【作者】 丁文渊；

【导师】 张薇；

【作者基本信息】 山东大学 ， 内科学， 2014， 博士

【摘要】 背景糖尿病心肌病(Diabetic Cardiomyopathy, DCM)是导致糖尿病病人罹难率升高的重要原因,迄今为止依然是人类心脏疾病中危险性最大的疾病之一。其作为一种独立并且特异性的心肌病变受到越来越多的关注。DCM的病理变化复杂多端,涉及到许多领域,其中主要涉及到心肌细胞和心肌间质两个领域。对于DCM的研究是一个逐渐演变的过程,在过去几十年的的研究中,研究领域重点主要放在心肌细胞的变化上,比如心肌细胞的肥大、凋亡和坏死；最近几年来,越来越多的学者把研究的重点转移到以前涉足较少的领域上来-心肌间质上来。许多研究发现,心肌间质领域--心肌间质纤维化在DCM的发生和发展中起着举足轻重的作用。研究心肌间质纤维化的病理生理过程以及影响机制或许成为攻克糖尿病心肌病的一个新的研究靶点。心肌间质纤维化指的是心肌组织成纤维细胞(cardiac fibroblasts)过度增生从而导致细胞外基质(extracellular matrix, ECM)大量沉积而形成的病理演变。心脏正常的成年人处在健康平衡生理状态下,心肌成纤维细胞细胞分泌和产生的胶原,是一个动态平衡过程。ECM主要成分包括胶原Ⅰ和胶原Ⅲ。生理学角度上来说,ECM的作用是作为“脚手架蛋白”从而把心肌、成纤维细胞、内皮细胞和血管联系起来的桥梁,促使形成网状结构进而使心肌产生抗张强度,这也就使得心肌产生了收缩和舒张功能。当处于病理状态下,比如氧化应激,高糖状态下,由于成纤维细胞受到外界的不适刺激,产生和分泌的胶原打破了动态平衡,导致ECM的过量累积。ECM的过量沉积会导致心脏壁松弛性的降低和僵硬度的升高,这将首先影响到心肌的舒张功能,进一步将会出现类似于限制性心肌病的改变。在病理情况下,过度累计的ECM在临床上的主要表现为：早期发生舒张功能的障碍,晚期发生收缩功能的障碍。详细说来,在心脏中,包括心肌细胞和非心肌细胞。非心肌细胞主要是心肌成纤维细胞,约占细胞总数的60%-70%。心肌成纤维细胞产生和分泌的Ⅰ型、Ⅲ型胶原是最重要的细胞外基质。现已证实,给予心肌成纤维细胞高糖刺激可以引起胶原Ⅰ/Ⅲ分泌增加。但是,在体外模拟糖尿病状态下,引起心肌成纤维细胞胶原沉积的分子机制仍所知甚少。所以研究心肌成纤维细胞分泌胶原的分子机制是迫在眉睫的问题。胰岛素抵抗(insulin resistance,IR)在DCM的发生和发展中起着重要作用,许多研究发现,胰岛素抵抗所引起的高糖血症和高胰岛素血症与纤维化密切相关。胰岛素和其受体胰岛素受体结合后的信号传导通路包括两条：其中一条是胰岛素底物受体(Insulin Receptor Subtract-IRS/phosphatidylinositol3-kinase-P13k/protein kinase B(Akt/PKB)信号通路,主要是影响糖脂代谢和蛋白质合成,这一信号通路称为代谢信号通路；另外一条信号通路是(mitogen-activated protein kinase-MAPK(促分裂原活化蛋白激酶)信号通路途径,主要参与对基因的转录过程和细胞的增殖过程的调控,称为生长信号通路。在胰岛素抵抗时,IRS/P13K/Akt信号通路途径出现明显的受损[11],而MAPK途径功能仍旧保持其正常[12],或许出现功能增强,这就是我们所谓的“选择性”胰岛素抵抗。MAPK活性的增强可以促进很多生长因子发挥作用。最为经典的是刺激转化生长因子(TGF-β)的表达[13]。TGF-β信号分子是典型的致纤维化因子[14]。但是国内学者对于TGF-β的大量研究发现,对于其的干预,甚至基因敲除,只能减轻不能完全遏制心肌纤维化的进程[15,16]。这提示着在致心肌纤维化中还存在其他信号通路。Oga等证实,在肺问质纤维化形成过程中,PGF2α-FP通路是除了TGF-β外另一重要的致纤维化的途径,它独立于TGF-β经典通路而直接作用于肺成纤维细胞促进其增殖和胶原的合成,导致肺间质纤维化[17,18]。而PGF2α-FP通路对于心肌成纤维细胞的研究尚未有人涉足。在DCM胰岛素抵抗状态下,PGF2α/FP受体信号通路是否参与了心肌成纤维细胞胶原的合成研究甚少。PGF2α是花生四烯酸在环氧化酶作用下的代谢产物。近几年来发现,在胰岛素抵抗病人的尿液中,或肥胖病人的体内,PGF2α的水平是明显增加的。所以在对于糖尿病胰岛素抵抗状态下一些病理因子和炎症因子的研究中,PGF2α的地位受到越来越多人的关注,日趋重要起来。最近,在对PGF2α的研究中发现,当PGF2α存在时,可以与其特异受体FP受体结合,两者结合后可以引起一系列生理活性反应。不仅激活PKC、 Rho激酶信号通路,同时还抑制PI3K的活性。PGF2α与PI3K竞争性结合FP受体。PI3K位于细胞内,所以当PI3K活性受到抑制时,与其结合的包内FP受体量就会减少,使FP受体内化(internalization)减少,细胞膜表面上的FP受体量明显增多,从而可以结合更多的PGF2α[19-23],形成正反馈循环。在胰岛素抵抗状态下,上述正反馈是否依然存在?PGF2α-FP受体/PKC/Rho激酶信号通路能否诱导大鼠成纤维细胞分泌胶原的增加,目前尚未有人研究。签于此,本实验设定这样的假说前提：在正常糖和胰岛素抵抗状态下,PGF2α-FP受体/PKC/Rho激酶信号通路促进心肌成纤维细胞胶原的合成。为了验证这一假说,我们进行了下列实验：1.在正常糖和胰岛素抵抗状态下,PGF2a刺激对大鼠成纤维细胞的胶原Ⅰ、胶原Ⅲ、KPGF2a-FP受体相应的mRNA和蛋白质表达的影响；2.在正常糖和胰岛素抵抗状态下,PGF2α对PI3K/Akt、FP受体/PKC/Rho激酶信号通路的检测；3.在正常糖和胰岛素抵抗状态下,应用分子阻断剂技术,检测PGF2a/FP受体/PKC/Rho激酶信号通路对心肌成纤维细胞合成胶原功能的影响中的作用；4.PGF2α-FP受体信号通路独立于TGF-β1信号通路的检测。本研究的主要目的在于,在正常糖条件和胰岛素抵抗条件下,PGF2α对心肌成纤维细胞胶原合成功能的影响及可能的信号介导机制。研究目的1.在正常糖和胰岛素抵抗状态下,PGF2α对心肌成纤维细胞FP受体、Ⅰ型和Ⅲ型胶原mRNA和蛋白质表达的影响；2.在正常糖和胰岛素抵抗状态下,PGF2a对PI3K/Akt、 FP受体/PKC/Rho激酶信号通路的检测；3.在正常糖和胰岛素抵抗状态下,应用分子阻断剂技术,检测PGF2a/FP受体/PKC/Rho激酶信号通路对心肌成纤维细胞合成胶原功能的影响中的作用；4.PGF2a-FP受体信号通路独立于TGF-β1信号通路的检测研究方法本研究以原代培养的新生乳鼠心肌成纤维细胞为对象。采用实时定量逆转录聚合酶链反应(real time RT-PCR),蛋白免疫印迹(western blotting)、酶联免疫吸附(ELISA)等实验方法,分别观察：1.在体外模拟胰岛素抵抗状态,分为正常糖组(5.5mmol/L)、正常糖+PGF2α(5.5mmol/L+PGF2α)、胰岛素抵抗模型组+PGF2α (15mmol/L+104μU/ml+PGF2a),检测FP受体mRNA以及Ⅰ型和Ⅲ型胶原mRNA和蛋白含量；2.