Zaocy S Type Ⅱ Collagen Regulated The Balance Of Treg/Th17Cells In Mice With Collagen-induced Arthritis

BackgroundRheumatoid arthritis (RA) is a chronic immune-mediated rheumatic disease which is characterized by synovial inflammation, and cartilage destruction and bone erosion subsequently. Though the exact etiology of RA still remains unclear, genetic and environmental factors are confirmed to play important roles in it. RA comes along with articular and systemic effects, its pathophysiological change is known as the combined action of T cells, B cells and the pro-inflammatory cytokines.Studies have already identified Th17cell and Treg cell, and gradually revealed their effects on immune-pathogenesis of RA, which was a breakthrough for the classical view of Thl/Th2being main effectors in RA. It is well-known that cytokines are great contributory to the cause of RA. And Th17cell can selectively secrete the pro-inflammatory cytokine IL-17A and other cytokines, including IL-21and tumor necrosis factor (TNF)-a, etc. Therefore, the hyperactivity and the increasing population of Th17cell is believed to be involved in the formation and development of RA, which is related to the hyper-production of IL-17A. As a result,IL-17A was supported as an important promoter in the development and pathogenesis of RA by the studies in rodents and mammalian cell culture experiments. Moreover, Treg cell counts and their function have also been considered as a new examination item in patients with RA since the last few years. Some studies have revealed that patients with RA had a decreased number of Treg cell in their serum, demonstrating that the reduction and hypo-function of Treg cell were also important in the immune imbalance that results in RA. Treg cells, comprising5-10%of CD4+T cells and marked by forkhead box P3protein (Foxp3) expression, have a pivotal role in controlling excessive immune reaction by suppressing pathogenic T cells. Treg cells suppress the immune responses via the release of cytokines such as TGF-β and IL-10, as well as up-regulating the expression of suppressive immune-cell surface molecules and inhibiting the expression of genes that activate T cells.What’s more, Transformation can happen between Treg and Th17cells under particular circumstance. TGF-β is a critical differentiation factor for the generation of Treg cell. With the help of TGF-β separately, naive CD4+T cells could differentiate into Treg cell. Instead, when TGF-P and IL-6exist together, they could induce the expression of RORyt, which will promote the differentiation of naive T cells into Th17cell. However, although we agree that the significance of Th17cell and Treg cell in experimental animal models of arthritis is beyond doubt, the limited data we own can’t yet prove the important roles that Th17/Treg cells and related cytokines have played in human arthritic diseases. But their suppression on arthritic responses in animal models of arthritis, makes them promising candidate for therapeutic targets in arthritic diseases like RA.For another, oral tolerance is a continuous natural immunological event which is driven by exogenous antigen. In this sense, it is of unique importance in immunology. In fact, it is considered that oral tolerance involves the activity of non-inflammatory/regulatory auto-reactive T lymphocytes present at a stable frequency in the normal immune repertoire. It is well known that the main mechanism of induction of oral tolerance is mediated by Treg cells via mediating suppression reactions. Treg cells can produceTGF-β, α cytokine with known regulatory functions. They can also exert function as important regulatory factors and express latency-associated peptide (LAP) on their surface. The expression of the LAP-TGF-p complex on the surface of Treg cells played a crucial role in controlling RA. Their major effect is to regulate the functions of other T lymphocytes, so as to avoid excessive immune activation, and further control autoimmune disease or auto-inflammatory reactions. In recent years, the induction of oral tolerance has been intensively investigated and applied to studies on autoimmune diseases in experimental animal models and in human condition.Zaocys has been applied to RA as a treatment in China for over1000years. It was widely used and its curative effect was quite recognized as well. But the medicative components of zaocys and the target are still unclear. According to modern pharmacology studies, zaocys has anti-inflammatory and analgesic effect. Previous researches from our team showed that ZCⅡ could ameliorate clinical signs of arthritis and suppressed the activity and level of the cytokines produced by synoviocytes. On the other hand, the model of CIA, which is the most common experimental animal model for studies on RA, has many similarities in clinical signs, pathological change and immunological features with RA.In order to further investigate the mechanisms of ZC Ⅱ’s therapeutical effect on RA and demonstrably explain the target, our study has analyzed the change of Treg cell, Th17cell and their cytokines in CIA mice, so as to achieve a further understanding about how ZCⅡ works on RA.Objective1Extracted and identified Analyzed the homology of ZCⅡ from Zaocys.2To investigate the possible mechanism by observing the effect of ZCⅡ on Treg/Th17cells in different parts of CIA mice.Methods1The homology oftype Ⅱ collagen from Zaocys.1.1Type Ⅱ collagen protein was abstracted and purified from Zaocys by Pepsin digestion;1.2Molecular weight of type Ⅱ collagen from Zaocys was detected by SDS-PAGE analysis.1.3Ultraviolet absorption of type Ⅱ collagen from Zaocys was detected by ultraviolet spectrophotometer.1.4The homology of type II collagen from Zaocys with other species was analyzed by Mass Spectrometry.2The effect of type II collagen from Zaocys on CIA mice2.1CIA was induced in C57BL/6mice by immunization with CCⅡ.2.2The mice were divided into the following groups at random:normal group, CIA model group, and three ZCⅡ groups, in which ZCⅡ (10,20, and40μg·kg-1·d-1) was intragastrically administrated to the mice with CIA from day21to28after immunization. The mice in the normal and CIA model groups received an equal volume