Glutathione and Lung Fibrosis

描述（由申请人提供）：肺纤维化是许多肺部疾病的特征性特征和终末期，目前尚无有效治疗方法。谷胱甘肽（GSH）是细胞内最丰富的游离硫醇，也是一种重要的抗氧化剂，其浓度在实验性纤维化和人类纤维化疾病中均下降。重要的是，雾化或口服GSH或n -乙酰半胱氨酸（NAC） （GSH的前体）可减轻实验性纤维化模型中的肺纤维化，减缓肺纤维化疾病患者肺功能的恶化，表明GSH/NAC对肺纤维化疾病具有潜在的治疗价值。尽管如此，GSH/NAC的治疗效果和机制仍然不明确。具体而言，尚不清楚GSH/NAC是否通过抑制早期炎症反应发挥其治疗作用，或者是否具有直接的抗纤维化活性，因此也可以阻断疾病晚期纤维化的进展。TGF-是一种最有效和普遍存在的促纤维化细胞因子。我们之前的研究表明，在成纤维细胞和肺纤维化模型中，TGF-(降低GSH并增加活性氧（ROS）的产生，而GSH/NAC补充通过阻断蛋白酶抑制剂纤溶酶原激活物抑制剂1 （PAI-1）的表达，从而刺激胶原降解，抑制TGF-（）刺激的胶原积累。四环素诱导的肺特异性谷氨酸半胱氨酸连接酶（GCL）转基因小鼠肺ELF中GSH升高也抑制TGF-(-)诱导的PAI-1表达和肺纤维化。初步研究进一步表明，TGF-(诱导蛋白巯基修饰，抑制JNK导向的磷酸酶活性，与JNK/p38磷酸化刺激有关，而GSH阻断TGF-(激活JNK和p38 MAPKs，介导TGF-（诱导PAI-1表达）。因此，我们假设GSH/NAC可以通过减弱TGF-的纤维化信号和刺激胶原降解来阻断纤维化进程。我们提出了三个特定的目标，将细胞和动物模型与药理学和遗传学方法结合起来，阐明GSH/NAC拮抗TGF-诱导的肺纤维化的分子机制。目的1将确定GSH/NAC是否可以阻断GCL转基因小鼠或NAC处理的野生型小鼠由组成型活性TGF-诱导的晚期肺纤维化的进展。Aim 2将确定GSH/NAC是否刺激TGF-处理野生型和PAI-1敲除小鼠的胶原降解以及纤溶酶和胶原酶的表达/活性。博莱霉素诱导的肺纤维化模型将用于进一步验证我们的假设。Aim 3将关注GSH/NAC在小鼠和人原代肺成纤维细胞中拮抗TGF-（）成纤维的分子机制。我们将系统研究GSH/NAC对TGF-诱导的硫醇修饰和JNK/p38定向磷酸酶活性的影响，以及磷酸酶的抑制与JNK/p38激活/PAI-1诱导的关系。这些研究结果将为TGF-（）纤维生成和GSH/NAC抗纤维化作用的分子机制提供新的见解，有助于设计更有效的预防和治疗这些纤维增生性疾病的策略。项目描述：肺纤维化是许多肺部疾病的特征性特征和终末期，没有有效的治疗方法。该项目将测试一个新的假设，即谷胱甘肽（细胞内最丰富的游离硫醇和重要的抗氧化剂）和n -乙酰半胱氨酸（NAC）（谷胱甘肽的前体）可以通过减弱TGF-（一种最有效和普遍存在的促纤维化细胞因子）的纤维化信号和刺激胶原蛋白降解来阻止纤维化进程。这些研究结果将为TGF-（）纤维生成和GSH/NAC抗纤维化作用的分子机制提供新的见解，有助于设计更有效的预防和治疗这些纤维增生性疾病的策略。

DESCRIPTION (provided by applicant): Lung fibrosis is a characteristic feature and terminal stage of many lung diseases with no efficacious treatment. The concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, is decreased in both experimental fibrosis and human fibrotic diseases. Importantly, aerosol or oral administration of GSH or N-acetylcysteine (NAC), a precursor of GSH, attenuates lung fibrosis in experimental fibrosis models and slows the deterioration of lung functions in patients with lung fibrotic diseases, indicating a potential therapeutic value of GSH/NAC for pulmonary fibrotic diseases. Nonetheless, the efficacy and mechanism underlying the therapeutic effects of GSH/NAC remains equivocal. Specifically, it is unclear whether GSH/NAC exerts its therapeutic effects by suppressing early-stage inflammatory response or whether it has direct antifibrotic activity and therefore can also block the progression of fibrosis in later stages of the disease. TGF-( is a most potent and ubiquitous profibrogenic cytokine. Our previous studies showed that TGF-( decreased GSH and increased reactive oxygen species (ROS) production in fibroblasts and in a lung fibrosis model while GSH/NAC supplementation inhibited TGF-(-stimulated collagen accumulation by blocking the expression of plasminogen activator inhibitor 1 (PAI-1), a protease inhibitor, and thus stimulating collagen degradation. Increasing GSH in lung ELF in tetracycline inducible lung specific glutamate cysteine ligase (GCL) transgenic mice also inhibits TGF-(-induced PAI-1 expression and lung fibrosis. Preliminary studies further show that TGF-( induces protein thiol modifications and inhibits the activity of JNK-directed phosphatases, associated with a stimulation of JNK/p38 phosphorylation while GSH blocks TGF-( activation of JNK and p38 MAPKs, which mediate TGF-( induction of PAI-1 expression. Therefore, we hypothesize that GSH/NAC can block fibrosis progression by attenuating TGF-('s fibrotic signaling and stimulating collagen degradation. Three specific aims are proposed that couple cell and animal models with pharmacologic and genetic approaches to elucidate the molecular mechanisms whereby GSH/NAC antagonizes TGF-(-induced lung fibrosis. Aim 1 will determine whether GSH/NAC can block the progression of late stage lung fibrosis induced by constitutively active TGF-( in GCL transgenic mice or NAC treated wild type mice. Aim 2 will determine whether GSH/NAC stimulates collagen degradation as well as the expression/activities of plasmin and the collagenases in TGF-( treated wild type and PAI-1 knockout mice. The bleomycin-induced lung fibrosis model will be used to further test our hypothesis. Aim 3 will focus on the molecular mechanisms whereby GSH/NAC antagonizes TGF-( fibrogenesis using mouse and primary human lung fibroblasts. Effect of GSH/NAC on TGF-(-induced thiol modifications and the activity of JNK/p38 directed phosphatases as well as the relationship between inhibition of the phosphatases and JNK/p38 activation/PAI-1 induction will be studied systematically. The results from these studies will provide new insight into the molecular mechanisms underlying TGF-('s fibrogenesis and GSH/NAC antifibrogenic effects, which will enable the design of more efficacious strategies for prevention and treatment of these fibroproliferative disorders. PROJECT NARRATIVE: Lung fibrosis is a characteristic feature and terminal stage of many lung diseases with no efficacious treatment. This project will test a novel hypothesis that glutathione, the most abundant intracellular free thiol and an important antioxidant, and N-acetyl cysteine (NAC), a precursor of GSH, can block fibrosis progression by attenuating the fibrogenic signals of TGF-(, a most potent and ubiquitous profibrogenic cytokine, and stimulating collagen degradation. The results from these studies will provide new insight into the molecular mechanisms underlying TGF-('s fibrogenesis and GSH/NAC antifibrogenic effects, which will enable the design of more efficacious strategies for prevention and treatment of these fibroproliferative disorders.