The invasive activation and apoptosis resistance of fibroblast-like synoviocytes (FLS) are primary drivers of synovial inflammation and joint destruction in rheumatoid arthritis (RA), yet therapeutic strategies that can selectively reprogram these pathogenic cells remain limited.

After identifying the GAS6/MERTK axis as a pivotal transcriptomic regulator in RA synovial tissues, we employed a "dual-key inhibition" strategy by combining Xinfeng Capsules (XFC)—a traditional Chinese medicine formula—with siRNA-mediated knockdown of GAS6 and MERTK in patient-derived RA-FLS. The therapeutic efficacy was evaluated through CCK-8, scratch wound, and flow cytometry assays to assess cell viability, migration, and apoptosis, respectively. Cytokine profiles were quantified via ELISA, and the underlying signaling cascades (AXL, CRK, DOCK1, ERK, FAK, and Rac1) were analyzed by Western blotting. Network pharmacology and molecular docking were further integrated to pinpoint the bioactive components and their molecular targets.

XFC-containing serum significantly suppressed RA-FLS viability and migration while augmenting apoptosis in a dose- and time-dependent manner. Treatment with XFC effectively recalibrated the cytokine balance by reducing pro-inflammatory markers (IL-1β, IL-6, and TGF-β1) and upregulating the anti-inflammatory cytokine IL-10. Crucially, the silencing of GAS6 or MERTK exacerbated these effects, with the triple combination (si-GAS6 + si-MERTK + XFC) yielding the most potent synergistic inhibition of the pathogenic FLS phenotype. Mechanistically, this synergy was driven by the downregulation of the AXL/CRK/DOCK1/ERK/FAK/Rac1 pathway. Molecular docking revealed that triptolide, a key constituent of XFC, exhibits robust binding affinity for DOCK1 (-8.9 kcal/mol) and other hub proteins within the GAS6/MERTK signaling axis.

XFC attenuates the aggressive phenotype of RA-FLS by orchestrating a multi-pathway modulation centered on the GAS6/MERTK axis. This study highlights the innovative "dual-key inhibition" strategy as a promising therapeutic paradigm for RA, providing a strong mechanistic rationale for integrating herbal formulations with targeted molecular interventions to restore synovial homeostasis.

In summary, this study identifies the GAS6/MERTK axis as a critical driver of the pathogenic transformation in RA-derived fibroblast-like synoviocytes (FLS). Our findings demonstrate that Xinfeng Capsules (XFC) exert potent anti-inflammatory, pro-apoptotic, and anti-migratory effects, effectively suppressing the "tumor-like" behavior of RA-FLS. By integrating transcriptomic profiling with experimental validation, we establish a novel 'dual-key' blockade strategy—simultaneously targeting the GAS6 ligand and its MERTK receptor—to effectively truncate the pathogenic signaling flow. This study reveals that XFC acts as a pharmacological sensitizer that synergistically enhances the efficacy of molecular-targeted interventions. Furthermore, network pharmacology and molecular docking confirm that triptolide, a primary bioactive constituent of XFC, possesses high binding affinity for hub proteins within this axis, particularly DOCK1(Fig. 7). These results provide a robust mechanistic foundation for the clinical application of XFC and suggest that targeting the GAS6/MERTK-mediated matrix remodeling pathway represents a promising adjunct strategy to conventional DMARDs for achieving sustained remission in rheumatoid arthritis.