NF-?B Signaling Pathway Mediated By BAFF Involved In The Activation Of B Cells In Rheumatoid Arthritis And The Regulation Of CP-25

Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by synovial inflammation and joint erosion.The pathogenesis of RA has been still unclear.Studies show that B cells play an important role in the pathogenesis of RA.B cell activating factor of TNF family(BAFF)is an important cytokine to maintain B cells homeostasis in vivo,which can promote the survival,differentiation and activation of B cells.BAFF has three receptors: B cell-activating factor of the TNF family receptor(BAFF-R),transmembrane activator and calciummodulator and cyclophilin ligand interactor(TACI)and B cell maturation antigen(BCMA).Excessive expression of BAFF is closely related to the pathogenesis and progression of autoimmune diseases such as RA.BAFF plays a biological role by activating NF-?B signaling pathways through binding to its receptors.However,whether does BAFF involve in B cell activation through NF-?B signaling pathways mediated by its receptor in RA? It is rarely reported.CP-25 is structural modification monomer compounds of paeoniflorin.Previousstudies suggested that CP-25 had obvious therapeutic effect on adjuvant arthritis rats.CP-25 also inhibited the maturation of dendritic cells stimulated by PGE2 or TNF-? by down-regulating the expression of CD40,CD80,CD83,CD86 and MHC-II.Whether is the anti-inflammatory effect of CP-25 related to the regulation of B cell function?Whether does CP-25 regulate B cells through NF-?B signaling pathway mediated by BAFF / BAFF receptors? What are the differences of regulation on B cell functions between CP-25 and biological agents for the treatment of RA in clinic(Etanercept or Rituximab)? It is still not clear.In this study,we observed the BAFF level in serum of RA patients,BAFF receptors expression and activation of NF-?B signaling pathways in B cells of RA patients and analyzed the association between BAFF levels and clinical or laboratory indicators.The activated B cell model stimulated by BAFF was established in vitro.We examined the changes of BAFF receptor and BAFF-mediated NF-?B signaling pathways in B cells stimulated by BAFF.At the same time,we also observed the effect of CP-25 on B cells function stimulated by BAFF and NF-?B signaling pathways mediated by BAFF.We also compared the differences of regulation of CP-25 on B cell functions with biological agents for the treatment of RA in clinic(Etanercept or Rituximab).Aim:1.To illustrate BAFF-mediated NF-?B signaling pathway involved in the activation of B cells in RA.2.To investigate the effect of CP-25 on NF-?B signaling pathway mediated by BAFF and its receptor.3.To compare the differences of regulation of CP-25 on B cell functions with Etanercept or Rituximab.Methods: RA patients were enrolled.Clinical and laboratory indicators were evaluated at enrollment.Blood samples from RA patients and healthy controls were collected.Peripheral blood mononuclear cells(PBMCs)were isolated by human lymphocyte separation fluid.B cells were isolated by a positive selection using magnetic cell separation.The correlation between BAFF levels and clinical or laboratory indicatorswas analyzed by linear regression.Serum BAFF level was detected by ELISA.The levels of immunoglobulin and cytokines were detected by protein chip technology.In vitro,PBMCs in normal human were isolated by human lymphocyte separation fluid and treated with(100 ?g/ml)and CP-25(10-5 mol/L)or Etanercept(10 ?g/ml)or Rituximab(5 ?g/ml)).B cell proliferation was detected by CCK-8.B cell subsets and BAFF receptors(BAFFR,BCMA and TACI)were analyzed by flow cytometry.The expression of MKK3,MKK6,P-p38,P-p65,TRAF2 and p100/52 was analyzed by Western blotting.Results:1.NF-?B signaling pathways mediated by BAFF and its receptor involved in B cell activation in RA1.1.The levels of immunoglobins and cytokines in serum of RA patients were higher than that in HCsCompared with HCs,the levels of IgA,IgE,IgD,IgG1,IgG2 and IgG4 in serum of RA patients were high.The levels of IL-1alpha,IL-1beta,IL-6,IL-8,TNF-alpha,IFN-gama,MCP-1,IL-4 