【作者】 宋辉；

【导师】 安丰双；

【作者基本信息】 山东大学 ， 内科学， 2008， 博士

【摘要】 背景心血管疾病是糖尿病患者的主要死亡原因,在西方国家约70%的糖尿病患者死于糖尿病性心血管疾病。自1972年Rubler等首次提出糖尿病性心肌病（Diabetic Cardiomyopathy,DCM）概念以来,流行病学、病理学以及实验研究的结果均提示糖尿病性心肌病变的存在。糖尿病性心肌病变与糖尿病特有的代谢异常有关。其临床特征早期以心脏舒张功能不全、晚期以收缩功能不全为主,容易发生充血性心力衰竭。近年来国内外学者对DCM进行了大量的基础和临床研究,取得了阶段性的成果。但DCM的发病机制、早期诊断和治疗,仍处在研究探索中。深入研究DCM的发病机制,阻断DCM的发病途径对DCM的防治具有重要意义。DCM发生受多种因素影响。心肌细胞代谢紊乱、微血管病变和氧化应激被认为是其发生和发展的可能机制。研究表明,DCM早期就可以出现心肌细胞的凋亡和坏死。高血糖时氧化应激增加,信号转导途径改变,引起异常基因表达,激活细胞程序化死亡。心肌细胞凋亡的信号转导机制可能是:①通过死亡受体转导通路介导心肌细胞凋亡;②丝裂原活化蛋白激酶（MAPK）转导通路;③磷脂酰肌醇3-激酶/Akt信号转导通路。DCM病理表现为心肌肥厚和心肌纤维化等。心肌成纤维细胞增生和其合成分泌胶原量及种类的变化,即心肌间质重构。心肌细胞的凋亡和心肌纤维化在DCM的发生发展中起重要作用。FoxO（Forkhead box O transcription factors）蛋白家族是2000年才正式发布统一命名的蛋白质家族,共可以分为17个亚家族,其分别被命名为FoxA～Q,在这17个亚家族中,Fox0亚家族主要通过转录调控信号传导途径在动物的生长发育、细胞分化、代谢、凋亡和免疫等方面起重要作用。依据DNA结合结构域之外氨基酸序列的相似性,脊椎动物的Fox0亚家族成员又可进一步分为Fox01、Fox03和Fox04。Fox0被生长因子激活的PI3K通路激活,产生Akt依赖磷酸化作用,磷酸化的Fox0激活或抑制细胞凋亡或细胞分裂周期相关的基因如:Bim、P27kipl、FasL、Bcl26、TRA IL、FLIP、MnS0D、GADD45（Za）,进而促进或抑制凋亡。研究表明,很有可能Fox0因子依赖于细胞类型而激活不同的基因,Fox0可能依赖于适当的辅助因子而对细胞起不同的作用,或者引起凋亡,或者不引起凋亡,或仅在应激条件下诱导凋亡,从而引起不同的效应。不同的研究对Fox0所诱导的凋亡基因表达的结果是有差别的,Fox0影响凋亡的机制目前尚远未清晰。文献提示Fox0转录因子对细胞及细胞外基质的病理生理学变化有转录调节作用。国外已有学者证实Fox03a转录因子在内皮细胞中通过PI3K/Akt/Fox03a途径调节内皮细胞凋亡。Fox03a转录因子是否参与了心肌细胞的凋亡国内外尚未见报道。研究表明,糖尿病目前的治疗,包括血糖的控制,可以延缓疾病的发展,但仅仅控制血糖并不能充分减少糖尿病患者的心血管事件的发生和进程。因此探讨该疾病发生和发展的潜在机制,寻找糖尿病性心肌病防治的新靶点已成为减少糖尿病性心肌病病死率方面的亟待解决的问题。他汀类（Statins）药物是细胞内源性胆固醇合成的限速酶—羟甲基戊二酸单酰辅A（3-hydroxy-3-methylglutaryl-coenzymeA,HMG-CoA）还原酶的抑制剂,能有效抑制胆固醇的合成,临床已普遍用于治疗高脂血症。近年来有研究报道他汀类药物对内皮细胞有抑制凋亡作用,但其作用机制尚不完全清楚。Carmen Urbich等证实阿托伐他汀和美伐他汀可通过抑制PI3K/Akt/Fox03a/Bim途径调节内皮细胞的凋亡。他汀类药物是否可通过抑制PI3K/Akt/Fox03a途径调节心肌细胞的凋亡、进而防治糖尿病性心肌病,目前国内外亦尚未见报道。本研究在分析糖尿病性心肌病细胞信号导途径的基础上,综合运用分子生物学、细胞生物学、组织病理学、超声心动图技术和计算机图像分析等方法,首次提出并探讨了PI3K/Akt/Fox03a信号传导途径在糖尿病性心肌病心肌细胞凋亡中的作用,进而应用HMG-CoA还原酶的抑制剂瑞舒伐他汀对糖尿病性心肌病模型动物进行了干预治疗,旨在探索糖尿病性心肌病药物防治的新靶点。目的（1）建立Wistar大鼠DCM动物模型;（2）观察DCM动物模型及瑞舒伐他汀干预组心肌组织病理学、超微结构和心脏形态结构的变化;（3）检测DCM动物模型及瑞舒伐他汀干预组心肌细胞的凋亡;（4）运用RT-PCR、Western-Blot、免疫组化等技术在大鼠DCM模型中验证PI3K/Akt/FoxO3a通路的存在,探讨该通路与心肌细胞凋亡的关系;（5）评价瑞舒伐他汀在防治DCM中的潜在作用。方法1.DCM动物模型的构建雄性Wistar大鼠（200～240g）46只,适应性喂养1周后,随机分为三组:正常对照组（n=10只）,DCM组（n=18只）,DCM+瑞舒伐他汀组（n=18只）。DCM组和DCM+瑞舒伐他汀组给予高糖、高热量饮食,4周后一次性腹腔注射链脲佐菌素（STZ,30mg/kg,溶解于10mM冷柠檬酸盐缓冲液中,PH值4.2）。正常对照组以标准大鼠饲料喂养,4周后腹腔注射同等剂量的柠檬酸盐缓冲液。注射STZ 72小时后取尾静脉血测血糖,糖尿病大鼠成模标准为:连续2次空腹血糖≥11.1mmol/L,未达成模标准者剔除。达成模标准者进入下一步实验,此后DCM+瑞舒伐他汀组大鼠每天以瑞舒伐他汀20mg/kg灌胃,DCM组和正常对照组大鼠以等量生理盐水灌胃,继续饲养20周。2.体重和血生化检查实验过程中,进行以下检测:（1）每隔1周称量体重1次,每隔2周检测空腹血糖1次。（2）分别于STZ注射前、STZ注射后1周、实验末抽血测定空腹血糖、空腹胰岛素、甘油三酯、胆固醇。3.超声心动图检测分别于糖尿病建模前、实验末进行常规超声心动图检查,测定如下指标:M型超声心动图测定左室收缩末期内径（LVIDs）、左室舒张末期内径（LVIDd）、射血分数（EF）、短轴缩短率（FS）;彩色多普勒超声心动图观察瓣膜返流情况;脉冲波及连续波多普勒超声心动图测定二尖瓣口E波最大速度、A波最大速度、E/A比值、E波减速时间（EDT）、等容舒张时间（IVRT）、主动脉血流最大速度（APV）。根据心动周期,计算校正的EDT′(EDT′=EDT/（心动周期）^1/2)及IVRT′(IVRT′=IVTR/（心动周期）^1/2)。4.透射电镜观察超微结构的改变5.心肌组织病理学检查处死实验动物后,进行组织取材、固定、脱水、透明、浸蜡、石蜡包埋、切片,常规HE染色和Masson三色染色,观察心肌细胞形态及胶原分布并拍片。6.采用TUNEL法检测心肌细胞凋亡染成棕色的细胞核即为凋亡阳性细胞。计数方法:在×400物镜下计算阳性细胞数占细胞总数的比例,选择细胞总数200以上的视野,每个标本观察5张切片,取其平均值,从中计算阳性细胞率,取其平均值即凋亡指数（apoptosis index,AI）。7.实时定量RT-PCR法检测从心肌组织提取总RNA,经逆转录反应（RT）得到cDNA,以管家基因β-actin作为参照,通过实时定量RT-PCR技术分别检测三组PI3K、Akt、FoxO3a各因子的mRNA的表达。8.免疫组织化学法检测取组织切片,免疫组织化学法分别检测三组的PI3K、Akt、FoxO3a的蛋白表达。9.免疫印迹检测取心肌组织,提取总蛋白,经过SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离、转膜、蛋白印记、DAB显色等步骤,分别检测三组的PI3K、Akt、FoxO3a的蛋白表达。结果1.实验动物基本情况实验过程中,正常对照组大鼠精神状态良好,体重增加明显,反应敏捷,毛色白而光泽。DCM组和DCM+瑞舒伐他汀组大鼠出现多饮、多尿、多食和消瘦等症状,体重增加迟缓,精神萎靡,皮毛无光泽,视力下降等。整个实验过程中3只大鼠死亡,其中DCM组2只,DCM+瑞舒伐他汀组1只,死亡原因可能与糖尿病酮症酸中毒、感染或其他相关并发症有关。DCM+瑞舒伐他汀组有1只大鼠血糖未达成模标准,予以剔除。最终共42只完成实验,其中正常对照组10只,DCM组16只,DCM+瑞舒伐他汀组16只。2.体重与生化指标高糖高热量饮食喂养4周后,与正常对照组相比,DCM组和DCM+瑞舒伐他汀组大鼠体重、空腹胰岛素、甘油三酯和胆固醇水平较高（P＜0.05～0.01）,空腹血糖无明显差异（P＞0.05）。STZ注射后1周,与正常对照组相比,DCM组和DCM+瑞舒伐他汀组大鼠体重、空腹胰岛素水平无差异（P＞0.05）,空腹血糖、甘油三酯和胆固醇水平明显升高（P＜0.01）。实验末,与正常对照组相比,DCM组和DCM+瑞舒伐他汀组大鼠体重显著下降（P＜0.01）;空腹胰岛素水平降低（P＜0.05）,空腹血糖、甘油三酯水平明显升高（P＜0.01）,DCM组胆固醇水平明显升高（P＜0.01）,DCM+瑞舒伐他汀组组胆固醇水平无差异（P＞0.05）。3.超声心动图检测实验初,三组大鼠超声心动图检测各项指标（包括LVIDs、LVIDd、EF、FS、瓣膜返流、E波最大速度、A波最大速度、E/A比值、EDT′、IVRT′及APV）无显著性差异（P＞0.05）。