Role of Glutathione Redox Status in Hepatotoxicity

谷胱甘肽氧化还原状态在肝毒性中的作用

• 批准号: 7274120
• 负责人: NEIL KAPLOWITZ
• 金额: $ 31.3万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-15 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7274120
• 关键词: Role; Glutathione; Redox; Status; Hepatotoxicity

DESCRIPTION (provided by applicant): Tumor necrosis factor (TNF) plays an important role in liver injury. Normal hepatocytes are resistant to TNF cytotoxicity. However, we have recently found that selective depletion of extramitochondrial GSH sensitizes to TNF induced apoptosis which is accompanied by sustained c-jun-N-terminal kinase (INK) activation in cytosol and impaired NF-kB transactivation in the nucleus. In addition, we have found that inhibition of JNK protects against acetaminophen (APAP)-induced necrosis in vitro and in vivo. The following aims are planned: 1. To determine the influence of rate, duration, and extent of GSH depletion on dual redox-dependent changes in JNK and NF-kappaB and sensitization to TNF-induced apoptosis in cultured hepatocytes. In this aim we will define the time window of sensitization to TNF and the relation to GSH/GSSG, compare rapid versus gradual GSH depletion, and determine the effect of GSH depletion on the levels and compartmentation of IkappaB isoforms in response to TNF. 2. To determine the role of JNK and the mechanism for protection by JNK inhibitor against APAP-induced necrosis in vitro and in vivo. We showed that GSH depletion by APAP activates JNK and JNK inhibitor protects against APAP-induced in vitro and in vivo necrosis, so we will examine the role of JNK in APAP toxicity using other approaches to inhibiting JNK and the effects on the Bcl2-family. 3. To determine the effect of APAP or AMAP in vivo on sensitization to TNF. We will compare sensitization to endogenous and exogenous TNF in vivo after treatment with APAP and AMAP (nontoxic regioisomer). 4. To determine the mechanism of the effect of GSH depletion on NFkappaB transactivation. We will assess nuclear compartmentalization of GSH, GSSG, and redox regulatory proteins in response to GSH perturbants and TNF. The chromatin immunoprecipitation (ChIP) assay will be employed to determine NF-kappaB binding to the iNOS gene promoter and redox-dependent, post-translational modifications of NF-kappaB, including disulfide formation, glutathionylation, phosphorylation, and acetylation, will be examined. Overall, this research will increase our understanding of the role of GSH and redox modifications in sensitizing the liver to injury. The findings will be broadly applicable to drug, viral, alcohol and metabolic liver diseases

描述(申请人提供)：肿瘤坏死因子在肝脏损伤中起重要作用。正常肝细胞对肿瘤坏死因子的细胞毒作用具有抵抗力。然而，我们最近发现，线粒体外GSH的选择性耗竭增加了对肿瘤坏死因子诱导的细胞凋亡的敏感性，同时伴随着胞浆中c-jun-N末端激酶(INK)的持续激活和核内核因子-kB的反式激活。此外，我们在体外和体内都发现，抑制JNK对对乙酰氨基酚(APAP)诱导的坏死具有保护作用。1.确定GSH耗竭的速度、持续时间和程度对培养肝细胞JNK和NF-kappaB双重氧化还原依赖性变化的影响以及对肿瘤坏死因子诱导的肝细胞凋亡的增敏作用。为此，我们将定义对肿瘤坏死因子增敏的时间窗口及其与GSH/GSSG的关系，比较快速和渐进性GSH耗竭，并确定GSH耗竭对肿瘤坏死因子对IkappaB亚型水平和区划的影响。2.研究JNK在体内外对APAP诱导的大鼠肝细胞坏死的保护作用及JNK抑制剂的保护作用机制。我们发现APAP耗尽GSH激活了JNK，而JNK抑制剂在体外和体内都能保护APAP诱导的坏死，所以我们将用其他方法抑制JNK以及对Bcl2家族的影响来研究JNK在APAP毒性中的作用。3.测定APAP或AMAP体内对肿瘤坏死因子的增敏作用。我们将比较APAP和AMAP(无毒区域异构体)治疗后对体内内源性和外源性肿瘤坏死因子的增敏作用。4.探讨GSH耗竭对NFkappaB反式激活的影响机制。我们将评估GSH、GSSG和氧化还原调节蛋白对GSH扰动和肿瘤坏死因子的反应。染色质免疫沉淀法(ChIP)将被用来确定NF-kappaB与iNOS基因启动子的结合，并将检测依赖于氧化还原的、翻译后的NF-kappaB的修饰，包括二硫键形成、谷胱甘肽基化、磷酸化和乙酰化。总体而言，这项研究将增加我们对GSH和氧化还原修饰在使肝脏对损伤敏感的作用的理解。这些发现将广泛适用于药物、病毒、酒精和代谢性肝病。