| Rheumatoid arthritis (RA) is a common autoimmune disease, marked with chronic systemic inflammatory and joint erosions. The pathological manifestations of RA include various immune cells infiltration, persistent synovitis and fibroblast-like synoviocytes (FLS) hyperplasia. Dendritic cells (DCs) are powerful professional antigen presenting cells (APC), which play important roles in modulating the immune response. B cell activating factor (BAFF) is an important directly stimulating factor in activation of the adaptive immune cells. Elevated levels of BAFF have been detected in serum and synovial fluid from RA patients. Moreover, the levels of BAFF increased in autoimmune diseases and were correlated with disease activity. DCs, mononuclear cell, macrophagocyte and FLS are all the source of BAFF, and these cells also express different BAFF receptors, these clues suggest that functions of DCs and FLS may be affected by BAFF from autocrine or paracrine, participating in the occurrence and development of RA. In addition, the studies of our group showed that the expression of COX2 and the secretion of PGE2 could be increased in DCs and FLS, the abnormal of PGE2-EP4-cAMP signal pathway could result in dysfunction of FLS. However it is still unclear that whether BAFF can decrease the cell inner adenosine monophosphate (cAMP) level in FLS and influence PGE2-cAMP signal pathway, also we are not clear the interaction between BAFF signal and PGE2 signal.A novel compound, paeoniflorin-6’-O-benzene sulfonate (code:CP-25) (patent number in China:ZL201210030616.4) that is a newly ester derivatives of Pae which is the active monomers in radix paeoniae alba, then it was synthesized, separated, purified and identified. In our preliminary experiment, CP-25 showed strong anti-inflammatory and immunomodulatory activity. But, the therapeutic effects and antiarthritic mechanism of CP-25 remains unclear when compared to Pae and TGP. In this reseach, we describe the anti-arthritic effect and characteristic of CP-25 in the rat adjuvant-induced arthritis (AA) model of human RA. In addition, on the basis of our previous research work, we further applied ELISA, QRT-PCR, flow cytometry, immunofluorescence, western blot, laser scanning confocal microscopy and co-immunoprecipitation method to study the roles of BAFF and its receptors in the function of DCs and FLS, and the effects of CP-25. We further study the interaction between BAFF signal and PGE2 signal in FLS, and the mechanism of CP-25, which may provide more experiment basis for exploration BAFF and its receptors participating in the pathomechanism of abnormal inflammatory immune response in RA and the target of CP-25.Objective:To clear BAFF and its receptors participate in the pathomechanism of abnormal inflammatory immune response in AA rats, and the therapeutic effects of CP-25. Further reveal the mechanism of BAFF and its receptors signal involved in the function of DCs and FLS, which provide important experimental basis for clarification therapeutic target of CP-25.Methods:1. The AA model was induced by complete Freund’s adjuvant, then they were treated with CP-25 by intragastric administration. The footpad volume was measured with a water replacement plethysmometer and arthritis sores were performed daily. Joint and spleen pathology were observed by HE dyeing. The levels of BAFF and IL-1β were detected by ELISA. The express of three BAFF receptors BAFF-R, TACI, BCMA in the spleen from AA rats were detected by western blot and QRT-PCR. The localization and expression of CD 103 were investigated by immunohistochemistry in spleen. The expression of activation markers (CD80, CD86 and CD40 and MHC II molecules) measured through flow cytometric analysis.2. Bone marrow cells were derived from the femurs and tibias of rats and were stimulated by rGM-CSF, rIL-4, which could be induced as bone marrow-derived DCs (BMDCs). BAFF-treated DCs promote allogeneic CD4+T cell proliferation (MLR) were determined by CCK-8 method. The express of BAFF-R, TACI, BCMA in DCs, and the influence of CP-25 in the expression of COX2 and BAFF in BAFF-DCs were measured by RT-PCR. The influence of CP-25 in the expression of IL-1β and TNF-a in BAFF-DCs were measured by QRT-PCR. The influence of CP-25 in the level of PGE2 in the supernatant of BAFF-DCs was detected by ELISA. The expression of three BAFF receptors BAFF-R, TACI, BCMA in BAFF-DCs and the influence of CP-25 in the expression of activation markers (CD40, CD86 and MHCII molecules), phagocytosis and apoptosis rate of BAFF-DCs were all measured through flow cytometric analysis. The influence of CP-25 on the expression of TACI, BAFF-R, apoptosis-related protein and ERK/NF-kB signal pathway proteins were detected by western blot.3. BAFF-treated DCs promote proliferation of FLS from AA rats and the influence of CP-25 in proliferation of MH7A cell stimulated by BAFF were determined by CCK-8 method. Migration and invasion function of AA FLS co-cultured with BAFF-DCs and the effects of CP-25 were studied by transwell methods. The expression of MMP9, in the AA FLS co-cultured with BAFF-DCs and the