| ObjectiveRheumatoid arthritis(RA), a multiple and severe auto-immunological disease, is characteristics by proliferation of synovial cells, growing of new blood vessel, infiltrating of inflammatory cell and destruction of joint cartilage. It is major disease of high damage and disability for individual as its easy recurring and chronic inflammation program. The pathogenesis of RA is still unclear. However, a large number of studies have demonstrated that activation of many kinds of immunocytes, formation of pannus, participation of cytokine and mediators of inflammation play important roles in RA. However, previous studies always observed the changes in inflammatory factors of animal models at a point of time, but dynamic observation on a series of time. Now there is not an ideal medicine for RA patients in the world. Rhizoma Dioscoreae Nipponicae (RDN) can increase blood flow and inhibite cough. Preliminary work shows that total saponin of RDN has an anti-inflammatory and immunomodulate effect. Collagen-induced arthritis(CIA) is the best animal model to study RA, because it approaches to RA with many symptoms including histipathological and immunopathological changes.So we choose CIA as animal model to study the changes of inflammatory cytokine in CIA rats and the relationship with disease severity dynamically, and to determine the influence on inflammatory cytokines in serum, and the possible mechanism of total saponin of RDN in treatment of RA.Methods1. Dynamic observation of inflammatory cytokines in CIA ratsThe CIA model was prepared by the mixture of bovine typeⅡcollagen (2mg/ml) and Freund's completed adjuvant injected intracutaneously on the back and tail root of Wistar rats with several points and the same procedure repeated on the 7th day after the first immunization. The general state was observed after injection. The body weight, volume and thickness of hind paws, arthritic index, spleen index and thymus index were measured; the level of TNF-α, IL-1βand IL-6 in the serum were detected by Enzyme-linked immunoadsordent assay (ELISA); the hitocology of arthral synovial tissues was observed by HE stainning with light microscope on the day 10, 20, 30, 40, 50 and 60 after the first immunization.2. The effect of total saponin from RDN on inflammatory cytokines in CIA ratsThe CIA model was established by typeⅡcollagen (2mg/ml) injected intracutaneously. On the 14th day after the first immunization, the CIA rats were divided into 5 groups, the CIA model, the high dose of RND (100 mg/ kg/ d), the middle dose of RND (50 mg/ kg/ d), the low dose of RND (25 mg/ kg/ d) and the Tripterygium group (12 mg/ kg/ d), and orally administrated with three doses of total saponin of RDN and Tripterygium for 21 days. The rats of model group and control group were given the same volume of solvent (0.5% CMC). The body weight, volume and thickness of hind, arthritic index, spleen index and thymus index were measured; the level of TNF-α, IL-1βand IL-6 in the serum were detected by Enzyme-linked immunoadsordent assay (ELISA); the hitocology of arthral synovial tissues were observed by HE staining with light microscopy or electron microscopy on the day 21 of total saponin of RDN treatment.Results1. The dynamic changes of inflammatory cytokines in CIA rats1.1 General StatusSome small ulcers on the back and tail were found after the first immunization, and healed after about one week. The inflammatory reaction was approximately appeared on the 14th day, the most seriously state on the 30th day, and then gradually decreased following the time. The body weight of CIA rats grow slowly and lower than that of control rats (P<0.01). 1.2 The swelling changes of hind pawsCompared with the control group, CIA rats'right hind paws volume and thickness significantly increased 0.15ml and 1.37mm on the 7th day after the first immunization, 0.78ml and 4.62mm on the 30th day, respectively. And then the volume and thickness were gradually lessened following the time. These results still significantly higher than those of control group until the 60th day, which were 0.65ml for volume and 2.56mm for thickness (P<0.01). The left hind paws volume and thickness significantly increased 0.70ml and 3.78mm on the 20th day after the first immunization, 0.89ml and 4.4mm on the 30th day, respectively. And then the volume and thickness were gradually lessened following the time. These results still significantly higher than those of control group until the 60th day, which were 0.66ml for volume and 2.50mm for thickness (P<0.01). The arthritis index (AI) was 0 of control groups at all time. The AI of CIA rats were consistent with the change of volume and thickness of hind paws.1.3 The changes of spleen and thymus indexCompared with the control group, the spleen index (SI) and thymus index (TI) of CIA rats were significantly increased on the 20th, 30th, 40th, 50th and 60th day after the first immunization (P<0.01). 