实验末,DCM组和DCM+瑞舒伐他汀组比正常对照组大鼠LVIDs及LVIDd明显增加（P＜0.01）,房室瓣瓣膜返流发生率明显增加（P＜0.05）,E波最大速度下降,A波最大速度增快,E/A比值下降,IVRT′延长,FS降低,APV降低（P＜0.05）,EF降低（P＜0.01）,EDT′无显著性差异（P＞0.05）。实验末,DCM+瑞舒伐他汀组比DCM组大鼠LVIDs及LVIDd减小（P＜0.05）,房室瓣瓣膜返流发生率减少（P＜0.05）,E波最大速度增快,A波最大速度下降,E/A比值升高,IVRT′缩短,FS升高,APV增快（P＜0.05）,EF升高（P＜0.05）,EDT′无显著性差异（P＞0.05）。4.透射电镜观察超微结构的改变正常对照组心肌组织细胞排列整齐,心肌细胞质膜连续、完整;粗、细肌丝排列整齐,细胞间质可见成纤维细胞和少量胶原纤维;微血管管腔大小正常,内皮细胞结构正常。DCM组心肌组织细胞排列紊乱,质膜模糊、断裂;肌原纤维呈灶性溶解,肌丝扭曲、断裂,肌节对位不齐;间质可见大量胶原纤维分布;微血管管腔狭窄,内皮细胞肿胀明显,呈柱状向管腔突起。DCM+瑞舒伐他汀组较DCM组心肌组织细胞排列较整齐,心肌细胞质膜较规则,间质胶原纤维堆积明显减少。5.组织病理观察HE染色:正常对照组心肌细胞排列整齐,细胞核大小均一,胞浆染色均匀;DCM组心肌细胞排列紊乱,细胞核大小不甚规则,毛细血管基底膜的增厚,间质纤维化,心肌细胞肥大、坏死;DCM+瑞舒伐他汀组较DCM组显著改善。Masson三色染色:心肌细胞染色呈红色,间质胶原呈蓝绿色,红细胞呈橘黄色。正常对照组心肌细胞排列整齐,细胞核大小均一,胞浆染色均匀,心肌胶原组织分布均匀;DCM组心肌细胞排列紊乱,细胞核大小不甚规则,心肌内胶原组织明显增多,粗大胶原纤维相互连接成网状,排列紊乱,分布不匀,紧密围绕于心肌细胞周围及小血管周围;DCM+瑞舒伐他汀组较DCM组心肌细胞排列较整齐,细胞核大小较均一,胞浆染色较均匀,胶原纤维明显减少。6.采用TUNEL法检测心肌细胞凋亡与正常对照组相比,DCM组和DCM+瑞舒伐他汀组心肌细胞凋亡指数（%）明显增加（P＜0.0001）。与DCM组比较,DCM+瑞舒伐他汀组的心肌细胞凋亡指数（%）明显降低（P＜0.01）。相关性分析显示DCM组空腹血糖与心肌细胞凋亡率呈明显的正相关（r=0.906,P＜0.0001）。7.RT—PCR检测PI3K、Akt、FoxO3a mRNA的表达水平与正常对照组相比,DCM组和DCM+瑞舒伐他汀组PI3K、Akt、FoxO3a mRNA表达水平明显升高（P＜0.05～0.01）。与DCM组相比,DCM+瑞舒伐他汀组PI3K、Akt、FoxO3a mRNA表达水平明显减低（P＜0.05）。相关性分析显示（1）DCM组空腹血糖与PI3K（r=0.396,P＜0.05）、Akt（r=0.534,P＜0.01）、FoxO3a（r=0.837,P＜0.001）mRNA表达水平呈明显的正相关。（2）DCM组心肌细胞凋亡率（%）与PI3K（r=0.48,P＜0.05）、Akt（r=0.593,P＜0.01）、FoxO3a（r=0.872,P＜0.001）mRNA表达水平呈明显的正相关。8.免疫组织化学染色分析PI3K、Akt、FoxO3a;染色阳性信号为棕色颗粒,主要定位于心肌细胞及内皮细胞。正常对照组心肌组织内可见少量、分布均匀、稀疏的浅棕色颗粒;DCM组心肌细胞胞浆内可见浓密的深棕色颗粒;DCM+瑞舒伐他汀组较DCM组明显稀疏,可见浅淡的棕色颗粒。9.各组大鼠PI3K/Akt/FoxO3a通路各关键分子蛋白质表达水平检测与正常对照组相比,DCM组和DCM+瑞舒伐他汀组PI3K、Akt、FoxO3a蛋白质表达水平明显升高（P＜0.05～0.01）。与DCM组相比,DCM+瑞舒伐他汀组PI3K、Akt、FoxO3a蛋白质表达水平明显减低（P＜0.05）。相关性分析显示（1）DCM组空腹血糖与PI3K（r=0.468,P＜0.01）、Akt（r=0.574,P＜0.01）、FoxO3a（r=0.731,P＜0.001）蛋白质表达水平呈明显的正相关。（2）DCM组心肌细胞凋亡率（%）与PI3K（r=0.571,P＜0.01）、Akt（r=0.645,P＜0.01）、FoxO3a（r=0.891,P＜0.001）蛋白质表达水平呈明显的正相关。结论1应用高热量饮食喂饲和小剂量STZ注射,成功建立了糖尿病性心肌病大鼠模型,为DCM发病机制的研究提供了可靠的平台;2心肌细胞凋亡,间质纤维化,是DCM的主要组织病理改变;3运用RT-PCR、Western-Blot、免疫组化等技术证实了在大鼠DCM模型中PI3K/Akt/FoxO3a通路的存在,并在心肌细胞的凋亡中起了重要作用;4瑞舒伐他汀可能通过影响PI3K/Akt/FoxO3a通路,抑制心肌细胞凋亡进而对DCM大鼠具有明显的保护效应。背景糖尿病性心肌病早期就可以出现心肌细胞的凋亡和坏死。这可能是由于高血糖时氧化应激增加,信号转导途径改变,引起异常基因表达,激活细胞程序化死亡。心肌细胞凋亡的信号转导机制可能是:①通过死亡受体转导通路介导心肌细胞凋亡;②丝裂原活化蛋白激酶（MAPK）转导通路;③磷脂酰肌醇3-激酶/Akt信号转导通路。FoxO在多种复杂疾病如:糖尿病、肿瘤中起重要的调节作用。FoxO被生长因子激活的PI3K通路激活,产生Akt依赖磷酸化作用,磷酸化的FoxO激活或抑制细胞凋亡或细胞分裂周期相关的基因如:Bim,P27^kip1,FasL,Bcl26,TRA IL,FLIP,MnSOD,GADD45（Za）,或者促进凋亡,或者抑制凋亡。目前Carsten Skurk等证实在人脐静脉内皮细胞可通过激活PI3K/Akt/FoxO3a/FLIP途径导致Caspase8增多而促进凋亡。论文一在大鼠DCM模型中证实PI3K/Akt/FoxO3a通路的存在。本研究以Wistar大鼠乳鼠心肌细胞为实验对象,以不同浓度高糖为处理因素,观察高糖对心肌细胞FoxO3a表达及细胞凋亡的影响,进一步探讨其作用的分子机制,为DCM的防治提供实验与理论依据。目的1.观察高糖对心肌细胞凋亡的影响;2.探讨PI3K/Akt/FoxO3a信号转导通路在高糖对心肌细胞凋亡中的作用;3.探索PI3K/Akt/FoxO3a信号转导通路及其下游分子作用可能机制。方法1.心肌细胞分离及培养无菌条件下取出Wistar（2～3天）乳鼠心脏,剪碎、胰酶消化、差速贴壁,获取心肌细胞,生长至亚融合状态,取原代细胞进行实验。2.实验设计在体外模拟糖尿病状态,D-葡萄糖处理组:不同浓度（5、15、30 mmol/L）D-葡萄糖处理24小时对心肌细胞凋亡的影响;甘露醇对照组:甘露醇浓度（30 mmol/L）处理24小时对心肌细胞凋亡的影响。3.心肌细胞凋亡的检测应用流式细胞仪检测心肌细胞的细胞周期的变化及细胞凋亡率,应用Annexin V细胞凋亡试剂盒检测凋亡的阳性细胞。4.实时定量RT-PCR检测将所收集的细胞提取总RNA,经逆转录反应得到cDNA,以管家基因β-actin作为参照,通过RT-PCR技术检测指标基因PI3K、Akt、Foxo3a、FLIP、Bim、Caspase8、Caspase9等因子的mRNA表达水平。5.Western-Blot检测将所收集的细胞提取总蛋白,经过SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离、转膜、蛋白印记、DAB显色等步骤,检测PI3K、Akt、Foxo3a、FLIP、Bim、Caspase8、Caspase9各指标蛋白质表达水平。结果1.心肌细胞凋亡的检测不同浓度（5、15、30 mmol/L）D-葡萄糖刺激心肌细胞24h,细胞凋亡率（%）分别为1.38±0.32,8.41±0.99,13.12±1.14,与5mmol/L浓度组比较,15mmol/L,30mmol/L浓度组细胞凋亡率明显升高（P＜0.001～43.0001）;30 mmol/L（甘露醇mannitol）刺激心肌细胞24h,细胞凋亡率（%）为1.69±0.35,与5mmol/L浓度组比较,30 mmol/L（甘露醇）组细胞凋亡率无明显变化（P＞0.05）。相关性分析显示D-葡萄糖浓度与心肌细胞凋亡率呈明显的正相关（r=0.871,P＜0.01）。2.实时定量RT—PCR检测PI3K、Akt、FoxO3a、FLIP、Bim、Caspase8Caspase9等因子的mRNA表达水平与5mmol/L浓度组相比,15mmol/L,30mmol/L浓度组PI3K、Akt、FoxO3a、Bim、Caspase8、Caspase9 mRNA表达水平明显升高（P＜0.05～0.01）;FLIPmRNA表达水平降低（P＜0.05）。相关性分析显示（1）D-葡萄糖浓度与PI3K（r=0.578,P＜0.01）、Akt（r=0.369,P＜0.05）、FoxO3a（r=0.776,P＜0.01）、Bim（r=0.417,P＜0.05）、Caspase8（r=0.663,P＜0.01）、Caspase9（r=0.453,P＜0.05）mRNA表达水平呈明显的正相关;与FLIP mRNA表达呈负相关（r=-0.483,P＜0.05）。