Machine learning reveals targets of Gnaphalium hypoleucum DC. flavonoids against rheumatoid arthritis through gut microbiota and anti-inflammation

Background Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and gut microbiota dysbiosis. Gnaphalium hypoleucum DC. total flavonoids (GHTFs) exhibit anti-inflammatory and immunomodulatory properties. Methods and results In this study, we employed an integrated strategy combining machine learning (ML), molecular docking, and molecular dynamics simulations to identify active compounds within GHTFs. The therapeutic mechanisms of these compounds were further investigated using LPS-stimulated RAW264. 7 macrophages and a collagen-induced arthritis mouse model. Differential expression analysis identified 2, 676 RA-associated genes. A glmBoost + LDA model demonstrated robust diagnostic performance (AUCₜrain = 0. 959; AUCᵥal ≥ 0. 837) and prioritized five key genes (POLB, EGFR, MMP13, VEGFA, and KMT2D). Molecular docking and dynamics simulations confirmed the stable binding of amentoflavone (AF), a primary constituent of GHTFs, to core targets MMP9, MMP13, TOP2A, and ALOX5. In vitro, both GHTFs and AF inhibited proliferation, migration, and nitric oxide release in LPS-stimulated RAW264. 7 macrophages, and suppressed IL-17, TNF-α, and NF-κB signaling pathways, with AF showing more potent effects (P 0. 05). In vivo, GHTFs treatment reduced clinical arthritis scores by over 40%, alleviated synovial hyperplasia, preserved collagen volume fraction, and lowered serum levels of TNF-α and IL-1β, demonstrating superior overall efficacy compared to AF and methotrexate (P 0. 05). Gut microbiota analysis revealed that GHTFs enriched beneficial Lactobacillus species (e. g. , L. johnsonii, L. intestinalis), reduced the abundance of pro-inflammatory taxa, and restored microbial metabolic functions. Conclusions Collectively, our findings identify GHTFs as a promising therapeutic candidate for RA, ameliorating disease progression through modulation of inflammatory responses and microbiota-mediated immune regulation.