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损伤。脂褐素是细胞质氧化损伤的产物之一，在溶酶体中积累。先前，我们发现患有杜氏型肌营养不良症（DMD）的2至7岁患者和10周龄mdx小鼠（DMD的模型动物）的肌营养不良蛋白缺陷的肌肉比年龄匹配的正常对照更早地积累老化色素脂褐素，其是氧化应激的产物（Nakae等人，Histochemistry and cell Biology，2001，115，205-214 ; Nakae等人，Journal of Molecular Histology，2005，35，489-499）。此外，mdx肌肉中的大多数凋亡肌纤维含有脂褐质颗粒（Nakae等人，Histochemistry and Cell Biology，2001，115，205-214）。鸟嘌呤是DNA中的四种碱基之一，具有最低的氧化电位，优先受到低水平氧化应激的攻击并被氧化。 ...更多信息 到8-氧代鸟嘌呤。因此，8-氧代鸟嘌呤是细胞核中氧化应激的敏感标志物。在本研究中，我们开发了一种用于定量评估核DNA中作为氧化应激标志物的8-氧代鸟嘌呤的新技术，并将其应用于DMD和mdx肌肉（Nakae等人，Histochemistry and Cell Biology，2005，124，335-345）。使用中性红DNA染色技术、8-氧代鸟嘌呤特异性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与DNA含量无关的氧化指数。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组高14%。8周龄mdx小鼠的膈肌肌核平均指数比同龄正常对照组高30%。然而，mdx和正常对照的舌肌中肌核的平均指数类似地低（Nakae等人，日本解剖学学报，2006，81（增刊），214）。脂褐素颗粒在mdx膈肌中丰富，在mdx舌肌中少见，在正常膈和舌肌中缺失。在mdx膈肌中观察到局灶性变性和再生，但在mdx舌肌中未观察到。这些结果表明，氧化应激与肌营养不良蛋白缺陷型肌营养不良症的病理学有关。我们的核DNA原位氧化损伤的定量评估技术被证实适用于生物医学研究。在本研究中，我们还发现了一种有效改善mdx小鼠肌营养不良症的化合物“A”。该化合物可用于DMD的药物治疗。正在研究该化合物在营养不良肌肉中作用的分子机制。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