【作者】 杨晓红；

【导师】 崔炜；

【作者基本信息】 河北医科大学 ， 内科学， 2008， 博士

【摘要】 慢性、亚临床性的炎症,以及炎症导致的胰岛素抵抗是2型糖尿病和动脉粥样硬化的共同病理基础。其特征一是产生炎症因子和炎症标志物。如CRP、TNF-a、IL-6和脂联素、抵抗素及瘦素等。其特征二是激活炎症信号通路。核因子κB抑制物激酶(IκB-kinase, IKK)是核因子κB(NF-κB)抑制物(IκB)激酶,而NF-κB是炎症启动、调节的关键因子。NF-κB与抑制物IκB结合,以无活性的形式存在细胞浆内,IKK经TNF-α、IL-1、IL-6等炎症因子激活后,使IκB磷酸化并与NF-κB解离,解除抑制的活性NF-κB进入细胞内,调节一系列炎症因子及炎症相关物质的基因转录和蛋白合成。糖尿病心肌病是一种独立的糖尿病心血管并发症,与糖尿病患者的高心力衰竭发生率和高死亡率密切相关。心肌间质重构及纤维化是糖尿病心肌病的特征性病理表现,现已明确参与糖尿病心肌纤维化的调控因素有肾素-血管紧张素-醛固酮系统(RAAS)、内皮素、NO、糖基化终末产物(AGEs)、基质金属蛋白酶(matrix metalloproteinases, MMPs)、TGF-β1等。其中最为关键的是TGF-β1/Smad信号转导通路的激活。目前关于TGF-β1/Smad信号通路的研究多集中于糖尿病肾病,有关TGF-β1/Smad信号通路在糖尿病心肌病中的研究,国内外尚未见报道。进一步需要了解炎症是否参与了糖尿病心肌纤维化的调控。已有证据表明,炎症对所有的心血管相关疾病的发生和发展都起着重要的作用,糖尿病心肌病作为糖尿病的心血管并发症可能亦是一种慢性低级别炎症或称亚临床炎症状态。因此本研究的目的是找出炎症NF-κB/IκB通路与纤维化TGF-β1/Smad通路的内在联系,以阐明炎症在糖尿病心肌纤维化中的作用机制,对制定糖尿病心肌病治疗策略,减少其发病率、病残率和死亡率具有重要意义。他汀类药物即3-羟基-3-甲基-戊二酸单酰辅酶A还原酶抑制剂(HMG-CoA reductase inhibitors)作为调脂药物的一种,广泛应用于心血管疾病的预防和治疗中。近年来,他汀类药物的非降脂作用及与过氧化酶体增殖物激活型受体(peroxisome proliferators-activated receptors, PPAR)的关系越来越引起人们的关注。噻唑烷二酮类药物(thiazolidinediones, TZDs)是近年来应用于临床的一类新型胰岛素增敏剂,是过氧化酶体增殖物激活型受体γ(peroxisome proliferators-activated receptors gamma, PPAR-γ)的人工合成激动剂。TZDs不但可以改善胰岛素抵抗,降低血糖,还可通过抑制炎症反应,保护内皮功能等非降糖途径来发挥心脏保护作用。最新研究显示吡格列酮与阿托伐他汀在减少梗塞面积时有协同作用,有关单独应用他汀及联合噻唑烷二酮类药物干预糖尿病心肌纤维化的研究,目前国内外尚无报道。本研究通过高糖,高脂饮食加腹腔注射小剂量STZ的方法,制成2型糖尿病大鼠模型。我们选择目前广泛应用于临床的噻唑烷二酮类药物―罗格列酮(Rosiglitazone, Rsg)和他汀类药物-阿托伐他汀(Atorvastatin, Ato),观察它们对该模型心肌组织中炎症细胞浸润程度、炎症因子、NF-κB/IκB通路、TGF-β1/Smad通路及胶原代谢的影响。第一部分阿托伐他汀、罗格列酮及二者联合对2型糖尿病大鼠心肌的保护作用目的:了解阿托伐他汀和罗格列酮是否对2型糖尿病大鼠心肌胶原堆积产生影响,探讨阿托伐他汀、罗格列酮及二者联合是否对2型糖尿病大鼠心肌有保护作用。方法:用成年SD大鼠,通过高糖,高脂饮食加腹腔注射小剂量STZ的方法,制成2型糖尿病大鼠模型。采用光镜、电镜观察对照组、糖尿病组大鼠心脏的形态学改变以确定模型建立情况及炎症细胞浸润程度,用Masson三色染色观察心肌胶原沉积情况,生化法检测心肌羟脯氨酸含量以确定心肌胶原蛋白含量。用Western blot技术检测大鼠心肌MMP-2、MMP-9、TIMP-1蛋白表达,用RT-PCR检测MMP-2、MMP-9、TIMP-1mRNA水平,用免疫组化法检测Ⅰ型、Ⅲ型胶原及MMP-2、MMP-9、TIMP-1蛋白表达和组织定位。12w时开始分成阿托伐他汀(20mg/kg/d)组、罗格列酮(2mg/kg/d)组及阿托伐他汀加罗格列酮组,用上述药物给大鼠每天一次灌胃至实验结束(干预12w),观察药物对上述指标的变化。结果:1.一般情况:对照组大鼠饮食水情况、精神状态、日常活动均正常。DM组大鼠给予高脂,高糖饮食后,体重增加明显,并出现脂肪的再分布-腹型肥胖,到6w时(注射STZ前)已明显高于对照组。给药后两周(8w,此时血糖已明显升高)体重开始下降,但到实验结束是仍明显高于对照组。罗格列酮及阿托伐他汀治疗组大鼠体重较DM组显著下降(p<0.01),但仍明显高于对照组。两种药物组之间相比,无显著性差异,但两药联合治疗组大鼠体重下降较单药组更显著(p<0.05)。糖尿病组大鼠在注射STZ后一周内出现多尿,多饮,多食症状,随病程的延长,上述症状逐渐加重并出现毛发干燥,色泽黯淡,反应迟钝,活动减少。罗格列酮及阿托伐他汀及两药联合治疗组大鼠的上述症状无明显改善。DM大鼠的左心室重量及左心室重量/体重比值明显高于同期对照组大鼠(p<0.01),罗格列酮及阿托伐他汀治疗组大鼠的左心室重量、左心室重量/体重比值与DM大鼠相比均明显下降(p<0.01)。两种药物组之间相比,无显著性差异,但两药联合治疗组大鼠体重下降较单药组更显著(p<0.01)。2.生化指标的改变:DM组大鼠的空腹血糖、胰岛素,甘油三酯、胆固醇水平较同期对照组大鼠均显著升高(P<0.01)。罗格列酮治疗组大鼠胆固醇、甘油三酯水平较DM组有下降但无统计学意义,胰岛素和空腹血糖水平则明显下降(P<0.01),阿托伐他汀治疗组大鼠胰岛素和空腹血糖水平较DM组有下降但无统计学意义,胆固醇、甘油三酯水平则明显下降(P<0.01)。双药组胰岛素、空腹血糖及胆固醇、甘油三酯水平均较DM组显著下降(P<0.01)。3.大鼠心肌组织的病理改变:HE染色可见正常对照组大鼠心肌细胞排列整齐,致密,结构清晰,细胞外间质较少,可见少量成纤维细胞。DM大鼠心肌细胞排列紊乱,心肌细胞肥大,扭曲,细胞间隙增大,间质和血管周围细胞外基质增多,成纤维细胞增多,并有炎症细胞浸润。电镜观察:正常大鼠心肌细胞排列整齐,结构清晰,细胞间质胶原含量很少,毛细血管内皮细胞及基底膜结构正常。DM大鼠心肌细胞肌丝纤维稀疏,扭曲,线粒体肿胀,数量减少,排列紊乱,空泡变性,部分嵴断裂,糖原减少,间质胶原增生,毛细血管内皮细胞肿胀,毛细血管基底膜增厚。罗格列酮及阿托伐他汀治疗组心肌病变较DM组明显减轻,间质胶原沉积较少,毛细血管基底膜增厚减轻,介于对照组和DM组之间。4.大鼠心肌组织胶原代谢的改变:Masson染色结果:对照组大鼠心肌细胞之间、血管周围有少量胶原纤维,DM大鼠心肌间质胶原纤维明显增多,排列紊乱,毛细血管周围基质增多。罗格列酮及阿托伐他汀治疗组心肌胶原积聚明显减轻,排列也趋于规则;两种药物组之间相比,无显著性差异,但双药组较单药组更明显(p<0.01)。DM大鼠心肌组织羟脯氨酸含量明显高于对照组(p<0.01),罗格列酮及阿托伐他汀治疗组羟脯氨酸含量明显低于DM组(p<0.01)。两种药物组之间相比,无显著性差异,但双药组较单药组降低更明显(p<0.01)。免疫组化显示:DM大鼠心肌Ⅰ、Ⅲ型胶原的表达较对照组显著增加,且Ⅰ/Ⅲ也较对照组显著增加(p<0.01),阿托伐他汀和罗格列酮干预后Ⅰ、Ⅲ型胶原水平明显下降(p<0.01),且Ⅰ/Ⅲ也明显下降(p<0.01)。两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。5.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织MMP-9蛋白表达的影响:免疫组化显示:正常对照大鼠的MMP-9阳性细胞主要是心肌细胞,而成纤维细胞有少量表达,血管内皮细胞则无表达。DM大鼠MMP-9阳性染色细胞为间质成纤维细胞和心肌细胞,血管内皮细胞亦有微量表达,阳性染色细胞数明显增多,染色加深。阿托伐他汀和罗格列酮干预后DM大鼠心肌MMP-9阳性染色细胞数减少(p<0.01)。两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。Western Blot结果显示:DM大鼠心肌MMP-9蛋白水平与同期正常对照大鼠比较明显升高(p<0.01),药物干预后上述蛋白水平明显下降(p<0.01),且双药组较单药组降低更明显(p<0.01)。6.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织MMP-2蛋白表达的影响:免疫组化显示:正常对照大鼠MMP-2在血管内皮细胞、成纤维细胞和心肌细胞均有明显表达。阳性信号呈黄色或棕黄色颗粒状,分布于上述细胞的胞浆内。DM大鼠MMP-2在血管内皮细胞、成纤维细胞阳性染色较强,但心肌细胞表达相对较弱。DM大鼠阳性染色光密度积分明显低于同期对照大鼠(p<0.01)。阿托伐他汀和罗格列酮干预后DM大鼠心肌MMP-2阳性染色细胞数增多(p<0.01)。两种药物干预组相比,无统计学差异,但双药组较单药组增加更明显(p<0.01)。Western Blot结果显示:DM大鼠心肌MMP-2蛋白水平与同期正常对照大鼠比较明显下降(p<0.01),阿托伐他汀和罗格列酮干预后上述蛋白水平明显升高(p<0.01),且双药组较单药组升高更明显(p<0.01)。7.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织TIMP-1蛋白表达的影响:免疫组化显示:TIMP-1在正常对照组大鼠心肌细胞、成纤维细胞少量表达, DM大鼠TIMP-1阳性染色细胞明显增多,分布于心肌细胞、成纤维细胞和血管内皮细胞,阳性染色光密度积分明显高于对照组大鼠(p<0.01),阿托伐他汀和罗格列酮干预后DM大鼠心肌TIMP-1阳性染色光密度积分明显减低(p<0.01)。