| Rheumatoid arthritis (RA) is a chronic autoimmune disease which characterized by persistent and progressive synoviocyte inflammatory, cartilage and osseous tissue destruction, ultimate lead to joint deform and functional incapacitation. It is one of the common disease which affecting approximately 1%~5% of people nationwide. synoviocyte inflammatory is the etiology foundation that result in cartilage and osseous tissue destruction. So control synoviocyte inflammation, protect cartilage is the key point of improving symptom and decreaseing Mutilation rate. Occurrence of RA is related to many cytokine,tumor necrosis factor-α(TNF-α)and prostaglandin E2(PGE2)is one of the important cytokine involved in RA .PGE2 plays an important role in the pathology of RA. PGE2 binding with prostaglandin E receptor (EP), coupled with G-protein, provoke the level of cAMP diversified,then generate wide cascade effect. Total glucosides of paeony (TGP) is an active compound extracted from roots of paeonia lactiflora Pall which has been recognized as the valuable tractional herbs used in treatment for RA with less side effect. Therefore, Collagen-induced arthritis(CIA) was used to investigate the diversity of prostaglandin E2-receptor-G-protein coupled signal transduction in RA and the effect of TGP on RA.OBJECTIVE According to the changes of paw swelling and histological morphological of rat CIA. This study tends to clarify therapeutic effects of TGP on rats CIA. We want to understand the key of therapeutically effect of TGP on CIA rat by analyzing the correlation of synoviocytes secretion with joint swelling and of synoviocyte sproliferation with level of intracellular cyclic adenosine monophosphates (cAMP).To investigate effects of TGP on the PGE2 and IL-1βproduction of synoviocyte stimulated by TNF-α, identifying the effect of TNF-αon synoviocyte proliferation. At the same time, we designed the prostaglandin E2-receptor (EP) as breakthrough target, investigated effects of TGP on the expression of EP and the G-protein coupled signal transduction pathway in synoviocytes and its anti-proliferation action.METHODS The model of rat CIA was induced by injection of chicken typeⅡcollagen(CCⅡ) emulsion.Treat rats with TGP (25,50,100mg·kg-1,d14～d21) by intragastric administration after immunization. GTW (40mg·kg-1) were given as positive control. Secondary paw swelling of CIA rats was measured with volume meter. The proliferation of FLS was detected with 3-(4,5-dimethylthiazol-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT). PGE2, cAMP and IL-1βproduction of synoviocytes were measured by radioimmunoassay assay. The expression of Gαi3, EP2 and EP4 were detected by Western blot analysis.RESULTS1 Effects of TGP on secondary inflammatory reaction and immune functions in CIA ratsInflammatory reaction occurred on d14 after immunization. There was a marked secondary inflammatory response in this model, which accompanied with paw swelling, pain, polyarthritis, decrease of body weight and the development of inflammatory lesions. TGP (25,50,100 mg·kg-1, ig, d14-d28) and GTW(40mg·kg-1·d-1,ig,d14～d28)diminished the right hind paw swelling and polyarthritic symptoms. The articular histopathological changes were improved, and ConA-induced T lymphocytes proliferation in CIA rats was also increased.2.The effect of TGP on relieving joints swelling of CIA rats is correlated intimatly with its effect on inhibiting PGE2 and IL-1βof synopviocytes induced by TNF-α. It is an important aspect that TGP to exert its anti-proliferation action through elevating level of intracellular cAMPFrom this study, we found that the lever of IL-1β,PGE2 in synoviocytes increased when induced by TNF-a(0.2,2.0,20μg·L-1), and TGP(62.5,125,250mg·L-1) could reduce the high level of IL-1β,PGE2. TGP(31.25,62.5,125,250mg·L-1) in vitro could increase the low level of cAMP induced by TNF-α( 0.2,2.0,20μg·L-1). TGP (15.625,31.25,62.5,125,250 mg·L-1) in vitro could suppressed proliferation of cultured fibroblast-like synoviocytes (FLS)-induced by TNF-α.3. Modulating the PGE2-EP-G protein-AC-cAMP signal transduction pathway of synoviocytes in CIA rats,and recovering the abnormal expression of EP4 probablly one of the most important molecule mechanisms of TGP preventing RA.The result of western blot analysis demonstrated that TGP (50,100 mg·kg-1, ig, d14-d28) and GTW(40mg·kg-1·d-1,ig,d14～d28)obviously inhibited the expression of Gαi3,and recovered the balance of Gαs/Gαi3. Stimulation by TNF-α(2.0,20μg·L-1)could inhibit the EP2 and EP4 subtypes, TGP(62.5,125,250 mg·L-1) could recover the expression of EP4. These results demonstrated that modulating the expression of EP4 subtypes, maintaining the balance of Gαs/Gαi, recovering the PGE2-EP-G protein-AC-cAMP signal transduction is probablly one of the most important molecule mechanisms of TGP preventing RA. CONCLUSIONS1.The effect of TGP on relieving joints swelling of CIA rats is correlated intimatly with its effect on inhibiting PGE2 and IL-1βof synopviocytes.It is a principal characteristic of TGP which inhibiting synovium hyperplasia of CIA rats through elevating intracellular cAMP level.2.TGP prevent RA through modulating the balance of the PGE2-EP-G protein-AC-cAMP signal transduction... |
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