在正常糖组、胰岛素抵抗模型组,加入PGF2α刺激0min,30min、120min,观察P13K/Akt、FP受体/PKC/Rho激酶信号通路的变化；3.在正常糖组、胰岛素抵抗模型组,加入FP受体,PKC, Rho激酶抑制剂后,观察对PGF2α刺激Ⅰ型和Ⅲ型胶原ⅠnRNA和蛋白表达的影响；4.在正常糖组、胰岛素抵抗模型组,加入FP受体,TGF-β1抑制剂后,观察对PGF2。或者TGF-β1刺激Ⅰ型和Ⅲ型胶原mRNA和蛋白表达的影响。结果1.在正常糖和胰岛素抵抗模型状态下加入PGF2α刺激lh:PGF2a刺激下,与正常糖对照组相比,模型+PGF2。组和正常糖+PGF2。组心肌成纤维细胞FP受体mRNA的表达均显著增高(15.0±0.02vs.1.00+0.00；1.8±0.50vs.1.00±0.00,P<0.05),且模型+PGF2α组高于正常糖+PGF2α组；模型+PGF2α组和正常糖+PGF2α组心肌成纤维细胞FP受体蛋白的表达也显著增高(1.13±0.16vs.0.2±0.03；0.69+0.08vs.0.29+0.03,P<0.05)；刺激4h：与正常糖对照组相比,模型+PGF2α组和正常糖+PGF2α组心肌成纤维细胞FP受体mRNA的表达均显著增高(9.36±0.74vs.1.00±0.00；1.28±0.05vs.1.00+0.00,P<0.05),且模型+PGF2a组高于正常糖+PGF2α组；模型+PGF2α组和正常糖+PGF2α组心肌成纤维细胞FP受体蛋白的表达也显著增高(0.56±0.03vs.0.39±0.01；0.47±0.02vs.0.3±0.01,P<0.05)。模型+PGF2α组和正常糖+PGF2α组心肌成纤维细胞胶原Ⅰ/Ⅲ的表达也显著增高(胶原Ⅰ：18.97±0.87vs.1.00±0.00,3.48±0.70vs.1.00±0.00；胶原Ⅲ：3.72±0.21vs.1.00±0.00,1.62±0.14vs.1.00±0.00)；刺激24h：(胶原Ⅰ：29.46±3.00vs.6.81+0.47,17.95+0.79vs.6.81±0.47；胶原Ⅲ：5.10±±0.10vs.1.23±0.027,3.20±±0.033vs.1.23±0.027)。2.在正常糖条件下和胰岛素抵抗状态下,PGF2a不仅使心肌成纤维细胞P13K、AKT磷酸化水平均显著下降；还使FP受体、PKC、 Rho激酶活化程度均显著增加。3.正常糖条件下,与PGF2α组相比,FP受体抑制剂AL-8810、 PKC抑制剂LY-333531、Rho激酶抑制剂Y-27632可明显抑制PGF2α引起的胶原I、 ⅢmRNA和蛋白水平的增加(P<0.05),胶原ⅠmRNA下降了47.59%、49.11%、49.00%,胶原ⅢmRNA下降了21.5%、27.25%、21.76%；胶原Ⅰ蛋白下降了14.83%、19.31%、14.11%,胶原Ⅲ蛋白下降了9.5%、13.45%、17.89%。胰岛素抵抗状态下,与PGF2a组相比,AL-8810、LY-333531、Y-27632可明显抑制PGF2。引起的胶原Ⅰ、Ⅲ mRNA和蛋白水平的增加(P<0.01-0.001),胶原Ⅰ mRNA下降了80.48%、82.03%、83.50%,胶原Ⅲ mRNA下降了26.49%、29.60%、36.08%,胶原Ⅰ蛋白下降了41.62%、35.07%、41.71%,胶原Ⅲ蛋白下降了22.33%、18.13%、18.20%。4.在正常糖条件下和胰岛素抵抗模型条件下,AL-8810、AL-8810+SB-505124可明显抑制PGF2a引起的胶原I、ⅢmRNA水平的增加(P<0.05),但是SB-505124对PGF2a引起的胶原I、 III mRNA水平的增加无明显抑制作用(P>0.05)；增加SB-505124的浓度至l0μmol/,仍然对PGF2α引起的胶原I、Ⅲ mRNA水平的增加无明显抑制作用；同样SB-505124、AL-8810+SB-505124可明显抑制TGF-β1引起的胶原I、ⅢmRNA水平的增加(P<0.05),但是AL-8810对TGF-β1引起的胶原I、ⅢmRNA水平的增加无明显抑制作用(P>0.05)。结论1.胰岛素抵抗状态下PGF2α可显著增高心肌成纤维细胞FP受体和胶原Ⅰ、Ⅲ的表达水平2.在正常糖条件下和胰岛素抵抗状态下,PGF2α不仅使FP受体/PKC/Rho激酶信号传导通路激活还钝化PI3K/Akt信号通路。3.在正常糖条件和胰岛素抵抗状态下,PGF2。可通过FP受体/PKC/Rho激酶信号通路刺激大鼠心肌成纤维细胞胶原I、ⅢmRNA和蛋白质的表达。4.正常糖和胰岛素抵抗状态下,PGF2α刺激胶原表达Ⅰ、Ⅲ的增加均是通过FP受体信号通路实现的,其作用独立于TGF-β1信号通路。背景糖尿病心肌病是由糖尿病引起的心肌的结构和功能的改变的代谢紊乱性疾病。现有的流行病学和临床资料标明,糖尿病心肌病亦是升高糖尿病患者罹难率和罹患率的首要病因,因此作为一种独立的,特异的心肌病变,受到越来越多人的关注[1，2]。最近十几年来,心血管领域众多学者对它的发病的复杂机制进行大量的临床实验研究和基础研究,对于其最终的发病机制的探讨仍然不成熟。故而,对于更进一步探讨糖尿病心肌病的最终发病的机制,研究遏制糖尿病心肌病的发病的途径对其防治有历史性意义。糖尿病心肌病的发生与众多因素复杂相关,是一个极其复杂的病理过程。其中,代谢紊乱(高糖血症和高胰岛素血症),心肌细胞的自噬和凋亡,心肌间质纤维化,微血管的病变等都是糖尿病心肌病的病理过程中发挥着至关重要的作用[2]。病理学研究发现,在糖尿病心肌组织形态学上主要表现为细胞外基质(ECM)沉积和心肌纤维化[3]。所以,心肌纤维化在糖尿病心肌病中的作用日益受到重视,但是其具体机制仍未完全阐明。心肌间质纤维化的机制错综复杂,胰岛素抵抗,炎症,激素的作用都会引起心肌间质纤维化。目前为止,许多学者逐一研究了上述各个原因与纤维化的关系[4，5]。但是,胰岛素抵抗与激素相互作用与心肌纤维化的研究,尚未有人涉足。因此,深入研究胰岛素抵抗与各种相关激素之间的相互作用与心肌纤维化之间的深层链接的关系具有深远的意义。胰岛素抵抗在纤维化的发生和发展中起着重要作用[5]。在胰岛素抵抗状态下,PI3K/Akt这一代谢信号通路受损[6,7]。PI3K/Akt信号通路的弱化会导致PGF2α与FP受体的过度结合,因为PGF2α与PI3K竞争性结合FP受体[8]。近几年来,PGF2α作为一个新的致纤维化的激素受到越来越多的关注。Oga已经证实,PGF2a与FP受体结合可以促进肺间质胶原沉积,从而导致肺间质纤维化[9,10]。另一方面,PGF2α与FP受体结合后可以活化PKC[11]或者Rho激酶[10]；而PKC和Rho激酶是经典的致纤维化因子,两者之间不仅能相互作用[12],而且能引起纤维化[13,14]。但是,在胰岛素抵抗状态下,PGF2α-FP受体/PKC/Rho激酶信号通路能否引起心肌间质纤维化,目前未见相关研究。