Sishen pill alleviates DSS induced colitis through AGE/RAGE/NLRP3 pathway based on transcriptomics analysis

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is a chronic inflammatory disorder of the colon associated with symptoms of abdominal pain, diarrhea, and rectal bleeding. Sishen Pill (SSP), a classical formulation documented in "The Secret Biography of Hua Tuo Divine Doctors ", has been widely utilized for over 1600 years in the treatment of gastrointestinal disorders, yet its precise pharmacological mechanisms require systematic elucidation. AIM OF STUDY: The therapeutic efficacy of SSP in UC and its underlying pathways were investigated using integrative approaches. MATERIALS AND METHODS: The phytochemical components of SSP were evaluated using UPLC-MS/MS. A mouse UC model was developed using 72 male C57BL/6 mice administered 2.5 % dextran sulfate sodium (DSS) to evaluate SSP's therapeutic effects. Comprehensive assessments included clinical manifestations (changes in body weights, colon lengths, and disease activity indices), systemic inflammatory responses (serum IL-6, IL-1β, and IL-13 levels), intestinal barrier integrity (occludin and claudin-1 expression via immunohistochemistry), and histopathological changes analyzed by H&E staining. Mechanistic exploration combined RNA sequencing with network pharmacology to identify pivotal pathways, which were further verified in mice and cells using pharmacological interventions and investigating of the AGE-RAGE-NLRP3 axis, respectively. RESULTS: UPLC-MS/MS analysis identified 100 chemical constituents in SSP. The treatment group showed significant mitigation of colitis manifestations compared to DSS controls, including restored body weights and colon lengths, and reduced disease activity indices and histopathological damage. SSP administration lowered production of pro-inflammatory cytokines (IL-6, IL-1β) while increasing that of the anti-inflammatory IL-13, as well as restoration of tight junction protein levels (occludin, claudin-1). Network pharmacology predictions combined with RNA-seq profiling revealed AGE-RAGE-NLRP3 axis as the core regulatory pathway, which was functionally verified through both animal and cell-based experiments. CONCLUSIONS: SSP exhibited a protective effect against UC and ameliorated the condition by inhibiting the AGEs-RAGE-NLRP3 signaling pathway.