Department of Ophthalmology, The First Hospital of Peking University, Beijing 100034, P.R. China.
Int J Mol Med. 2018 Nov;42(5):2315-2328. doi: 10.3892/ijmm.2018.3820. Epub 2018 Aug 10.
The aim of the present study was to examine whether activation of the nuclear factor E2‑related factor 2 (Nrf‑2)/heme oxygenase‑1 (HO‑1) antioxidant pathway in the cornea was involved in the protective effect of sulforaphane (SF) following keratoconus (KC) injury. Following epithelial debridement, collagenase type II was applied in KC groups at room temperature for 30 min. Following this, rabbits were administered with a subconjunctival (s.c.) injection of SF or placebo (maize oil) daily for a total of 2 weeks. To investigate whether HO‑1 was involved in the Nrf‑2‑related antioxidant pathway, rabbits were injected with zinc (II) protoporphyrin IX (ZnPP IX, s.c.) treatment in combination with SF 24 h following the application of collagenase type II. The protective effects of SF were evaluated by examining the mean keratometry (Km) and central cornea thickness (CCT), measuring reactive oxygen species (ROS) production using immunofluorescent staining, and analyzing the protein expression of NADPH oxidase (Nox) family members Nox‑2 and Nox‑4, and Nrf‑2 and HO‑1 using immunohistochemistry and western blot analysis. The mRNA levels of Nox‑2, Nox‑4, Nrf‑2 and HO‑1 were quantitatively detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) analysis. No significant difference in Km or CCT was observed among groups prior to surgery (P=0.700 and P=0.982, respectively). KC induced an apparent increase of ROS generation, and caused a significant increase in Km and a significant decrease in CCT. These changes were neutralized or reversed by SF treatment. Simultaneously, SF treatment decreased the expression of Nox‑2 and Nox‑4, and enhanced the expression of Nrf‑2 and HO‑1 in the KC corneas. The RT‑qPCR results indicated that SF induced downregulation of the mRNA expression of Nox‑2 and Nox‑4, and upregulation of the mRNA expression of Nrf‑2 and HO‑1 following KC injury. The HO‑1 inhibitor, ZnPP IX, counteracted the protective effects of SF on KC corneas. Therefore, the present study provided evidence that activation of the Nrf‑2/HO‑1 signal transduction pathway may partially promote the protective effect of the antioxidant SF in the KC cornea.
本研究旨在探讨核因子 E2 相关因子 2 (Nrf-2)/血红素加氧酶-1 (HO-1) 抗氧化途径在萝卜硫素 (SF) 对圆锥角膜 (KC) 损伤后的保护作用中的作用。在进行上皮清创术之后，KC 组在室温下应用 II 型胶原酶处理 30 分钟。之后，兔子每日接受结膜下 (s.c.) SF 或安慰剂 (玉米油) 注射，共 2 周。为了研究 HO-1 是否参与 Nrf-2 相关抗氧化途径，在应用 II 型胶原酶后 24 小时，兔子接受锌 (II) 原卟啉 IX (ZnPP IX，s.c.) 治疗与 SF 联合注射。通过检查平均角膜曲率 (Km) 和中央角膜厚度 (CCT)、使用免疫荧光染色测量活性氧 (ROS) 产生、以及通过免疫组化和 Western blot 分析分析 NADPH 氧化酶 (Nox) 家族成员 Nox-2 和 Nox-4、Nrf-2 和 HO-1 的蛋白表达，评估 SF 的保护作用。通过反转录-定量聚合酶链反应 (RT-qPCR) 分析定量检测 Nox-2、Nox-4、Nrf-2 和 HO-1 的 mRNA 水平。手术前各组之间 Km 或 CCT 无显著差异 (P=0.700 和 P=0.982)。KC 诱导 ROS 生成明显增加，并导致 Km 明显增加，CCT 明显降低。SF 治疗可中和或逆转这些变化。同时，SF 治疗降低 KC 角膜中 Nox-2 和 Nox-4 的表达，增强 Nrf-2 和 HO-1 的表达。RT-qPCR 结果表明，SF 诱导 KC 损伤后 Nox-2 和 Nox-4 的 mRNA 表达下调，Nrf-2 和 HO-1 的 mRNA 表达上调。HO-1 抑制剂 ZnPP IX 拮抗 SF 对 KC 角膜的保护作用。因此，本研究提供的证据表明，Nrf-2/HO-1 信号转导途径的激活可能部分促进了抗氧化 SF 在 KC 角膜中的保护作用。