因此,我们提出这样的假设,在胰岛素抵抗状态下,PGF2α/FP受体PKC/Rho激酶信号通路可能参与了糖尿病心肌病心肌间质纤维化的过程。所以,我们建立了二型糖尿病动物模型,运用FP受体基因沉默技术来研究PGF2a-FP受体对糖尿病心肌病心肌间质纤维化影响及可能的信号传导机制。目的1.建立二型糖尿病动物模型和糖尿病基因沉默动物模型2.探讨在糖尿病状态下和基因沉默状态下,PGF2α-FP受体对糖尿病心肌病心肌间质纤维化影响及可能的信号传导机制方法雄性SD大鼠40只,随机分为两组：正常对照组(n=8只),糖尿病组(n=32只),正常对照组大鼠以基础饲料喂养；糖尿病组大鼠以高脂高糖高热量饲料喂养。四周后,糖尿病组大鼠禁食12h后,腹腔内注射链脲菌素(STZ)27.5mg/kg,正常对照组大鼠注射等量柠檬酸钠／柠檬酸缓冲液。注射STZ一周后,连续三次通过尾静脉取血检测动物血糖,若空腹血糖>7.1或者随机血糖>11.1者纳入糖尿病模型组。此时,糖尿病组大鼠分为四个亚组：糖尿病组(n=7),糖尿病+空载体注射组(n=7),糖尿病+腺病毒(FP受体sh-RNA)注射组(n=7),糖尿病+腺病毒(FP受体sh-RNA)注射+PGF2a注射组(n=7)。动物血糖升高后,继续喂养14周。在第19周末,采用颈静脉注射法给予腺病毒组大鼠注射腺病毒2.5*1010PFU,给予空载体组大鼠注射同等量的空载体。两周后,即第21周末,重复注射同等剂量的腺病毒；同时,最后一组,即糖尿病+腺病毒(FP受体sh-RNA)注射+PGF2α注射组,给予PGF2α(0.1mg/kg)连续肌肉注射2周。第23周,处死动物,留取动物标本。实验过程中,进行以下检测：①实验末大鼠体重测量和心脏重量测量,血压,饮水,饮食,以及尿量检测。②分别于实验开始基线水平,注射STZ前(四周高脂饮食喂养后)、注射STZ后1周、糖尿病中期(第17周)、注射腺病毒前和实验末颈静脉取血测定空腹血糖、空腹胰岛素、甘油三酯、胆固醇,PGF2α,并计算胰岛素敏感指数(1SI)。③分别于实验开始基线水平、四周高脂喂养后,实验末进行胰岛素耐量和糖耐量检测。④于实验末以心导管法进行左室舒张末压(LVEDP)检测。⑤应用H&E, Masson,天狼猩红染色进行心肌组织间质胶原定量。⑥实时定量RT-PCR法和WB法检测FP受体mRNA和蛋白的表达以及PKC和Rho激酶的表达。⑦采用免疫组织化学法检测胶原Ⅰ/Ⅲ蛋白的表达。结果1.实验动物基本情况与正常对照组相比,糖尿病大鼠心脏重量,饮水,饮食以及尿量明显增加。给予FP受体基因沉默治疗后,与空载体组相比,单纯腺病毒注射组和腺病毒+PGF2α注射组饮水和尿量明显减少,心脏重量和饮食量稍微减少。2.大鼠生化指标检测高脂高热量饲料喂养4周后,与正常对照组相比,糖尿病组血清甘油三酯和空腹血糖明显上升(P<0.05),血清胆固醇,PGF2α和空腹胰岛素升高,但没有统计学意义(P>0.05),胰岛素敏感指数(ISI)明显下降(P<0.05)。STZ注射后1周,与正常对照组相比,糖尿病组血清胆固醇,甘油三酯,空腹血糖,PGF2α和空腹胰岛素明显升高(P<0.01)；胰岛素敏感指数(ISI)明显下降(P<0.01),这种状态一直持续到实验末。实验末,与糖尿病组相比：单纯腺病毒注射组和腺病毒+PGF2α注射组,血清胆固醇,甘油三酯,空腹血糖和空腹胰岛素明显下降(P<0.05),胰岛素敏感指数(ISI)也明显上升(P<0.05),PGF2α水平仍然很高(P<0.05)。3.大鼠糖耐量和胰岛素耐量检测与基线水平相比,高脂高热量饲料喂养4周后,糖尿病组大鼠胰岛素耐量和糖耐量开始受损,糖耐量和胰岛素耐量曲线下面积AUC明显增加,这种状态一直持续到实验末。实验末,与糖尿病组相比,单纯腺病毒注射组和腺病毒+PGF2α注射组胰岛素耐量和糖耐量明显改善,糖耐量和胰岛素耐量曲线下面积AUC明显降低。4.大鼠心导管检测与对照组相比,糖尿病组大鼠舒张末期左室内压力(LVEDP)明显增加；给予腺病毒注射后,单纯腺病毒注射组和腺病毒+PGF2。注射组LVEDP明显下降。5.组织形态学观察正常对照组心肌左室和血管周围胶原组织分布均匀,糖尿病组心肌内和管周胶原含量明显增多。定量分析结果显示：与正常对照组相比,糖尿病组左室心肌组织胶原容积分数和血管周围纤维化指数明显升高(P<0.05)。给予腺病毒治疗后,单纯腺病毒注射组和腺病毒+PGF2。注射组,心肌胶原含量显著下降,血管周围纤维化程度减弱。定量分析结果显示：与空载体组相比,单纯腺病毒注射组和腺病毒+PGF2α注射组,心肌组织胶原容积分数和血管周围纤维化指数明显下降(P<0.05)。天狼猩红染色也观察到同样的结果。6.信号通路检测与对照组相比,糖尿病大鼠体内FP受体mRNA和蛋白质的含量明显增加,磷酸化PKCβ2和MYPT-1水平也明显增加,磷酸化Akt活性明显下降；给予FP受体基因沉默治疗后,不仅FP受体mRNA和蛋白质的含量明显下降,磷酸化PKCβ2和MYPT-1水平也明显下降；受损的Akt活性逐渐恢复。7.免疫组化检测心肌组织FP受体分布从免疫组织化学图片上显示,我们可以看出,与对照组相比,糖尿病状态下,FP受体的分布广泛而密集,表达量增加；给予FP受体腺病毒注射后,FP受体分布相对稀疏而零散,表达量减少,同样FP受体腺病毒注射后,即使同时又给予PGF2α注射,FP受体分布与单纯FP受体腺病毒干预组相比没有明显变化；统计学结果亦得到同样结果。8.免疫组化检测胶原Ⅰ/Ⅲ免疫组化显示,与正常对照组相比,糖尿病组胶原Ⅰ/Ⅲ的蛋白表达明显上调；FP受体基因沉默治疗后,与空载体组相比,单纯腺病毒注射组和腺病毒+PGF2α注射组胶原Ⅰ/Ⅲ表达明显下降。结论1.四周高脂高热量饮食,糖尿病组大鼠体内出现代谢紊乱,这种代谢紊乱一直持续到实验末；四周腺病毒注射后,代谢紊乱得到了缓解。2.糖尿病组大鼠出现糖耐量受损和胰岛素抵抗；FP受体基因沉默治疗后,这种糖耐量受损和胰岛素抵抗得到了一定程度的改善。3.在糖尿病大鼠体内出现心脏舒张功能障碍,给予FP受体腺病毒基因沉默治疗后,心脏舒张功能障碍得到一定程度的改善。4.在2型糖尿病动物体内存在心肌间质纤维化现象,给予FP受体基因干预治疗后,这一现象得到改善。5.FP受体/PKC/Rho激酶信号通路参与了糖尿病病心肌病的发生和发展。6.在糖尿病状态下,胶原Ⅰ/Ⅲ的沉积加速；FP受体的基因沉默治疗,可以缓解胶原Ⅰ/Ⅲ的沉积过程。更多还原

