减压病血管内皮损伤机制研究

Decompression sickness (DCS) is a pivotal medical problem in underwater and hyperbaric interventions and bubble formation is a direct etiology. Bubbles can cause damage not only via physically pressing tissues or embolising vessels, but also through inducing biochemical cascades. However, the underlying pathogenesis is still poorly understood. Recent studies suggest that bubbles activate vascular endothelial cells (VECs) during moving in the vessel lumen and induces endothelium damage and inflammatory reactions,with a close correlation with the bubble ammount. Further in vitro study found that, with the increasing of the intensity of bubble perfusion, the moderately-constricted rat pulmonary arteries presented initial dialation and subsequent constriction. We speculate that the activation of shear stress-sensitive ion channel TRPV4 and subsequent increasing of intracellular calcium might be the start of VEC activation by DCS bubbles. Mild contact of bubbles stimulates the synthesis/release of vessel relaxing factors such as NO, prostacyclin. Persistent or severe irritation would result in intracellular calcium overload, endoplasmic reticulum stress and dysfuction of mitochondria, which activate vasoconstriction via inhibiting NO and enhancing endothelin-1 synthesis, and at last VEC apoptosis. In this study, rat DCS models, pulmonary artery segments and VECs were employed to investigate the bubble-induced damages to VECs and its underlying molecular mechanisms. In vivo bubble detection, in vitro vascular perfusion with bubbles, multi- and single-bubble contact with a layer or a single VEC and other techniques will be adopted. The study may provide theoretical basis for the elusidation of mechanisms of VEC injury and then the prevention and treatment of DCS.

减压病（DCS）是潜水作业中的关键医学问题，致病原因是体内形成气泡。除通过阻塞、压迫等物理作用造成损伤外，气泡还可促发一系列生化反应，但机制尚未完全明了。我们前期研究证实，血管内气泡可激活血管内皮细胞（VEC），引起损伤和炎症反应，程度与气泡量密切相关；进一步实验发现，随着气泡灌流强度的由低及高，适度收缩的离体肺动脉段出现先舒张后进一步收缩的现象。我们推测，气泡引起VEC应力敏感离子通道TRPV4开放、胞内钙离子浓度升高很可能是DCS气泡激活VEC的起始环节；气泡轻度刺激促进VEC生成舒血管物质；而在高强度刺激后，胞内钙超载，导致内质网应激、线粒体功能障碍，促发缩血管反应和VEC损伤。本项目拟以大鼠DCS模型、肺动脉血管段及VEC为研究对象，通过活体气泡探测、离体血管气泡灌流、多气泡和单气泡触碰等技术，系统研究DCS VEC损伤效应和机制，探索DCS有效防治措施。

背景：减压病（DCS）是妨害潜水和高气压作业的关键医学问题，由于后果严重，预防是关键。气泡是减压病发病的原因，除了通过阻塞、压迫等物理作用造成损伤外，气泡还可触发一系列生化反应。我们前期研究发现，血管内气泡可损伤血管内皮细胞（VEC），但机制尚未完全明了。.主要研究内容：（1）动物水平，模拟潜水后大鼠VEC损伤指标明显变化，程度同气泡量相关。β-七叶皂苷素可显著降低大鼠减压病发病率和死亡率，VEC保护是主要机制。（2）血管段水平，微气泡灌流导致肺动脉段VEC损伤，程度同气泡密度和灌流时间成正比。Ca2+螯合剂BAPTA、TRPV4通道阻滞剂HC-067047、IP3R通道阻滞剂xestospongin C、IKCa通道阻滞剂TRAM-34、ROS清除剂NAC可显著减轻VEC损伤。（3）细胞水平，多气泡触碰大鼠肺动脉内皮细胞（PAEC）一定时间导致VEC损伤，程度同触碰时间成正比。轻度触碰后，PAEC胞内Ca2+浓度升高，激活CaMMK-β和AMPK，诱导适度自噬，对抗气泡损伤。较高强度触碰时，Ca2+超载，一方面，促进ROS生成，减少NO合成，激活CaMMK-β、AMPK，抑制AKT和mTOR，过度激活自噬，促进VEC损伤；另一方面，激活Rho/ROCK通路，引起细胞膜骨架蛋白重构，抑制翻转酶Flippase活性，促进内皮微粒（EMPs）释放，间接损伤VEC。（4）细胞水平，单气泡触碰PAEC，被触碰细胞及毗邻细胞胞内Ca2+浓度升高，HC-067047、xestospongin C和TRAM-34可抑制这一变化。.结论：DCS VEC损伤由气泡引起并在疾病进程中发挥重要作用。气泡刺激对VEC功能存在双向作用，主要同Ca2+和自噬水平相关。本研究阐明了DCS VEC损伤效应和机制，为后续探索DCS防治措施提供了理论依据。同时，本研究首次观察到不同强度气泡刺激对VEC发挥双向作用，可能与潜水实践中经常安全潜水不易罹患DCS现象有关，值得深入探索。