| Rheumatoid arthritis(RA)is a chronic,systemic autoimmune disease,and accompanied by synovial inflammation and bone and cartilage destruction.The pathogenesis of RA is still unclear,but a large number of studies have shown that B cells and synovial cells are involved in the occurrence and development of RA.The abnormal activation of B cells is regarded as an early stage marker of RA.B cell-activating factor(BAFF)is essential for T/B cell function.BAFF can regulate B cell proliferation,maturation,activation,activation of assist T cell,and maintain plasmablast survival and proliferation.The signal transduction pathway mediated by BAFF and its receptors promotes B cell activation,proliferation,and inhibits apoptosis,and are also involved in abnormal inflammatory response and immune response of RA.Fibroblast-like synoviocytes(FLSs)are effector cells of RA.BAFF is expressed on FLSs and plays a key role in the course of RA.The impaired apoptosis of FLSs can lead to synovial hyperplasia and promote the destruction of RA cartilage.FLSs proliferation in affected joints is a feature that distinguishes RA from other arthritis.Tumor necrosis factor receptor-associated factors 2(TRAF2),as an adaptor protein,mainly activates nuclear factor of kappa B(NF-?B)signaling pathway and c-Jun N-terminal kinase(JNK)signaling pathway,thereby regulating immune cells and maintaining homeostasis.Under the stimulation of tumour necrosis factor-?(TNF-?),K48 and K63 linked polyubiquitination occurred in TRAF2.The lysine at position 31 of the TRAF2 protein is the main modification site of the K63 polyubiquitin chain and is an essential amino acid for TRAF2 to play the role of a linker protein.TRAF2 can be modified by K48 and K63 linked polyubiquitin chains to regulate NF-?B activation.NF-?B is a nuclear transcription factor that regulates the inflammatory process,innate immune response and adaptive immune response,participates in cell survival,apoptosis,and controls gene proliferation and tumor cell invasion.Is the BAFF-BAFFR-TRAF2-NF-?B signaling pathway activated in RA B cells? Is it involved in the pathogenesis of RA? How about the expression of BAFF-BAFFR-TRAF2-NF-?B signaling protein? Is TRAF2 a key signaling molecule? There is no report at this time.Paeoniflorin-6-oxo-benzenesulfonate(CP-25)is a Paeoniflorin(Pae)structure-modified monomer compound.Previous studies have found that CP-25 has a good therapeutic effect on collagen induced-arthritis(CIA)mice and adjuvant arthritis(AA)rats.CP-25 can inhibit the maturation of dendritic cells and regulate the function of dendritic cells by effecting on TNF-?-mediated signaling pathways.The efficacy of CP-25(50mg/kg)combined with low-dose Leflunomide(LEF)/Methotrexate(MTX)in the treatment of AA rats is consistent with that of high-dose MTX/LEF alone.CP-25 can reduce the expression of BAFF-stimulated BAFF receptor,TRAF2 and NF-?B signaling protein in human peripheral blood B cells.Does CP-25 treat mice CIA by regulating B cell function? Does CP-25 regulate B cells through the BAFF-BAFFR-TRAF2-NF-?B signaling pathway? Does CP-25 regulate BAFF-mediated NF-?B signaling by affecting TRAF2 phosphorylation and ubiquitination activity?In this study,the CIA mice,HEK293 transfected cells and MH7 A synovial cells were used as research objects.Flow cytometry,immunofluorescence assay,co-immunoprecipitation,TRAF2-si RNA silencing TRAF2,plasmid overexpression TRAF2 and western blot were used as research methods.To observe the therapeutic effect of CP-25 on CIA mice and its effect on B cell function,and to clarify the mechanism by which CP-25 inhibits B cell activation and differentiation by regulating the BAFF-BAFFR-TRAF2-NF-?B signaling pathway.To clarify that CP-25 regulates BAFF-mediated NF-?B signaling by affecting TRAF2 phosphorylation and ubiquitination,and provides an experimental basis for studying the mechanism of RA and drug targets.Objective: 1.To observe the therapeutic effect of CP-25 on CIA mice and its effect on B cell function.2.To study that CP-25 inhibits B cell activation and differentiation by regulating the BAFF-BAFFR-TRAF2-NF-?B signaling pathway.3.To