【Abstract】 BackgroundIn the last30years, diabetic cardiomyopathy (DCM) has attracted people’s attention for the increased morbidity and mortality in diabetes population. Emerging data now reveal that, the initial change of DCM is considered to be myocardial fibrosis. Fibrosis, in general, is a process which is characterized by cardiac fibroblasts accumulation and excess extracellular matrix (ECM) deposition. ECM is composed of a complex fibrillar collagen network, comprising mainly collagen types I and III secreted by cardiac fibroblasts, and acts as a scaffold for the myocytes. However, the mechanism mediating collagen deposition in DCM remains incompletely understood. Insulin resistance is considered to play a causal role in the pathogenesis and development of DCM. In an insulin-resistant state, the IRS/PI3K/AKT pathway was drastically blunted, whereas MAPK pathway was fully activated. This situation is known as "pathway-selective insulin resistance". Activated MAPK could induce overproduction of transforming growth factor-β(TGF-β, which has been implicated in the fibrosis. Previous studies have shown that antagonizing TGF-βdoes not prevent fibrosis completely; indicating an additional pathway also has a key role in fibrogenesis. Recently, Oga has confirmed that PGF2α-FP signaling facilitates pulmonary fibrosis independently of TGF-β. However, the role of PGF2α-FP in the development of myocardial fibrosis has not been investigated.PGF2αexerts its biological effect via activation of FP receptor. Apart from leading to a decrease of PI3K activity, activated FP receptor also leads to the activation of the PKC and Rho signaling pathways. Furthermore, inhibition of PI3K increases membrane association of FP receptor by blocking constitutive internaization of FP receptor. In turn, accumulation of the FP receptor on the cell surface membrane binds to PGF2a, thereby establishing a positive feedback loop. In an insulin-resistant state, it has not been explored whether the positive feedback loop still exists and whether PGF2a/FP receptor/PKC/Rho signaling pathway can implicate in the regulating of collagen synthesis in cardiac fibroblasts.In light of the relationship between PGF2a-FP receptor, PKC and Rho, we hypothesized that PGF2a regulates collagen expression through an FP receptor/PKC/Rho signaling pathway in cardiac fibroblasts.Objective1.To determine the effect of PGF2a on FP receptor and collagen expressions in normal and insulin-resistant cellular model2. To determine the effect of PGF2a on signaling pathways in normal and insulin-resistant cellular model3.To determine the effects of FP/PKC/Rho inhibitors on collagen synthesis induced by PGF2αin normal and insulin-resistant cellular model4.To determine the effects of FP receptor inhibitor and TGF-β inhibitor on collagen synthesis induced by PGF2a or TGF-β in normal and insulin-resistant cellular modelMethods1.Cardiac fibroblasts were separated into:normal groups, LG+PGF2a (5.5mM glucose + PGF2a) group, IR+PGF2a (insulin-resistant cellular model + PGF2α group. After treatmenting with PGF2a for1h, 4h and24h, cells were collected, and both the mRNA and protein level of FP receptor, collagen types I and III by real time PCR and enzyme linked immunosorbent