of acetic acid at the same time.2.3Mice were monitored for signs of arthritis by2independent observers on day14、17、20、23、26、29after immunization, and a macroscopic scoring system was used to evaluate the severity of arthritis. Each paw was evaluated and scored individually on a scale of0-4:0=no swelling and focal redness,1=swelling of finger joints,2=mild swelling of ankle or wrist joints,3=severe inflammation of the entire paw, and4=eformity or ankylosis.2.4Lymphocytes were isolated from splenocytes and MLNLs in mice by routine method.2.5Treg cell was labeled with Anti-Mouse CD4FITC and anti-mouse CD25APC and stained the cells with anti-mouse Foxp3PE or PE-Rat IgG2a. Th17cell was stimulated with50ng/ml PMA/Ionomycin mixture plus500ng/ml BFA/Monensin Mixture, then stained with Anti-Mouse CD4FITC and then fixed and permeabilized, followed by intracytoplasmic staining using anti-IL-17A PE or PE-Rat IgG2a. After staining, the percentages of cells with different positive labels were analyzed on a FACSCalibur flow cytometer with CellQuest software (Becton Dickinson)2.6For intracellular cytokine analysis from splenocytes, MLNLs and serum, cells were stimulated with50ng/ml PMA plus500ng/ml ionomycin for4-5hours in the presence of lμg/ml BFA. The level of TGF-β and IL-17A were measured by enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions.2.7The paws from each mouse were collected under sterile conditions and then stained with hematoxylin and eosin. The histologic analysis was assessed microscopically by2independent observers. The severity of arthritis was evaluated and scored individually on a scale of0-4.2.8The changes of Treg/Th17cells were measured by direct immunofluorescence. After de-waxing and hydration, tissue slices were incubated with3%BSA to block the non-specific binding sites. Joint tissue sections were fixed in acetone, and stained for Treg cell with FITC-labeled anti-CD4(1:100) and PE-labeled anti-Foxp3(1:100), while Th17cell were stained by FITC-labeled anti-CD4(1:100) and PE-labeled anti-IL-17A (1:100). After incubation overnight at4℃, cell nucleus was stained with DAPI. Anti-fluorescence quenching agent was used as a mounting medium. We observed the results of double immunofluorescence with Leica inverted fluorescence microscope and took pictures after observation.3Statistical analysisData analyses were performed using SPSS software of version13.0. Results are expressed as the mean±SD. Statistical differences of the percentage of Treg and Th17cells were analyzed using analysis of variance (ANOVA) with LSD adjustment. And the differences of scores for histopathologic changes were analyzed by ANOVA with Tamhane adjustment.P values less than0.05were considered significant.Results1The homology of type Ⅱ collagen from Zaocys1.1Molecular weight of CⅡ type Ⅱ collagen from Zaocys is about110KD-140KD detected by SDS-PAGE analysis, the same as the molecular weight of the standard CⅡ.1.2The UV absorption peak of type Ⅱ collagen from Zaocys is about230nm detected by ultraviolet spectrophotometer, the same as that of the standard CⅡ.1.3The peptide mass fingerprinting (PMF) of type Ⅱ collagen from Zaocys by Mass spectrometry analysis showed that it had at least4peptides matched with other species, and the protein score was greater than95%, demonstrating that the collagen was ZCⅡ.2The effect of type Ⅱ collagen from Zaocys on mice with CIA2.1CIA was induced in C57BL/6mice by immunization with CCⅡ. After immunization, ZC Ⅱ (H、M、L;40,20and10μg/kg/d)was administered orally to mice from day21to28accordingly. The severity of the arthritis in each limb was evaluated using a macroscopic scoring system, and ZC II groups showed significant difference against the CIA model group on therapeutic effect, suggesting that ZC Ⅱ can remarkably suppressed the severity of arthritic paws. The therapeutic effect of ZCⅡ on mice with CIA was further verified by histologic examination. Compared with normal group, mice of model group had obvious clinical signs including inflammation of synovial tissue, pannus formation, cartilage destruction, and bone erosion. In contrast, remarkable reduction in inflammatory cell infiltration and pannus formation could be seen within the joint tissues of ZCⅡ treated mice. Besides that, ZCⅡ could also ameliorated clinical signs including cartilage destruction and bone erosion significantly.2.2Treatment with ZCⅡ increased Treg cell but reduced Th17cell frequencies.The percentage of Treg cell from splenocytes and MLNLs of CIA mice was up-regulated, compared with normal group. Treatment with ZCⅡ to CIA mice could significantly increase the percentage of Treg cell more. Compared with normal group, the frequency of Th17cell from splenocytes and MLNLs of CIA mice was also increased, but ZCⅡ treated mice with CIA showed a decrease in Th17cell in contrast. We determined the counts of CD4+Foxp3+Treg cell and CD4+IL-17A+Th17cell in jointtissues from mice treated with ZCⅡ. The results demonstrated that ZCⅡ increased the number of Treg cell and reciprocally decreased the number of Th17cell, compared with CIA model group.2.3The effect of ZCⅡ on TGF-β and IL-17A in mice with CIACompared to normal group, the mice with CIA had a higher level of TGF-β from serum, splenocytes and MLN lymphocytes. And mice treated with ZC II also had a higher level of TGF-β than the model group significantly. Meanwhile, the CIA mice had a higher level of IL-17A compared with normal group. Whereas treatment with ZCⅡ to the mice with CIA resulted in a decreased production of IL-17A compared with model group.ConclusionThe bands of CⅡ samples extracted by Pepsin digestion and purified from Zaocys could be seen as same as the bands of the standard CⅡ.The oral administration of ZCⅡ suppressed hind paw secondary swelling, and remarkable reduction in inflammatory cell infiltration and pannus formation, etc. could be seen in ZC Ⅱ-treated mice.Oral administration of ZCⅡ could enhance the expression of TGF-P, a immunosuppressive cytokine, in the serum and lymphocyte culture supernatant of MLNLs and splenocytes from the CIA mice, so as to inhibit the function of T cells and antigen presenting cells(APC) indirectly, and suppress the generation of IL-17A.