Autophagy and Drug-Induced Liver Injury

描述（由申请人提供）：对乙酰氨基酚（APAP）诱导的肝损伤是美国和许多其他国家急性肝衰竭的最常见原因。APAP通过一系列事件产生肝细胞死亡，这些事件需要早期形成反应性代谢物N-乙酰基-对苯醌亚胺（NAPQI），随后形成细胞和线粒体蛋白质的加合物，最后这些加合物触发线粒体功能障碍和坏死细胞死亡。在早期阶段给予N-乙酰半胱氨酸（NAC）可以有效地增加细胞内谷胱甘肽水平和NAPQI，并防止肝损伤。然而，患者通常仅在该阶段结束后才出现，并且没有可用的治疗方法来预防随后发病阶段的肝损伤。我们最近的工作表明，细胞在损伤后期的存活依赖于自噬途径，这可能是因为自噬作用去除了受损的线粒体。我们的初步数据表明，p62/SQSTM 1（以下简称为p62）被招募到APAP-AD和受损的线粒体，并诱导其通过自噬选择性去除。我们发表的数据还表明，肝脏特异性Atg 5基因敲除小鼠对APAP诱导的肝损伤具有抗性，即使它们具有缺陷的自噬。在这种情况下，抗性是由p62的积累引起的，p62的积累导致抗氧化剂转录因子Nrf 2的持续激活。这些数据表明，p62的自噬和非自噬作用都参与了对APAP诱导的肝损伤的保护。该提议的中心假设是，选择性p62介导的APAP-AD和受损线粒体的自噬去除以及Nrf 2的非自噬激活对于防止APAP诱导的肝损伤的后期至关重要。为了检验我们的假设，提出了三个具体目标：1）确定p62在体内以及在原代小鼠和人肝细胞中自噬去除APAP-AD和受损线粒体中的中心作用，2）确定p62在保护免受APAP诱导的肝损伤中的自噬和非自噬作用，和3）在APAP治疗后小鼠模型中确定Nrf 2的p62介导的自噬和非自噬激活的诱导可以促进APAP诱导的肝损伤的恢复。这项研究的结果将导致对p62介导的自噬和非自噬机制在APAP诱导的发病机制的晚期阶段保护细胞免受损伤的深入理解，该晚期阶段没有可用的治疗方法，NAC治疗不再有效。最终，这些知识具有确定用于治疗晚期APAP诱导的肝损伤和肝衰竭的新治疗方法的潜力。

DESCRIPTION (provided by applicant): Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the US and many other countries. APAP produces liver cell death by a sequence of events that requires the early formation of a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), a subsequent phase where adducts of cellular and mitochondrial proteins are formed, and a final phase where these adducts trigger mitochondrial dysfunction and necrotic cell death. Administration of N-acetyl cysteine (NAC) at the early phase can efficiently increase intracellular glutathione levels and scavenge NAPQI and protect against liver injury. However, patients frequently present only after this phase is over and there are no treatments available to prevent liver injury in the subsequent phases of pathogenesis. Our recent work has shown that cell survival in the later injury phases is dependent on the autophagy pathway, presumably because of the autophagic removal of damaged mitochondria. Our preliminary data show that p62/SQSTM1 (hereafter referred to as p62) is recruited both to APAP-AD and damaged mitochondria and it induces their selective removal by autophagy. Our published data also show that liver-specific Atg5 knockout mice are resistant to APAP-induced liver injury even though they have defective autophagy. In this case, resistance results from the accumulation of p62 which results in persistent activation of the antioxidant transcription factor Nrf2. These data suggest that both autophagic and non-autophagic roles of p62 are involved in protecting against APAP-induced liver injury. The central hypothesis of this proposal is that selective p62-mediated autophagic removal of APAP-AD and damaged mitochondria and non-autophagic activation of Nrf2 are crucial to protect against the later phase of APAP-induced liver injury. To examine our hypothesis, three specific aims are proposed: 1) determine the central role of p62 in autophagic removal of APAP-AD and damaged mitochondria in vivo and in primary mouse and human hepatocytes, 2) determine the autophagic and non-autophagic role of p62 in protection against APAP-induced liver injury, and 3) determine the induction of p62-mediated autophagy and non- autophagic activation of Nrf2 can promote the recovery from APAP-induced liver injury in a post-APAP treatment mouse model. Results from this study will lead to the in-depth understanding of p62-mediated autophagy and non-autophagic mechanisms in protecting against cell injury in the late phase of APAP-induced pathogenesis, which no treatment is available and NAC treatment is no longer effective. Ultimately, such knowledge has the potential of identifying novel therapeutic approaches for treating the late phase of APAP- induced liver injury and liver failure.