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The Mechanisms of β2 Adrenergic Receptor Signal Transduction and Desensitization on Mesenteric Lymph Node Lymphocytes in Rats with Rheumatoid Arthritis and the Effect of Paeoniflorin

Author: WuHong
Tutor: SongLiHua;WeiWei
School: Anhui Medical University,
Course: Pharmacology
Keywords: adjuvant arthritis paeoniflorin β2-adrenergic receptor mesenteric lymph node lymphocytes immune tolerance desensitization signal transduction
CLC: R285
Type: PhD thesis
Year: 2007
Downloads: 199
Quote: 0
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Abstract


Rheumatoid arthritis (RA) is a chronic, inflammatory and systemic autoimmunedisease. Growing evidences suggest that T lymphocyte plays an important role in thedevelopment and pathogenesis of RA. The imbalance of Th1/Th2/Th3 cytokines mayplay an important role in the pathogenesis of RA. The balance is skewed toward Th1and excess of Th1 cytokines. The evaluation of this imbalance leads up to thepossibility of discrimination in this disease. An increasing number of investigationssuggest that pro-inflammatory cytokines play a pivotal role in the pathology of RAbecause of their biological functions as mediators of inflammation, cell growth, andactivation. In recent years the involvement of sympathetic nervous system (SNS) inthe pathogenesis of RA has also become evident. It is not surprising as lymphoidtissues are densely innervated with postganglionic noradrenergic and peptidergicneurons, suggesting that sympathetic innervation may play an important role in the inflammatory response. But a major obstacle to the development of rational treatmentstrategies is that the disease mechanisms and the causative environmental and geneticfactors remain largely unknown.Total glucosides of paeony (TGP) is an active compound extrated from roots ofpaeonia lactiflora Pall which has been recognized as the valuable traditional herbsused in treatment for RA with less side effect. Paeoniflorin, a monoterpene glucoside,is one of the main bioactive components of TGP. Pae was extracted and purified fromTGP by methods of solvent extraction, column chromatography, and the structure wasidentified by physicochemical properties and spectroscopic analysis, and then thecontent was determined by high performance liquid chromatography (HPLC).Anti-inflammatory, analgesia, antioxidative, antihepatic injury and immunoregulatoryactivities of TGP have been extensively proved in our laboratory for many years. TGPnot only inhibits secondary inflammatory reaction, bone destruction and ultrastructurechange of synoviocytes in AA rats, but also significantly decreases the production ofIL-1, prostaglandin E2 (PGE2) and TNF-alpha by macrophage-like synoviocytes(MLS) in AA rats. Furthermore, the increased phosphorylation of mitogen-activatedprotein kinases (MAPKs), cell proliferation, and matrix metalloproteinases (MMPs)expression in fibroblast-like synoviocytes (FLS) stimulated by supernatants of MLSin AA rats can also be inhibited by TGP. Subsequent study found that the expressionof inhibitory alpha subunit of G protein (Gαi) increased in synoviocytes of AA rats,while the expression of stimulatory alpha subunit of G protein (GαS) decreased, cyclicadenosine monophosphate (cAMP) reduced. These results demonstrated that Gprotein-AC-cAMP signal transduction pathway has been involved in the progressionof RA.Many G protein-coupled receptors (GPCRs) are important in determining the outcome of inflammatory responses. Beta 2-adrenergic receptor (β2AR) is animportant GPCR for the inflammatory responses.