两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。Western Blot结果显示:DM大鼠心肌TIMP-1蛋白水平与同期正常对照大鼠比较明显升高(p<0.01),阿托伐他汀和罗格列酮干预后上述蛋白水平明显下降(p<0.01),且双药组较单药组降低更明显(p<0.01)。结论:阿托伐他汀及罗格列酮可减轻糖尿病大鼠心肌组织炎症细胞浸润和胶原沉积,改善糖尿病大鼠心肌MMPs/TIMPs平衡,减轻糖尿病心肌病变的严重程度。阿托伐他汀联合罗格列酮可提高抗炎、抗纤维化程度。第二部分炎症因子与2型糖尿病大鼠心肌胶原代谢的关系及阿托伐他汀、罗格列酮干预研究目的:观察实验性2型糖尿病大鼠心肌胶原的积聚情况与血浆、心肌TNF-α、IL-6、脂联素、CRP和AngⅡ、Ald的变化及其关系,了解阿托伐他汀、罗格列酮对它们的影响。方法:采用放免法检测血浆和心肌局部TNF-α、IL-6及AngⅡ、Ald的含量;应用ELISA法检测血浆和心肌中脂联素、hs-CRP的含量。结果:1.症状、体征、生化指标的改变:HE染色结果;超微结构的改变;Masson染色结果;心肌组织羟脯氨酸含量:均同第一部分。2. DM大鼠血浆、心肌hs-CRP、TNF-α、IL-6及AngⅡ、Ald的含量明显高于对照组大鼠(p<0.001),罗格列酮及阿托伐他汀治疗组血浆、心肌hs-CRP、TNF-α、IL-6及AngⅡ、Ald的含量较同期DM大鼠均显著下降(p<0.01),且双药组较单药组降低更明显(p<0.01);DM大鼠血浆、心肌Adi含量明显低于对照组大鼠(p<0.001),罗格列酮及阿托伐他汀治疗组血浆、心肌脂联素含量较同期DM大鼠均显著升高(p<0.01),且双药组较单药组升高更明显(p<0.01)。3.心肌胶原含量与CRP、TNF-α、IL-6、脂联素、AngⅡ、Ald相关分析表明:心肌局部CRP、TNF-α、AngⅡ、Ald、IL-6含量与心肌胶原面密度(r=0.623、0.679、0.622、0.684,p<0.01;0.535,p<0.05)呈正相关,脂联素与胶原面密度(r=-0.562,p<0.05)呈负相关。结论:DM大鼠无论血浆中还是心肌组织中致炎因子CRP、IL-6、TNF-α增多,抗炎因子脂联素减少,表明炎症参与了DM心肌病变的发生与发展。DM大鼠心肌组织局部AngⅡ、Ald明显增高,表明心肌中RAAS激活参与了DM心肌病变的发生与发展。阿托伐他汀和罗格列酮可通过抑制炎症因子及RAAS激活,增加抗炎因子,使糖尿病大鼠心肌组织胶原沉积和纤维化程度明显减轻,延缓糖尿病心肌病变的进程。阿托伐他汀联合罗格列酮可提高抗炎、抗纤维化程度。第三部分阿托伐他汀、罗格列酮及二者联合对2型糖尿病大鼠心肌NF-κB/IκB信号转导通路的影响目的:了解NF-κB/IκB通路在糖尿病大鼠心肌中的表达及阿托伐他汀、罗格列酮对它的干预作用。方法:用Western blot技术检测大鼠心肌TNF-α、NF-κB亚单位P65、阻抑物IκBα蛋白的表达,用RT-PCR检测NF-κB、TNF-αmRNA水平,用免疫组化法检测上述蛋白表达和组织定位。结果:1.症状、体征、生化指标的改变:HE染色结果;超微结构的改变;Masson染色结果;心肌组织羟脯氨酸含量:均同第一部分。2. RT-PCR结果:糖尿病组大鼠心肌组织中NF-κB和TNF-αmRNA表达水平较对照组明显增加(p<0.01),罗格列酮及阿托伐他汀干预组较糖尿病组NF-κB和TNF-αmRNA表达水平明显减低(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组减低更明显(p<0.01)。3.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织NF-κBp65蛋白表达的影响:免疫组化结果显示:NF-κBp65在对照组心肌细胞核中弱表达,在糖尿病组心肌细胞核中表达显著增加(p<0.01),阿托伐他汀和罗格列酮干预组较糖尿病组表达水平明显减低(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。Western Blot结果显示:DM大鼠NF-κB p65蛋白表达与同期正常对照大鼠比较明显升高(p<0.01) ,阿托伐他汀和罗格列酮干预后NF-κB p65蛋白水平明显下降(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。4.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织IκBα蛋白表达的影响:免疫组化结果显示:IκBα在糖尿病组心肌细胞核中表达显著减少(p<0.01),阿托伐他汀和罗格列酮干预后IκBα表达水平明显增加(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组增加更明显(p<0.01)。Western Blot结果显示:DM大鼠心肌组织IκBα的蛋白水平与同期正常对照大鼠比较明显降低(p<0.01),药物干预后上述蛋白水平明显升高(p<0.01),且双药组较单药组升高更明显(p<0.01)。5.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织TNF-α蛋白表达的影响:糖尿病大鼠心肌组织TNF-α含量(放免法)较对照组明显升高(p<0.01);免疫组化显示:TNF-α在对照组心肌细胞浆内弱表达,在糖尿病组心肌细胞浆中表达显著增加(p<0.01),阿托伐他汀和罗格列酮干预后TNF-α蛋白水平明显下降(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。用Western Blot检测也得到相同结果。9.相关分析:对糖尿病成模大鼠心肌组织中NF-κB与TNF-α阳性细胞染色的积分吸光度值(A)值作相关分析表明糖尿病成模大鼠心肌组织中NF-κB与TNF-α(r=0.950,p<0.01)呈明显正相关,两者均与心肌胶原面密度呈正相关(r=0.617;0.646;0.900,p<0.01);IκBα与心肌胶原面密度呈负相关(r=-0.736,p<0.01)。结论:NF-κB/IκB通路在糖尿病大鼠心肌中被激活,从而参与糖尿病心肌病变的发病。阿托伐他汀和罗格列酮可通过抑制糖尿病大鼠心肌NF-κB/IκB信号转导通路的激活,减轻糖尿病大鼠心肌组织胶原沉积和纤维化程度,从而延缓糖尿病心肌病变的进展。两药联合应用对NF-κB/IκB通路的干预作用具有叠加效应。第四部分TGF-β1/Smad信号转导通路在2型糖尿病大鼠心肌病变中的表达及阿托伐他汀、罗格列酮干预研究目的:观察2型DM大鼠心肌纤维化形成过程中TGF-β1/Smad信号通路的变化及阿托伐他汀、罗格列酮对它的干预作用。方法:用RT-PCR检测TGF-β1、c-fos mRNA表达,用Western blot技术检测大鼠心肌TGF-β1、Smad2/3、Smad7、c-fos的表达,用免疫组化法检测上述蛋白表达及组织定位。结果:1.症状、体征、生化指标的改变:HE染色结果;超微结构的改变;Masson染色结果;心肌组织羟脯氨酸含量:均同第一部分。2.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织TGF-β1、c-fos mRNA表达的影响:糖尿病组大鼠心肌组织中TGF-β1、c-fos mRNA表达水平较对照组明显增加(p<0.01),阿托伐他汀和罗格列酮干预组较糖尿病组TGF-β1、c-fos mRNA表达水平明显减低(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。3.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织TGF-β1蛋白表达的影响:免疫组化显示:TGF-β1在对照大鼠成纤维细胞、血管内皮细胞和心肌细胞均有表达,但DM大鼠阳性染色光密度值明显高于同期对照大鼠的光密度值(p<0.01),阿托伐他汀和罗格列酮干预后TGF-β1蛋白水平明显下降(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。用Western Blot检测也得到相同结果。4.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织Smad2/3蛋白表达的影响:免疫组化显示:Smad2/3在DM大鼠心肌中的表达明显升高(p<0.01),阿托伐他汀和罗格列酮干预后Smad2/3蛋白水平明显下降(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。Western Blot结果显示:DM大鼠心肌Smad2/3蛋白水平与同期正常对照大鼠比较明显升高(p<0.01),药物干预后Smad2/3蛋白水平明显下降(p<0.01),且双药组较单药组降低更明显(p<0.01)。5.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织Smad7蛋白表达的影响:免疫组化显示:Smad7在DM大鼠心肌中的表达明显降低(p<0.01),阿托伐他汀和罗格列酮干预后Smad7蛋白水平明显升高(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组升高更明显(p<0.01)。用Western Blot检测也得到相同结果。6.阿托伐他汀、罗格列酮及二者联合对2型DM大鼠心肌组织c-fos蛋白表达的影响:免疫组化显示:c-fos在DM大鼠心肌中的表达明显升高(p<0.01),阿托伐他汀和罗格列酮干预后c-fos蛋白水平明显下降(p<0.01),两种药物干预组相比,无统计学差异,但双药组较单药组降低更明显(p<0.01)。Western Blot结果显示:DM大鼠心肌c-fos蛋白水平与同期正常对照大鼠比较明显升高(p<0.01),药物干预后c-fos蛋白水平明显下降(p<0.01),且双药组较单药组降低更明显(p<0.01)。7.心肌胶原纤维的含量与TGF-β1、c-fos的关系:以胶原面密度为因变量, TGF-β1、c-fos(Western灰度积分光密度值)为自变量,进行直线相关分析,显示心肌胶原含量与TGF-β1、c-fos表达呈正相关,相关系数为0.542;0.507 (p<0.05)。8. c-fos与NF-κB、TGF-β1的相关性分析:以c-fos(Western灰度积分光密度值)为因变量,NF-κB、TGF-β1为自变量,进行直线相关分析,显示c-fos与NF-κB、TGF-β1均呈正相关,相关系数分别为0.955;0.924, (p<0.01)。结论: DM大鼠心肌组织TGF-β1及Smad2/3的mRNA和蛋白表达明显增高,说明糖尿病心肌病的病理改变与TGF-β1系统启动有关。阿托伐他汀和罗格列酮可抑制糖尿病大鼠心肌TGF-β1/Smad通路。阿托伐他汀和罗格列酮可下调糖尿病大鼠心肌c-fos mRNA和蛋白水平。更多还原

