【作者】 周浩；

【导师】 郭志刚；

【作者基本信息】 南方医科大学 ， 心血管内科（专业学位）， 2016， 博士

【摘要】 研究背景慢性心衰是目前全世界范围内危及健康的主要问题之一。心脏重构是心力衰竭的基本机制,也是其进展的病理生理学基础;近年来,肾脏重构在心衰中的相互作用日益受到人们重视。心力衰竭的直接或间接效应可导致急性肾损伤、肾脏重构和功能障碍。肾功能损害可增加患者短期和远期的死亡率,而这种改变与心脏、肾脏的纤维化存在密切联系。心、肾纤维化是心衰发展重要机制之一。纤维化机制研究和开发有效的抗纤维化药物是目前心衰研究领域的难点和热点。心、肾纤维化是指在细胞外基质中胶原纤维过量积聚,胶原含量显著升高或胶原成分发生改变,是机体对负荷、炎症或者细胞坏死作出的一种适应性的反应,常见于高血压或者缺血性心脏病的病理生理状况。在这些病理情况下,心、肾组织释放一系列生长因子如血管紧张素原、TGF-β、ET1、TNF-α等最终导致细胞外基质的沉积[1];另一方面神经体液因子的激活,尤其是循环和局部肾素-血管紧张素-醛固酮系统也参与了心、肾纤维化的应答。心、肾纤维化是受损组织僵硬度的结构基础,是心、肾功能由代偿期向失代偿期转变的关键过程。因此,如何阻断这个环节显得十分重要。内皮细胞-间质化现象(endothelial-mesenchymal transition EndMT)是指细胞逐渐失去其内皮细胞特性,包括极性、细胞与细胞之间的特殊连接,而获得从内皮细胞池中向外周组织迁移等间质细胞具有的能力的现象。其在胚胎发育、伤口愈合、组织再生和癌症的进程中的重要作用已经被阐明[2]。而新近的研究则发现,EndMT现象存在各类器官纤维化的过程中,如肺脏,肾脏,肠系膜动脉等。转化生长因子-β(transcriptiongrower factor-β,TGF-β)具有促进及诱导EndMT现象产生的作用[3,4]。Elisabeth M Zeisberg 1等则在纤维化的心脏中发现EndMT现象,从而为心脏纤维化机制研究揭开了新的一页[5]。那么内皮细胞间质化可能是通过什么信号途径实现的呢?最近研究表明,Notch信号通路以及微卫星RNA在EndMT过程中发挥着关键的调节作用。研究表明Jagged-1激活上皮细胞内Notch信号后,可抑制上皮细胞特异性蛋白分子(如E-cadherin、β-catenin、内皮细胞NO合成酶等)表达、诱导间充质细胞特异性蛋白分子表达升高,并伴随上皮细胞形态、功能等向间充质细胞方向分化,提示Jagged-1激活的Notch信号通路在EndMT过程中发挥重要作用。醛固酮——肾上腺皮质球状带细胞分泌的盐皮质激素,作用于肾脏盐皮质激素受体,对水盐代谢及钠、钾平衡起着重要的调节作用。RAAS激活,醛固酮水平升高,可通过上调基质金属蛋白酶的活性[6],刺激TGF-β1的合成[7]等途径促进心肌胶原的合成,导致心脏纤维化并呈剂量依赖关系[8]。因此,醛固酮拮抗剂逐渐引起人们的重视。螺内酯——选择性醛固酮受体拮抗剂现已成为抗心衰研究的重点药物之一[9]。近来发现心血管系统独立存在的醛固酮形成系统,心血管局部有肾素mRNA的表达,并不受全身RAAs的影响[10]。醛固酮与心肌细胞、内皮细胞、心血管成纤维细胞等细胞胞浆盐皮质激素受体(mineralocorticoid receptor,MR)结合,形成激素-受体复合物;后者通过核膜,与核中的DNA醛固酮反应基因结合,调节特异性mRNA转录,最后合成多种醛固酮诱导蛋白,从而影响心血管系统、泌尿系统和植物神经系统等,并参与组织修复,调节水、电解质和血容量[12]。螺内酯与醛固酮有相似的化学结构,它可与醛固酮竞争结合MR,阻止醛固酮-受体复合物的形成。越来越多的证据表明螺内酯可以防止或逆转心、肾纤维化的形成,成为抗纤维化治疗的非常重要药物,但其机制仍不明确。本研究拟在心衰动物模型上,探讨螺内酯抑制心、肾纤维化的作用以及与EndMT的关系,并在细胞上进一步研究Notch信号通路在其中的调控作用,为其在临床上应用提供新的理论依据,为探索EndMT成为心衰抗纤维化治疗新靶点提供实验依据,也为进一步新药研发提供新思路。第一部分:螺内酯抑制内皮间质化改善心力衰竭心脏重构的实验研究在异丙肾上腺素诱导的心力衰竭大鼠模型上研究螺内酯对心脏功能,结构,心肌纤维化的影响以及内皮细胞-间质细胞转化现象是否得到抑制。在原代培养的人脐静脉内皮细胞实验中研究:TGF-β诱导内皮细胞-间质细胞转化,螺内酯作为肾素-血管紧张素-醛固酮阻滞剂对其形态和功能变化的影响;同时进一步研究Notch通路在内皮细胞-间质细胞转化诱导纤维化过程中的作用机制。材料与方法:一.动物实验部分1.动物模型建立及实验分组1.1实验分组:雄性SD大鼠随机分为4组:①正常对照组(NS control group;10只)、②模型组(Isogroup;10只)、③低剂量螺内酯组(Low dose spironolactone group;30mg/kg/d,10 只)、④高剂量螺内酯组(High dose spironolactone group;60mg/kg/d,10 只)1.2动物模型制作模型组:雄性SD大鼠背部皮肤处皮下注射异丙肾上腺素,5mg/kg/d,共1周。对照组给予等同量的生理盐水。螺内酯药物组:大鼠背部皮下注射异丙肾上腺素1周建立心肌纤维化模型,造模开始后连续螺内酯溶液灌胃(30mg/kg/d、60mg/kg/d)共3周。2.取材和观察指标2.1心功能检测多媒体生物信号记录仪的压力换能器,记录LVSP、LVEDP、HR、+dp/dtmax及-dp/dtmax。2.2标本采集收集血液标本。开胸,冰上处理心脏组织。测定右心室游离壁及左心室质量。取少量左心室心尖部组织(约80mg),甲醛固定,常规脱水、石蜡包埋。2.3心脏质量指数测定右心室质量除以体重计为右心室质量指数(RVWI)。左心室质量除以体重计为左心室质量指数(LVWI)。2.4 苏木精-伊红染色法(HematoxyHn-eosinstaining,HE)心肌组织行HE染色,观察心肌病理学变化。2.5 Masson 染色(Masson trichrome staining)石蜡切片行Masson染色后胶原呈蓝色。显微镜下观察心肌纤维化情况,采用Image-Pro Plus软件,测量心肌胶原面积,取平均值.2.6 ELISA(Enzyme-linked immunoassay)测心脏组织中Ⅰ型胶原及Ⅲ型胶原浓度2.7免疫荧光(Immunofluorescence methods,IFA)免疫荧光法检测内皮细胞中α-SMA(纤维性指标)及CD31(内皮性指标)的表达,观察EndMT现象。二.