clarify that CP-25 regulates BAFF-mediated NF-?B signaling by affecting TRAF2 phosphorylation and ubiquitination activity.Method: 1.DBA/1 male mice were selected to establish CIA model and randomly divided into 5 groups,namely the normal group,the CIA model group,the CP-25 group,the rituximab group and the etanercept group respectively.CP-25(35mg/kg)was oral,and rituximab(5mg/kg)and etanercept(5mg/kg)were intraperitoneal injection.The global score and arthritis index of CIA mice were assessed,the paw swelling was counted,body weight was measured,and spleen and thymus index were measured.CCK-8 method was used to detect T and B lymphocyte proliferation response,HE staining was performed on mice spleen and ankle joints,and pathological changes of mice spleen and ankle joints were observed.2.Flow cytometry was used to detect the percentage and number of peripheral blood and spleen B cell subsets and BAFF receptors in mice at different stages of inflammation,including CD19+ total B cell,CD19+CD27+ activated B cells,CD27+CD19–CD138+ plasma cell,CD19+BAFFR+ and CD19+TACI+ cell? 3.Enzyme-linked immunosorbent assay(ELISA)and protein chip were used to detect BAFF and immunoglobulin Ig G1,Ig A,Ig G3,Ig E,Ig D,Ig M,Ig G2 a and Ig G2 b expression in mice serum.4.The expression of TRAF2 in the spleen of mice was observed by immunohistochemistry,and the expression of TRAF2,MKK3,MKK6,p-P38 and p-P65 in mice spleen B cells was detected by western blot.5.CCK-8 and flow cytometry were used to detect the proliferation and percentage of CD19+TRAF2+ cells in mice spleen.6.The CCK-8 and transwell were used to detect the proliferation and migration of MH7 A cells which stimulated by different concentrations of BAFF.7.The expression of TRAF2 in BAFF-stimulated MH7 A cells was detected by immunohistochemistry.Western blot was used to detect the BAFF-mediated NF-?B signaling protein expression change in MH7 A cells after TRAF2-si RNA interference TRAF2.8.Using CP-25 treatment BAFF-stimulated MH7 A cells,the co-expression of TRAF2 and BAFFR,TRAF2 and GRK2,TRAF2 and NIK was detected by co-immunoprecipitation and immunofluorescence assay.9.Flow cytometry was used to detect the percentages of BAFFR,TRAF2,and GRK2 cells in CP-25 treatment BAFF-stimulated MH7 A cells.10.Constructed the p IRES2-EGFP-TRAF2-wt,p IRES2-EGFP-TRAF2-K31 R,p IRES2-EGFP-TRAF2-K320 R,p IRES2-EGFP-TRAF2-K31RK320 R plasmids,and transfected them into HEK293 cells and MH7 A cells.Western blot was used to detect the over-expressed TRAF2 levels in HEK293 cells and MH7 A cells.Results: 1.CP-25 had obvious therapeutic effect on CIA mice D29 was started every three days to evaluate the whole body of mice and the score of arthritis index,counted the number of swelling of paw and measured the weight index.The results showed that the foot of CIA mice became red and swollen.The arthritis index and swollen joint counts increased with the progression of disease,and the body weight was significantly lower than that of the normal group.The number of paws swelling of CIA mice in CP-25 group gradually decreased from d63,and the arthritis index d65 gradually decreased.The number of paw swelling in the rituximab group gradually decreased from d55,and the arthritic index d63 gradually decreased.In the etanercept group,the number of paw swelling and arthritic index of the mice gradually decreased from d55.CP-25,rituximab and etanercept significantly restored abnormally reduced body weight in CIA mice.Compared with mice in the CP-25 group,rituximab and etanercept significantly reduced the arthritis index and the number of paw swelling in CIA mice.2.In CIA mice,CP-25 can reduce the thymus and spleen index,inhibit the proliferation of spleen B lymphocytes and T lymphocytes,and improve pathological changes in spleen and joint tissues The spleen index and thymus index of CIA mice were significantly higher than that of normal group,and the proliferation ability of thymic T cells and spleen B cells was significantly increased.In the CIA model group,the germinal center of the spleen was blurred and a large amount,the number of lymphoid follicles increased