1.4 The changes of inflammatory cytokines'level in serumThe levels in TNF-αwere 19.20-23.04pg/ml of control rats from 0 to 60th day and increased significantly in CIA rats of the time. TNF-αlevel was 28.79pg/ml on the 10th day after the first immunization, 46.09pg/ml on the 30th day, 29.30pg/ml on the 60th day (P<0.01). The levels in IL-1βwere 141.46-51.75pg/ml of control rats from 0 to 60th day and increased significantly in CIA rats of the time. IL-1βlevel was 65.35pg/ml on the 20th day after the first immunization, 301.89pg/ml on the 30th day , 117.72pg/ml on the 60th day(P<0.01).The levels in IL-6 were 42.52-51.05pg/ml of control rats from 0 to 60th day and increased significantly in CIA rats of the time. IL-6 level was 63.55pg/ml on the 20th day after the first immunization, 130.41pg/ml on the 30th day, 62.38pg/ml on the 60th day (P<0.01).1.5 Pathologic change of synoviumSynovium and articular surface were not observed any changes in control group rats, and proliferation of synovial cells, growing of new blood vessel, infiltrating of inflammatory cell and destruction of joint cartilage were found in CIA rats.2. The effect of total saponin from RDN on inflammatory cytokines in CIA rats2.1 The effect of total saponin from RDN on body weight, feet swelling, spleen and thymus indexCIA rats were daily and orally treated with total saponin of RDN for 21 days on the day 14 after the first immunization. Compared with the control group, the body weight of CIA model group was significantly reduced (P <0.01), the volume and thickness of hind paws, and index of arthritis, spleen and thymus were significantly increased (P <0.01). However, the changes of volume and thickness of hind, body weight, and index of arthritis, spleen and thymus appeared improving tendency with three dose of total saponin of RDN for 21 days, but no statistical significance (P> 0.05). The same result was gotten from Tripterygium group.2.2 The effect of inflammatory cytokine in serumThe levels of TNF-α, IL-1βand IL-6 in serum were significantly increased in CIA model group, as compared with the control group (P <0.01). The level of TNF-α, IL-1βand IL-6 in CIA model were 47.39pg/ml, 260.21pg/ml and 133.54pg/ml, respectively. However, the levels of TNF-α, IL-1βandIL-6 in CIA rats were marked decreased after treatment with total saponin of RDN for 21 days (P <0.01). The levels were 26.57-30.17 pg/ml for TNF-α, 44.98-65.12pg/ml for IL-1βand 89.96-100.34pg/ml for IL-6, respectively. In the same, the levels of TNF-α, IL-1βand IL-6 in serum were marked decreased after treatment with Tripterygium for 21 days (P <0.01). The levels were 28.34pg/ml for TNF-α, 58.26pg/ml for IL-1βand 101.52pg/ml for IL-6, respectively. 2.3 The effect of the pathological changes of synoviumCompared with the control group, proliferation of synovial cells, growing of new blood vessel, infiltrating of inflammatory cell and destruction of joint cartilage were found in CIA model group. Synovial cells'rough endoplasmic reticulum (RER) expansion, mitochondria (MCa) decrease and lysosomes (LYS) increase were also observed by electro microscropy. The synovium thickened slightly, the number of inflammatory cell decreased, the destruction of joint cartilage lighten after treatment with total saponin of RDN for 21 days. Synovial cells'RER dilated slightly.Conclusion1. The inflammatory reaction was approximately appeared on the 14th day and the most seriously state was on the 30th day. And then the volume and thickness of hind paws gradually decreased following the time. The concentration of TNF-αwere increased on the 10th day, and the concentration of IL-1βand IL-6 increased on the 20th day after the first immunization. The peak concentrations of TNF-α, IL-1βand IL-6 were all on the 30th day, and then gradually decreased following the time. The concentration of TNF-α, IL-1βand IL-6 were consistent with the inflammatory reaction.2. Total saponin of RDN could obviously decrease the level of TNF-α, IL-1βand IL-6 in the serum, protected synovial tissues. The results suggested that total saponin of RDN may be benificial for treatment of RA.
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