Elucidating the Mechanism of the Liqi Yangyin Formula in Treating Depression–Constipation Comorbidity: An Integrative Approach Using Network Pharmacology and Experimental Validation

Background: The traditional formula Liqi Yangyin (LQYY) has shown clinical and preclinical efficacy for depression with constipation, yet its molecular mechanisms remain incompletely defined. This study aimed to elucidate its mechanisms using an integrative approach. Methods: Constituents of LQYY were profiled by UPLC-MS/MS and integrated with network pharmacology and molecular docking to identify brain-accessible components and putative targets. A chronic unpredictable mild stress (CUMS) model was used for experimental validation. Outcomes included behavioral tests (sucrose preference test, open field test, and forced swimming test), gastrointestinal indices, including fecal water content, time of first black stool, and intestinal propulsion rate, histopathology of the prefrontal cortex (PFC) and colon, TUNEL staining, NeuN immunofluorescence, Western blotting, and qRT-PCR. Results: LQYY attenuated CUMS-induced weight loss and depressive-like behaviors and improved intestinal transit metrics. It reduced neuronal apoptosis in the PFC and ameliorated colonic injury. Mechanistically, docking and enrichment analyses highlighted hub targets (STAT3, AKT1, ESR1, IL-6, TNF, TP53) and the JAK/STAT pathway. In vivo, LQYY decreased IL-6, TNF-α, ESR1, TP53, and STAT3, and increased AKT1 in the PFC and colon; it also reduced the TUNEL-positive rate and restored NeuN labeling, upregulated Bcl-2, and downregulated p-JAK2/JAK2 and p-STAT3/STAT3 ratios, and the expression of Bax and cleaved-caspase-3 in the PFC, consistent with the suppression of pro-inflammatory and apoptotic signaling. Conclusions: LQYY exerts antidepressant and pro-motility effects in CUMS mice by modulating JAK2/STAT3-centered networks and inhibiting neuronal apoptosis, thus supporting a multi-component, multi-target strategy for treating depression with constipation, and providing a defined molecular hypothesis for future investigation.