| Rheumatoid arthritis (RA) is a chronic heterogeneit systemic autoimmune disease and characterized by chronic inflammation of the synovial tissues in multiple joints that leads to joint destruction going with immune function disorder. Macrophage plays an important role in the inflammatory response process. During the systemic inflammafory response process, macrophage is activated, which produces many inflammatory mediators, such as tumor necrosis factor alpha (TNF-α), interleukin-1(IL-1), interleukin-6( IL-6), prostaglandin E2(PGE2). The signal transduction pathways mediated by guanine nucleotide- binding proteins (G-protein) is important in the inflammafory autoimmune disease. Cytokines, neurotransmitters, hormones can activite G-protein by G-protein-coupled receptors (GPCRs)and cause downstream signaling transcription. Abnormal function changes of peritoneal macrophage from rats of adjuvant-induced arthritis(AA) may have some relation to the change of the G-protein-cAMP signal transduction pathways. Glucosides of Chaenomeles speciosa (GCS), extracted from the fructus of Chaenomeles speciosa,,is an active compound. Previous studies from our laboratory showed that GCS possesses anti-inflammatory and analgesic properties. Paeoniflorin (PF) is the principal constituent of total glucosides of paeony (TGP). The present study was therefore designed to investigate the effects of GCS and PF on abnormal function changes of peritoneal macrophage from rats of adjuvant-induced arthritis and its relative mechanisms on the signal transduction of G protein-adenylate cyclase(AC)-cyclic adenosine monophosphate (cAMP) of peritoneal macrophage. Thus, this study would provide references for the better exploitation and utilization of traditional Chinese medicine. OBJECTIVE In this study, we investigated the effects of GCS and PF on abnormal function changes of peritoneal macrophage from rats AA. We also investigated some mechanisms of GCS and PF on them.METHODSFreund's complete adjuvant was used to induce AA in rats. Rats were divided into nine groups, in which the rats with AA were given intragastrically GCS(30,60,120 mg?kg-1?day-1), PF (25, 50, 100mg?kg-1?day-1) and glucosides of tripterygium wifordii (GTW) (40 mg?kg-1?day-1) from d 17 to 24 after immunization. And for the groups of normal and AA model, rats were given an equal volume of vehicle.Noninjected hind paw volumes of rats were measured by volume meter. The phagocytotic activity of peritoneal macrophage in AA rats was determined by natural red method, and interleukin-1(IL-1) activity in peritoneal macrophage supernatant was measured by thymocyte proliferation assay. Tumor necrosis factor alpha(TNF-α) , cyclic adenosine monophosphate(cAMP)and prostaglandin E2(PGE2) produced by peritoneal macrophage were determined by radioimmunoassay.The production of G protein-coupled receptor kinase2(GRK2),β-arrestin2,E-prostanoid2(EP2)in peritoneal macrophage were detected by Western blot analysis.RESULTS1. GCS and PF had therapeutic effects on rat AA The AA model in rats was induced by FCA. There was a marked secondary inflammatory response in this model similar characteristics to RA, which accompanied with paw edema, pain, polyarthritis, decrease of body weight and the development of inflammatory lesions. Therapeutic treatment of GCS(30,60,120mg?kg-1d-1,ig) and PF (25,50,100 mg?kg-1,ig) suppressed significantly the secondary paw swelling. This suggested that GCS and PF might be effective on chronic autoimmune disease such as RA. This provided the further consideration that GCS might be a new class of effective anti-inflammatory agents.2. GCS and PF ameliorated abnormal phagocytotic activity of peritoneal macrophage from rat AAGCS (60,120mg?kg-1d-1,ig)and PF (25,50,100 mg?kg-1,ig) decreased markedly abnormal phagocytotic activity of peritoneal macrophage from rat AA. It was probably a specific method of suppressing abnormal activity of peritoneal macrophage of GCS and PF in treating rheumatoid arthritis.There was no influence in the level of IL-1 and TNF-αat the dose of GCS(30mg·kg-1). GCS (60,120mg?kg-1d-1,ig)and PF (50,100 mg?kg-1,ig)decreased IL-1, TNF-α, PGE2 activity of peritoneal macrophage,which was demonstrated that the treatment of GCS on rat AA through modulating immune functions. It also suggested that GCS and PF may regulate AA peritoneal macrophage by acting directly and via involvement in the effects of proinflammatory cytokines.3. Effects of GCS and PF on the signal transduction pathway of G protein -AC-cAMP of peritoneal macrophageTreatment with GCS (60,120 mg·kg-1,ig) and PF (25,50,100 mg?kg-1,ig)could increase the cAMP level of peritoneal macrophage. This study showed the changes of cAMP might reflect changes in G-protein levels affecting signal transduction pathways.The further aim of this study was to examine association between the EP2,GRK2,β-arrestin1 expression in peritoneal macrophage after treatment with GCS(30,60,120mg?kg-1d-1,ig)and PF (25,50,100 mg?kg-1,ig)and increasing cAMP level of peritoneal macrophage. The study showed GCS decreased the expression of EP2 andβ-arrestin1 of peritoneal macrophage. It indicated that signaling mediated by G-protein-AC-cAMP playing critical roles in the inflammation in AA. The effects of GCS increasing cAMP level of peritoneal macrophage might be related to its inhibiting Gi-protein. It was therefore likely that the therapeutic effects of GCS on AA rats dued to the coupled AC-cAMP signal transduction of peritoneal macrophage.CONCLUS1ONS1. GCS and PF had therapeutic effects on paw swelling in rats with AA. GCS and PF ameliorated the secondary inflammatory reaction of AA via modulating hemorheology and immune functions.2. GCS and PF had inhibitory effects on the reduction of IL-l,TNF-α,PGE2 from peritoneal macrophage.3. GCS and PF decreased the expression of EP2 andβ-arrestin1 of peritoneal macrophage, increased cAMP level of peritoneal macrophage so as to decrease the secretion of peritoneal macrophage. Regulation of G protein- AC-cAMP signal transduction pathway of peritoneal macrophage might be one of the important mechanisms by which GCS and PF exert their effects on rat AA.
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