SERPINA3K Protects against Oxidative Stress via Modulating ROS Generation/Degradation and KEAP1-NRF2 Pathway in the Corneal Epithelium

Abstract

We recently reported that SERPINA3K (SA3K), a member of the serine proteinase inhibitor (SERPIN) family, has antiangiogenic and anti-inflammatory activities. Here we investigated the antioxidant effects of SA3K in the corneal epithelium and the mechanism underlying its action. We established the oxidative stress models induced by hydrogen peroxide (H₂O₂) in cultured human corneal epithelial (HCE) cells and in rat corneal epithelium in vivo. Cell viability, flow cytometry, and TUNEL analysis were conducted to detect viable cells and cell death; reactive oxygen species (ROS) and 3-Nitrotyrosine fluorescent assay was applied to measure ROS levels. Activity assay, immunostaining, Western blot, and quantitative RT-PCR were performed to analyze the factors of the ROS generation/degradation system and pathway. SA3K protected the HCE cells from H₂O₂-induced oxidative stress in a dose- and time-dependent manner. SA3K also significantly reduced the production of ROS. Regarding the mechanism underlying these effects, SA3K downregulated ROS generation by inhibiting NOX4 and upregulated ROS degradation by increasing the activity of superoxide dismutases and catalase. Furthermore, H₂O₂ induced activation of the Kelch-like ECH-associated protein 1 (KEAP1)/NF-E2-related factor-2 (NRF2) pathway, while SA3K inhibited H₂O₂-induced activation of KEAP1 and NRF2 and their downstream factors, including NAD(P)H quinone oxidoreductase and glutathione S-transferase. In the H₂O₂-induced rat corneal epithelium, SA3K alleviated the oxidative stress and downregulated NOX4 and NRF2. Collectively, SA3K protects against oxidative stress by targeting the ROS generation/degradation system and modulating the KEAP1-NRF2 signaling pathway.