【Abstract】 Chronic or subclinical inflammation and insulin resistance induced by inflammation are common pathologic foundation. The one characteristic of the pathology is production of proinflammatory cytokines and markers, such as CRP, TNF-α, IL-6, adiponectin, resistin, leptin and so on. The other one is activating inflammatory signals pathways. IκB-kinase(IKK) is an inhibitor(IκB) kinase of NF-κB, which is a key factor for initiation and regulation of inflammation. NF-κB binds with IκB, presenting in cytoplasm in inactive pattern. When IKK is activated by such proinflammatory cytokines as TNF-α, IL-1, IL-6, etc, IκB is phosphorylated and dissociated with NF-κB, then causing nuclear translocation of NF-κB. Once translocated, NF-κB transcriptional activates genes involved in the immune and inflammatory response and regulates the genetic transcription and protein synthesis for a series of proinflammatory cytokines and related substances of inflammation.Diabetic cardiomyopathy is an independent cardiovascular complication of diabetes, which is closely associated with high incidence of heart failure and high mortality of the diabetes mellitus. Special pathological lesions of the diabetic cardiomyopathy are reconstruction and fibrosis of the myocardial matrix. It is well known that the regulating factors of myocardial fibrosis include in the renin-angiotensin-aldosterone system, endothelin, NO, AGEs, matrix metalloproteinases(MMPs),TGF-β1,etc.Among which the signal transduction pathway of TGF-β1/Smad is the most critical. As for as the pathway is concerned, majority of relevant studies have been concentrated in the field of diabetic nephropathy, whereas studies in the field of diabetic cardiomyopathy have not been seen both in domestic and abroad.It is further needed whether inflammation takes part in regulation of myocardial fibrosis or not . The evidence is shown that inflammation may play an important role in genesis and development of all cardiovascular diseases. As a cardiovascular complication, the diabetic cardiomyopathy perhaps is a low-grade chronic or subclinical inflammatory state. The purpose of the present study is to find out the internal association between NF-κB/IκB pathway of inflammation and TGF-β1/Smad pathway of fibrosis, to elucidate what role inflammation may play in diabetic myocardial fibrosis. It may be important for formulating therapeutic strategies of the diabetic cardiomyopathy, reducing its incidence, disability and mortality.Statins are HMG-CoA reductase inhibitors as one kind of drug that adjusts the level of blood-lipids, and widely utilizes in prevention and treatment of cardiovascular diseases. It has been closely paid attention recently for statins to relate their non-reducing lipids role with peroxisome proliferators-activated receptors(PPAR). Thiazolidinediones(TZDs) have been applied in clinical treatment as a new kind of euglycemic agent of insulin, is an artificial-synthesized ligand for peroxisome proliferators-activated receptors gamma(PPAR-γ). TZDs could not only ameliorate insulin resistance and decrease glycemia, but also inhibit the inflammatory reactions, protect function of the endothelium, which exerts a protective role for the heart by way of non-decreasing glycemia. The newest study shows that Pioglitazone and atorvastatin may have synergism in reduction of infarct size. It has not yet known both in China and abroad that any study on single usage of statins and combined usage with TZDs has been carried out in intervention of the diabetic myocardial fibrosis.The present study made rat modal of type 2 diabetes by high glucose and fat diet combined with intraperitoneal injection of STZ in low dose. Therapeutically effects of rosiglitazone and