【作者】 王晓玉；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2007， 硕士

【摘要】 类风湿关节炎(rheumatoid arthritis, RA)是一种慢性、异质性、系统性的自身免疫性疾病。其主要特征是关节软骨和骨质的进行性破坏,并伴有免疫功能的紊乱。巨噬细胞在炎症反应过程起着十分重要的作用。在局部或全身炎症反应过程中,巨噬细胞活化,其功能发生改变,同时产生许多炎症介质,其中包括TNF-α、IL-1、PGE2、IL-6等。鸟苷酸结合蛋白(Guanine nucleotide- binding proteins, G-protein),简称G蛋白,其介导的信号转导通路在炎症免疫性疾病中发挥重要作用。细胞因子、神经递质、激素、磷脂、光刺激、嗅觉及味觉特异性配基和丝裂原均可通过跨膜的G蛋白偶联受体(G-protein-coupled receptors,GPCRs)引发G蛋白活化及其下游信号传递。佐剂性关节炎(adjuvant arthritis,AA)模型大鼠腹腔巨噬细胞功能的异常改变可能与G蛋白-cAMP信号转导通路的异常改变有关。本研究在建立大鼠AA模型的基础上,研究从安徽道地药材木瓜中制取的有效部位木瓜苷(glucosides of chaenomeles speciosa,GCS)和白芍总苷的主要成分芍药苷(paeoniflorin,PF)对AA大鼠腹腔巨噬细胞异常功能变化及其部分作用机制,为进一步筛选高选择性的RA有效治疗药物、开发和利用中药及天然药物资源提供理论依据。目的:研究GCS和PF对AA大鼠继发性炎症反应的治疗作用;以腹腔巨噬细胞为突破口,阐明大鼠AA状态下腹腔巨噬细胞功能异常变化并明确GCS和PF在这方面的作用。方法:大鼠足跖皮内注射弗氏完全佐剂(Freund’s completed Adjuvant, FCA),诱发大鼠AA模型,以GCS30、60、120mg?kg-1(ig,d17~d24)治疗给药,PF25、50、100 mg?kg-1(ig,d17~d24)、雷公藤多苷(glucosides of tripterygium wifordii,GTW)作阳性对照40 mg?kg-1(ig,d17~d24)。足容积法测量大鼠继发侧足爪肿胀度(△ml)。吞噬功能采用中性红法测定,白细胞介素-1(interleukin-1, IL-1)活性的测定采用小鼠胸腺细胞增殖法,肿瘤坏死因子α(tumor necrosis Factor alpha, TNF-α)、前列腺素E2(prostaglandin E2, PGE2)和环磷酸腺苷(cyclic adenosine monophosphate,cAMP)含量的测定采用放射免疫测定法,Western blot检测腹腔巨噬细胞中G蛋白偶联受体激酶2(G protein-coupled receptor kinases, GRK2)、视紫红质抑制蛋白(arrestin)中的β-arrestin1、前列腺素E2受体(E-prostanoid, EP2)的表达水平。结果:1. GCS和PF对大鼠AA具有治疗作用应用FCA诱导大鼠AA模型,其继发性反应表现为以多发性关节炎为特征的关节肿胀和触痛、消瘦、活动障碍等类似RA的症状。AA模型组在致炎后d17、d21、d24继发侧足肿胀度与正常组比较明显增加。GCS30、60、120mg?kg-1(ig,d17~d24)和PF25、50、100 mg?kg-1(ig,d17~d24)体内给药可不同程度明显减轻AA大鼠的继发性足肿胀。表明GCS和PF对大鼠AA具有治疗作用。2.改善AA大鼠腹腔巨噬细胞异常吞噬功能是治疗大鼠AA的重要作用环节AA模型组腹腔巨噬细胞吞噬中性红的量明显高于正常组。同时,GCS60,120mg·kg-1(ig,d17~d24)和PF25、50、100 mg?kg-1(ig,d17~d24)能明显抑制腹腔巨噬细胞过强的吞噬功能。表明GCS和PF均可通过改善AA大鼠异常吞噬功能从而发挥其治疗作用。AA大鼠腹腔巨噬细胞中IL-1、TNF-α、PGE2的生成明显高于正常组。GCS(30mg·kg-1)与AA组比较,对IL-1及TNF-α的生成,没有明显改变;对PGE2的生成有显著性改变。GCS60,120mg·kg-1可明显抑制PMф中IL-l、TNF-α、PGE2的生成。PF25mg·kg-1与AA组比较,对IL-1、TNF-α及PGE2的生成,均没有显著性差异。PF50,100mg·kg-1可明显抑制PMф中IL-l、TNF-α、PGE2的生成。对AA大鼠腹腔巨噬细胞产生过高的IL-1、TNF-α及PGE2水平有抑制作用,表明GCS和PF可能通过直接作用或抑制腹腔巨噬细胞分泌炎性细胞因子,调节AA大鼠免疫功能是其发挥作用的重要途径之一。3. GCS和PF影响AA大鼠滑膜细胞G蛋白-AC-cAMP信号转导通路GCS60,120 mg·kg-1和PF25、50,100 mg·kg-1显著提高腹腔巨噬细胞cAMP水平。GCS和PF对腹腔巨噬细胞cAMP水平的影响提示G蛋白-AC-cAMP信号转导通路可能参与其过程。为了进一步探讨GCS和PF影响AA大鼠腹腔巨噬细胞G蛋白水平与升高cAMP含量之间的关系,对GCS 30,60,120 mg·kg-1和PF25,50,100 mg·kg-1治疗给药(ig,d 17~d24)后大鼠腹腔巨噬细胞EP2、GRK2、β-arrestin1蛋白表达水平进行检测,结果表明,AA大鼠腹腔巨噬细胞中EP2和β-arrestin1表达较正常组大鼠升高,GRK2表达较正常组均无明显差异。而GCS (60,120 mg·kg-1)可不同程度地下调EP2和β-arrestin1的表达;PF(25,50,100 mg·kg-1)可明显下调β-arrestin1的表达;对EP2的表达有下调的趋势,但不显著。提示:G蛋白-AC-cAMP信号转导通路介导了大鼠AA的发病过程,降低EP2受体的表达、调节GRK2/β-arrestin1的平衡、影响AA大鼠腹腔巨噬细胞PGE2-EP-G蛋白-cAMP信号的正常转导可能是GCS和PF发挥治疗作用的机制之一。结论:1. GCS和PF对大鼠AA关节局部肿胀具有明显的治疗作用,改善其异常的吞噬功能是发挥作用的重要途径之一。2. GCS和PF不同程度上均可抑制AA大鼠腹腔巨噬细胞分泌IL-l、TNF-α、PGE2水平。3. GCS和PF不同程度下调AA大鼠腹腔巨噬细胞中β-arrestin1和EP2的蛋白表达,促进cAMP生成增加,影响了G蛋白-AC-cAMP信号转导通路,这可能是GCS和PF治疗大鼠AA的另一机制。更多还原

【Abstract】 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.更多还原