Exploring the Anti-Rheumatoid Arthritis Inflammatory Mechanism of Shikonin Based on Network Pharmacology and Experimental Validation

Introduction: Shikonin (SKN) is known for its anti-inflammatory effects; however, the molecular mechanisms underlying its therapeutic action in rheumatoid arthritis (RA) remain unclear. This study aimed to investigate the anti-inflammatory effects of SKN in RA using an integrative approach combining network pharmacology and experimental validation. Methods: Potential targets for SKN and RA were identified using multiple databases and network pharmacology approaches. The overlapping targets were subsequently analyzed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Meanwhile, a protein-protein interaction (PPI) network was constructed to screen for core targets. Molecular docking was performed to evaluate binding affinities between SKN and the core targets. In vitro, the effects of SKN on RA fibroblast-like synoviocytes (RA-FLSs) were assessed using MTT assays, ELISA, qRT-PCR, and Western blot analyses. Results: We identified 194 potential targets for SKN and 942 targets associated with RA. Among these, 87 overlapping targets were considered potential targets of SKN against RA and were primarily involved in cytokine-mediated signaling pathways, regulation of inflammatory responses, and the PI3K-AKT and NF-κB signaling pathways. Nine core targets—AKT1, BCL2, CCL2, EGF, INS, MMP9, NFκB1, TNF, and TP53—were screened through protein-protein interaction (PPI) network analysis. Molecular docking revealed stable binding affinities between SKN and all nine core targets (< -5 kcal/mol). In vitro, SKN significantly inhibited RA fibroblast-like synoviocyte (RA-FLS) proliferation and reduced the production of IL-17, IL-8, TNF-α, MMP3, and MMP9 in a dose-dependent manner. Furthermore, SKN downregulated the mRNA expression of most core targets and suppressed the PI3K-AKT and NF-κB signaling pathways. Discussion: This article offers a methodological framework for the future development of natural products with anti-RA properties, while also incorporating data and insights from recent studies on biologics and JAK inhibitors in RA treatment, to enhance the evidence base for SKN's role in managing RA. We believe that our research provides novel insights into the therapeutic potential of SKN for RA. Conclusion: SKN has shown potential as a therapeutic agent for RA by acting on multiple core targets and signaling pathways.