Effect And Mechanism Of Tripterygium Glucosides On Antiangiogenesis About Treating Rheumatoid Arthritis

ObjectiveRheumatoid arthritis (RA) is a chronic systemic autoimmune disorder that is characterized by arthrosynovitis. RA synovium lead to the destruction of cartilage and bone and functional incapacitation of joints, even to physical disability. Its prevalence in adults in China is about 0.32%-0.36%, which is lower than that in other places 1%. And it is one of major diseases causing disability in our nation.The major pathological change of RA is synovialis hyperplasia, formation of pannus and the erosion of periarticular tissues. The mechanism of RA is still unclear. Vascular endothelial growth factor (VEGF) plays an important role in the mechanism of RA. At present, there is few specifed therapy except for anti-inflammatory and symptomatic treatment. With the increasing acquaintance of angiogenesis and RA, anti-angiogenesis treatment has been a novel research point at issue. Tripterygium glucosides are Chinese drugs which have an effect on RA. At present, Tripterygium wilfordii has effects on anti-inflammatory, immunological inhibition, anti-tumor, antibiosis and antifertility. There have been further research on the two effects above, however, there is few report on angiogenesis of tripterygium glucosides.The aim of the study is to investigate the anti-angiogenesis effect of tripterygium glucosides and illuminate the mechanism by observing the influence of tripterygium glucosides on collogen-induced arthritis (CLA). The results may lay the theory foundation on the clinical application of tripterygium glucosides.Methods1. The primers and probes of VEGF and VEGFR2 were used to evaluate the specificity as well as reproducibility by real-time PCR.2. Effects of tripterygium glucosides were studied on CLA rat models. Thalidomide was the positive control.3. To establish a models of CLA and observe the influence of tripterygium glucosides on the expression of VEGF and VEGFR2 by real-time PCR. The contents of VEGF and VEGFR2 in serum were detected by ELISA.4. Specific expressions of VEGF gene in the normal tissues like lung, heart, liver, renal, brain, stomach, intestine and glandula angularis were analyzed.5. The expressions of VEGF and VEGFR2 were investigated after treatment of tripterygium glucosides by the synovim cells culture in vitro.Results 1. Detected by DNA sequencing and electrophoresis, the amplification of VEGF and VEGFR2 was specific with the coefficient correlation >0.98. The reproducibility was presented by 2.0%～6.7% and 2.0%～6.4% in intra-assay CV,3.5%～10.6%和3.6%～10.2% in inter-assay CV.2. Assessment scores and degree of swelling had significant correlation between arthrosis model groups and normal groups. In arthrosis groups, appearances in the pathology of synovim tissues were cell degeneration and hyperplasy in synovium of joint, inflammatory cells infiltrating in matrix of joint, as well as formation of granulation tissues and damage of arthrodial cartilage. Furthermore, fibroplasia and fibrosis emerged, loss of joint at serious time. Delayed allergy was induced by collagen typeⅡin periarticuar underlying soft tissues where epithelioid cells appeared nodosity.3. The mRNA expressions of VEGF and VEGFR2 decreased after treatment of tripterygium glycosides or thalidomide. However, there was no significant difference between each treatment group.4. The mRNA expression of VEGF was the highest in the lung tissues in normal rats compared with any other groups, P<0.05, with statistical difference.5. In model serum groups, the contents of VEGF and VEGFR2 were higher compared to control groups (P<0.05) as well as the experiment groups (0.05).6. The mRNA levels of VEGF and VEGFR2 decreased markedly after the treatment of tripterygium glycosides (P<0.05).ConclusionAccurate quantitative analysis method of the mRNA expression of VEGF and VEGFR2 by real-time PCR was successful established. The results of in vivo and in vitro research showed that tripterygium glycosides have curative effect on RA, and the mechanism maybe relate with the decrease the expression of VEGF and VEGFR2 so that to inhibit angiogenesis.The innovation of this research including.1) There have been many reports on VEGF, but the methods used to study the expression are limited to hybridization and RT-PCR, which have the deficiency on accuracy and great variation and then influent the reliability of the conclusion. The method to detect the mRNA of VEGF and VEGFR2 by real-time PCR was established in this study. It is the first report that the combination of absolute and relative quantitation makes the accuracy and sensitivity of the methods further higher than previous.2) The specific expression of VEGF in various tissues of normal rats was investigated to lay the foundation for research on models, which has not been reported.3) We first reported the mRNA expression of VEGFR2 by using real-time PCR.4) Tripterygium wilfordii has been comfirmed as the most effective single Chinese traditional medicine on RA treatment. The research on its effect of antiinflammatory and immunosuppression was relative thorough, however, there are few report on the effect of angiogenesis. In our study, the anti-angiogenesis effect of tripterygium glycosides was analyzed systematically, and it is demonstrated that down regulation of VEGF and VEGFR2 may be the mechanism.