| B cell stimulating factor (BAFF) also called B cell activating factor, is a member of the tumor necrosis factor ligand superfamily, mainly derived from bone marrow cells such as monocytes, macrophages, dendritic cells and so on. It combines with B cell activating factor receptor (BAFF-R), transmembrane activator and calcium-modulating cyclophylin ligand-interactor (TACI), B cell maturation antigen (BCMA),promoting B-cell survival, growth, antigen-presenting as well as different stages of immunoglobulin class switching and restructuring, which plays an important role in B cell survival, proliferation and activation.Total glucosides of paeonia (TGP) is the first drug which can treat RA anti-inflammatory and immunomodulatory approved for marketing in our country. New active monomer:Paeoniflorin-6’-O-tosylate (code:CP-25) is structural modification monomer of TGP effective active ingredient paeoniflorin (Pae). Compared with Pae, CP-25 has better lipid-soluble and its absolute bioavailability is significantly improved, and effect of anti-inflammatory immunomodulatory is obviously. CP-25 which has been obtained patents in China (ZL 2012 1 0030616.4). However, the regulation mechanism of CP-25 about immune response and immune cell functions on rheumatoid arthritis (RA) has not been reported.In this study, adjuvant arthritis in rats (AA) rats were used to explore the expression of BAFF and its receptor of spleen and serum in AA rats and effects of CP-25 on BAFF-R and its downstream signal molecule on AA rats. It also discussed the effect and mechanism.of CP-25 on B cells function.Objective:To observe the expression of BAFF/BAFF-R of spleen B cells in AA rats and to investigate the effect of CP-25 on AA rats.It is also investigated that C P-25 regulating B cells function and BAFF-R downstream signal molecules.Methods:The model of AA rats was induced by intradermal injection of 0.1ml complete Freund adjuvant into the right hind metatarsal footpad of SD rats. The normal group as control injected with saline instead of CFA by same method. Animals which were induced successfully were randomly divided into eight groups including normal group, model group, CP-25 groups(25,50,100mg/kg), MTX (0.5mg/kg), TGP (50mg/kg) and Pae (50mg/kg).Paw swelling of AA rats was measured. Spleen and thymus index were recorded. The expression of BAFF-R mRNA in spleen were determined by Q-PCR. The protein expression of BAFF-R and TRAF2 in spleen were assayed by Western Blot and immunohistochemistry. BAFF-R, TACI and BCMA expression of CD45R+B cells in spleen tissues were measured by flow cytometry. B cells proliferation in spleen were measured by using CCK-8 assay. The BAFF and IgM level in serum and the supernatant were assayed by ELISA.Results:1. Variety of serum BAFF and IgM level in AA rats of different periods of inflammation.Compared with the normal group, serum BAFF levels of AA rats in inflammatory reaction period(D9) were significantly increased. Serum BAFF levels of AA rats in The fore-period(D16),the peak(D23) and the paracmasis(D30) of inflammation were higher but have not statistically significant compared with normal rats; serum IgM levels og different periods of AA rats were significantly higher than normal rats.2. The expression of BAFF receptor BAFF-R, TACI, BCMA in spleen CD45R+B cells of AA ratsCompared with the normal group, BAFF-R expression was significantly higher in AA rat spleen tissue and BCMA, TACI expression did not change. Q-PCR assay showed that BAFF-R mRNA expression was significantly increased in AA rat spleen; Western Blot and immunohistochemical examination of tissues showed that compared with the normal group, BAFF-R expression in spleen tissue of AA rats was significantly higher.3. The effect of CP-25 on paw swelling number and immune organ index of AA ratsThe AA rats was induced by CFA and while the secondary inflammatory reaction occurred after the d 14 in AA rats, there was a polyarthritis characterized by multiple joint swelling deformation and movement disorder. It reached to the peak of inflammation after the d21. CP-25 in each dose group can inhibit the secondary paw swelling in rat, reducing the number of paw swelling of rats. Compared with normal group, AA rat expressed obviously enlargement of the spleen and spleen index were conspicuously increased.CP-25 in each dose group was reduced to varying degrees spleen index of AA rats, which significantly inhibited. But thymus index of AA rats were no significant change compared with normal rats and each treatment group had no significant effect on thymus index.4. Expression of BAFF-R in spleen on AA rats and the effect of CP-25 in vivoCompared with normal group,the expression of baff-r in spleen on AA rats increased significantly.CP-25(25,50, 100mg/kg) reduced the expression of baff-r mRNA to varying degrees detected by Q-PCR.Low expression of BAFF-R in spleen on normal rats and CP-25(25,50, 100mg/kg) can reduced the expression of BAFF-R protein to varying degrees detected by Immunohistochemistry and Western Blot.5. The effect of CP-25 on regulating B cells function in vivoCompared with normal group,level of serum IgM on AA rats is significantly increased.The proliferation of B cells is promoted on AA rats.CP-25(25,50, 100mg/kg) reduced the serum IgM level and inhibit the proliferation of B cells significantly.6. Expression of BAFF-R,TRAF2,Bax/Bcl-2 in spleen on BAFF-stimulated rats and effect of CP-25 in vitroUse BAFF(500ng/ml) to stimulate splenic lymphcyte of normal rats for 48h and the expression of BAFF-R,TRAF2 and Bcl-2 were increased significantly,and the expression of BAX was decrased.CP-25(10-6mol/L) reduced the expression of BAFF-R,TRAF2 and Bcl-2 and increased Bax expression.7. Effect of CP-25 on regulating B cells function of normal rats in vitroUse BAFF to stimulate splenic lymphcyte for 48h.Level of IgM increased and BAFF promoted the proliferation of splenic lymphcyte significantly.CP-25(10-8,10-7, 10-6mol/L)reduced IgM level to varying degrees and inhibit the proliferation of splenic lymphcyte.Conclusions:1.Secondary inflammatory lesions of AA in rats began to appear dl4 after immunization and d21 to reach the peak.The secondary side paw began to swell. Spleen index increased;Serum BAFF, IgM levels higher than normal rats.Proliferation of splenic lymphocytes increased; Expression of BAFF-R on splenic CD45R+B cell increased. Thymus index and expression of TACI, BCMA on splenic CD45R+B cell did not change significantly.2.Both IgM levels and proliferation of splenic lymphcyte increased after BAFF stimulated; spleen lymphocytes BAFF-R, TRAF2, Bcl-2 expression increased, Bax expression decreased.3.CP-25 can reduce the number of paw swelling of AA rats, improve the immune organ index, reduced serum IgM levels, inhibited splenic lymphocyte proliferation, reduced expression of BAFF-R on splenic lymphcyte; CP- 25 induced IgM levels and inhibited the proliferation of splenic lymphcyte after BAFF stimulated, reduced splenic lymphocytes BAFF-R, TRAF2, Bcl-2 expression, increased Bax expression. Tip that therapeutic effect of CP-25 on rat AA related to regulating lymphocyte receptors BAFF and BAFF-R signal transduction. |
PDF Full Text Request