Zhuche Pill alleviates DSS-induced colonic epithelial injury in mice: involvement of IL-17/NF-κB and Wnt/β-catenin signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Zhuche Pill (ZCP), a traditional Chinese medicine formula first documented in Qianjin Yaofang during the Tang Dynasty, has been used for centuries to treat dysentery-associated gastrointestinal disorders, including abdominal pain, diarrhea, hematochezia, and tenesmus. Despite the long-standing clinical application of ZCP, the pharmacological basis underlying its therapeutic effects remains incompletely understood. AIM OF THE STUDY: This study aims to evaluate the protective effects of ZCP against colitis and explore its potential underlying mechanisms. METHODS: The efficacy of ZCP was evaluated in a DSS-induced mouse model of ulcerative colitis (UC), with mesalazine as the positive control. Macroscopic and histological parameters were assessed to evaluate the effects of ZCP on intestinal inflammation and epithelial barrier integrity. To investigate the potential underlying mechanisms, this study integrated Gene Expression Omnibus (GEO) dataset mining, network pharmacology prediction, and RNA-seq transcriptomic profiling, followed by molecular validation in colonic tissues of DSS mice using reverse transcription quantitative PCR (RT-qPCR), Western blotting (WB), and immunofluorescence (IF) staining. In parallel, primary crypt-derived colonic organoids were established as an in vitro UC surrogate model to corroborate the in silico and in vivo findings. Additionally, the chemical composition of ZCP was characterized by ultra-performance liquid chromatography coupled with quadrupole-Orbitrap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS). RESULTS: ZCP treatment significantly ameliorated weight loss, diarrhea, and hematochezia in UC mice, as reflected by a reduced disease activity index (DAI) score. Histologically, ZCP attenuated inflammatory cell infiltration and reduced colonic levels of TNF-α, IL-6, and IL-1β, while increasing the expression of the tight junction protein Occludin and the mucin MUC2. Integrative analyses encompassing GEO data mining, network pharmacology, and RNA-seq identified the IL-17/NF-κB and Wnt signaling pathways as prominently implicated in UC pathogenesis. In contrast, ZCP administration was associated with attenuation of IL-17/NF-κBIL-17 signaling and enhancement of Wnt/β-catenin signaling, as evidenced by reduced IL-17RA, ACT1, TRAF6, and a decreased p-p65/p65 ratio, together with increased levels of β-catenin, Cyclin D1, c-Myc, Pcna, Yap1 and Axin2. These alterations were associated with an increased BCL-2/BAX ratio, consistent with reduced epithelial apoptosis and enhanced regenerative responses. Consistent with these in vivo observations, organoid-based in vitro experiments suggested that ZCP downregulated IL-17RA, ACT1, TRAF6 and p-p65, while upregulating ZO-1, Occludin and E-cadherin. Moreover, ZCP treatment was associated with enhanced Wnt/β-catenin signaling and increased numbers of LGR5+ and Ki-67+ cells, consistent with enhanced epithelial regeneration and reduced apoptosis. Finally, UPLC-Q-Orbitrap HRMS identified 90 compounds in ZCP, with berberine and palmatine as the major constituents, providing a phytochemical basis for further investigation. CONCLUSION: ZCP ameliorates DSS-induced epithelial injury and is associated with attenuation of IL-17/NF-κB inflammatory signaling and enhancement of Wnt/β-catenin-associated regenerative responses.