Neuroprotective effects of Chaihu-Longgu-Muli Decoction in an insomnia-depression comorbidity model via Galectin-3-dependent JAK2/STAT3 inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaihu-Longgu-Muli Decoction (CLMD) is a classical Traditional Chinese Medicine (TCM) formula from the Shanghan Lun comprising Bupleureum chinense DC., Os Draconis, Concha Ostreae, and the other herbs. Traditionally used to harmonize Shaoyang, calm Shen (spirit), and treat emotional disturbances and insomnia related to liver qi stagnation. AIM OF THE STUDY: To evaluate CLMD's therapeutic efficacy in insomnia-depression comorbidity and elucidate molecular mechanisms through bioinformatics and experimental validation. MATERIALS AND METHODS: Differentially expressed genes were identified from the GEO database, and key targets were screened using machine-learning approaches. Functional enrichment and single-cell analyses were performed to explore biological relevance. Molecular docking was used to assess compound target interactions. A mouse model of insomnia-depression comorbidity was established using chronic unpredictable mild stress combined with sleep deprivation, followed by CLMD treatment and behavioral evaluation. In addition, in vitro experiments were conducted in astrocytes with Galectin-3 (Gal-3) overexpression to further investigate the underlying mechanisms. RESULTS: Bioinformatics identified Gal-3 as the core target, and KEGG/GSEA highlighted JAK2/STAT3 signaling. Docking indicated strong binding of major CLMD constituents to Gal-3. In a CUMS plus sleep deprivation mouse model, CLMD improved depressive-like behaviors and sleep architecture, reduced Gal-3/JAK2/p-STAT3 expression, and regulated inflammatory and neurochemical markers. In vitro, CLMD improved cell viability, reduced apoptosis and ROS, and suppressed Gal-3-induced JAK2/STAT3 activation. CONCLUSIONS: CLMD alleviates insomnia-depression comorbidity by inhibiting Gal-3-mediated JAK2/STAT3 signaling, reducing neuroinflammation and enhancing neuroprotection.