自噬在气泡损伤血管内皮细胞中的双向作用及机制研究

Decompression sickness (DCS) is a pivotal concern for people who take part in diving and hyperbaric practice, and genesis of inert gas bubbles is the primary etiology.The prevention of DCS is the first issue that should be treated due to its severe consequences. Increased tolerance to DCS has been reported in regular divers, of which the mechanism remains unclear.Previously, we have proved that vascular endothelial cells (VEC) damage that triggered by bubbles played a key role in the development of DCS. Meanwhile, further studies revealed that autophagy played a biphasic role in bubble-induced endothelial injury, which may be related to bubble stimulus intensities.Therefore,we hypothesize that bubbles may regulate autophagy activities by changing intracellular calcium concentration. Mild bubble contaction may result in activating of calcium channels and subsequent moderate autophagy activation that could alleviate the VEC injury. Severe bubble irritation would result in intracellular calcium overload, which would cause overactivation of autophagy and finally promote VEC damage. This may be related to the mechanism of regular diving prevents DCS. Our study aims to investigate the above hypothesis in multiple levels, including cells, vessel segments and animals, reveal the effects and mechanisms of bubble contaction on autophagy activities and demonstrate biphasic role of autophagy in bubble-induced endothelial injury, which could provide theorical evidence for exploring new preventive measures.

减压病（DCS）是妨害潜水和高气压作业的关键医学问题，其致病原因是快速减压形成气泡。由于引起的后果常比较严重，因此预防是关键。潜水实践表明，经常潜水者不易罹患DCS，但机制未明。我们证实，血管内气泡可损伤血管内皮细胞（VEC）并在DCS进程中发挥重要作用。进一步研究发现，自噬参与气泡损伤VEC进程，并在其中发挥保护或促进的双向作用，气泡刺激强度不同可能是基本原因。我们推测，气泡刺激通过胞内钙离子调节自噬活性。少量气泡激活钙通道，适度激活自噬，可增强VEC对抗损伤的能力，可能与常潜水不易罹患DCS有关；大量气泡则可引起钙超载，过度激活自噬，加剧VEC损伤。本项目拟在细胞、器官和动物水平研究上述假说，通过控制气泡刺激强度，系统研究气泡触碰对VEC自噬活性的影响及机制，在不同水平证实自噬在气泡导致VEC损伤中的双向作用，为阐明DCS机制、探索有效防治措施积累理论依据。

背景：随着海洋开发的持续推进，潜水医学保障日益重要。减压病（DCS）是潜水安全的核心威胁，预防是关键，但措施匮乏。潜水实践表明，经常潜水者不易罹患DCS，明确其机制可能有助于探索DCS预防措施。气泡是DCS致病因素，其引起的血管内皮细胞（VEC）损伤是DCS发生的重要环节。我们前期研究发现，自噬参与VEC损伤进程，但具体机制尚未完全明了。.主要研究内容：（1）在细胞水平发现微气泡触碰诱导大鼠肺动脉内皮细胞自噬，其程度同气泡刺激强度及VEC损伤程度密切相关。（2）在细胞、血管段和动物水平发现强度较重的气泡刺激直接损伤VEC，自噬抑制剂3-MA可有效抑制这一损伤效应；强度较轻的微气泡刺激预处理可减轻后续较重微气泡刺激所导致的VEC损伤，这一保护作用同样可被3-MA有效抑制。在此基础上，在动物水平证实连续轻度高气压暴露预处理，即只产生微量气泡的高气压暴露预处理可有效降低大鼠DCS发病率，3-MA可抑制这一预防效应。（3）在细胞水平明确微气泡触碰对不同位置VEC胞内Ca2+的影响，发现轻度气泡触碰通过升高胞内Ca2+浓度激活CaMKK-β/AMPK/ERK/mTOR通路诱导自噬；较重气泡触碰进一步升高胞内Ca2+水平，除激活上述通路外，还通过诱导ROS生成进一步提高ERK活性并抑制mTOR和AKT活性，增强自噬。同时观察到气泡触碰同步诱导VEC焦亡，也同胞内Ca2+浓度升高有关。.结论：本研究发现气泡触碰可激活VEC自噬并在其损伤进程中发挥双向作用，可能同自噬激活程度相关。明确了自噬对VEC的保护效应是连续潜水预防DCS的重要机制，进一步阐明了气泡通过Ca2+诱导VEC自噬的具体机制，为后续探索DCS防治措施提供了理论依据。同时，本研究首次观察到气泡刺激同时诱导VEC焦亡，可能与DCS炎性损伤有关，值得深入探索。