| ObjectiveThis study aimed to detect the frequency of circulating T follicular helper(Tfh)cells and the level of serum interleukin(IL)-21 in patients with new-onset rheumatoid arthritis(RA);To analyse the relationship between the frequency of circulating Tfh cells and the clinical characters of patients measured in the laboratory;to study the regulation of oxymatrine(OMT)on the frequency of spleen Tfh cells in mice with arthritis induced by collagen,and the regulation of oxymatrine on the levels of serum IL-21 and CII-Ab(Collagen type II antibody),the expression of synovial tissue TLR2(Toll-like receptor 2),TLR9 and CXCR5;and to explore the regulation mechanism of oxymatrine on rheumatoid arthritis via Tfh cells and provide experimental evidence for the clinical treatment to rheumatoid arthritis in the future.Methods(1)Peripheral blood of new-onset RA patients(n=30)was collected from the General Hospital of Ningxia Medical University from November 2016 to July 2017,age and sex matched to healthy controls(HC,n=30)that was obtained from the medical examination center,the frequency of Tfh cells was detected by flow cytometry.(2)The level of serum IL-21 was examined by enzyme-linked immunosorbent assay(ELISA).(3)The relationship between the frequency of Tfh cells and clinical measures include erythrocyte sedimentation rate(ESR),rheumatoid factor(RF)and anti-citrullinated protein antibody(ACPA)in RA patients was analysed with GraphPad prism5.(4)Forty two DBA/1 mice were prepared and the mice were divided into six groups(each group has seven mice):normal control group,CIA group,dexamethasone(DEX)group,high dose group of oxymatrine(OMT-H),medium dose group(OMT-M)and low-dose group(OMT-L).Six groups mice were immunized by subcutaneous multi-site injection in the back of the mice with 0.1ml mixture of bovine type II collagen and complete Freund’s adjuvant.The second immunity was did after 21 days.After15 days,the CIA mice were treated with different dose of OMT intraperitoneally once a day,the DEX group with dexamethasone sodium phosphate injection and the normal control group with saline for 45 days.The body weight,paw volume and arthritic score were surveyed dynamically.The joint histopathology were measured with HE staining.(5)The frequency of spleen Tfh cells were detected by flow cytometry.(6)The expression of synovial tissue TLR2,TLR9 and CXCR5 was observed by immunohistochemistry.(7)The levels of the IL-21 and CII-Ab were checked by ELISA.Results(1)The frequency of CD4~+T cells and CD4~+CXCR5~+PD-1~+Tfh cells was increased significantly in the peripheral blood of new-onset RA patients compared with healthy control(p<0.01).(2)The expression level of serum IL-21 in RA patients increased higher than the healthy controls(p<0.01).(3)The frequency of circulating CD4~+CXCR5~+PD-1~+Tfh cells and the serum level of IL-21 in RA patients showed the positive relation to ESR,RF and ACPA.(4)The paw swelling and arthritic scores of CIA mice treated with different dose of OMT were relieved obviously compared with the normal control group(p<0.05).(5)The frequency of spleen CD4~+CXCR5~+ICOS~+Tfh cells decreased significantly treated with the OMT,compared with healthy control(p<0.01).(6)The result of HE staining showed that high dose and mediate dose OMT relieved synovial hyperplasi and inflammation of CIA mice.(7)The expression of TLR2 and CXCR5 of ankle synovial tissue in model group were higher than the OMT group measured by IHC.The expression of TLR9 may be stimulated by high dose and mediate dose OMT,but expression of TLR9 was not observed in model group.(8)OMT decreased the serum expression levels of IL-21 and CII-Ab in the model mice(Progressive p<0.05).Conclusion1.Tfh cells participated in the occurrence of rheumatoid arthritis.2.Oxymatrine anti-rheumatoid arthritis via regulating Tfh cells,which may be associated with the influence on the expression of CXCR5,TLR2 and TLR9. |
