Exogenous 1,25(OH)2D3/VD3 counteracts RSL3-Induced ferroptosis by enhancing antioxidant capacity and regulating iron ion transport: Using zebrafish as a model

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.