【作者】 徐健；

【导师】 李俊；

【作者基本信息】 安徽医科大学 ， 药理学， 2008， 博士

【摘要】 目的:充血性心力衰竭（congestive heart failure,CHF）是一种复杂的临床症候群,是多种心血管疾病的终末期表现。心力衰竭是心脏对负荷和刺激产生连续的结构和功能重构的一种动态过程,因而对心室重构的防治就成为阻止心力衰竭发生的重要环节。本研究的目的,是探讨氯沙坦（losartan,Los）联合辛伐他汀（simvastatin,Sim）对压力负荷心力衰竭大鼠心脏重构的作用,并从神经内分泌激素、细胞因子、心肌细胞凋亡以及心肌纤维化等方面,探讨其协同作用的可能机制,为临床上心力衰竭的药物治疗提供新的思路。方法:降主动脉缩窄（ascending aortic constriction,AAC）术建立大鼠压力负荷心力衰竭模型;模型建立后将SD雄性大鼠随机分成6组:正常对照组（control group,不用药物干预）)、假手术组（sham group,不用药物干预）)、模型组（ascending aorticconstriction,AAC,不用药物干预）、氯沙坦组（AAC+Los;Los,5mg/kg）、辛伐他汀组（AAC+Sim;Sim,2 mg/kg）、氯沙坦组与辛伐他汀合用组（AAC+Los+Sim;Los,5mg/kg;Sim,2 mg/kg）。术后第5天开始灌胃,每天一次,共35天。正常对照组、假手术组、模型组每天灌胃等容积蒸馏水一次;应用经胸壁超声心动图（transthoracic echocardiography,TTE）检查,心、肺重量测定,光镜、透射电镜分析细胞形态学以及心肌胶原的改变,对AAC模型进行评价并观察药物治疗各组对压力负荷大鼠心脏重构的影响;概率总和检验评价氯沙坦联合辛伐他汀对压力负荷大鼠心脏重构的协同作用;心电图检查计算心率,颈总动脉插管测定血压;直接清除法,测定血清TCH、TG、LDL-C和HDL-C的浓度;放射免疫法测定外周血浆AngⅡ、ET和ANP浓度;双抗体夹心ELISA方法检测外周血清TNF-α、IL-6、BNP和CRP的浓度;心室组织切片TUNEL法检测细胞凋亡;体外实验,Ⅱ诱导心肌细胞凋亡,应用3-（4,5-二甲基噻唑）-2,5-二苯基四氮唑溴盐（MTT）测定心肌细胞活力,荧光显微镜观察凋亡细胞形态,FITC-Annexin V和PI双重染色流式细胞仪检测心肌细胞凋亡率,流式细胞仪检测Bax和Bcl-2蛋白的表达,免疫组化测定心肌细胞中Caspase-3蛋白的表达;Western印迹法测定心室组织ERK1/2、P-ERK1/2蛋白质含量;实时荧光定量PCR,探针法检测大鼠心室组织中AT₁R mRNA表达,SYBRgreenⅠ染料法检测大鼠心室组织中MMP2、MMP9、TIMP1、TIMP2、AT₂R以及Collagen-1、Collagen-3 mRNA的表达。结果:1.压力负荷心力衰竭大鼠模型的评价TTE检查,AAC术后3周模型组即出现心脏重构现象,术后5周更加明显,表现为左心室扩大、室壁增厚、心脏射血功能下降以及左心室质量及左心室质量指数增加;术后5周器官重量,AAC组心脏重量、心脏重量指数、左心重量、左心重量指数、肺脏重量以及肺脏重量指数明显高于control、sham组;AAC组血液BNP浓度明显升高;光学显微镜下,AAC组心肌和肺组织HE染色显示:心肌纤维排列紊乱,大部分心肌细胞浊肿变性,部分心肌纤维溶解,肺泡腔缩窄,肺泡腔有少量出血,部分肺泡腔内积液,肺间质水肿,肺间质淤血,有少量炎症细胞浸润;投射电镜下,AAC组心肌纤维排列紊乱,部分Z带消失,线粒体肿胀,嵴突模糊不清,可见大量空泡变性,闰盘结构紊乱,有少量细胞核破裂,可见少量凋亡小体,胶原纤维增生明显。以上结果表明,应用AAC术能成功建立压力负荷心力衰竭大鼠模型。2心脏重构的检测TTE检查,氯沙坦联合辛伐他汀,在减轻心脏质量及心脏质量指数方面优于两药单用;药物治疗5周,各组收缩压及心率比较无显著性差异;氯沙坦和辛伐他汀均能缓解AAC导致的心脏重量、心脏重量指数、肺脏重量以及肺脏重量指数的增加,氯沙坦联合辛伐他汀效果更明显;概率总和检验,LV Mass/Body Weight、HW/BW Ratio、LV Weight/BW Ratio和Lung Weight/BW Ratio的q值分别是1.17、1.17、1.16和1.26,均大于1.15。结果表明,氯沙坦联合辛伐他汀对压力负荷心力衰竭大鼠心脏重构可能具有协同作用。3.血脂分析药物治疗5周,TCH、LDL-C、HDL-C和TG,各组比较无显著性差异。4.AngⅡ、ET-1、ANP、BNP、CRP、IL-6和TNF-α的血液浓度压力负荷心力衰竭大鼠AngⅡ、ET-1、ANP、BNP、CRP、IL-6和TNF-α血液浓度明显升高,氯沙坦、辛伐他汀以及两药合用可以明显降低压力负荷心力衰竭大鼠ET-1、ANP、BNP、CRP、IL-6和TNF-α血液浓度,而对AngⅡ的血液浓度无明显影响,两药联合比氯沙坦、辛伐他汀单用对ET-1、ANP、BNP和TNF-α血液浓度的降低作用更明显。5.AT₁R、AT₂R mRNA的表达压力负荷心力衰竭大鼠心室组织AT₁R mRNA的表达明显升高,氯沙坦对AT₁R mRNA的表达无明显影响,辛伐他汀和两药联合对压力负荷心力衰竭大鼠高表达的AT₁R mRNA有明显的下调作用;压力负荷心力衰竭大鼠心室组织AT₂R mRNA的表达与正常对照组和假手术组比较无显著性差异,辛伐他汀对AT₂R mRNA的表达无明显影响,而氯沙坦以及两药联合对AT₂R mRNA的表达有明显的上调作用。6.ERK、P-ERK蛋白表达压力负荷心力衰竭大鼠ERK1/2的磷酸化程度增加,氯沙坦、辛伐他汀以及两药合用均能部分抑制ERK1/2的磷酸化,而氯沙坦联合辛伐他汀作用更明显。7.心肌细胞凋亡心室肌组织切片TUNEL染色结果显示,药物治疗各组均能减少压力负荷造成的心肌细胞凋亡,而氯沙坦联合辛伐他汀比两药单用效果更显著;体外实验,用10（-7）mol/L的AngⅡ培养心肌细胞48小时,增加了心肌细胞的凋亡,降低了细胞的存活能力。氯沙坦(10^-5mol/L)和辛伐他汀(10^-5mol/L),无论是单独还是联合使用,均显著的减少了由AngⅡ诱导的心肌细胞凋亡,提高了心肌的存活能力。概率总和检验,细胞活力和心肌细胞凋亡q值分别是1.31和1.21,提示,氯沙坦联合辛伐他汀对提高细胞活力以及抑制AngⅡ诱导的心肌细胞凋亡具有协同作用。AngⅡ可引起心肌细胞Bax蛋白表达的显著增加,而Bcl-2蛋白表达减少。氯沙坦和联合用药可以有效地下调Bax蛋白表达,上调Bcl-2蛋白表达,而辛伐他汀没有类似的作用。AngⅡ可引起心肌细胞Caspase-3蛋白的表达显著增加,氯沙坦、辛伐他汀以及联合用药均可以有效地下调Caspase-3蛋白表达,而联合用药作用更显著。8.