The Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.
J Physiol Pharmacol. 2012 Apr;63(2):137-42.
Nonsteroidal anti-inflammatory drugs (NSAIDs) often cause gastrointestinal complications such as gastric ulcers and erosions. Recent studies on the pathogenesis have revealed that NSAIDs induce lipid peroxidation in gastric epithelial cells by generating superoxide anion in mitochondria, independently with cyclooxygenase-inhibition and the subsequent prostaglandin deficiency. Although not clearly elucidated, the impairment of mitochondrial oxidative phosphorylation, or uncoupling, by NSAIDs is associated with the generation of superoxide anion. Physiologically, superoxide is immediately transformed into hydrogen peroxide and diatomic oxygen with manganese superoxide dismutase (MnSOD). Rebamipide is an antiulcer agent that showed protective effects against NSAID-induced lipid peroxidation in gastrointestinal tracts. We hypothesized that rebamipide may attenuate lipid peroxidation by increasing the expression of MnSOD protein in mitochondria and decreasing the leakage of superoxide anion in NSAID-treated gastric and small intestinal epithelial cells. Firstly, to examine rebamipide increases the expression of MnSOD proteins in mitochondria of gastrointestinal epithelial cells, we underwent Western blotting analysis against anti-MnSOD antibody in gastric RGM1 cells and small intestinal IEC6 cells. Secondly, to examine whether the pretreatment of rebamipide decreases NSAID-induced mitochondrial impairment and lipid peroxidation, we treated these cells with NSAIDs with or without rebamipide pretreatment, and examined with specific fluorescent indicators. Finally, to examine whether pretreatment of rebamipide attenuates NSAID-induced superoxide anion leakage from mitochondria, we examined the mitochondria from indomethacin-treated RGM1 cells with electron spin resonance (ESR) spectroscopy using a specific spin-trapping reagent, CYPMPO. Rebamipide increased the expression of MnSOD protein, and attenuated NSAID-induced mitochondrial impairment and lipid peroxidation in RGM1 and IEC6 cells. The pretreatment of rebamipide significantly decreased the signal intensity of superoxide anion from the mitochondria. We conclude that rebamipide attenuates lipid peroxidation by increasing the expression of MnSOD protein and decreasing superoxide anion leakage from mitochondria in both gastric and small intestinal epithelial cells.
非甾体抗炎药（NSAIDs）常引起胃肠道并发症，如胃溃疡和糜烂。最近的发病机制研究表明，NSAIDs 通过在 线粒体中生成超氧阴离子，独立于环氧化酶抑制和随后的前列腺素缺乏，诱导胃上皮细胞中的脂质过氧化。虽然尚未明确阐明，但 NSAIDs 对线粒体氧化磷酸化的损害或解偶联与超氧阴离子的产生有关。在生理上，超氧阴离子会立即被锰超氧化物歧化酶（MnSOD）转化为过氧化氢和双原子氧。瑞巴派特是一种抗溃疡剂，它显示出对胃肠道中 NSAID 诱导的脂质过氧化的保护作用。我们假设瑞巴派特可以通过增加线粒体中 MnSOD 蛋白的表达和减少 NSAID 处理的胃和小肠上皮细胞中超氧阴离子的漏出，来减轻脂质过氧化。首先，为了检查瑞巴派特是否增加了胃肠道上皮细胞中线粒体中 MnSOD 蛋白的表达，我们用抗 MnSOD 抗体进行了 Western 印迹分析胃 RGM1 细胞和小肠 IEC6 细胞。其次，为了检查瑞巴派特预处理是否减少 NSAID 诱导的线粒体损伤和脂质过氧化，我们用或不用瑞巴派特预处理这些细胞，然后用特定的荧光指示剂进行检查。最后，为了检查瑞巴派特预处理是否能减轻 NSAID 诱导的超氧阴离子从线粒体中漏出，我们用特定的自旋捕获试剂 CYPMPO 对吲哚美辛处理的 RGM1 细胞进行了电子自旋共振（ESR）光谱分析，检查了线粒体中的超氧阴离子。瑞巴派特增加了 MnSOD 蛋白的表达，并减轻了 RGM1 和 IEC6 细胞中 NSAID 诱导的线粒体损伤和脂质过氧化。瑞巴派特预处理显著降低了来自线粒体的超氧阴离子信号强度。我们的结论是，瑞巴派特通过增加胃和小肠上皮细胞中线粒体中 MnSOD 蛋白的表达和减少超氧阴离子从线粒体中的漏出，来减轻脂质过氧化。