atorvastatin were investigated, such index as inflammatory cell infiltration, proinflammatory cytokines, NF-κB/IκB pathway, TGF-β1/Smad pathway, and collagen metabolism in myocardium.Part 1 The Myocardial protection by rosiglitazone, atorvastatin, and their combination in rats with type 2 diabetesObjective: To study whether rosiglitazone, atorvastatin, and their combination can affect collagen accumulation in rats with type 2 diabetes.Methods: The experimental type 2 diabetic rats were yielded by injecting streptozotocin(STZ, 30mg/kg) and fed with high fat and glucose food, then we observed morphology by light and electron microscope and collagen accumulation of cardiac tissue by Masson staining. The hydroxyproline content of cardiac tissue was examined by biochemistry technique. The protein expression of MMP-2, MMP-9, TIMP-1 were detected with Western blot technique and immunnohistochemistry.The mRNA expression of MMP-2, MMP-9, TIMP-1 were measured with RT-PCR. The animals were divided into three groups: group with administration of atorvastatin(20mg/kg/d), group with administration of rosiglitazone(2mg/kg/d), and group with administration both of atorvastatin and rosiglitazone after 12 weeks, once per day via intragastric administration until the termination of the experiments, and the index mentioned above was observed.Results:1. Symptoms and Signs: in the control group drink and food, mental state, and daily activities were all normal. In the experimental group the body weight gained markedly and the fatty tissue was redistributed as the abdominal obesity after drink and food with high fat and high glucose, both indications were obviously higher than that of the control group by 6 weeks before injection of STZ. Two weeks after administration of the drugs (8 weeks ,when their hyperglycemia was significantly enhanced) their body weights began to lose, however they were still higher than that of the control group until termination of the experiments.The losing of the body weight in the atorvastatin and rosiglitazone groups were all significant compared with that of the DM group(p<0.01), whereas their body weights were still heavier than that of the control group. furthermore, the losing of the body weight in group of combined administration of the both drugs even markedly than that of single drug groups(p<0.05). One week after injection of STZ, the diabetic rats began to have polyuria, polydipsia, and polyphagia, all of which were gradually aggravated as the course of the disease prolongated, and the other manifestations were as trichoxerosis, hair colour faint, reaction dullness, and reduced activities. The symptoms mentioned above were present without obvious amelioration in all groups of the drug administration. The weight of the left ventricle and the ratio of the weight of left ventricle/body weight in DM group were significantly higher than that of the same periods of the control group(p<0.01). The weight of the left ventricle and the ratio of the weight of left ventricle/body weight in atorvastatin and rosiglitazone treatment groups were dropped down compared with DM group(p<0.01), and the both indications in the combined drug group were dropped down even further(p<0.01).2. Changes of biochemical indexes: fasting blood glucose, insulin, triglyceride and cholesterol in DM group were significantly enhanced compared with that of the control group(p<0.01). Cholesterol and triglyceride in rosiglitazone administration group were dropped down but had no statistical significance, insulin and fasting blood glucose were decreased markedly (p<0.01). Insulin and fasting blood glucose in atorvastatin administration group were decreased but had no statistical significance whereas cholesterol and triglyceride were decreased markedly(p<0.01).3. Pathologic changes in myocardium: in the control group the myocardial cells arranged regularly and densely, clear structures, scanty extracellular matrix, and a few fibroblasts were observed. In DM group the myocardial cells arranged irregularly, they may be hypertrophy and distortion; intercellular spaces were enlarged, extracellular matrix was enhanced, and inflammatory cells infiltration, there were fibroblasts infiltration between the myocardial cells.Electron microscopic observations: in the control group the myocardial cells arranged regularly, ultrastructure of them were clearly seen, collagen was rare in cellular matrix, and the endothelial cells and basement membrane were normal of the blood capillaries. In DM group the myofilaments of myocardium were rare and distortion; the mitochondria were swollen, reduced in number, disarranged, vacuolar degenerated, partial cristae were disruptured, glycogens were reduced, the interstitial collagen were proliferated, the endothelial cells were swollen and basement membrane was thicken. The pathologic changes of the myocardium in the atorvastatin and rosiglitazone administration groups were obviously relieved compared with that of the DM group, interstitial collagen deposition and inflammatory cells infiltration were reduced, the thicken basement membrane was relieved to degree between that of the control and DM groups.4. Changes of collagen metabolism in myocardium: the result of Masson` s stain: there were a few collagen fibers between the myocardial cells and around the blood vessels in the control group. However in the DM group the interstitial collagen fibers were increased obviously and disarranged, the pericapillary substance was increased. In the atorvastatin and rosiglitazone administration groups, collagen accumulation in myocardium was markedly lessened, its arrangement tend to more regular; the combined drug group was even better than that of the single drug groups(p<0.01). In DM group the content of hydroxyproline was significantly enhanced compared with that of the control group(p<0.01); the content of hydroxyproline in the drugs (atorvastatin and rosiglitazone) administration groups were obviously reduced compared with that of DM group(p<0.01), and the combined drug group was even lowered than that of the single drug groups(p < 0.01). Immunohistochemistry: as far as expressions of collagenⅠandⅢwere concerned, in the control group there were as linear distribution among the myocardial cells, interstitial cells in myocardium, and around the blood vessels, in DM group they distributed as focal or plaque-like increase, the expression levels were 23.25 and 6.69 times as those in the control group(p<0.01), and ratio of collagenⅠ/Ⅲwas higher than that of the control group. after the rosiglitazone and atorvastatin intervention, the expressions of collagenⅠandⅢwere obviously reduced(p<0.01), and that of the combined rosiglitazone and atorvastatin group was even lower than that of the single rosiglitazone and atorvastatin groups(p<0.01).All indicators mentioned above, atorvastatin administration groups compared with that of rosiglitazone administration groups, it was no statistical significance.5. Results of RT-PCR: the expression MMP-9 and TIMP-1mRNA in the DM group were all obviously increased compared with that of the control group(p<0.01). The expression levels of MMP-9 and TIMP-1mRNA in the atorvastatin and rosiglitazone administration groups were obviously reduced compared with that of the DM group(p<0.01), and that of the combined drug group was even lower than that of the single drug groups(p<0.01). The mRNA expression of MMP-2 and MMP-9/TIMP-1 in DM group were significantly reduced compared with that of the control group, whereas were obviously enhanced in the atorvastatin and rosiglitazone groups(p<0.01), and that of the combined drug group were even higher than that of the single drug group(p<0.01).6. The effects on protein expression of MMP-9 by rosiglitazone, atorvastatin, and their combination:Immunohistochemistry: The positive cells of MMP-9 are concerned, it was present in myocardial cells in large amount in the control group, whereas no such expression in the fibroblasts and vascular endothelium; in DM group the positive cells were mainly the myocardial cells, a few in the fibroblasts and vascular endothelial cells, and the positive stain size was increased, whereas that of the atorvastatin and rosiglitazone groups were reduced in number(p<0.01),and that of the combined drug group was even dropped down compared with that of the single drug groups(p<0.01).Western Blot: just as that of immunohistochemistry.7. The effects on protein expression of MMP-2 by rosiglitazone, atorvastatin, and their combination:Immunohistochemistry: The