细胞实验部分1.HUVECs细胞分离、HUVEC的培养、细胞的传代、Ⅷ因子相关抗原荧光染色细胞鉴定2.HUVECs实验分组:HUVECs随机分为5组:①空白对照组②DAPT处理组(DAPT dissolved in DMSO)、③TGF-β处理组④螺内酯+TGF-β处理组(SP+TGF-β)⑤螺内酯+TGF-β+DAPT(TGF-β+SP+DAPT)。螺内酯在 TGF-β24h 前预处理,DAPT 在螺内酯1h前预处理。3.Transwell小室检测各实验组细胞迁移能力4.免疫荧光(Immunofluorescence methods,IFA)免疫荧光法检测内皮细胞中vimentin(纤维性指标)及CD31(内皮性指标)的表达,观察EndMT现象。5.Werstern-blot蛋白免疫印迹测定各组细胞vimentin,CD31和Notch 1的表达。6.统计学处理SPSS16.0统计分析软件用于数据处理,所得数据用均数士标准差((?)±S)表示,用单因素方差分析(One-wayANOVA)进行多组样本间的均数比较。P<0.05判定为差异具有显著性意义。结果:一.动物实验部分1.心功能检测:模型组LVSP、HR、+dp/dtmax、-dp/dtmax低于空白组,LVEDP高于空白组,且差别均具有统计学意义。螺内酯治疗改善模型组心功能,且差别均具有统计学意义。2.心脏质量指数测定:模型组LVWI,RVWI高于空白组,且差别均具有统计学意义。螺内酯治疗减少LVWI及RVWI的增加,且差别均具有统计学意义。3.心肌病理学检查:模型组心肌可见广泛的间质增生,心肌排列紊乱、心肌细胞肥厚、空泡形成,炎细胞浸润。螺内酯治疗明显改善上述改变。4.心肌纤维组织纤维染色:模型组心肌组织间可见广泛的蓝色纤维组织,胶原面积测定高于空白组,且差别均具有统计学意义。螺内酯治疗改善模型组心肌纤维化,且差别均具有统计学意义。5.心肌组织中Ⅰ型和Ⅲ型胶原蛋白含量测定:模型组心脏组织胶原Ⅰ、胶原Ⅲ浓度高于空白组,且差别均具有统计学意义。螺内酯治疗组胶原Ⅰ、胶原Ⅲ浓度低于模型组,且差别均具有统计学意义。6.EndMT指标测定:模型组心肌组织中内皮细胞CD31(内皮性标志)表达低于空白组,α-SMA(纤维性标志)表达高于空白组,且差别均具有统计学意义。螺内酯治疗后CD31表达高于模型组,α-SMA表达低于模型组,且差别均具有统计学意义。二.细胞实验部分1.Transwell小室检测各实验组细胞迁移能力TGF-β对HUVECs迁移能力的影响:与对照组相比,TGF-β显著增强HUVECs的增殖、迁移、黏附能力,差异有统计学意义。SP对TGF-β诱导的HUVECs迁移能力的影响:与TGF-β诱导组相比,SP预处理24 h,对TGF-β诱导HUVECs迁移有抑制作用。两组相比差异有统计学意义。DAPT对SP干预TGF-β诱导的HUVECs迁移能力的影响:加入Notch1蛋白抑制剂(DAPT)处理1h后可减弱SP抑制TGF-β诱导迁移的作用。与SP+TGF-β处理组相比,HUVECs迁移能力有所增强。两组相比差异有统计学意义。2.荧光单染在各实验组的表达与阴性对照组相比,TGF-β组内皮细胞特异性蛋白CD31表达下调,间质细胞标志性蛋白vimentin明显上升;与TGF-β组相比,SP+TGF-β组CD31表达上调而vimentin表达明显下降;与SP+TGF-β组对比,SP+DAPT+TGF-β组CD31表达下降而vimentin表达明显上升。各组相比均差异有统计学意义。3.Werstern-blot 对各实验组 CD31、vimentin、Notch-1 蛋白的检测:与对照组相比,TGF-β组CD31表达显著下调,而vimentin显著上升;与TGF-β组相比,SP+TGF-β组CD31表达上调,而vimentin表达下降;与SP+TGF-β相比,SP+DAPT+TGF-β组CD31表达下调,而vimentin表达上调。各组相比均差异有统计学意义。与对照组相比,Notch蛋白抑制剂(DAPT)单独作用组Notch1蛋白表达显著下降;与TGF-β诱导组相比,SP+TGF-β组Notch1蛋白上调,与SP+TGF-β组相比,TGF-β+SP+D APT组Notch1蛋白显著下降。各组相比均差异有统计学意义。结论:醛固酮拮抗剂螺内酯通过Notch信号通路抑制内皮间质化而改善心衰心室重构。第二部分:螺内酯抑制内皮间质化改善心力衰竭肾脏重构的实验研究本部分实验的目的是探讨螺内酯对异丙肾上腺素(ISO)诱导的心力衰竭大鼠的肾功能、EndMT、TGF-β表达的影响,以及这些变化和肾纤维化之间的关系材料与方法:1.动物模型建立及实验分组1.1实验分组:雄性SD大鼠随机分为4组:①正常对照组(NS control group;10只)、②模型组(Iso group;10只)、③低剂量螺内酯组(Low dose spironolactone group;30mg/kg/d,10 只)、④高剂量螺内酯组(High dose spironolactone group;60mg/kg/d,10 只)1.2动物模型制作模型组:雄性SD大鼠背部皮肤处皮下注射异丙肾上腺素,5mg/kg/d,共1周。对照组给予等同量的生理盐水。螺内酯药物组:大鼠背部皮下注射异丙肾上腺素1周建立心肌纤维化模型,造模开始后连续螺内酯溶液灌胃(30mg/kg/d、60mg/kg/d)共3周。2取材和观察指标2.1心功能检测多媒体生物信号记录仪的压力换能器,记录LVSP、LVEDP、HR、+dp/dtmax及-dp/dtmax。2.2标本采集进行腹开胸并冰上处理肾脏组织。肾脏PBS洗净并用滤纸吸干后称重。取少量肾脏组织(约80mg),甲醛固定,常规脱水、石蜡包埋。2.3肾脏质量指数测定左右肾脏称重后除以体重计为肾脏质量指数。2.4 苏木精-伊红染色法(HematoxyHn-eosinstaining,HE)肾脏组织行HE染色,观察肾脏病理学变化。2.5 Masson 染色(Masson trichrome staining)石蜡切片行Masson染色后胶原呈蓝色。显微镜下观察肾脏间质纤维化情况,200倍视野下随机选取5个不重复的视野进行拍照。采用Image-Pro Plus软件,测量肾脏胶原面积,取平均值。2.6 ELISA(Enzyme-linked immunoassay)测肾脏组织中Ⅰ型胶原及Ⅲ型胶原浓度2.7 免疫荧光(Immunofluorescence methods,IFA)免疫荧光法检测肾脏组织中内皮细胞中α-SMA(纤维性指标)及CD31(内皮性指标)的表达,观察EndMT现象。2.8Werstern-blot蛋白免疫印迹测肾脏组织中CD31、α-SMA及TGF-β1表达3.统计学处理SPSS16.0统计分析软件用于数据处理,所得数据用均数士标准差(x土S)表示,用单因素方差分析(One-wayANOVA)进行多组样本间的均数比较。