Revealing the mechanism of suanzaoren tang against insomnia via integrated metabolomics and gut microbiota analysis.

Abstract

Suanzaoren Tang (SZRT), a classic prescription from the Jingui Yaolue, is commonly used to treat insomnia associated with liver-blood deficiency. However, the mechanisms underlying SZRT's treatment of insomnia remain poorly understood. Using an integrated multi-omics approach, this research aimed to systematically explore the underlying mechanisms through which SZRT exerted its alleviating efficacy in p-chlorophenylalanine (PCPA)-induced insomnia. UPLC-ESI-MS/MS analysis was performed to identify the compounds in SZRT. Intraperitoneal administration of PCPA was utilized to develop an insomnia model in rats. Behavioral evaluation, ELISA, immunofluorescence, untargeted metabolomics and 16S rRNA sequencing were applied to assess how SZRT affected sleep quality, neurotransmitters, inflammatory markers, gut microbiota composition, and metabolic pathways. 30 compounds were identified in SZRT. PCPA treatment significantly prolonged sleep latency and altered neurochemical profiles, including decreased γ-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) levels, and increased glutamate (Glu), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and CYP2E1 expression. Immunofluorescence revealed reduced expression of glial fibrillary acidic protein (GFAP), ionized calcium-binding adapter molecule 1(Iba-1), 5-HTA, and 5-HTA receptors, indicating glial cell dysfunction and serotonergic dysregulation in PCPA group. SZRT intervention reversed these changes. Microbiota analysis showed that PCPA disrupted gut microbial composition, with decreased Prevotellaceae and Butyricicoccus abundance, both of which were restored by SZRT. Metabolomics identified key differential metabolites enriched in branched-chain amino acid metabolism, D-amino acid metabolism, α-linolenic acid metabolism, and arachidonic acid pathways. SZRT restored neurotransmitter homeostasis and reduced inflammation via microbiota-metabolite interactions. Suanzaoren Tang may improve insomnia by modulating the gut microbiota-metabolome-brain axis and glial cell activity, thereby restoring neurotransmitter balance, attenuating oxidative stress, and suppressing neuroinflammation. These findings provide experimental support for the clinical application of SZRT in insomnia treatment.