Anti-angiogenic effect of Shikonin in rheumatoid arthritis by downregulating PI3K/AKT and MAPKs signaling pathways.

Zicao is the dried root of Lithospermum erythrorhizon Sieb, et Zucc, Arnebia euchroma (Royle) Johnst, or Arnebia guttata Bunge and commonly used to treat viral infection, inflammation, arthritis and cancer in China.Shikonin (SKN) is a major active chemical component isolated from zicao. Previous research showed that SKN has anti-inflammatory, immunomodulatory and analgesic effects, and inhibits the development of arthritis and the condition of collagen arthritis (CIA) mice; nevertheless, its role in the angiogenesis of rheumatoid arthritis (RA) has not been elucidated. The purpose of this study was to investigate the antiangiogenic activity of SKN in CIA rats and various angiogenesis models. The anti-arthritic effect of SKN on CIA rats was tested by arthritis score, arthritis incidence, radiological observation and histopathology evaluation of inflamed joints. Vessel density evaluated with CD31 immunohistochemistry/immunofluorescence in joint synovial membrane tissues of CIA rats, chick chorioallantoic membrane assay, rat aortic ring assay, and the migration, invasion, adhesion and tube formation of human umbilical vein endothelial (HUVEC) cells induced by tumor necrosis factor (TNF)-α were used to measured the antiangiogenenic activity of SKN. Moreover, the effect of SKN on the expression of angiogenic mediators, such as vascular endothelial growth factor (VEGF), VEGFR2, TNF-α, interleukin (IL)-1β, platelet derived growth factor (PDGF) and transforming growth factor (TGF)-β in sera and joint synovia of rats, and in TNF-α-induced MH7A/HUVEC cells were measured by immunohistochemistry, enzyme linked immunosorbent assay, Western blot and/or real-time polymerase chain reaction (PCR). Through the analysis of protein and mRNA levels of phosphoinositide 3-kinase (PI3K), Akt and PTEN, and the autophosphorylation of ERK1/2, JNK and p38 in joint synovia of rats and in TNF-α-induced HUVEC cells, the molecular mechanism of its inhibition was elucidated by using Western blot and/or real-time PCR. SKN significantly reduced the arthritis score and arthritis incidence, and inhibited inflammation, pannus formation, cartilage and bone destruction of inflamed joints in CIA rats. Partially, SKN remarkably decreased the immature blood vessels in synovial membrane tissues of inflamed joints from CIA rats. It also suppressed in vivo angiogenesis in chick embryo and VEGF165-induced microvessel sprout formation ex vivo. Meanwhile, SKN inhibited TNF-α-induced migration, invasion, adhesion and tube formation of HUVEC cells. Moreover, SKN significantly decreased the expression of angiogenic activators including VEGF, VEGFR2, TNF-α, IL-1β, PDGF and TGF-β in synovia of CIA rats and/or in MH7A/HUVEC cells. More interestingly, SKN downregulated PI3K and Akt, and simultaneously upregulated PTEN both at protein and mRNA levels in synovia tissues and/or in TNF-α-induced HUVEC cells. It also suppressed the phosphorylation and gene level of TNF-α-induced signaling molecules, as ERK1/2, JNK, and p38 in synovium and/or in TNF-α-induced HUVEC cells. These findings indicate for the first time that SKN has the anti-angiogenic effect in RA in vivo, ex vivo and in vitro by interrupting the PI3K/AKT and MAPKs signaling pathways.

Liu C ，He L ，Wang J ，Wang Q ，Sun C ，Li Y ，Jia K ，Wang J ，Xu T ，Ming R ，Wang Q ，Lin N ... -  《-》

Wen Luo Yin inhibits angiogenesis in collagen-induced arthritis rat model and in vitro.

