| Background:Rheumatoid arthritis(RA)is a common systemic autoimmune joint disease,characterized by chronic synovial inflammation,significant synovial membrane proliferation,infiltration of inflammatory cells,abnormal secretion of cytokines and destruction of articular cartilage and bone.Rheumatoid arthritis synovial cells(RASF)showed characteristics of abnormal cell proliferation and decreased apoptosis.A large number of immunological studies have found that regulatory T cells(Treg)play important roles in RA.Treg cells are differentiated from CD4+ T cell subsets,accounting for 5%-10%of CD4+ T cells.Treg cells specifically express interleukin(IL)-2 receptor a chain CD25 and can secrete IL-10,IL-35 and transforming growth factor-?(TGF-?).Treg cells have strong immunosuppressive functions and are closely related to the pathogenesis of RA.Defective numbers and functions of peripheral blood Treg cells in RA patients lead to over-activation of effector T cells and promote the development of RA.Helper T(Th)17 cells also differentiated from CD4+ T cells.In contrast to Treg cells function,they exert pro-inflammatory effects in RA.Treg cells and Th17 cells form a dynamic balance to preserve the body in normal immunity.An imbalance of Treg and Th17 cells is the typical feature of RA.It is reported that the frequency of Treg cells are decreased,while that of Th17 cells are increased in RA patients.Following anti-rheumatism treatment,the level of Treg cells in RA patients increases and the Th17 cells level decreases,and joint inflammation relieved significantly.Thus,increasing Treg cells level in RA and improving Treg/Th17 cell imbalance may be the key to RA treatment.Additionally,Th1/Th2 lymphocyte subsets imbalance also play an important role in the development of RA.Studies showed that joint inflammation in RA is dominated by Th1 cells,and the ratio of Th1/Th2 is elevated in actived RA.Decreasing the Th1/Th2 ratio and improving Th1/Th2 imbalance also have a positive effect on RA treatment.Therefore,this study suggests that supplementation of exogenous Treg cells or the use of drugs to increase Treg cells level may have potential therapeutic effect on RA,by increasing Treg cells level and reducing Th17 cells level and even improving Th1/Th2 imbalance.Our study was conducted in two parts.The first part directly observed the effect of exogenous Treg cells on RA.Human Treg cells were prepared from the peripheral blood of healthy donors.RASF cells were cocultured with the Treg cells and the effects of Treg cells on RASF cells were observed.Meanwhile,Treg cells were injected into collagen ?-induced arthritis(CIA)rats,a well established model of human RA.And the therapeutic effect of exogenous Treg cells on CIA was observed.The effect of Treg cells on RA was investigated from experimental zoological results.In the second part,RA synovial fluid(SF)samples were collected and stimulated with XueBiJing(XBJ)to observe the effect of XBJ on RA in vitro.At the same time,XBJ were used to treat CIA rats and RA patients,and the changes of Treg cells level and the therapeutic effects of increased Treg cells level on CIA rats and RA patients were observed.We hypothesized that Treg cells could inhibit the abnormal proliferation and apoptosis of RASF cells in vitro.Supplementation therapy with exogenous Treg cellsor XBJ treatment could increase Treg cells level,reduce Th17 cells level and might even affect Th1/Th2 balance to treat RA.XBJ is a Chinese medicine preparation,which implements the effects of activating blood circulation to dissipate blood stasis and cooling blood to remove toxic substances.XBJ is mainly used in the treatment of sepsis and multiple organ dysfunction syndromes.It is reported that XBJ injection upregulates the level of Treg cells and downregulates serum levels of TNF-? and IL-6 in septic mice which result in improving survival rate.Clinical studies showd that XBJ injection reduces mortality and incidence of complications and improves prognosis of sepsis patients.Based on thses studies,we hypothsized that XBJ treatment might also be effective to RA by increasing Treg cells level,reducing Th17 cells level and affecting Th1/Th2 balance.The present study