effects of CP-25 were studied by QRT-PCR, also it was used to measure the expression of IL-1β, IL-6 and TNF-a in the MH7A stimulated by BAFF. The effect of CP-25 on the expression of COX2 in the MH7A stimulated by BAFF was detected by western blot. The effect of CP-25 on the level of PGE2 in the supernatant of MH7A and the level of cAMP intracellular stimulated by BAFF was detected by ELISA.4. The express of three BAFF receptors BAFF-R, TACI, BCMA in MH7A were measured through RT-PCR, flow cytometry and immunofluorescence. The co-localization of BCMA and TRAF2 in MH7A were detected by laser scanning confocal microscopy. The effect of CP-25 on the expression of BCMA, TRAF2 in the MH7A stimulated by BAFF was detected by western blot. The effect of CP-25 on the co-localization of TRAF2 and GRK2 in MH7A stimulated by BAFF were detected by co-immunoprecipitation assay. The express of four subtypes of EPs in MH7A were detected by immunofluorescence. The influence of PGE2-EP4 signal pathway in the secretion of MH7A cell stimulated by BAFF were measured by QRT-PCR. Cytoplasm membrane proteins were separated by ultracentrifugation, the effect of CP-25 in the expression of EP4 and GRK2 on the cell membrane in the MH7A stimulated by BAFF and PGE2 were detected by western blot. The influence of EP4 selective agonist on the expression of EP4 on the membrane of MH7A after pretreatment by BAFF and the effect of CP-25 were detected by flow cytometry. The influence of EP4 selective agonist on the level of cAMP in MH7A after pretreatment by BAFF and the effect of CP-25 were detected by ELISA. The effect of CP-25 on the co-localization of GRK2 and EP4 in MH7A stimulated by BAFF and PGE2 were detected by co-immunoprecipitation assay.Results:1. The change of BAFF and its receptors during the inflammation process of AA rats and the effects of CP-25In the study of inflammation process of AA, we found that the levels of BAFF in the serum and spleen homogenate were increased during the inflammation process, and was highest in the inflammatory reaction period (d7), BAFF levels in spleen were definite positive correlation with status of spleen lesion. In the three receptors of BAFF, TACI and BAFF-R were increased obviously from early phase of arthritis (d14), whereas there no significant difference in BCMA. DCs are the main source of BAFF, the number of DCs in spleen increased significantly from d7, and the number was the maximum. The expression of costimulatory molecules CD40, CD80, CD86 and MHCII were all upregulated on spleen DCs especially on day 7 and day 14. BAFF-treated DCs strongly augment CD4+T cell proliferation. Taken together, the findings support the pathogenic role of DCs, BAFF, and its receptors in the development of experimental arthritis.CP-25 treatment attenuated the the paw volume of AA rats and decreased the levels of BAFF, IL-1βin serum, alleviated joint and spleen histopathological manifestations. CP-25 intragastric administration significantly decreased the expression of CD40, CD86 and MHCII on the DCs in spleen. These results showed that CP-25 inhibited the progression of arthritic rats and possessed potent anti-arthritic activity. At the same time it could regulate the function of DCs and attenuate the inflammation-immune response in AA rats.2. The influence of BAFF on DCs and the effect of CP-25BAFF inhibited the apoptosis of DCs in vitro, up-regulated the expression of Bcl2 and down-regulated the expression of Bax. At the same time, BAFF could strongly inhibit the phagocytosis of DCs and induce the expression of CD40, CD86, and MHCII. BAFF promoted the expression of COX2, BAFF, IL-1β and TNF-a, increased the secretion of PGE2. TACI and BAFF-R were the main receptors expressed on DCs, after BAFF stimulating the expression of TACI, BR3, p-ERK, p-IκBa and p-P65 on DCs was increased obviously. These results showed that the activation and maturation of DCs stimulated by BAFF mainly through TACI-ERK/NF-κB and BR3-ERK/NF-κB signal pathway.CP-25 promoted the apoptosis of BAFF-DCs, up-regulated the expression of Bax and down-regulated the expression of Bcl2. The phagocytosis of BAFF-DCs was increased and the expression of CD40, CD86, and MHCII were decreased after treatment with CP-25. CP-25 also decreased the expression of COX2 and BAFF, inhibited the secretion of IL-10, TNF-a and PGE2 in BAFF-DCs. CP-25 reduced the expression of TACI, BR3, p-ERK, p-MBa and p-P65 in BAFF-DCs, inhibited TACI-ERK/NF-kB and BR3-ERK/NF-kB signal pathway. These all showed that CP-25 could promote the apoptosis of BAFF-DCs, regulate the maturation and pro-inflammation cytokins secretion of BAFF-DCs.3. The influence of BAFF on FLS and the effect of CP-25BAFF promoted DCs secreted pro-inflammation cytokins such as BAFF and PGE2. The ability of proliferation, migration and invasiness in AA-FLS were increased after co-cultured with BAFF-DCs. The expression of MMP9 was increased in AA-FLS after co-cultured with BAFF-DCs. These clues showed inflammatory mediator such as BAFF and PGE2 probably participate the immuno-inflammatory reactions of joint synovium in AA rats. We found BAFF stimulated