Wen Luo Yin (WLY) is a traditional Chinese formula, which has the traditional use of scattering cold pathogen, draining dampness, freeing the flow of network vessels and relieving pains. It is extensively used in the treatment of rheumatoid arthritis (RA) patients for more than 2000 years, but its actions on angiogenesis of RA have not been clarified. The present study aims to determine the anti-angiogenic activity of WLY on collagen-induced arthritis (CIA) rat model and in human fibroblast-like synoviocytes of RA (HFLS-RA) and human umbilical vein endothelial cells (HUVEC). For in vivo experiment, arthritis was induced by immunization with bovine II collagen in DA rats. Treatment with WLY (3.45, 6.9, 13.8 g/kg, p.o., daily), or vehicle began from day 1 to day 28 of first immunization. The arthritis score, arthritis incidence, microfocal computed tomography analysis and histopathology evaluation of inflamed joints were assessed. Angiogenesis was measured by synovial vessel density with immunohistochemistry and histomorphometric analysis in synovial membrane tissues of joints. For in vitro experiments, HFLS-RA and HUVEC were used. Assays to determine HFLS-RA migration and adhesion were performed in the presence of vascular endothelial growth factor (VEGF)165 or interleukin (IL)-1β and/or the WLY (8, 16, 32 mg/ml). Angiogenesis was assessed by measuring the migration, adhesion, and tube formation of HUVEC. Further the effect of treatment with WLY on expression levels of angiogenic activators in sera of CIA rats and in IL-1β-induced HFLS-RA were evaluated by enzyme linked immunosorbent assay. WLY significantly decreased the arthritis score and arthritis incidence, and inhibited inflammation, pannus formation, cartilage and bone destruction of inflamed joints in CIA rats. More interestingly, doses of 3.45-13.8 g/kg WLY could markedly reduce the capillaries, small, medium and large vessel density in synovial membrane tissues of inflamed joints. Moreover, WLY suppressed the VEGF-induced chemotactic migration of HFLS-RA and HUVEC, and inhibited matrigel-induced cell adhesion of them. It also disrupted tube formation of HUVEC on matrigel. Furthermore, WLY significantly reduced the expression of angiogenic activators including tumor necrosis factor-α, IL-1β, IL-17, VEGF, VEGFR, angiopoietin (Ang)-1, Ang-2 and Ang-2 receptor in sera of CIA rats and/or in IL-1β-induced HFLS-RA/HUVEC. Our data suggest for the first time that WLY posses the anti-angiogenic effect in RA both in vivo and in vitro by downregulating angiogenic activators.

Liu C ，Kong X ，Li X ，Guo W ，Zhang C ，Sun Y ，Su X ，Liu X ，Lu A ，Lin N ... -  《-》

Wu-Tou Decoction Inhibits Angiogenesis in Experimental Arthritis by Targeting VEGFR2 Signaling Pathway.

He L ，Liu C ，Sun C ，Wang J ，Zhi K ，Sun D ，Wang H ，Wang Q ，Lin N ... -  《-》

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.

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. This study explored the therapeutic roles of SKN on rat adjuvant-induced arthritis (AIA) and cellular inflammation, migration and invasion of TNF-α-induced RA FLS (MH7A cells), and further demonstrated the involved mechanisms. 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-α-induced migration and invasion in RA FLS. The involvement of Wnt/β-catenin pathway was checked by immunohistochemistry or immunofluorescence assay for β-catenin and western blot for pathway-related proteins. 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-1β, IL-6, IL-8, TNF-α, MMP-2 and MMP-9) in sera and synovium of AIA rats, and in TNF-α-induced MH7A cells. Gelatin zymography result revealed the suppression of SKN on TNF-α-induced MMP-2 activity in vitro. Moreover, SKN inhibited TNF-α-induced migration, invasion and cytoskeletal reorganization in MH7A cells. Mechanistically, SKN suppressed the activation of Wnt/β-catenin signaling in AIA rat synovium and in TNF-α-induced MH7A cells, indicated by the reduced protein levels of Wnt1, p-GSK-3β (Ser9) and β-catenin, the raised protein level of GSK-3β and the decreased nuclear translocation of β-catenin. Interestingly, the combination of LiCl (Wnt/β-catenin agonist) canceled the therapeutic functions of SKN on cellular inflammation, migration and invasion in TNF-α-induced MH7A cells, whereas XAV939 (Wnt/β-catenin inhibitor) enhanced the therapeutic roles of SKN. SKN showed therapeutic effects on rat AIA and cellular inflammation, migration and invasion of TNF-α-stimulated RA FLS via interrupting Wnt/β-catenin pathway.

Liu FY ，Wang MQ ，Liu MM ，Li T ，Wang XH ，Jiang F ，Wu XJ ，Cheng J ，Cai L ，Li R ... -  《-》

Anti-angiogenic effect of triptolide in rheumatoid arthritis by targeting angiogenic cascade.

Kong X ，Zhang Y ，Liu C ，Guo W ，Li X ，Su X ，Wan H ，Sun Y ，Lin N ... -  《PLoS One》