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。CYP2E1受乙醇诱导，是活性氧的活性产生者，在乙醇性肝损伤中发挥作用。自噬是溶酶体介导的清除和循环长寿命蛋白质、细胞器和脂滴的途径。本R21应用的目的是评估急性和慢性乙醇治疗后，自噬是否可以调节体外和体内cyp2e1依赖性乙醇毒性。其基本原理是CYP2E1在乙醇诱导的氧化应激和脂肪肝中起作用

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.