【作者】 杨永曜；

【导师】 李隆贵；

【作者基本信息】 第三军医大学 ， 内科学， 2007， 博士

【摘要】 研究背景:近年研究表明,心力衰竭发生、发展的基本机制是心室重构（ventricular remodel）。心室重构是由一系列复杂的分子和细胞机制引起的心肌细胞结构、功能及表型的改变,然而对其复杂的适应性和非适应性分子调控机制还知之甚少。进一步探讨心力衰竭过程中心室重构的分子机制,将对临床预防和治疗心力衰竭具有重要意义。过氧化物酶体增殖激素型受体（peroxisome proliferator activated receptors,PPARs）是一类由配体激活的核转录因子,属核受体超家族成员之一。PPARs具有多种生物学效应,可促进脂肪细胞分化和脂肪生成,增强机体对胰岛素的敏感性,调节体内糖平衡,抑制炎症因子生成及炎症形成,影响肿瘤生长,近年来,还发现其对心血管产生保护效应。PPARα、γ的配体Fibrates类和TZDS（thiazolidinediones）类药物已在临床应用多年,分别用于治疗高脂血症和II型糖尿病。文献及本室的研究表明,心肌肥厚时PPARα活性被抑制,导致心肌脂质及能量代谢紊乱。但PPARs途径激活对左室心肌重构的影响及机制仍不清楚。本课题旨在阐明心室重构时心肌细胞肥大、凋亡和心肌间质重塑进程中PPARs的活性变化及其规律和意义,探讨PPARs活性改变与心肌细胞肥大、凋亡、心肌间质重塑、RAAS激活以及心力衰竭的关系,探索心力衰竭的发生、发展机制和新的干预策略。研究方法:1、细胞培养:1)、取1天龄wistar乳鼠心脏,分离消化后在普通培养基上培养,加入AngII（终浓度10-7mol/L）孵育,介导心肌细胞肥大、凋亡;2)、取1天龄wistar乳鼠心脏,分离消化后在普通培养基上培养,分别加入AngII+非诺贝特（5、10、20μmol/L）、AngII+吡格列酮（5、10、20μmol/L）及AngII +非诺贝特+吡格列酮共同孵育,观察心肌细胞肥大、凋亡、间质重塑情况。2、动物实验:1)、实验动物:成年雄性wistar大鼠;2)、建立CAA模型:将大鼠麻醉后开腹,束扎腹主动脉后饲养4、8周;3)、分组:取术后存活的大鼠按4、8周二个时间段喂养,每个时间段又各随机分为4组:（1）手术组(CAA_4w组,n=10;CAA_8w组,n=9);（2）非诺贝特组(F_4w组,n=10;F_8w组,n=9):30mg/（kg·d ）;（3）吡格列酮组(P_4w组,n=10;P_8w组,n=9):3 mg/（kg·d ）;（4）非诺贝特和吡格列酮合用组(F+P_4w组,n=10;F+P_8w组,n=9):30 mg/（kg·d ）和3 mg/（kg·d ）。另取10只大鼠,只穿线不节扎为假手术组（SH组）作对照。治疗组均予药粉末溶于水中灌胃。SH、CAA组以等量蒸馏水灌胃,每天一次,各组喂饲标准鼠食,自由饮水; 3、观察指标:1)、TUNNEL染色、流式细胞仪Annexin FITC/PI染色观察心肌细胞和心肌凋亡;2)、Western Blot检测心肌细胞和心肌PPARαγ、Bcl-2、Bax、Fas、Fas-L的含量;3)、RT-PCR检测心肌细胞和心肌PPARαγ、α/β-MHC的mRNA丰度;4)、流式细胞仪及MTT测定成纤维细胞增殖,RT-PCR、天狼星红染色观测心肌间质I/III型胶原含量;5)、经颈动脉插管测定动物模型的血流动力学指标（心率（HR）、左心室收缩压（LVSP）、舒张末压（LVEDP）、±dp/dt、主动脉收缩压及舒张压等）;6)、测量动物模型的左心室重量、左心室重量/体重、左心室容积、左心室长、短轴及室壁厚度等心肌病理学指标;7)、电镜观察心肌细胞、心肌超微结构;8)、RT-PCR检测心肌细胞AT1R mRNA丰度;9)、放免法测定心肌、血浆RAAS系统的含量及活性。结果:1、AngII激活心肌细胞胚胎基因β-MHC mRNA再表达,使心肌细胞的面积增加,表明已发生心肌细胞肥大,心肌细胞肥厚模型制作成功;2、与对照组相比,非诺贝特、吡格列酮预处理24h显著逆转了AngII诱导的心肌细胞肥大,抑制AngII引起细胞活力改变,增加α-MHC mRNA表达,降低胚胎基因β-MHC mRNA的表达,α/β-MHC mRNA比值明显增加;3、非诺贝特、吡格列酮显著逆转了AngII诱导的心肌细胞肥大,抑制AngII诱导的心肌细胞凋亡,降低Fas/ FasL蛋白的表达,增加Bcl-2蛋白表达,降低Bax蛋白的表达,Bcl-2/Bax蛋白水平比值明显增加;4、非诺贝特、吡格列酮显著降低了AngII诱导的心肌成纤维细胞增殖及基础胶原合成;5、与假手术组相比,各手术处理组左室湿重/体重（LVW/BW）、平均动脉压（MAP）、左室收缩压（LVSP）、左室舒张末期压（LVEDP）、最大上升及下降速率（+dp/ dt）及血浆和心肌血管紧张素II、醛固酮活性及心肌AT1 mRNA的表达均显著增加（ P < 0.05～0.01） ,而心率（HR）、左室内压最大下降速率（- dp/ dt）均显著降低（ P均< 0.05）,各组右室湿重/体重（RVW/BW）无显著差异,表明手术后已出现左室重塑;与手术对照组相比,非诺贝特、吡格列酮及其合用三个治疗组在8周时明显降低了LVW/BW、MAP、LVSP、LVEDP、HR（P均< 0.05）,-dp/ dt增高。血浆和心肌血管紧张素II、醛固酮活性却无明显变化。除非诺贝特组外,吡格列酮和两药合用组降低了心肌AT1 mRNA的表达（ P < 0.05）。除AT1 mRNA外,上述各指标在三个治疗组间差异均无显著性（ P均> 0.05）;5、手术组电镜观察心肌细胞出现凋亡超微结构特征;与假手术组相比,8周时非诺贝特、吡格列酮显著减轻了压力超负荷诱导的心肌肥厚,改善了心率（HR）、平均动脉压（MAP）、左室收缩压（LVSP）、左室舒张末期压（LVEDP）等血流动力学指标,抑制了心肌肥厚过程中的心肌细胞凋亡,上调Bcl-2/Bax蛋白水平比值,降低Fas/ FasL蛋白的表达;6、相对于假手术组,各手术处理组心肌I/III型胶原mRNA和VG染色光密度比值增加,非诺贝特、吡格列酮及其合用三个治疗组8周时可明显降低上述变化。相对两药处理组,上述指标与两药合用组无显著差异（P <0.05）。结论:1、PPARα、γ信号通路激活后参与抑制了心肌细胞肥大和活力的改变;对成纤维细胞增殖有显著抑制作用;2、非诺贝特、吡格列酮长时间预处理（24h）激活PPARα和γ除能逆转心肌细胞肥大外,还可抑制心肌细胞凋亡,并能改变凋亡相关基因Bcl-2/Bax、Fas/Fas-L的表达;3、经长时间处理（8周）,PPARα、γ信号通路激活能改善压力超负荷大鼠血流动力学和左心室重构指标;4、PPARα和γ信号通路激活除能减轻压力超负荷大鼠的心肌肥厚外,还可抑制心肌细胞凋亡,对凋亡相关基因Bcl-2/Bax、Fas/ FasL的表达有调控作用;5、PPARα、γ信号通路激活后能明显改善压力超负荷左室心肌间质胶原重塑;6、PPARα、γ配体对血浆和心肌血管紧张素II、醛固酮活性无明显影响,但PPARγ途径激活能抑制AT1mRNA的表达;7、PPARα、γ配体合用对心室重构无叠加效应。更多还原

