Physiological and transcriptomic analysis revealed the alleviating effect of 1,25(OH) 2 D 3 on environmental iron overloading induced ferroptosis in zebrafish.

Iron overload in the aquatic environment can cause damage in fish bodies. Vitamin D3 (VD3 ) has been proven to have antioxidant and regulatory effects on iron transport. The current research investigated the effects of environmental iron overload on larval zebrafish and explored the effects of 1,25(OH)2 D3 on ferroptosis in zebrafish larvae and zebrafish liver cells (ZFL) caused by iron overload in the environment and its possible regulatory mechanisms. The results showed that 1,25(OH)2 D3 alleviated liver damage in zebrafish larvae and mitochondrial damage in ZFL after excessive ammonium ferric citrate (FAC) treatment, and improved the survival rate of ZFL. 1,25(OH)2 D3 cleared and inhibited excessive FAC induced abnormal accumulation of ROS, lipid ROS, MDA, and Fe2+ in zebrafish larvae and ZFL, as well as enhanced the activity of antioxidant enzyme GPx4. Transcriptomic analysis showed that 1,25(OH)2 D3 can regulate ferroptosis in ZFL by regulating signaling pathways related to oxidative stress, iron homeostasis, mitochondrial function, and ERS, mainly including ferroptosis, neoptosis, p53 signaling pathway, apoptosis, FoxO signaling pathway. Validation of transcriptome data showed that 1,25(OH)2 D3 inhibits ferroptosis in zebrafish larvae and ZFL caused by excessive FAC via promoting the expression of slc40a1 and hmox1a genes and increasing SLC40A1 protein levels. In summary, 1,25(OH)2 D3 can resist ferroptosis in zebrafish caused by iron overload in the environment mainly via regulating antioxidant capacity and iron ion transport.