【作者】 魏群；

【导师】 杨萍；

【作者基本信息】 吉林大学 ， 内科学， 2010， 博士

【摘要】 慢性心力衰竭(chronic heart failure,CHF)是心血管疾病发展到晚期的病理综合征,是当前危害人类健康的主要疾病之一。目前的研究认为心室重构是慢性心力衰竭发生发展的主要病理基础,既包括心肌细胞的重构又包括心肌间质的胶原沉积和纤维化,即心肌纤维化(myocardial fibrosis,MF)。MF是指在心肌细胞外基质(extracellular matrix,ECM)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,主要表现为心肌细胞肥大、心肌成纤维细胞(cardiac fibroblasts,CFb)增殖并分泌大量胶原蛋白。研究表明,心肌纤维化是不同病因的心脏疾病发展到一定阶段的共同病理改变,可导致心肌僵硬度增加、心室舒张功能减退、心肌电生理紊乱、心律失常及冠状动脉储备下降甚至引起猝死,是心室重构的主要表现之一。由于梗死区的心肌组织被胶原代替形成瘢痕,非梗死区占心脏的大部分,其重构的特点与梗死区不同,梗死后非梗死区的变化对心脏长期预后的影响可能更大。而且目前国外的研究也表明非梗死区在心衰的心室重构中占有重要地位,因此本实验着眼于对非梗死区心室重构的研究。研究发现某些细胞因子在MF的形成中发挥了关键作用。转化生长因子β(Transforming growth factorβ,TGF-β)即是与MF有关的最重要的细胞因子,而激活素A(Activin A)属于TGF-β超家族成员,是机体参与急性期应答以及纤维化形成的重要因子。卵泡抑素(Follistatin,FS)作为Activin A特异结合蛋白,可以阻断Activin A的生物学作用。FS与Activin A常共同存在、共同表达构成一个维持组织器官正常生长的平衡系统,其中一项的过度表达或另一项的减少引起的Activin A-FS系统的失衡将伴随某些组织器官生理过程的失调和病理过程的形成,而且Activin A-FS系统被认为是重要的、以复合体形式调节多种细胞功能的系统。尽管有研究报道Activin A在心梗后心衰模型组左室缺血区及非缺血区表达升高,但有关Activin A-FS系统在心梗后心衰形成及纤维化中的作用仍不清楚。因此,本研究通过检测心力衰竭患者血清中Activin A、FS及BNP的表达水平,探讨其表达水平变化及临床意义,同时采用结扎Wistar大鼠左冠状动脉前降支制备心梗后心衰模型,分析Activin A-FS系统的表达在心衰后左心室重构中的作用机制及雷米普利与替米沙坦的干预机制。主要研究内容如下:1、临床研究:选择2008年3月~2008年12月门诊和住院的充血性心力衰竭(congestive heart failure,CHF)病人。CHF诊断标准参照2005年ESC《成人慢性心力衰竭的诊断与治疗指南》,心功能分级参照美国纽约心脏病协会1994年修定标准NYHA分类法。健康对照组(control)44例,为随机选取的同期健康者,男20例,女24例,年龄63~84(平均73.27±7.07)岁,排除心肺、肝肾、糖尿病及其他内分泌疾病。CHF病人87例,其中男性46例,女性41例,年龄63~85(平均72.88±7.35)岁,排除肺、肝、肾、糖尿病及其他内分泌疾病,按照美国纽约心脏病学会(NYHA)心功能分级分为3组:心功能II级组(NYHA II,30例)、心功能III级组(NYHA III,26例)和心功能IV级组(NYHAIV,31例)。清晨空腹采集静脉血3ml,1500r/min离心15min后,分装血清于?80℃冰箱冻存。人血清Activin A、FS及BNP水平检测具体操作步骤按照人Activin A、FS及BNP的ELISA检测试剂盒说明书(美国ADL公司)进行。临床研究发现,与健康对照组比较,证实心衰病人血清BNP水平升高,同时心衰病人血清Activin A水平也明显升高,差异显著(P<0.05～0.001),并且心衰病人血清Activin A的水平与心衰程度及BNP水平呈正相关,相关系数及P值分别为(R=0.9350,P<0.001),(R=0.8918,P<0.001)。NYHA II组及NYHA III组血清FS的水平无明显变化(P>0.05),NYHA IV组血清FS水平呈下降趋势,但差异无显著性(P>0.05)。2、结扎Wistar大鼠左冠状动脉前降支6周建立心梗后心衰模型,同步灌胃给予雷米普利与替米沙坦干预5周后,观察各组大鼠心肌形态学的变化、血流动力学、心肌肥厚指数,分析Activin A-FS系统的表达变化。应用RT-PCR法检测左室非梗死区心肌组织Activin A及其相关受体IIA、IIB,FS,col- I及col-III mRNA表达水平,以及测定col-I / col-III比值。ELISA方法检测大鼠血清及左室非梗死区心肌组织Activin A、血管紧张素II(Ang II)蛋白表达,左室非梗死区心肌组织FS蛋白表达及血清BNP水平。行HE染色及Masson染色分析左室非梗死区心肌胶原沉积。进一步行免疫组织化学染色检测左室非梗死区心肌组织Activin A及FS蛋白表达情况。研究结果表明心梗后心衰模型组大鼠Activin A-FS系统存在明显的失衡。与假手术组比较,心梗后心衰模型组左室非梗死区心肌组织Activin A mRNA及蛋白表达水平均显著升高,激活素II型A受体(ActRIIA)和II型B受体(ActRIIB)、胶原蛋白I及III(col-I & col-III)mRNA表达水平,以及col-I / col-III比值亦增高,同时与心室重构相关介质(BNP和AngII)水平升高,并伴随心功能降低及心肌肥厚指数升高,Masson染色可见大量胶原沉积。有意义的是,我们发现心梗后心衰模型组左室非梗死区心肌组织FS mRNA及蛋白表达水平下降。给予雷米普利与替米沙坦同步干预后,可以下调左室非梗死区心肌组织Activin A mRNA及蛋白表达水平,上调FS mRNA及蛋白表达水平,同时可见ActR II及胶原蛋白mRNA表达水平下降,心功能明显改善,BNP和AngII水平及心肌肥厚指数亦明显降低,心肌胶原沉积减轻。综上所述,本研究结果显示,临床检测血清Activin A水平有望作为心力衰竭诊断、治疗和监控的又一临床血清学指标,同时心梗后心衰的发生发展过程中Activin A-FS系统失衡,调节Activin A/FS比率可能成为改善心衰的治疗靶点。雷米普利与替米沙坦可通过调节Activin A-FS系统,改善左心室重构,本实验为其作用机制作一补充。更多还原

【Abstract】 Activin A is a member of the transforming growth factor-βsuperfamily. It is involved in the acute-phase response and fibrosis . Follistatin is an activin-binding protein and is measurable in extracellular matrix and serum . Activins trapped by follistatin are then internalized by endocytosis and are subsequently degraded by proteolysis.It has been reported that the expressions of activin A and its receptors in ischemic and nonischemic regions markedly increased after myocardial infarction (MI) and that activin A increased gene expression of atrial natriuretic peptide(ANP), brain natriuretic peptide (BNP), and matrix metalloproteinase-9(MMP-9), the factors known to play an important role in myocardial remodeling and interstitial fibrosis. The coexpression of activin A and follistatin is vital in tissue remodeling and repair. The imbalance of activin A-follistatin system may induce pathological changes in tissues. Overexpression of activin in the skin of transgenic mice caused dermal fibrosis