Rush University Medical Center, Chicago, Illinois.
Division of Digestive Disease and Nutrition, Department of Internal Medicine, Section of Gastroenterology, Rush University Medical Center, Chicago, Illinois.
Alcohol Clin Exp Res. 2017 Dec;41(12):2007-2014. doi: 10.1111/acer.13513. Epub 2017 Oct 30.
Alcoholic liver disease (ALD) is commonly associated with intestinal permeability. An unanswered question is why only a subset of heavy alcohol drinkers develop endotoxemia. Recent studies suggest that circadian disruption is the susceptibility factor for alcohol-induced gut leakiness to endotoxins. The circadian protein PER2 is increased after exposure to alcohol and siRNA knockdown of PER2 in vitro blocks alcohol-induced intestinal barrier dysfunction. We have shown that blocking CYP2E1 (i.e., important for alcohol metabolism) with siRNA inhibits the alcohol-induced increase in PER2 and suggesting that oxidative stress may mediate alcohol-induced increase in PER2 in intestinal epithelial cells. The aim of this study was to elucidate whether a mechanism incited by alcohol-derived oxidative stress mediates the transcriptional induction of PER2 and subsequent intestinal hyperpermeability.
Caco-2 cells were exposed to 0.2% alcohol with or without pretreatment with modulators of oxidative stress or PKA activity. Permeability of the Caco-2 monolayer was assessed by transepithelial electrical resistance. Protein expression was measured by Western blot and mRNA with real-time polymerase chain reaction. Wild-type C57BL/6J mice were fed with alcohol diet (29% of total calories, 4.5% v/v) for 8 weeks. Western blot was used to analyze PER2 expression in mouse proximal colon tissue.
Alcohol increased oxidative stress, caused Caco-2 cell monolayer dysfunction, and increased levels of the circadian clock proteins PER2 and CLOCK. These effects were mitigated by pretreatment of Caco-2 cells with an antioxidant scavenger. Alcohol-derived oxidative stress activated cAMP response element-binding (CREB) via the PKA pathway and increased PER2 mRNA and protein. Inhibiting CREB prevented the increase in PER2 and Caco-2 cell monolayer hyperpermeability.
Taken together, these data suggest that strategies to reduce alcohol-induced oxidative stress may alleviate alcohol-mediated circadian disruption and intestinal leakiness, critical drivers of ALD.
酒精性肝病(ALD)通常与肠道通透性有关。一个尚未解决的问题是,为什么只有一部分大量饮酒者会发生内毒素血症。最近的研究表明,昼夜节律紊乱是酒精引起肠道通透性增加导致内毒素易位的易感因素。暴露于酒精后,昼夜节律蛋白 PER2 增加,体外 siRNA 敲低 PER2 可阻断酒精诱导的肠道屏障功能障碍。我们已经表明,用 siRNA 阻断 CYP2E1(即酒精代谢的重要酶)可抑制酒精诱导的 PER2 增加,并表明氧化应激可能介导酒精诱导的肠上皮细胞中 PER2 的增加。本研究旨在阐明由酒精衍生的氧化应激引发的机制是否介导 PER2 的转录诱导和随后的肠道高通透性。
将 Caco-2 细胞暴露于 0.2%酒精中,或用氧化应激或 PKA 活性调节剂预处理。通过跨上皮电阻评估 Caco-2 单层的通透性。通过 Western blot 和实时聚合酶链反应测量蛋白质表达和 mRNA。用含 29%总热量(4.5% v/v)酒精的饮食喂养野生型 C57BL/6J 小鼠 8 周。用 Western blot 分析小鼠近端结肠组织中 PER2 的表达。
酒精增加了氧化应激,导致 Caco-2 细胞单层功能障碍,并增加了昼夜节律钟蛋白 PER2 和 CLOCK 的水平。用抗氧化剂清除剂预处理 Caco-2 细胞可减轻这些作用。酒精衍生的氧化应激通过 PKA 途径激活 cAMP 反应元件结合(CREB),并增加 PER2 mRNA 和蛋白。抑制 CREB 可防止 PER2 增加和 Caco-2 细胞单层通透性增加。
综上所述,这些数据表明,减少酒精诱导的氧化应激的策略可能减轻酒精介导的昼夜节律紊乱和肠道通透性增加,这是 ALD 的关键驱动因素。