in vitro did not promote the proliferation of MH7A, but it could enhance the proliferation promotion effect of PGE2 after combined with PGE2. The expression of COX2 in MH7A was increased stimulated by BAFF for 2h in vitro, indometacin (INN) inhibited the produce of IL-1β, IL-6 and TNF-a in MH7A stimulated by BAFF, PGE2 stimulation restored the produce of IL-1β, IL-6 and TNF-ain MH7A stimulated by BAFF. These results suggested that BAFF play important roles in the abnormal function of FLS, PGE2 could facilitate the role of BAFF in pro-inflammation.CP-25 inhibited the ability of proliferation, migration and invasiness in AA-FLS which were co-cultured with BAFF-DCs. The mechanism of inhibition of AA-FLS migration and invasion maybe related with the reduced expression of MMP9. CP-25 concentration-dependent inhibited the proliferation of MH7A stimulated by BAFF combined with PGE2. CP-25 also inhibited the production of IL-1β,IL-6 and TNF-a induced by BAFF. These results demonstrate that CP-25 could inhibit the abnormal proliferation and secretion of MH7A stimulated by inflammation factors.4. The influence of BAFF signal on the expression of related-receptor and related-signal molecules on MH7A and the effect of CP-25We found that BCMA was the major BAFF receptor on MH7A. BAFF stimulated in vitro at different time, the expression of COX2, BCMA and adaptor proteinTRAF2 were all increased, activating NF-kB signal pathway. The level of cAMP in MH7A was decreased obviously stimulated by BAFF for 24h in vitro. CP-25 (0.1 μM, 1μM, 10μM) could increase the level of cAMP in MH7A. These results showed that BAFF stimulation lead to the abnormal of PGE2-cAMP signal pathway, CP-25 could restore the intracellular level of cAMP and regulate the function of MH7A. Further analysis the results, we found that the effect of TACI Ig (1μg/ml) on restore level of cAMP was more significantly than INN (1μM), These hinted that promoting PGE2 secretion was not the only way which reduced intracellular level of cAMP by BAFF stimulation. EP4 is the major EPs receptor on MH7A. ONO-AE3-208 is a specific antagonist of EP4, which could decrease the expression of TNF-a and IL-1β stimulated by BAFF for 24h. However, TSC 2510 a specific agonist of EP4 could increased the expression of TNF-a and IL-1β influenced by ONO-AE3-208 after stimulated by BAFF. These results demonstrated that PGE2-EP4 signal pathway maybe play important role in the abnormal function of MH7A stimulated by BAFF. Pretreatment with BAFF for 30min, then TSC 2510 a specific agonist of EP4 was used to stimulate at different time, the results showed the expression of EP4 on the cytomembrane in the group pretreatment with BAFF were lower than the group TSC 2510 alone. Membrane EP4 receptors were increased after stimulus 5-10 min and decreased from 15 min. The results of intracellular level of cAMP also showed that cAMP level increased at 5-10 min and reduced from 15 min. The EP4 membrane expression decreased and GRK2 membrane expression increased after stimulating by BAFF and PGE2 at the same time for 24h. We further found TRAF2-GRK2 association was increased by BAFF stimulation through co-immunoprecipitation assay. In BAFF pre-treatment cells, PGE2 induced increase association in EP4-GRK2 was more obvious than stimulated alone. These results demonstrated that BAFF facilitated PGE2-dependent EP4 receptor desensitization, which may be relate to recruitment of TRAF2, promoting activation of GRK2 and further phosphorylation EP4 receptors.CP-25 decreased the expression of BCMA, TRAF2 and inhibited the activity of NF-kB signal pathway. CP-25 restored the membrane expression of EP4, and decreased the membrane expression of GRK2. Co-immunoprecipitation assay showed that CP-25 decreased association in TRAF2-GRK2 stimulated by BAFF, also it could decrease association in GRK2-EP4 stimulated by BAFF and PGE2. Thus we speculate that CP-25 could improve synovial cell dysfunction by regulating the influence of BAFF in the balance of PGE2-EP4-cAMP signal transduction through affecting the activity of GRK2.Conclusion:1. BAFF and its receptors mediate the dysfunction of DCs which participate in the inflammation process of AA;2. CP-25 intragastric administration can regulate function of DCs, inhibit development of paw inflammation, the improvement of CP-25 in bone destruction is better than that of the TGP and Pae in AA rats;3. BAFF influence the function of DCs through TACI-ERK/NF-kB and BR3-ERK/ NF-kB signal pathway, CP-25 inhibit the abnormal activation of AA-FLS co-cultured with BAFF-DCs by regulating the function of DCs.4. The interaction between BAFF signal and PGE2 signal promote the dysfunction of FLS, BAFF facilitate PGE2-dependent EP4 receptor desensitization, which may be relate to promote secretion of PGE2, and recruitment of TRAF2 promoting activation of GRK2 and further phosphorylation EP4 receptors; CP-25 could improve synovial cell dysfunction by regulating the influence of BAFF in the balance of PGE2-EP4-cAMP signal transduction through affecting the activity of GRK2. |
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