| Objective:To establish collagen induced arthritis model and in vitro and in vitro angiogenesis model,to observe the intervention effect of classical prescription WTD on synovial angiogenesis of RA,and explore the relevant mechanism through the VEGFR2/AKT/ERK/JNK/p38 signaling pathway.Method:In vivo experiments,a rat model of Ⅱ type collagen induced arthritis(CIA)was established to observe the effects of WTD in different dosage groups(0.95-3.8 g/kg)on synovial hyperplasia,angiogenesis and joint destruction in CIA rats.After 28 days of administration,joint swelling degree of CIA rats was analyzed by foot swelling degree test,pathological changes were observed by HE staining,and protein expression levels of CD31 andαSMA in synovium tissue of inflammatory joints were detected by IHC and IF double standard test.The direct effect of WTD on angiogenesis was tested by rat thoracic aorta ring and chicken embryo allantoic membrane test.The effects of WTD on the function of HUVEC cells were detected by transwell migration and invasion,adhesion and lumen formation tests.The protein expression levels of VEGF and VEGFR2 in rat synovium were detected by IHC assay.ELISA was used to detect VEGFR2 protein expression in VEGF-induced HUVEC.The expression levels of VEGFR2,AKT,ERK1/2,JNK and p38 and their corresponding phosphorylated proteins in VEGF-induced HUVEC were detected by WB assay.Results:1.Pharmacodynamic study of WTD on CIA induced RA in ratsThe model success rate of SD rats immunized with type Ⅱ collagen was close to 100%.When the concentration of WTD is 0.95-3.8 g/kg,it can reduce the degree of swelling in CIA rats’inflammatory joints dose-dependent,reduce the degree of synovial cell proliferation in inflammatory joints,and inhibit pannus formation.IHC results showed that WTD could inhibit the positive expression of vascular endothelial cell marker CD31 in joint synovium when the concentration was 0.95-3.8g/kg.The results of IF double labeling showed that WTD could reduce the generation of CD31~+/αSMA~-immature blood vessels and total blood vessels(sum of immature blood vessels and mature blood vessels)in joint synovium at the concentration of 0.95-3.8 g/kg,and there was no statistical difference on the effect of CD31~+/αSMA~+mature blood vessels.These results indicate that WTD can effectively treat CIA by inhibiting angiogenesis in the synovial tissue of inflammatory joints.2.Effect of WTD on synovial angiogenesis in CIA ratsThe medium and high dose groups of WTD(1 and 5μg/m L)could significantly inhibit VEGF induced bad angiogenesis in the thoracic aorta of rats,and showed a downward trend when the concentration was 0.2μg/m L,but there was no statistical difference.WTD can inhibit angiogenesis of chicken embryo allantoic membrane when the concentration is 0.2-5μg/m L.These results indicate that WTD can directly inhibit angiogenesis,which provides a direct basis for its inhibition of angiogenesis.3.Effects of WTD on migration,invasion,adhesion and lumen formation of HUVECThe results of MTS test showed that there was no statistical difference in the effect of WTD on HUVEC and MH7A cell viability when the concentration was 0.2-5μg/m L,that is,no cytotoxicity.WTD can dose-dependently inhibit VEGF or MH7A-induced transwell migration,invasion,adhesion and lumen formation of HUVEC at 0.2-5μg/m L.These results indicated that WTD could significantly inhibit angiogenesis in all links,and thus inhibit angiogenesis.4.Effects of WTD on angiogenesis stimulating factor and VEGFR2/AKT/ERK/JNK/p38 signaling pathway in CIA rats and HUVEC cells in vitroIHC assay results showed that WTD could inhibit the expression levels of VEGF and VEGFR2 in synovium in a dose-dependent manner at the concentration of 0.95-3.8 g/kg.ELISA results showed that the VEGFR2 expression level in HUVEC cells induced by VEGF could be decreased dose-dependent at 0.2-5μg/m L WYD.WB analysis showed that when the concentration of WTD was 0.2-5μg/m L,The expression levels of p-VEGFR2/VEGFR2,p-AKT/AKT,p-ERK/ERK,p-JNK/JNK and p-p38/p38 in VEGF-induced HUVEC cells were dose-dependent.Conclusions:WTD has antiangiogenic effects on CIA rat model,in vitro chick embryo allallic membrane and thoracic aorta ring model,and in vitro VEGF-induced HUVEC cell model,and its mechanism may be related to the VEGFR2/AKT/ERK/JNK/p38 signaling pathway.WTD down-regulates the abnormal activation of VEGFR2,AKT,ERK1/2,JNK and p38 by angiogenesis stimulating factors,and reduces the migration,adhesion,invasion and lumen formation of vascular endothelial cells,thus playing a role in inhibiting RA synovial angiogenesis. |
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