Ishii Hisanari, Arai Toshiyuki, Mori Hiroko, Yamada Hiroko, Endo Nobuyuki, Makino Keisuke, Fukuda Kazuhiko
Department of Anesthesia, Kyoto University Hospital, Kyoto 606-8507, Japan.
Life Sci. 2005 Jul 8;77(8):858-68. doi: 10.1016/j.lfs.2004.11.038. Epub 2005 Mar 17.
The effects of 6-formylpterin on tumor necrosis factor (TNF)-alpha-induced apoptotic cell injury were studied in cultured rat hepatocytes. The incubation of the hepatocytes with TNF-alpha and actinomycin D (ActD) induced the apoptotic cell injury. The level of aspartate transaminase (AST) in the culture supernatant increased, and the cell viability, estimated by mitochondrial respiration (MTT assay), decreased. The DNA fragmentation and the caspase 3-like activity, which are characterized to apoptosis, increased. When the hepatocytes were incubated with 100-500 microM 6-formylpterin, the intracellular formation of reactive oxygen species (ROS) was observed, and the ratio of reduced and oxidized glutathione (GSH/GSSG) of whole cell lysate decreased. The co-incubation of the TNF-alpha/ActD-treated hepatocytes with 100-500 microM 6-formylpterin attenuated the TNF-alpha/ActD-induced apoptotic cell injury. The level of AST decreased and the cell viability increased. Both the DNA fragmentation and the caspase 3-like activity decreased. The caspases, executors of apoptosis, are known to require a reduced cystein in their active site to function, and the intact intracellular GSH/GSSG is essential for the caspase activation. Therefore, our findings suggest that intracellular ROS generated by 6-formylpterin decline the intracellular redox state to an oxidant state, which suppresses the caspase activity and prevents the apoptotic cell injury of hepatocytes.
在培养的大鼠肝细胞中研究了6-甲酰蝶呤对肿瘤坏死因子（TNF）-α诱导的凋亡细胞损伤的影响。用TNF-α和放线菌素D（ActD）孵育肝细胞可诱导凋亡细胞损伤。培养上清液中天冬氨酸转氨酶（AST）水平升高，通过线粒体呼吸（MTT法）估计的细胞活力降低。具有凋亡特征的DNA片段化和半胱天冬酶3样活性增加。当肝细胞与100-500 microM 6-甲酰蝶呤孵育时，观察到细胞内活性氧（ROS）的形成，全细胞裂解物中还原型和氧化型谷胱甘肽（GSH/GSSG）的比例降低。将用TNF-α/ActD处理的肝细胞与100-500 microM 6-甲酰蝶呤共同孵育可减轻TNF-α/ActD诱导的凋亡细胞损伤。AST水平降低，细胞活力增加。DNA片段化和半胱天冬酶3样活性均降低。半胱天冬酶是凋亡的执行者，已知其活性位点需要一个还原型半胱氨酸才能发挥作用，完整的细胞内GSH/GSSG对于半胱天冬酶的激活至关重要。因此，我们的研究结果表明，6-甲酰蝶呤产生的细胞内ROS将细胞内氧化还原状态降低至氧化状态，从而抑制半胱天冬酶活性并防止肝细胞的凋亡细胞损伤。
