Sulforaphane protects rabbit corneas against oxidative stress injury in keratoconus through activation of the Nrf-2/HO-1 antioxidant pathway

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.