Melatonin ameliorates NEC-associated brain injury by suppressing pro-inflammatory (M1-like) microglia via the AMPK-IL-1β axis

Background Necrotizing enterocolitis (NEC) is a severe neonatal gastrointestinal disease. Its associated brain injury involves neuroinflammation and long-term cognitive impairment, yet specific therapies are lacking. Melatonin (MEL) has shown neuroprotective effects, but its role in NEC-associated brain injury remains unclear. Methods Neonatal C57BL/6 J mice were subjected to NEC induction and studied at short-term (acute) and long-term (chronic, up to 9 weeks). Cognitive function was assessed by Y-maze and open field tests. Microglial activation, inflammatory cytokine expression, and AMPK pathway activity were analyzed in mouse brain tissue and in LPS/IL-1β-stimulated peritoneal macrophages with pharmacological interventions. Results Prophylactic MEL improved survival and intestinal injury, and reversed NEC-induced long-term spatial memory deficits. NEC triggered a sustained M1 microglial polarization in the hippocampus that persisted into adulthood, which was durably attenuated by MEL. IL-1β was identified as the most prominently upregulated cytokine, and transcriptomic profiling confirmed enrichment of IL-1β-related pathways. In vitro, recombinant IL-1β directly drove M1 polarization, while IL-1β neutralization blocked this effect. Mechanistically, MEL restored AMPK phosphorylation in NEC brains, and AMPK inhibition abolished the MEL-mediated suppression of M1 polarization. Conclusions Prophylactic MEL ameliorates NEC-associated brain injury by activating AMPK to suppress IL-1β production, thereby inhibiting chronic M1 microglial polarization. This AMPK–IL-1β axis represents a key therapeutic target, with MEL providing dual gut–brain protection.