| Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmune disease. T lymphocyte plays an important role in the development and pathogenesis of RA. Helper T cell 1 (Th1), Th2, Th17 and regulatory T-cells (Treg), which are the main subtypes of helper T cell, playing pivotal role in the pathology of RA because of their biological functions as mediators of inflammation, cell growth, and activation. A major obstacle to the development of rational treatment strategies is that the mechanisms and the causative environmental and genetic factors of RA remain largely unknown. Although much of the RA pathogenesis remains to be elucidated, it has been reported that joint proteins, probably type II collagen (CII), play a key role in the instigation of T-cell mediated immune responses. Chicken type II collagen (CCII) is a protein extracted from the cartilage of chicken breast and exhibits intriguing possibilities for the treatment of autoimmune diseases by inducing oral tolerance. Trentham firstly reported that oral CII could relieve joint swelling and tenderness index in a random, contrast cinical studies containing 28 subjects. An early study of our topic group found that CCII has an immune regulation in rats with adjuvant arthritis (AA), it could lower the proliferative response of lymphocytes, decrease the level of pro-inflammatory cytokine (IL-2), and lower the secretion of IL-1 in peritoneal macrophages. Furthermore, studies of our topic group about II and III phase of CCII clinical trials, which contained 236 and 503 subjects, respectively, suggested that CCII could reduce pain, morning stiffness, and the incidence of adverse events of CCII is lower compared with Methotrexate (MTX). Based on the former studies of our group, this experiment focus on the mesentric lymph node lymphocytes (MLNLs), to study the mechanisms of how could CCII induce immune balance and its effects.OBJECTIVEAccording to the changes of paw swelling, polyarthritis index, and histological morphological of mesenteric lymph nodes (MLNs) and synovium of CIA rats, we observed the effects of CCII on the production of IL-2, IL-17, IL-4 and TGF-βof CIA rats through analyzing the correlation of paw swelling with the level of pro-inflammatory cytokines and the level of anti-inflammatory cytokines; to confirm the actions of therapeutic effects of CCII on CIA rats at levels of organ, cell and molecule, respectively. Based on the former studies of our group, to study the possibilities function of pro-inflammatory and anti-inflammatory cells in CIA, to further confirm the actions of therapeutic effects and immuno-regulations in the MLNLs of main subtype of T helpers of CCII.METHODSThe model of CIA rats were induced by injection of chicken type II collagen (CCII) emulsion, after immunization, blood serum was collected, then the rats were divided into the following groups: normal group with IgG anti-CII negative, CIA model group with IgG anti-CII positive , CCII (10, 20 and 40μg/kg/d) groups with IgG anti-CII positive. CCII (10,20,40μg·kg-1·d-1, ig, d18-d25) was administrated intragastricly to CIA rats. Secondary paw swelling of CIA rats was measured by volume meter, the arthritic severity in each paw was evaluated by using a macroscopic scoring system. Histopathological change of MLNs and synovium were observed by light microscope. IL-2 activity in MLNLs supernatant sample was measured by ConA-induced splenocyte proliferation of C57BL/6J mice assay. IL-4, IL-17 and TGF-βlevels in MLNLs supernatant sample was measured by enzyme-linked immunosorbent assay (ELISA), the proportion of CD4+CD25+ Treg cells and Th17 cells were labeled by double-color for flow cytometry analysis.RESULTS1. CCII relieves joints swelling , changes inflammation of MLNs and synovium in CIA ratsThe peak of the inflammatory reaction occurred on the d26 after inmmunization, there was a marked secondary inflammatory response in the models, which accompanied with paw swelling, pain, polyarthritis. Compare to the normal rats, the histological morphological of rats knee joints exammination in CIA showed hyperplastic synovium, synovial lining hyperplasia, inflammatory cells infiltration, pannus formation; the MLNs of CIA represented that lymphatic follicle was hyperplastic and the number of inflammatory cells. It was found that CCII (10,20,40μg·kg-1·d-1, ig, d18-d25) significantly suppressed secondary hind paw swelling and polyarthritis index, as well as improved arthritic status histological of mesenteric lymph node and synovium in CIA rats.2. CCII inhibited the pro-inflammatory cytokines IL-2 and IL-17 production, enhanced the anti-inflammatory cytokines IL-4 and TGF-βproduction and induced immune balance of Th1/Th2 and Th17/Treg.CCII (10,20,40μg·kg-1·d-1, ig, d18-d25) significantly diminished the production of IL-2 and IL-17, and increased the lowered levels of IL-4 and TGF-βof MLNLs in rats with CIA, and the results of flow cytometry analysis indicated that the the MLNs from CIA presented with a high ratio of Th17/Treg comparing to normal group, when the administration of CCII significantly increased the proportion of Treg and decreased the proportion of Th17. CCII inhibited the pro-inflammatory cytokines IL-2 and IL-17 production, enhanced the anti-inflammatory cytokines IL-4 and TGF-βproduction and induced immune balance of Th1/Th2 and Th17/Treg.CONCLUSIONS1. CCII significantly suppressed secondary hind paw swelling and polyarthritis index, as well as improved arthritic status histological of MLNs and synovium in CIA rats. CCII significantly reduced the production of IL-2 and IL-17, and increased the production of IL-4 and TGF-βof MLNLs supernatants. The correlation analysis demonstrated that the effects of CCII on relieving joints swelling and inflammation of mesenteric lymph node in CIA rats were correlated intimately with its effects on inhibiting IL-2 and IL-17 production and enhancing IL-4 and TGF-βprodution in MLNLs.2. CCII significantly reduced the production of IL-2 and IL-17, and increased the production of IL-4 and TGF-βof MLNLs supernatants, actived the suppressed anti-inflammatory cells, inhibited the proliferation of pro-inflammatory cells, and then induced the immune balance of Th1/Th2 and Th17/Treg. |
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