【作者】 游敏生；

【导师】 温进坤；

【作者基本信息】 河北医科大学 ， 中西医结合基础， 2009， 博士

【摘要】 急性心肌梗死(acute myocardial infarction, AMI)后心肌,特别是左室心肌细胞及细胞外基质(extracellular matrix, ECM),在结构、功能、形态等方面而出现的适应性、增生性变化称为AMI后心室重塑。AMI后心室重塑是发生心力衰竭的重要病理生理基础,也是影响AMI患者预后的主要原因之一。研究表明,AMI后心室重塑的发生发展与激素和体液因素,如肾素-血管紧张素-醛固酮系统、循环-内分泌系统平衡失调密切相关。临床上用血管紧张素转化酶抑制剂(ACEI)或血管紧张素受体拮抗剂(ARB)防止AMI后心室重塑取得较好效果,一些单味中药、复方制剂、重要注射剂也能有效逆转心室重塑。近年发现,AMI早期心肌缺血和损伤部位各种细胞释放大量促炎细胞因子,这些因子所引发的炎性反应在AMI诱发心室重塑中发挥重要作用。因此,寻找有效的早期炎症干预措施,对于减缓心室重塑具有重要意义。已经证明,核因子κB(nuclear factorκB, NF-κB)是调节促炎细胞因子、趋化因子、黏附分子等基因表达的关键转录因子,参与激活上百种促炎反应相关基因的表达,介导炎性反应、免疫应答、细胞增殖等多种细胞生物学事件。促炎细胞因子有白细胞介素(IL)、肿瘤坏死因子(TNF-α)和干扰素(IFN)等多种,它们大多均为NF-κB的靶基因。近年证明,TNF-α在梗死后的心肌组织中表达上调,并进而介导胶原和基质金属蛋白酶基因的表达,被认为在AMI后心室重塑中发挥重要作用。转化生长因子β(TGF-β),虽然可抑制促炎细胞因子的生成,但依据细胞所处环境的不同,兼有抗炎和促炎双重作用。现有研究结果表明,TGF-β在心肌缺血损伤部位表达增加,在心肌纤维化早期和心室重塑过程中起重要作用。基于这些研究结果,TNF-α和TGF-β拮抗剂作为抗炎药物在动物模型上对防治AMI后心室重塑取得一定效果。通心络是基于络病理论研制的防治心血管病变的代表方,该方可通过降低心肌局部血管紧张素Ⅱ水平、调节降钙素基因相关肽(CGRP)和内皮素(ET)之间的平衡、抑制胶原纤维合成等多条途径防治AMI后心室重塑。但该方对AMI引发的早期炎性反应是否具有拮抗效应及其作用机制目前尚不清楚。本研究以急性心肌梗死诱发心室重塑的炎性反应机制为切入点,建立大鼠AMI模型,以促炎细胞因子为主要观测指标,系统研究通心络对AMI诱发心肌炎性反应的拮抗效应及其作用机制。1通心络对急性心肌梗死诱发的NF-κB表达的影响结扎大鼠冠状动脉左前降支制备AMI动物模型后,分为大剂量通心络组、小剂量通心络组、卡托普利组和对照组。给药4周后处死动物,TCC染色法测量心肌梗死面积,组织切片经HE染色后观察形态学变化,电镜观察超微结构改变,RT-PCR检测NF-κB基因表达。1.1大鼠AMI模型的鉴定大鼠行冠脉左前降支结扎术后,取心脏常规固定、切片后,进行TCC染色,测灰白色区域的面积,灰白色区域的面积与左心室重量比值,结果表明,各组梗死面积无显著差异,证实AMI动物模型制备成功。1.2通心络对缺血心肌形态结构和炎性反应的影响形态学分析结果显示,假手术组心肌细胞核呈紫红色,细胞质淡染,呈有序性排列;模型组缺血区心肌细胞结构模糊,炎性细胞浸润明显,增多的胶原组织呈紫蓝色,纤维母细胞增多,提示心肌开始出现纤维化趋势;大剂量通心络组的心肌细胞形态改变较轻微,只有少量中性粒细胞浸润和心肌细胞轻度肿胀变性,胶原纤维明显少于模型组;卡托普利组与大剂量通心络组变化一致形态学分析表明,通心络和卡托普利都可改善缺血和坏死引发的炎性细胞浸润,减轻缺血区的炎症反应和心肌纤维化程度。免疫组化染色结果显示,假手术组心肌细胞内的NF-κB阳性染色颗粒很少且染色较浅;模型组的NF-κB阳性染色细胞数量明显多于假手术组;大剂量通心络组和卡托普利组的NF-κB表达较模型组明显减少;而小剂量通心络组对NF-κB蛋白表达无明显影响。1.3通心络对缺血心肌细胞超微结构的影响扫描电镜观察可见,假手术组心肌细胞膜连续、完整;粗细肌丝排列整齐,肌小节及明暗各带清晰可见;线粒体丰富、大小均一、圆形或椭圆形,基质浓密一致,肌节排列整齐,横纹清楚。模型组心肌线粒体超微结构可见明显空泡化,部分双层膜融合,模糊不清,糖原颗粒减少,肌节排列紊乱,横纹消失;核多见深的切迹,核形状极不规则,染色质成簇状聚集在核一端。大剂量通心络组,心肌组织的破坏程度较模型组有明显减轻,线粒体明显增多,大小较一致,排列较规整,仅有轻度空泡化现象,部分双层膜熔合,模糊不清,但仍显示肌节排列稍紊乱,横纹不清。卡托普利组心肌线粒体轻度空泡化,线粒体轻度水肿,部分或大部分嵴融合模糊不清,有的嵴断裂,有的嵴缺失,糖原数量轻度减少,肌节横纹不清。小剂量通心络组效果不明显,线粒体部分或大部分嵴融合,有的线粒体部分或大部分空泡化,有的完全空泡化。肌节横纹消失。线粒体排列极不规则。核多见深的切迹,核形状极不规则,核染色质不均匀,染色质变淡。1.4通心络对NF-κB表达的影响RT-PCR结果显示,与假手术组相比,模型组NF-κB mRNA表达明显升高(P<0. 01);大剂量通心络组和卡托普利组NF-κB mRNA的表达明显低于模型组(P<0.05)。免疫组化染色结果显示,假手术组心肌细胞内的NF-κB阳性染色颗粒很少且染色较浅,模型组的NF-κB阳性染色细胞数量明显多于假手术组;大剂量通心络组和卡托普利组的NF-κB表达较模型组明显减少;而小剂量通心络对NF-κB蛋白表达无明显影响。2通心络对转化生长因子-β1表达的影响TGF-β1过度表达与心肌纤维化和心室重塑密切相关,是心肌梗死后心室重塑的主要诱发因子。因此,抑制TGF-β1的表达,已成为防治心肌纤维化和心室重塑的重要策略之一。在第一部分的研究中,已经证实,给予急性心梗的大鼠通心络后,可明显减轻缺血心肌的炎症反应,减少胶原纤维的合成。提示该药物具有抗炎和抗心肌纤维化的功效。在此基础上,本部分研究以TGF-β1为观测点,观察通心络对该基因表达的影响,旨在评价该药物抑制心肌缺血后炎症反应的继发效应并探讨该药物抗心肌纤维化的作用机制。2.1通心络对胶原纤维合成的影响心肌组织切片经Masson染色观察可见,假手术组的心肌细胞浆呈淡红均一着色,细胞间质中,仅见少量稀疏的、蓝色淡染的胶原纤维,散在分布于间质中。模型组心肌细胞胞浆着色不均一,间质中蓝色条索状的胶原纤维弥漫性分布,心肌细胞数量明显减少,可见粗大胶原纤维相互连接成网状,排列紊乱,心肌发生明显的纤维化。大剂量通心络组的心肌细胞胞浆着色均匀,蓝色胶原纤维呈局限或网状分布,心肌纤维化程度较模型组明显减轻,呈现散在片状的心肌纤维化;而小剂量通心络组较模型组比较,无明显变化。