assay (ELISA).2.After the cardiac fibroblasts were incubated in normal and insulin-resistant cellular model for Omin, 30min, 60min and120min, the activity of PI3K/Akt and FP receptor/PKC/Rho cascades were measured respectively.3.According to the effect of PGF2α on FP receptor/PKC/Rho cascades, specific inhibitors of FP/PKC/Rho signal pathway were used to detect the collagen types I and ⅢmRNA expression and protein secretion.4.To confirmed the TGF-βindependent, FP receptor-mediated collagen production by PGF2a, specific inhibitors of FP receptor and TGF-βwere used to detect the collagen types I and III mRNA expression.Results1.As shown, after treatment with PGF2α for1h, FP receptor mRNA expression and protein content were significantly elevated in IR+PGF2α compared with normal and LG+PGF2α. The same to stimulation with PGF2αfor4h. In comparison with normal and LG+PGF2α, the stimulation of PGF2αfor4h in IR+PGF2αdisplayed remarkably increases in collagen types I and III mRNA expression. After treatment with PGF2α for24h, collagen types I and III protein secretion were notably up-regulated in IR+PGF2α compared with normal and LG+PGF2α.2.PGF2a induced time-dependent decreases in phosphorylation of PI3K and Akt and time-dependent increases in FP receptor, PKCβ and p-MYPT-1in normal and insulin-resistant cellular model.3.1n normal, pretreatment of cardiac fibroblasts with AL8810, LY-333531, Y27632, respectively, reduced the PGF2α-induced increases in mRNA level of collagen type I by47.59%,49.11%,49.00% and type III by21.50%,27.25%,21.76% at4h; which is also decreased PGF2a-induced effects on protein level of collagen type I by14.83%,19.31%,14.11% and type III by9.5%,13.45%,17.89% at24h. In insulin-resistant cellular model, pretreatment of cardiac fibroblasts with AL8810, LY-333531, Y27632, respectively, remarkably down-regulated the PGF2a-induced effects on mRNA level of collagen type I by80.48%,82.03%,83.50% and type III by26.49%,29.60%,36.08%at4h; which also reversed the PGF2α-induced increases in protein level of collagen type I by41.62%,35.07%,41.71% and type III by22.33%,18.13%,18.20%at24h.4.The effect of PGF2a was inhibited by AL8810but not by SB-50124alone in normal and insulin-resistant cellular model; the effect of TGF-βwas inhibited by SB-50124but not by AL8810alone in normal and insulin-resistant cellular model.Conclusions １.PGF2a induces higher levels of FP receptor, collagen types I and III in insulin-resistant cellular model2.PGF2a not only activates FP receptor/PKC/Rho cascades, but also further blunts PI3K/Akt pathways3.These data revealed that FP receptor/PKC/Rho pathway mediates the role of PGF2a in regulating collagen types I and III mRNA expression and protein secretion in normal and insulin-resistant cellular model4.PGF2a facilities synthesis of collagen types I and III in cardiac fibroblasts via FP receptor signaling independently of TGF-β BackgroundDiabetic cardiomyopathy (DCM) is described as the structural and functional