Orexinergic modulation by Uwhangchungsimwon restores homeostasis of GABA/BDNF signaling in insomnia-depression comorbidity.

ETHNOPHARMACOLOGICAL RELEVANCE: Uwhangchungsimwon (UCW) is a traditional East Asian multi-herbal formulation, used for managing stroke, convulsions, and mental confusion. Given the close association between stress, sleep disturbance, and mood disorders, UCW may hold therapeutic potential for insomnia-depression comorbidity, although its underlying mechanisms remain unclear. This study aims to explore the therapeutic potential of UCW for insomnia-depression comorbidity by targeting the orexinergic and GABAergic systems and identifying its bioactive compounds. METHODS: In vivo experiments were performed in caffeine (CAF)-induced insomnia models of ICR mice, treated with CAF and UCW for 1 or 6 days. Sleep-wake patterns were monitored with the PiezoSleep system, and depression-like behaviors assessed using the Open Field Test (OFT) and Tail Suspension Test (TST). Hypothalamus and hippocampus from chronically exposed mice were analyzed for biomarker changes. To identify UCW's bioactive compounds, network pharmacology, molecular docking, molecular dynamics, DARTS assay, and calcium influx assay were employed. RESULTS: UCW (400-800 mg/kg, po) markedly improved sleep-wake patterns, circadian rhythm, and depression-like behaviors in CAF-treated mice. CAF elevated serum corticosterone, hypothalamic orexin A, and OX1R, OX2R, GABABR1, BDNF, TNF-α, IL-6, and IL-1β levels in the hypothalamus and hippocampus, which were all reversed by UCW. In silico, in vitro, and in vivo analyses identified stigmasterol as a key UCW compound that reduced orexin-A-induced calcium influx and ERK1/2 phosphorylation and modulated GABABR1 and BDNF expression. CONCLUSION: UCW exerts sleep-promoting and antidepressant-like effects by modulating inflammatory markers and OX2R-GABABR1-BDNF-ERK signaling. UCW and stigmasterol are potential therapeutic agents for insomnia and depression.