P<0.05判定为差异具有显著性意义。结果:1.肾脏质量指数测定:模型组肾脏质量指数(KWI)高于空白组,且差别具有统计学意义。螺内酯治疗减少模型组KWI,且差别具有统计学意义。2.肾脏病理学检查:模型组肾脏也可见较多的间质增生,炎细胞浸润。螺内酯治疗明显改善上述改变。3.肾脏纤维组织纤维染色:模型组肾脏组织间可见较多的蓝色纤维组织,胶原面积测定高于空白组,且差别具有统计学意义。螺内酯治疗改善模型组肾脏纤维化,且差别具有统计学意义。4.肾脏组织中Ⅰ型和Ⅲ型胶原蛋白含量测定:模型组肾脏组织的胶原Ⅰ、胶原Ⅲ浓度高于空白组,且差别均具有统计学意义。螺内酯治疗组胶原Ⅰ、胶原Ⅲ浓度低于模型组,且差别均具有统计学意义。5.肾脏组织中EndMT指标免疫荧光测定:模型组肾脏组织中内皮细胞CD31(内皮性标志)表达低于空白组,α-SMA(纤维性标志)表达高于空白组,且差别均具有统计学意义。螺内酯治疗后CD31表达高于模型组,α-SMA表达低于模型组,且差别均具有统计学意义。6.肾脏组织中EndMT指标Werstern-blot测定:模型组肾脏组织中内皮细胞CD31(内皮性标志)表达低于空白组,α-SMA(纤维性标志)表达高于空白组,且差别均具有统计学意义。螺内酯治疗后CD31表达高于模型组,α-SMA表达低于模型组,且差别均具有统计学意义。7.肾脏组织中TGF-β1表达:模型组肾脏组织中TGF-β1表达高于空白组,且差别具有统计学意义。螺内酯治疗组TGF-β1表达低于模型组,且差别具有统计学意义。结论醛固酮拮抗剂螺内酯可能通过抑制肾脏内皮间质化而改善心衰肾脏重构。更多还原

【Abstract】 Background:Chronic heart failure is one of the major issues that jeopardize healthworldwide.Cardiac remodeling is the pathophysiological basis of the occurrence and development of heart failure.Recently,the interaction of renal remodeling in heart failure has been increasingly emphasized.The direct and indirect effects of heart failure are identified as leading to acute kidney injury and dysfunction.The degree of renal function impairmentrelates to increased short-and long-term mortality.It has been linked to cardiac and renal fibrosis.Thecardiac and renal fibrosis play a key role in its development.Exploring the mechanisms of heart failure and developing effctive anti-fibrosis drugs have become the tough problems as well as hot topics in current research.Cardiac and renal fibrosisis characterized by the excess accumulation of collagen fibers,increased collagen concentration or collagen component alterationin extracellular matrix(ECM).Fibrosis a adaptive response of the organism to harmful stimulilike load,inflammation or necrosis that are common in hypertension and ischemic heart disease.In these pathological conditions,heart and kidney release a series of growth factors such as angiotensinogen,TGF-β,ET-1,TNF-α,and eventually lead to theexcess deposition of a collagen-rich extracellularmatrix[1].On the other hand,neurohormonal activation,especially circulating and local renin-angiotensin-aldosterone system,is also involved in the response of cardiac and renal fibrosis.Fibrosis results in increased tissue stiffness,promotes cardiac and renal function shifting from compensation to decompensation phase.Therefore,how to block this process is very important.Endothelial-mesenchymal transition(EndMT)is a process in which endothelial cells lose their polarityand cell-to-cell contacts and acquire mesenchymal phenotype.The important role of EndMT in theprogression of embryogenesis,wound healing and cancer have been elucidated[2].Recently,increasing evidence had suggested that EndMT contribute to the development of organ fibrosis,such as lung,kidney,mesenteric artery.Transforming growth factor β(TGF-β)is the