| Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by progressive joint destruction and functional impairment. In the treatment of autoimmune disease, early effective treatment is the key to prevent the disease development. Methotrexate (MTX) is widely used in treating rheumatoid arthritis (RA) principally via up-regulating adenosine production. However, the ACR20 response rate to MTX treatment is between 46%-65% which is considered to associate with the polymorphisms of targets and metabolic enzymes of MTX, but there is no firm evidence shows whether signaling mediated by adenosine receptor can influence the efficacy of MTX. There are four adenosine receptor subtypes, A1AR, A2AAR, A2BAR, and A3AR, among them, the A2AAR plays pivotal role in immunlolic responses. However, whether the activity of A2AAR signaling pathway influences the effects of MTX treatments on RA is still unclear. This study was carried out to investigate the effect of MTX on collagen induced arthritis (CIA) rats and reveal the potential mechanism of MTX function.OBJECTIVE To explore the reason of the different treatment effect of MTX on rats CIA, and to reveal the role of A2AAR signaling pathway in MTX treatment.METHODS A preparation of 0.2mg of heterologous chicken type II collagen (CII) was injected intradermally into the back and the base of the tail of SD rat to induced CIA on day 0 and day 7. CIA rats were sacrificed on day 0, day 14, day 28 and day 42. Another group of CIA rats were treated with MTX (0.5mg/kg) by intragastrically from day 14, once every 3 days for 15 days. The effects of MTX on CIA rats were evaluated by arthritis index, swollen joints count, globle assessment and paw swelling. After treatment the histology of joints were observed, and indices of thymus and spleen were evaluated. Levels of IL-1β, L-6, TNF-a in serum and cAMP concentration in FLS were tested by ELISA. Serum level of adenosine was detected by HPLC. The protein expression of A2AAR, β-arrestin2, and IκBα in FLS, as well as the activity of Gas were determined by Western blot. The combination between β-arrestin2, A2AAR and Gas were determined by Co-Immunoprecipitation.RESULTS1. Effects of MTX in the treatment of CIA rats.In the process of the CIA development, the arthritis index, the globle assessment, paw swelling and swollen joints count were gradually increased from d14 after immulization, and were decreased after reaching to the inflammatory peak at d28, most of the CIA rats got remited at d42. After MTX treatment, not all the CIA rats were recovered, there are 50% CIA rats didn’t show obvious response to the treatment. Compared with CIA group, the arthritis index, the globle assessment, paw swelling and swollen joints count were significantly attenuated in another 50% rats treated with MTX.2. Effects of MTX on the joints histopathology of CIA rats.In CIA rats, histology examination for joints showed that the synoviocytes abnormally proliferated, more lymphocytes infiltrated in the synovium, and the articular cartilages were destroyed. The hyperplastic synovium in CIA rats included a large number of fibroblasts and new blood vessels. The histopathology of the joints was obviously ameliorated in rats which can be effectively treated by MTX (MTX Eff group), while the MTX treatment ineffective rats have no changes (MTX Ineff group).3. Effects of MTX on indices of thymus and spleen in CIA rats.Results showed that thymus index and spleen index of CIA model rats were increased compared with normal rats. Comparing with the CIA rats, both spleen index and thymus index were reduced in MTX Eff group, but the indices were not markedly changed in MTX Ineff group.4. Effects of MTX on serum cytokines levels in CIA rats.In the process of CIA, the levels of IL-1β, L-6 and TNF-a were increased gradually and the peak levels were reached aroud d28, following by a recovery at aroud d42. Compared with CIA rats, the serum levels of IL-1β, L-6 and TNF-a were decreased significantly in MTX Eff group, but not in the MTX Ineff group.. Effects of MTX on serum adenosine level in CIA rats.We found that the serum level of adenosine was decreased along with the process of CIA development. The trough level was shown around d28. Comparing with the CIA cats, the level of serum adenosine in MTX Eff rats was increased obviously, however, there is no change in serum adenosine concentration in MTX Ineff rats.6. Effects of MTX on cAMP level in the FLS.With the CIA progressing, on d14 and d28 after immunilization, the cAMP level in FLS of rats were decreased and returned to normal level until d42. Comparing with CIA rats and the MTX Ineff group, the cAMP level in FLS was upregulated in MTX Eff group. The cAMP level in FLS was decrease in MTX Ineff group than that of the normal rats.7. Expression of A2AAR, β-arrestin2 and IκBa in MTX treated rats.The Expression of A2AAR, β-arrestin2 and IκBa in FLS of CIA rats was increased along with the CIA procession and still was in high level on d42. Comparing with the normal rats, the expression of A2AAR and IκBα was increased while β-arrestin2 expression was decreased in MTX Eff group. However, A2AAR and IκBα expression was downregulated and β-arrestin2 level was elevated in the FLS from MTX Ineff group.8. Effects of MTX on the Gas activation of FLS from CIA rats.With the developing of CIA, the activation of Gas was gradually decreased and dropped to the lowest level around d28. There was a lower activation of Gas in CIA FLS on d42 than that of the normal FLS. Compared with normal rats, the Gas activation was obviously decreased in MTX Eff group, while no changes were observed in MTX Ineff group.9. Location and relationship between β-arrestin2, A2AAR and Gas in the FLS.Compared with the MTX-Ineff group, the corelation between A2AAR and β-arrestin2 was decreased, but the combination of A2AAR and Gas was increased in MTX Eff group.CONCLUSIONS1. CIA rats response differently to MTX treatment.Under MTX treatment, only part of the CIA rats got obvious therapeutic effect, the effective rate was 50%.2. The effective treatment of MTX on CIA rats maybe associated with the modulation of A2AAR signaling pathway.In the FLS of MTX Eff group, A2AAR expression was increased, Gas activity was enhanced, cAMP production was promoted, and the proimflammatory cytokines such as IL-1-β, IL-6 and TNF-α were reduced. The different activity of A2AAR signaling pathway may be one of the mechanisms to explain the different responses of CIA rats to the MTX treatment. |
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