NCOA4-mediated ferritinophagy is involved in ionizing radiation-induced ferroptosis of intestinal epithelial cells

Ferroptosis is a newly recognized form of regulated cell death that is characterized by severe lipid peroxidation initiated by iron overload and the generation of reactive oxygen species (ROS). However, the role of iron in ionizing radiation (IR)-induced intestinal injury has not been fully illustrated yet. In this study, we found that IR induced ferroptosis in intestinal epithelial cells, as indicated by the increase in intracellular iron levels and lipid peroxidation, upregulation of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA, reduced glutathione peroxidase 4 (GPX4) mRNA and glutathione (GSH) levels, and significant mitochondrial damage. In addition, the iron chelator deferoxamine (DFO) attenuated IR-induced ferroptosis and intestinal injury in vitro and in vivo. Intriguingly, pharmacological inhibition of autophagy with 3-methyladenine (3-MA) mitigated IR-induced ferritin downregulation, iron overload and ferroptosis. IR increased the levels of nuclear receptor coactivator 4 (NCOA4) mRNA and protein. NCOA4 knockdown significantly inhibited the reduction of ferritin, decreased the level of intracellular free iron, and mitigated ferroptosis induced by IR in HIEC cells, indicating that NCOA4-mediated autophagic degradation of ferritin (ferritinophagy) was required for IR-induced ferroptosis. Furthermore, cytoplasmic iron further activated mitoferrin2 (Mfrn2) on the mitochondrial membrane, which in turn increased iron transport into the mitochondria, resulting in increased ROS production and ferroptosis. In addition, mice fed with an iron-deficient diet for 3 weeks showed a significant reversal in the intestinal injury induced by abdominal IR exposure. Taken together, ferroptosis is a novel mechanism of IR-induced intestinal epithelial cytotoxicity, and is dependent on NCOA4-mediated ferritinophagy.