【作者】 孙颖颖；

【导师】 葛志明；

【作者基本信息】 山东大学 ， 内科学， 2009， 博士

【摘要】 研究背景左室肥厚是高血压最常见的靶器官损害之一。而左室肥厚最终可进展至心功能不全和/或心力衰竭（HF）。在高血压的早期即可出现左室舒张功能障碍,且发生在收缩功能受损之前。但高血压左室肥厚致左室舒张功能障碍的发生机制目前尚未完全阐明。研究发现,高血压主要靶器官（心、脑、肾）中均存在细胞凋亡现象,而且心肌细胞的凋亡和心肌肥厚、心功能障碍均密切相关。因此认为,大量心肌细胞的凋亡可能是促使心肌肥厚走向心功能障碍的重要原因。内质网应激（endoplasmic reticulum stress,ERS）是内质网功能紊乱时出现错误折叠与未折叠蛋白在腔内聚集以及Ca²⁺平衡紊乱的状态。多种生理或病理条件,如分泌细胞过度分泌、分泌性蛋白基因突变、化学物质致内质网损伤等均可引起ERS。目前已经明确的ERS介导的凋亡通路有三条:①CHOP/GADD153（growtharrest and DNA damage-inducible gene 153）基因的激活转录;②JNK（c-JunNH2-terminal protein kinase）的激活通路;③内质网特有的半胱氨酸蛋白酶Caspase-12的激活通路。经研究发现ERS作为一条应激通路,参与了多种疾病的发生,同时也发现ERS参与介导心肌细胞的凋亡。作为应激状态下普遍存在的一种亚细胞器病理过程,ERS是否也在高血压诱导的左室肥厚致左室舒张功能障碍的过程中发挥作用目前仍尚不清楚。全身及心肌局部肾素-血管紧张素系统（RAS）的激活是高血压重要的病理生理改变。血管紧张素Ⅱ（AngⅡ）是RAS系统中最重要的效应分子。越来越多的实验证实,AngⅡ是促使心肌细胞凋亡的重要诱因之一。AngⅡ受体拮抗剂是高血压,特别是高血压伴左室肥厚患者的一线药物。但该类药物改善左室重构是否与参与调节ERS有关尚未明确。本研究希望能为高血压的左室重构在亚细胞器和分子水平引入新的概念和理论,并为左室肥厚以及左室舒张功能障碍的预防和治疗提供新的靶点。研究目的（1）验证高血压左室肥厚致左室舒张功能障碍过程中存在细胞凋亡的增加,明确凋亡在高血压致心肌功能障碍过程中发挥的作用。（2）明确高血压左室肥厚致左室舒张功能障碍过程中是否存在ERS的激活。（3）明确高血压左室肥厚致左室舒张功能障碍过程中是通过哪条或哪几条与ERS相关的途径介导心肌细胞凋亡。（4）明确AT1受体拮抗剂是否通过参与调节ERS发挥逆转左室肥厚、延缓心衰的出现和发展的作用。研究方法1动物模型构建1.1高血压所致左室肥厚以及左室舒张功能障碍模型的建立购买普通Wistar-Kyoto（WKY）大鼠（8周龄）30只和自发性高血压大鼠（SHRs）（8周龄）30只。随机各取10只8周龄SHRs和10只WKY大鼠处死后保留心脏标本,余给予普通饲料分别喂养至16周、32周。应用超声心动图等技术判定各组大鼠左室肥厚程度以及左室舒张功能。大鼠符合实验要求后全部处死,留取标本。1.2缬沙坦的干预实验选用SHRs和WKY大鼠为研究对象。购买普通WKY大鼠（16周龄）10只和SHRs（16周龄）20只。随机选取10只SHRs大鼠,给予缬沙坦进行药物干预（20mg/kg/d）,其余大鼠均给予等量生理盐水,1次/d。所有大鼠均普食喂养至32周,全部处死后留取标本。2大鼠收缩压的测定大鼠40℃预热5-10分钟,采用RBP-1型大鼠无创尾动脉血压测定分析系统,测量大鼠安静、清醒状态下尾动脉收缩压和心率,连测三次,取其平均值。3大鼠左室重量指数（left ventricular mass index,LVMI）的比较LVMI=左心室重量(LVM,mg/体重（BM,g）。用分析天平称取LVM（包括室间隔）,计算LVMI。4超声心动图检测分别于高血压左室肥厚、左室舒张功能障碍建模前以及建模后进行常规超声心动图检查。测定如下指标:室间隔（Interventricular septum,IVS）和左室后壁（LVPosterior wall,LVPW）厚度:左室射血分数（left ventricular ejective fraction,LVEF）;左室短轴缩短率（fractional shortening,FS）;二尖瓣E波最大速度、A波最大速度、E/A比值和等容舒张时间（Isovolumic relaxation time,IVRT）并观察瓣膜返流情况。5心肌组织病理学检查大鼠处死后,进行组织取材、固定、脱水、透明、浸蜡、石蜡包埋、切片,常规HE染色,观察心肌细胞形态并拍片。6 TUNEL染色法鉴定凋亡细胞取组织切片,进行TUNEL染色,观察细胞凋亡水平。7免疫组织化学检测取组织切片,进行GRP78、CHOP、p-JNK、Caspase-12、Caspase-3的免疫组织化学检测。所用一抗分别为:GRP78多克隆抗体（SantaCruz公司,1:200稀释）;CHOP多克隆抗体（SantaCruz公司,1:200稀释）;p-JNK多克隆抗体（SantaCruz公司,1:300稀释）;Caspase-12多克隆抗体（SantaCruz公司,1:100稀释）;Caspase-3多克隆抗体（SantaCruz公司,1:150稀释）。8免疫印迹检测取左室组织,提取总蛋白,经过SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离、转膜、蛋白印记、ECL显色等步骤,检测GRP78、CHOP、p-JNK、JNK、Caspase-12、Caspase-3的蛋白表达。9 real time RT-PCR法检测分别从左室组织提取总RNA,经逆转录反应（RT）得到cDNA,以GAPDH作为参照,通过real time RT-PCR技术检测GRP78、CHOP、p-JNK、Caspase-12 mRNA的表达。研究结果1实验动物基本情况及血压检测（1）最终全部大鼠完成了实验。SHRs血压呈持续升高趋势。与同周龄的WKY大鼠相比,8周龄、16周龄、32周龄组SHRs的血压均显著升高（P＜0.05）。（2）32周龄缬沙坦干预组大鼠较32周龄组SHRs血压显著降低（P＜0.05）,与32周龄组WKY大鼠相较无显著性差异。2左室重量指数（left ventricular mass index.LVMI）的比较（1）与同周龄的WKY大鼠相比,8周龄组SHRs的LVMI未见明显变化;16周龄、32周龄组SHRs的LVMI均显著增加（P＜0.05）。与8周龄组SHRs相比,16周龄、32周龄组SHRs的LVMI均显著增加（P＜0.05,P＜0.01）。