most important mediator in this process and can promote and induceEndMT[3,4].The discovery of EndMT in heart fibrosis by Elisabeth M Zeisberg opened a new chapter of myocardial fibrosis mechanism research[5].So what signal pathway may be involved in EndMT?Recent studies have confirmed,Notch signaling pathway and micro satellite RNA play a vital role in regulating EndMT.Activation of Notch signaling by Jagged-1 in the epithelial cells inhibited the expression of epithelial cell specific protein molecules(such as E-cadherin,β-catenin,endothelial cell nitric oxide synthetase),increased the expression of mesenchymal cell specific protein molecules,and induced the acquisition ofinesenchymal morphology and phenotype,suggesting that activation of Notch signaling pathway by Jagged-1 plays an important role in EndMT process.Aldosterone,a kind of mineralocorticoid secreted by Adrenal glomerulosa cells,regulates water-electrolytes metabolism and sodium and potassium homeostasis through mineralocorticoid receptors(MR)-dependentinteractions.Elevated aldosterone levels can upregulated the activity of matrix metalloproteinase[6],increased TGF-β1 levels[7],stimulated the synthesis of myocardial collagen,thereby resulting in cardiac fibrosis and in a dose-dependent manner[8].Therefore,aldosterone antagonist has gradually attracted people’s attention.The selective aldosterone receptor antagonist-spironolact,has become one of the key anti-heart failure drugs[9].In recent years,researchers found the cardiovascular system has its own way to produce aldosterone.The renin mRNA expression was detected in local cardiovascular tissue and was not affected by circulating RAAs[10].Aldactone combines with MRs expressed in cardiomyocytes,vascular smooth muscle cells,fibroblasts and renal tubular epithelial cells to form hormone-receptor complex,it binds to hormone-response elements in the promoter of target genes to regulate gene transcription,then produce a variety of aldosterone induced protein(including luminal tubular membrane sodium channel protein,cell membrane sodium pump,mitochondrial ATPase)[11],thus affecting the cardiovascular system,urinary system and autonomic nervous system etc,participating in tissue repair,regulation of water,electrolyte and blood volume[12]With structural similarity to aldosterone,spironolactone competitively inhibit the binding of aldosterone to the mineralocorticoid receptor.As evidence mounted that spironolactone can prevent or reverse thecardiaac renal fibrosis process,spironolactonebecomes an important drug of ant-fibrosis therapy,but its pathophysiology remains unclear.We aimed to investigate the inhibitory effectsof spironolactone on cardiac and renal fibrosis and its relationships with EndMT in heart failure animal model,study the role of Notch signaling pathway in EndMT at the cellular level.Part 