used in vitro experiments,animal models and clinical trials to verify the above hypothesis and explore the effects and mechanisms of Treg cells on RA.Methods:1.Primary RASF cells were prepared from synovial tissues and Treg cells were prepared from peripheral blood lymphocytes of healthy donors.Mature Treg cells were collected and cocultured with RASF cells.The cell proliferation and apoptosis of RASF were determined by the CCK-8 assay and flow cytometry,respectively.2.Lymphocytes in SF of RA patients were isolated and stimulated with or without XBJ.Flow cytometry was used to detect changes in Treg cells level and Th1/Th2 ratio in SF before and after stimulation to investigate weather XBJ injection could increase Treg cells level and improve Th1/Th2 imbalance in SF.3.The CIA rat model was established,human Treg cells derived from the peripheral blood of healthy donors or XBJ were injected into the CIA rats via the tail vein.The joint inflammation of normal control(NC)group,CIA group,Treg treatament group and XBJ treatment group was observed and analyzied with inflammatory curve and histochemistry analyses to determine the therapeutic effect of exogenous Treg cells and XBJ on CIA rats.4.Treg cells,nature killer cell(NK)and B lymphocyte subsets of peripheral blood and spleen were detected by flow cytometry to investigate the changes of lymphocyte subsets before and after Treg cell or XBJ treatment.5.Cytokines including interleukin(IL)-2,IL-4,IL-5,IL-6,IL-10,IL-13,IL-17A,tumor necrosis factor-?(TNF-?),interferon-?(IFN-?)and granulocyte-macrophage colony-stimulating factor(GM-CSF)in the peripheral blood were observed by flow cytometry to investigate the changes of cytokines level before and after Treg cell orXBJ treatment.6.IL-4 and IFN-y in peripheral blood of CIA rats were detected by flow cytometry and the ratio of Thl/Th2 was calculated to investigate the changes of Thl/Th2 balance before and after Treg cell or XBJ treatment.7,Four RA patients were recruited and treated with XBJ.The erythrocyte sedimentation rate(ESR),C-reactive protein(CRP),rheumatoid factor(RF)and 28 joint disease activity scores(DAS 28)were measured before and after treatment to investigate the effectiveness of XBJ treatment on RA.8.Treg cells,Thl7 cells level and Thl/Th2 ratio of peripheral blood were detected by flow cytometry to investigate the changes of immue imbalance in RA patients before and after XBJ treatment.Results:1.After coculture with Treg cells,CCK-8 assay showed that RASF cell proliferation was decreased(P<0.001),and RASF cell apoptosis was increased(P =0.035).These results indicated that Treg cells inhibited RASF cell proliferation and promoted apoptosis.2.After stimulate with XBJ,flow cytometry showed that Treg cells level in SF were significantly increased(P<0.001),and the Thl/Th2 ratio was slightly decreased but showed no significant difference(P =0.241).These results indicated that XBJ could increase the Treg cells level in SF,but had no effect on Thl/Th2 balance.3.Inflammation curves showed that Treg cell treatment and XBJ treatment had significantly alleviated arthritis symptoms(P<0.001,P<0.001),which was close to the joints of NC group(P=0.491,P=0.639).HE staining showed that Treg cell treatment and XBJ treatment signif-icant decreased synovial hyper:plasia,inflammatory cell infiltration and bone damage occurred in CIA rats.These results indicated that Treg cell treatment and XBJ treatment could inhibite CIA.4.Flow cytometry showed that the proportions of rat Treg cells in the peripheral blood and spleen of the CIA group were lower(P =0.021,P<0.001)and B cells were higher(P =0.001,P =0.036)than that of the NC group.After treatment with exogenous human Treg cells,the proportion of rat Treg cells in peripheral blood and spleen(P=0.007,P<0.001)and B cells in spleen(P =0.031)went back to normal compared with the CIA group.The proportions of NK cells in the peripheral blood and spleen of the CIA group(P =0.012,P=0.017)and Treg treatment group(P =0.003,P =0.001)were higher than that of the NC group.These results indicated that Treg cell therapy could increase the level of Treg cells and decrease the level of B cells in CIA rats,but had no significant effect