Zeng H, Ding M, Chen X-X, Lu Q
Beijing Tongren Eye Center, Beijing Tongren Hospital, Capital Medical University, Beijing Ophthalmology & Visual Science Key Laboratory, Beijing 100730, China.
Department of Ophthalmology Teaching and Research Section of Wei Fang Medical University, Weifang 261053, China.
Neuroscience. 2014 Sep 5;275:54-61. doi: 10.1016/j.neuroscience.2014.05.065. Epub 2014 Jun 12.
Accumulating evidence supports that nicotinamide adenine dinucleotide phosphate (NADPH) oxidase contributes to microglia-mediated neurotoxicity in the CNS neurodegenerative diseases. Several studies, including ours, suggest that microglial activation is involved in the retinal degeneration in the animal models of retinitis pigmentosa (RP). In the present study, we investigated the activation of NADPH oxidase in the rod degeneration in rd mice and further explored its role in the microglia-mediated photoreceptor apoptosis. Expression of gp91phox protein, a major subunit of NAPDH oxidase in the whole retina of rd mice at postnatal days (P) 8, 10, 12, 14, 16 and 18 was assessed by western blot analysis. Location of gp91phox in the rd retina at each age group and its cellular source were studied by immunohistochemical analysis and double labeling respectively. The generation of superoxide radicals in the rd retinas was demonstrated by intraperitoneal injection of hydroethidine. Apocynin was applied intraperitoneally in the rd mice from P8 to P14 to inhibit the activity of NAPDH oxidase and the outer nuclear layer (ONL) thickness was measured before and after apocynin treatment. Our results demonstrated that during the rod degenerative process, the expression of gp91phox started to increase in the outer part of rd retina at P10 and reached a peak at P14. Double labeling of gp91phox with CD11b showed co-localization of gp91phox in the retinal microglial cells. Increasing generation of superoxide radicals visualized by hydroethidine was noted at P8 and reached a peak at P14. Apocynin markedly reduced the production of superoxide radicals and preserved the rod cells. The results suggested that NADPH oxidase might play an important role in the rod degeneration in the rd mice. Inhibition of NAPDH oxidase could be a possible approach to treat RP in the early degenerative stage.
越来越多的证据支持烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶在中枢神经系统神经退行性疾病中促成小胶质细胞介导的神经毒性。包括我们的研究在内的多项研究表明，小胶质细胞激活参与色素性视网膜炎（RP）动物模型中的视网膜变性。在本研究中，我们研究了rd小鼠视杆细胞变性过程中NADPH氧化酶的激活情况，并进一步探讨了其在小胶质细胞介导的光感受器细胞凋亡中的作用。通过蛋白质印迹分析评估出生后第（P）8、10、12、14、16和18天rd小鼠整个视网膜中NAPDH氧化酶的主要亚基gp91phox蛋白的表达。分别通过免疫组织化学分析和双重标记研究每个年龄组rd视网膜中gp91phox的定位及其细胞来源。通过腹腔注射氢化乙锭证明rd视网膜中超氧自由基的产生。从P8到P14对rd小鼠腹腔注射阿朴吗啡以抑制NAPDH氧化酶的活性，并在阿朴吗啡治疗前后测量外核层（ONL）厚度。我们的结果表明，在视杆细胞变性过程中，gp91phox的表达在P10时开始在rd视网膜外层增加，并在P14时达到峰值。gp91phox与CD11b的双重标记显示gp91phox在视网膜小胶质细胞中共定位。通过氢化乙锭观察到的超氧自由基生成在P8时增加，并在P14时达到峰值。阿朴吗啡显著减少了超氧自由基的产生并保护了视杆细胞。结果表明，NADPH氧化酶可能在rd小鼠的视杆细胞变性中起重要作用。抑制NAPDH氧化酶可能是治疗早期变性阶段RP的一种可行方法。
