| Objective:Rheumatoid Arthritis(RA), a common and multiple auto-immunolo-gical disease, is characterized by proliferation of synovial cells, growing of new blood vessel, formation of pannus. Its major characteristic is mutiple and progressive joint synovitis. In recent years, the former results showed that growing of new blood vessel and formation of pannus play important roles in the development of RA. Inhibiting angiogenesis may be a new therapeutic target of treating RA. Preliminary works showed that total saponin from Rhizoma Dioscreae Nipponicae (RDN) could relieve swelling of joint in rats, reduce infiltrating of inflammatory cells and proliferation of synovium; inhibit inflammatory cytokines IL-1(3, IL-6, TNF-a and etc. The purpose of the research is based on preliminary works, choosing collagen-induced arthritis (CIA) rat as animal model to explore the effects of total saponin from RDN on cytokines (VEGF,Ang-2) and receptors (Flk-1,Tie-2) about angiogenesis in synovium. The research will provide experimental basement to further clarify the mechanism of treating RA of RDN, and to make RDN a new medicine of treating RA.Methods:1Sixty-five Wistar rats were randomly divided into five groups, normal control group, CIA model group, RDN treament group, Tripterygium treament group and Diosgenin treament group, twelve rats in each group. The rats in CIA model group, RDN treament group, Tripterygium treament group and Diosgenin treament group were established CIA rat model. After the CIA rat model was successfully established, the rats in CIA model group had no more treatment; the rats in RDN treament group were lavaged with total saponin from RDN(25mg/kg/d,21d); the rats in Tripterygium treament group were lavaged with tripterygium(TP)(12mg/kg/d,21d); the rats in Diosgenin treament group were lavaged with diosgenin(50mg/kg/d,25mg/kg/d,21d). The rats of normal control group and CIA model group were given the same volume of solvent (0.5%CMC).1.1The rats were sacrificed after blooding from abdominal vein. The synovium of the bilateral knee joints were taken. The rats of Diosgenin treatment group(50mg/kg/d) were killed. Immunohistochemical staining was used to observe micro-vessel density (MVD), vascular endothelial growth factor (VEGF), fetal liver kinase-1(Flk-1), angiopoietin-2(Ang-2), Tie-2expression in synovium. The pictures were analyzed by MiVnt image system.1.2The rats were sacrificed after blooding from abdominal vein. The synovium of the bilateral knee joints were taken. The rats of Diosgenin treatment group(25mg/kg/d) were killed. The synovium total RNA was extracted by Trizol and reversed transcriptoion cDNA. VEGFmRNA expression in synovium was examined by PrimeScriptTM real-time quantitative PCR detection kit.2Use SPSS11.5statistical software to analyze the above mentioned data. Results are presented as Mean±E. Differences between groups were analyzed using q-test by one-way ANOVA. a=0.01is the standard of significant test.RESULTS:1General statusSome small ulcers on the back and tail were found after the first immunization. Swelling and erythema of the hind paws were appeared on the12th day, then swelling and erythema were invased on hind knees and fore paws. The rats in CIA model group were eating less and apathetic. Swelling of joint and deformity of paw were found in rats of CIA model group. The rats in RDN treament group, Tripterygium treament group and Diosgenin treament group were relieved after the corresponding treatment. The rats in normal control group were all normal.2The result of MVD in rat synovium of different groups Many and intensive new blood vessels were appeared in synovium of rats in CIA model group. Compared with rats in normal control group(3.00±0.71), MVD of rats in CIA model group (9.20±1.30) incressed obviously(P<0.01). MVD of rats in RDN treament group(4.80±0.45), Tripterygium treament group(5.00±0.82) and Diosgenin treament group(5.20±0.84), which was significantly lower than that of CIA model group(P<0.01); moreover, there had no obvious differences among every treatment group(P>0.05).3VEGF expression in rat synovium of different groupsVEGF immunopositive products were yellow or brown exquisite granules and located at cytoplasm. Compared with normal control rats, VEGF expression in synovium of rats in CIA model group increased remarkably(P<0.01). VEGF expression in synovium of rats in RDN treament group, Tripterygium treament group and Diosgenin treament group were remarkably lower than that of CIA model group(P<0.01); moreover, there had no obvious differences among every treatment group(.P>0.05).4Flk-1expression in rat synovium of different groupsFlk-1immunopositive products were yellow or brown exquisite granules and located at cytoplasm or cell membrane. Compared with normal control rats, Flk-1expression in synovium of rats in CIA model group increased remarkably(P<0.01). Flk-1expression in synovium of rats in RDN treament group, Tripterygium treament group and Diosgenin treament group were remarkably lower than that of CIA model group(P<0.01); moreover, there had no obvious differences among every treatment group(P>0.05).5Ang-2expression in rat synovium of different groupsAng-2immunopositive products were yellow or brown exquisite granules and located at cytoplasm or nucleus. Compared with normal control rats, Ang-2expression in synovium of rats in CIA model group increased remarkably(P<0.01). Ang-2expression in synovium of rats in RDN treament group, Tripterygium treament group and Diosgenin treament group were remarkably lower than that of CIA model group(P<0.01); moreover, there had no obvious differences among every treatment group(P>0.05). 6Tie-2expression in rat synovium of different groupsTie-2immunopositive products were yellow or brown exquisite granules and located at cytoplasm or cell membrane. Compared with normal control rats, Tie-2expression in synovium of rats in CIA model group increased remarkably(P<0.01). Ang-2expression in synovium of rats in RDN treament group, Tripterygium treament group and Diosgenin treament group appeared declining tendency; but had no statistical significance(P>0.05); moreover, there had no obvious differences among every treatment group(P>0.05).7VEGFmRNA expression in rat synovium of different groups by real-time quantitative PCRCompared with normal control rats(0.736±0.162), the level of VEGFmRNA expression in synovium of rats in CIA model group(1.056±0.342)increased remarkably(P<0.01). The level of VEGFmRNA expression in synovium of rats in RDN treament group(0.433±0.233), Tripterygium treament group(0.711±0.160), Diosgenin treament group(0.750±0.199)were remarkably lower than that of CIA model group(P<0.01); moreover, compared with Tripterygium treament group and Diosgenin treament group, the level of VEGFmRNA expression in synovium of rats in RDN treament group decreasd remarkably(P<0.05).Conclusion:1Many and intensive new blood vessels were found in synovium of rats in CIA model group. VEGF, Flk-1, Ang-2and Tie-2all play importmant roles in synovium angiogenesis of CIA model rats.2Total saponin from RDN could decrease the number of new blood vessels.3Total saponin from RDN could obviously decrease VEGF, VEGF-mRNA, Flk-1, Ang-2and Tie-2expression in synovium of CIA model rats. Inhibiting the above cytokines and receptors may be one of the reasons of inhibiting angiogenesis with total saponin from RDN. |
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