1:spironolactone improves cardiac remodeling in heart failure via inhibition of Endothelial-Mesenchymal TransitionWe aim to investigate the influence ofspironolactone on cardiac function,structure,the myocardial fibrosis and EndMT in isoprenaline(Iso)-induced heart failure rats.In the experiment of primary cultured human umbilical vein endothelial cells:We investigate whether aldosterone receptor-blocker spironolactone affects cell morphology and function in TGF-β-induced EndMT,further study the role of Notch signaling pathway in EndMT induced fibrosis and the underlying mechanism.一、Animalexperimental sectionMaterial and Methods:1.Modelbuilding and group design1.1 Group design:rats were randomly divided into four groups(ten per group)for treatment:control;myocardial fibrosis(isoproerenol[Iso]);low-and high-dose spironolactone(30mg·kg-1·d-1 and 60mg·kg-1·d-1,respectively).1.2 Rat model of cardiac fibrosisFor Iso-model,Iso(5 mg·kg-1·d-1)was injected subcutaneously in male SD rats for 1 week,The same volume of saline was injected for controls.In the therapeutic groups,Iso administration was the same as in the Iso-model group,intragastric administration of spironolactone solution(30mg·kg-1·d-1 and 60mg·kg-1·d-1)for 3 weeks at the same time as Iso injection.2.1 Measurement of hemodynamicsRats were connected with a pressure-transducer input signal recorder for recording heart rate,the LV mean systolic pressure,LV end diastolic pressure,and maximum rate of change in LV pressure(+dp/dtmax,-dp/dtmax).2.2 Sample collectionAfter being humanely euthanized by bleeding.The heart was rapidly excised and rinsed in cold normal saline.Subsequently,the left and right ventricles were separated and weighed.A piece of left ventricular apical tissue(about 80mg)were cut and fixed in formalin,embedded in paraffin and sectioned.2.3 Left Ventricular Weight Index(LVWI)and Right Ventricular Weight Index(RVWI)After the left and right ventricles were separated and weighed,and the left and ight ventricular weight indices(LVWI and RVWI)were calculated as the left and right ventricular free wall mass(mg)divided by body mass(g),respectively.2.4 hematoxylin and eosin stainingHematoxylin and eosin(HE)staining was performed to eval-uate pathological and morphological changes of myocardial tissue.2.5 Masson trichrome stainingMasson’s trichrome staining was performed to assess myo-cardial fibrosis.The sections were examined using light microscopy,and photographs were taken at×200 magnification.Five non-repeating visual fields were randomly selected,myocardial collagen areas were measured using Image-Pro Plus and the areas were averaged.2.6 ELISA(Enzyme-linked immunoassay:measurement of collagen Ⅰ and Ⅲ content in myocardial tissue.2.7 Immunofluorescence stainingα-SMA(fibrous maker)and CD31(endothelial maker)expression was observed by Immunofluorescence staining to investigate EndMT phenomenon.