changes in the myocardium that are associated with diabetes, which is one of the leading causes of morbidity and mortality worldwide. Several mechanisms have been implicated in the pathophysiology of diabeticcardiomyopathy. Of these, metabolic disturbances, myocardial fibrosis, small vessel disease, and cardiac autonomic neuropathy are the major players in the pathophysiology of diabeticcardiomyopathy.One major pathophysiology player of DCM is myocardial fibrosis. In this regard, myocardial fibrosis is multifactorial and can be caused by several processes including insulin resistance, inflammation, and various hormones. Many studies revealed the relationship of the above processes with myocardial fibrosis respectively; however, the crosstalk between insulin resistance and hormones is unclear. Hence, there is a need to understand the detailed mechanisms and factors associated with myocardial fibrosis in DCM.PGF2a, a metabolism of arachidonic acid, attracts people’s attention as a new fibrosis hormone. Ogo have found that PGF2α/FP receptor signaling facilitates pulmonary fibrosis. What’s more, PGF2α-FP receptor could mediate protein kinase C (PKC) or Rho kinase signaling molecules resulting in a series of biological activity. Furthermore, PKC and Rho kinase molecules can participate in the progression of fibrosis respectively. However, whether PGF2a-FP receptor/PKC/Rho kinase pathway could play an important role in the development of fibrosis has been little known. In our previous study, we have demonstrated that PGF2α regulated collagen I and III expression by an FP receptor/PKC/Rho kinase pathway in insulin-resistant cardiac fibroblasts, but the extrapolation of these finding to myocardial fibrosis in the diabetic animal model warrants further investigation.Objective1.To established the type2diabetic’s model.2.To explore the mechanisms of PGF2a-FP receptor involved in DCM by FP-receptor gene silencing in vivo.MethodsThis study enrolled forty male Sprague-Dawley (SD) rats at4weeks. All the rats were randomly assigned to control group (n =8) and diabetic group (n =32). The control group received normal chow; the diabetic groups were fed an HF diet. After four weeks, IPGTT and IPITT were performed again. The control group received citrate buffer (intraperitoneally) alone. The rats with insulin resistance in diabetic group were rendered with an intraperitoneal injection of a single dose of STZ (Sigma, St. Louis, MO;27.5mg/kg i.p. in0.1mol/L citrate buffer, pH4.5). Tail vein blood glucose was measured every3days in the first week and those with plasma glucose levels≥11.1mmol/L were considered to be diabetic. The diabetic group (n =32) was redivided into three groups: diabetes + vehicle group (n =8), diabetes + FP receptor-shRNA group (n =8) and diabetes + FP receptor-shRNA+PGF2αgroup (n8). After14weeks of diabetes, the last two groups were injected with2.5 *1010plaque-forming units of an adenovirus harboring FP receptor gene (FP receptor-shRNA) and