and epidermal hyperthickening, and in the epidermis of transgenic mice in which the activin antagonist follistatin was overexpressed, the skin was characterized by a mild dermal and epidermal atrophy, significantly reduced granulation tissue formation, and smaller scar area. These studies implicate an important function of endogenous activin in wound repair and scar formation. Follistatin, the activin antagonist, may prevent activin A-induced fibrosis and play an important role in the treatment of fibrosis.The purpose of this study was to determine whether the imbalance of activin A-follistatin systme is involved in the myocardial fiborsis and the subsequent heart failure after MI. For this purpose, we analyzed the relationship between the imbalance of activin A-follistatin system and the left ventricular remodeling in the rat model of heart failure after MI. Non-infarcted ventricular remodeling in heart failure plays an important role, so this study focused on non-infarcted area of left ventricular remodeling.1 Clinical study.1.1 MethodsBetween March and December 2008, we enrolled 87 inpatients and outpatients with congestive heart failure (CHF) and 44 healthy control subjects. The CHF patients were 63 to 85 years of age (mean age, 72.88±7.35 years); 46 were men and 41 were women. CHF was diagnosed according to ESC Guidelines for the Diagnosis and Management of Chronic Heart Failure in the Adult 2005. According to the New York Heart Association (NYHA) functional classification system released in 1994, the CHF patients were classified into NYHA II group (n=30), NYHA III group (n=26), and NYHA IV group (n=31). The healthy control subjects were 63 to 84 years of age (mean age, 73.77±7.07 years); 20 were men and 24 were women. None of the participants had concomitant disease such as lung, liver, and kidney diseases, diabetes, and other endocrine diseases. Written informed consent was obtained from each participant.The venous blood of 3 ml was drawn from each fasting individual in the morning and centrifuged at 1500 r/min for 15 minutes. The serum was stored at -80℃until analyzed. Serum levels of activin A,FS and BNP were analyed by enzyme-linked immunosorbent assay according to the instruction (ADL ELISA Kit, USA).1.2 ResultsThe levels of activin A and BNP were significantly higher in sera of all patients with NYHA II, NYHA III, and NYHA IV than that in healthy control subjects (P<0.05～P<0.001); and it was significantly higher in NYHA IV groups, compared with NYHA II group (P<0.05). Although the levels of follistatin increased slightly in sera of all patients with NYHA III, the differences in all proteins were not significant, compared with that in healthy control subjects (P>0.05). The data show that patients with heart failure significantly increased the levels of Activin A in serum, Activin A levels and degree of heart failure and BNP levels were positively correlated, the correlation coefficient and P values were (R = 0.9350, P <0.001), (R = 0.8918 , P <0.001).2 The role of activin A and follistatin in left ventricular remodeling in a rat model of heart failure2.1 MethodsFemale Wistar rats were subjected to the ligation of the anterior descending branch of the left coronary artery or sham operation during halothane anesthesia (1% halothane in a mixture of one third O2 and two thirds N2O) anesthesia as previously described . Post-operated rats were divided into 6 groups (n=6 each group): SO group, sham-operated rats receiving 0.5% CMC; SO-ramipril group, sham-operated rats treated with ramipril (3 mg/kg/day); SO-telmisartan group, sham-operated rats treated with telmisartan (30 mg/kg/day); MI group, MI rats receiving 0.5% CMC; MI-ramipril group, MI rats treated with ramipril (3mg/kg/day), and MI-telmisartan group, MI rats treated with telmisartan (30 mg/kg/day). All