二、Cell experimental section1.Primary HUVECs separation,culture and identification of HUVECs2.HUVECs Experimental designHU-VECs were randomly divided into 5 groups for treatment:blank control;vehicle control(DAPT dissoolved in DMSO);TGF-β(10 ng/ml);spironolactone+TGF-β;spironolactone+DAPT +TGF-β.HUVECs were exposed to spironolactone 24h before TGF-β.In selected experiments,cells were treated with DAPT 1h before spironolactone.3.Transwell assay were performed to observe cell migration.4.Immunofluorescence stainingvimentin(fibrous maker)and CD31(endothelial maker)expression was observed by Immunofluorescence staining in endothelial cells to investigate EndMT phenomenon.5.CD31;vimentin and Notch protein level were examined by Western blot analysis.6.Statistical analysisData are expressed as mean ± SEM.Statistical analyses involved use of SPSS v16.0 by Student’s t-test for comparing the two groups or one-way ANOVA.P,0.05 was considered statistically significant.Results:一、Animalexperimental section1.Cardiac function:Iso significantly decreased LVSP,heart rate,+dp/dtmax,-dp/dtmax and increased LVEDP compared with controls(P<0.01).Spironolactone attenuated ventricular function(P<0.05).2.Organ weight index(LVWI,RVWI):LVWI,RVWI were significantly higher with Iso than control treatment(P<0.01);treatment with spironolactone decreased LVWI,RVWI as compared with Iso treatment(P<0.05).3.Histopathological observations:Heart tissues from Iso-treated rats showed collagen fibre hyperplasia,disordered myocardial structure and leukocyte infiltration compared with controls.Treatment with spironolactone alleviated these changes4.Masson’s trichrome staining:Blue fibrous tissue can be seen widely in the heart tissues from of Iso-treated rats.The area of collagenwas higher with Iso treatment than control treatment(P<0.01).Treatment with spironolactone alleviated these changes.5:Types Ⅰ and Ⅲ collagen content:Content of type Ⅰ,Ⅲ collagen in cardiactissue homogenate was higher with Iso treatment than control treatment(P<0.01).Treatment with spironolactone significantly decreased the content as compared with Iso treatment(P<0.01).6.Expression of CD31 and a-SMA:Iso decreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01),but spironolactone reversed these changes as compared with Iso treatment(P<0.05).二、Cell experimental section1.Transwell assayEffect ofTGF-β1 oncell migration:TGF-β significantly enhanced HUVECs’ ability of proliferation,migration and adhesion as compared with the control group(P<0.01).Effect ofSPon cell migration:Pretreatment with SP for 24h inhibited cell migration stimulated by TGF-βas compared with the TGF-βgroup(P<0.01).Effect ofDAPTon cell migration with SP treatment:SP could inhibit HUVEC cell migration stimulated by TGF-β,and DAPT could abrogate the effect.After co-culture