（2）32周龄缬沙坦干预组大鼠较32周龄组SHRs的LVMI显著降低（P＜0.05）。3超声心动图检测（1）选用IVS和LVPW评价大鼠的左室肥厚程度;E/A比值、IVRT评价大鼠左室舒张功能;EF、FS评价大鼠左室收缩功能。与同周龄的WKY大鼠相比,16周龄和32周龄组SHRs的IVS和LVPW均明显增加（P＜0.05）:16周龄组SHRs的IVRT明显延长（P＜0.05）,E/A比值明显降低（P＜0.05）;32周龄组SHRs的E/A比值显著增加（E/A＞＞2）,出现左室舒张功能障碍的E/A比值变化趋势:早期降低,晚期升高,提示32周龄组的SHRs大鼠的心脏舒张功能己明显受损。EF以及FS在各组大鼠间均无显著性差异。（2）同样选用上述指标评价表明:缬沙坦干预组大鼠较同周龄的SHRs相比,IVS和LVPW,E/A比值以及IVRT均显著降低（P＜0.05）;EF以及FS在两组间均无显著性差异。4光镜标本观察（HE染色）（1）HE染色显示,对照组大鼠心肌细胞排列整齐,细胞核大小均一,胞浆染色均匀;8周龄组SHRs与对照组比较未见显著性变化,仍可见心肌细胞排列整齐,细胞核大小均一,胞浆染色均匀;16周龄组SHRs心肌细胞肥大,细胞核出现增大、畸形等改变,肌纤维排列较紊乱;32周龄组SHRs可出现心肌细胞的变性,呈局灶性、片状坏死,心肌纤维粗大、断裂等。微血管管壁增厚,官腔狭小,管周纤维组织增多。（2）较32周龄组SHRs相比,32周龄缬沙坦干预组大鼠心肌细胞肥大,轻度变性,心肌纤维无断裂,心肌细胞核形状略不整,微血管管壁、管腔均正常,肌纤维排列较整齐。5 TUNEL染色（1）与同周龄的WKY大鼠相比,8周龄组SHRs的心肌细胞凋亡无明显增加;16周龄组SHRs的心肌细胞凋亡明显增加（P＜0.05）;32周龄组SHRs的心肌细胞凋亡显著增加（P＜0.01）。与8周龄组的SHRs相比,16周龄、32周龄组SHRs心肌细胞凋亡均显著增加（P＜0.05,P＜0.01）。（2）较32周龄组SHRs相比,32周龄缬沙坦干预组大鼠心肌细胞凋亡率明显降低（P＜0.05）。6免疫组织化学检测（1）GRP78、CHOP、Caspase-12、Caspase-3蛋白均定位于心肌细胞胞浆内,呈浅棕色颗粒。与同周龄的WKY大鼠相比,各组SHRs心肌细胞胞浆内GRP78、CHOP蛋白的表达均显著增加（P＜0.05）;Caspase-12、Caspase-3蛋白只在16周龄和32周龄组SHRs心肌细胞胞浆内的表达显著增加（P＜0.05）;p-JNK蛋白的表达在各组间未见明显差异。（2）较32周龄组SHRs相比,缬沙坦干预组大鼠心肌细胞胞浆内的GRP78、CHOP、Caspase-3蛋白表达均显著减少（P＜0.05）;p-JNK和Caspase-12蛋白表达未见明显变化。7免疫印迹检测（1）与同周龄的WKY大鼠相比,各组SHRs心肌组织的GRP78、CHOP蛋白表达均显著增加（P＜0.05,P＜0.01）;Caspase-12和Caspase-3蛋白只在16周龄和32周龄组SHRs心肌组织中表达增多（P＜0.05,P＜0.01）;JNK和p-JNK的蛋白表达各组间均无显著差异。（2）较32周龄组SHRs相比,32周龄缬沙坦干预组大鼠心肌组织蛋白GRP78、CHOP的蛋白表达显著减少（P＜0.05）。Caspase-12的蛋白表达两组间未见明显变化。8 real time RT-PCR法检测（1）与同周龄的WKY大鼠相比,各组SHRs心肌组织的GRP78、CHOP mRNA表达均显著增加（P＜0.05,P＜0.01）;Caspase-12 mRNA表达只在16周龄和32周龄组SHRs心肌组织中明显增多（P＜0.01）;p-JNK mRNA表达在各组间无显著变化。（2）较32周龄组SHRs相比,32周龄缬沙坦干预组大鼠心肌组织的GRP78、CHOP mRNA表达显著减少（P＜0.05）;Caspase-12 mRNA表达两组间未见显著变化。研究结论（1）验证了高血压左室肥厚致左室舒张功能障碍过程中存在心肌细胞凋亡的增加,证实凋亡在高血压致心肌功能障碍过程中发挥重要作用。（2）SHRs心肌组织GRP78的表达升高,证实高血压左室肥厚致左室舒张功能障碍过程中存在ERS的激活。（3）SHRs心肌组织CHOP、Caspase-12表达增高,证实高血压左室肥厚致左室舒张功能障碍过程中是通过ERS相关的CHOP和Caspase-12通路介导心肌细胞凋亡。（4）缬沙坦可能通过抑制CHOP通路的激活来减少心肌细胞的凋亡,从而发挥其逆转左室肥厚、延缓心衰出现和发展的作用。更多还原

【Abstract】 PartⅠEndoplasmic Reticulum Stress Involved with Myocardial Apoptosis in Spontaneously Hypertensive RatsBackgroundIt is well known that left ventricular hypertrophy commonly occurs in hypertensive patients.However,the mechanisms in the progression from left ventricular hypertrophy to left ventricular diastolic dysfunction resulting from hypertension are not fully known.Emerging data now indicate that apoptosis occurs in the critical organs(heart,brain,or kidney) in hypertension.Therefore,we hypothesized that myocardial apoptosis may contribute the incidence of the left ventricular diastolic dysfunction.Endoplasmic reticulum(ER) is an organelle involved in the intrinsic pathway of apoptosis and it is involved in several important functions such as the folding of secretory and membrane proteins.Various conditions can disturb the functions of the ER and result in ER stress(ERS).When