on NK cells level.5.Flow cytometry showed that the levels of IL-4,IL-5,IL-6,IL-10,IL-13,IFN-?,GM-CSF and TNF-a in the peripheral blood of CIA group were increased compared with that of the NC group(P =0.043,P =0.002,P =0.002,P =0.002,P =0.030,P=0.019,P =0.016,P<0.001).And the levels of IL-5,IL-6,IL-10,IL-17A and TNF-a in the Treg treatment group were decreased compared with that of the CIA group(P=0,013,P=0.009,P =0.006,P =0.002,P =0.032).These results indicated that Treg cell therapy could reduce IL-5,IL-6,IL-10,IL-17A and TNF-a level,but had no significant effect on IL-4,IL-13,IFN-y and GM-CSF level.6.Flow cytometry showed that the Thl/Th2 ratio in Treg treatment group was decreased significantly compared with that of the CIA group(P =0.012).These results indicated that Treg cell therapy could affect Thl/Th2 balance in peripheral blood of CIA rats.7.Flow cytometry showed that the Treg cells level in the peripheral blood and spleen of the CIA group were lower(P =0.002,P<0.001)and B cells were higher(P =0.015,P =0.016)than that of the NC group.After XBJ treatment,Treg cells(P<0.001,P<0.001)and B cells(P<0.001,P =0.046)level in peripheral blood and spleen went back to normal compared with CIA group.The NK cells level in the peripheral blood and spleen of the CIA group(P=0.005,P =0.013)were higher than that of the NC group.The level of NK cells in peripheral blood of XBJ treatment group was further increased,but showed no significant difference compared with CIA group(P =0.652).The level of NK cells in spleen was slightly decreased but showed no significant difference compared with CIA group(P=0.252),either.These results indicated that XBJ treatment could increase the level of Treg cells in CIA rats and decrease the level of B cells,but had no significant effect on NK cells level8.Flow cytometry showed that the levels of IL-5,IL-6,IL-10,IL-13,IFN-?,GM-CSF and TNF-a in the peripheral blood of CIA group were significantly increased compared with that of the NC group(P=0.007,P<0.001,P =0.005,P=0.034,P=0.002,P =0.004,P =0.005,P =0.013).And the levels of IL-2,IL-4,IL-5,1L-6,IL-10,IL-13,IL-17A,IFN-y,GM-CSF and TNF-a in the XBJ treatment group were decreased compared with that of the CIA group(P<0.001,P =0.012,P =0.001,P<0.001,P<0.001,P =0.002,P<0.001,P<0.001,P<0.001,P =0.003).These results indicated that XBJ treatment could reduce the above 10 cytokines level.9.Flow cytometry showed that the Thl/Th2 ratio in XBJ treatment group showed no significant difference compared with that of the CIA group(P =0.992).These results indicated that XBJ treatment had no effect on the balance of Thl/Th2 in CIA rats.10.Clinical analysis showed that the ESR,CRP,RF and DAS28 scores of the 4 RA patients were reduced after XBJ treatment.Only 2 patients had no significant changes in RF levels after XBJ treatment.These results indicated that XBJ treatment was effective on RA.11.Flow cytometry showed that the Treg cells level of the 4 RA patients increased after XBJ treatment,while the Thl7 cells level decreased.Thl/Th2 ratio of 3 patients decreased after XBJ treatment,only 1 patient increased slightly.These results indicated that XBJ treatment could improve the imbalance of Treg/Thl7 and Thl/Th2 in peripheral blood of RA patients.Conclusions:The above results demonstrated that:1.Treg cells inhibit the proliferation of RASF cells and promote apoptosis.2.XBJ stimulation can increase Treg cells level in the SF of RA patients.3.Treg cell and XBJ treatment have therapeutic effect on CIA.4.Treg cell and XBJ may treat CIA by restoring Treg/Th17 and Thl/Th2 cell balance.5.Treg cell and XBJ may treat CIA by increasing IL-6,IL-17A and TNF-? level.These findings suggest that Treg cells can regulate the proliferation and apoptosis of RASF cells.Exogenous human Treg cell derived from the peripheral blood of healthy donors and XBJ may treat CIA by regulating immune balance and inhibiting the secretion of inflammatory cytokines.These findings are helpful to explain the cellular immunological mechanisms of Treg cells on RA therapy.It is suggested that Treg cell supplementation and XBJ treatment may be potential strategies for RA therapy. |
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