【作者】 熊日波；

【导师】 罗炳德；

【作者基本信息】 南方医科大学 ， 营养与食品卫生学， 2013， 博士

【摘要】 研究背景及目的：类风湿关节炎(Rheumatoid Arthritis, RA)是一种自身免疫性疾病,以非化脓性增生性滑膜炎为特点,逐渐导致关节软骨的破坏,最后造成关节功能的障碍。RA是全世界范围内倍受关注的公共卫生问题,流行病学调查显示我国患病率约为0.32～0.36%,欧洲成年人RA患病率为0.2%-0.8%,其中南欧地区(意大利、希腊)患病率较低,为0.2%-0.4%,患病率随着年龄的增长而逐渐增加,研究显示老年人(65岁以上或70岁以上)患病率最高,其中女性高于男性。RA发病机制复杂,涉及遗传、营养、感染等多个因素。大量研究表明,细胞因子的异常产生和相互作用在形成RA炎症、黏附、新生血管形成和骨质减少方面起着重要的作用。细胞因子是活化的免疫细胞和某些基质细胞分泌的具有生物活性的小分子多肽物质的总称,它包括淋巴细胞产生的淋巴因子和单核巨噬细胞产生的单核因子,作为细胞间信号转导分子,主要调节免疫应答、参与免疫细胞分化发育、介导炎症反应、刺激造血功能并参与组织修复等。RA的启动是通过递呈至今未明的抗原或自身抗原给CD4+淋巴细胞,两者相互作用激活了淋巴细胞,而活化的淋巴细胞可以刺激单核/巨噬细胞释放细胞因子,这些细胞因子能介导RA炎症反应和关节损伤。RA中存在复杂的细胞因子网络,网络的生物学效应则依靠于细胞因子及其抑制物的相对浓度。瘦素(leptin)主要是由白色脂肪组织分泌的一种非糖基化蛋白类激素,分子量为16000,传统观点认为leptin主要与脂肪储存、能量摄入以及机体对食物的摄入有关。越来越多的证据显示瘦素是机体营养状况和免疫系统之间相互影响、相互联系的重要中介,与自身免疫性疾病有密切关系,可作为炎症介质网络中的一种细胞因子发挥作用。leptin主要通过JAK/STAT信号通路在调节细胞增殖、分化、免疫调节、炎症等多种过程中发挥重要作用,瘦素受体属于I类细胞因子超家族,包括细胞外结构域、跨膜结构域和细胞内结构域。作为JAK家族的成员,胞质酪氨酸激酶结合于瘦素受体上,其识别位点分布于受体特异的膜近端区域,STATs与细胞膜上的瘦素受体特异性结合,酪氨酸被JAKs磷酸化后,STATs与受体解离,形成同型或异型二聚体,进入细胞核内调节特异的基因转录和蛋白合成。由于该通路中的STATs是一组多效能蛋白质,在不同条件下可表现出不同的生物学功效。在RA中,参与信号转导的主要是STAT1和STAT3.红细胞沉降率(Erythrocyte sedimentation rate, ESR)、C反应蛋白(C-reactive protein, CRP)、类风湿因子(Rheumatoid arthritis, RF)是RA常用的辅助诊断项目。ESR增快表明机体处于病理状态,广泛用于监测感染、炎症及一些癌症患者的疾病活动程度；CRP是一种急性时相反应蛋白,很多急性和慢性炎性反应时CRP均表现为增高；RF是目前最常用的血清学检测项目,RF的敏感性虽高,特异性却不高,在RA以外的其他疾病如系统性红斑狼疮、原发性干燥综合征、恶性肿瘤甚至一些正常的老年人也可出现升高,经常容易造成误诊和漏诊,给早期诊断带来困难,从而延误病情,错过治疗时机,因此RF阳性不能作为诊断RA唯一标准。近年的研究表明,RF与临床表现和关节损伤程度密切相关,是损伤指标中最强有力的预后因子之一,但不是RA的高特异性血清标志物,其他指标如CRP、ESR等也不能取代RF在RA诊断和预后判断中的地位。对RA的治疗目前尚缺乏特效手段,主要采用改善病情的抗风湿药物、非甾体类抗炎药、生物制剂等,由于用药周期长加上药物的一些副反应,病人依从性较差。一项调查显示有20%～50%的患者希望通过膳食减轻RA症状,约有2/3的RA患者使用过膳食补充剂。尽管RA的病因尚不明确,大量研究表明,富含鱼类、橄榄油、蔬菜的饮食能抑制RA病情的发展,而富含红肉等的食物能加重病情。研究表明,具有抗氧化功能的维生素可能对RA具有治疗作用,VitA、VitE具有抗氧化、免疫调节、抗肿瘤等多种生物功能。体外实验显示VitA、 VitE具有抑制某些炎症因子的作用。本研究拟通过建立类风湿关节炎大鼠模型,观察瘦素在RA发病过程中所起的作用；灌胃给予类风湿关节炎大鼠VitA、VitE,取血清及滑膜组织,观察VitA、VitE对类风湿关节炎大鼠血清细胞因子如瘦素、TNF-α,、IL-6、IL-4、IL-10水平的影响,同时观察其对疾病活动指标ESR、CRP、RF的影响。采用Western blot研究VitA、VitE对CIA大鼠关节滑膜组织STAT1、STAT3蛋白表达水平的影响,从分子水平探讨VitA、VitE在RA中所起的作用。材料与方法：选体重为(147±15g)的健康雄性Wistar大鼠作为研究对象,饲养于恒温,恒湿条件下并自由摄取食水,饲养一周左右开始实验。随机分为正常组和模型组。将牛II型胶原与弗氏不完全佐剂用三通管在低温条件(<3℃)下按1：1的比例充分乳化(约1h),形成乳白色粘稠样液体备用。用5%水合氯醛5ml/kg常规腹腔麻醉,将配置好的试剂按3点/只,0.05ml/点皮内注射进行初次免疫,以皮肤出现白色丘疹样隆起为度。初次免疫动物2周后,加强免疫一次,剂量：2点/只,0.05ml/点,空白对照组用生理盐水0.2ml腹腔注射。初次免疫第一天起,每天观察大鼠的生长情况,重点观察四肢足关节肿胀情况,并按0-4级进行评分,四肢评分之和为关节炎指数(Arthritis index, AI), AI越高,表明关节炎程度越严重。0分,无关节炎；1分,关节表面有红色斑点或关节部位轻度肿胀；2分,关节部位中度肿胀；3分,关节部位严重肿胀；4分,关节部位严重肿胀,且不能负重。四周后将模型制作组中AI≥6的大鼠保留。模型组大鼠于第4周末随机分为4组,即模型组；模型组+VitA42.86μgRE/kg.bw;模型组+VitE200mg/kg.bw;模型组+布洛芬50mg/kg.bw。药物均为灌胃给药,正常对照组予等体积生理盐水腹腔注射,每日一次,持续4周。4周后,小鼠禁食24h,6%水合氯醛麻醉后,腹主动脉取血处死,分离血清,置-20℃保存。打开膝关节腔,完整剥离滑膜组织。用ELISA法按试剂盒操作,检测血清瘦素、TNF-α、IL-6、IL-4、IL-10水平,以Western blot印迹分析STAT1、STAT3蛋白表达水平。结果：1.CIA大鼠模型的建立牛Ⅱ型胶原与弗氏不完全佐剂建立CIA大鼠模型,以关节炎指数为评分标准,AI>6的大鼠保留。模型组与空白对照组同期比较,主要表现为关节红肿,累及踝关节、趾间关节及膝关节,此可以得出结论,CIA大鼠模型建立成功。2.CIA大鼠血清leptin、TNF-α、IL-6、IL-4、IL-10及ESR、CRP、RF的水平。模型组大鼠血清leptin、TNF-α、IL-6水平明显高于对照组(P=0.000)；IL-4、IL-10水平低于对照组,差异有显著性(P=0.000,0.001)；模型组大鼠血清ESR、CRP、RF高于对照组,差异有显著性(P=0.000)；3.VitA、ViE对CIA大鼠关节炎指数、细胞因子、疾病活动指标的影响。与模型组相比,VitA组与VitE组关节炎指数降低,差异有显著性(P=0.008,0.014)；与模型组相比,VitA组leptin、TNF-α、IL-6水平降低,差异有显著性(P=0.000,0.038,0.000),VitE组leptin、TNF-α、IL-6水平亦降低,差异有显著性(P=0.004,0.033,0.000)；VitA、VitE组与模型组比,IL-10含量升高,差异有显著性(P=0.006,0.001)。