β2AR and signal transductionpathway may be an important target of investigating drug for treatment RA. Tumornecrosis factor alpha (TNF-α) plays a pivotal role in the pathology of RA because ofits functions as mediators of inflammation, cell growth, and activation. This studydesigns to investigate the effects of TNF-αon the mechanisms ofβ2ARdesensitization andβ2AR-AC-cAMP transmembrane signal transduction, and theeffect of Pae on RA via the model of AA.OBJECTIVE According to the changes of paw swelling and histologicalmorphological of rat AA, this study tends to clarify therapeutic effects of Pae on ratsAA. We observed the effects of Pae on MLNcs proliferation and the production ofIL-2, TNF-α, IL-4 and TGF-β1 of AA rats through analyzing the correlation of pawswelling with the level of pro-inflammatory cytokines and the level ofanti-inflammatory cytokines, and of the level of intracellular cAMP with MLNcssecretions. Meanwhile, we designed theβ2AR as breakthrough target, to investigateeffects of Pae on the expression ofβ2AR, GRK2 andβ-arrestin1, 2. The goal of thisstudy was to extend our understanding of the roles played by Pae in the arthritisdisease by intervening withβ2-AR desensitization. To further confirm the actions oftherapeutic effects of Pae on rats AA, in this paper we investigated the effects andmechanisms of Pae on beta 2-AR-AC-cAMP transmembrane signal transduction atlevels of organ, cell, molecule, and protein, respectively.METHODS Freund’s completed adjuvant (FCA) was used to induce AA in rats.Treatment with Pae (25, 50, 100 mg·kg-1, d17-d24) by intragastric administrationafter immunization, GTW (40 mg·kg-1) were given as positive control. Secondary paw swelling of AA rats was measured with volume meter.Histopathological Change of mesenteric lymph node was observed by lightmicroscope. The proliferation of MLNcs was detected with MTT assay. TNF-αandcAMP productions of MLNcs in AA rats were measured by radioimmunoassay. IL-2activity in MLNcs supernatant sample was measured by ConA-induced splenocyteproliferation of C57BL/6J mice assay. IL-4 and TGF-β1 activity in MLNcssupernatant sample were measured by enzyme-linked immunosorbent assay (ELISA).The expressions ofβ2AR, GRK2 andβ-arrestin1, 2 were detected by Western blotand immunohistochemistry analysis, respectively.The expression diversity ofβ2AR, GRK2 andβ-arrestin1, 2 in membrane andcytoplasm were detected by Western blot in rTNF-α-induced MLNcs that treated withβ2AR agonist isoprenaline. To observe the effects of Pae on discussed above.RESULTS1. The effects of Pae on relieving joints swelling and inflammation ofmesenteric lymph node in AA rats were correlated intimately with its effects oninhibiting IL-2 and TNF-αproduction and enhancing IL-4 and TGF-β1production from MLNcs.It was found that Pae (50, 100 mg·kg-1, d17~d24) significantly suppressedsecondary hind paw swelling and polyarthritis index, as well as improved arthriticstatus histological of mesenteric lymph node in AA rats. Pae (50, 100 mg·kg-1,d17~d24) significantly reduced the elevated IL-2 and TNF-α, and increased thelowered IL-4 and TGF-β1 production of MLNcs supernatants. The correlation analysisdemonstrated that the effect of Pae on relieving the joint swelling of AA rats wascorrelated intimately with its effect on inhibiting the secretion of IL-2, TNF-αandenhancing IL-4, TGF-β1 production from MLNcs. 2. Pae inhibited the pro-inflammatory cytokines IL-2 and TNF-αproduction,enhanced the anti-inflammatory cytokines IL-4 and TGF-β1 production andinduced immune tolerance.Pae (50, 100 mg·kg-1, d 17~d 24) significantly diminished the production ofIL-2 and TNF-α, and increased the lowered levels of IL-4 and TGF-β1 of MLNcs inAA rats.Pae (12.5, 62.5, 312.5μg·ml-1) significantly diminished the production of IL-2and TNF-α, and increased the lowered levels of IL-4 and TGF-β1 in rTNF-α-inducedMLNcs in vitro. This led to the suggestion that Pae exerts tonic inhibition on theproduction of Th1 pro-inflammatory cytokines, causes a shift toward Th2,Th3-mediated anti-inflammatory and immunoregulatory effects, and recovers thebalance of Th1/Th2/Th3 in AA rats.3. The effects of Pae on inhibiting IL-2 production, enhancing IL-4 and TGF-β1production of MLNcs in rats were correlated intimately with its effect ondecreasing the elevated cAMP level.Pae (50, 100 mg·kg-1, d17~d24) significantly diminished the production ofIL-2 and cAMP level, and increased the lowered levels of IL-4 and TGF-β1 of MLNcsin AA rats. The correlation analysis demonstrated that the effect of Pae on decreasingcAMP level is correlated intimately