Esculin exerts Nrf2-mediated antioxidant response in DrF cell lines and zebrafish larvae

Nuclear factor E2-related factor 2 (Nrf2) plays a major role in cellular defense by regulating antioxidant response element (ARE)-driven genes in response to oxidative stress. The present study aimed to identify and validate AREs throughout the zebrafish genome in response to Esculin (ESC). Danio rerio fin (DrF) cell lines were treated with ESC and the positive control, sulforaphane (SFN) to assess cytotoxicity and Nrf2 activation. Cytotoxicity was measured using MTT and Alamar Blue assays. Nrf2-mediated ARE activation was analyzed using immunofluorescence and luciferase reporter assays. In addition, Chromatin immunoprecipitation sequencing (ChIP-Seq) was employed to identify Nrf2 binding sites in the genome of the zebrafish larvae, while qRT-PCR and ChIP-qPCR were used to validate Nrf2 binding to specific AREs in annotated genes. ESC treatment, similar to SFN, significantly increased Nrf2 expression and ARE-driven luciferase activity, confirming activation of the Nrf2-ARE pathway. ChIP-Seq analysis in ESC-treated zebrafish larvae revealed Nrf2 binding sites at various genomic regions, with few AREs near the proximal promoters. qRT-PCR further validated the upregulation of key genes, including ash1 and arhgef1a in treated larvae, suggesting direct regulation by Nrf2. In conclusion, our findings confirmed that ESC effectively activates Nrf2 via ARE-mediated mechanisms in 3 dpf zebrafish larvae. These findings provide valuable insights into the conserved mechanism of Nrf2 in vertebrates and serve as a therapeutic option in oxidative-stress-mediated conditions.