ERS conditions persist,initiation of apoptotic processes are promoted by CHOP/GADD153(growth arrest and DNA damage-inducible gene 153),the Caspase-12-dependent pathway,and activation of the c-Jun NH2-terminal kinase(JNK)-dependent pathway.Recent studies have also demonstrated that the apoptosis of cardiocytes is bound up with ERS.However,the mechanisms by which ERS lead to cell death remain enigmatic,particularly in the progression from left ventricular hypertrophy to left ventricular diastolic dysfunction resulting from hypertension.Valsartan is a kind of angiotensis receptor blocker which not only ameliorate hypertension but also protect target organ uniquely.Based on Val-HeFT,valsartan significantly reduces the combined end point of mortality and morbidity and improves clinical signs and symptoms in patients with heart failure,when added to prescribed therapy.The mechanisms of valsartan’s effects are complicated,and whether or not the mechanism related to ERS is also unclear.Objectives1.To definite the function of apoptosis in the progress of myocardial dysfunction induced by hypertension.2.To observe the expression of ERS-relative factors in the myocardium in SHRs and controls.3.To observe the relationship between the interventive effect of valsartan and ERS.Methods1.Establishment of animal model1.1 Establishment of cardiac hypertrophy and animal model of the left ventricular diastolic dysfunctionThirty SHRs(8 weeks old,SPF,male) and thirty WKY rats(8 weeks old,SPF,male) were taken from vital river laboratories.The rats were housed in cages,and had free access to standard rat diet and tap water of the center.They were maintained under conditions of standard lighting(alternating 12h light/dark cycle),temperature (22±0.5℃) and humidity(60±10%) for at least 1 week before the experiments.Then, ten SHRs or ten WKY rats were randomly killed in each group when they were 8 weeks,16 weeks and 32 weeks old.All the rats drank freely and their blood pressure was measured every 2 weeks.1.2 the Interventive Effect of valsartanTwenty SHRs(16weeks old,SPF,male) and ten WKY rats(16weeks old,SPF,male) were taken from vital river laboratories.Under the same raising conditions,they were feed for at least 1 week before the experiments.Twenty SHRs were randomly divided into two groups of ten.The first group of SHRs and the ten WKY rats were fed normally to grow for 32 weeks and the other group was fed valsartan(20mg/kg/d) until they were 32 weeks old.All the rats drank freely and their blood pressure was measured every 2 weeks.Both groups of SHRs were killed after the defined period of growth.2.Measure Blood PressureAll the rats drank freely and their blood pressure was measured every 2 weeks.3.Blood pressure and left ventricular mass index(LVMI)LVMI=left ventricular mass(LVM,mg)/body