Cellular Mechanisms of Hepatotoxicity.

描述（由申请人提供）：对乙酰氨基酚（APAP）毒性是急性肝衰竭的主要原因。APAP肝毒性小鼠模型具有高度可重复性，被广泛用于阐明药物和毒素引起的肝损伤机制。最近，信号转导通路在调节APAP代谢下游毒性中的主要作用已被使用该模型确定。c-jun- n末端激酶（JNK）的激活及其对线粒体的影响已被发现在导致apap诱导的坏死的信号通路中至关重要。本研究的目的是阐明APAP诱导MAPK激酶激活的机制，以及激活JNK靶向线粒体在肝细胞中实施APAP致死作用的意义、靶点和后果。目的如下：(1)确定已确定的P-JNK与特定线粒体靶蛋白结合在APAP肝毒性发病机制中的作用和意义：结合伙伴将通过免疫沉淀和蛋白质组学方法鉴定，线粒体JNK靶点的重要性将通过腺病毒- shrna敲低来确定；将评估纯激活JNK对线粒体功能和完整性的影响：(2)确定导致APAP毒性中JNK早期和持续激活的信号转导途径；各种上游通路如GSK32、Ras/Raf-1、MLK-3、MEKK-1、PKC1、Akt 1+2的相互作用，将使用反义寡核苷酸沉默和/或敲除小鼠进行检测，以阐明随后依赖ASK-1的持续JNK激活所需的初始ASK-1非依赖性JNK激活；(3)确定内质网应激在APAP毒性中的作用：内质网应激对导致线粒体崩溃的信号转导途径的可能贡献将通过敲除或过表达关键内质网应激传感器GRP78来评估；(4)确定RIP激酶在APAP诱导的坏死中的作用及其与GSK/JNK/Sab通路的关系：RIP可能在导致APAP坏死的信号转导通路中发挥关键作用，将通过特异性抑制剂和对RIP-1的反义沉默来评估。这四个目标的阐明将导致对信号转导途径和线粒体在肝毒性中的作用和相互作用的更好理解，并将确定新的治疗靶点。

DESCRIPTION (provided by applicant): Acetaminophen (APAP) toxicity is the leading cause of acute liver failure. The mouse model of APAP hepatotoxicity is highly reproducible and widely utilized to elucidate the mechanisms of liver injury, due to drugs and toxins. Recently, a major role for signal transduction pathways in modulating toxicity downstream of APAP metabolism has been identified using this model. The activation of c-jun-N-terminal kinase (JNK) and its effects on mitochondria have been found to be critical in the signaling pathway leading to APAP-induced necrosis. The goal of this proposal is to elucidate the mechanism of APAP-induced activation of MAPK kinases and the significance, targets, and consequences of the targeting of activated JNK to mitochondria in carrying out the lethal effects of APAP in hepatocytes. The aims are as follows: (1) Determine the role and significance of binding of P-JNK to specific mitochondrial target protein which has been identified in the pathogenesis of APAP hepatotoxicity: Binding partners will be identified by immunoprecipitation and proteomic approaches and the importance of the mitochondrial JNK target will be determined by adenovirus-shRNA knockdown; the effects of pure activated JNK on mitochondrial function and integrity will be assessed: (2) Determine the signal transduction pathways that lead to both early and sustained activation of JNK in APAP toxicity: the interplay of various upstream pathways such as GSK32, Ras/Raf-1, MLK-3, MEKK-1, PKC1, Akt 1+2, will be examined using antisense oligonucleotide silencing and/or knockout mice to elucidate the initial ASK-1 independent activation of JNK required for subsequent ASK-1 dependent sustained JNK activation; (3) Determine the role of ER stress in APAP toxicity: the plausible contribution of ER stress to the signal transduction pathways leading to mitochondrial collapse will be assessed by exploiting knockout or over expression of the key ER stress sensor, GRP78; (4) Determine the role of RIP kinases in APAP-induced necrosis and the relationship to the GSK/JNK/Sab pathway: RIP may play a key role in signal transduction pathways leading to APAP necrosis which will be assessed using specific inhibitors and antisense silencing of RIP-1. The elucidation of these four aims will lead to an improved understanding of the role and interplay of signal transduction pathways and mitochondria in hepatotoxicity and which will identify new therapeutic targets.