with the Notch inhibitor DAPT,the number of migrating cells was increased as compared with TGF-β+ SP(P<0.01).2.Immunofluorescence staining results:TGF-βdecreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01);compared with TGF-β,CD31 protein level was increasedand a-SMA level was decreased in SP+TGF-βgroup(P<0.01);compared with SP+TGF-β group,CD31 protein level was decreasedand a-SMA level was increased in TGF-β+SP+DAPT group(P<0.01).3.Werstern-blot for CD31、vimentin、Notch-1TGF-βdecreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01);compared with TGF-β,CD31 protein level was increasedand a-SMA level was decreased in SP+TGF-β group(P<0.01);compared with SP+TGF-β group,CD31 protein level was decreasedand a-SMA level was increased in TGF-β+SP+DAPT group(P<0.01).Notch inhibitor DAPT significantly decreased Notch1 level as compared with the control(P<0.01);compared with TGF-β,Notchl protein level was increased in SP+TGF-β group(P<0.01);compared with SP+TGF-β group,Notchl protein level was decreased in TGF-β+SP+DAPT group(P<0.01).Conclusion:Spironolactone inhibits endothelial mesenchymal transition to improve cardiac remodeling in heart failure via Notch pathway.Part 2:spironolactone improves renal remodeling in heart failure via inhibition of Endothelial-Mesenchymal TransitionFrom previous observations,we aimed to investigate whether spirolactone affects renal function in isoprenaline(Iso)-induced heart failure in rats,affects EndMT and the expression of TGF-β as well as the relation between these changes and renal fibrosis.1.Material and Methods:model building and group design1.1 Group design:rats were randomly divided into four groups(ten per group)for treatment:control;myocardial fibrosis(isoproerenol[Iso]);low-and high-dose spironolactone(30mg·kg-1·d-1 and 60mg·kg-1·d-1,respectively).1.2 Rat model of cardiac fibrosis For Iso-model,Iso(5 mg·kg-1·d-1)was injected subcutaneously in male SD rats for 1 week,The same volume of saline was injected for controls.In the therapeutic groups,Iso administration was the same as in the Iso-model group,intragastric administration of spironolactone solution(30mg·kg-1·d-1 and 60mg·kg-1·d-1)for 3 weeks at the same time as Iso injection.2.1 Measurement of hemodynamicsRats were connected with a pressure-transducer input signal recorder for recording heart rate,the LV mean systolic pressure,LV end diastolic pressure,and maximum rate of change in LV pressure(+dp/dtmax,-dp/dtmax).2.2 Sample collectionAfter being humanely euthanized by bleeding.The kidney was rapidly excised and rinsed in cold normal saline.A piece of kidney tissue(about 80mg)were cut and fixed in formalin,embedded in paraffin and sectioned.The remaining tissue were frozen in liquid nitrogen and then preserved in-80 C refrigerator for measurement.2.3 Left renal Weight Index(LVWI)and Right renal Weight