| Rheumatoid arthritis (RA) is one of the most serious medical problems affecting approximately 1 ~ 1.5 % of all people worldwide. A major obstacle to the development of rational treatment strategies is that the disease mechanisms remain largely unknown and clues may come from experimental arthritis. The disease is autoimmune in nature and characterized by chronic inflammation of the synovial tissues in multiple joints that leads to joint destruction. Although palliative treatments are widely prescribed, there are currently only a few treatments that can modify the progression of the disease.Total glucosides of paeony (TGP) is an active compound extracted from roots of paeonia lactiflora Pall which have been recognized as the valuable traditional herbs used in treatment for rheumatoid arthritis (RA) and hepatitis with a long history in traditional Chinese medicine. TGP contains more than 40% percentage of paeoniflorin and other components such as hydroxy-paeoniflorin, paeonin, albiflorin, benzoylpaeoniflorin, et al. It has anti-inflammatory and immune-regulatory effects and has been approved by State Food and Drug Administration (SFDA) for the treatment of RA as a disease-modifying drug.Signaling through stress- and mitogen-activated protein kinases (SAPK/MAPK) pathways in FLS is a typical feature of chronic synovitis in RA. As well known, various MAPK cascades can be activated by guanine nucleotide binding regulatory proteins (G proteins)-coupled receptors (GPCRs)/G-protein components. Thus, thecytokine-signaling including G protein transmembrane signal and MAPKs pathway became new targets for treatment of RA. Interleukin-1 (IL-1) is an important pro-inflammation cytokine which mediates bone resorption and cartilage destruction by inducing MAPK signal transduction in RA. The present studies investigated the relationship between G protein associated signal transduction pathway and MAPK cascades in AA-derived FLS stimulated by IL-1 and the effects of TGP on Adjuvants arthritis (AA).OBJECTIVE MAPKs cascades in fibroblast-like synoviocytes (FLS) induced bypro-inflammatory cytokines such as IL-1 can be activated by G-proteins -coupled signal ways in RA. This study was to elucidate the relationship between G protein-associated transmembrane cascades and MAPKs signal transduction pathways in recombinant rat IL-1 a (rIL-1 a )-induced FLS from rats with AA. This study was to investigate the relationship between ultrastructure of synoviocytes and its secretory function and the relationship between different types of synoviocytes. The study was also to extend our understanding of the roles played by TGP in the pathogenesis of arthritic diseases and to develop a new therapeutic strategy for the treatment of rheumatoid arthritis (RA) by blockade of cytokine-signaling pathways in synoviocytes.MATHODS (1) The expressions of MAPKs phosphorylation, matrixmetalloproteinases (MMPs), cyclooxygenase (COX) , the stimulatory subunit of G alpha protein (Gs) and the inhibitory subunit of G alpha protein (G;) were detected by Western blot in rIL-1 a -induced FLS that treated with cholera toxin (CT), pertussis toxin (PT), SB 203580(a selective inhibitor of c-Jun N-terminal kinase (JNK)/p38 cascades) and U 0126(a selective inhibitor of extracellular regulating kinase (ERK) cascade), respectively. While cAMP accumulation was measured by enzyme-linked immuno-absorbant assay (ELISA) and prostaglandin E2 (PGE2) was measured by radioimmunoassay. The activities of protein kinase A (PKA) and PKC were detected by ELISA.(2) AA in rats was established and hind paw volumes of rats were measured byvolume meter. Before the onset of arthritis, animals were divided into eight groups randomly. Rats were given intragastrically TGP (25, 50, 100 mg â– kg'1, once per day) or given an intracutaneous injection of IL-lra (2.5, 10, 40 mg ? kg"1, thrice per day) from days 14 to 21 after immunization. For the normal and AA model, rats were given an equal volume of vehicle. Synoviocytes proliferation and the activity of IL-1 were determined by MTT assay. Tumor necrosis factor alpha (TNF-a) and PGE2 were measured by radioimmunoassay. Ultrastructure of synovioctes was observed under transmission electron microscope. The phosphorylation of JNK, ERK and p38 kinase and the expressions of MMPs, Gs and Gi were detected by Western blot analysis. The activities of PKA and PKC were detected by ELISARESULTS1. Relationship between G- proteins associated transmembrane cascades and MAPK cascades in rat AA-derived FLS stimulated by rIL-la(1) Relationship between a subunits of G- proteins and MAPK cascades induced by rIL-la in rat AA-derived FLSrIL-la induced phosphorylation of ERK, JNK, and p38 at 20 min in rat AA-derived FLS. PT(1 11 g - ml"1) and CT (10 u g . ml"1) inhibited phosphorylation of ERK, JNK, and p38 induced by rIL-la. Further studies found that U 0126 (25 and 50 V M) and SB 203580(2.5 and 25 u M)decreased the expressions of Gu, Gj2, Gj3 and Gs induced by rIL-1 a in rat AA-derived FLS.