Exogenous 1,25(OH)₂D₃/VD₃ counteracts RSL3-Induced ferroptosis by enhancing antioxidant capacity and regulating iron...

RSL3 is a common inhibitor of glutathione peroxidase 4 (GPx4) that can induce ferroptosis. Ferroptosis is an iron ion-dependent, oxidative-type of programmed cell death. In this study, larval/adult zebrafish were stimulated with RSL3 to construct a ferroptosis model, and CYP2R1^-/- zebrafish was used as a 1,25(OH)₂D₃ knock-down model to explore the regulatory effect and mechanism of 1,25(OH)₂D₃/VD₃ on RSL3-induced ferroptosis. The results showed that 1,25(OH)₂D₃/VD₃ alleviated RSL3 induced mitochondrial damage in liver of larval/adult zebrafish, reversed the decline of GPx4 activity, and reduced the accumulation of ROS, LPO and MDA. VD₃ also inhibited hepcidin (HEPC) in adult fish liver, promoted the production of ferroportin (FPN), and reduced the aggregation of Fe²⁺. Exogenous 1,25(OH)₂D₃ increased the CYP2R1^-/- survival and liver GPx4 activity after RSL3 treatment. At the gene level, 1,25(OH)₂D₃/VD₃ activated Keap1-Nrf2-GPx4 and inhibited the NFκB-hepcidin axis. In the ferroptosis context, deletion of the cyp2r1 gene resulted in a more severe decline in gpx4 expression, but the exogenous 1,25(OH)₂D₃ increased the expression of the GPx4 gene and protein in CYP2R1^-/- zebrafish liver after RSL3 treatment. The collective results indicated that 1,25(OH)₂D₃/VD₃ can inhibit ferroptosis induced by RSL3 in liver of larval/adult zebrafish by improving the antioxidant capacity and regulating iron ion transport. Exogenous 1,25(OH)₂D₃ reverses the downregulation of GPx4 in the CYP2R1^-/- zebrafish liver in the ferroptosis state. Compared with the ferroptosis inhibitor Fer-1, the mechanism of action of 1,25(OH)₂D₃/VD₃ is diversified and nonspecific. This study demonstrated the resistance of VD₃ to RSL3-induced ferroptosis at different developmental stages in zebrafish.