2.2通心络对间质肥大细胞浸润的影响甲苯氨蓝染色显示心肌缺血区的肥大细胞,镜下可见,假手术组大鼠心肌细胞间质中仅存在少量肥大细胞(3±1.06个/mm2),模型组心肌组织中肥大细胞的数量明显增多(22.17±3.87/mm2),大剂量通心络组的心肌组织中,肥大细胞数量(9.47±1.39/mm2)较模型组明显减少(P<0.05),但小剂量通心络组(21.17±4.54/mm2)与模型组比较肥大细胞数量无明显差异。2.3通心络对TGF-β1表达的影响将心肌组织匀浆后,提取上清液,用ELISA法检测组织中TGF-β1的含量。结果显示,在模型组的心肌组织中,TGF-β1 230.56±20.67 ng/ml,与假手术组78.30±45.43 ng/ml比较,具有明显差异(p<0.05);给予大剂量通心络后,TGF-β1水平降低至接近假手术组(p<0.05),但小剂量通心络对TGF-β1无明显影响。从各组大鼠心肌组织中提取总RNA,采用RT-PCR扩增TGF-β1 mRNA。扩增产物经电泳分离和EB染色后,于紫外光下,观察扩增产物的长度并对其进行相对定量分析。结果显示,各组心肌组织中均有不同程度的TGF-β1基因表达。密度扫描分析显示,模型组中TGF-β1 mRNA水平明显升高(0.773±0.09),约为假手术组(0.114±0.02)的7倍,给予大剂量通心络,其心肌组织中TGF-β1 mRNA水平(0.378±0.07)明显低于模型组(p<0.05)。这些结果提示,通心络通过抑制心肌组织TGF-β1基因表达,进而减缓心肌缺血后心肌纤维化的进程。3通心络抑制肿瘤坏死因子基因表达第一部分的研究已经证实,大剂量通心络能够在急性心梗早期抑制NF-κB。NF-κB是多种致炎基因表达的转录激活因子,其表达上调意味着炎性基因表达活性增强。TNF-α是心肌缺血诱导释放的致炎细胞因子,为探讨通心络的抗炎的抗炎效应是否与抑制TNF-α表达有关,本部分研究通过观察急性心肌梗死以及通心络干预后,心肌组织中TNF-α含量及基因转录活性的变化,进一步明确通心络的抗炎效应,为通心络的提供新的研究证据和作用机制。3.1通心络对心脏功能的影响结果显示,各组治疗前,各组大鼠的心肌梗死面积无明显差异,除假手术组外,其他各组大鼠的EDD明显增大,FS和EF明显减小,表明急性心梗模型是成功的。药物治疗后,大剂量通心络组和卡托普利组的EDD与治疗前相比明显减小(p<0.05),而FS和EF与治疗前相比明显增大(p<0.05)。提示通心络具有改善心肌收缩功能、提高心肌收缩力和心脏射血能力的作用。3.2通心络对TNF-α表达的影响用ELISA法检测心肌组织中TNF-α的水平。结果显示,在模型组的心肌组织中,TNF-α含量为245.56±0.67 ng/ml,与假手术组180.30±20.46 ng/ml比较,具有明显差异(p<0.01);给予大剂量通心络后,TNF-α水平降低致接近至假手术组(p<0.01);但小剂量通心络对TNF-α水平无明显影响。实验结束时从各组动物心肌组织中提取总RNA,用RT-PCR检测TNF-α基因表达水平。结果显示,各组大鼠心肌组织中均可扩增出TNF-αcDNA片段。密度扫描分析表明,模型组TNF-α表达水平较假手术组明显增高(p<0.05),大剂量通心络组和卡托普利组的TNF-αmRNA表达量明显减少(p<0.05),而小剂量通心络组TNF-αmRNA表达较模型组无明显变化。4通心络抑制急性心梗炎症反应和改善心功能的临床研究4.1通心络对急性心梗患者心脏功能的影响治疗前,常规治疗组和通心络治疗组的患者心脏超声心动图均显示EDD增大,FS和EF减小。通心络治疗8周后,心脏超声心动图EDD与常规治疗组相比明显减小(p<0.05)。治疗前,常规治疗组和通心络治疗组比较,心脏超声心动图EDD和EF无明显差异,通心络治疗8周后,左室收缩功能(LVEF、FS、PER)明显增强,左室舒张功能(PFR、1/3FF)明显改善。4.2通心络对患者血浆TNF-α水平的影响用ELISA法检测患者血浆TNF-α的水平。结果显示,治疗前常规治疗组和通心络治疗组的TNF-α水平明显升高,通心络治疗组8周后TNF-α水平明显降低。结论1大鼠AMI模型缺血区心肌细胞形态及超微结构发生改变,炎性细胞浸润明显,胶原纤维合成增加,心脏功能受损。2通心络能有效抑制缺血心肌组织的炎性反应,改善心肌缺血和心肌结构,抑制胶原合成,减缓心肌纤维化进程。3通心络抗炎和抗纤维化的作用机制与其抑制NF-κB、TNF-α和TGF-β1表达,降低心肌组织中的TNF-α和TGF-β1有关。4通心络治疗能降低急性心梗患者血浆TNF-α水平,改善患者心脏功能。更多还原

【Abstract】 Ventricular remodeling is the result of molecular, cellular and interstitial changes in response to acute myocardial infarction, representing adverse alterations in the size, shape and function of the left ventricle. Ventricular remodeling exists during the whole pathophysiological course of acute myocardial infarction. Recently, some scientists have proposed that ventricular remodeling may be one of the main reasons for the recovery in patients with acute myocardial infarction. Cardiac remodeling after myocardial infarction is a significant pathological basis contributing to eventual heart failure. The systolic, diastolic function damages and structure changes in heart usually affect ventricular remodeling. The progressed cardiac function deterioration is very correlative with ventricular remodeling. The recent studies show that myocardial inflammatory response induced by acute myocardial infarction plays an important role in