【Abstract】 Background and ObjectivesCurrent studies have demonstrated that in the progression of cardiac insufficiency caused by various cardiac disorders, such as chronic ischemic heart disease, myocardial infarction, valvular heart disease,and myocarditis,there are a series of changes including myocardium hypertrophy and vascular hyperplasia. These changes, called ventricular remodeling, are one of the major causes that eventually induce heart failure. In particular, left ventricular hypertrophy in the course of ventricular remodeling has been proved as an independent risk factor for heart failure . In addition, hypertrophic ventricular cardiomyocytes have been found to undergo transition from compensatory hypertrophy to apoptosis. It has been shown that during the progression of compensatory left ventricular hypertrophy to failure, loss of cardiomyocyte plays an important role and the development of cardiac disorders is usually accompanied with cardiomyocyte apoptosis . Meanwhile,myocardial fibrosis act an importment role in ventricular collagen remodeling .It has become a new target of heart failure therapy.Peroxisome proliferator activated receptors (PPARs) is one kind of nuclear transcription factors, the activation of which is mediated by their corresponding ligands. There are three subtypes of PPARs: PPARα,PPARβ(also known as PPARδ) and PPARγ. Their ligands are both receptor- and tissue-specific, and show various biological effects. PPARβ/δis found in almost all tissues, but its physiologic functions remain unclear. PPARαand PPARγcan promote adipocyte differentiation and fat production, enhance the sensitivity of body to insulin, modulate sugar balance, inhibit inflammatory factor production and inflammation occurrence, etc. In recent years, PPARαand PPARγhave also been found to have a capacity of protecting the cardiovascular system, which, however, is paradoxically.The aim of this study is to research the effects of Fenofibrate and Pioglitazone, two activators of PPARαand PPARγ, on the cardiac hypertrophy in vitro and in vivo. To explore the role of the PPARs signal pathway in hypertrophy, apoptosis and fibrosis of cardiomyocytes during ventricular remodeling.Methods1. Cell culture:1).The neonatal rat cardiomyocytes were cultured by the method of digestion step by step and adherence with different speeds,and with Angiotensin II(final concentration10-7mmol/L ) stimulation ,a model of hypertrophy and apoptosis of neonatal rat cardiac myocytes was established. PPARαligands, Fenofibrate and PPARγligands,pioglitazone pretreatment ,10μmol/L,24h prior to AngII. With the aid of Leica Qwin Image software, the surface area of cardiac myocytes was analysis. The mRNA expression of PPARα,PPARγ,α-MHC andβ-MHC was measured by reverse transcription-polymerase chain reaction(RT-PCR) and the cultured myocyte viability was estimated by MTT assay .Annexin V-FITC and PI staining and then Flow cytometry was used to monitor the apoptosis cells. Using Western blot , the proto-oncogene BCL-2/Bax and Fas/Fas-L expression was observed; 2). Cardiac fibroblasts(CFs) were isolated by trypsin digestion method. CFs in third passage in this study. Then the cultured rat cardiac fibroblasts were divided randomly into five groups :control group ,AngII stimulation group(10-7mol/L), Fenofibrate pretreatment group(10μmol/L), Pioglitazone pretreatment group(10μmol/L) and concomitant fenofibrate and pioglitazone group . Cell cycle kinetics of CFs was analyzed by flow cytometry. Collagen synthesis rate was determined by measuring