心肌胶原含量及MMPs、TIMPs mRNA的表达压力负荷大鼠心肌胶原含量、Ⅰ型和Ⅲ型胶原mRNA表达的比值明显增加,氯沙坦、辛伐他汀以及两药合用均能减少压力负荷引起的心肌胶原增生,降低Ⅰ型和Ⅲ型胶原mRNA表达的比值,尤其两药合用效果更明显;压力负荷心力衰竭大鼠MMP2、MMP9 mRNA的表达明显升高,氯沙坦、辛伐他汀以及两药合用对其无明显影响,压力负荷心力衰竭大鼠TIMP1、TIMP2mRNA的表达亦明显升高,而氯沙坦、辛伐他汀以及两药联合均能明显下调TIMP1、TIMP2 mRNA的表达,两药联合的下调作用更明显;结论:1.应用降主动脉缩窄术能成功建立压力负荷心力衰竭大鼠模型。主要表现为左心室扩大、室壁增厚、心脏射血功能下降、左心重量增加、肺间质水肿淤血、线粒体肿胀,嵴突模糊不清、胶原纤维增生以及血液BNP浓度明显升高。2.氯沙坦联合辛伐他汀对压力负荷心力衰竭大鼠心脏重构的抑制作用优于两药单用。3.氯沙坦联合辛伐他汀抑制压力负荷心力衰竭大鼠心脏重构的可能机制a辛伐他汀对压力负荷心力衰竭大鼠高表达的AT₁ R mRNA有明显的下调作用,氯沙坦对AT₂R mRNA的表达有明显的上调作用;b氯沙坦联合辛伐他汀比两药单用能更明显抑制ERK1/2的磷酸化;c氯沙坦联合辛伐他汀比两药单用能更有效地抑制心肌细胞凋亡、提高细胞活力、下调Caspase-3蛋白的表达;d氯沙坦联合辛伐他汀比两药单用能更明显地抑制心肌胶原增生、降低Ⅰ型和Ⅲ型胶原mRNA表达的比值;e氯沙坦联合辛伐他汀比两药单用更能明显下调TIMP1、TIMP2 mRNA的表达。更多还原

【Abstract】 Objective:Congestive heart failure（CHF） is a complex clinical syndrome that features a failing heart together with advanced signs and symptoms arising from various cardiovascular diseases.Heart failure is a successive dynamic structure and function remodeling process,which is characterized by the heart responses to overloading and some other stimulation.Thus,target at cardiac remodeling is very important therapies in preventing the progress of heart failure.In this study,we aimed to investigate the effects of combined treatment with simvastatin and losartan on cardiac remodeling in Rats. Furthermore,the mechanisms of these effects were investigated focus on neuroendocrine hormonal activation,proinflammatory cytokines,apoptosis of myocardial cell and myocardial fibrosis.This maybe can lead to the notion that new drug therapy in clinical practice for heart failure.Methods:The pressure overload dependent heart failure model in rats was induced by ascending aortic constriction（AAC）.After AAC,the animals were randomly separated 6 groups:normal control group,no operation and drug treatment;control sham group, no drug treatment;model group（AAC）,no drug treatment,losartan treatment group（AAC+Los,Los,5mg/kg）;simvastatin treatment group（AAC+Sim,Sim,2 mg/kg）; combination of losartan and simvastatin（AAC+Los+Sim;Los,5mg/kg;Sim,2 mg/kg）. Different drug was intragastric administrated to the animals daily from day 5 after operation to the end of experiment for 35 days.The control group was administered with the same volume of vehicle.The heart and lung weight was measured by transthoracic echocardiography.Cell morphology and myocardium collagen change was checked by light microscope and transmission electron microscope.These methods were used for investigating the effects of drug treatment on cardiac remodeling.The probability sum test（q test） was used to evaluate the synergism effects of simvastatin and losartan on pressure overload dependent heart failure in rats.The heart rate was calculated by electrocardiography and blood pressure was determined by carotid arterial cannula.The concentration of TCH,TG,LDL-C and HDL-C in serum was measured by the method of directly clearance.The concentration of AngⅡ,ET and ANP in blood plasma was measured by the method of radioimmunity.The concentration of TNF-α, IL-6,BNP