significantly,the density of lymph sheaths around the arteries and the cell density in the marginal area increased,and the red pulp was congested.H&E staining results showed that the articular surface was clearly visible,smooth and intact,joint space was uniform,and neatly arranged single or double synovial cell layers were visible on the outside of cartilage in normal mice joint tissue sections.Compared with the normal group,in the CIA model group the synovial cells had obvious proliferation,increased number of layers,disordered arrangement,synovium hyperplasia,inflammatory cell infiltration and vascular pannus formation,and severe cartilage tissue damage.CP-25,rituximab and etanercept significantly inhibited thymus index and spleen index,reduced abnormal proliferation of thymic T cells and spleen B cells in CIA mice,and reduced the number of lymphoid follicles and germinal centers in CIA mice,reduce red pulp congestion,improve synovial proliferation and bone erosion in CIA mice.Rituximab and etanercept have stronger inhibitory effects on T cell proliferation and suppress T lymphocytes below normal levels.Rituximab and etanercept can also inhibit the periarteriolar lymphoid sheaths and the density of cells in the marginal zone,and inhibit inflammatory conditions such as angiogenesis and cartilage destruction in CIA mice.3.CP-25 reduced the percentage of B cell subsets in peripheral blood of CIA mice at different stages of inflammation The results showed that the percentage of CD19+ total B cells,CD19+CD27+ activated B cells,and CD27+CD19–CD138+ plasma cells between the mice in each group before modeling were not statistically significant.The percentage of CD19+ total B cells,CD19+CD27+ activated B cells,and CD27+CD19–CD138+ plasma cells between each groups at the time of re-immunization on day 21 was not statistically significant,but compared with the normal group,the percentage of CD19+B cells,CD19+CD27+activated B cells,and CD27+CD19–CD138+ plasma cells in CIA group were increased.On day 35,the B cell subsets in the peripheral blood of the mice in each group were compared.The results showed that compared with the mice in the normal group,the percentage of CD19+ total B cells and CD27+CD19–CD138+ plasma cells in the CIA model group increased significantly.The percentage of CD27+CD19–CD138+ plasma cells in the CP-25 group,the rituximab group,and the etanercept group significantly decreased.On the 45 th and 65 th days,the B cell subsets in the peripheral blood of the mice in each group were compared.The results showed that compared with the mice in the normal group,the percentage of CD19+ total B cells,CD19+CD27+ activated B cells,and CD27+CD19–CD138+ plasma cell in CIA group were significant increased.The results at day 45 showed that compared with the CIA model group,CP-25 reduced the percentage of CD19+CD27+activated B cells and CD27+CD19–CD138+ plasma cells in CIA mice,but rituximab and etanercept reduced the percentage of CD19+total B cells,CD19+CD27+activated B cells,and CD27+CD19–CD138+ plasma cells.Compared with CP-25 group,etanercept more significantly reduced the percentage of CD27+CD19–CD138+ plasma cells in CIA mice.On day 65,the results showed that compared with mice in the CIA model group,the percentages and numbers of CD19+ total B cells,CD19+CD27+ activated B cells,and CD27+CD19–CD138+ plasma cells in the CP-25,rituximab and etanercept group were reduced to varying degrees.Compared with CP-25,etanercept to a greater extent inhibited the percentage of CD19+ total B cells,CD19+CD27+ activated B cells and CD27+CD19–CD138+ plasma cells in CIA mice.Compared with the normal group of mice,etanercept inhibited the percentage of CD19+CD27+ activated B cells and CD27+CD19–CD138+ plasma cells and made cell numbers of CIA mice below normal levels.4.CP-25 reduced the percentage of CD19+BAFFR+ cells and CD19+TACI+ cells in peripheral blood of CIA mice at different stages of inflammation The results showed that the percentage of CD19+BAFFR+,CD19+TACI+ cells in the peripheral blood of mice increased significantly after successful modeling,and the analysis results showed