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加重 正常情况下，反应和组织修复不会消失。这一应用中的研究将检验这一假说 肺损伤后修复受损是2型肺泡型GSH/AKT信号失衡的结果 上皮细胞(AEC2)受细胞保护和促进生存的转录因子Nrf2调节。我们 发现Nrf2基因的整体缺失损害了亚致死性促氧化剂(高氧血症)诱导的肺修复 阿里。用条件突变型Nrf2小鼠模型确定肺的贡献 驻留细胞应激对急性肺的修复作用，我们发现Nrf2在肺上皮细胞中缺失 以与在Nrf2基因缺失小鼠中观察到的类似的方式损害ALI的分辨率，这表明 肺上皮细胞-Nrf2信号调节促分解反应和肺修复。同意这一点 结果发现，缺乏Nrf2的原代AEC2(Nrf2-/-AEC2)由于氧化作用而增殖不良 应激和G2/M(不是G1/M)细胞周期停滞。有趣的是，减轻NRF2-/-AEC2的氧化应激 外源性N-乙酰半胱氨酸，但不能挽救G2/M期阻滞。相反，外源性GSH 减轻应激，激活AKT信号转导通路，恢复Nrf2-/-AEC2细胞增殖。初步3D 细胞培养实验显示AEC2可减少“肺泡球”的形成 将NRF2+/+小鼠暴露在亚致死(48小时)高氧环境中与室内空气中的对应小鼠进行比较。AEC2来自 NRF2-/-暴露在室内空气或高氧环境中的小鼠形成无序和数量减少的 “肺泡球”。添加GSH的NRF2-/-AEC2促进了肺泡球的形成， 但AEC2/1转分化效率不高。在这个项目中，我们测试了AEC2特定的假设 NRF2调节的信号对于使平衡向任何一个倾斜以获得最佳GSH/AKT-是必不可少的。 依赖AEC2增殖和GSH/AKT非依赖性AEC2/1转分化。我们将使用 为这一假说提供机械检验的多种方法，包括使用AEC2-组织- 特异性功能丧失(Nrf2-/-AEC2)和功能获得(Nrf2抑制剂Keap1-/-AEC2)小鼠模型和 小分子NRF2激活剂。具体目标是：1)确定机制 Nrf2调节GSH/AKT介导的信号转导在AEC2调控机制中的作用 增殖和AEC2/1反式分化，2)在体内解决AEC2特异性Nrf2作为一种 ALI后的促生存和促再生机制，以及3)检验Nrf2激活的假设 将加速AEC损伤后的修复。高氧被广泛应用于肺疾病的治疗 疾病(如COPD和ARDS)，但它对这些患者肺泡上皮修复的影响 对病人的理解不是很清楚。同样，肺泡上皮细胞的异常修复也是由 细菌感染是一个主要的健康问题。因此，拟议的研究具有重大的科学和 危重ALI/ARDS患者的临床重要性。

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.