Dachengqi decoction alleviates constipation by inhibiting the 5-HT pathway-mediated PANoptosis and improving metabolic abnormalities.

ETHNOPHARMACOLOGICAL RELEVANCE: Dachengqi decoction (DCQ), a well-known Traditional Chinese Medicine (TCM) formula, has been widely applied for anti-constipation in clinical practice for thousands of years. Nevertheless, its pharmacological mechanisms remain not fully elucidated.

AIM OF THE STUDY: This research employed a comprehensive approach integrating network pharmacology and metabolomics, supplemented with molecular docking techniques and both in vitro and in vivo experimental validation, to systematically elucidate the bioactive constituents, critical targets, and underlying mechanisms of DCQ in alleviating constipation.

MATERIALS AND METHODS: This investigation utilized a comprehensive pharmacology integration strategy to forecast the principal therapeutic pathways through which DCQ alleviates constipation. The establishment of a loperamide-induced constipation model in rats served to evaluate the interventive effects of orally administered DCQ. In addition, ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) was used to detect the changes of metabolites in plasma and urine of rats, and the metabolic related targets were analyzed. Combined with network pharmacology, the potential mechanism of DCQ in ameliorating constipation was revealed and further verified by molecular docking. According to the results of joint analysis, the intervention of DCQ mainly involved the serotonin (5-HT) pathway, including the inflammatory pathways of arachidonic acid and cAMP downstream of 5-HT. Therefore, the mRNA and protein expression levels of key factors associated with the 5-HT pathway, including phospholipaseA2 (PLA2), cAMP/PKA/AQPs, and PANoptosis, were detected. Furthermore, an inflammatory model of Caco-2 cells was induced to investigate the effect of DCQ on the 5-HT pathway. The expression of related targets (PLA2, PKA and ZBP1) was detected by reverse transcription quantitative PCR and immunofluorescence to further verify the molecular mechanism of DCQ.

RESULTS: DCQ demonstrated efficacy in ameliorating constipation symptoms induced by loperamide in rats and reducing inflammatory responses in Caco-2 cells, while normalizing aberrant plasma and urinary metabolic profiles and regulating arachidonic acid metabolism in constipated rats. Integrated metabolomics and network pharmacology analyses revealed that DCQ mainly alleviated constipation via the 5-HT pathway, markedly suppressing the PLA2-mediated inflammation and PANoptosis while concurrently activating the cAMP-PKA-AQPs cascade to notably increase the expression of aquaporins (AQPs).

CONCLUSIONS: DCQ could restore the homeostasis of metabolic profiles in plasma and urine in model rats, activate the 5-HT pathway to alleviate inflammation, and may be involved in the regulation of PANoptosis-related mechanisms in intestinal epithelial cells, providing a new perspective for constipation treatment.