Role of Glutathione Redox Status in Hepatotoxicity

描述（由申请人提供）：肿瘤坏死因子（TNF）在肝损伤中起重要作用。正常肝细胞对TNF细胞毒性具有抗性。然而，我们最近发现，选择性耗尽线粒体外GSH敏感TNF诱导的细胞凋亡，这是伴随着持续的c-jun-N-末端激酶（INK）在胞质溶胶中的激活和受损的NF-κ B在核中的反式激活。此外，我们还发现，抑制JNK可以在体外和体内保护对乙酰氨基酚（APAP）诱导的坏死。计划实现以下目标：1.确定GSH耗竭的速率、持续时间和程度对JNK和NF-κ B的双重氧化还原依赖性变化以及对培养肝细胞中TNF诱导的细胞凋亡的敏感性的影响。在这个目标中，我们将定义的时间窗口致敏TNF和GSH/GSSG的关系，比较快速与渐进的GSH消耗，并确定GSH消耗的水平和区室的IkappaB亚型响应TNF的影响。2.在体内外研究JNK的作用及JNK抑制剂对APAP诱导的细胞坏死的保护机制。我们发现，APAP激活JNK和JNK抑制剂保护APAP诱导的体外和体内坏死的GSH消耗，所以我们将使用其他方法来抑制JNK和Bcl 2-家族的影响，检查JNK在APAP毒性中的作用。3.确定APAP或AMAP在体内对TNF致敏的影响。我们将比较用APAP和AMAP（无毒区域异构体）处理后对内源性和外源性TNF的体内致敏性。4.确定GSH耗竭对NF κ B反式激活影响的机制。我们将评估GSH，GSSG和氧化还原调节蛋白在GSH干扰物和TNF反应的核区室化。将采用染色质免疫沉淀（ChIP）试验测定NF-κ B与iNOS基因启动子的结合，并检查NF-κ B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。总的来说，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中的作用的理解。这一发现将广泛适用于药物、病毒、酒精和代谢性肝病

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