the vehicle group was injected with a control empty virus (vehicle) by the jugular vein. Adenoviru transfer was repeated in2weeks. The FP receptor-shRNA + PGF2α group not only repeated adenovirus transfer but also injected with PGF2a (0.1mg/kg) for two weeks. Four weeks after the first adenovirus injection, rats were sacrificed. The heart was excised and weighed. During the experiments, we carried out:①Measurement of body weight and biochemical indexes.②fter rats deprived of food overnight, we collected blood sample through the jugular vein. Total cholesterol, triglyceride levels, FBG, PGF2α and fasting insulin levels were measured.③IPGTT was performed to access glucose tolerance; IPITT was carried out to evaluate insulin sensitivity.④Changes in the left ventricular end diastolic diameter pressure (LVEDP) were measured.⑤The tissue sections underwent HE staining, Masson’s trichrome staining and Picrosirius red staining. The myocyte cross-sectional area, the collagen volume fraction (CVF) and perivascular collagen area/luminal area (PVCA/LA) were analyzed.⑥The mRNA expression of FP receptor, the protein expression of FP receptor, PKC and Rho were measured.⑦Tissue sections (4μm) were subjected to immunohistochemistry.Results1.Animal characteristicsThe heart weight, water intake, food intake and urine volume were significantly higher in diabetic group than the control group (P<0.05). In FP receptor-shRNA group and FP receptor-shRNA + PGF2α group, the water intake and urine volume were significantly reduced (P<0.05) compared to vehicle group, thus the heart weight and food intake were slightly reduced with no significantly differences.2.Total cholesterol, triglyceride, FBG and PGF2α concentrationsAfter4weeks of a HF diet, serum cholesterol, PGF2α and insulin began to slightly increase in the diabetic group (P>0.05). The level of total triglyceride and FBG were significantly elevated in the diabetic group; the ISI was also decreased in the diabetic group (P<.05). One week after STZ injection, the levels of serum total cholesterol, triglyceride, FBG, PGF2α and insulin were remarkably elevated in the diabetic group and remained elevated until the end of the experiment. With a4-week transfection of FP receptor shRNA, the elevated serum levels of total cholesterol, triglyceride, FBG, and insulin were greatly reduced (P<0.05).3. Glucose and insulin toleranceIPGTT revealed that, the levels of blood glucose were significantly higher at week4in diabetic group at0,30min compared to control group. The AUC across the time for glucose level was higher at week4than at baseline. Similarly, IPITT showed impaired insulin sensitivity.At the end of the experiment, the diabetic group showed elevated levels of blood glucose and higher AUC on both IPGTT and IPITT compared with the control (P<0.05). In FP receptor shRNA group and FP receptor shRNA + PGF2a group, the IPGTT and IPITT showed the levels of blood glucose were significantly lower to vehicle group; the AUC