treatment regimens were initiated 6 weeks after surgery and continued for 5 weeks. Ramipril (C7002) and telmisartan (602328) were provided by Sanofi-Aventis Pharma. Beijing Co., Ltd and Boehringer Ingelheim International Trade (Shanghai) Co., respectively.After 11 weeks,the Hemodynamic measurements were observed ,the rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (30mg/kg). A catheter was inserted into the right carotid artery and then further advanced into the left ventricular chamber to record left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), and rate of contraction and relaxation (±dp/dt). The systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) were also recored. After the cardiac function was recorded, 3 ml of the arterial blood was drawn from abdominal aorta and centrifuged at 2500 r/min for 10 minutes. The serum was stored at -70℃. The rats were then killed by decapitation, and the hearts were immediately removed. The heart weight and left ventricular mass were measured, and the ratio of heart weight to body weight (HW/BW) and the ratio of left ventricular mass to body weight (LVM/BW) were calculated.The infarcted and noninfarcted left ventricular tissue was separated. The noninfarcted left ventricular tissue was cut into two parts along the longitudinal axis of the left ventricle. One part were fixed in 4% paraformaldehyde, embedded in paraffin, and sliced into 5-μm sections. morphological changes in the myocardium, sections (5μm) were stained by hematoxylin and eosin (H&E) and Masson’s trichrome.The expressions of activin A and follistatin proteins were detected immunohistochemical stain. Another part was stored at -70℃for subsequent determination of the protein expressions of activin A, follistatin, and angiotensin II (Ang II) and the mRNA expressions of activin A, type II A and II B activin receptor (ActR II A and ActR II B), follistatin, and type I and III collagen (col-I and col-III).2.2 results2.2.1 Gross morpholgical evidenceRamipriland telmisartan could decrease the volume of heart of VR rats, limit the infarct area, lower the rate of aneurysm and reduce the expansion of ventricular,and could increase the number of residual cardialcytes in infarct area and reduce the cytopathic around the infarct area.2.2.2 Morphological changes in the myocardiumH&E stain showed that in SO, SO-telmisartan, and SO-ramipril groups, the cardiomyocytes were intact, and the arrangement of myocardial fibers was regular. In MI group, the arrngement of myocardial fibers were irregular, and various degrees of cardiomyocyte degeneration and hypertrophy and interstitial fibrosis were found. Compared with MI group, the above changes were less severe in MI-telmisartan and MI-ramipril groups.2.2.3 Cardiac functionHW/BW and LVM/BW were significantly higher in MI group than in SO group (both P<0.001) 11 weeks after the surgery, and they were also significantly higher in MI-telmisartan and MI-ramipril groups than in MI group (P<0.001, P<0.01). There were significant differences in both HW/BW and LVM/BW between MI-telmisartan and MI-ramipril groups (both P<0.05). Compared with SO group, SBP, DBP, LVSP, and±dp/dt significantly decreased and LVEDP significantly increased in MI group (P<0.01, P<0.01, P<0.05, P<0.01, and P<0.01, respectively). In MI-telmisartan and MI-ramipril groups, LVSP, +dp/dt, and -dp/dt were significantly higher and LVEDP significantly lower, as compared with MI group (P<0.05, P<0.01, P<0.05, and P<0.05, respectively),but no significant between-group difference was found in HR, SBP, and DBP.2.2.4 Expressions of activin A, ActR II A, ActR II B, follistatin, col-I, and col-III mRNA in the noninfarcted area of the left ventricleThe expression levels of activin A, ActRIIA, ActRIIB, col-I, and col-III mRNA in the noninfarcted area of the left ventricle and