and IL-13 were also elevated.Especially,the levels of IL-1alpha,IL-4 and TNF-alpha were increased significantly.The level of IL-10 was not different between RA patients and HCs.1.2.Abnormal activation of B cell subsets were observed in RA patientsCompared with HCs,the percentage of CD19+B cells,CD19+CD27+B cells,CD19+CD20+CD27+B cells and CD19+CD20-CD27+B cells was increased significantly in RA patients.1.3.The expressions of BAFFR,BCMA and TACI on B cells in RA patients were elevatedCompared with HCs,the percentage of CD19+BAFFR+ B cells,CD19+BCMA+ B cells,and CD19+TACI+ B cells was increased significantly in RA patients.1.4.BAFF level was association with clinical and laboratory indicators in RA patientsBAFF level in RA was higher than that in HCs.The mean values of BAFF in RA patients and HCs were 270 ± 24 pg/ml and 192 ± 9 pg/ml respectively.There was a significant difference between the two groups(p=0.008).There were significantcorrelations between BAFF level and RF(p=0.03)or CRP(p=0.003).An association was also showed between BAFF level and DAS28,SJC or TJC.But BAFF level was not associated with anti-CCP.1.5.Expression of signal molecules in BAFF mediated signaling pathway in the B cells of RA Patients The expression of MKK3,MKK6,P-p38,P-p65,TRAF2 and p52 in B cell RA patients was higher than that in HCs.Compared with HCs,the expression of p100 in RA was decreased.2.CP-25 down-regulated B cell function by regulating NF-?B signaling pathway mediated by BAFF and its receptor.2.1.CP-25 inhibited B cells proliferationCompared with control group,the proliferation of B cell was increased in BAFF group.Compared with BAFF group,CP-25,Rituximab and Etanercept inhibited B cells proliferation.No significant difference between CP-25 and Rituximab or Etanercept.2.2.CP-25 down-regulated B cell subsetsCompared with the control group,BAFF increased the percentage and number of CD19+B cells,CD19+CD20+B cells,CD19+CD27+B cells and CD19+CD20+CD27+B cells.Compared with BAFF group,CP-25 reduced the percentage and number of CD19+B cells,CD19+CD20+B cells,CD19+CD27+B cells and CD19+CD20+CD27+B cells.There was no significant difference compared with control group.Compared with CP-25,Rituximab and Etanercept excessively suppressed the percentage and number of CD19+B cells,CD19+CD20+B cells,CD19+CD27+B cells and CD19+CD20+CD27+B cells,which lead the above B cell subsets to be below the control and CP-25 group significantly.2.3.CP-25 down-regulated the percentage of CD19+BAFFR+ B cell,CD19+BCMA+B cell and CD19+TACI+B cellCompared with the control group,BAFF increased the percentage of CD19+BAFFR+B cell ? CD19+BCMA+B cell and CD19+TACI+B cell.Compared with BAFF group,CP-25 down-regulated the percentage of CD19+BAFFR+B cell ?CD19+BCMA+B cell and CD19+TACI+B cell.There was no significant differencecompared with control group.Rituximab and Etanercept excessively suppressed the percentage of CD19+BAFFR+B cell and CD19+BCMA+B cell,which lead CD19+BAFFR+B cell to be below the control and CP-25 group.The difference was statistically significant.2.4.The effect of CP-25 on TRAF2,P-p38,P-p65,p100/52 expression in B cellCompared with control group,BAFF promoted the expression of TRAF2,P-p38,P-p65 and p100/52.Compared with BAFF group,CP-25 and Rituximab could down-regulate the expression of MKK3,P-p38,P-p65,TRAF2 and p52 in B cells stimulated by BAFF.Etanercept also reduced the expression of MKK3,P-p38,P-p65 and p52.Conclusion:1.High levels of BAFF involved in the activation of B cells through NF-?B signaling pathways.2.Elevated levels of BAFF in serum of RA are associated with RA disease activity.3.BAFF may be a new biomarker for the diagnosis and treatment of RA.4.CP-25 can moderately downregulated B cell function stimulated by BAFF,which is related to its regulation of NF-?B signaling pathways5.Etanercept and Rituximab down-regulated stronger B cell function stimulated by BAFF than CP-25,which suggests that this may be one of the adverse reactions mechanisms of Etanercept and Rituximab for clinical treatment.6.CP-25 may be a promising anti-inflammatory immune and soft regulation drug.