（2）心肌细胞凋亡率与PI3K（r=0.749,P＜0.01）、Akt（r=0.423,P＜0.05）、FoxO3a（r=0.822,P＜0.01）、Bim（r=0.521,P＜0.05）、Caspase8（r=0.698,P＜0.01）、Caspase9（r=0.506,P＜0.05）mRNA表达水平呈明显的正相关;与FLIP mRNA表达呈负相关（r=-0.582,P＜0.05）。3.Western-Blot检测PI3K、Akt、FoxO3a、FLIP、Bim、Caspase8、Caspase9各指标蛋白质表达水平与5mmol/L浓度组相比,15mmol/L,30mmol/L浓度组PI3K、Akt、FoxO3a、Bim、Caspase8、Caspase9蛋白表达水平明显升高（P＜0.05～0.01）;FLIP蛋白质表达水平降低（P＜0.05）。相关性分析显示（1）D-葡萄糖浓度与PI3K（r=0.395,P＜0.05）、Akt（r=0.436,P＜0.05）、FoxO3a（r=0.804,P＜0.01）、Bim（r=0.542,P＜0.05）、Caspase8（r=0.658,P＜0.01）、Caspase9（r=0.507,P＜0.05）蛋白质表达水平呈明显的正相关;与FLIP蛋白质表达水平呈负相关（r=-0.696,P＜0.01）。（2）心肌细胞凋亡率与PI3K（r=0.484,P＜0.05）、Akt（r=0.531,P＜0.05）、FoxO3a（r=0.846,P＜0.01）、Bim（r=0.570,P＜0.05）、Caspase8（r=0.705,P＜0.01）、Caspase9（r=0.543,P＜0.05）蛋白质表达水平呈明显的正相关;与FLIP蛋白质表达水平呈负相关（r=-0.709,P＜0.01）。结论1.不同浓度的高糖对Wistar大鼠离体培养的心肌细胞均具有致凋亡的作用,且符合浓度—效应的一般对应规律;2.FoxO3a通过PI3K/Akt信号转导通路参与了Wistar大鼠离体培养的心肌细胞的凋亡;3.FoxO3a通过其下游的凋亡基因FLIP、Bim、Caspase8、Caspase9参与了心肌细胞的凋亡。更多还原

【Abstract】 BackgroundCardiovascular disease is the major reason for Diabetic Patient mortality.In western countries,about 70 percents of Diabetic Patients die of diabetic cardiovascular（DCM）disease.Since the first time of DCM was proposed by Rubler et at.in 1972,all epidemiological,pathological and laboratory research results implied the existence of Diabetic Cardiomyopathy lesion.Great correlation between Diabetic Cardiomyopathy lesion and the unique metabolic disorders of diabetes exists. Typical clinical symptoms includes defect of diastolic function in early stage,and systolic in late stage.Congestive heart failure（CHF）is prone.Recent basic and clinical researches done by scholars at home and abroad have made abundant progress.However,the pathogenesis,early diagnosis and treatment of DCM is still under active investigation.It has great value for the cure of DCM to study its pathogenesis in-depth and block its prevalence.DCM prevalence and development is affected by many factors,among which are disorder of metabolism of cardiocytes,micro-angiopathy and oxidative stress.It is shown that,the apoptosis and necrosis of myocardial cells may arise in early stage of DCM.Hyperglycemia could result in oxidative stress increases and signal transduction changes,which in turn leads to abnormal gene expression and activates programmed cell death.The potential mechanism of the apoptosis of cardiocytes and signal pathways is:1.myocardial apoptosis mediated by death receptor pathway;2. Mitogen-Activated Protein Kinase（MAPK）transduction pathway and 3. Phosphatidylinositol-3 kinase/Akt signal transduetion pathway.Pathological manifestations of DCM includes hypertrophic cardiomyopathy and myocardial fibrosis etc.Cardiac fibroblasts proliferation and the changes of the type and quantity of collagen synthesis and secretion,i.e.myocardial interstitial reconstruction, myocardial apoptosis and myocardial fibrosis play important roles in DCM development.Forkhead box O transcription factors（FoxO）protein family,which has just been formally denominated in 2000,consists of 17 subfamilies that are named Fox A to Q. Among them,the FoxO subfamily plays an important part in the development, differentiation,metabolism,apoptosis,and immunity process by signal transduction pathway.According to the homology of amino acid sequence excluding the DNA binding domain,the vertebrate FoxO subunit can be further divided into three subgroups-FoxO1,FoxO3 and FoxO4.FoxO is activated in the PI3K pathway by growth factor.Phosphorylagted FoxO activates or inhibits the genes related to the apoptosis or the cell division cycle,such as Bim,P27kip1,FasL,Bc126,TRA IL,FLIP, MnSOD,GADD45（Za）etc.It has been found that FoxO may activate different genes in different cell types. Its cell type specificity,apoptosis or not,or only inducing apoptosis in stress condition may depend on suitable cofactors.The expression of apoptosis genes induced by FoxO is different in various studies,leading to different