Background: Raf kinase inhibitory protein (RKIP) is a critical signaling modulator with well-characterized roles in oncology; however, its involvement in the pathogenesis of rheumatoid arthritis (RA) remains largely unexplored. Identifying novel upstream regulators that control synovial inflammation and bone destruction is essential for developing effective therapeutic strategies.

Objectives: The aim of this study was to identify RKIP as a potential therapeutic candidate for RA by integrating AI-driven structure prediction with experimental validation. We specifically sought to elucidate the mechanism by which RKIP modulates inflammatory signaling pathways in fibroblast-like synoviocytes (FLS) and to evaluate its therapeutic efficacy in vivo.

Methods: Differentially expressed genes (DEGs) were identified from RA synovial tissue datasets in the Gene Expression Omnibus (GEO). We utilized bioinformatics pipelines, including Protein-Protein Interaction (PPI) networks and pathway enrichment analyses, to identify the intersection between RKIP-interactors and RA-DEGs. AI-based AlphaFold protein structure prediction and docking simulations were employed to predict binding affinities between RKIP and key targets. Therapeutic mechanisms were validated in vitro using RA-FLS and in vivo through collagen-induced arthritis (CIA) and SCID mouse co-implantation models.

Results: Computational analysis identified 5,342 DEGs, 46 of which intersected with known RKIP-associated genes, primarily mapping to MAPK/ERK and PI3K/Akt pathways. AlphaFold docking simulations confirmed high-affinity binding interactions between RKIP and these intersection proteins. In vitro, RKIP knockdown activated NF-κB and ERK1/2 signaling, whereas its overexpression inhibited these pathways, significantly reducing pro-inflammatory gene expression and osteoclastogenic potential. In vivo, RKIP overexpression in RA-FLS significantly attenuated synovial invasion, bone erosion, and reduced overall clinical severity scores in RA mouse models.

Conclusions: Our findings establish RKIP as a key upstream regulator that suppresses RA pathogenesis via the inhibition of NF-κB and ERK signaling. This study highlights the synergy of bioinformatics and AI in therapeutic target discovery, positioning RKIP restoration as a promising strategy to modulate inflammation and preserve joint integrity in RA.

REFERENCES: NIL.

Acknowledgments: NIL.

Disclosure of Interests: None declared.