血管内皮对肺移植供肺保存过程中炎症和损伤的调控作用及机制

A major impediment in the field of lung transplantation is oxidative injury that arises in the donor lung during the ischemia or storage period and later post-transplant. This damage is the result of ischemia-reperfusion injury. We have observed in clinical practice that inflammation and injury occur prior to the transplant and may be triggered during the ischemic episode itself. We propose to here to focus on the donor lung endothelium before transplant, instead of the standard clinical practice of controlling inflammation by immunosuppressive therapy to the recipient, to eliminate the signals that emanate from the endothelium and promote inflammation and damage. Our preliminary data have demonstrated that 1) Ischemia induecd Nox2-dependent ROS production in pulmonary endothelial cells; 2) The protein level of HMGB1 and RAGE was increased in lung tissues after ischemia; 3) Ischemia activated NLRP3 inflammasome in mouse lung tissue; 4) Ischemia induced Nox2-dependent oxidative damage in mouse lung tissue. Based on those data, we propose that ROS generated by the pulmonary endothelium during ischemia (i.e. storage before transplant) drives the production of HMGB1-RAGE and NLRP3 inflammasomes eventually causing pyroptosis (programmed inflammatory cell death) and oxidative injury in the stored lung. In the current study we will determine the role of vascular endothelium in the regulation of inflammation and injury during donor lung storage as well as its related molecular mechanism through cell, animal and human donor lung experiments. This study will provide a theoretical basis for developing lung protection strategies in clinic, increasing donor lung availability, thus promoting long-term survival of grafts after lung transplantation.

肺移植领域的一个重要难题是供体保存期缺血和移植后的氧化损伤，其本质是缺血-再灌注导致的炎症反应。我们在临床实践中观察到有些供肺在移植前已经发生炎症和损伤，可能是缺血本身所致。与目前肺移植常用的受体免疫抑制控制炎症的方式不同，我们将把研究重点放在移植前的供体肺内皮，从根源上消除诱发炎症和损伤的信号。我们前期发现，缺血致①血管内皮NOX2依赖的ROS生成②肺组织HMGB1和RAGE水平升高③肺NLRP3炎症小体表达④小鼠肺组织依赖NOX2的氧化损伤，我们推测：供肺保存期间肺内皮由于缺血产生的ROS可致HMGB1-RAGE信号通路的激活和NLRP3炎症小体的生成，最终导致细胞焦亡和氧化损伤。本研究从细胞、动物、临床样本层面阐明血管内皮对肺移植供肺保存过程中炎症和损伤的调控作用及可能机制，为临床制定供肺保护策略，增加供肺可利用性，进而提升肺移植后移植物以及受体的长期生存提供理论依据。

随着脑死亡供肺成为我国肺移植的唯一合法供肺来源，供肺质量问题已成为肺移植取得长足进步需要逾越的最大障碍。如何改善脑死亡供肺质量，是目前肺移植领域亟待解决的问题。我们在临床实践中观察到有些供肺在移植前已经发生炎症和损伤，我们因此将研究重点放在移植前的供肺组织，拟从根源上消除诱发炎症和损伤的信号。本项目主要研究结果显示：1）缺血致供肺血管内皮NOX2依赖的ROS生成及肺组织HMGB1和RAGE水平的升高，缺血同时诱导供肺NLRP3炎症小体的生成及氧化损伤；2）抑制NOX2表达后供肺中ROS的生成减少，同时供肺中HMGB1，RAGE和NLRP3的表达也减少，肺组织损伤减轻；3）NLRP3抑制剂MCC950能够抑制缺血诱导的血管内皮细胞（HUVEC）的死亡和焦亡，亦能够改善体内肺缺血诱导的肺损伤。我们的研究结果提示，供肺保存期间肺血管内皮细胞由于缺血产生的 ROS 可导致HMGB1-RAGE信号通路的激活和 NLRP3 炎症小体的生成，最终导致细胞焦亡和肺组织的氧化损伤。本研究从分子水平、细胞水平、整体动物模型和临床样本等多层次展开研究，基本明确血管内皮细胞对供肺冷缺血保存过程中炎症和损伤的调控作用，同时基本阐明相关分子机制，为临床制定供肺保护策略，增加供肺可利用性，进而提升肺移植后移植物以及受体的长期生存提供了理论依据。