effect.Still,the apoptosis mechanism induced by FoxO has been far from clear.Literatures suggest that FoxO transcription factor can regulate the pathology and physiological changes of cell and ECM.Some scholars from abroad attest to that FoxO3a can regulate the apoptosis of endothelial cells through PI3K/Akt/ FoxO3a /Bim pathway.There is no report about the effect of FoxO3a on apoptosis of cardiocytes yet.Now,medication for diabetes includes hypoglycemic agents,can slow down the development of the disease but not decrease the process of cardiovascular events of diabetes patients.It has great value for the cure of DCM to study its pathogenesis in-depth and block its prevalence,to open up new drug intervention mechanism in pathogenesis of DCM.Statins is the inhibitor of the rate-limiting enzyme for the synthesis of cellular endogenous cholesterin:3-hydroxy-3-methylglutaryl-coenzymeA, HMG-CoA reductase,which can effectively inhibit the synthesis of cholesterin,is widely used clinically to cure hyperlipemia.These years,research reported that statins can inhibit apoptosis of endothelial cells,but the mechanism is not completely clear. Carmen Urbich et al proved that atorvastatin and mevastatin can inhibit the PI3K/Akt/ FoxO3a/Bim pathways to regulate the apoptosis of endothelial cells.Can they treat DCM by inhibiting PI3K/Akt/FoxO3a pathways and regulating the apoptosis of cardiac muscle cells?Based on systematic analysis of signal transduction pathways in DCM,we combined molecular biology,cell biology,histopathology,Echocardiography and computer image analysis,explored the effect of glucose/PI3K/Akt/FoxO3a signal transduction pathway in initiation and development of DCM.Using Resovastatin,an inhibitor of the rate limiting enzyme 3-hydroxy-3-methylglutaryl-coenzymeA （HMG-CoA）in endogenous synthesis,we treat DCM animal models to explore new target of DCM therapy.Objectives（1）To establish an animal model of DCM;（2）To observe the changes of histopathology and ultra structure in DCM model;（3）To discuss cardiac morphology reconstruction of DCM animal model and observing the role of crestor on the intervention of DCM with application of Echocardiography Detection Technology;（4）To confirm the existence of P13K/Akt/FoxO3a pathway in the rat DCM model through technology of RT-PCR、Western-Blot、Immunohistochemistry etc;（5）To evaluate the role of Resovastatin in DCM prevention.Materials and Methods 1.Construction of DCM animal modelMale Wistar rats,（200-400g）forty-six,after adaptive feeding one week,which were randomly divided into three groups:normal control（n=10）,DCM（n=18）and DCM plus crestor group（n=18）.For DCM and DCM plus crestor group,high-glucose and high-calorie diet was given.Four weeks later,giving a single intraperitoneal injection with Streptozotocin（STZ,30mg/kg,dissolve in 10mM cold citrate buffer, PH4.2）.Normal control group were given standard rat feed,four weeks later,inject the same dose of citrate buffer to Intraperitoneal.Injecting STZ and measuring blood glucose using tail vein blood after 72 hours,Die-diabetic rat’s standards:two consecutive fasting blood glucose≥11.1mmol/L.Those haven’ treach those standards Die were removed.Those reach the standard were used to next procedure,and then, the DCM plus resovastatin group was fill stomach with Resovastatin（20mg/kg）.The other two groups were given a gavage with Physiological saline,feeding another 20 weeks.2.Body weight and biochemical indicesFollowing observations on animals were performed during the whole experiments:（1）body weight（BW）was documented every week,and FBG was detected every two weeks.