Top Institute Food and Nutrition, Wageningen, the Netherlands; Division of Gastroenterology-Hepatology, Department of Internal Medicine, Maastricht University Medical Center, Maastricht, the Netherlands; School for Nutrition, Toxicology and Metabolism of Maastricht University Medical Center, Maastricht, the Netherlands; and
Top Institute Food and Nutrition, Wageningen, the Netherlands; Division of Gastroenterology-Hepatology, Department of Internal Medicine, Maastricht University Medical Center, Maastricht, the Netherlands; School for Nutrition, Toxicology and Metabolism of Maastricht University Medical Center, Maastricht, the Netherlands; and.
Am J Physiol Gastrointest Liver Physiol. 2014 Aug 1;307(3):G286-94. doi: 10.1152/ajpgi.00103.2014. Epub 2014 Jun 5.
There is compelling evidence indicating that ethanol and its oxidative metabolite acetaldehyde can disrupt intestinal barrier function. Apart from the tight junctions, mucins secreted by goblet cells provide an effective barrier. Ethanol has been shown to induce goblet cell injury associated with alterations in mucin glycosylation. However, effects of its most injurious metabolite acetaldehyde remain largely unknown. This study aimed to assess short-term effects of acetaldehyde (0, 25, 50, 75, 100 μM) on functional characteristics of intestinal goblet-like cells (LS174T). Oxidative stress, mitochondrial function, ATP, and intramitochondrial calcium (Ca(2+)) were assessed by dichlorofluorescein, methyltetrazolium, and bioluminescence, MitoTracker green and rhod-2 double-labeling. Membrane integrity and apoptosis were evaluated by measuring lactate dehydrogenase (LDH), caspase 3/7, and cleavage of cytokeratin 18 (CK18). Expression of mucin 2 (MUC2) was determined by cell-based ELISA. Acetaldehyde significantly increased reactive oxygen species generation and decreased mitochondrial function compared with negative controls (P < 0.05). In addition, acetaldehyde dose-dependently decreased ATP levels and induced intramitochondrial Ca(2+) accumulation compared with negative controls (P < 0.05). Furthermore, acetaldehyde induced LDH release and increased caspase3/7 activity and percentage of cells expressing cleaved CK18 and increased MUC2 protein expression compared with negative controls (P < 0.0001). ATP depletion and LDH release could be largely prevented by the antioxidant N-acetylcysteine, suggesting a pivotal role for oxidative stress. Our data demonstrate that acetaldehyde has distinct oxidant-dependent metabolic and cytotoxic effects on LS174T cells that can lead to induction of cellular apoptosis. These effects may contribute to acetaldehyde-induced intestinal barrier dysfunction and subsequently to liver injury.
有确凿的证据表明，乙醇及其氧化代谢物乙醛可破坏肠道屏障功能。除了紧密连接外，杯状细胞分泌的粘蛋白也提供了有效的屏障。已有研究表明，乙醇可诱导与粘蛋白糖基化改变相关的杯状细胞损伤。然而，其最具危害性的代谢物乙醛的影响在很大程度上仍不清楚。本研究旨在评估乙醛（0、25、50、75、100 μM）对肠类杯状细胞（LS174T）功能特性的短期影响。通过二氯荧光素、甲基噻唑基四唑和生物发光、MitoTracker 绿色和 rhod-2 双重标记评估氧化应激、线粒体功能、ATP 和线粒体内钙离子（Ca(2+))。通过测量乳酸脱氢酶（LDH）、半胱天冬酶 3/7 和细胞角蛋白 18（CK18）的裂解来评估细胞膜完整性和细胞凋亡。通过细胞基础 ELISA 测定粘蛋白 2（MUC2）的表达。与阴性对照相比，乙醛显著增加了活性氧的产生，降低了线粒体功能（P<0.05）。此外，与阴性对照相比，乙醛呈剂量依赖性地降低了 ATP 水平并诱导了线粒体内 Ca(2+)的积累（P<0.05）。此外，与阴性对照相比，乙醛诱导了 LDH 的释放，增加了 caspase3/7 的活性和表达裂解 CK18 的细胞比例，并增加了 MUC2 蛋白的表达（P<0.0001）。抗氧化剂 N-乙酰半胱氨酸可显著防止 ATP 耗竭和 LDH 释放，表明氧化应激起着关键作用。我们的数据表明，乙醛对 LS174T 细胞具有独特的依赖氧化剂的代谢和细胞毒性作用，可导致细胞凋亡的诱导。这些影响可能导致乙醛诱导的肠道屏障功能障碍，进而导致肝损伤。
