NADPH氧化酶抑制剂对遗传性视网膜色素变性感光细胞凋亡的抑制作用

Inhibitor of NADPH oxidase slow photoreceptor cell death in the retinal degeneration of rd mice

背景 我们先前的研究表明,rd小鼠遗传性视网膜色素变性（RP）过程中小胶质细胞活化与感光细胞的凋亡密切相关.研究显示,小胶质细胞中烟酰胺二磷酸腺苷（NADPH）氧化酶的活化在小胶质细胞活化及神经元损伤中发挥重要作用,但NADPH氧化酶在RP过程中作用机制及其抑制剂的作用有待探讨.目的 探讨rd小鼠发生RP过程中NADPH氧化酶产生活性氧簇（ROS）的活化反应及其抑制剂对感光细胞的保护作用. 方法 按抛掷硬币法将60只SPF级rd小鼠随机分为香荚兰乙酮注射组和PBS对照组,香荚兰乙酮注射组小鼠于出生后9d（P9）腹腔内注射NADPH氧化酶抑制剂香荚兰乙酮10 mg/kg（0.01 ml/kg）,每日1次,连续5d（至P13）;PBS对照组rd小鼠以同样方式注射等容量的PBS;C57 BL/6N小鼠10只不注射任何药物作为rd小鼠的野生对照鼠.各组小鼠于出生后14 d（P14）处死并制备视网膜冰冻切片,采用二氢乙锭（DHE）荧光染色法检测3个组小鼠视网膜中ROS的表达;采用实时定量PCR（real-time PCR）法测定2个组rd小鼠视网膜感光细胞中视紫红质mRNA的定量表达;采用苏木精-伊红染色法检查2个组rd小鼠视网膜外核层厚度.结果 DHE荧光染色表明,小鼠视网膜中ROS表达呈红色荧光,注药组小鼠视网膜外核层中ROS的红色荧光明显强于C57BL/6N野生鼠,但明显弱于PBS对照组.Real-time PCR检测表明,香荚兰乙酮注射组小鼠感光细胞中视紫红质mRNA相对表达量为4.21 ±0.33,明显低于PBS对照组的0.93±0.24,差异有统计学意义（t=2.360,P=0.000）;香荚兰乙酮注射组小鼠视网膜外核层厚度为（35.95±1.63） μm,明显厚于PBS对照组的（23.17±1.38） μm,差异有统计学意义（t=3.850,P=0.016）.结论 在rd小鼠视网膜感光细胞变性过程中,NADPH氧化酶生成ROS的活化反应明显增强;香荚兰乙酮能够延缓rd小鼠感光细胞的凋亡过程.

Background Our previous study demonstrated that microglial activation is closely associated with photoreceptor apoptosis in rd mice.Recent studies on central nervous system （CNS） showed that activation of nicotinamide adenine dinucleotide phosphate （NADPH） oxidase plays a key role in the microglia activation and neural cell death.However,the mechanism of NADPH oxidase during the retinal degeneration and the effect of NADPH oxidase inhibitor on photoreceptor apoptosis are concerned.Objective The aim of this study was to further explore the production of reactive oxygen species （ROS） by NADPH oxidase in the retinal degenerative process of rd mice and protection of NADPH oxidase inhibitor on photoreceptors.Methods Sixty rd mice at postnatal day 9 （P9） were randomized into the experimental group and the control group by throwing coins method.Apocynin,a NADPH oxidase inhibitor,was intraperitoneally injected in the dose of 10 mg/kg （0.01 ml/kg） once daily for 5 days （P13） in the experimental group,and the equal amount of PBS was used in the same way in the control group,and 10 C57BL/6N mice without injection of any drugs served as the wild type mice group.All the mice were sacrificed in P14 for the preparation of retinal sections.The expression of ROS in the retina was detected by dihydroethidium （DHE） fluorescence staining.Expression level of rhodopsin mRNA in the photoreceptor of the mice was determined by real-time PCR,and the thickness of retinal outer nuclear layer （ONL） in the mice of the experimental group and the control group was measured using hematoxylin ＆amp; eosin staining.The use and care of the animals complied with the Statement of Association for Research in Vision and Ophthalmology.Results DHE staining showed that the ROS presented with the red fluorescence in the mouse retinas.In the rd mice of the experimental group,the ROS fluorescence intensity was dramatically enhanced in comparison with C57BL/6N mice,but weakened in comparison with the rd mice of the control group.Real-time PCR revealed that the relative expressing level of rhodopsin mRNA in the photoreceptor was （4.21±0.33） in the experimental group and （0.93±0.24） in the control group,showing a significant difference between them （t =2.360,P =0.000）.The thickness value of retinal ONL was （35.95±1.63）μm in the mice of the experimental group,which was significantly higher than that in the mice of the control group （[23.17±1.38] μm） （t=3.850,P=0.016）.Conclusions In the retinal degeneration of rd mice,activation of NADPH oxidase increases the ROS production.Apocynin can slow the apoptosis procedure of photoreceptor cells of rd mice.