线粒体DNA介导的肺结构细胞焦亡调控急性肺损伤肺部液体失衡的机制研究

The excess lung fluid accumulation regulated by injured alveolar epithelial cell and lung microvascular endothelial cell promotes the progress of acute lung injury (ALI). Our preliminary studies showed that the copies of mitochondrial DNA (mtDNA) in serum and bronchoalveolar fluid of acute respiratory distress patients was sharply increased when comparing with healthy subjects. Our in-vivo studies showed that mtDNA amplified lung fluid imbalance of ALI, and enhanced pyroptosis in ALI lungs. Recent studies revealed that the pyroptosis of immune cells and lung microvascular endothelial cell was involved in ALI. Therefore, we assume that mtDNA may regulate the alveolar fluid imbalance through acting on the pyroptosis of alveolar epithelial cell and lung microvascular endothelial cell. In the present proposal, we plan to perform clinical studies, in-vivo and in-vitro studies based on primary cells and gene knockout mice, to study the clinical relationship between cell-free mtDNA, pyroptosis-related cytokines, and human lung fluid clearance, to investigate the role of mtDNA-regulated pyroptosis in ALI and potential mechanisms, and to explore whether the intervention of mtDNA-related pyroptosis could alleviate ALI, providing novel pathways for treating ARDS.

肺泡上皮细胞及肺微血管内皮细胞损伤所致的肺部液体异常集聚是急性肺损伤（ALI）重要发病机制，我们前期研究发现急性呼吸窘迫综合征患者血清及肺泡灌洗液中线粒体DNA（mtDNA）拷贝数增高，而mtDNA能够增加小鼠肺部细胞焦亡，放大ALI小鼠肺部液体失衡。新近研究提示细胞焦亡介导了ALI免疫细胞及肺微血管内皮细胞损伤，提示mtDNA可能通过调控肺泡上皮细胞及肺微血管内皮细胞焦亡，参与放大ALI肺部液体失衡。本项目拟：①招募ARDS患者，分析mtDNA与ARDS患者焦亡炎症介质及肺部液体重吸收的相关性；②利用细胞焦亡caspase-1/11及mtDNA受体TLR-9、DNA受体AIM-2基因敲除鼠，及原代细胞培养，明确mtDNA介导的肺泡上皮细胞及肺微血管内皮细胞焦亡是否参与调控ALI液体失衡，及其具体机制；③靶向关键受体或信号分子进行干预实验，探索阻断上述通路是否可能作为ALI的潜在治疗靶点。

肺泡上皮细胞损伤所致的肺部液体异常集聚是急性肺损伤（ALI）重要发病机制，我们前期研究发现急性呼吸窘迫综合征（ARDS）患者血清及肺泡灌洗液中线粒体DNA（mtDNA）拷贝数增高，而mtDNA能够放大ALI小鼠肺部液体失衡。新近研究提示细胞焦亡介导了ALI免疫细胞及肺微血管内皮细胞损伤，提示mtDNA可能通过调控肺泡上皮细胞及肺微血管内皮细胞焦亡，参与放大ALI肺部液体失衡。本项目：（1）募集了急性呼吸窘迫综合征患者和健康受试者，采集其血清及肺泡灌洗液标本，检测mtDNA、炎症介质、细胞程序性死亡相关的蛋白、血管内皮损伤相关的蛋白、肺损伤修复相关的蛋白等指标，结合患者的临床指标及临床结局，进行统计分析，发现ARDS患者肺泡灌洗液中mtDNA的拷贝数与炎症介质正相关，与ARDS疾病严重程度指标氧合指数负相关；（2）引入了toll样受体9（TLR-9）基因敲除小鼠，利用气道雾化LPS及mtDNA，构建ALI模型，研究发现，敲除TLR-9基因后，mtDNA所致的急性肺损伤的放大作用被明显削弱，进一步的蛋白检测提示细胞间紧密连接蛋白和细胞离子通道可能参与上述过程，提示mtDNA/TLR-9通过细胞间紧密连接蛋白和细胞离子通道，调控ALI液体失衡；（3）利用大鼠mtDNA刺激大鼠II型肺泡上皮细胞，检测细胞焦亡相关信号分子的变化情况，发现mtDNA可能通过诱导II型肺泡上皮细胞焦亡，参与调控ALI肺部液体失衡；（4）使用福多司坦干预急性肺损伤小鼠，检测急性肺部炎症反应和肺部液体失衡的变化情况，发现福多司坦可以通过抑制小鼠肺部细胞焦亡，减轻急性肺损伤肺部液体失衡及急性肺部炎症反应。本项目研究结果，共发表SCI论文5篇，获得授权国家发明专利2项，支持博士后出站1名，协助培养博士研究生1名，参与国内学术交流1次。项目研究结论为急性肺损伤的发病机制提供了新的思路，为ARDS的临床诊治提供了新的潜在靶点。