Cao Yongxing, Zhang Xin, Shang Wei, Xu Jiejia, Wang Xianhua, Hu Xiaoqing, Ao Yingfang, Cheng Heping
Institute of Sports Medicine, Peking University Third Hospital, Beijing, China.
PLoS One. 2013 Jun 19;8(6):e66444. doi: 10.1371/journal.pone.0066444. Print 2013.
Mitochondria play important roles in many types of cells. However, little is known about mitochondrial function in chondrocytes. This study was undertaken to explore possible role of mitochondrial oxidative stress in inflammatory response in articular chondrocytes.
Chondrocytes and cartilage explants were isolated from wild type or transgenic mice expressing the mitochondrial superoxide biosensor - circularly permuted yellow fluorescent protein (cpYFP). Cultured chondrocytes or cartilage explants were incubated in media containing interleukin-1β (10 ng/ml) or tumor necrosis factor-α (10 ng/ml) to stimulate an inflammatory response. Mitochondrial imaging was carried out by confocal and two-photon microscopy. Mitochondrial oxidative status was evaluated by "superoxide flash" activity recorded with time lapse scanning.
Cultured chondrocytes contain abundant mitochondria that show active motility and dynamic morphological changes. In intact cartilage, mitochondrial abundance as well as chondrocyte density declines with distance from the surface. Importantly, sudden, bursting superoxide-producing events or "superoxide flashes" occur at single-mitochondrion level, accompanied by transient mitochondrial swelling and membrane depolarization. The superoxide flash incidence in quiescent chondrocytes was ∼4.5 and ∼0.5 events/1000 µm(2)*100 s in vitro and in situ, respectively. Interleukin-1β or tumor necrosis factor-α stimulated mitochondrial superoxide flash activity by 2-fold in vitro and 5-fold in situ, without altering individual flash properties except for reduction in spatial size due to mitochondrial fragmentation.
The superoxide flash response to proinflammatory cytokine stimulation in vitro and in situ suggests that chondrocyte mitochondria are a significant source of cellular oxidants and are an important previously under-appreciated mediator in inflammatory cartilage diseases.
线粒体在多种类型的细胞中发挥着重要作用。然而，关于软骨细胞中线粒体功能的了解却很少。本研究旨在探讨线粒体氧化应激在关节软骨细胞炎症反应中的可能作用。
从野生型或表达线粒体超氧化物生物传感器——环形排列黄色荧光蛋白（cpYFP）的转基因小鼠中分离软骨细胞和软骨外植体。将培养的软骨细胞或软骨外植体在含有白细胞介素-1β（10 ng/ml）或肿瘤坏死因子-α（10 ng/ml）的培养基中孵育，以刺激炎症反应。通过共聚焦显微镜和双光子显微镜进行线粒体成像。通过延时扫描记录的“超氧化物闪光”活性评估线粒体氧化状态。
培养的软骨细胞含有丰富的线粒体，这些线粒体表现出活跃的运动性和动态形态变化。在完整的软骨中，线粒体丰度以及软骨细胞密度随离表面距离的增加而下降。重要的是，在单个线粒体水平上会发生突然的、爆发性的超氧化物产生事件或“超氧化物闪光”，同时伴有短暂的线粒体肿胀和膜去极化。在体外和原位，静止软骨细胞中的超氧化物闪光发生率分别约为4.5次和0.5次事件/1000 µm²×100秒。白细胞介素-1β或肿瘤坏死因子-α在体外刺激线粒体超氧化物闪光活性增加2倍，在原位增加5倍，除了由于线粒体碎片化导致空间尺寸减小外，未改变单个闪光特性。
体外和原位对促炎细胞因子刺激的超氧化物闪光反应表明，软骨细胞线粒体是细胞氧化剂的重要来源，并且是炎症性软骨疾病中一个重要但此前未得到充分认识的介质。