Index(RVWI)After the left and right kidney were separated and weighed,and the left and right renal weight indices(LVWI and RVWI)were calculated as the left and right renal mass(mg)divided by body mass(g),respectively.2.4 hematoxylin and eosin staining Hematoxylin and eosin(HE)staining was performed to eval-uate pathological and morphological changes of kidney.2.5 Masson trichrome stainingMasson’s trichrome staining was performed to assess renal fibrosis.The sections were examined using light microscopy,and photographs were taken at x200 magnification.Five non-repeating visual fields were randomly selected,renal collagen areas were measured using Image-Pro Plus and the areas were averaged.2.6 ELISA(Enzyme-linked immunoassay):measureent of collagen Ⅰ and Ⅲ content in kidney2.7 Immunofluorescence staininga-SMA(fibrous maker)and CD31(endothelial maker)expression was observed by Immunofluorescence stainingin in endothelial cells to investigate EndMT phenomenon.2.8.CD31,α-SMA and TGF-β1 level were examined by Western blot analysis.3.Statistical analysisData are expressed as mean ± SEM.Statistical analyses involved use of SPSS v16.0 by Student’s t-test for comparing the two groups or one-way ANOVA.P,0.05 was considered statistically significant.Results:1.renal weight index(KWI):KWI were significantly higher with Iso than control treatment(P<0.01);treatment with spironolactone decreased KWI as compared with Iso treatment(P<0.05).2.Histopathological observations:Kidney tissues from Iso-treated rats showed collagen fibre hyperplasiaand leukocyte infiltration compared with controls.Treatment with spironolactone alleviated these changes.3.Masson’s trichrome staining:Blue fibrous tissue can be seen widely in the kidney tissues from of Iso-treated rats.The area of collagenwas higher with Iso treatment than control treatment(P<0.01).Treatment with spironolactone alleviated these changes(P<0.01).4.Types I and III collagen content:Content of type Ⅰ,Ⅲ collagen in kidney tissue homogenate was higher with Iso treatment than control treatment(P<0.01).Treatment with spironolactone significantly decreased the content as compared with Iso treatment(P<0.01).5.Immunofluorescence staining results:Iso decreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01),but spironolactone reversed these changes as compared with Iso treatment(P<0.05).6.Expression of CD31、α-SMAin kidney:Iso decreased CD31 protein level and increased a-SMA level as compared with the control(P<0.01),but spironolactone reversed these changes as compared with Iso treatment(P<0.05).7.Expression ofTGF-β1in kidney:Iso increased TGF-β1 level as compared with the control(P<0.01),spironolactone treatment decreasedTGF-β1 level as compared with Iso treatment(P<0.01).Conclusion:Spironolactone inhibits endothelial mesenchymal transition to improve renal remodeling in heart failure.更多还原