ventricular remodeling.Nuclear factor NF-κB, a protein that binds specifically to an enhancerκB sequence ofκB immunoglobulin light chain gene, can activate transcription and expression of multiple cytokines by specific binding to their promoter or enhancer regions, and promote inflammation and immunological response, and has been closely correlated with certain important pathological and physiological processes including cell proliferation, transformation and apoptosis. NF-κB plays an important role in the development and progress of some diseases, thus becoming a new research hot spot.The proinflammatory cytokines play an important role in ventricular remodeling. Ventricular remodeling is the result of not only myocardial cell changes but also cardiac interstitial collagen changes. Nuclear factor NF-κB is a proinflammatory transcription factor. Tumor necrosis factor-alpha （TNF-α） is a proinflammatory cytokine that has been implicated in the pathogenesis of cardiovascular diseases, including acute myocardial infarction, chronic heart failure,atherosclerosis and viral myocarditis. Angiotensin converting enzyme inhibitor ACEI can improve cardiac function and inhibit ventricular remodeling and myocardial fibrosis.The ventricular remodeling is closely correlated with the proinflammatory cytokine activity TGF-β1 plays an important role in ventricular remodeling. The TGF-β1 participates in the early period of myocardial fibrosis. Many studies show that the TGF-β1 expression increases after infarction , indicating that TGF-β1 plays key role in ventricular remodeling and myocardial fibrosis.The occurrence and development of ventricular remodeling after myocardial infarction are correlated with the increased expression and activation of TNF-αresulting from secondary immune reaction triggered by myocardium injury and ischemia. TNF-αis not expressed or expressed in low level in normal myocardium, but its expression increases significantly in myocardium after infarction. The role of TNF-αin promoting ventricular remodeling lies in promoting the proliferation of fibroblast and secretion of collagen, as well as regulating the expression of matrix metalloproteinase in some extent. Meanwhile, TNF-αhas some correlation with left ventricular function.Under large amount of experiments and clinical studies, some regulation factors of ventricular remodeling have been found from modern medicine, and the prevention of ventricular remodeling by modern medicine and traditional medicine has got great progress. The development of ventricular remodeling after infarction is a complex course, and it is an effective method to prevent it with combination of traditional medicine and western medicine.The main contents of this thesis are as follow:1 The inhibitory effects of tongxinluo on NF-κB expression after myocardial infarction in ratTo investigate the inhibitory effects of tongxinluo on NF-κB expression after infarction in the rats, 80 rats were randomly divided into two groups: Sham operation group and coronary artery ligation group. Sham group was fed with 0.9 % NaCl（ n=10, after 4 weeks, n=8）, and killed after 4 weeks. 