Xinfeng Capsule ameliorates RA joint injury by inhibiting cuproptosis via the METTL3/miR-221/222-3p/ATP7A Axis

BACKGROUND: Xinfeng Capsule (XFC), a traditional Chinese herbal formulation, has long been used clinically for rheumatoid arthritis (RA); however, its underlying mechanism remains unclear. OBJECTIVE: This study explored whether XFC alleviates RA-induced joint damage by suppressing cuproptosis via modulation of the METTL3/miR-221/222-3p/ATP7A axis. METHODS: Potential therapeutic targets of XFC against RA were predicted using bioinformatic analyses. Adjuvant-induced arthritis (AA) rat models and in vitro chondrocyte injury models were established and allocated to different treatment groups. Dual-luciferase reporter assay confirmed the interaction between miR-221/222-3p and ATP7A. Optimal XFC treatment conditions (10 % for 24 h) were determined with a CCK-8 assay. The expression levels of METTL3, miR-221/222-3p, ATP7A, ATP7B, and key cuproptosis-related markers were assessed by RT-qPCR and western blotting. Functional assays were performed to evaluate cell morphology, reactive oxygen species (ROS), inflammatory cytokines, and intracellular copper accumulation. RESULTS: Bioinformatic analysis identified METTL3 as a pivotal target. In both animal and cell models, XFC markedly downregulated METTL3 and miR-221/222-3p while upregulating ATP7A and ATP7B. The direct targeting of ATP7A by miR-221/222-3p has been previously validated. Molecular docking revealed favorable binding between active XFC components and METTL3. Functionally, XFC suppressed the METTL3/miR-221/222-3p/ATP7A path...