Insomnia is a multifactorial sleep-wake disorder associated with altered neurotransmitter homeostasis, systemic inflammatory changes, and gut microbial dysbiosis. Recombinant bovine αs1-casein (RC) is a scalable milk-protein ingredient, but its effects on insomnia-like physiological and behavioral abnormalities have not been systematically evaluated. We investigated RC in a p-chlorophenylalanine (PCPA)-induced mouse model. Male ICR mice received RC (100, 300, or 500 mg kg-1 day-1), melatonin, Naoxinshu oral liquid, natural casein, or vehicle for 7 days after PCPA modeling. Body weight, open-field behavior, serum cytokines, brain neurotransmitters, hippocampal histology, organ indices, and cecal microbiota were assessed. RC improved body-weight recovery and open-field performance without altering major-organ indices. It increased brain 5-HT, 5-HIAA, dopamine, GABA, and glutamate concentrations and reduced the elevated norepinephrine concentration. High-dose RC reduced serum IL-6 and was associated with better-preserved hippocampal morphology. 16S rRNA gene sequencing showed RC-associated changes in bacterial richness and community structure. The parallel physiological, neurochemical, inflammatory, and microbial changes support an association between RC administration and improvement of PCPA-induced insomnia-like abnormalities. These results identify recombinant bovine αs1-casein as a promising food-derived ingredient for further investigation in sleep-health applications, while the microbiota-gut-brain axis is presented as a testable mechanistic framework rather than a demonstrated causal pathway.
