Role of Nrf2 in Alveolar Epithelial Cell Regeneration During Lung Repair

急性肺损伤（ALI）后肺泡上皮修复受损可导致炎症反应增强， 反应和组织修复通常不能解决。本申请中的研究将检验这一假设 损伤后受损的肺修复是2型肺泡上皮细胞中GSH/AKT信号失衡的结果， 上皮细胞（AEC 2）由细胞保护和促生存转录因子Nrf 2调节。我们 发现Nrf 2的整体缺失会损害亚致死促氧化剂（高氧）诱导的肺修复。 阿里使用条件性突变Nrf 2（“floxed”）小鼠模型来确定肺Nrf 2的贡献。 急性肺损伤后，肺上皮细胞Nrf 2的缺失， 以类似于在Nrf 2缺失小鼠中观察到的方式损害ALI的消退，这表明， 肺上皮Nrf 2信号传导调节促消退反应和肺修复。同意这一 结果，我们发现缺乏Nrf 2的原代AEC 2（Nrf 2-/-AEC 2）由于氧化损伤而增殖不良， 应激和G2/M（而非G1/M）细胞周期阻滞。有趣的是，减轻Nrf 2-/-AEC 2中的氧化应激 外源性N-乙酰半胱氨酸可使G_2/M期阻滞恢复，但不能挽救G_2/M期阻滞。相反，外源GSH 减轻应激，激活AKT信号传导并恢复Nrf 2-/-AEC 2的增殖。初步3D 细胞培养实验表明，从大肠杆菌中分离的AEC 2形成的“肺泡球”尺寸减小， Nrf 2 +/+小鼠暴露于亚致死（48小时）高氧与室内空气对应物相比。AEC 2从 Nrf 2-/-小鼠暴露于室内空气或高氧形成紊乱和减少的数量， “肺泡球”补充有GSH的Nrf 2-/-AEC 2表现出改善的肺泡球形成， 但没有有效的AEC 2/1转分化。在这个项目中，我们测试假设，AEC 2特异性 Nrf 2调节的信号传导对于将平衡向最佳GSH/AKT-1或最佳GSH/AKT-2倾斜是必不可少的。 依赖性AEC 2增殖和GSH/AKT非依赖性AEC 2/1转分化。我们将使用 多种方法来提供该假设的机制检验，包括使用AEC 2-组织- 特异性功能丧失（Nrf 2-/-AEC 2）和功能获得（Nrf 2抑制剂Keap 1-/-AEC 2）小鼠模型， 小分子Nrf 2激活剂。具体目标是：（1）确定机制 Nrf 2调节GSH/AKT介导的信号传导在最佳AEC 2机制中的作用 增殖和AEC 2/1转分化，2）在体内阐明AEC 2特异性Nrf 2作为一种细胞因子的作用， ALI后的促存活和促再生机制，以及3）测试Nrf 2激活 将加速AEC损伤后的修复。高氧治疗是目前广泛应用的肺动脉高压治疗方法 在慢性阻塞性肺疾病（如COPD和ARDS）中， 患者并不清楚。同样地，肺内炎症引起的肺泡上皮细胞的异常修复， 细菌感染是主要的健康问题。因此，拟议的研究具有重大的科学意义， 对重症ALI/ARDS患者的临床意义。

Impaired repair of alveolar epithelium after acute lung injury (ALI) can lead to heightened inflammatory response and tissue repair do not normally resolve. Studies in this application will test the hypothesis that impaired lung repair after injury is the result of GSH/AKT signaling imbalance in type 2 alveolar epithelial cells (AEC2s) regulated by cyto-protection and pro-survival transcription factor, Nrf2. We found that global deletion of Nrf2 impairs lung repair after sub-lethal pro-oxidant (hyperoxia)-induced ALI. Using conditional mutant Nrf2 ("floxed") mouse model to determine the contribution of lung resident cellular stress to acute lung and repair, we found that deletion of Nrf2 in lung epithelium impaired the resolution of ALI in a manner similar to that observed in Nrf2-null mice, suggesting that lung epithelial-Nrf2 signaling regulates pro-resolution response and lung repair. In agreement with this result, we found that primary AEC2s lacking Nrf2 (Nrf2-/-AEC2s) proliferate poorly due to oxidative stress and G2/M (not G1/M) cell cycle arrest. Interestingly, mitigating oxidative stress in Nrf2-/-AEC2s by exogenous N-acetyl-cysteine, but it failed to rescue G2/M arrest. In contrast, exogenous GSH mitigated stress, activated AKT signaling and restored proliferation in Nrf2-/-AEC2s. Preliminary 3D cell culture experiments showed reduced size of "alveolospheres" formation by AEC2s isolated from Nrf2+/+ mice exposed to sub-lethal (48-h) hyperoxia compared to room air counterparts. AEC2s from Nrf2-/- mice exposed to either room air or hyperoxia formed disorganized and reduced number of "alveolospheres". Nrf2-/-AEC2s supplemented with GSH exhibited improved alveolosphere formation, but not efficient AEC2/1 trans-differentiation. In this project, we test hypothesize that AEC2-specific Nrf2 regulated signaling is essential for tipping the equilibrium towards either for optimal GSH/AKT- dependent AEC2 proliferation and GSH/AKT-independent AEC2/1 trans-differentiation. We will use multiple approaches to provide a mechanistic test of this hypothesis including the use of AEC2-tissue- specific loss-of-function (Nrf2-/-AEC2) and gain-of-function (Nrf2 inhibitor Keap1-/-AEC2) mouse models and small molecule Nrf2 activators. The Specific Aims to be pursued are: 1) to determine the mechanisms and role of Nrf2 regulated GSH/AKT-mediated signaling in the mechanisms of optimal AEC2 proliferation and AEC2/1 trans-differentiation, 2) to address in vivo the role of AEC2-specific Nrf2 as a pro-survival and pro-regenerative mechanism after ALI, and 3) to test the postulate that Nrf2 activation will accelerate AEC repair post-injury. Hyperoxia is used widely in the treatment of pulmonary diseases (such as COPD and ARDS), but its effects on the repair lung alveolar epithelium in these patients are not clearly understood. Likewise, abnormal repair of lung alveolar epithelium caused by bacterial infection is a major health concern. Thus, the studies proposed are of major scientific and clinical importance to critically ill ALI/ARDS patients.