MTT.2. Animal experiment: To study associated pathologic and pathophysiologicchanges of myocardial hypertrophy, the pressure overloading model was established by the constriction of abdominal aorta in Wistar rats. Forty-eight hours after the procedure , the surviving rats were randomly divided into three time stages to breed, that is, 4, 8 weeks.Then the rats in every time stage were also randomly divided into five groups: (1) coarctation of abdominal aorta(CAA)controls group, (2) fenofibrate(F group,30 mg·kg- 1·d - 1 ), (3) pioglitazone(P group, 3 mg·kg- 1·d - 1) , (4) concomitant fenofibrate and pioglitazone (F+P group, 30mg·kg- 1·d - 1and 3mg·kg- 1·d - 1)groups ,and (5) sham-operated(SH) group was selected to serve as non-pressure overload controls.There are ten groups in all.Fenofibrate and pioglitazone were delivered by direct gastric gavage.Heart rate (HR), left ventricular end diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), mean arterial pressure (MAP) and left ventricular pressure maximal rising and declining velocities(±dp/dtmax), and the ratio of left ventricular weight to body weight (LVW/BW) and the ratio of right ventricular weight to body weight(RVW/BW). And the rennin activity,level of angiotensin II and aldosteron in Plasma and myocardial were detected by radioimmunity. The morphologic features of myocardiac cells were observed by transmission electron microscopy. DNA fragmentations were determined semiquantitatively by in situ TDT-mediated dUTP nick end labeling ( TUNEL). After Sirius red trinitrophenol staining, myocardial collagen volume fraction (CVF) were determined by Leica analysis software. PPARα,PPARγ,α-MHC/β-MHC , AT1, types I and III collagen mRNA expressions were assessed with reverse transcription polymerase chain reaction (RT-PCR). Using Western blot, the proto-oncogene Bcl-2/Bax and Fas/FasL expression were observed.Results1.Fenofibrate and pioglitazone pretreatment 24h prior to AngII, Significantly (p<0.01-0.05) reduced AngII-induced cardiac hypertrophy,increased expression ofα/β-MHC mRNA,and inhibited the effect of Ang II on the cardiac myocyte viability .There were no significant differences in the above mentioned indices between fenofibrate and pioglitazone group(P >0. 05) .2.Fenofibrate and pioglitazone pretreatment significantly(P<0.05) reduced angiontensinII-induced cardiocyte apoptosis by Flow cytometry analysis.Significantly inhibited the expression of Fas/FasL and Bax, while enhanced the expression of Bcl-2 and Bcl-2/Bax ratio.3. AngII significantly increased the proliferation index [(S+G2/M )/(S+G2M +G0/G1 )] of CFs with low concentration (final concentration10-7mmol/L ). Fenofibrate and pioglitazone pretreatment ,10μmol/L,24h prior to AngII, inhibited the effects of the proliferation induced by AngII (P<0. 01). Fenofibrate and pioglitazone pretreatment decreased the OD numerus of MTT assay induced by AngII for 24 h (P < 0. 05).4.In CAA8W group characteristic features of apoptotic myocardiac cells was recognized by transmission electron microscopy. We discovered the two drugs treatment significantly(P<0.05) reduced cardiocyte apoptosis in pressure-overload induced when they treatment 8 weeks. Compared with sham-operated rats ,the Bax/Bcl-2 and Fas/ FasL expressions increased at protein levels in all CAA groups (all P < 0. 