and CRP in serum was measured by ELISA.Myocardial cell apoptosis was checked by TUNEL method.In vitro experiment,AngⅡwas used to induce apoptosis of myocardial cell.The activity of myocardial cell was checked by MTT method.Cell morphology of apoptotic cell was observed by fluorescence microscope.Double staining for FITC-Annexin V binding and for cellular DNA using propidium iodide（PI） was performed to check the ratio of apoptotic cell using flow cytometry.Bax and Bcl-2 protein expressions were measured by flow cytometry detection.The expression of caspase-3 was in myocardial cell was examined by immunohistochemistry.The protein expression of ERK1/2 and P- ERK1/2 in ventricular tissue was measured by Western blot.The mRNA expression of MMP2,MMP9,TIMP1,TIMP2,AT₂R,Collagen-1 and Collagen-3 in ventficular tissue was measured by real-time PCR.Results1.The evaluation of pressure overload dependent heart failure model in ratsThe results of TTE showed that the phenomenon of cardiac remodeling was emerged 3 weeks after AAC and more clear in 5 weeks after operation.The signs showed that left ventricular enlargement,ventricle wall thickening,the dysfunction of cardiac ejection and increasing of left ventricular mass or index of left ventricular mass. weight of heart,index of heart weight,left heart weight,index of left heart weight, weight of lung and index of lung weight of AAC group is significantly high than the group of control and sham.The concentration of BNP in serum significantly increased in AAC group.For AAC model group,the HE staining of heart and lung tissue showed as follows:The derangement structure of cardiac muscle fibers,bulk cloudy swelling degeneration of myocardial cell,fibronolysis in part of cardiac muscle fibers, coarctation of alveolar space,Congestion and edema in lung mesenchyma,small amounts bleeding and fluidify in alveolar space and inflammatory cell infiltration. Observed by projection electron microscope showed that in AAC group as follows:The derangement structure of cardiac muscle fibers,Partly disappearance in zone Z,unclear of crista mitochondriales,magnanimous vacuolar degeneration,disorder structrue of intercalary disc,apoptosis body and hyperplasy of collagen fibers.The results above showed that the pressure overload dependent heart failure model in rat was successful established by AAC.2.The assessment of cardiac remodelingThe results of TTE showed that combination of simvastatin and losartan significantly decreased the weight of heart and index of heart weight when compared with single drug treatment.There were no significant differences about systolic pressure and heart rates among all the groups with drug treatment for 5 weeks.Simvastatin and losartan alone relieved the increased of weight of heart,index of heart weight,weight of lung and index of lung weight induced by AAC.Moreover,the effects of combination of simvastatin and losartan on these indexs were much better.Using the probability sum test,the q value of LV Mass / Body Weight,HW / BW Ratio,LV Weight / BW Ratio and Lung Weight/BW Ratio were 1.17,1.17,1.16 and 1.26.All of the values were higher than 1.15.These results showed that synergism effects of simvastatin and losartan on cardiac remodeling. 