Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA.
Alcohol Clin Exp Res. 2012 Jan;36(1):14-23. doi: 10.1111/j.1530-0277.2011.01602.x. Epub 2011 Jul 25.
Many alcoholic patients have serum protein deficiency that contributes to their systemic problems. The unfolded protein response (UPR) is induced in response to disequilibrium in the protein folding capability of the endoplasmic reticulum (ER) and is implicated in hepatocyte lipid accumulation and apoptosis, which are associated with alcoholic liver disease (ALD). We investigated whether alcohol affects ER structure, function, and UPR activation in hepatocytes in vitro and in vivo.
HepG2 cells expressing human cytochrome P450 2E1 and mouse alcohol dehydrogenase (VL-17A) were treated for up to 48 hours with 50 and 100 mM ethanol. Zebrafish larvae at 4 days postfertilization were exposed to 350 mM ethanol for 32 hours. ER morphology was visualized by fluorescence in cells and transmission electron microscopy in zebrafish. UPR target gene activation was assessed using quantitative PCR, in situ hybridization, and Western blotting. Mobility of the major ER chaperone, BIP, was monitored in cells by fluorescence recovery after photobleaching (FRAP).
VL-17A cells metabolized alcohol yet only had slight activation of some UPR target genes following ethanol treatment. However, ER fragmentation, crowding, and accumulation of unfolded proteins as detected by immunofluorescence and FRAP demonstrate that alcohol induced some ER dysfunction despite the lack of UPR activation. Zebrafish treated with alcohol, however, showed modest ER dilation, and several UPR targets were significantly induced.
Ethanol metabolism directly impairs ER structure and function in hepatocytes. Zebrafish are a novel in vivo system for studying ALD.
许多酒精性肝病患者存在血清蛋白缺乏，这导致了他们的全身问题。未折叠蛋白反应（UPR）是在内质网（ER）蛋白折叠能力失衡时被诱导的，与肝细胞脂质积累和凋亡有关，而这些正是酒精性肝病（ALD）的特征。我们研究了酒精是否会影响体外和体内肝细胞的 ER 结构、功能和 UPR 激活。
用 50 和 100 mM 乙醇处理表达人细胞色素 P450 2E1 和小鼠醇脱氢酶（VL-17A）的 HepG2 细胞，时间长达 48 小时。用 350 mM 乙醇处理受精后 4 天的斑马鱼幼虫 32 小时。通过细胞内荧光和斑马鱼透射电子显微镜观察 ER 形态。通过定量 PCR、原位杂交和 Western blot 评估 UPR 靶基因的激活。通过荧光恢复后光漂白（FRAP）监测细胞中主要 ER 伴侣 BIP 的迁移。
VL-17A 细胞代谢酒精，但在乙醇处理后仅轻微激活一些 UPR 靶基因。然而，免疫荧光和 FRAP 表明 ER 片段化、拥挤和未折叠蛋白的积累表明，尽管没有 UPR 激活，但酒精还是引起了一些 ER 功能障碍。然而，用酒精处理的斑马鱼仅显示出适度的 ER 扩张，并且几个 UPR 靶基因明显被诱导。
乙醇代谢直接损害肝细胞的 ER 结构和功能。斑马鱼是研究 ALD 的新型体内系统。
