| ObjectiveRheumatoid arthritis is an autoimmune disease characterized by chronic inflammation,which affects joints and cartilage.Additionally,rheumatoid arthritis could cause different degrees of joint pain,stiffness,swelling and deformity,even could cause different degrees of disability.At present,the main purpose of treating rheumatoid arthritis is relieved pain and inflammation,reduced joint injury and disability,maintained or improved body function,and ensured normal quality of life.China is a big country of Chinese herbal medicine.Chinese medicine treatment of rheumatoid arthrit has a long history and rich treatment experience and strategy.Hence,Chinese medicine is widely used in the clinical treatment of rheumatoid arthritis.Under the guidance of the concept of “treating according to local conditions” and“treatment based on syndrome differentiation”,our laboratory has worked out the Dutengyimu decoction(KDYMF),which was an effective compound for clinical.Previous laboratory studies have confirmed that KDYMF has a good effect on rheumatoid arthritis.And as a Vietnamese doctor,I has promoted KDYMF in Vietnam and achieved certain clinical results.but its mechanism in the treatment of rheumatoid arthritis is still largely unknown.In recent years,with the deepening understanding of the pathogenesis of rheumatoid arthritis,it was found that the functional imbalance between T helper 17 cells(Th17)and regulatory T cells(Treg)is one of the pathogenesis of rheumatoid arthritis.Therefore,collagen induced arthritis(CIA)animal model and cell experiment were used to investigate the effects of KDYMF on Th17 / Treg imbalance,inflammatory factors,and micro RNAs expression level.The conclusion was provided a new theoretical basis for the mechanism of KDYMF in the treatment of rheumatoid arthritis.Methods(1)Twenty-four SPF DBA/1 male mice,nine weeks old,were divided into four groups: normal group(Normal),model group(Model),methotrexate group(MTX),and Duanteng Yimu Decoction group(KDYMF)according to random number table method.Normal mice were used as normal group and MTX group as positive control group.The sample size of each group was 6.Mice were immunized with bovine collagen II(CII)to induce arthritis.From the 21 st day after the first immunization,the corresponding treatment as follow: KDYMF(0.2 m L/10g),once a day for 2 weeks;MTX group,2 mg/kg,gavage twice a week for 2 weeks;the normal control group was given the same volume of normal saline,once a day,for 2 weeks.Clinical scores of limbs were observed every 2 days.On the 35 th day,the mice were killed by cervical spondylectomy.Blood,spleen,synovium,and cartilage of mice were collected on day 35.The histopathological changes of ankle joint was detected using HE staining.(2)The m RNA expression levels of the specific transcription factor forkhead box protein 3(Foxp3)in Treg cells and retinoid related orphan nuclear receptor(RORγt)in Th17 cells in peripheral blood mononuclear cells(PBMC)and joint synovial tissue was measured using RT-q PCR.The m RNA expression levels of Toll like receptors 4(TLR4)and signal transducers and activators of transcription(STAT3)in serum,synovial tissue and articular cartilage tissue were detected using RT-q PCR;The expression levels of miR-21-5p in serum and synovial tissues were detected using RT-q PCR.The expression levels of interleukin-1β(IL-1β),IL-17,tumor necrosis factor-α(TNF-α)and IL-10 in PBMC were measured by ELISA.(3)T cells were isolated from spleen of mice and cultured in the polarized environment of IL-6(30 ng/m L),and then treated with KDYMF(600 μg/m L).The experiment was divided into four groups: control group(Control),KDYMF group(KDYMF),IL-6 group(IL-6),and IL-6+KDYMF group(IL-6+KDYMF).After 48 hours of culture,the expressions of IL-17,IL-1β,TNF-α and IL-10 were detected by RT-q PCR.Then,anti-CD4 antibody(2 μg/m L)and anti-CD25 antibody(2 μg/m L)were used to measured the ratio of Th17 and Treg cells in flow cytometry.