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: 6967084
• 负责人: NEIL KAPLOWITZ
• 金额: $ 32.91万
• 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/6967084
• 关键词: BCL2 gene /protein; JUN kinase; SDS polyacrylamide gel electrophoresis; acetaminophen; apoptosis; autoradiography; chromatin immunoprecipitation; drug metabolism; enzyme activity; enzyme inhibitors; glutathione; hepatotoxin; laboratory mouse; nuclear factor kappa beta; oxidation reduction reaction; phosphorylation; posttranslational modifications; protein isoforms; protein structure function; thiols; tissue /cell culture; tumor necrosis factor alpha; western blottings

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（TNF）在肝损伤中发挥重要作用。正常肝细胞对 TNF 细胞毒性具有抵抗力。然而，我们最近发现，线粒体外 GSH 的选择性消耗会对 TNF 诱导的细胞凋亡敏感，同时伴随着胞浆中 c-jun-N 末端激酶 (INK) 的持续激活和细胞核中 NF-kB 反式激活的受损。此外，我们发现抑制 JNK 可以防止对乙酰氨基酚 (APAP) 诱导的体外和体内坏死。计划实现以下目标： 1. 确定 GSH 消耗的速率、持续时间和程度对 JNK 和 NF-κB 双重氧化还原依赖性变化以及培养肝细胞中 TNF 诱导的细胞凋亡的敏感性的影响。在此目的中，我们将定义对 TNF 敏感的时间窗口以及与 GSH/GSSG 的关系，比较快速与渐进的 GSH 消耗，并确定 GSH 消耗对响应 TNF 的 IkappaB 同工型水平和区划的影响。 2. 确定JNK的作用以及JNK抑制剂对体内外APAP诱导的坏死的保护机制。我们表明，APAP 消耗的 GSH 会激活 JNK，而 JNK 抑制剂可防止 APAP 诱导的体外和体内坏死，因此我们将使用其他抑制 JNK 的方法来研究 JNK 在 APAP 毒性中的作用以及对 Bcl2 家族的影响。 3.确定体内APAP或AMAP对TNF致敏的影响。我们将比较用 APAP 和 AMAP（无毒区域异构体）治疗后体内对内源性和外源性 TNF 的敏感性。 4. 确定GSH消耗对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 扰动物和 TNF 的反应的核区室化。将采用染色质免疫沉淀 (ChIP) 测定来确定 NF-kappaB 与 iNOS 基因启动子的结合，并检查 NF-kappaB 的氧化还原依赖性翻译后修饰，包括二硫键形成、谷胱甘肽化、磷酸化和乙酰化。总体而言，这项研究将加深我们对 GSH 和氧化还原修饰在使肝脏对损伤敏感中的作用的理解。研究结果将广泛适用于药物、病毒、酒精和代谢性肝病