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

Ethnopharmacological relevance and aim of the study 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). Materials and methods 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. Results 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. Conclusions 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.