Glutathione and Lung Fibrosis

描述(申请人提供)：肺纤维化是许多肺部疾病的特征和终末期，没有有效的治疗方法。谷胱甘肽(GSH)是细胞内含量最丰富的游离硫醇，也是一种重要的抗氧化剂，在实验性纤维化和人类纤维化疾病中，其浓度都会降低。重要的是，雾化或口服GSH或GSH前体N-乙酰半胱氨酸(NAC)可减轻实验性纤维化模型中的肺纤维化，并延缓肺纤维化疾病患者的肺功能恶化，提示GSH/NAC对肺纤维化疾病具有潜在的治疗价值。然而，GSH/NAC的疗效和作用机制仍不明确。具体地说，目前尚不清楚GSH/NAC是否通过抑制早期炎症反应发挥其治疗效果，或者它是否具有直接的抗纤维化活性，从而也可以阻止疾病后期的纤维化进展。转化生长因子-β是一种最有效、最普遍的促纤维化细胞因子。我们以前的研究表明，在成纤维细胞和肺纤维化模型中，GSH-1降低了GSH并增加了ROS的产生，而GSH/NAC的补充通过抑制纤溶酶原激活物抑制物1(PAI-1)的表达而抑制了由TGF-1刺激的胶原堆积，从而刺激了胶原的降解。在四环素诱导的肺特异性谷氨酸半胱氨酸连接酶(GCL)转基因小鼠中，增加肺ELF中的GSH也能抑制转化生长因子-1诱导的PAI-1的表达和肺纤维化。初步研究进一步表明，转化生长因子-1可诱导蛋白硫醇修饰，抑制JNK-1导向的磷酸酶活性，从而刺激JNK/p38的磷酸化；而谷胱甘肽抑制转化生长因子-1的激活，从而诱导PAI-1的表达。因此，我们推测GSH/NAC可以通过抑制转化生长因子-S的纤维化信号和刺激胶原降解来阻止纤维化的进展。将细胞和动物模型与药理学和遗传学方法相结合，以阐明GSH/NAC拮抗转化生长因子诱导的肺纤维化的分子机制。目的1确定GSH/NAC能否阻止GCL转基因小鼠或NAC处理的野生型小鼠由结构性活性的转化生长因子-1诱导的晚期肺纤维化的进展。目的研究GSH/NAC是否刺激转化生长因子基因敲除的野生型和PAI-1基因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被用来进一步检验我们的假设。目的3利用小鼠和原代人肺成纤维细胞，重点研究GSH/NAC拮抗转化生长因子-β纤维形成的分子机制。系统研究GSH/NAC对转化生长因子诱导的硫醇修饰和JNK/p38直接磷酸酶活性的影响，以及抑制磷酸酶活性与JNK/p38活化/PAI-1诱导的关系。这些研究的结果将为研究转化生长因子-S纤维形成和谷胱甘肽/N-乙酰半胱氨酸抗纤维化作用的分子机制提供新的见解，这将使设计更有效的预防和治疗这些纤维增生性疾病的策略成为可能。项目简介：肺纤维化是许多肺部疾病的特征和终末期，没有有效的治疗方法。该项目将测试一个新的假说，即细胞内最丰富的游离硫醇和重要的抗氧化剂谷胱甘肽和谷胱甘肽的前体N-乙酰半胱氨酸(NAC)可以通过减弱转化生长因子-(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降解来阻止纤维化的进展。这些研究的结果将为研究转化生长因子-S纤维形成和谷胱甘肽/N-乙酰半胱氨酸抗纤维化作用的分子机制提供新的见解，这将使设计更有效的预防和治疗这些纤维增生性疾病的策略成为可能。

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.