| 1. Background and objectivesRheumatoid arthritis (RA) is an autoimmune disease characterized by joint synovitis. Synovial hyperplasia and angiogenesis are the key factors resulting in cartilage and bone detruction. In the past years, most of researches on RA pathomechanism focused on on the process of autoimmune inflammatory reactions mediated by T cells. On the contrary, synovial fibroblasts (SFs) are considered as a "passive participant", proliferating and infiltrating under the irritation of T cells and inflammatory factors. In recent years, the role of RA synovial fibroblasts (RASFs) in the RA pathological process gradually comes to a highlight. Because RASFs is somewhat like transformants, RASFs alone can proliferate and infiltrate into the cartilage, even without the irritation of T cells and inflammatory factors. For this reason, it is pointed out that RASFs are actually a "positive invader" playing an important role in the RA pathomechanism. This fully explains why some immunosuppressive agents and anti-inflammatory analgesics can merely improve clinical symptoms in the clinical treatment, however, finally fail to inhibit the development of disease, and the destruction of cartilages and bones. As a result, in recent years, RA researches focus on the drug discovery aiming to inhibiting synovial hyperplasia and preventing the destruction of bone and cartilage.Idea animal model, as a technical platform, is essential for this purpose. However, in some animal models usually be used such as collagen-induced arthritis, adjuvant-induced arthritis, etc., the synovial hyperplasia and cartilage invasion can not be isolated, because these pathological changes are totally drived by immune and inflammatory responses. Therefore, since the middle 90s of last century, much attention has been quickly paid to new research fields. In these studies, RA synovial membrane was transplanted into immunodeficient animals to bulid animal-human rheumatoid arthritis grafts (HuRAg) chimera model. Such kind of model is widely used in exploring the pathomechanism of RA, especially in screening new drugs or medications for purpose of inhibiting synovial hyperplasia and cartilage invasion. However, in China, the development of this kind of animal model is just on its first step. Immunodeficient animals include nude mice, mice with severe combined immune deficiency (SCID) of T cells and B cells, BNX mice with immune deficiency of T cells, B cells and natural killer cells. In the present time, SCID is widely employed. As for whether BNX mice can be used as a carrier of RA synovial grafts or not, no report is found.Our previous animal experiments showed that methanol-extract of Celastrus orbiculatu (MECO) had an fine effect of anti-inflammation, analgesia andimmune suppression. But as a RA medication, in vivo experiment is still needed to explore its actual effects on RA, and investigate its working mechanisms. Beside that, Etanercept is the best accepted TNF-a blocking agent in the current time. It plays an important role in the RA treatment. However, as a TNF-a receptor fusion protein, there is no direct evidence for its real effects on inhibiting synovial hyperplasia and cartilage invasion.For the above-mentioned reasons, the aims of this study is to explore the pathomechanisms of synovial hyperplasia and cartilage destruction in RA, investigate the feasibility of the chimera model of BNX-HuRAg, observe the effects and discuss the mechanism of Celastrus orbiculatu to inhibit the RA synovial hyperplasia and cartilage invasion, further more, explore the overall pharmaco-mechanisms of Etanercept on RA.2. MethodsThis study consists of three parts: first, setting up NOD/SCID-HuRAg model, and comparing its characters with those of NOD/SCID-HuOAg model; second, assessing the effect of Celastrus orbiculatu on NOD/SCID-HuRAg with the control of leflunomide, and discussing its mechanism of action; third, evaluating the effect of Etanercept on BNX-HuRAg and its mechanism of action.2.1 Method for set up the Model The synovial tissues sampled from the RA patients or OA patients were trimmed to 0.3×0.5cm. The normal articular cartilages from the patients with amputation were sliced to 0.5×0.8cm. Under general anesthesia, the NOD/SCID mice or BNX mice were operated in the back with a longitudinal incision. The prepared cartilage slice was planted under the skin of the back, then, the synovial tissues was planted on the slice. The skin was sutured. All of the above processes were performed under the sterile environment. The mice were raised in specified-pathogens-free (SPF) envirement.2.2 Administration method NOD/SCID-HuRAg mice were radomized into three groups with 8 mice in each group. 29 days after implantation, the mice were intragastricly administered with MECO (30mg/d), leflunomide (500μg/d) and distilled water (as control), respectively, once a day, lasted for 4 weeks. BNXHuRAg mice were radomized into two groups with 10 mice/group. 