Abstract

Radiation-induced intestinal injury involves catastrophic loss of intestinal stem cells (ISCs), but the mechanisms enabling their rescue remain elusive. Here, we demonstrated that melatonin, a potent mitigator of mitochondrial oxidative stress, robustly promoted the regeneration of intestinal crypts and organoids following irradiation. Using in vivo lineage tracing with Lgr5 CreERT2 ; Rosa26 -tdTomato, Mist1 CreERT2 ; Rosa26 -tdTomato, and Bmi1 CreER ; Rosa26 -tdTomato models, we pinpointed Bmi1 + cells as the source of the melatonin-induced proliferating cells responsible for regeneration. Genetic ablation of Bmi1 + lineages abolished the regenerative benefits of melatonin. Mechanistically, melatonin was associated with a shift in mitochondrial metabolism in Bmi1 + ISCs, in part through the suppression of Ca 2+ /CaMKⅡ-driven phosphorylation of DRP1, which contributed to reduced reactive oxygen species (ROS) production. This attenuation of oxidative stress subsequently reduced DNA damage and cell cycle arrest, while enhancing mitochondrial oxidative phosphorylation. Crucially, pharmacological inhibition of p-DRP1 phenocopied the effects of melatonin, promoting Bmi1 + ISC-mediated regeneration. In conclusion, our work revealed that melatonin acts as a guardian of Bmi1 + ISCs and promotes regeneration by imposing a critical checkpoint on CaMKII/DRP1-dependent metabolic dysfunction. Targeting this pathway with melatonin or specific p-DRP1 inhibitors represents a novel and highly promising therapeutic strategy for radiation-induced intestinal injury.