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要的全球性健康问题，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。CYP 2 E1由乙醇诱导，是活性氧的活性产生者，在乙醇诱导的肝损伤中起作用。自噬是溶酶体介导的清除和回收长寿命蛋白质、细胞器和脂滴的途径。这项R21应用的目的是评估自噬是否可以调节急性和慢性乙醇治疗后的CYP 2 E1依赖性乙醇毒性的体外和体内研究。其基本原理是CYP 2 E1在乙醇诱导的氧化应激、脂肪肝 和肝损伤。在某些情况下，自噬可以保护细胞免受损伤，而在其他情况下，自噬可以促进细胞毒性。如果自噬对乙醇/CYP 2 E1毒性具有保护作用，那么刺激自噬可能有助于降低乙醇诱导的肝损伤。如果自噬促进乙醇/CYP 2 E1毒性，则自噬抑制剂可能有助于改善乙醇肝毒性。我们将处理表达CYP 2 E1的HepG 2细胞，（E47细胞）或不（C34细胞）与乙醇（0- 100 mM，1-10天），并测定以下：细胞活力、细胞凋亡、氧化应激、CYP 2 E1的水平和活性、线粒体功能障碍、脂肪变性、促分裂原活化蛋白激酶的活化，肝保护防御、自噬和自噬调节剂如Bcl-2、AMPK、mTOR。对于体内研究，野生型SV 129小鼠、SV 129 CYP 2 E1敲除（KO）小鼠和其中人CYP 2 E1已“敲入”的SV 129 CYP 2 E1敲入（KI）小鼠将用乙醇（3g/kg，体重）急性处理。每天两次，持续1、2和4天）或盐水，或喂食含有乙醇或等热量葡萄糖的高脂肪Lieber-DeCarli饮食2至8周。一些小鼠还将用自噬抑制剂3-甲基腺嘌呤或自噬激活剂雷帕霉素治疗。将评价在不存在和存在自噬调节剂的情况下，急性和慢性乙醇对具有“正常”小鼠CYP 2 E1水平的WT小鼠、不具有CYP 2 E1的KO小鼠和具有升高的人CYP 2 E1水平的KI小鼠的上述反应的影响。时程实验旨在帮助定义自噬被抑制或激活时乙醇和CYP 2 E1之间相互作用的事件顺序。时间过程实验也可以提供关于急性或慢性乙醇喂养是否可以最初激活自噬作为对乙醇的适应性反应的信息，随后不被更长时间的急性乙醇治疗或慢性乙醇喂养所持续。

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.