(4)Mice T cells were cultured with IL-6(30 ng/m L)at the same time with Fibroblast-like synoviocytes cells(FLSs),and then treated with KDYMF(600μg/m L).The experiment was divided into four groups: T cells+FLSs group(Control),T cells+FLSs+KDYMF group(KDYMF),T cells+FLSs+IL-6 group(IL-6),T cells+FLSs+IL-6+KDYMF group(IL-6+KDYMF).After 48 hours of culture,the proliferation of FLSs was detected by CCK8 method.After 48 hours of culture,the migration and invasion of FLSs were assessed using Transwell;and the expression levels of miR-21-5p,Foxp3,and RORγt were detected using RT-q PCR.Results(1)From the 25 th day,obvious clinical symptoms of rheumatoid arthritis were observed in both ankle joints of model group.On the 35 th day,most mice showed ankylosing changes of ankle joint.There was no obvious swelling of ankle joint in MTX group and KDYMF group,and there was no significant difference in joint range of motion between two groups and Normal groups.(2)The pathological results of HE staining showed that the ankle joint structure of CIA mice was seriously damaged,inflammatory cells infiltration was obvious,synovial hyperplasia,leading to cartilage and bone damage.In MTX group and KDYMF group,ankle joint was relatively intact,synovial thickening was not obvious,and a small amount of inflammatory cells infiltrated.(3)On the 35 th day of immunization,the relative m RNA expression of Foxp3 in PBMC and synovium of mice in MTX group and KDYMF group was up-regulated,but it was more significant in KDYMF group.However,the relative m RNA expression of RORγt in PBMC and the synovial tissues of mice in MTX group and KDYMF group was down regulated,but it was more significant in KDYMF group.(4)On the 35 th day of immunization,compared with model group,the expression levels of IL-1β,IL-17 and TNF-α in PBMC of MTX group were up-regulated,while the expression of IL-10 was significantly down regulated.The expression levels of IL-1β,IL-17 and TNF-α in PBMC of KDYMF group were lower than those of MTX group,which were closer to the expression level of normal group;while the expression level of IL-10 was higher than that of MTX group and closer to that of normal group.(5)On the 35 th day of immunization,compared with model group,the relative expression levels of miR-21-5p in serum and synovial tissue of mice in MTX group and KDYMF group were up-regulated,but more significantly in KDYMF group.(6)On the 35 th day of immunization,compared with model group,the relative m RNA expression levels of TLR4 and STAT3 in serum,synovial tissue and articular cartilage tissue of mice in MTX and KDYMF groups were down-regulated.But it was more significant in KDYMF group and close to normal group.(7)KDYMF could inhibit the expression of inflammatory factors IL-17,IL-1β and TNF–α in T cells,while promote the expression of IL-10.IL-6stimulation could promote the expression of inflammatory factors IL-17,IL-1βand TNF-α in T cells,but inhibit the expression of IL-10;KDYMF could reduce the expression of inflammatory factors IL-17,IL-1β and TNF-α in T cells stimulated by IL-6,but up regulate the expression of IL-10.(8)KDYMF could up-regulate the proportion of Treg cells and reduce the proportion of Th17 cells.IL-6 significantly increased the number of Th17 cells and decreased the number of Treg cells;Additionally,KDYMF could increase the proportion of Treg cells,down-regulate the proportion of Th17 cells,and make Treg cells and Th17 cells closer to the normal level.(9)KDYMF could significantly inhibit the proliferation,migration,and invasion of FLSs.At the same time,it could significantly inhibit the proliferation,migration,and invasion of FLSs co-cultured with T cells stimulated by IL-6.(10)KDYMF could significantly promote the relative expression of miR-21-5p and Foxp3 m RNA in FLSs under normal conditions,and reduce the expression levels of RORγ m RNA.At the same time,it could significantly up regulate the relative expression of miR-21-5p and Foxp3 m RNA in FLSs co-cultured with T cells stimulated by IL-6,and decrease the expression level of RORγt m RNA.Conclusions(1)There was imbalance of Th17/Treg cell function in rheumatoid arthritis mice.(2)In vivo animal model and in vitro cell model confirmed that KDYMF could significantly increase the level of IL-10 in serum or cells,and down regulate the levels of IL-1β,IL-17 and TNF-α.(3)In vivo animal model and in vitro cell model confirmed that KDYMF could significantly inhibit the differentiation of Th17 cells,promote the generation of Treg cells,and improve the imbalance of Treg/Th17.(4)KDYMF could inhibit the proliferation,migration,and invasion of FLSs.(5)KDYMF could promote the relative expression levels of miR-21-5p. |
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