The Effects Of PGE2 And Receptors Signal On Dendritic Cells And Fibroblast-like Synoviocytes In Adjuvant-induced Arthritis Rats And The Therapeutic Effects Of CP-25

Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by abnormal Fibroblast-like synoviocytes (FLS) proliferation. Dendritic cells (DCs) are one of the most powerful antigen presenting cells, which plays an important roles in modulating the immune response. All prostaglandin receptor (EP) subtypes were coexpressed in DCs, but the expression of different EP subtypes in DCs and its regulation of DCs function in RA is unclear. In particular, abnormal FLS proliferation has long been explained by the over-production of prostaglandin E2 (PGE2) in synovial tissue. PGE2 mediates its biological activities through four different receptors, which couple to different guanine-nucleotide binding (G)-proteins and regulation signal transduction systems. G-protein-coupled receptor kinases (GRKs) are regulator of G protein-coupled receptors (GPCRs), which can activate GPCRs and result in receptor desensitization. However, the effect of GRKs in regulating EP receptor need to be elucidated.A novel compound Phenylsulfinyl-paeoniflorin (paeoniflorin-6’-O-benzene sulfonate, code is CP-25) showed anti-inflammatory effect. CP-25 comes from the structure modification of Paeoniflorin (Pae), which is one of the main bioactive components of total glucosides of paeony (TGP). Whether the treatment of autoimmune arthritis by CP-25 and what’s the mechanism are still not clear. In this reseach, we established the model of adjuvant arthritis rats (AA), and adopted QRT-PCR, flow cytometry, western blot, co-immunoprecipitation, laser scanning confocal microscopy and small interfering RNA (siRNA) method, and discussed the effects of PGE2 on DCs and FLS from AA rats and the effects of CP-25, which may provide a scientific basis for revealing the EP signal transduction involved in abnormal inflammatory immune response of RA and CP-25 targets with the treatment of RA.Objective:To investigate the function of PGE2 signal on DCs and FLS from rats, and the therapeutic effects and mechanism of CP-25 on AA rats, which reveal the mechanism of PGE2 signal transduction involved in abnormal inflammatory immune response of RA, and provide an important basis for CP-25.Methods:The model of AA was induced by complete Freund’s adjuvant. Bone marrow cells of rat were stimulated by rIL-4, rGM-CSF, which could be induced as bone marrow-derived DCs. EPs mRNA level were determined by QRT-PCR. The expression of EP4 and GRK2 on FLS, the phenotypic of DCs and antigen uptake function were detected by flow cytometry. FLS proliferation and MLR were determined by CCK-8 method. The levels of inflammatory cytokines, PGE2 and cAMP were detected by ELISA. AA rats were treated with CP-25 by intragastric administration and inspected daily for signs of arthritis by arthritis index. Joint pathology was observed by HE dyeing. The distribution and association with EP4 and GRK2 in FLS were detected by laser scanning confocal microscopy and co-immunoprecipitation assay. GRK2 gene was silenced by siRNA transfection. The expression of EP4 and GRK2 in the cell membrane was detected by Western blot.Results:1. The effects of PGE2 signal on DCs from AA ratsFour EP subtypes were coexpressed in bone marrow-derived DCs. Compared with normal group, the expression of EP4 mRNA of AA model was increased significantly. The level of PGE2, TNF-α, IL-23 significantly increased on d23, d28. The expression of CD40 and CD86 of Peripheral blood CD103+ DCs was increased on d7, and the expression of CD80 and MHCII was increased on d17. However, the expression MHCⅡ, CD86 and CD40 was increased on d7.To observe the function of PGE2 on DCs, we used TNF-a to stimulate DCs, which in part simulate AA inflammatory environment in vitro. PGE2 obviously enhanced MLR, decreased the antigen uptake function, and increased the secretion of IL-23 and IL-12 of DCs.EP4 agonist CAY10598 obviously enhanced MLR and increased the expression of CD80. EP2 agonist Butaprost and EP4 agonists CAY10598 increased the level of IL-23 and the expression of MHCII and CD86. But the EP1 and EP3 agonist Sulprostone had no significant effect on these functions. A stable analog of cAMP, dbcAMP increased IL-23 and IL-12 production and the expression of MHCⅡ and co-stimulatory (CD80, CD86). These results indicated that EP4 receptor activation utilize a cAMP-dependent signaling pathway to promote DC maturation and activation. Furthermore, a chemical inhibitor of PI3K, wortmannin, instead of reducing IL-23 and IL-12 expression, the PI3K inhibitors actually increased IL-23 and IL-12 expression, but inhibited the