（2）FBG,fasting insulin,triglyceride（TG）and cholesterol（Chol）were analyzed in each of the three group before STZ injection,one week after STZ injection and at the end of the experiment.3.Echocardiogram examinationAt the beginning and at the end of the study,transthoracic echocardiogram was performed in diabetic and control animals.Rats were placed supine and the anterior chest wall was shaved.Echocardiograms were performed with a Hewlett-Packard Sonos 7500 sector equipped with a 7.5-MHz phased-array transducer.Conventional images induced 2-dimensional,M-mode,and continuous wave and pulsed Doppler images.4.Histopathologic examination of myocardialAfter the animals were killed,draw the material from tissue then fix,dehydrate, transparent,wax dip,paraffin-embedded,slice,conventional HE stain and Masson stain,observe cardiac morphology and collagen distribution then take photos.5.Detect cardiocyte apoptosis index（AI）by TUNELAmber-coloured caryon is apoptosis masculine Cell.count method:calculate masculine cells and total cellular score under×400 object glass,select campus visuals which total cellular score above 200,observe five blades each type,take mean value,calculate apoptosis index（AI）.6.Real-time PCRExtract total RNA from myocardial,from which we could get cDNA through reverse transcription PCR.Taking house-keeping geneβ-actin as a reference and detect expression of the three factors’ mRNA（P13K,Akt and FoxO3a）in each of the three group,which were carried out through quantitative real time RT-PCR technology.7.ImmunohistochemistryTake tissue slice and detect protein expression of P 13K,Akt and FoxO3a through immunohistochemistry.8.Western-Blot analysisTake myocardial and extract total protein.Detect ptotein expression of P13K, Akt and FoxO3a through the following procedures:SDS-PAGE segregate, membrane-trans,protein marking,DAB color development etc.Results1.General features of the experimental ratsAmong experiments,the rats in normal control group have high spirit,body weight added noticeably,react cute and have a colorful white fur.The rats in group of DCM and DCM plus Resovastatin emerge symptom of more drink,polyuria,more food and wasting.Aside from this,the slowly added body weight,spirit languish,matt fur and decreased visual acuity are also found.Three rats died in the whole experiment.Two belong to the group of DCM and the left one was from DCM plus Resovastatin group.The reason of death may relate to diabetic ketoacidosis,infection or other complications.DCM plus Resovastatin group removed a rat that hasn’t reached the standard.A total of 42 completed this study,10 of them in normal control group,16 in DCM group and the rest from DCM plus Resovastatin group.2.Body weight and biochemical indicesGive four weeks’ high glucoser and high calorie diet and then compare with control group.It is found that the DCM plus Resovastatin group had a higher level in aspect of body weight,insulin of limosis,triglyceride and cholesterol than other two groups（P＜0.05-0.01）.There was no remarkable difference in terms of fasting blood glucose（P＞0.05）.One week after STZ injection,compared with normal control group,DCM group and DCM plus Resovastatin group had no difference on fasting insulin level,but had a increasing on levels of fasting blood glucose,triglyceride and cholesterol（P＜0.01）.At end,compared with normal control group,the body weight of other two groups declined noticeably（P＜0.01）and fasting insulin level declined too（P＜0.05）.The level of fasting blood glucose,triglyceride and increased（P＜0.01）,The level of cholesterol of DCM group increased（P＜0.01）,but there was no remarkable difference in terms of cholesterol of DCM plus Resovastatin group（P＞0.05）.3.Eehoeardiogram examinationAt the beginning,the Echocardiogram examination index（including LVIDs, LVIDd,EF,FS,valvular regurgitation,Max E wave speed,Max A wave speed,ratio of E/A,EDT’,IVRT’ and APV）of the three groups had no uncommon difference （P＞0.05）.At the end of experiment,compared with normal control group,the DCM group and DCM plus Resovastatin group had uncommon increase on value of LVIDs and LVIDd（P＜0.01）,as the incidence of valvular regurgitation（P＜0.05）.The Max E wave speed declined,Max A wave speed increased,E/A