Glutathione transport, oxidative stress and lung injury

描述（由申请人提供）：本申请的总体目标是了解谷胱甘肽（GSH）转运在氧化性肺损伤亚细胞机制中的作用。CF是一种遗传性疾病，可导致持续的肺部炎症和慢性感染，与进行性肺损伤有关。令人兴奋的初步研究表明，囊性纤维化跨膜传导调节剂（CFTR）的遗传缺陷通过减少GSH向肺上皮衬里液（ELF）的转运并可能改变GSH向线粒体的转运而产生抗氧化失衡。CFTR基因对GSH转运的调节可能使CF患者易受外源性和内源性氧化应激的影响，从而导致CF肺部表型。虽然已有证据支持氧化应激在CF患者肺中的作用，但CFTR基因缺陷与氧化应激的病因和致病关系尚未确定。拟议的实验将描述CF相关肺损伤中GSH转运改变与氧化应激之间的关系。CFTR KO小鼠提供了一种独特的方法来研究GSH转运缺陷导致肺抗氧化失衡和氧化应激反应的病理生理机制。CFTR KO反映了CF患者的肺GSH失衡和氧化应激。假设肺GSH转运和代谢的改变导致肺氧化损伤的加重和宿主防御的改变。该假设的目的是：(1)表征CFTR KO患者肺部GSH转运、代谢、利用和相关氧化应激的改变；(2)确定GSH转运体的调节是否可以纠正CFTR KO中GSH失衡、氧化应激和宿主防御反应；(3)确定GSH转运改变的CFTR KO小鼠是否对急性肺损伤更敏感。为了达到上述目的，我们测定了CFTR KO和野生型小鼠肺中GSH、GSSG和GSNO的稳态水平及相关酶活性。此外，对蛋白质、脂质和DNA的氧化损伤标志物进行了定量分析。谷胱甘肽通过ABC盒蛋白的运输将被表征、调节，并与氧化应激和宿主防御的变化相关。利用感染和氧化应激的肺损伤模型来评估GSH转运改变在肺损伤反应中的作用。催化抗氧化金属卟啉和吸入GSH被用于减少CF中氧化负担和纠正夸大的肺氧化损伤反应。这些研究可能强调氧化性空气污染物对已有肺部疾病的敏感人群的氧化应激的潜在不利影响，因为大量肺部疾病（包括COPD、ARDS、哮喘和肺纤维化）也存在肺ELF GSH缺陷

DESCRIPTION (provided by applicant): The overall goal of this application is to understand the role of glutathione (GSH) transport in the subcellular mechanisms of oxidative lung damage. (CF) is a genetic disorder that results in persistent lung inflammation and chronic infection that is implicated in progressive lung injury. Exciting preliminary studies indicate that the genetic defect in the cystic fibrosis transmembrane conductance regulator (CFTR) produces an antioxidant imbalance by decreasing GSH transport into the pulmonary epithelial lining fluid (ELF) and possibly altering GSH transport into the mitochondria. Modulation of GSH transport by the CFTR gene may render the CF patient vulnerable both exogenous and endogenous oxidative stress and thus contribute to the CF pulmonary phenotype. Although evidence exists supporting the role of oxidative stress in the lungs of CF patients, the etiologic and pathogenic relationship between the CFTR gene deficits to oxidative stress has yet to be established. Proposed experiments will delineate the relationship between altered GSH transport and oxidative stress in pulmonary injury associated with CF. The CFTR KO mouse provides a unique way to study pathophysiological mechanisms by which defective GSH transport contributes to antioxidant imbalance and oxidative stress responses in the lung. The CFTR KO recapitulates pulmonary GSH imbalance and oxidative stress of CF patients. It is hypothesized that altered lung GSH transport and metabolism contributes to exaggerated pulmonary oxidative injury and altered host defense. The hypothesis is addressed by the AIMS: (1) To characterize the altered GSH transport, metabolism, utilization, and associated oxidative stress in the lungs of the CFTR KO; (2) Determine whether modulation of GSH transporters can correct the GSH imbalance, oxidative stress, and host defense responses in the CFTR KO; (3) Determine if CFTR KO mice with altered GSH transport are more sensitive to acute lung injury. To accomplish the above aims, the steady-state levels of GSH, GSSG, and GSNO and associated enzyme activities are determined in the lungs of CFTR KO and wild type mice. In addition, markers of oxidative damage to protein, lipid, and DNA are quantitated. GSH transport through ABC cassette proteins will be characterized, modulated, and correlated with changes in oxidative stress and host defense. Lung injury models of infection and oxidative stress are utilized to assess the role of altered GSH transport in lung injury responses. Catalytic antioxidant metalloporphyrins and inhaled GSH are employed to reduce oxidant burden and correct the exaggerated pulmonary oxidative injury response in CF. These studies may emphasize the potential adverse effects of oxidative stress from oxidant air pollutants in sensitive populations with pre-existing lung disease, since a large number of pulmonary diseases (including COPD, ARDS, asthma and pulmonary fibrosis) also have deficits pulmonary ELF GSH