ORIGINAL RESEARCH article
Front. Immunol.
Sec. Nutritional Immunology
Machine learning-guided discovery of Gnaphalium hypoleucum DC Flavonoids as multitarget rheumatoid arthritis therapeutics via anti-inflammatory effects and gut microbiota regulation
Provisionally accepted
- 1School of Basic Medicine, Youjiang Medical University for Nationalities, Baise, China
- 2Xinjiang University, Urumqi, China
- 3Guangdong University of Technology, Guangzhou, China
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Abstract: 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. 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 (AUC_train = 0.959; AUC_val ≥ 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. 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.
Keywords: intestinal microflora, machine learning, molecular docking, rheumatoid arthritis (RA), Total flavonoids of G. hypoleucum DC. (GHTFs)
Received: 26 Oct 2025; Accepted: 16 Feb 2026.
Copyright: © 2026 Li, Chu, Xu, Wei, Nong, Luo, Wang and Zeng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Hong Zeng
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