VitA组与模型组比,ESR、CRP含量明显降低,差异有显著性(P=0.025,0.001),VitE组与模型组比,ESR、CRP含量亦明显降低,差异有显著性(P=0.001,0.017)4. VitA、VitE对CIA大鼠关节滑膜组织p-STAT1、p-STAT3表达水平的影响与对照组相比,模型组p-STAT1、p-STAT3蛋白表达水平升高,差异有显著性(P=0.000)。VitA、VitE给药4周后,p-STAT3蛋白表达水平较模型组降低,差异有显著性(P=0.000)。结论：1.应用牛Ⅱ型胶原加弗氏不完全佐剂建立方法可以成功制作大鼠RA模型,该模型是国内外最常用的RA动物模型。2.在RA发病过程中存在一个失衡的细胞因子网络,促炎性细胞因子相对增多,抑炎性细胞因子相对减少,leptin参与了RA的发病过程,可作为一种促炎性细胞因子发挥作用。3.VitA、VitE能抑制促炎性细胞因子leptin、TNF-α、IL-6的水平,同时提高抑炎性细胞因子IL-10的水平。能降低CIA大鼠的关节炎指数、降低ESR和CRP。VitA、VitE通过下调关节滑膜组织中STAT3蛋白的表达水平阻断JAK/STAT信号转导通路,这就为RA的治疗开辟了一条新的途径。更多还原

【Abstract】 Background and objectiveRheumatoid arthritis(RA) is an autoimmune inflammatory disease characterized by proliferative synovitis which has been shown to be associated with the destruction of articular cartilage and deprivation of joint’s function.RA is a common disease worldwide.Epidemiological investigations revealed the incidences of RA in different regions,for example, the incidence in Chinese is0.32～0.36%,while in European adults,the incidence ranged from0.2～0.8%. People in Southern European regions including Italy and Greece had a low incidence of0.2～0.4%. Reaseachers had found that the incidence of RA is increased with the growth of age which culminated in people over65or70years.Females are more susceptible to RA than males.The mechanism of RA is complex involving heredity、nutrition、infection et al which has not been fully elucidated. Plenty of experiments showed that a complex cytokine networks exsited in RA, the biological effects of which is associated with the relative serum concentrations of inflammatory cytokines and their inhibitors.The interactions of cytokines played an important role in the inflammation、adhesion、 neovascularization and decreased bone density.Leptin is a type of hormone secreted by adipose tissue with a molecular weight of16000.It has long been regarded with relation to feeding and energy storage.It has been shown that leptin may act as a bridge between nutrition and immunology.Leptin has been indentified as an important cytokine in the inflammatory networks of RA. JAK/STAT signaling transduction pathway is a major way of leptin’s biological effects such as cell proliferation and differentiation、 immunological regulation and inflammation.Once leptin is combined to leptin receptor,corresponding moleculars are activated and transported to cell nucleus which promote the transcribing of target genes involving two key moleculars:STAT1and STAT3.RA is a chronic disease which require the intake of drugs including untirheumatics、NAIDs and biological agents. Patients are prone to drop out due to the side effects. It has been reported that33to75%of RA patients believe food plays an important role in their symptom’severity and20to50%will have tried dietary manipulation in an attempt to relieve their suffering. Anti-oxidants such as VitA and VitE manifested the inhibitory effects on inflammatory cytokines in vivo. It’s indicated vitamins with anti-oxidation property may be curable to RA.Therefore,our study aimed to observe the role of leptin in CIA mice, to examine the