Antioxidant effect of esculin and sulforaphane in zebrafish larvae: A comparative study

Antioxidants are crucial for maintaining cellular redox homeostasis by neutralizing free radicals, including reactive oxygen species (ROS). Nuclear factor erythroid-2-related factor 2 (Nrf2) is a transcription factor that activates various cytoprotective genes to counteract oxidative damage. The present study aimed to compare the antioxidant potential of natural phytochemicals, esculin (ESC), and sulforaphane (SFN) in zebrafish larvae, focusing on Nrf2 activation. Zebrafish larvae were treated with ESC (100 μM) and SFN (40 μM) along with control for 6 h. The levels of ROS, lipid peroxidation (LPO), nitric oxide (NO), glutathione S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), and reduced glutathione (GSH) were measured. Further, RT-PCR and Western blotting methods were employed to determine the antioxidant genes and protein levels, respectively. In addition, Nrf2 localization within the cell was assessed using nuclear/cytosolic fractionation assay and wholemount immunohistochemistry method. Our results demonstrate that ESC treatment significantly increased antioxidant enzyme activities (GST, GPx, CAT), similar to the effects observed with SFN. Further, ESC enhanced the mRNA levels of antioxidant genes expression, such as nrf2, gstp1, hmox1a, prdx1, nqo1, gss, gsr, sqstm1, hsp90aa1.2 than SFN. Antioxidant proteins Nrf2, Gstp1, Cat, and Sod2 were upregulated in the ESC-treated groups. Subsequently, nuclear localization of Nrf2 was detected in both SFN- and ESC-treated groups, confirming Nrf2 translocation into the nucleus. Our findings suggest that ESC possesses potent antioxidant activity as SFN in 72-hpf zebrafish larvae through the Keap1-Nrf2 signaling pathway.