Role of autophagy on the modulation of CYP2E1 alcohol liver toxicity

描述(由申请人提供)：酒精性肝损伤是一个重大的全球健康问题，也是导致死亡的主要原因。乙醇处理导致细胞死亡的机制尚不清楚。乙醇诱导的细胞色素P450-2E_1是活性氧的产生者，在酒精性肝损伤中起重要作用。自噬是溶酶体介导长寿命蛋白质、细胞器和脂滴移除和再循环的途径。此R21应用的目的是评估在急性和慢性乙醇处理后，自噬是否能够在体外和体内调节依赖于CYP2E1的乙醇毒性。其基本原理是在乙醇诱导的氧化应激、脂肪肝中起作用 以及肝脏损伤。在某些情况下，自噬可以保护细胞免受伤害，而在其他情况下，自噬可以促进细胞毒性。如果自噬对乙醇/CYP2E1毒性具有保护作用，那么刺激自噬的尝试可能被证明有助于降低乙醇诱导的肝损伤。如果自噬促进乙醇/CYP2E1毒性，自噬抑制剂可能有助于减轻乙醇的肝毒性。我们将在无自噬抑制剂或自噬激活剂的情况下，用乙醇(0-100 mM，1-10天)处理表达CYP2E1的HepG2细胞(E47细胞)或不表达该基因的细胞(C34细胞)，并检测下列指标：细胞存活率、细胞凋亡率、氧化应激、细胞色素P450-2的水平和活性、线粒体功能障碍、脂肪变性、丝裂原活化蛋白激酶的激活、肝保护防御、自噬和自噬调节因子，如Bcl-2、AMPK、mTOR。在体内研究中，野生型SV129小鼠、SV129 CYP2E1基因敲除(KO)小鼠和人CYP2E1基因敲除(KI)小鼠将接受乙醇(3g/kg，体重，每天两次，持续1、2和4天)或生理盐水的急性治疗，或给予含乙醇或等卡路里葡萄糖的高脂肪Lieber-DeCarli饮食2至8周。一些小鼠还将接受自噬抑制剂3-甲基腺嘌呤或自噬激活剂雷帕霉素的治疗。在没有和存在自噬修饰剂的情况下，急性和慢性乙醇对上述反应的影响将被评估在没有和存在自噬修饰剂的WT小鼠中具有“正常”水平的小鼠、在没有CYP2E1的KO小鼠中以及在KI小鼠中具有高水平的人CYP2E1的小鼠。时间进程实验旨在帮助确定自噬被抑制或激活时乙醇和CYP2E1之间相互作用的事件序列。时间进程实验也可能提供信息，关于急性或长期乙醇喂养是否可能最初激活自噬作为对乙醇的适应性反应，随后更长时间的急性乙醇处理或长期乙醇喂养不能维持这种自噬。

DESCRIPTION (provided by applicant): Alcohol-induced liver injury is a significant global health problem and a leading cause of death. The mechanisms by which ethanol treatment causes cell death are not clear. CYP2E1 is induced by ethanol, is an active producer of reactive oxygen species and plays a role in ethanol-induced liver injury. Autophagy is a lysosomal-mediated pathway for removal and recycling of long-lived proteins, cellular organelles and lipid droplets. The goal of this R21 application is to evaluate whether autophagy can modulate CYP2E1-dependent ethanol toxicity in vitro and in vivo after acute and chronic ethanol treatment. The rationale is that CYP2E1 plays a role in ethanol-induced oxidant stress, fatty liver and liver injury. Autophagy, in some settings is protective against cell injury, while in other settings autophagy can promote cell toxicity. If autophagy is protective against ethanol/CYP2E1 toxicity, attempts to stimulate autophagy may prove to be helpful in lowering ethanol-induced liver injury. If autophagy promotes ethanol/CYP2E1 toxicity, inhibitors of autophagy may help to ameliorate ethanol hepatotoxicity. We will treat HepG2 cells which express CYP2E1 (E47 cells) or do not (C34 cells) with ethanol (0-100mM, 1-10 days) in the absence and presence of inhibitors of autophagy or activators of autophagy and assay the following: cell viability, apoptosis, oxidant stress, levels and activity of CYP2E1, mitochondrial dysfunction, steatosis, activation of mitogen activated protein kinases, hepatoprotective defense, autophagy and autophagy regulators such as Bcl-2, AMPK, mTOR. For in- vivo studies, wild type SV129 mice, SV129 CYP2E1 knockout (KO) mice, and SV129 CYP2E1 knockin (KI) mice in which human CYP2E1 has been "knocked" in will be treated acutely with ethanol (3g/kg, body wt. twice a day for 1, 2 and 4 days) or saline or be fed the high fat Lieber-DeCarli diet containing ethanol or isocaloric dextrose for 2 to 8 weeks. Some mice will also be treated with the autophagy inhibitor 3-methyladenine or the autophagy activator rapamycin. The effects of acute and chronic ethanol in the absence and presence of modifiers of autophagy in WT mouse with "normal" levels of mouse CYP2E1, in KO mice without CYP2E1 and in KI mice with elevated levels of human CYP2E1 on reactions described above will be evaluated. Time course experiments are designed to help define the sequence of events from the interactions between ethanol and CYP2E1 when autophagy is inhibited or activated. Time course experiments may also be informative as to whether the acute or chronic ethanol feeding may initially activate autophagy as an adaptive response to ethanol, which subsequently is not sustained with more prolonged acute ethanol treatment or chronic ethanol feeding.