Mulberry fruit anthocyanin extract modulates insomnia-related behavioral and neurochemical alterations via the gut–brain axis: A multi-omics and network pharmacology study

Insomnia and related neurological disorders have emerged as major global public health problems. Although mulberry fruit exhibits neuroprotective properties, its underlying mechanisms in sleep regulation remain poorly understood. This study systematically investigated the therapeutic effects and molecular mechanisms of mulberry fruit anthocyanin extract MFE by using an integrative approach of animal experiments, multi-omics analyses, and network pharmacology in an insomnia mouse model. We evaluated behavioral changes, neurotransmitter levels, inflammatory cytokines, gut microbiota composition via 16S rRNA sequencing, and serum metabolomics, and further validated key regulatory pathways and genes through RT-qPCR. MFE treatment significantly attenuated insomnia-related behavioral abnormalities, as evidenced by improved locomotor activity and attenuated body weight loss in model mice. Mechanistically, MFE restored the GABA/Glu balance and elevated 5-HT levels in brain tissue while mitigating neuroinflammation. Gut microbiota analysis revealed that MFE improved the gut microbiota composition, reducing the Firmicutes/Bacteroidetes (F/B) ratio and enriching beneficial taxa such as Akkermansia and Muribaculaceae. Serum metabolomic analysis identified marked elevations in multiple metabolites, including amino acid derivatives, glycosides, organic acids, and aliphatic compounds. Network pharmacology predictions, corroborated by RT-qPCR, implicated the involvement of the TNF and IL-17 signaling pathways and the serotonergic synapse pathway. Consistently, MFE intervention downregulated the hippocampal mRNA expression of key pro-inflammatory genes (PTGS2, IL-6, and IL-1β) in insomnia model mice. These results reveal multifaceted association mechanism by which MFE modulates insomnia-associated neurochemical and behavioral alterations, concurrently involving the modulation of neurotransmitter homeostasis, neuroinflammation, gut microbiota ecology, and systemic metabolism. The findings suggest a potential involvement of the gut-brain axis as a mechanistic hypothesis that warrants further causal validation that warrants further causal validation. This study provides mechanistic insights and a scientific rationale for developing MFE as a functional food for promoting sleep health.