the ratio of col-I to col-III were higher in MI group than in SO group, but the expression level of follistatin mRNA in MI group was significantly lower than that in SO group. Compared with MI group, the expression levels of activin A, ActR II A, ActR II B, col-I, and col-III mRNA and the ratio of col-I to col-III were lower and but the expression level of follistatin mRNA was higher in MI-telmisartan and MI-ramipril groups.2.2.5 Serum level of activin A, Ang II, and BNPThe serum level of activin A, Ang II, and BNP in MI group were significantly higher than those in SO group, and compared with MI group, their levels significantly decreased in MI-telmisartan and MI-ramipril groups. In MI-telmisartan and MI-ramipril groups, the expressions of these 3 proteins were down-regulated. In all group, the serum level of activin A was positively correlated to the levels of Ang II and BNP. The data indicated that expression of activin A protein increased after myocardial infarction, ramipril and telmisartan could improved cardiac function which may be associated with down-regulated expression of activin A protein.2.2.6 Levels of activin A, follistatin, and Ang II in the noninfarcted area of the left ventriucle.The levels of activin A and Ang II in MI group were significantly higher than those in SO group 11 weeks after the surgery, but the level of follistatin was signficantly lower in MI group than in SO group. Compared with MI group, the expressions of activin A and Ang II were down-regulated and the expression of follistatin was up-regulated in MI-telmisartan and MI-ramipril groups.The differences in the expression of these 3 proteins were more signficant between MI-telmisartan and MI groups .2.2.7 Expressions of activin A and follistatin identified by immunohistochemistry.Low expressions of activin A and follistatin were found in the noninfarcted area of the left ventricle in SO, SO-ramipril, and SO-telmisartan groups . In MI group, the expression level of activin A was up-regulated, and the expression of follistatin was down-regualted, while the converse was true in MI-telmisartan and MI-ramipril groups .2.2.8 Myocardial fibrosis after MIMasson’s trichrome stain demonstrated the formation of myocardial fibrosis after MI. In SO,SO-ramipril, and SO-telmisartan groups, the arrangement of cardiomyocytes was regular, and no obvious green collagen fiber was found. In the noninfarcted area of rats in MI group, collagen fiber deposition and cardiomyocyte hypertrophy was found, the number of cardiomyocytes decreased, and the arrangement of cardiomyocytes was irregular. The above changes in MI-telmisartan and MI-ramipril groups were less severe than those in MI group.Conclusions1.Serum activin A was in a state of high expression in patients with heart failure,which was positively correlated with the degree of heart failure,furthermore,activin A level could be expected as a clinical serological indicator to make a diagnosis of heart failure, therapy and monitor this disease.2.The imbalance of activin A-follistatin system which is characterized by up-regulated expression of activin A is an important predisposing factor for myocaridal fibrosis and ultimately heart failure after MI. The down-regulated expression of follistatin could promote the development of heart failure after MI. Thus, up-regulating follistatin expression or exogenous follistatin and reversing the imbalance of activin A-follistatin system may be the effective ways to improve heart failure and prevent activin A-induced myocardial fibrosis.3.Activin A and type II activin receptors (ActR II A and ActR II B) were down-regulated, the expression of follistatin increased, and the collagen deposition decreased by using telmisartan and ramipril,which may be a new mechanism of action to telmisartan and ramipril in heart failure.更多还原