Sishen pill alleviates ulcerative colitis via the NLRP3/ASC/Caspase-1 signaling pathway: Comprehensive validation through UPLC-Q-TOF/MS, network pharmacology, molecular docking, and in vivo experiments.

BACKGROUND

The "Sishen Pill" (SSP) is a traditional Chinese medicinal formulation traditionally employed in the treatment of diarrhea attributed to spleen-kidney yang deficiency, and it has exhibited notable clinical efficacy in managing ulcerative colitis (UC). Nevertheless, the bioactive compounds and the underlying mechanisms by which SSP exerts its therapeutic effects on UC remain inadequately elucidated.

OBJECTIVES

This study sought to systematically elucidate the bioactive constituents and the mechanism of action of SSP in the treatment of UC through the application of ultra-high-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS), network pharmacology, molecular docking, and animal experimentation.

METHODS

The chemical constituents of SSP were characterized using UPLC-Q-TOF/MS. Network pharmacology was employed to predict the principal chemical constituents and core targets of SSP in the context of UC treatment, while molecular docking was utilized to assess their binding affinities. Subsequently, a rat model of UC was established, and mechanistic validation was performed using a range of techniques, including hematoxylin and eosin (HE) staining, transmission electron microscopy, immunofluorescence staining, Western blotting, reverse transcription-polymerase chain reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA).

RESULTS

In this study, a total of 79 chemical constituents were identified in SSP. Through network pharmacology analysis, Citric acid, Eugenol, Daidzein, 7-Hydroxycoumarin, 4-Hydroxycoumarin, Astragalin, and Octadecenoic acid were determined to be the core chemical constituents, with Caspase-1 and IL-1β identified as the primary targets. Molecular docking studies indicated a strong affinity between these core targets and chemical constituents. In vivo experiments demonstrated that SSP significantly mitigated weight loss symptoms, enhanced the disease activity index (DAI), and reduced colonic tissue damage in UC rats. Furthermore, SSP was found to decrease the protein expression levels of NLRP3, ASC, and Caspase-1 in colonic tissue, as well as downregulate serum levels and mRNA expression of IL-18 and IL-1β in colonic tissue.

CONCLUSIONS

SSP may exert its therapeutic effects on UC by modulating the NLRP3/ASC/Caspase-1 signaling pathway, thereby attenuating intestinal inflammatory responses and facilitating the repair of the intestinal mucosal barrier.