positive cells of MMP-2 in DM group mainly distributed in the vascular endothelial cells , fibroblasts, myocardial cells, number of the positive cells were fewer than that in the control group, shallower in stain, and reduced size (p<0.01); the positive cells of MMP-2 in the atorvastatin and rosiglitazone groups in such cells as those mentioned above were expressed higher than that in DM group, especially in the vascular endothelial cells(p<0.01), and that of the combined drug group was even higher than that of the single drug groups(p<0.01).Western Blot: just as that of immunohistochemistry.8. The effects on protein expression of TIMP-1 by rosiglitazone, atorvastatin, and their combination:Immunohistochemistry: The expression of TIMP-1 was concerned, in the control group there were very weak stain in the myocardial cells and fibroblasts, in DM group the positive cells were obviously increased, distributing in myocardial cells, fibroblasts, and vascular endothelial cells, which were stained darker and larger; their optic density of the positive cells were obviously higher than that of the control group. The positive cells of TIMP-1 in the atorvastatin and rosiglitazone groups in such cells as those mentioned above were expressed lower than that of DM group(p<0.01), and that of the combined drug group was even dropped down compared with that of the single drug groups(p<0.01).Western Blot: just as that of immunohistochemistry.All indicators mentioned above, atorvastatin administration groups compared with that of rosiglitazone administration groups, it was no statistical significance.Conclusions: atorvastatin and rosiglitazone may obviously relieve the collagen accumulation and inflammatory cells infiltration, improvement of balance of MMPs/TIMPs, thus delay the progress of the diabetic cardiomyopathy. Atorvastatin combined with rosiglitazone may enhance abilities of anti-inflammation and anti-fibrosis.Part 2 The expression of proinflammatory cytokines and collagen metabolism in type 2 diabetic rat hearts and intervention by rosiglitazone, atorvastatin and their combination Objective: To Study rosiglitazone, atorvastatin and their combination whether can affect myocardial collagen accumulation and TNF-α, IL-6, adiponectin, CRP and AngⅡ, Ald changes in blood plasma and myocardium of the rat with type 2 diabetes.Methods: The local levels of TNF-α, IL-6 and AngⅡ, Ald in serum and myocardium were detected with radioimmunity assay(RIA). The levels of adiponectin and hs-CRP both in blood plasma and myocardium were detected with ELISA.Results: The levels of hs-CRP, TNF-α, IL-6 and AngⅡ, Ald in serum and myocardium in DM group were significantly higher than that of the control group(p<0.001). The contents of hs-CRP, TNF-αand IL-6 and AngⅡ, Ald in blood plasma and myocardium in the atorvastatin and rosiglitazone administration groups were significantly dropped compared with that of same periods of the DM group(p<0.01), and those in the combined drug group were even dropped down further than that of the single drug groups(p<0.01). The contents of adiponectin in blood plasma and myocardium in DM group were dropped down obviously compared with that of the control group(p<0.001). The contents of adiponectin in blood plasma and myocardium in the atorvastatin and rosiglitazone administration groups were significantly enhanced compared with that of the same periods of DM group(p<0.01), and that of the combined drug group was even higher than that of the single drug groups(p<0.01). Further correlation analysis suggested that the contents of hs-CRP, TNF-α, , AngⅡ, Ald and IL-6 in local myocardium were positive correlation with that of myocardial collagen area density, and their coefficient correlation(r=0.623、0.679、0.622、0.684,p<0.01;0.535,p<0.05) ;the content of adiponectin was negative correlation with myocardial collagen area density (r=-0.562,p<0.05).All indicators mentioned above, atorvastatin administration groups compared with that of rosiglitazone administration groups, it was no statistical significance.Conclusion: The pathologic changes in rats with the diabetic cardiomyopathy include in collagen accumulation and fibrosis in myocardium, proinflammatory cytokines such as hs-CRP, IL-6, and TNF-αincreased, anti-inflammatory factors