effect of VitA、VitE on leptin and other related experimental and clinical index and to explore the mechanisms based on JAK/STAT signaling transduction pathway.Materials and methodsAnimals and treatmentsMale Wistar mice weighed (147±15g) were used in the experiments. Both the animal care and study protocol employed were in accordance with Institutional Animal Care and Use Committee (IACUC) and the Organization for Economic Cooperation and Development (OECD) guidelines. The mice were housed in cages in a climate-controlled room with a12-hr light-dark cycle. Throughout the study, animals were fed with regular standard mice chow and water ad libitum. After1-week acclimation period, animals were given an intradermal injection of bovine type II collagen emulsified in incomplete Freund’s adjuvant or0.9%normal saline(the model and control groups, respectively). Two weeks later, the mice were given a booster intradermal injection. At the end of the4th week, arthritis index were applied to evaluate the paw swelling. Each paw was graded with a maximum score of4:0, normal,without any macroscopic signs of arthritis;1, mild, but definite redness and swelling of the ankle, or apparent redness and swelling limited to individual digits, regardless of the number of affected digits;2, moderate redness and swelling of the ankle;3, redness and swelling of the entire paw including digits;4, maximally inflamed limb with involvement of multiple joints. The4paw scores for each mouse were summed.Mice with a score higher than6were kept.Model group was divided into five subgroups:1) model group (n=6);2) VitA (42.86μgRE/kg.bw) group (n=6);3) VitE (200mg/kg.bw group (n=6); and4) Ibuprofen (50mg/kg.bw) group (n=6). Mice in the VitA、VitE and Ibuprofen groups received an intragastric administration of VitA、VitE、Ibuprofen respectively. At the end of the8th week, all the mice were anaesthetized and sacrificed, and blood samples and joint synovium tissue were extracted.The tissue was stored at-80℃until it was used for western blotting.1.Determination the level of serum leptin and other related experimental and clinical index.Blood samples were isolated by centrifugation at1000r/min for10min and then kept at-20℃before the following assay. The levels of leptin、TNF-α、IL-6、IL-10、 IL-4、CRP、RF were measured by ELISA using commercial kits.2.Western blot for STAT1and STAT3expression.Tissue samples were homogenized in complete RIPA lysis buffer. Total protein was quantified with the BCA protein assay kit. All preparations were carried out at 4℃. For western blotting, A Total of15μl of the mixture of protein and sample buffer was loaded per lane and electrophoretically separated on an SDS-PAGE gel. Protein bands were transferred to a PVDF membrane using a Trans-Blot SD Semi-Dry Electrophoresis Transfer Cell.The gels were then incubated with a primary antibody for p-STAT1and p-STAT3overnight at4℃. Finally, gels were incubated in secondary antibodies for1hr at room temperature.Statistical analysisData are expressed as means±SD. Statistical difference between two groups were measured by t test of two independent-samples.Statistical differences between groups were compared