Lu Yongke, Cederbaum Arthur I
Department of Structural and Chemical Biology, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Department of Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
Biomolecules. 2015 Oct 16;5(4):2659-74. doi: 10.3390/biom5042659.
Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT), CYP2E1 knockout (KO) or CYP2E1 humanized transgenic knockin (KI), mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA), an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These results suggest that autophagy is protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. We speculate that autophagy-dependent processes such as mitophagy and lipophagy help to minimize ethanol-induced CYP2E1-dependent oxidative stress and therefore the subsequent liver injury and steatosis. Attempts to stimulate autophagy may be helpful in lowering ethanol and CYP2E1-dependent liver toxicity.
自噬是一种细胞内途径，通过该途径溶酶体降解并回收长寿命蛋白质和细胞器。乙醇对自噬的影响很复杂，但最近的研究表明，自噬对乙醇诱导的肝损伤具有保护作用。在表达CYP2E1的HepG2细胞体外实验以及急性酒精/CYP2E1依赖性肝损伤模型的体内实验中，发现自噬对CYP2E1依赖性毒性也具有保护作用。本报告的目的是将先前的体外和急性体内实验扩展到慢性乙醇模型，以评估在慢性乙醇喂养的小鼠模型中自噬是否也对CYP2E1依赖性肝损伤具有保护作用。野生型（WT）、CYP2E1基因敲除（KO）或CYP2E1人源化转基因敲入（KI）小鼠被给予乙醇液体饮食或对照葡萄糖饮食四周。在最后一周，一些小鼠接受生理盐水或自噬抑制剂3-甲基腺嘌呤（3-MA）或刺激自噬的雷帕霉素。3-MA抑制自噬增强了WT和KI小鼠中乙醇诱导的血清转氨酶和甘油三酯水平升高，但对KO小鼠没有影响，而雷帕霉素预防了乙醇肝损伤。3-MA处理增强了WT小鼠中乙醇诱导的脂肪积累，并在KI小鼠中导致坏死；在乙醇喂养的KO小鼠或任何葡萄糖喂养的小鼠中几乎没有发现影响。3-MA处理进一步降低了WT和KI小鼠肝脏中乙醇诱导的谷胱甘肽水平降低，并进一步增加了丙二醛的形成，而雷帕霉素减弱了乙醇的这些作用。3-MA和雷帕霉素处理均未影响CYP2E1的催化活性或含量，也未影响乙醇对CYP2E1的诱导。3-MA处理降低了乙醇喂养的WT和KI小鼠中Beclin-1和Atg 7的水平，但增加了p62的水平，而雷帕霉素具有相反的作用，分别验证了自噬的抑制和刺激。这些结果表明，在慢性乙醇喂养的小鼠模型中，自噬对CYP2E1依赖性肝损伤具有保护作用。我们推测，自噬依赖性过程，如线粒体自噬和脂质自噬，有助于将乙醇诱导的CYP2E1依赖性氧化应激降至最低，从而减少随后的肝损伤和脂肪变性。刺激自噬的尝试可能有助于降低乙醇和CYP2E1依赖性肝毒性。