70 rats underwent the left descending coronary artery ligation, 38 surviving rats were randomly assigned to one of the following groups: AMI model group （M group, the rats were fed with 0.9 % NaCl, n=9）; high dose tongxinluo group (TH group, 1.0 g kg^-1 d^-1, n=10), low dose tongxinluo group (TL group, 0.05 g kg^-1 d^-1); Captopril treatment group (C group, 15 mg kg^-1 d^-1, n=10). Latter three of all the groups were fed with the medicine for 4 weeks. At the end of the experiment, the animals were weighted. Myocardial infarction areas were quantified by TCC. Electrocardiograms of animals were recorded on the operation. The rat hearts were fixed and pathologically analyzed. Morphological characteristics were evaluated with HE staining. Myocardial ultra-structural and histopathological changes were observed. NF-κB mRNA was detected by reverse transcription- polymerase chain reaction （RT-PCR）.Body weight of M group was significantly lower than sham operation group. TH group and C group were significantly higher than M group. Myocardial infarction areas were different. Electrocardiograms （ECG） of 10 animals in the control group were all normal. ECG of 38 animals in the myocardial infarction rats was not normal. ST segment elevated in rats of the myocardial infarction.The myocytes from the control group arranged regularly. The size of the nucleus was uniform. The staining of cytoplasm was homogeneous. The myocytes from the M group arranged irregularly. The nucleus was irregular and the interrupted myofibril was observed. The TH group and C group were obviously improved. The myocytes from TH group and C group arranged more regularly than those from the M group. The interrupted myofibril was not common. The nuclear shape was more regular than the M group.The left ventricular myocytes from the control group arranged regularly. The thick and thin myofilaments arranged regularly. The sarcomere and light-dark band were clear. The pericellular membrane was uninterrupted and intact. The uniformly sized mitochondrion was abundant and showed round or oval shape.The left ventricular myocytes from the M group arranged irregularly. The pericellular membrane was interrupted and unclear. The local myofibril was disintegrated. The mitochondrial shape was abnormal with deep notch. The swelling mitochondria increased and accumulated. The nuclear shape was abnormal with deep notch. The mitochondrion was presented in the form of blank space.Compared with the M group, the ultrastructural shapes of mitochondria in the TH group and C group were obviously improved. The myocytes from the TH group arranged more regularly than