mass(BM,g).4.Echocardiographic examinationAt the beginning and the end of the study,transthoracic echocardiogram was performed in hypertensive and control animals.Rats were placed supine and the anterior chest wall was shaved.Echocardiograms were performed with a Hewlett-Packard Sonos 7500 sector scanner equipped with a 7.5-MHz phased-array transducer.Conventional images included 2-dimensional,M-mode,and continuous wave and pulsed Doppler images.5.HE stainingHE staining was used to study the pathological changes in this study.6.TUNEL stainingTUNEL staining was used to detect the expression of apoptotic cells.7.ImmunohistochemistryImmunohistochemistry was used to detect the protein expression of GRP78,CHOP, p-JNK,Caspase-12,Caspase-3.8.Western-blot analysisWestern-blot analysis was used to determine the protein expression of GRP78,CHOP, p-JNK,JNK,Caspase-12,Caspase-3.9.real time RT-PCR analysisThe mRNA expressions of GRP78,CHOP,p-JNK,Caspase-12 were determined by real time RT-PCR.Results1.Blood pressure and LVMISystolic blood pressure of SHRs group were kept on at high level.Blood pressure and LVMI differed significantly(P＜0.05) in the animals of SHRs compared with the sex-matched control WKY rats.After treatment of valsartan,the level of blood pressure was obviously decreased(P＜0.05) compared with the sex-matched SHRs.2.Establishment of the left ventricular hypertrophy and left ventricular diastolic dysfunctional model(1).Left ventricular diastolic function variables expressed by the ratio of E-wave(early diastolic filling,early peak velocity) and A-wave(late atrial filling,atrial peak velocity) differed significantly(P＜0.05) in SHRs(16 and 32 weeks) compared with those in WKY rats(16 and 32 weeks).A significant decrease in the E-wave velocity,significant increase in the A-wave velocity,and remarkable decrease in the E/A ratio was found in SHRs(16 weeks) and remarkable increase in the E/A ratio was found in SHRs(32 weeks).Left ventricular systolic function parameters including fractional shortening (%),and ejection fraction(%) of the SHRs were no significant difference compared with the WKY rats(32 weeks).Forthermore,the thickness of interventricular septum and left ventricular posterior wall and LVMI also differed significantly(P＜0.05) compared with those in WKY rats at 16 weeks.These results demonstrated that SHRs had cardiac hypertrophy at 16 weeks of age and had left ventricular diastolic dysfunction at 32 weeks old.