血红素加氧酶-1/Nrf-2/ARE通路在小鼠海水淹溺性肺损伤自身修复中的作用及机制研究

Drowning is the main cause of accidental death. Heme oxygenase-1 (HO-1) plays an extremely important role in the process of lung injury repair. We found that in the surviving mouse after drowning HO-1 expression increased continuously, and the lung injury was able to repair itself within 15 days; inhibition of HO-1 function aggravated pulmonary injury. Pre-experimental studies have found that Nrf-2 siRNA can inhibit the promotion of the expression of anti-oxidation-related genes by heme. Moreover, HO-1 siRNA decrease the nuclear factor E2-related factor 2 (Nrf-2) transcriptional activities by 39% through bioinformatics analysis. Therefore, we hypothesize that the HO-1/Nrf-2/ARE pathway plays a key role in self-repair of seawater drowning-induced acute lung injury. In order to test the hypothesis, HO-1loxP/loxP SPC; Cre+ mice, which knockout HO-1 gene in alveolar type II epithelial cells, were specifically prepared to establish acute lung injury in seawater drowning. In this study, we will investigate the repair ability of seawater-drowning lung injury in HO-1 knockout mice. Next, we test the effect of HO-1 on Nrf-2 translocation and transcriptional activity and the differentiation of alveolar type II epithelial cell. With the completion of this study, we will explore the exact role and mechanisms of HO-1 on the self-repair of seawater drowning-induced acute lung injury and lay a foundation for the treatment of drowning-induced acute lung injury.

淹溺是意外死亡主要原因。血红素加氧酶-1（HO-1）在肺损伤修复过程中有极为重要的作用。我们发现溺水抢救后存活的小鼠HO-1持续表达增高，肺损伤15天内能够自身修复；抑制HO-1功能则加重损伤。预实验发现Nrf-2siRNA可抑制血晶素的促进抗氧化相关基因表达的新现象，据此本研究假说HO-1/Nrf-2/ARE通路在海水淹溺性肺损伤自身修复中发挥关键作用。本研究特制备肺泡II型上皮细胞HO-1基因缺失的HO-1Cre/loxP基因条件性敲除小鼠，建立海水淹溺性肺损伤动物模型和海水孵育的肺泡II型上皮细胞损伤模型，观察HO-1促II型上皮细胞增殖和转分化及外源性补充HO-1对急性肺损伤的保护作用，明确HO-1促Nrf-2核转位、Nrf-2/ARE转录活性及抑制核内降解的作用；本研究旨在阐明HO-1/Nrf-2/ARE通路在淹溺性肺损伤自身修复过程中的作用及机制，为治疗淹溺性肺损伤奠定基础。

海上生产作业、旅游意外、军队海上训练和作战等均可能出现溺水事故。尽管采用糖皮质激素、肺泡表面活性物质和人工呼吸机等综合治疗措施，海水淹溺诱导的急性肺损伤患者预后还是很差。因此，急需寻找新的特异性措施治疗海水淹溺诱导的急性肺损伤。本课题组前期研究发现血红素加氧酶-1（heme oxygnase-1, HO-1）在肺损伤修复过程中有极为重要的作用，但具体分子机制不清楚。本项目制备肺泡II型上皮细胞HO-1基因缺失的HO-1Cre/loxP基因条件性敲除小鼠模型、髓系细胞特异性HO-1基因敲除（HO-1loxp/loxpLysM Cre）小鼠、Nrf-2基因敲除小鼠模型3种基因敲除的小鼠模型；利用人工海水刺激的小鼠肺上皮细胞株MLE-12、A549细胞和RAW264.7细胞模型，研究的重要结果如下：（1）海水淹溺3小时后，小鼠肺、心脏、脑和肾脏等肿胀、大量水肿液形成，提示海水淹溺可以导致多脏器血管内皮细胞损伤；（2）海水淹溺可以导致肺组织存在严重的线粒体功能障碍、氧化应激损伤和铁死亡等多种病理过程；（3）溺水抢救后存活的小鼠红系衍生核因子相关因子-2（Nrf-2）/HO-1持续表达增高，小鼠15天内肺组织可以进行自身修复，出现自愈现象；（4）使用Nrf-2激动剂或者HO-1激动剂可以有效减轻海水淹溺诱导的急性肺损伤；使用Nrf-2激动剂或者HO-1抑制剂则使肺损伤加重；（5）Nrf-2或者HO-1基因敲除小鼠海水淹溺后，肺损伤程度明显加重，死亡率增高。本项目的研究成果明确了Nrf2/HO-1信号通路在淹溺性肺损伤自身修复中的作用重要作用，提示Nrf2/HO-1信号通路激活剂极有可能是治疗海水淹溺诱导的急性肺损伤患者的新方法。