Effects Of Prostaglandin E₂ Receptors And G Protein Coupled Signal Transduction On Synoviocytes Of Rats With Adjuvant Arthritis And The Effect Of Total Glucosides Of Paeony

Rheumatoid arthritis(RA) is a chronic autoimmune disease which characterized by joint destruction of local and system.It is one of the common disease which affecting approximately 0.3%-0.4%and 1%of people nationwide and west nations respectively. The pathologic characterizes of RA are chronic joint inflammation,inflammatory cell infiltrate,cartilage and osseous tissue destruction,accompany by neovascularity hyperplasy and system inflammation,ultimate lead to joint deform and functional incapacitation.Up to date,the etiology of RA is not fully understood.Most of anti-rheumatic drugs only stay in controlling inflammation symptom and can't modify the progression of the disease,simultaneously accompany with severe side adverse 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.Our previous study has been found that TGP has effects of inhibiting the inflammation of rat with carrageenan-induced and of rat with adjuvant arthritis(AA);exerting two-ways regulation of immunocyte and cytokines,such as interleukin-1(IL-1),interleukin-2(IL-2),tumor necrosis factor et (TNFet),in activity-dependent mode.Subsequent study found that the expression of inhibitory et subunit of G protein(Geti) mRNA increased in AA rats' synoviocytes,while the expression of stimulatory a subunit of G protein(Gets) decreased,level of prostaglandin E₂(PGE₂) increased,cyclic adenosine monophosphate(cAMP) reduced, synoviocytes hyperplasy accentuated.These results demonstrated that G protein-AC-cAMP signal transduction pathway has been involved in the progression of AA.We have been found that TGP could inhibit the production of PGE2 by synoviocytes and the synovium proliferation of AA rats.TGP could inhibit the expression of Gαi1,Gαi2,Gαi3 of fibroblast-like synoviocytes(FLS) of AA rats induced by recombinant rat IL-1α(rIL-1α) in vitro.In CIA rats,FLS pre-treated with interleukin-1β(IL-1β),TGP could inhibit the ADP fibosylation catalyzed by pertussis toxin(PT) and increase the level of ADP fibosylation catalyzed by cholera toxin(CT), modulate the activity of G protein.It has been known that PGE₂ plays an important role in the pathology of RA.PGE₂ binding with prostaglandin E receptor(EP),coupled with G-protein,then activating adenylate cyclase(AC),provoke the level of cAMP diversified.The changes of PGE₂ level and the expression of EP,Gαi and Gets all affected the level of cAMP.Is TGP affecting these elements? The present study design to investigate the diversity of EP -G-protein coupled signal transduction in RA and the effect of TGP on RA through the model of AA.AIM According to the changes of paw swelling and histological morphological of rat AA,this study tends to clarify therapeutic effects of TGP on rats AA.We want to understand the key of therapeutically effect of TGP on AA rat by analyze the correlation of synoviocytes secretion with joint swelling and of synoviocyte sproliferation with level of intracellular cAMP.At the same time,we designed the EP as breakthrough target,to investigate effects of TGP on the expression of EP and the G-protein coupled signal transduction pathway in synoviocytes and exert its anti-proliferation action.METHODS Freund's complete adjuvant(FCA) was used to induce AA in rats.Treat rats with TGP(25,50,100 mg·kg^-1,d14～d21) by intragastric administration after immunization.GTW(8 mg·kg^-1) were given as positive control.Secondary paw swelling of AA rats was measured with volume meter. Histopathological change of joint was observed by light microscope.Activity of IL-1 was measured by ConA-induced thymocyte proliferation of mice assay.PGE₂,cAMP, TNFαand IL-1βproduced by synoviocytes were measured by radioimmunoassay.The proliferation of FLS was detected with 3-(4,5-dimethylthiazol-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide(MTT) assay.The expression of Gαs,Gαi1,EP2 and EP4 were detected by Western blot analysis and immunohistochemistry analysis respectively.Pathology of