Melatonin ameliorates heatstroke-induced liver injury by inhibiting neutrophil recruitment and differentiation.

Heatstroke (HS) is a systemic and critical disease caused by high fever, known as a "sepsis-like reaction". Liver injury is one of the most common complications in HS and is closely related to its occurrence and development. This study utilized single-cell RNA sequencing (scRNA seq) to reveal the regulatory effects of Melatonin on the hepatic cellular landscape and immune environment changes in HS mice. Our results indicated that melatonin could effectively inhibit the inflammatory response, oxidative stress response, and liver cell apoptosis. scRNA seq showed that the large recruitment and infiltration of neutrophils mediated by the Anxa1-Fpr1 ligand-receptor interaction is the most significant immune environment change feature of early liver injury in HS. Our data suggest that melatonin is associated with alleviated neutrophil recruitment and decreased expression of Anxa1 on neutrophils, whereas Fpr1 expression showed differential regulation; the observed upregulation of Fpr1 in the melatonin group may represent a compensatory response, though this hypothesis requires further investigation. Neutrophils in HS liver injury exhibit significant heterogeneity, with Cd177+ neutrophils as the dominant subgroup. Cd177+ neutrophils are associated with highly activated features and correlate with liver injury as suggested by excessive neutrophil extracellular traps (NETs). Melatonin is associated with reduced infiltration of Cd177+ neutrophils in HS liver injury, correspondingly reducing the expression level of NETs in HS mice and associating with alleviated liver injury. The potential mechanism of action of Melatonin may involve inhibition of the key transcriptional regulator Cebpe of Cd177+ neutrophils, which may inhibit the differentiation of Cd177- neutrophils into Cd177+ neutrophils.