ratio declined,IVRT’ prolonged,FS declined and AVP declined too（P＜0.05）.EF declined（P＜0.01）,But there had no obvious difference on value of EDT’（P＞0.05）.Experimental end,compared with DCM group,the group of DCM plus Resovastatin declined on value of LVIDs and LVIDd（P＜0.05）,the incidence of valvular regurgitation declined（P＜0.05）,Max E wave speed declined,Max A wave speed increased,E/A ratio declined,IVRT’ prolonged,FS declined and AVP declined too（P＜0.05）.EF increased.But there had no obvious difference on value of EDT’.4.Ultrastructural changes observed by transmission electron microscopyThe cardiocytes from the normal control group arranged regularly,a little fibroblast and collagenous fibers distributed in extra-cellular matrix.The cardiocytes from the DCM group arranged irregularly.The pericellular membrane was interrupted and unclear.The local myofibrillar was disintegrated.The myofilament was distorted and interrupted,a lot of collagenous fibers distributed in extra-cellular matrix.The microvessel lumen was narrow.Compared with the DCM group,the cardiocytes in DCM plus Resovastatin were more neatly arranged,the quality of inter fiber piled up noticeably decreaseed.5.pathological observationHE staining:In normal control group,cardiocytes arranged neatly,an uniform nuclear and cytoplasmic staining;in DCM group,cardiocytes arranged disorderly, irregular nuclei size,capillary basement membrane thickening,interstitial fibring, myocardial cells overgrowth and mortify;the DCM plus Resovastatin group had a signifant improvement.Masson staining:Cardiocytes arranged neatly,the collagen tissue was appropriate arranged among cardiocytes in normal control group;however,the cardiocytes arranged irregularly,collagen tissue increased markedly,and disrupted in some area in DCM group;the DCM plus Resovastatin group had a signifant improvement.6.Detect cardiocytes apoptosis index by TUNELCompared with normal control group,the DCM group and DCM plus Resovastatin group had uncommon increase on cardiocytes apoptosis rate（P＜0.0001）.Compared with DCM group,the group of DCM plus Resovastatin had obvious declined on cardiocytes apoptosis rate（P＜0.01）.Correlative analysis showed that In DCM group,fasting blood glucose had positive correlation with cardiocytes apoptosis index（r=0.906,P＜0.0001）.7.RT-PCRCompared with normal control group,the expression levels of P13K,Akt and FoxO3a increased evidently in DCM group and DCM plus Resovastatin group respectively（P＜0.05-0.01）.Compared with the DCM group,mRNA expression levels of P13K、Akt、FoxO3a in DCM plus Resovastatin group reduced obviously respectively（P＜0.05）.Correlative analysis showed thatIn DCM group,mRNA expression levels ofPI3K（r=0.396,P＜0.05）、Akt（r= 0.534,P＜0.01）、FoxO3a（r=0.837,P＜0.001）had positive correlation with fasting blood glucose respectively.In DCM group,mRNA expression levels ofPI3K（r=0.48,P＜0.05）、Akt（r= 0.593,P＜0.01）、FoxO3a（r=0.872,P＜0.001）had positive correlation with cardiocytes apoptosis rate respectively.8.ImmunohistoehemistryP13K、Akt、FoxO3a:brown granules showed the positive signal of coloration,which mainly located at cardiocytes and the Endothelial cells.In normal control group,a small quantity of light brown granules exsited in the tissue of cardiac muscle which distributed equably,sparsely.In DCM group dark brown granules exsited in the cytoplast of cardiocytes which distributed densely.Compared with the DCM group,light brown granules exsited in the DCM plus Resovastatin group which distributed much sparsely.9.Western-Blot analysisCompared with the normal control group,protein expression levels of PI3K、Akt、FoxO3a in DCM group and DCM plus Resovastatin group increased obviously respectively（P＜0.05-0.01）.Compared with the DCM group,protein expression levesl of PI3K、Akt、FoxO3a in DCM plus Resovastatin group decline clearly respectively（P＜0.05）.Correlative analysis showed that Protein expression levels