reduced, suggesting that inflammation may take part in genesis and development of he diabetic cardiomyopathy. The levels of AngⅡand Ald in local myocardium were significantly enhanced in the DM group, suggesting that RAAS in local myocardium may be activated and participated in genesis and development of the diabetic cardiomyopathy. Atorvastatin and rosiglitazone may reduce proinflammatory cytokines; increase anti-inflammatory factors. Atorvastatin combined with rosiglitazone may enhance abilities of anti-inflammation and anti-fibrosis.Part 3 The expression of signal transduction pathway of NF-κB/IκB in the rat cardiac tissue with type 2 diabetes and intervention by rosiglitazone, atorvastatin, and their combinationObjective: To understand whether pathway of NF-κB/IκB is activated or not in the rat cardiac tissue with type 2 diabetes, and explore NF-κB/IκB pathway may play some role in genesis and development of the diabetic cardiomyopathy. To observe preventive effect of rosiglitazone, atorvastatin, and their combination.Methods: The protein expressions of TNF-α, subunit P65 of NF-κB and IκBαwere detected with Western blot technique. The mRNA expression of NF-κB and TNF-αwere measured with RT-PCR. The expressed proteins mentioned above were observed and localized in tissues with the relevant immunnohistochemistry.Results :1. Results of RT-PCR: the expression of NF-κB and TNF-α, mRNA in the DM group were all obviously increased compared with that of the control group(p<0.01). The expression levels of NF-κB and TNF-αmRNA in the atorvastatin and rosiglitazone administration groups were obviously reduced compared with that of the DM group(p<0.01), and that of the combined drug group was even lower than that of the single drug groups(p<0.01).2. The effects on protein expression of NF-κBp65 by rosiglitazone, atorvastatin, and their combination:Immunohistochemistry: The expression of NF-κB was weaker within the nuclei of myocardial cells in the control group whereas was significantly enhanced in DM group(p<0.01), that of the atorvastatin and rosiglitazone groups were obviously reduced compared with that of DM groups(p<0.01), and that in the combined drug group was even lower than that of the single drug groups(p<0.01).Western Blot: compared with that of same periods of the control group the protein expressions of NF-κB was all obviously enhanced(p<0.01), however all the proteins were reduced markedly after atorvastatin and rosiglitazone intervention(p<0.01), and that of the combined drug group was even lower than that of the single drug groups(p<0.01).3. The effects on protein expression of IκBαby rosiglitazone, atorvastatin, and their combination:Immunohistochemistry: The expression of IκBαwithin the nuclei of myocardial cells in DM group was obviously reduced(p<0.01), whereas that of the atorvastatin and rosiglitazone groups were markedly enhanced compared with that of the DM group(p<0.01), and that in the combined drug group was even higher than that of the single drug groups(p<0.01). Western Blot: just as that of immunohistochemistry.4. The effects on protein expression of TNF-αby rosiglitazone, atorvastatin, and their combination:Immunohistochemistry: The expression of TNF-αin cytoplasm of myocardial cells is concerned, that in the control group was weaker, that in the DM group was enhanced obviously(p<0.01), that in the atorvastatin and rosiglitazone groups were obviously reduced compared with that in the DM group(p<0.01), and that in the combined drug group was reduced further (p<0.01).Western Blot: just as that of immunohistochemistry.All indicators mentioned above, atorvastatin administration groups compared with that of rosiglitazone administration groups, it was no statistical significance.5. Correlation analysis suggested that the protein expression(by immunohistochemistry) of NF-κB and TNF-αin local myocardium were positive correlation (r=0.950, p<0.01); both were positive correlation with collagen area density(r=0.617; 0.646, p<0.01); IκBαwas negative correlation with collagen area density(r=-0.736, p<0.01).Conclusions: By way of inhibition of activations of signal transduction pathways of NF-κB/IκB, atorvastatin and rosiglitazone may obviously relieve the collagen accumulation and fibrosis in myocardium, thus