by one-way analysis of variance (ANOVA) with SPSS16.0software for statistical analysis. P<0.05was considered statistically significant.Results1.The induction of collagen-induced arthritis(CIA)Measured by arthritis index, mice with a score higer than6were regarded as a CIA model which is parallel with the pathogenesis of rheumatoid arthritis in human.2. Measurement on the levels of serum leptin、TNF-α、IL-6、IL-10、IL-4and ESR、 CRP、RFAs shown in Fig2,compared with the control,the serum leptin levels were significantly increased in model animals(P=0.000); Fig3showed the alterations in CIA mice assessed by serum TNF-α、IL-6、IL-4、IL-10levels.There was a significant increase in serum TNF-α、IL-6levels compared with control group(P=0.000);The level of serum IL-4、IL-10was significantly reduced compare with the control group(P=0.000); ESR、CRP and RF were makers of disease activity index in RA, Fig4-6. showed the changes.Compared with control group,the level of ESR、CRP and RF was significantly increased(P=0.000).3. The effects of VitA and VitE on arthritis index and the levels of serum leptin、 TNF-α、IL-6、IL-10、IL-4and ESR、CRP、RFFig7.showed the effect of VitA、VitE on arthritis index.Four-week administration of VitA and VitE significantly decreased arthritis index(P=0.008,0.014);As shown in Fig(8-12), the serum leptin、TNF-α、IL-6levels were significantly reduced in VitA and VitE group compared to model groupfor VitA group; the level of IL-10was significantly increased compared with model group(P=0.006,0.001); Fig(13-15). showed the effects of VitA, VitE on ESR、CRP and RF.Compared with model group,VitA、VitE can reduce the level of ESR and CRP significantly (P=0.025,0.021for VitA group,P=0.001,0.017for VitE group).4The effects of VitA、VitE on p-STAT1and p-STAT3protein expression levelp-STAT1and p-STAT3are the molecules of JAK/STAT signaling transduction pathway.Fig(16-17) showed the effects of VitA、VitE on p-STATl and p-STAT3protein expression level.The model group showed a significant up-regulation of p-STAT1and p-STAT3protein expression compared to model group(P=0.000).Mice treated with VitA、VitE for4weeks showed a a significant reduction of p-STAT3protein expression level(P=0.000).ConclusionOur study suggests the role of leptin on the pathogenesis of collagen-induced arthritis mice and the effects of VitA、VitE on cytokine network and other related index, the study also examined the effects of VitA、VitE on p-STAT1and p-STAT3protein expression level,the marker of lepin’s signaling transduction pathway. An unbalanced cytokine network exsisted in the pathogenensis of RA which manifested increased inflammatory cytokines and decreased anti-inflammatory cytokines. Leptin played an important role in RA and can be indentified as a inflammatory cytokine. Compared with model group,VitA、VitE can reduce the level of inflammatory cytokines such as leptin、TNF-α、IL-6and improve the level of anti-inflammatory cytokine such as IL-10. VitA、VitE can also decrease arthritis index and the level of ESR、CRP in CIA mice.There were reduced p-STAT1and p-STAT3protein expression levels in VitA and VitE group.The present study may provide a new way for curing RA.更多还原