ofPI3K（r=0.468,P＜0.01）、Akt（r=0.574,P＜0.01）、FoxO3a（r=0.731,P＜0.001）had obviously positive correlation with fasting blood glucose in DCM group respectively.Protein expression levels of PI3K（r=0.571,P＜0.01）、Akt（r=0.645,P＜0.01）、FoxO3a（r=0.891,P＜0.001）had obviously positive correlation with cardiocytes apoptosis rate in DCM group respectively.Conclusions1.An animal model of DCM with specific metabolic characteristics was established by high-calorie diet and small dose STZ injection.This model is valuable for the study of the mechanism of DCM;2.Cardiocytes apoptosis and interfibrosis are the main tissue pathology changes in DCM;3.The existence of PI3K/Akt/FoxO3a access in rat DCM model was confirmed with the technique of RT-PCR,Western-Blot、Immunohistochemistry,etc.This pathway may play important role in cardiocytes apoptosis;4.Resovastatin can treat DCM by inhibiting PI3K/Akt/FoxO3a pathway and regulating the apoptosis of cardiocyte cells. IntroductionEarly phase of diabetic cardiomyopathy will present apoptosis and necrosis of cardiocyte.The reasons may be when high blood glucose appears,oxidative stress increses,signal transduction pathway changes,which induce abnormal gene express, active cell programmed death.The signal transduction mechanism of cardiocyte apoptosis may be:①death receptor transduction pathway induced cardiocyte apoptosis;②mitogen-activated protein kinases（MAPK）transduction pathway; phosphatidylinositol-3kinase/Akt signal transduction pathway.FoxO play an important regulation role in many complicated diseases such as diabetes and cancers.FoxO is activated by growth factor activated PI3K pathway, which induce Akt dependent phosphorylation.Phosphorylated FoxO activates or inhibits cell apoptosis or cell division cycle related genes,such as Bim,P27kipl, FasL,Bcl26,TRAIL,FLIP,MnSOD,GADD45（Za）,then activates or inhibits cell apoptosis.At present Carsten Skurk etc have proved Caspase8 increase by activation of PI3K/Akt/FoxO3a/FLIP pathway in human umbilical veins endothelial cells so as to promote apoptosis.The rat DCM model in the paper one has proved the present of PI3K/Akt/FoxO3a pathway.This research treats Wistar rat suckling mouse cardiocytes as experiment object, different concentration D-glucose as process factors,observes the influence of different concentration D-glucose on cardiocytes FoxO3a expression and cell apoptosis,fatherly discusses the molecular mechanism of the effect.Purpose:1.To observe the influence of high glucose on cardiocytes apoptosis.2.To explore the role of PI3K/Akt/FoxO3a signal transduction pathways on cardiocytes apoptosis when they were treated with high glucose.3.To Make sure the possible downstream molecular mechanism of PI3K/Akt/FoxO3a signal transduction pathways.Methods1.Cell cultureNeonatal rat cardiac fibroblasts were prepared by the following procedures:three to four hearts from 2- 3-day-old Wistar rats were finely minced and placed together in 0.25%trypsin.Pooled cell suspensions were centrifuged and resuspended in Dulbecco’s modified Eagle’s medium（DMEM）supplemented with 10%fetal bovine serum,100 U/ml penicillin and 100μg/ml streptomycin.The resuspension was plated onto culture flasks for 90 min,which allowed for attachment of cardiocyte to the bottom of the culture flask.Non-adherent and weakly attached cells were removed and the medium was changed.Cell cultures were incubated at 37°C in a humidified atmosphere of 5%CO₂/95%air.Studies were conducted on original passage cardiocyte that were grown to subconfluence in serum-containing media and then growth arrested for 24h in serum-free medium before treatment.2.Study designThe simulation of the diabetes mellitus in vitro,group handed with D-glucose and mannitol:To observe the effect on the myocardial cells’ apoptosis of treatment by different concentration D-glucose（5、15、30 mmol/L）and mannitol（30 mmol/L） 24 hours. 