(2) Effects of MAPKs cascades on G protein-cAMP signal pathway in rat AA-derived FLS activated by rIL-la.rIL-1 a decreased intracellular cAMP levels from Omin to 30 min, and it increased intracellular cAMP levels from 30 min to 150 min. rIL-1 a also induced accumulation of extracellular PGE2 production by FLS. SB 203580(2.5 and 25 u M) and U0126 (25 u M) decreased the cAMP and PGE2 accumulation induced by rIL-la. Exogenously PGE2 (10, 50 and 100 pg ? ml'1) increased the cAMP levels inhibited by U0126 and SB203580 in a concentration-dependent manner. The data ruled out the possibility that the effects of U 0126 and SB 203580 may be due to inhibit the rIL-1 a-induced production of PGE2(3) Effects of MAPKs cascades on G protein-PKA signal and G protein-PKC signal in rat AA-derived FLS activated by rIL-la.Compared with those in rat AA-derived FLS, the activities of PKA PKC and PKC /PKA increased significantly in AA FLS induced by rIL-1 a (lOng . ml*1) for 150 min. SB 203580 (2.5 and 25 u M) and U0126 (25 u M) not only decreased the activities of PKC and PKA but also decreased the PKC/PKA in rat AA-derived FLS stimulated by rIL-1 a .(4) Effects of G- proteins associated cascades and MAPK cascades induced by rIL-la on function of rat AA-derived FLSThe expression of MMP-K MMP-3 and COX-2 increased significantly in rat AA-derived FLS induced by rIL-1 a and was inhibited by PT, CT, SB203580 or 10126. The inhibitors had no significant effect on expression of COX-1. The results demonstrate that G- proteins associated cascades and MAPK cascades make important roles on function of rat AA-derived FLS.2. Effects of TGP on G- proteins associated cascades and MAPK cascades in synoviocytes from rats with AA(1) Effects of TGP on secondary inflammatory reaction, body weight and histopathology of AA ratsInflammatory polyarthritis was induced in all immunized rats. The paw swelling occurred on d 13 after immunizatioa Treatment with TGP (25, 50, 100 mg ? kg'1, ig, days 14-21) diminished the right hind paw swelling and polyarthritic symptoms on d 17 and d21 after immunization. The administration of TGP (50 and 100 mg ? kg'1, ig, days 14-21) increased significantly the body weight of AA rats on d 17 and the same efficacy of TGP (50 mg ? kg"1) were observed on d 21 after immunization.In AA rats, synoviocytes proliferated three to four layers and became ovalis types, and articular cartilages were destructed and infiltrated with inflammatory cells. The hyperplastic synovium in AA rat formed a large number of fibroblasts and new blood vessels. Proliferation of collagen fribrils was found under synoviums of AA rats. In AA rats given TGP intragastrically, the synovial hyperplasia was inhibited and thedestruction of articular cartilages was alleviated.(2) Effect of TGP on ultrastructure of synoviocytes and cytokines production by Macrophage-like synoviocytes (MLS) from AA ratsThe intracellular changes were observed in AA rats, including that extracellular matrix and collagen increased, Golgi bodies reduced in size and curled, mitochondria swelled with ridges decreasing, rough endoplasmic reticulum (RER) increased and dilated, dense bodies and vacuoles increased. TGP (50 and 100 mg . kg'1, ig, days 14-21) repaired the injury mentioned above to some extent and decreased significantly the production of IL-1, PGE2and TNF-a by MLS from AA. The results suggested that TGP reduced the second inflammatory in AA rats, which was associated with prevention of ultrastructural of synoviocytes and inhibition of secretion of proimfiammatory cytokines.(3) Effects of TGP on phosphorylation of MAPKs, cell proliferation and MMPs production in rat-AA derived synoviocytesTGP (50 and lOOmg . kg"1, ig, days 14-21) inhibited phosphorylation of MAPKs in synovial membrane of AA rats. Furthermore, supernatants of MLS from AA rats induced more phosphorylation of MAPKs including JNK, ERK and p38 in AA FLS than that from normal rats. The surpernatents of MLS from AA rats treated with TGP (50 and 100 mg ? kg"1, ig, days 14-21) induced less phosphorylation of MAPKs in AA FLS than that from AA rats.Compared with supernatants of normal MLS, the supernatants of AA MLS increased cell proliferation and expression of MMP-1 and MMP-3 in AA FLS. The surpernatents of MLS from AA rats treated with TGP (50 or 100 mg . kg"1, ig, days 14-21) induced less production of MMP-1 and MMP-3 and decreased cell proliferation in AA FLS than that from AA rats.(4) Effects of TGP on G protein associated signal pathways in rat-AA derived FLS stimulated by rIL-1 aTGP (2.5 and 12.5 mg â– L"1) decreased expressions of Gil, Gi2 and Gi3 in AA FLS stimulated by rIL-1 a . Furthermore, TGP (2.5 and 12.5 mg - L'1) not only increased the the activities of PKA but also decreased the activities of PKC and...
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