Tianwang Buxin Dan regulated PI3K/Akt/mTOR signaling pathway to improve constipation and emotional disorders in rats

Functional constipation (FC) is frequently comorbid with anxiety and depression, yet pharmacological strategies targeting both gastrointestinal and emotional symptoms remain limited. This study investigated the therapeutic effects and molecular mechanisms of Tianwang Buxin Dan (TWBXD), a classical Chinese herbal formula, in treating FC with emotional disturbances using an integrated strategy combining network pharmacology, molecular docking, and in vivo validation.

TWBXD chemical constituents were characterized exclusively by UHPLC-MS/MS, yielding 650 compounds for network pharmacology. Targets were predicted via TargetNet and intersected with disease-associated genes from GeneCards. Common targets underwent PPI network analysis, GO/KEGG enrichment, and molecular docking. In vivo, a rat model was established by CUMS plus diphenoxylate and treated with TWBXD (low, medium, high doses) or positive drugs (mosapride plus fluoxetine) for 28 days. Therapeutic effects and pathway regulation in colon and hippocampus were evaluated by behavioral tests, gastrointestinal function assessment, histopathology, transmission electron microscopy, Western blotting, and qPCR.

Network pharmacology identified 473 drug targets and 308 disease-overlapping targets. KEGG enrichment highlighted PI3K-Akt, MAPK, IL-17, and neuroactive ligand-receptor interaction pathways, while PPI analysis identified Akt1, IL-6, and TNF as hub genes. Molecular docking showed quercetin, luteolin, and tanshinone IIA bound stably to Akt1, IL-6, and JUN (binding energies ≤ -6.6 kcal/mol). In vivo, TWBXD dose-dependently improved fecal water content, intestinal transit, sucrose preference, and open-field performance, and attenuated colonic mucosal injury, hippocampal neuronal damage, and mitochondrial disruption. Mechanistically, TWBXD was associated with increased PI3K/Akt/mTOR phosphorylation in colonic and hippocampal tissues and with reduced JNK expression in the hippocampus. No organ toxicity was observed.

TWBXD alleviates constipation-related gastrointestinal dysfunction and comorbid anxiety/depression-like behaviors in rats, and these effects are associated with activation of PI3K/Akt/mTOR signaling in colonic and hippocampal tissues and with modulation of JNK signaling in the hippocampus. Although the concordance between computational predictions and multi-level in vivo validation supports TWBXD as a multi-target therapeutic candidate for functional constipation with emotional comorbidities, the causal contribution of PI3K/Akt/mTOR signaling, and the extent to which the peripheral and central changes are functionally coupled through the gut-brain axis, remain to be established.