Hirokawa M, Miura S, Yoshida H, Kurose I, Shigematsu T, Hokari R, Higuchi H, Watanabe N, Yokoyama Y, Kimura H, Kato S, Ishii H
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Alcohol Clin Exp Res. 1998 May;22(S3 Pt 1):111S-114S. doi: 10.1111/acer.1998.22.s3_part1.111s.
Although it has been speculated that active oxidants and mitochondrial membrane damages play roles in ethanol-induced gastric mucosal damages, its detail remains unknown. The present study was designed to investigate whether ethanol induces oxidative stress and mitochondrial permeability transition (MPT) before cell death of gastric mucosal cells. Rat gastric mucosal cells (RGM-1) were kept in serum-free Dulbecco's modified Eagle's medium before addition of various concentrations of ethanol. Nuclear morphological aftemations and membrane barrier dysfunction of RGM-1 cells were assessed by staining with Hoechst 33342 and propidium iodide, respectively. To assess the contribution of oxygen-derived free radicals and intracellular glutathione, scavenger of hydrogen peroxide and the hydroxyl radical, N,N-dimethylthiourea, glutathione precursor, N-acetyl-L-cysteine, and an inhibitor of alcohol dehydrogenase, 4-methylpyrazole were added before treatment with ethanol. To investigate MPT, calcein and tetramethylrhodamine methyl ester were loaded before addition of ethanol, and the changes of fluorescence intensity were monitored using a laser scanning confocal microscope. Ethanol (>5% v/v) dose-dependently increased the number of propidium iodide-positive cells, suggesting a diminished barrier function of cell membrane. After addition of ethanol, mitochondria were filled quickly with calcein indicating MPT, which was accompanied by mitochondrial depolarization, as shown by loss of tetramethylrodamine methyl ester before cell death. Ethanol-induced cell death was significantly attenuated by simultaneous incubation with either N,N-dimethylthiourea or N-acetyl-L-cysteine, suggesting the importance of intracellular redox states in inducing cellular damage, whereas such change was not attenuated by 4-methylpyrazole. Present results suggest that ethanol treatment induces intracellular oxidative stress and produces MPT and mitochondrial depolarization, which are preceding cell death in gastric mucosal cells. Intracellular antioxidants, such as glutathione, may have a significant protective action against ethanol in gastric mucosal cells.
尽管有人推测活性氧化剂和线粒体膜损伤在乙醇诱导的胃黏膜损伤中起作用，但其具体细节仍不清楚。本研究旨在调查乙醇在胃黏膜细胞死亡之前是否会诱导氧化应激和线粒体通透性转换（MPT）。在添加不同浓度的乙醇之前，将大鼠胃黏膜细胞（RGM-1）置于无血清的杜尔贝科改良伊格尔培养基中。分别用Hoechst 33342和碘化丙啶染色来评估RGM-1细胞的核形态变化和膜屏障功能障碍。为了评估氧衍生自由基和细胞内谷胱甘肽的作用，在乙醇处理前加入过氧化氢和羟自由基清除剂N，N-二甲基硫脲、谷胱甘肽前体N-乙酰-L-半胱氨酸以及酒精脱氢酶抑制剂4-甲基吡唑。为了研究MPT，在添加乙醇之前加载钙黄绿素和四甲基罗丹明甲酯，并使用激光扫描共聚焦显微镜监测荧光强度的变化。乙醇（>5% v/v）剂量依赖性地增加了碘化丙啶阳性细胞的数量，表明细胞膜屏障功能减弱。加入乙醇后，线粒体迅速被钙黄绿素充满，表明发生了MPT，同时伴随着线粒体去极化，这在细胞死亡前表现为四甲基罗丹明甲酯的丧失。与N，N-二甲基硫脲或N-乙酰-L-半胱氨酸同时孵育可显著减轻乙醇诱导的细胞死亡，表明细胞内氧化还原状态在诱导细胞损伤中的重要性，而4-甲基吡唑并不能减轻这种变化。目前的结果表明，乙醇处理会诱导细胞内氧化应激并产生MPT和线粒体去极化，这些都先于胃黏膜细胞死亡。细胞内抗氧化剂如谷胱甘肽可能对胃黏膜细胞中的乙醇具有显著的保护作用。