that percentage of CD19+BAFFR+ and CD19+TACI+ cells in blood before modeling the mice peripherals between the each groups on day 0,21 and 35 was not statistically significant.On day 45,the percentages of CD19+BAFFR+ and CD19+TACI+ cells in the peripheral blood of the mice in each group were compared.The results showed that compared with the mice in the normal group,the percentage of CD19+BAFFR+ and CD19+TACI+ cells in the CIA model group increased significantly.Compared with mice in the CIA model group,CP-25 and etanercept began to reduce the percentage of CD19+BAFFR+ cells on day 45.On day 65,the percentages and numbers of CD19+BAFFR+ and CD19+TACI+ cells in the peripheral blood of the mice in each group were compared.The results showed that compared with the mice in the normal group,the percentage of CD19+BAFFR+ and CD19+TACI+ cells in the CIA model group were increased significantly.Compared with the CIA model group,CP-25,rituximab and etanercept could reduce the percentages and the numbers of CD19+BAFFR+ and CD19+TACI+ cells in CIA mice.5.CP-25 reduced the percentage of B cell subsets,CD19+BAFFR+,CD19+TACI+ cells in spleen of CIA mice Compared with normal mice,the percentages and numbers of CD19+ total B cells,CD19+CD27+ activated B cells,CD27+CD19–CD138+ plasma cells,CD19+BAFFR+ and CD19+TACI+ cells in spleen of CIA mice were significantly increased.CP-25 can reduce the percentage and number of CD19+ total B cells,CD19+CD27+ activated B cells,CD27+CD19–CD138+ plasma cells,CD19+BAFFR+ and CD19+TACI+ cells in spleen of CIA mice.Etanercept reduced the percentages and numbers of CD19+CD27+ activated B cells and CD27+CD19–CD138+ plasma below normal levels in spleen of CIA mice.Compared with the CP-25 group,rituximab and etanercept had stronger inhibitory effects on B cell subsets. 6.CP-25 reduced serum BAFF and immunoglobulin Ig A,Ig D,Ig G1,Ig G2 a and Ig G2 b levels in CIA mice Compared with normal mice,the serum BAFF levels of CIA mice increased,and the expression of serum immunoglobulins Ig G1,Ig A,Ig E,Ig D,Ig G2 a and Ig G2 b increased significantly.CP-25 reduced the levels of Ig A,Ig D,Ig G1,Ig G2 a and Ig G2 b proteins in CIA mice,and down-regulated the expression of Ig A,Ig G1,Ig D,Ig G2 a and Ig G2 b in CIA mice to physiologically balanced levels.Rituximab inhibited the expression of Ig A and Ig G2 a proteins in CIA mice to below normal levels.Etanercept inhibits the expression of Ig A,Ig G1 and Ig G2 a proteins in CIA mice to below normal levels.CP-25,rituximab and etanercept did not affect Ig G3 and Ig M protein expression in CIA mice.7.CP-25 reduced the expression of TRAF2,MKK3,MKK6,p-P38 and p-P65 in the spleen of CIA mice Western blot results showed that compared with normal mice,the expressions of TRAF2,MKK3,MKK6,p-P38 and p-P65 in spleen B cells of CIA mice were significantly increased.The results of immunohistochemistry showed that the number of TRAF2 in spleen B cells of CIA mice was significantly increased.CP-25,rituximab and etanercept could reduce the expressions of TRAF2,MKK3,MKK6,p-P38 and p-P65 in spleen B cells of CIA mice.Compared with CP-25 mice,rituximab inhibited the expressions of TRAF2,MKK6 and p-P38 in CIA mice to a greater extent,and etanercept inhibited TRAF2,MKK3,MKK6,p-P38 and p-P65 expressions of CIA mice more violently.Compared with normal mice,rituximab and etanercept inhibited the expressions of MKK6,p-P38 and p-P65 of CIA mice to below normal levels to varying degrees.CP-25 can down-regulate MKK6 and p-P38 expressions in CIA mice to normal levels.8.CP-25 reduced the proliferation response of mice spleen lymphocytes and inhibited the expression of CD19+TRAF2+ cells in spleen BAFF can stimulate the proliferation of mice spleen lymphocytes and promote the expression of spleen CD19+TRAF2+ cells.CP-25(10-5mol/L)reduced BAFF-stimulated mice spleen lymphocytes proliferation and spleen CD19+TRAF2+ cell expressions.Etanercept inhibits BAFF-stimulated mice spleen lymphocytes proliferation and spleen CD19+TRAF2+ cells expression to a greater extent,which can reduce mice spleen CD19+TRAF2+ cells expression to below normal levels.9.CP-25 