Therapeutic effects of shikonin on adjuvant-induced arthritis in rats and cellular inflammation, migration and invasion of rheumatoid fibroblast-like synoviocytes via blocking the activation of Wnt/βcatenin pathway

Background: Shikonin (SKN), the main bioactive component isolated from Lithospermum erythrorhizon Sieb et Zucc, has multiple activities including anti-rheumatic effect, but its specific roles and the precise mechanisms in regulating biological properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) are unclear and need further clarification. Purpose: This study explored the therapeutic roles of SKN on rat adjuvant-induced arthritis (AIA) and cellular inflammation, migration and invasion of TNF-alpha-induced RA FLS (MH7A cells), and further demonstrated the involved mechanisms. Methods: SKN was intraperitoneally given to AIA rats and its therapeutic role was valued. The effects of SKN in vivo and in vitro on the production of pro-inflammatory factors were examined by ELISA and western blot. Wound-healing, transwell and phalloidin staining assay were carried out to evaluate the effects of SKN on TNF-alpha-induced migration and invasion in RA FLS. The involvement of Wnt/beta-catenin pathway was checked by immunohistochemistry or immunofluorescence assay for beta-catenin and western blot for pathway-related proteins. Results: SKN treatment in AIA rats reduced paw swelling, arthritis index and pathological damage of ankle joints, indicating its anti-arthritic effect in vivo. SKN had anti-inflammatory roles in vivo and in vitro, evidenced by inhibiting the production of pro-inflammatory factors (like IL-1 beta, IL-6, IL-8, TNF-alpha, MMP-2 and MMP-9) in sera and synovium of AIA rats, and in TNF-a-induced MH7A cells. Gelatin zymography result revealed the suppression of SKN on TNF-alpha-induced MMP-2 activity in vitro. Moreover, SKN inhibited TNF-alpha-induced migration, invasion and cytoskeletal reorganization in MH7A cells. Mechanistically, SKN suppressed the activation of Wnt/beta-catenin signaling in AIA rat synovium and in TNF-a-induced MH7A cells, indicated by the reduced protein levels of Wnt1, p-GSK-3 beta (Ser9) and beta-catenin, the raised protein level of GSK-3 beta and the decreased nuclear translocation of beta-catenin. Interestingly, the combination of LiCl (Wnt/beta-catenin agonist) canceled the therapeutic functions of SKN on cellular inflammation, migration and invasion in TNF-a-induced MH7A cells, whereas XAV939 (Wnt/beta-catenin inhibitor) enhanced the therapeutic roles of SKN. Conclusion: SKN showed therapeutic effects on rat AIA and cellular inflammation, migration and invasion of TNF-alpha-stimulated RA FLS via interrupting Wnt/beta-catenin pathway.