with its effect on inhibiting the secretion of IL-2and enhancing the secretion of IL-4, TGF-β1, respectively.Pae (12.5, 62.5, 312.5μg·ml-1) decreased the elevated cAMP level, significantlydiminished the production of IL-2, and increased the lowered levels of IL-4 andTGF-β1 in rTNF-α-induced MLNcs of AA rats in vitro. The correlation analysisdemonstrated that the effect of Pae on decreasing cAMP level is correlated intimately with its effect on dragon-regulating the production of IL-2 and up-regulating the levelsof IL-4, TGF-β1, respectively.4. One of characteristic effects of Pae on inducing immune tolerance andexerting therapy was further decreasing the proliferation of MLNcs anddown-regulating the level of cAMP.Pae (50, 100 mg·kg-1, d17~d24) further decreased the lowered proliferation ofMLNcs, and significantly diminished the level of cAMP of MLNcs in AA rats.Dynamic examinations of the level of cAMP from MLNcs in AA rats were doneby radioimmunoassay. The results showed that the level of cAMP significantlyincreased afterβ2AR had combined with its agonist isoprenaline for 30 min, and Pae(12.5, 62.5, 312.5μg·ml-1) decreased the elevated cAMP level in rTNF-α-inducedMLNcs of AA rats in vitro. The above results suggested that one of characteristiceffects of Pae on inducing immune tolerance and exerting therapy rats AA is furtherdecreasing the proliferation of MLNcs and down-regulating the level of cAMP.5. Enhancingβ2AR desensitization, and recovering the balance of the expressionof GRK2/β-arrestin1, 2 proteins from MLNcs in AA rats was one of the mostimportant mechanisms of the therapeutic effects of PaeThe results of Western blot and immunohistochemistry analysis demonstratedthat Pae markedly increased the lowered protein expression ofβ2AR and GRK2, anddecreased that ofβ-arrestin1, 2 in MLNcs of AA rats for the first time. The resultssuggested that enhancingβ2AR desensitization, and recovering the balance of theexpression of GRK2/β-arrestin1, 2 proteins from MLNcs in AA rats is one of the mostimportant mechanisms of the therapeutic effects of Pae.6. The administration of Pae reversed the inhibiting effect of TNF-α-inducing on β2AR desensitization of MLNcs in AA rats in vitroThe results showed that the expression levels ofβ2AR and GRK2 protein inmembrane decreased, while that ofβ-arrestin1, 2 protein in membrane increased inTNF-α-induced MLNcs of AA rats, and Pae significantly reversed above change. Itwas found that inflammatory cytokine TNF-αinhibited GRK2 protein membranetranslocation activity, further inhibitedβ2AR desensitization. Pae markedly reversedthe inhibiting effect of TNF-α-inducing onβ2AR desensitization of MLNcs in AA ratsin vitro.CONCLUSIONS1. Pae inhibited the production of the anti-inflammatory cytokines IL-2 and TNF-α,enhanced the production of the anti-inflammatory cytokines IL-4 and TGF-β1, anddiminished the level of cAMP of MLNcs in AA rats. These results suggested that Paemight induce the Th1 cells immune tolerance, which then shift to Th2, Th3 cellsmediated activities to effect the therapeutic effects on AA of rats.2. Pae significantly enhanced the lowered expression levels ofβ2AR and GRK2protein, inhibited the elevated expression level ofβ-arrestin1, 2 protein, enhancedβ2AR desensitization, and recovered the balance of GK2/β-arrestin1, 2 in MLNcsof AA rats. Enhancingβ2AR desensitization, and recovering the balance of theexpression of GRK2/β-arrestin1, 2 proteins from MLNcs in AA rats is one of the mostimportant mechanisms of the therapeutic effects of Pae.3. Pae markedly increased the lowered expression levels ofβ2AR and GRK2 proteinin membrane, inhibited the elevated expression level ofβ-arrestin1, 2 protein inmembrane in TNF-α-induced MLNcs of AA rats in vitro. Inflammatory cytokineTNF-αinhibited GRK2 protein membrane translocation activity, further inhibited β2AR desensitization. Pae significantly reversed the inhibiting effect ofTNF-α-inducing onβ2AR desensitization of MLNcs in AA rats, and enhancedβ2ARto take effect its normal regulatory effects.

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