expression of MHCII and CD80/86. These data indicate that the mechanism of PGE2-induced PI3K activity on DC function needs further research.2. The effects of PGE2 signal on FLS from AA ratsCompared with normal group, the levels of PGE2 and TNF-a obviously increased in serum. PGE2 stimulated the proliferation of FLS in concentration -dependent and the optimum concentration was 10 μM. The western blot and flow cytometry detection all showed that EP4 membrane espression in FLS of AA significantly decreased. After stimulated by PGE2, GRK2 expression in FLS was increased continuously after 10min under PGE2 stimulation. However, EP4 expression in FLS was increased at 20min, and was up to the peak at 2h, and then decreased compared to control level at 12h in response to PGE2 treatment. After pretreated for 30min by PGE2 (10 μM), EP4 membrane expression was increased, then decreased to basal level at 30min in response to EP4 agonist. However, GRK2 membrane expression was increased gradually. Compared with control group, the low expression of EP4 and high expression of GRK2 were more obvious in TNF-a group. The change of cAMP level was consistent with the change of EP4 membrane expression. Theses results indicated that under stimulation of inflammatory cytokine TNF-a, EP4 agonist further promemoted EP4 desensitization, which resulted in the level of cAMP decreasing.Laser seanning confocal microscopy detection showed that EP4 and GRK2 were mainly distributed in the cytoplasm and cell membrane, and the colocalization showed that Overlap Coeffcient was 0.6595, which indicated that GRK2 was associated with EP4 in FLS. Co-immunoprecipitation assay showed that without agonist stimulation little GRK2 was associated with EP4, CAY10598 stimulation clearly increased GRK2 association with EP4 in control cells. Further more, in TNF-a treated cells, the agonist induced increase in EP4-GRK2 association was more obvious. EP4 membrane expression in FLS transfected with GRK2 siRNA was increased gradually after stimulated by CAY10598, and abnomal FLS proliferrion induced by PGE2 was almost abrogated. Compared to normal cells, GRK2 gene silencing FLS showed no obviously response in the cAMP level upon CAY10598 stimulation after pretreatment with PGE2. These results demonstrate that GRK2 plays an important role in abnomal FLS proliferation, which may be through promoting EP4 receptor desensitization and decreasing the level of cAMP.3. The therapeutic effects and mechanisms of CP-25 on AA ratsCP-25 significantly decreased arthritis index and alleviated joint histopathological manifestations. These results showed that CP-25 had therapeutic effects on AA rat.CP-25 inhibited FLS proliferation and the secretion of PGE2 and TNF-α level. CP-25 salbutamol intragastric administration significantly decreased the expression of MHCⅡ, CD40 and CD80, but there was no significant effect on the expression of CD86. CP-25 improved the antigen uptake function of DCs, and down-regulated the expression of MHCⅡ, CD86 and CD80, and inhibited MLR and the secretion of IL-23 in vitro. CP-25 also inhibited the proliferation of FLS in concentration-dependent in vitro. CP-25 significantly increased EP4 and inhibited GRK2 membrane expression in FLS.The results of PGE2-treated DCs and FLS co-culture showed that PGE2-treated DCs significantly promoted the proliferation of FLS, increased the secretion of IL-1β, CP-25 can inhibit FLS proliferation and the secretion of cytokines in vitro. In addition, the results of PGE2-treated FLS and DCs co-culture showed that PGE2-treated FLS can significantly promote the DCs secreted TNF-α, and CP-25 can inhibit its secretion. In-vitro experiments further confirmed that the CP-25 can improve the function of the FLS and DCs. These results suggested that the therapeutic effects of CP-25 may be through regulating of AA rats abnormal immune function and inhibiting FLS abnormal proliferation.Conclusion:1. PGE2-EP4-cAMP signal hyperfunction led to DCs function abnormal activation, which correlated with the course of disease in AA rats;2. Abnomal high level of PGE2 resulted in a decline in EP4 membrane expression (excessive desensitization), which correlated with abnormal FLS proliferation and the activation of FLS and DCs;3. Abnomal high expression of GRK2 in AA rats and associated with EP4, which resulted in the enhancement of EP4 desensitization;4. The therapeutic effects of CP-25 may be through regulating DCs function, inhiting FLS abnormal proliferation and inflammatory secretion, which can be realized by decreasing GRK2 membrane expression and up-regulation EP4 and inhition the of AA rats abnormal immune function and inhibiting excessive desensitization of EP4.