Involvement of reactive oxygen species in indomethacin-induced apoptosis of small intestinal epithelial cells
Involvement of reactive oxygen species in indomethacin-induced apoptosis of small intestinal epithelial cells
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DOI：
10.1007/s00535-008-2293-3
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发表时间：
2009-01-01
影响因子：
6.3
通讯作者：
Yoshikawa, Toshikazu
中科院分区：
文献类型：
--
作者：
Omatsu, Tatsushi;Naito, Yuji;Yoshikawa, Toshikazu
背景资料。非甾体抗炎药所致小肠损伤的确切发病机制尚不清楚。在本研究中，我们利用体外小肠模型研究了吲哚美辛引起粘膜损伤的机制。方法：研究方法。采用从隐窝细胞开始表现为小肠表型并分化为绒毛表型的结肠癌细胞系Caco-2和大鼠肠上皮细胞系RIE。加入吲哚美辛诱导Caco-2和RIE单层细胞分化，用四甲基偶氮唑盐比色法和细胞培养上清液中乳酸脱氢酶释放量检测细胞死亡。荧光显微镜下的荧光探针检测也证实了吲哚美辛诱导的细胞死亡。作为一项功能研究，通过测量跨上皮电阻(TEER)和FITC标记的葡聚糖跨单层通量来评估Caco-2单层的通透性。用氧化还原灵敏的荧光探针和荧光计评价了吲哚美辛诱导的CaCO-2和RIE中的活性氧产生。在一些实验中，抗氧化剂被用来阐明活性氧在消炎痛诱导的Caco-2细胞死亡中的作用。结果。吲哚美辛以剂量和时间依赖的方式诱导Caco-2和RIE细胞死亡(主要是细胞凋亡)，并与Caco-2单层通透性增加相关。用消炎痛暴露于CaCO-2和RIE也导致显著的活性氧产生，而这些细胞被抗氧化剂预处理后被抑制。结论。综上所述，活性氧的产生是吲哚美辛引起小肠损伤的机制之一。
Background. The precise pathogenic mechanism of nonsteroidal antiinflammatory drug-induced small intestinal injury is still unknown. In the present study, we investigated the mechanism by which indomethacin induced mucosal injury by using an in vitro model of small intestine. Methods. The colon cancer cell line Caco-2, exhibiting a small intestinal phenotype starting as a crypt cell and differentiating to a villous phenotype, and RIE, a rat intestinal epithelial cell line, were employed. Indomethacin was added to differentiated the Caco-2 and RIE monolayer, and cell death was quantified by MTT assay and LDH release in the cell culture supernatant. Indomethacin-induced cell death was also qualified by fluorescent probes under the fluorescent microscope. As a functional study, the permeability of the Caco-2 monolayer was assessed by measuring transepithelial electrical resistance (TEER) and the flux of FITC-conjugated dextran across the monolayer. Indomethacin-induced reactive oxygen species production in Caco-2 and RIE was evaluated by redox-sensitive fluorogenic probes using a fluorometer. In some experiments, antioxidants were used to clarify the role of reactive oxygen species on indomethacin-induced Caco-2 cell death. Results. Indomethacin caused cell death (mainly apoptosis) of Caco-2 and RIE in a dose- and time-dependent manner that was correlated with increased permeability of the Caco-2 monolayer. Exposure of Caco-2 and RIE with indomethacin also resulted in a significant reactive oxygen species production that was inhibited by the pretreatment of these cells with antioxidants. Conclusions. Taken together, reactive oxygen species production is one of the mechanisms by which indomethacin induced small intestinal injury.