Oxidative damage of DNA and its protection in ageing and diseases of oral and maxillary muscles.

氧化应激参与了退行性疾病和衰老的机制。它引起亚细胞结构和生物分子的破坏。溶酶体中积累的与年龄相关的色素脂褐素是细胞质氧化损伤的产物之一。先前我们发现，2 - 7岁杜氏型肌营养不良症（DMD）患者和10周龄mdx小鼠（DMD模型动物）的肌营养不良蛋白缺乏，比年龄匹配的正常对照组更早积累老化的色素脂褐素，这是氧化应激的产物（Nakae等人，组织化学和细胞生物学，2001年，115,205 -214；Nakae等人，分子组织学杂志，2005年，35,489 -499）。此外，mdx肌肉中大多数凋亡肌纤维含有脂褐素颗粒（Nakae等人，组织化学和细胞生物学，2001,115,205-214）。作为DNA的四种碱基之一，鸟嘌呤具有最低的氧化电位，它优先受到低水平氧化应激的攻击，并被氧化成8-氧鸟嘌呤。因此，8-氧鸟嘌呤是细胞核氧化应激的敏感标志物。在本研究中，我们开发了一种定量评估核DNA中8-氧鸟嘌呤作为氧化应激标志物的新技术，并将其应用于DMD和mdx肌肉（Nakae et al., Histochemistry and Cell Biology, 2005,124, 335-345）。采用中性红DNA染色、8-氧鸟嘌呤单克隆抗体和细胞标记物层粘连蛋白抗体原位测定细胞核中独立于DNA含量的氧化指标。我们发现2- 7岁DMD患者肱二头肌肌核的平均指数比年龄匹配的正常对照高14%。8周龄mdx小鼠膈肌肌核平均指数比同龄正常对照组高30%。然而，mdx和正常对照舌肌肌核的平均指数同样较低（Nakae et al., Acta Anatomica Nipponica, 2006,81 (Suppl), 214）。脂褐素颗粒在膈肌中丰富，在舌肌中少见，在正常膈肌和舌肌中不存在。膈肌有局灶性变性和再生，舌肌无。这些结果提示氧化应激与肌营养不良症的病理有关。我们的核DNA原位氧化损伤定量评估技术被证实可用于生物医学研究。本研究还发现了一种能有效改善mdx小鼠肌营养不良的化合物a。该化合物可用于DMD的医学治疗。目前正在研究该化合物在营养不良肌肉中的作用的分子机制。少

Oxidative stress is involved in the mechanisms of degenerative diseases and ageing. It causes damages of subcellular structures and bio-molecules. An age-related pigment, lipofuscin, accumulated in lysosomes is one of products of the oxidative damage in the cytoplasm. Previously we found that dystrophin-deficient muscles of 2 to 7-year-old patients with Duchenne-type muscular dystrophy (DMD) and 10-week-old mdx mice, model animals of DMD, accumulate an ageing pigment lipofuscin, which is a product of oxidative stress, earlier than age-matched normal controls (Nakae et al., Histochemistry and cell Biology, 2001, 115, 205-214 ; Nakae et al., Journal of Molecular Histology, 2005, 35, 489-499). Further, most apoptotic myofibres in mdx muscles contain lipofuscin granules (Nakae et al., Histochemistry and Cell Biology, 2001, 115, 205-214). One of the four bases in DNA, guanine, which has the lowest oxidation potential, is preferentially attacked by low levels of oxidative stress and oxidised … More to 8-oxoguanine. Therefore, 8-oxoguanine is a sensitive marker of oxidative stress in nuclei. In the present study, we developed a new technique for the quantitative assessment of 8-oxoguanine in nuclear DNA as a marker of oxidative stress and apply it to DMD and mdx muscles (Nakae et al., Histochemistry and Cell Biology, 2005, 124, 335-345). The oxidative indices independent of DNA contents in cell nuclei were determined in situ using the technique with Neutral Red for DNA staining, a monoclonal antibody specific for 8-oxoguanine and a antibody for a cell marker, laminin. We found that the mean index for the myonuclei in bicepts brachii muscles of 2- to 7-year-old DMD patients was 14% higher than that in age-matched normal controls. The mean index for the myonuclei in diaphragm muscles of 8-week-old mdx mice was 30% higher than that in age-matched normal controls. However, the mean indices for the myonuclei in lingual muscles of mdx and normal controls were similarly low (Nakae et al., Acta Anatomica Nipponica, 2006, 81(Suppl), 214). Lipofuscin granules were abundant in mdx diaphragm muscle, rare in mdx lingual muscle and absent in normal diaphragm and lingual muscles. Focal degeneration and regeneration were observed in mdx diaphragm muscles but not in mdx lingual muscles. These results suggest that oxidative stress is related to the pathology of dystrofin-deficient muscular dystrophy. Our technique for the quantitative assessment of oxidative damage in nuclear DNA in situ was confirmed to be applicable in biomedical research.In the present study we also discovered a compound "A" effective to ameliorate muscular dystrophy of mdx mice. This compound may be useful for medical therapy of DMD. The molecular mechanism of the action of the compound in dystrophic muscles is being investigated. Less