Department of Biochemistry and Cell Biology, Graduate School of Medicine, Nippon Medical School, 1-396 Kosugi-machi, Nakahara-ku, Kawasaki-city, Kanagawa 211-8533, Japan.
Department of Materials and Life Science, Faculty of Science and Technology, Seikei University, 3-3-1 Kichijojikitamachi, Musashino-shi, Tokyo, 180-8633, Japan.
Can J Physiol Pharmacol. 2019 Oct;97(10):999-1005. doi: 10.1139/cjpp-2018-0741. Epub 2019 Jul 11.
Molecular hydrogen (H) was believed to be an inert and nonfunctional molecule in mammalian cells; however, we overturned the concept by reporting the therapeutic effects of H against oxidative stress. Subsequently, extensive studies revealed multiple functions of H by exhibiting the efficacies of H in various animal models and clinical studies. Here, we investigated the effect of H on free-radical-induced cytotoxicity using -butyl hydroperoxide in a human acute monocytic leukemia cell line, THP-1. Cell membrane permeability was determined using lactate dehydrogenase release assay and Hoechst 33342 and propidium iodide staining. Fatty acid peroxidation and mitochondrial viability were measured using 2 kinds of fluorescent dyes, Liperfluo and C11-BODIPY, and using the alamarBlue assay based on the reduction of resazurin to resorufin by mainly mitochondrial succinate dehydrogenase, respectively. Mitochondrial membrane potential was evaluated using tetramethylrhodamine methyl ester. As a result, H protected the cultured cells against the cytotoxic effects induced by -butyl hydroperoxide; H suppressed cellular fatty acid peroxidation and cell membrane permeability, mitigated the decline in mitochondrial oxidoreductase activity and mitochondrial membrane potential, and protected cells against cell death evaluated using propidium iodide staining. These results suggested that H suppresses free-radical-induced cell death through protection against fatty acid peroxidation and mitochondrial dysfunction.
分子氢（H）曾被认为是哺乳动物细胞中一种惰性和非功能性的分子；然而，我们通过报告 H 对氧化应激的治疗作用颠覆了这一概念。随后，广泛的研究通过展示 H 在各种动物模型和临床研究中的功效，揭示了 H 的多种功能。在这里，我们使用 - 丁基过氧化物（-butyl hydroperoxide）在人急性单核细胞白血病细胞系 THP-1 中研究了 H 对自由基诱导的细胞毒性的影响。使用乳酸脱氢酶释放测定法和 Hoechst 33342 和碘化丙啶染色来测定细胞膜通透性。使用两种荧光染料 Liperfluo 和 C11-BODIPY 测量脂肪酸过氧化和线粒体活力，并使用基于主要由线粒体琥珀酸脱氢酶将 Resazurin 还原为 Resorufin 的 alamarBlue 测定法来测量。使用四甲基罗丹明甲酯评估线粒体膜电位。结果表明，H 可保护培养细胞免受 - 丁基过氧化物诱导的细胞毒性作用；H 抑制细胞内脂肪酸过氧化和细胞膜通透性，减轻线粒体氧化还原酶活性和线粒体膜电位的下降，并通过碘化丙啶染色评估保护细胞免受细胞死亡。这些结果表明，H 通过防止脂肪酸过氧化和线粒体功能障碍来抑制自由基诱导的细胞死亡。
