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/β-catenin Pathway

Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by persistent synovial inflammation and cartilage destruction,ultimately leading to joint damage and irreversible disability.Fibroblast-like synoviocytes(FLS)are the main cell type in synovial tissue and play a crucial role in the pathological processes of synovial inflammation and cartilage injury in RA.Under normal conditions,FLS secrete synovial fluid to protect the joints.However,in the inflammatory environment of RA synovium,FLS are in an aberrantly activated state.Activated FLS exhibit tumor-like biological behaviors,such as abnormal proliferation,resistance to apoptosis,and excessive secretion of various inflammatory and chemotactic factors,further aggravating the condition of RA.Therefore,selectively inhibiting the aberrant biological characteristics of RA FLS may be an effective method for treating or controlling RA.Research has found a close association between abnormal activation of the Wnt/β-catenin signaling pathway and the pathogenesis of RA,suggesting that inhibiting the Wnt/β-catenin signaling pathway may have potential therapeutic significance for RA.Shikonin(SKN)is a natural compound isolated from the traditional Chinese herb Lithospermum erythrorhizon.It belongs to the naphthoquinone class of compounds and exhibits a wide range of pharmacological activities,such as anti-tumor,anti-inflammatory,and wound healing properties.Previous studies have shown that SKN can alleviate the development of RA in experimental animals and inhibit excessive proliferation of RA FLS.In this study,we elucidated the therapeutic effects of SKN on adjuvant-induced arthritis(AIA)in rats,revealed the anti-inflammatory effects of SKN on AIA rats and TNF-α stimulated RA FLS(MH7A cells),and evaluated the inhibitory effects of SKN on TNF-α-induced migration,invasion,and cytoskeletal rearrangement in MH7A cells in vitro.Furthermore,we clarified that SKN exerts its aforementioned therapeutic effects by inhibiting the Wnt/β-catenin signaling pathway.This study mainly conducted the following work:Objective: To elucidate the therapeutic effects of SKN on AIA rats,investigate the effects of SKN on TNF-α-stimulated migration,invasion,and inflammation in MH7A cells,and explore its underlying mechanism based on the Wnt/β-catenin pathway.Methods: AIA rat models were established by injecting complete Freund’s adjuvant(CFA)into the left paw.The severity of AIA in rats was evaluated by measuring the degree of secondary paw swelling and arthritis index changes.Ankle joints of rats were collected for HE staining and safranin O-fast green staining.Serum and synovial tissues of rats were collected,and the expression of inflammatory mediators such as IL-1β,IL-6,TNF-α,MMP-2,and MMP-9 was detected using ELISA and western blot.Immunohistochemistry(IHC)was used to detect the expression of β-catenin in synovial tissues.Western blot was performed to detect the expression of Wnt/β-catenin pathway-related proteins in synovial tissues.The MTT assay was used to screen for non-toxic doses of SKN.ELISA and western blot were conducted to determine the expression of inflammatory cytokines(IL-1β,IL-6,IL-8)and matrix metalloproteinases(MMP-2/9),and gelatin zymography was performed to measure MMP-2 activity.Scratch healing and Transwell assays were used to evaluate the migration and invasion of MH7A cells.Phalloidin staining was performed to examine the actin cytoskeleton in the cells.Immunofluorescence staining was used to detect β-catenin nuclear translocation,and western blot was used to measure the expression levels of Wnt/β-catenin pathway-related proteins.Results: SKN reduced the degree of paw swelling,arthritis index,and alleviated ankle joint pathological damage in AIA rats,indicating its anti-arthritic effects.SKN inhibited the expression of pro-inflammatory cytokines(such as IL-1β,IL-6,IL-8,TNF-α,MMP-2,and MMP-9)in the serum and synovial tissues of AIA rats,as well as in TNF-α-induced MH7A cells,indicating its anti-inflammatory effects in both in vivo and in vitro settings.Gelatin zymography results showed that SKN had inhibitory effects on MMP-2 activity in TNF-α-induced MH7A cells.Additionally,SKN inhibited the migration,invasion,and cytoskeletal rearrangement of TNF-α-induced MH7A cells.Mechanistically,SKN inhibited the activation of the Wnt/β-catenin pathway in synovial tissues of AIA rats and TNF-α-induced MH7A cells,manifested by decreased levels of Wnt1,p-GSK-3β(Ser9),and β-catenin proteins,increased levels of GSK-3β protein,and reduced nuclear translocation of β-catenin.Interventions targeting the Wnt/β-catenin pathway revealed that in TNF-α-induced MH7A cells,the combined use of Li Cl(a Wnt/β-catenin agonist)reversed the inhibitory effects of SKN on cell inflammation,migration,and invasion,while XAV939(a Wnt/β-catenin inhibitor)enhanced the inhibitory effects of SKN.Conclusion: Shikonin exerts therapeutic effects on AIA rats and inhibits TNF-α-stimulated migration,invasion,and expression of inflammatory factors in MH7A cells by inhibiting the Wnt/β-catenin pathway.