inflammatory joints was detected with hematoxylin-erosin (HE) staining.RESULTS1.The effect of TGP on relieving joints swelling of AA rats is correlated intimatly with its effect on inhibiting PGE₂,IL-1 and TNFαproduced by synopviocytes.It is an important aspect that TGP through elevating level of intracellular cAMP to exert its anti-proliferation action.The AA model in rats was induced by FCA;the secondary inflammatory response of AA rats was characterized by paw swelling,inflammatory nodule of ear or tail and acting disorder.The histological morphological of inflammatory joints of AA rats were showed synovial tissue thickening which composed of 4-6 layers synoviocytes.Some of synoviocytes became ovalis or villiform types and joint periphery tissue hyperemia, edema and infiltrated with inflammatory cells.Articular cartilages were destructed accompanied with collagen fiber hyperplasia and pannus formation.The administration of TGP(50,100 mg·kg^-1,ig,d14～d21) diminished the paw swelling of AA rats at different extent.In AA rats,the synovial hyperplasia was inhibited and the infiltrated of lymphocyte was reduced.The destruction of articular cartilages was alleviated after administration with TGP(50,100 mg·kg^-1,ig,d14～d21). IL-1,TNFαand PGE₂ are the important inflammatory cytokines or mediators.The experiment results demonstrated that TGP(50,100 mg·kg^-1,ig,d14～d21) significantly reduced the elevated IL-I,TNFαand PGE₂ levels of synoviocytes supernatants.The correlation analysis demonstrated that the effect of TGP on relieving the joint swelling of AA rats is correlated intimatly with its effect on inhibiting PGE₂,IL-1 and TNFαproduction by synoviocytes.The correlation coefficient are 0.9696,0.9639and 0.9384 respectively.TGP(50,100 mg·kg^-1,ig,d14～d21) significantly inhibited proliferation of fibroblast-like synoviocytes and increased the level of cAMP in synoviocytes.The experimental in vitro has the same results.The result of correlation analysis demonstrated that TGP inhibit the fibroblast-like synoviocytes hyperplasia through increasing the level of cAMP in synoviocytes.The correlation coefficient are -0.9956, -0.9877 respectively.2.One of characteristic effects of TGP anti-proliferation is inhibiting the IL-1βproduction of FLS induced by PGE₂ and increasing the level of cAMP in synoviocytesWhen FLS of AA rats was stimulated by 10⁷TM PGE₂ for one to four hours,the level of cAMP increased,while it decreased when FLS was stimulated by PGE₂ for 24 hours.TGP(62.5,125,250μg.ml^-1) inhibited the production of IL-1βinduced by PGE₂ in vitro.These results showed that TGP could inhibit synoviocytes action and decrease inflammatory cytokines or mediator's production.3.Recovering the PGE₂-EP-G protein-AC-cAMP signal transduction pathway of synoviocytes in AA rats is one of the most important mechanisms of TGP preventing RAThe results of western blot analysis demonstrated that TGP obviously inhibited the expression of Gαi1,improved the expression of Gαs,and recovered the balance of Gαs/Gαi1.And TGP significantly improved the expression of EP2 and EP4 subtypes. These results demonstrated that recovering the PGE₂-EP-G protein-AC-cAMP signal normal transduction is one of the most important mechanisms of TGP preventing RA.CONCLUSIONS1.It is a principal effective of TGP which relieving joints swelling and inhibiting synovium hyperplasia of AA rats through decreasing PGE₂,IL-1 and TNFαproduction and elevating intracellular cAMP level.2.TGP decreased the production of IL-1βinduced by PGE₂ and increased the low level of cAMP induced by PGE₂.It is an important characteristic effect of TGP inhibiting accentuates secretion of synoviocytes and exerting anti-proliferation action.3.TGP obviously inhibited the expression of Gαil,improved the expression of Gαs, EP2 and EP4 and recovered the balance of Gαs/Gαi.These results lead to increasing intracellular cAMP level and decreasing proliferation and secretion of synoviocytes of AA rats.These demonstrated that recovering the PGE₂-EP-G protein-AC-cAMP signal normal transduction is one of the most important mechanisms of TGP preventing RA.