05) . Compared with the CAA group , Bax/Bcl-2 and Fas/ FasL expressions were significantly decreased in 8 weeks treatment groups ( P < 0. 05 - 0. 001 ). There were no significant differences in the above mentioned indices among the four treatment groups in 4 weeks(all P > 0. 05) .5. At 4, 8 weeks after the operation, LVW/BW and CVF were increased significantly in CAA group,fenofibrate group and pioglitazone group compared with those in sham operated group, but those in fenofibrate and pioglitazone 8 weeks group were significantly lower than that of CAA group.Meanwhile,fenofibrate and pioglitazone significantly decreased the types I and III collagen mRNA expressions of the left ventricular myocardium induced by CAA. There were no significant differences in the above mentioned indices among the four treatment groups in 4 weeks(all P > 0. 05) .6. Compared with sham-operated rats , the left ventricular end diastolic pressure (LVEDP) , left ventricular systolic pressure(LVSP),mean arterial pressure(MAP)and the maximum left ventricular pressure rising rates (+dp/ dt),and the LVW/BW , mRNA of AT1 as well as plasma and myocardial angiotensin II and aldosteron were significantly increased in all CAA groups (all P < 0. 05) ,accompanied with heart rate(HR) and the maximum left ventricular pressure declining rates (-dp/ dt) were decreased.RVW/BW is no change in all groups, indicating that left ventricular remodeling(LVRM) occurred after CAA.Compared with the CAA group ,LVW/BW ,MAP ,LVSP ,LVEDP,HR were all significantly decreased when drug treatment 8 weeks ( P < 0. 05 - 0. 001 ), while corrected -dp/dt was significantly enhanced ( P < 0. 05 - 0. 001). The three drug treatment groups had no effect on plasma and myocardial angiotensin II and aldosteron activity(p>0.05). Pioglitazone treatment 8 weeks can downregulation the mRNA expression of AT1 while fenofibrate group had no effect on it. There were no significant differences in the above mentioned indices among the three drug treatment groups except AT1 mRNA (all P > 0. 05).Conclusions1. It is suggested that PPARα,PPARγactivators inhibit hypertrophy of cardiac myocytes and PPARs-dependent pathway be involved in the inhibitory course.2. Chronic treatment with the PPARαand PPARγagonist may be useful in preventing cardiac hypertrophy and apoptosis in vitro(24h) and vivo(8weeks), which is related the regulation of the changes of proto-oncogene Bax/Bcl-L , Fas and FasL expression.3. AngII can enhance proliferation in CFs and increase basal collagen syntheses. PPARαand PPARγactivators may attenuate myocardial fibrosis induced by AngII by inhibiting the above effects.4. PPARαligands(fenofibrate) and PPARγligands(pioglitazone) chronic treatment(8 weeks) can ameliorate pressure overload rat’s left ventricular hypertrophy and hemodynamic parameters. 5. PPARαand PPARγagonist can attenuate remodeling of myocardial collagen network in pressure overloaded rats.6. Peroxisome proliferator-activated receptors ligands had no effect on plasma and myocardial angiotensin II and aldosteron activity. But mRNA expression of AT1 was upregulated by activing PPARγsignaing pathway . PPARα、γligands can improve left ventricular remodeling (LVR).7. There is no additive effect when the two drugs were used in combination.更多还原