inhibited the migration of BAFF-stimulated MH7 A cells,but has no effect on the proliferation of BAFF-stimulated MH7 A cells Low concentration of BAFF(400ng/ml)has no effect on the proliferation of MH7 A cells,and high concentration of BAFF(800ng/ml)can stimulate the proliferation of MH7 A cells and promote the migration of MH7 A cells.CP-25 had no effect on the proliferation of MH7 A cells stimulated by BAFF.CP-25,etanercept,and MTX reduced MH7 A cell migration.10.TRAF2-si RNA interfered with TRAF2 to reduced BAFF-mediated NF-?B signaling protein in MH7 A cells The results showed that after TRAF2-si RNA interfered with the expression of TRAF2 in MH7 A cells,the protein expression of NIK,p-IKK? and P100/52-NF-?B in MH7 A cells stimulated by BAFF was significantly reduced.TRAF2-si RNA interfered with MH7 A cells had no effect on BAFFR expression.11.CP-25 reduced the percentage of BAFFR,TRAF2 and GRK2 cells in MH7 A cells Compared with the control group,the percentage of BAFFR,TRAF2 and GRK2 cells in BAFF-stimulated MH7 A cells increased significantly.Compared with the BAFF-stimulated group,CP-25,etanercept and MTX reduced the percentage of BAFFR,TRAF2 and GRK2 cells in BAFF-stimulated MH7 A cells.Compared with the CP-25 group,MTX reduced the BAFFR percentage in BAFF-stimulated MH7 A cells to a greater extent.12.CP-25 reduced BAFFFR,BCMA,TRAF2,GRK2,p-P38,p-P65,c IAP1,NIK,p-IKK? and P100/52-NF-?B proteins expressions in the BAFF-mediated NF-?B signaling pathway of MH7 A cells The expressions of BAFFR,BCMA,TRAF2,GRK2,p-P38 and p-P65 in the classical NF-?B signaling pathway of MH7 A cells stimulated by BAFF were significantly increased,and c IAP1,NIK,p-IKK? and P100/52-NF-?B protein expressions in the no-classical NF-?B signaling were significantly increased.CP-25,etanercept and MTX reduced BAFFFR,BCMA,TRAF2,GRK2,p-P38,p-P65,c IAP1,NIK,p-IKK? and P100/52-NF-?B proteins expressions in BAFF-stimulated MH7 A cells.Compared with the CP-25 group,etanercept and MTX inhibited the expressions of GRK2,p-P65,TRAF2 and c IAP1 in BAFF-stimulated MH7 A cells to a greater extent.13.CP-25 could reduce co-expressions of TRAF2 and BAFFR,TRAF2 and GRK2 in MH7 A cells,but had no effect on co-expression of TRAF2 and NIK Under normal circumstances,TRAF2-BAFFR,TRAF2-GRK2 and TRAF2-NIK have weak co-expression in MH7 A cells,and the co-expressions of TRAF2-BAFFR,TRAF2-GRK2 and TRAF2-NIK in MH7 A cells after BAFF stimulation are significantly enhanced.CP-25 attenuated the co-expression of TRAF2 and BAFFR,and also reduced the co-expression of TRAF2 and GRK2.CP-25 had a tendency to decrease the combination of TRAF2 and NIK stimulated by BAFF,but there was no statistical difference.14.Constructed the TRAF2 plasmid and TRAF2 recombinant mutant plasmid Using molecular biology methods to construct p IRES2-EGFP-TRAF2-wt,p IRES2-EGFP-TRAF2-K31 R,p IRES2-EGFP-TRAF2-K320 R and p IRES2-EGFP-TRAF2-K31RK320 R plasmids,and transfected them into HEK293 cells and MH7 A cells.Transfection efficiency of green fluorescent plasmid can be observed under a fluorescence microscope.Western blot analysis showed that TRAF2 protein was successfully expressed in HEK293 and MH7 A cells.15.CP-25 inhibited TRAF2 overexpression in HEK293 cells and MH7 A cells Overexpression of TRAF2 transfected into HEK293 cells and MH7 A cells,CP-25 could reduce the expression of p IRES-EGFP-TRAF2-wt in HEK293 cells and MH7 A cells.CP-25 had no effect on p IRES2-EGFP-TRAF2-K31 R and p IRES2-EGFP-TRAF2-K320 R expressions in HEK293 cells and MH7 A cells.Conclusion: 1.BAFF was involved in the pathogenesis of CIA.2.In B cells of CIA mice,BAFF-BAFFR-TRAF2-NF-?B signaling was abnormally activated and involved in the pathogenesis of CIA.3.CP-25 had therapeutic effect on CIA mice.4.CP-25 inhibited the function of B cells by regulating the BAFF-BAFFR-TRAF2-NF-?B signaling pathway,and further inhibited the activation,differentiation and immunoglobulin secretion of B cells.5.CP-25 regulated BAFF-mediated NF-?B signaling by affecting TRAF2 phosphorylation and ubiquitination.6.TRAF2-K31 and TRAF2-K320 may be the site of action of CP-25. |
PDF Full Text Request