Daily zeaxanthin supplementation prevents atrophy of the retinal pigment epithelium (RPE) in a mouse model of mitochondrial oxidative stress

Oxidative damage is implicated in the pathogenesis of age-related macular degeneration (AMD). The dry form of AMD (geographic atrophy) is characterized by loss of RPE, photoreceptors, and macular pigments. The cumulative effects of oxidative stress impact mitochondrial function in RPE. In Sod2flox/floxVMD2-cre mice, the RPE specific deletion of Sod2, the gene for mitochondrial manganese superoxide dismutase (MnSOD), leads to elevated oxidative stress in retina and RPE, and causes changes in the RPE and underlying Bruch’s membrane that share some features of AMD. This study tested the hypothesis that zeaxanthin supplementation would reduce oxidative stress and preserve RPE structure and function in these mice. Zeaxanthin in retina/RPE/choroid and liver was quantified by LC/MS, retinal function and structure were evaluated by electroretinogram (ERG) and spectral domain optical coherence tomography (SD-OCT), and antioxidant gene expression was measured by RT-PCR. After one month of supplementation, zeaxanthin levels were 5-fold higher in the retina/RPE/choroid and 12-fold higher in liver than in unsupplemented control mice. After four months of supplementation, amplitudes of the ERG a-wave (function of rod photoreceptors) and b-wave (function of the inner retina) were not different in supplemented and control mice. In contrast, the c-wave amplitude (a measure of RPE function) was 28% higher in supplemented mice than in control mice. Higher RPE/choroid expression of antioxidant genes (Cat, Gstm1, Hmox1, Nqo1) and scaffolding protein Sqstm1 were found in supplemented mice than in unsupplemented controls. Reduced nitrotyrosine content in the RPE/choroid was demonstrated by ELISA. Preliminary assessment of retinal ultrastructure indicated that supplementation supported better preservation of RPE structure with more compact basal infoldings and intact mitochondria. We conclude that daily zeaxanthin supplementation protected RPE cells from mitochondrial oxidative stress associated with deficiency in the MnSOD and thereby improved RPE function early in the disease course.

氧化损伤与年龄相关性黄斑变性（age-related macular degeneration, AMD）的发病机制密切相关。干性年龄相关性黄斑变性（geographic atrophy，地图样萎缩）以视网膜色素上皮（retinal pigment epithelium, RPE）、光感受器及黄斑色素的丢失为特征。氧化应激的累积效应会损害RPE的线粒体功能。在Sod2flox/floxVMD2-cre小鼠中，RPE特异性敲除Sod2基因——该基因编码线粒体锰超氧化物歧化酶（mitochondrial manganese superoxide dismutase, MnSOD）——会导致视网膜与RPE的氧化应激水平升高，并引发RPE及下方布鲁赫膜（Bruch’s membrane）出现具有AMD部分特征的病理改变。本研究验证了如下假说：补充玉米黄质可减轻此类小鼠的氧化应激，并维持RPE的结构与功能。研究通过液相色谱-质谱联用（liquid chromatography-mass spectrometry, LC/MS）定量检测了视网膜/RPE/脉络膜与肝脏中的玉米黄质含量；通过视网膜电图（electroretinogram, ERG）与光谱域光学相干断层扫描（spectral domain optical coherence tomography, SD-OCT）评估视网膜的功能与结构；通过逆转录聚合酶链式反应（reverse transcription polymerase chain reaction, RT-PCR）检测抗氧化基因的表达水平。补充干预1个月后，视网膜/RPE/脉络膜中的玉米黄质水平较未补充的对照组小鼠升高5倍，肝脏中则升高12倍。补充干预4个月后，ERG的a波（视杆光感受器功能的检测指标）与b波（内层视网膜功能的检测指标）振幅在补充组与对照组间无显著差异。与之相反，c波振幅——作为RPE功能的衡量指标——在补充组小鼠中较对照组升高28%。补充组小鼠的RPE/脉络膜中，抗氧化基因（Cat、Gstm1、Hmox1、Nqo1）及支架蛋白Sqstm1的表达水平均高于未补充的对照组。酶联免疫吸附实验（enzyme-linked immunosorbent assay, ELISA）证实，补充组的RPE/脉络膜中硝基酪氨酸含量有所降低。视网膜超微结构的初步评估结果显示，补充干预可更好地维持RPE的结构，表现为更致密的基底褶与完整的线粒体结构。本研究最终得出结论：每日补充玉米黄质可保护RPE细胞免受MnSOD缺陷相关的线粒体氧化应激，从而在疾病早期改善RPE的功能。