Abstract
<title>Abstract</title> <p>Objective Zhufu-Tongbi Granules (ZFTB), originated from the clinical experience of nationally renowned physician Professor Changchuan Bai, has presented satisfied clinical benefits in treating rheumatoid arthritis (RA), while the underlying acting mechanisms remain unclear. This study aims to clarify the anti-arthritic effects and related potential mechanisms of ZFTB. Methods Chemical constituents of ZFTB were profiled by UPLC-Q-TOF-MS/MS. Therapeutic efficacy was assessed in adjuvant-induced arthritis with cold-damp stimulation (AIA-C) rats via observing arthritis index, pain thresholds, ankle histopathology, micro-CT, blood counts, and serum cytokines. Corresponding mechanism was explored through integrated multi-omics (fecal metagenomics, synovial transcriptomics, serum SCFA metabolomics) and validated through western blot, ELISA, and multiplex immunofluorescence. Results ZFTB contained 109 constituents, which predominantly comprised 27 prenol lipids, 26 flavonoids, and 20 phenylpropanoids. ZFTB administration significantly reduced arthritis scores, paw edema, and serum TNF-α and IL-1β levels, elevated pain thresholds, and normalized hematological and histopathological changes. Multi-omics revealed that ZFTB reversed gut dysbiosis by modulating key phyla (e.g., Bacteroidota, Verrucomicrobiota, Bacillota) and normalizing the butyrate-producing microbiota, thereby replenishing serum butyrate. Correspondingly, synovial transcriptomics showed upregulation of Wnt antagonist Sfrp2 and gluconeogenic gene G6pc1, and downregulation of Wnt/glycolysis‑associated genes (e.g., Wnt16, Mmp11, Ldhb). Further protein validation confirmed increased SFRP2 and G6PC1, and suppressed β-catenin, Cyclin D1, VEGFA, MMP11, and LDHB. Conclusion ZFTB ameliorates RA by orchestrating a gut microbiota-butyrate-Wnt signaling axis, which restores microbial homeostasis and suppresses aberrant Wnt signaling, providing a strong pharmacological rationale for its clinical application.</p>