delay the progress of the diabetic cardiomyopathy. Atorvastatin combined with rosiglitazone may have additive effect on inhibiting of activations of signal transduction pathways of NF-κB/IκB.Part 4 The expression of signal transduction pathway of TGF-β1/Smad in the rat cardiac tissue with type 2 diabetes and intervention by rosiglitazone, atorvastatin, and their combinationObjective: Observation on changes of signal transduction pathway of TGF-β1/Smad in the course of myocardial fibrosis in the rat with type 2 diabetes, which may associate with collagen metabolism. To observe preventive effect of rosiglitazone, atorvastatin, and their combination.Methods: RT-PCR was used to observe the gene expression of TGF-β1 and c-fos in experiment rat hearts. The expression of TGF-β1, Smad2/3, Smad7 and c-fos were detected with Western blot. The protein expression and tissue localization of TGF-β1,Smad2/3, Smad7and c-fos were observed with the relevant immunohistochemistry.Results:1. The results of RT-PCR: the expression levels of TGF-β1 and c-fos mRNA in the DM group were all obviously increased compared with that of the control group(p<0.01). The expression levels of TGF-β1 and c-fos mRNA in the rosiglitazone and atorvastatin administration groups were obviously reduced compared with that of the DM group(p<0.01), and that of the combined rosiglitazone and atorvastatin group was even lower than that of the single rosiglitazone and atorvastatin groups(p<0.01).2. The effects on protein expression of Smad2/3, Smad7and TGF-β1 by rosiglitazone, atorvastatin and their combination:Immunohistochemistry: the positive expressions of Smad2/3 and TGF-β1 were present in fibroblasts, vascular endothelial and myocardial cells in the control group. The positive expressions of Smad2/3 and TGF-β1 were even darker and larger in size compared with that in the control group in view of their optic density(p<0.01). There were only the vascular endothelial cells and a few of fibroblasts in the rosiglitazone and atorvastatin groups were the Smad2/3 and TGF-β1 positive, but shallow in colour, the positive optic density was reduced obviously compared with that of DM group(p<0.01), and that in the combined rosiglitazone and atorvastatin group was reduced further(p<0.01) compared with that of the single rosiglitazone and atorvastatin groups(p<0.01). In DM group the positive cells of Smad7 mainly distributed in the vascular endothelial cells, fibroblasts, myocardial cells, the numbers of the positive cells was reduced compared with that of the control group(p<0.01). In atorvastatin and rosiglitazone groups the positive cells of Smad7 were increased in such cells as those mentioned above, especially in the vascular endothelial cells(p < 0.01), and that of the combined rosiglitazone and atorvastatin group even higher than that of the single rosiglitazone and atorvastatin groups(p<0.01).3. The effects on protein expression of c-fos by rosiglitazone, atorvastatin, and their combination:Immunohistochemistry: c-fos was markedly enhanced in DM group compared with that of the control group(p<0.01), and the rosiglitazone and atorvastatin groups were significantly reduced compared with that of the DM group(p<0.01), and that of the combined rosiglitazone and atorvastatin group even lowered than that of the single rosiglitazone or atorvastatin groups(p<0.01).Western Blot: just as that of immunohistochemistry.All indicators mentioned above, atorvastatin administration groups compared with that of rosiglitazone administration groups, it was no statistical significance.4. Correlation analysis suggested that the protein expression(by immunohistochemistry) of TGF-β1, c-fos were positive correlation with collagen area density(r= 0.542; 0.507, p<0.01).Conclusion: Both mRNA and protein expression of TGF-β1, Smad2/3 and c-fos in the DM group were significantly enhanced, suggesting that the pathologic changes of the diabetic cardiomyopathy may be associated with the initiation of TGF-β1 system. By way of inhibition activation of signal transduction pathways of TGF-β1/Smad and mRNA and protein expression of c-fos, atorvastatin and rosiglitazone may obviously relieve the collagen accumulation and fibrosis in myocardium, thus delay the progress of the diabetic cardiomyopathy.更多还原