29 days after implantation, the mice were administered by hypodermic injection with Etanercept (100μg) and distilled water (as control), respectively, twice a week, lasted for 4 weeks. The first-built animal models of NOD/SCID-HuRAg and NOD/SCID-HuOAg (10 of each) were kept as blank control.2.3 Detection method 61 days after operation, whole blood was sampled by enucleating eyeballs. The planted synovial tissues and cartilages were totally seperated. Radioimmunity was applied to detect serum TNF-a amouts. TUNEL method was applied to detect apoptosis status. Histological scores of grafts for synovial hyperplasia, cartilage invasion by synoviocyte and perichondroncytic cartilage degradation were evaluated. CRI micro color system was applied to observe and take photos. The professional image analysis software, Image Pro plus 6.0 was used to analyse the difference of the mean integrated optical density (MIOD) and mean value of stained area (MSA) in the two groups.2.4 Statistical analysis Independent-samples t test was used to compare the mean value of the first-built models in NOD/SCID-HuRAg group and NOD/SCIDHuOAg group. In the experiment of Celastrus orbiculatu and leflunomide, analysis of variance was used to analyse the result of the three groups. SNK test was used to compare the result between two groups. Provided with heterogeneity of variance, Games—HoweⅡtest was used. In the experiment of BNX-HuRAg, Independent samples t test was used to compare the mean value of the two groups of Etanercept and contorl.3. Results3.1 The grafts kept living in the mice till the end of experiment. No graft versus host reaction was observed. In comparison with NOD/SCID-HuOAg group, the scores of synovial hyperplasia in NOD/SCID-HuRAg group was significantly low (2.56±1.15 vs 3.7±0.52, p＜0.05), so was cartilage invasion (2.42±1.29 vs 1.15±0.51, p＜0.01), and cartilage degradation (2.44±1.04 vs 1.50±0.51, p＜0.01). The expression of VEGF in the synovium of NOD/SCID-HuRAg group is frequently observed, while in NOD/SCID-HuOAg group, little expression was observed. Beside that, apoptosis in NOD/SCID-HuRAg group was significantly lower in comparison with NOD/SCID-HuOAg group (MIOD: 168.371±12.866 vs 959.226±80.886, p＜0.01; MSA: 180.210±8.206 vs 1890.510±159.139, p＜0.01).3.2 MECO and leflunomide significantly decreased the scores of synovial hyperlasia (2.000±0.756, 2.250±0.886 vs 3.625±0.517, p＜0.01), cartilage invasion (1.687±0.799, 2.000±1.362 vs 3.750±0.535, p＜0.01), cartilage degradation (1.875±0.835, 2.125±0.835 vs 3.635±0.744, p＜0.01) and the serum amount of TNF-a (0.840±0.088, 0.803±0.068 vs 0.993±0.114, p＜0.01, ng/ml). The synovium apoptosis were increased significantly in both groups of MECO and leflunomide (MIOD: 350.595±253.354, 354.002±265.903 vs 75.138±44.793, P＜0.05; MSA: 932.324±715.187, 892.480±563.899 vs 191.331±115.550, P＜0.05). Furthermore, the expression of TNF-a was down-regulated by MECO, and in leflunomide group, no statistical significance was observed.3.3 In Etanercept group, the scores of synovial hyperplasia (2.100±0.994 vs 3.700±0.483, p＜0.01), cartilage invasion(1.550±0.726 vs 3.750±0.535, p＜0.05), cartilage degradation (1.300±0.823 vs 3.635±0.744, p＜0.01) and the serum amount of TNF-a (0.596±0.095 vs 0.694±0.112, p＜0.05, ng/ml) decreased significantly; The expressions of TNF-a (MIOD: 35.336±32.277 vs 162.664±142.269, P＜0.05; MSA: 127.185±117.824 vs 582.376±530.321, P＜0.05) and VEGF mRNA (MIOD: 75.580±34.115 vs 211.944±114.096, P＜0.01; MSA: 201.530±82.974 vs 552.965±413.223, P＜0.05) in the synovium also declined significantly. But the apoptosis in synovium did not show significant difference between Etanercept and control group.4. ConclusionHuman RA synovium and normal cartilage can grow well in both NOD/SCID mice and BNX mice. The transplanted synovium keep the ability of proliferation and cartilage invasion. Furthermore, the expression of TNF-a and VEGF is obvious in the transplanted synovium, whereas apoptosis is significantly inhibited. MECO can inhibit RA synovial hyperplasia, relieve synovial invasion in cartilage, and cartilage degradation mediated by chondrocytes. The mechanisms of action involve in inhibiting the production of TNF-ain synovium and promoting apoptosis of synovial cells. The effect is similar to that of leflunomide, while MECO has a better effect in inhibiting the expression of TNF-a than leflunomide. Etanercept also has an effect in inhibiting synovial hyperplasia, invasion and cartilage degradation. The mechanisms of action are probably associated with counteracting TNF-a in synovium and peripheral blood, and down-regulating the expression of VEGF in synovial cells. NOD/SCID-HuRAg and BNX-HuRAg are powerful tools for exploring RA pathomechanism and relative pharmacology researches.
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