胞GCX损伤调控新机制：“PLD2-PA-溶酶体Cathepsin L-HPA-GCX损伤”，为深入揭示ARDS的发病机制及相关治疗药物开发提供了新的理论依据。