(2).In the valsartan group,left ventricular diastolic function was obviously improved (P＜0.05) compared with SHRs group.3.HE stainingWith hematoxylin-eosin staining,we found that the cell size was increased significantly in the hearts of SHRs(16weeks) and cardiac muscle fibers(32weeks of SHRs) were disordered,and many of them were collapsed.In the valsartan group, myocyte hypertrophy was improved obviously.4.TUNEL stainingTo assess whether cardiac myocyte apoptosis in hypertensive heart,the tissue sections were labeled with an in situ TUNEL assay.Apoptosis was observed in both the cardiomyocyte and the endothelium of the hypertensive heart.More apoptotic cardiocytes(P＜0.05) were found from SHRs(16 weeks and 32 weeks) groups. Estimation of cardiac apoptosis revealed a nearly threefold increase in TUNEL-positive nuclei in hypertensive heart.In the valsartan group,the level of apoptosis was significantly(P＜0.05) decreased compared with SHRs group.5.ImmunohistochemistryImmunohistochemistry studies showed that GRP78,CHOP,Caspase-12,Caspase-3 were all abundantly expressed in the myocardium of SHRs.In contrast,WKY rats exhibited modest or weak immunoreactivity for this molecule.We also found that in the myocardium of SHRs,the increasing of GRP78,CHOP,Caspase-12 and Caspase-3 positivecells paralleled with the increasing of apoptotic cells.In the valsartan group, the number of cardiac myocyte apoptosis and the expression of GRP78,CHOP and Caspase-3 were significantly(P＜0.05) decreased.However,a similar level of p-JNK protein was observed in all hypertensive and normal rats.6.Western blot analysis of GRP78,CHOP,p-JNK,JNK,Caspase-12 and Caspase-3The densitometric analysis of bands for GRP78,CHOP,Caspase-12,Caspase-3 but not p-JNK,JNK revealed a significant(P＜0.05,P＜0.05) increase in relative protein content in myocardium from SHRs(16weeks,32weeks) in comparison with those from WKY rats.During the initial stage of hypertension,only the protein levels of GRP78 and CHOP were upregulated(P＜0.05).In the valsartan group,protein level of GRP78, CHOP but not Caspase-12 significantly(P＜0.05) decreased compared with SHRs group.7.real time RT-PCR analysis of GRP78,CHOP,p-JNK and Caspase-12 expressionThe mRNA levels of GRP78,CHOP,Caspase-12,but not p-JNK were found to be significantly(P＜0.01) upregulated in the hypertrophic and diastolic dysfunctional heart. However,during the initial stage of hypertension,only the mRNA levels of GRP78 and CHOP were upregulated.A similar level of p-JNK mRNA was observed in all hypertensive and normal rats.In the valsartan group,mRNA levels of GRP78,CHOP were diversity(P＜0.05) compared with SHRs group but not Caspase-12. Conclusions1.Myocardial apoptosis plays an important role in the progress of myocardial dysfunction induced by hypertension.2.Increase expression of GRP78,CHOP,Caspase-12 but not p-JNK in hypertensive heart paralleled with the increase of apoptotic cells.These results suggest that ERS can contribute to cardiac myocyte apoptosis in the progress of myocardial dysfunction induced by hypertension.3.Valsartan could significantly reduced cardiac myocyte apoptosis by inhibiting the expression of CHOP.更多还原