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

Abstract

Wu-tou decoction (WTD) is a classic traditional Chinese medicine formula and has been extensively used for the treatment of rheumatoid arthritis (RA). Previous reports indicate that WTD possesses anti-inflammatory and antinociceptive activities, and inhibits the development of arthritic joints and disease severity of collagen-induced arthritis (CIA) or adjuvant-induced rats; however, its action on angiogenesis of RA has not been clarified. This study aims to determine the anti-angiogenic activity of WTD in CIA rats and in various angiogenesis models. Our data showed that WTD (0.95, 1.9, and 3.8 g/kg) markedly reduced the immature blood vessels in synovial membrane tissues of inflamed joints from CIA rats. It also inhibited in vivo angiogenesis in chick embryo and VEGF-induced microvessel sprout formation ex vivo. Meanwhile, WTD suppressed VEGF-/MH7A-induced migration, invasion, adhesion, and tube formation of human umbilical vein endothelial cells (HUVECs). Moreover, WTD significantly reduced the expression of angiogenic activators, including vascular endothelial growth factor (VEGF), VEGFR2, interleukin (IL)-1β, IL-17, transforming growth factor-β, platelet-derived growth factor, placenta growth factor, angiopoietin (Ang) I and Ang II in synovium of CIA rats, and/or in HUVECs. More interestingly, WTD blocked the autophosphorylation of VEGF-induced VEGFR2 and consequently downregulated the signaling pathways of activated AKT, ERK1/2, JNK, and p38 in VEGF-induced HUVECs. These findings suggest for the first time that WTD possesses the anti-angiogenic effect in RA in vivo, ex vivo, and in vitro by interrupting the targeting of VEGFR2 activation.

ErMiao San Inhibits Angiogenesis in Rheumatoid Arthritis by Suppressing JAK/STAT Signaling Pathways

“…is assay was carried out as previously described [12]. In order to measure blood vessel in synovial membrane tissues of inflamed joints, the polyclonal antibody (rat antibody, dilution 1 : 50, Abcam, Cambridge, MA, USA) recognizing the CD31 panendothelial antigen was used for microvessel and single endothelial cell staining on 5 μm thick paraffin embedded sections of knee joints by immunohistochemical analysis as previously described [11].…”

“…is assay was performed as previously described [12]. Forty-eight well plates were covered by 100 μl of matrigel (5 mg/mL) and left to polymerize for 45 min at 37°C.…”

“…Cell Culture. We followed the methods of Liu et al [11], and they were carried out described as previously described [12]. Human umbilical vein endothelial cells (HUVECs) were purchased from ScienCell Inc. (Carlsbad, CA, USA).…”

“…We followed the methods of Liu et al [11], and they were carried out as previously described [12]. HUVECs (5 × 10 4 cells/mL) were seeded in 96-well plates and incubated in sterile H-DMEM supplemented with 5% FBS, 100 U/mL penicillin, and 80 U/mL streptomycin for 24 h. Cells were then incubated with or without VEGF (20 ng/mL) and 1 h later with different concentrations of EMS (0.2, 0.4, and 0.8 mg/mL) for 24 h. Cell viability was determined by MTT method according to the manufacturer's instructions.…”

“…Transwell Migration Assay. We followed the methods of Liu et al [11], and they were carried out as previously described [12]. e transwell migration assay was performed using a transwell chamber.…”