for glucose and insulin were also lower to vehicle group.4. LV function assessed by catheterizationWe found that left ventricular end diastolic diameter pressure (LVEDP) was higher in the diabetic group than control group. With FP receptor gene silencing, LVEDP was lower in FP receptor shRNA group and FP receptor shRNA + PGF2a group than the vehicle group.5. Pathology characteristicsThe diabetic group showed cardiac fibrosis, with a scattered, small, inordinate pattern, as well as damaged and irregular collagen network structure in the interstitial and perivascular area. CVF and PVCA/LA were higher in the diabetic group than control group. With FP receptor shRNA treatment, CVF and PVCA/LA were decreased in FP receptor shRNA group and FP receptor shRNA + PGF2a group compared to vehicle group.6. FP receptor/PKC/Rho kinase and Akt pathwaysThe relative mRNA and protein expression of FP receptor was significantly elevated in diabetic group. Accompanied by FP receptor overexpression, the phosphorylation of PKCβ2and MYPT-1were markedly increased. With FP receptor shRNA treatment, the relative mRNA and protein expression of FP receptor was significantly downregulated in FP receptor shRNA group and FP receptor shRNA + PGF2a group compared with vehicle treatment, as well as the phosphorylation of PKCβ2and MYPT-1.7. The distribution of FP receptorImmunohistochemistry revealed the protein distribution of the FP receptor was upregulated in the diabetic group. With FP receptor shRNA treatment, the FP receptor was downregulated in FP receptor shRNA group and FP receptor shRNA + PGF2a group compared with vehicle group.8. Collagen Ⅰ and ⅢImmunohistochemistry revealed the protein expression of collagen Ⅰ and Ⅲ content was upregulated in the diabetic group. With FP receptor shRNA treatment, collagen Ⅰ and Ⅲ was downregulated in FP receptor shRNA group and FP receptor shRNA+PGF2a group compared with vehicle group.Conclusion1.Metabolite disturbance appeared at week4in rats fed a HF diet. With a4-week transfection of FP receptor shRNA, the metabolite-disturbant states were ameliorated.2.The diabetic group showed impaired glucose tolerance and insulin sensitivity; with FP receptor shRNA treatment, the impaired glucose tolerance and insulin sensitivity were ameliorated.3.Diastolic dysfunction was developed and progressed during DCM. With FP receptor gene silencing, the diastolic dysfunction was improved, which also confirmed the beneficial effect of FP receptor shRNA on improving LV diastolic dysfunction.4.Our results revealed that severe cardiac fibrosis exists in type2diabetic rats; cardiac fibrosis may ameliorate with FP receptor gene silencing.5.FP receptor/PKC/Rho kinase signaling pathway involves in the pathogenesis of DCM.6.In diabetic states, the deposition of collagen Ⅰ and Ⅲ was accelerated. With FP receptor gene silencing, the excessive deposition of collagen Ⅰ and Ⅲ was ameliorated, which reconfirmed the beneficial effect of FP receptor shRNA in DCM.更多还原