3.Detection of the cardiocytes’ apoptosis indexThe change of the cell cycle and the rate of the apoptosis were detected by flow cytometer,the positive cells of the apoptosis was detected by the apoptosis kit.4.Real-time RT-PCRcDNA was achieved from the complete RNA which was abstracted from the selected cells by the reverse transcription,the mRNA expression of the marker genes PI3K,Akt,Foxo3a,FLIP,Bim,Caspase8 and Caspase9 were detected by the real-time RT-PCR with the reference-β-actin,a housekeeping gene.5.Western-Blot analysisThe sum protein collected from the harvested cells was treated with isolation, trarsmembrane,protein imprinting,DAB coloration and other steps of SDS-PAGE,in order to detect the expressing level of marker proteins such as PI3K、Akt、Foxo3a、FLIP、Bim、Caspase8、Caspase9.Results1.Detection of the apoptosis index of cardiomytesCardiocytes were stimulated with different concentration D-glucose（5、15、30 mmol/L）and mannitol（30 mmol/L）24 hours,the apoptosis index are 1.38±0.32,8.41±0.99,13.12±1.14,1.69±0.35 respectively,compared with the group treated by 5mmol/L D-glucose,the apoptosis index of the groups treated by 15、30mmol/L D-glucose increase obviously（P＜0.001-0.0001）;the apoptosis index of the groups treated by mannitol（30 mmol/L）had no uncommon difference（P＞0.05）.Correlative analysis showed that:The cardiocytes’ apoptosis index had a positive correlation with the concentration of D-glucose（r=0.871,P＜0.01）.2.Detection the mRNA expressions levels of PI3K、Akt、FoxO3a、FLIP、Bim、Caspase8、Caspase9Compared with the 5mmol/L concentration D-glucose group,the mRNA expression levels of those factors PI3K、Akt、FoxO3a、Bim、Caspase8、Caspase9 in 15mmol/L, 30mmol/L concentration groups increased significantly（P＜0.05-0.01）;the mRNA expression level of FLIP decreased（P＜0.05）.Correlative analysis showed that:（1）The concentration of D-glucose had obvious positive correlation with the mRNA expression levels of PI3K（r=0.578,P＜0.01）、Akt（r=0.369,P＜0.05）、Fox03a（r=0.776,P＜0.01）、Bim（r=0.417,P＜0.05）、Caspase8（r=0.663, P＜0.01）、Caspase9（r=0.453,P＜0.05）respectively;had negative correlation withthe mRNA expression levels of FLIP（r=-0.483,P＜0.05）（2）The cardiocytes’ apoptosis index had obvious positive correlation with the mRNA expression levels ofPI3K（r=0.749,P＜0.01）,Akt（r=0.423,P＜0.05）、FoxO3a（r=0.822,P＜0.01）、Bim（r=0.521,P＜0.05）、Caspase8（r=0.698, P＜0.01）、Caspase9（r=0.506,P＜0.05）respectively;had negative correlation with the mRNA expression levels of FLIP（r=-0.582,P＜0.05）.3.Detection of the protein expressions levels of PI3K、Akt、FoxO3a、FLIP、Bim、Caspase8、Caspase9 by Western-Blot.Compared with the 5 mmol/L concentration D-glucose group,the protein expression levels of PI3K、Akt、FoxO3a、Bim、Caspase8、Caspase9 in the 15mmol/L,30mmol/L concentration groups increased significantly（P＜0.05-0.01）; the protein expression level of.FLIP decreased（P＜0.05）.Correlative analysis showed that:（1）The concentration of D-glucose had obvious positive correlation with the protein expression levels of PI3K（r=0.395,P＜0.05）、Akt（r=0.436,P＜0.05）、FoxO3a（r=0.804,P＜0.01）、Bim（r=0.542,P＜0.05）、Caspase8（r=0.658, P＜0.01）、Caspase9（r=0.507,P＜0.05）respectively;had negative correlation with the protein expression level of FLIP（r=-0.696,P＜0.01）.（2）The cardiocytes’ apoptosis index had obvious positive correlation with the protein expression levels of PI3K（r=0.484,P＜0.05）、Akt（r=0.531,P＜0.05）、FoxO3a（r=0.846,P＜0.01）、Bim（r=0.570,P＜0.05）、Caspase8（r=0.705, P＜0.01）、Caspase9（r=0.543,P＜0.05）respectively;had negative correlation with the protein expression level of FLIP（r=-0.709,P＜0.01）.Conclusions1.High D-glucose concentration can result in the apoptosis of cardiocytes from neonate Wistar rat cultured in vitro,and according to the common rule of density-effect;2.FoxO3a engages in the apoptosis of cardiocytes by PI3K/Akt signal transduction pathway;3.FoxO3a engages in the apoptosis of cardiocytes by the downstream apoptosis genes: FLIP、Bim、Caspase8、Caspase9.更多还原