3.Results of lipoprotein cholesterol and triglyceride in bloodThere were no significant differences about TCH,LDL-C,HDL-C and TG among all the groups with drug treatment for 5 weeks.4.The concentration of AngⅡ,ET-1,ANP,BNP,CRP,IL-6 and TNF-αin bloodIn AAC group,the concentration of AngⅡ,ET-1,ANP,BNP,CRP,IL-6 and TNF-αin blood significantly increased.Treatment with simvastatin and losartan alone and in combination decreased the level of ET-1,ANP,BNP,CRP,IL-6 and TNF-α,but had no effect on AngⅡ.Combination of simvastatin and losartan significantly decreased the level of ET-1,ANP,BNP and TNF-αin blood when compared with single drug treatment.5.The mRNA expression of AT₁ R and AT₂RThe mRNA expression of AT₁ R in ventricular tissue significantly increased In AAC group.Treatment with losartan had no effect on the expression of AT₁R.Simvastatin or combination of simvastatin and losartan both significantly decreased the high level of AT₁R mRNA expression by AAC.There was no significant difference of mRNA AT₂R expression among the normal and model rats.Treatment with losartan had no effect on the expression of AT₂R. Losartan or combination of simvastatin and losartan both significantly increased the AT₂ R mRNA expression.6.ERK and P-ERK proteinIn pressure overload dependent heart failure rats,the degree of phosphorylation of ERK1/2 increased,Treatment with simvastatin,losartan or combination of simvastatin and losartan all partial inhibited the phosphorylation ERK1/2.The degree of inhibition of phosphorylation ERK1/2 was much higher when combination of simvastatin and losartan compared with single drug treatment.7.Cardiomyoeyte apoptosisCardiomyocyte apoptosis was checked by TUNEL method and the results showed that drug treatment decreased the cardiomyocyte apoptosis induced by AAC.The degree of anti-apoptotic effect was much higher in the combination of two drugs when compared with single drug treatment.In vitro experiment,incubation with 10^-7 mol/L AngⅡfor 48 h increased cardiomyocyte apoptosis and decreased cell viability.Losartan(10^-5 mol/L) and simvastatin(10^-5 mol/L),either alone or in combination,significantly decreased AngⅡ-induced cardiomyocyte apoptosis and increased cell viability.The q-values calculated by the probability sum test were 1.31 and 1.21 for cardiomyocyte apoptosis and cell viability,respectively.These demonstrated that there was a synergistic effect between losartan and simvastatin in inhibiting AngⅡ-induced cardiomyocyte apoptosis and increasing cell viability.AngⅡinduced a significant increase in Bax protein expression but decreased those of Bcl-2.Losartan decresed Bax expression and increased Bcl-2 expression while simvastatin