Effects Of Recombinant Human IL-1α On Function And Prostaglandin E₂ Receptors-G Protein-cAMP Signal Transduction In Fibroblast-like Synoviocytes From Human And The Effects Of Paeoniflorin

Rheumatoid arthritis (RA) is a chronic autoimmune disease which characterized by the persistent synovial inflammation, inflammatory cell infiltration, Synovial thickening and Pannus formation which can destructive cartilage and bone, ultimately lead to joint deformation and functional incapacitation. Fibroblast-like synoviocytes( FLS)play an important role in RA. On the stimulation of proinflammatory factors, they can release excess effector moleculars such as interleukin-1 (IL-1), tumor necrosis factor-α(TNF-α) and prostaglandin E2 (PGE2) that act on other cells to modulate joint inflammation and promote matrix degradation.Total glucosides of paeony (TGP) is an active compound, extracted from roots of paeonia lactiflora Pall, a Chinese traditional herbal medicine. TGP has been recognized as the valuable drug used in treatment for RA, and paeoniflorin (Pae) is the main active ingredients which has anti-inflammatory and immunoregulation. Preliminary studies on animals have found that TGP could inhibit synoviocytes proliferation and cytokines secretion, and these therapy effects may through modulating PGE2-EP-G protein-cAMP signal transduction pathway. To further clarify the mechanism, in this experiment we used recombinant human IL-1α(rhIL-1α)to stimulate FLS from normal human in vitro, and examined effects of Pae on synoviocytes proliferation and cytokines levels, the expressions of EP2 receptor , Gas protein and cAMP.OBJECTIVEClarify the effects and mechanisms of pae on FLS from human. In this article, we examine the proliferation and cytokines level such as PGE2,TNF-α, expression of EP2 receptor and Gas protein and cAMP of human FLS stimulated by rhIL-1αin vitro, explore the effect of Pae may be through modulating PGE2 receptors- G Protein -cAMP signal transduction pathway.METHODSThe joint synovium were isolated from the healthy humen and cultured as tissue culture method. The third generation of synovial cells was confirmed with FLS through the immunohistochemical identification. FLS was stimulated with rhIL-1αand culture with different concentrations of Pae, synoviocytes proliferation, cytokines level, expression of EP2 receptor and Gas protein were examined. The proliferation of FLS was assessed by MTT assay. Level of TNF-αwas examined by radio immunoassay (RIA). Levels of PGE2 and cAMP were determined by enzyme linked immunosorbent assay (ELISA). The expression of EP2 and Gαs protein were detected by immunofluorescence technique.RESULTS1.Effects of rhIL-1αon the proliferation and cytokines level of human FLS in vitro IL-1 is one of the most important proinflammatory cytokines in RA. So in this experiment we examined the proliferation and cytokines levels of human FLS stimulated with different concentrations of rhIL-1α(0.01, 0.1, 1, 10, 100μg·L-1) in vitro.The proliferation of FLS was stimulated with rhIL-1α, levels of TNF-αand PGE2 were increased. It suggested that rhIL-1αcan promote the proliferation and cytokines level of the human FLS in vitro.2. The effects of Pae on proliferation and cytokines levels of human FLS stimulated with rhIL-1αin vitroWe examined the proliferation and cytokines levels of human FLS culture with different concentrations of Pae (10-8, 10-7, 10-6, 10-5, 10-4 mol·L-1) stimulated with rhIL-1α. The result showed that Pae can inhibit the proliferation of FLS, down-regulate levels of TNF-αand PGE2 under the stimulation of rhIL-1α. It suggested that Pae may inhibit proliferation of FLS by regulating the secretion of cytokines.3. Effects of Pae on the expressions of EP2 receptor and Gas protein in human FLS under the stimulation of rhIL-1αin vitrorhIL-1αcan down-regulate level of cAMP in human FLS in vitro, Pae(10-8, 10-7, 10-6, 10-5, 10-4 mol·L-1) can up-regulate level of cAMP under the stimulation of rhIL-1α. In this experiment we detected the expressions of EP2 receptor and Gas protein in human FLS by immunofluorescence technique. Expressions of human FLS EP2 receptor and Gas protein were increased when stimulated with rhIL-1αfor 3～6h,and then decreased when stimulated with rhIL-1αfor 12～24h. Pae( 10-7, 10-6, 10-5, 10-4 mol·L-1)can elevate EP2 receptor and Gas protein expression of human FLS when stimulated by rhIL-1αwith 24h. Combining with the above results, it demonstrated that Pae may inhibited the proliferation and inflammatory cytokines of FLS by modulating PGE2– EP– G protein- cAMP signal transduction pathway.CONCLUSIONS1. rhIL-1αcan promotes the proliferation of normal human FLS in vitro, up-regulate cytokines levels such as TNF-αand PGE22. Pae can inhibits the proliferation of normal human FLS under the stimulation of rhIL-1αin vitro, down-regulate levels of TNF-αand PGE2. It suggested that Pae may inhibit the proliferation of FLS by regulating the secretion of cytokines.3. Pae may inhibited the proliferation and inflammatory cytokines of FLS by modulating PGE2-EP-G-protein-cAMP signal transduction pathway.