Molecular Gastroenterology and Hepatology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan.
J Clin Biochem Nutr. 2011 Nov;49(3):174-81. doi: 10.3164/jcbn.11-26. Epub 2011 Jun 17.
Protection of the small intestine from mucosal injury induced by nonsteroidal anti-inflammatory drugs including acetylsalicylic acid is a critical issue in the field of gastroenterology. Polaprezinc an anti-ulcer drug, consisting of zinc and L-carnosine, provides gastric mucosal protection against various irritants. In this study, we investigated the protective effect of polaprezinc on acetylsalicylic acid-induced apoptosis of the RIE1 rat intestinal epithelial cell line. Confluent rat intestinal epithelial cells were incubated with 70 µM polaprezinc for 24 h, and then stimulated with or without 15 mM acetylsalicylic acid for a further 15 h. Subsequent cellular viability was quantified by fluorometric assay based on cell lysis and staining. Acetylsalicylic acid-induced cell death was also qualified by fluorescent microscopy of Hoechst33342 and propidium iodide. Heat shock proteins 70 protein expression after adding polaprezinc or acetylsalicylic acid was assessed by western blotting. To investigate the role of Heat shock protein 70, Heat shock protein 70-specific small interfering RNA was applied. Cell viability was quantified by fluorometric assay based on cell lysis and staining and apoptosis was analyzed by fluorescence-activated cell sorting. We found that acetylsalicylic acid significantly induced apoptosis of rat intestinal epithelial cells in a dose- and time-dependent manner. Polaprezinc significantly suppressed acetylsalicylic acid-induced apoptosis of rat intestinal epithelial cells at its late phase. At the same time, polaprezinc increased Heat shock protein 70 expressions of rat intestinal epithelial cells in a time-dependent manner. However, in Heat shock protein 70-silenced rat intestinal epithelial cells, polaprezinc could not suppress acetylsalicylic acid -induced apoptosis at its late phase. We conclude that polaprezinc-increased Heat shock protein 70 expression might be an important mechanism by which polaprezinc suppresses acetylsalicylic acid-induced small intestinal apoptosis, a hallmark of acetylsalicylic acid-induced enteropathy.
保护小肠免受包括乙酰水杨酸在内的非甾体抗炎药引起的粘膜损伤是胃肠病学领域的一个关键问题。波利前列锌是一种抗溃疡药物，由锌和 L-肉碱组成，可提供胃粘膜对各种刺激物的保护。在这项研究中，我们研究了波利前列锌对 RIE1 大鼠肠上皮细胞系中乙酰水杨酸诱导的细胞凋亡的保护作用。将汇合的大鼠肠上皮细胞与 70 μM 的波利前列锌孵育 24 小时，然后再用或不用 15 mM 的乙酰水杨酸刺激 15 小时。随后通过基于细胞裂解和染色的荧光测定法来量化细胞活力。通过 Hoechst33342 和碘化丙啶的荧光显微镜来定性乙酰水杨酸诱导的细胞死亡。通过 Western blot 评估添加波利前列锌或乙酰水杨酸后热休克蛋白 70 蛋白的表达。为了研究热休克蛋白 70 的作用，应用了热休克蛋白 70 特异性小干扰 RNA。通过基于细胞裂解和染色的荧光测定法来量化细胞活力，并通过荧光激活细胞分选分析凋亡。我们发现，乙酰水杨酸以剂量和时间依赖的方式显著诱导大鼠肠上皮细胞凋亡。波利前列锌在其晚期显著抑制大鼠肠上皮细胞的乙酰水杨酸诱导的凋亡。同时，波利前列锌以时间依赖的方式增加大鼠肠上皮细胞的热休克蛋白 70 表达。然而，在热休克蛋白 70 沉默的大鼠肠上皮细胞中，波利前列锌不能抑制乙酰水杨酸诱导的晚期细胞凋亡。我们得出结论，波利前列锌增加热休克蛋白 70 的表达可能是波利前列锌抑制乙酰水杨酸诱导的小肠细胞凋亡的重要机制，这是乙酰水杨酸诱导的肠炎的一个标志。
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