had no such effect.AngⅡinduced the protein expression of caspase-3 in cardiomyocyte.Simvastatin,losartan or combination of simvastatin and losartan significantly decreased the protein expression of caspase-3.The degree of inhibition in expression of caspase-3 was much higher when combination of simvastatin and losartan compared with single drug treatment.8.Collagen production and the mRNA expression of MMPs and TIMPsIn AAC group,the mRNA expression of collagenⅠandⅢsignificantly increased. Treated with simvastatin and losartan alone and in combination,the collagen and the ratio of mRNA expression of collagenⅠandⅢsignificantly decreased.The degree of inhibition in the ratio of collagenⅠandⅢmRNA expression was much higher when combination of simvastatin and losartan compared with single drug treatment. In AAC group,the mRNA expression of MMP2 and MMP9 significantly increased. Treatment with simvastatin and losartan alone and in combination had no effects on the mRNA expression of MMP2 and MMP9.Also,the mRNA expression of TIMP1 and TIMP2 significantly increased In AAC group.Treatment Simvastatin and losartan alone and in combination significantly decreased the TIMP1 and TIMP2 mRNA expression. The degree of inhibition in the ratio of TIMP1 and TIMP2 mRNA expression,was much higher when combination of simvastatin and losartan compared with single drug treatment.Conclusions1.The pressure overload dependent heart failure model in rat was successful established by ascending aortic constriction.2.Combination of simvastatin and losartan significantly inhibited cardiac remodeling induced by AAC when compared with single drug treatment.3.Some mechanisms of effects of combination simvastatin and losartan on cardiac remodeling were investigated as follows.a,Combination of simvastatin and losartan significantly decreased the protein expression of caspase-3 when compared with single drug treatment.b,Combination of simvastatin and losartan significantly increased the activity of cells and inhibited the apoptosis of myocardial cell induced by AngⅡwhen compared with single drug treatment.c,Combination of simvastatin and losartan significantly inhibited myocardium collagen production induced by AAC when compared with single drug treatment.More interesting,Combination treatment significantly decreased the ratio of mRNA expression of collagenⅠand collagenⅢ.d,Combination of simvastatin and losartan significantly decreased the mRNA expression of TIMP 1 and TIMP2 when compared with single drug treatment.e,Simvastatin significantly decreased the high level mRNA expression of AT₁R by AAC;losartan significantly increased the mRNA expression of AT₂ R.f,Combination of simvastatin and losartan significantly inhibited the phosphorylation ERK1/2 when compared with single drug treatment.g,Combination of simvastatin and losartan significantly decreased the concentration of ET-1,ANP,BNP and TNF-αin blood when compared with single drug treatment.更多还原