The Role of Nrf2-Mediated Defense System in Corneal Epithelial Wound Healing

Abstract

Purpose: The Nrf2-mediated defense system plays a central role in protecting cells by activating genes against these types of stress. In the present study, we investigated the role of the Nrf2-mediated defense system in corneal epithelial wound healing by using Nrf2-knockout (KO) mice.

Methods: The corneal epithelium of wild type (WT) and Nrf2 KO mice were removed by treatment of n-heptanol for 1 minute under anesthesia. The epithelial defect was stained with 1% fluorescein solution and photographed at 0, 6, 12, 18, 24, 30, 36, 48, 60, and 72 h after epithelial debridement. Injured corneas healed at various time points were subjected to immunohistochemistry with Ki-67and Nrf2 antibody. Telomerase-immortalized corneal epithelial cell line (C/TERT) was used for cell migration assay and siRNA experiment.

Results: Nrf2 was expressed in the corneal epithelium of WT mice, but not in KO mice. Observation of wounds after 24 h of healing revealed that healing of the corneal epithelium was significantly delayed in the Nrf2 KO mice, while Nrf2 was activated in the corneal epithelium of WT mice. Ki-67-staining revealed that the number of Ki-67-positive proliferation cells was significantly lower in the Nrf2 KO mice than in the WT mice at 24-36 h after injury; however, these numbers were approximately equivalent by 48 h. To clarify the role of Nrf2 during wound healing, we performed in vitro experiments of siRNA for Nrf2. The result showed that Nrf2 knock-down significantly delayed corneal epithelial cell migration.

Conclusions: This study provides evidence that the Nrf2-mediated defense system plays a crucial role in corneal epithelial wound healing, by regulating the cell-migration activities of corneal epithelial cells.

Keywords: 482 cornea: epithelium • 480 cornea: basic science • 765 wound healing