Schepens Eye Research Institute, Massachusetts Eye and Ear, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Invest Ophthalmol Vis Sci. 2013 Oct 15;54(10):6724-34. doi: 10.1167/iovs.13-12699.
Fuchs endothelial corneal dystrophy (FECD) is an oxidative stress disorder that leads to age-related and gradual loss of corneal endothelial cells resulting in corneal edema and loss of vision. To date, other than surgical intervention, there are no treatment options for patients with FECD. We have shown that in FECD, there is a deficiency in nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated antioxidant defense due to decreased Nrf2 nuclear translocation and activation of antioxidant response element (ARE). In this study, we used sulforaphane (SFN) and D3T to investigate a strategy of targeting Nrf2-ARE in FECD.
FECD and normal ex vivo corneas and human corneal endothelial cell lines were pretreated with SFN or D3T and exposed to oxidative stress with tert-Butyl hydroperoxide (tBHP). Apoptosis was detected with TUNEL. Cellular localization of Nrf2 and p53 was assessed by immunohistochemistry. Effect of SFN was determined by using DCFDA assay, Western blot and real-time PCR.
After pretreatment with SFN, oxidative stress was induced with tBHP. In ex vivo FECD specimens, SFN decreased CEC apoptosis by 55% in unstressed group and by 43% in tBHP-treated specimens. SFN enhanced nuclear translocation of Nrf2 in FECD specimens and decreased p53 staining under oxidative stress. Pretreatment with SFN enhanced cell viability by decreasing intracellular reactive oxygen species production. Upregulation of Nrf2 levels led to increased synthesis of DJ-1, heme oxygenase 1, and nicotinamide adenine dinucleotide quinone oxidoreductase-1. SFN significantly upregulated major ARE-dependent antioxidants and ameliorated oxidative stress-induced apoptosis in FECD.
Our results suggest that targeting Nrf2-ARE pathway may arrest degenerative cell loss seen in FECD.
Fuchs 内皮角膜营养不良（FECD）是一种氧化应激疾病，导致与年龄相关的角膜内皮细胞逐渐丧失，从而导致角膜水肿和视力丧失。迄今为止，除了手术干预外，FECD 患者没有其他治疗选择。我们已经表明，在 FECD 中，由于 Nrf2 核易位减少和抗氧化反应元件（ARE）激活减少，核因子红细胞 2 相关因子 2（Nrf2）调节的抗氧化防御存在缺陷。在这项研究中，我们使用了萝卜硫素（SFN）和 D3T 来研究针对 FECD 中 Nrf2-ARE 的策略。
用 SFN 或 D3T 预处理 FECD 和正常离体角膜以及人角膜内皮细胞系，并使其暴露于叔丁基过氧化物（tBHP）中产生氧化应激。用 TUNEL 检测细胞凋亡。用免疫组织化学检测 Nrf2 和 p53 的细胞定位。通过 DCFDA 测定、Western blot 和实时 PCR 确定 SFN 的作用。
SFN 预处理后，用 tBHP 诱导氧化应激。在离体 FECD 标本中，SFN 在未处理组中使 CEC 凋亡减少 55%，在 tBHP 处理组中使 CEC 凋亡减少 43%。SFN 增强了 FECD 标本中 Nrf2 的核易位，并在氧化应激下减少了 p53 染色。SFN 预处理通过减少细胞内活性氧的产生来提高细胞活力。Nrf2 水平的上调导致 DJ-1、血红素加氧酶 1 和烟酰胺腺嘌呤二核苷酸醌氧化还原酶 1 的合成增加。SFN 显著上调了主要的 ARE 依赖性抗氧化剂，并改善了 FECD 中氧化应激诱导的细胞凋亡。
我们的研究结果表明，靶向 Nrf2-ARE 途径可能阻止 FECD 中所见的退行性细胞丢失。