the M group. The mitochondrial shape was uniform. The phenomenon indicates that the local myofibril disintegration was decreased. The mitochondria were presented in the form of slightly blank space.The mitochondrial shape was not uniform in TL group. The swelling mitochondria increased and accumulated. The nuclear membrane shape was abnormal with deep notch. The mitochondria were presented in the form of blank space.The positive reaction of NF-κB protein was stained brown and existed in myocardial cytoplasm. There was uniform distribution of weak brown granules in the control group. There were thick brown granules in the M group. The granule of the TH group was weaker than the M group. There was uniform distribution of weak brown granules on NF-κB in the control group. The granule of the M group was thicker than the TH group.Compared to sham operation group, the NF-κB expression was enhanced in M group. The NF-κB expression in the TH group and C group was significantly lower than M group.At M group, NF-κB expression on the myocardium increased after infarction, which suggests that NF-κB plays an important role in ventricular remodeling. In comparison with M group, the NF-κB expression was significantly lower （p<0.01） in the TH group and C group. The inhibitory effects of tongxinluo are based on its anti-oxidative action. The protective effect of tongxinluo on ventricular remodeling may be beneficial. Nuclear factor NF-κB is a proinflammatory transcription factor. The ventricular remodeling is closely correlated with the proinflammatory cytokine activity. 2 The inhibitory effects of tongxinluo on TGF-β1 expression after infarction in ratTo investigate inhibitory effects of tongxinluo on TIFF-β1 expression after infarction in rats, 60 rats were randomly divided into two groups: sham operation group and coronary artery ligation group: Sham operation group was fed with 0.9 % NaCl for 4 weeks （n=10, after 4 weeks, n=8）, and killed after 4 weeks; 50 rats underwent the left descending coronary artery ligation, 28 surviving rats were randomly assigned to one of the following groups: AMI model group （M group, the rats were fed with 0.9 % NaCl for 4 weeks, n=9）; high dose Tongxinluo group (TH group, 1.0 g kg^-1 d^-1, n=10), and low dose Tongxinluo group (TL group, 0.05 g kg^-1 d^-1, n=9). All the groups were fed with the medicine for 4 weeks. At the end of the experiment, the mRNA and protein expression of NF-κB was examined by reverse transcription- polymerase chain reaction （RT-PCR） and immunochemistry.Compared to sham operation rats, the expression of TGF-β1 mRNA and TGF-β1 protein showed significant increase in M group （p<0.01）. In comparison with the M group, the TGF-β1 expression was significant lower in the TH and C groups （p<0.01）.The TGF-β1 expression on the myocardium after infarction in rats indicates that TGF-β1 has proinflammatory activity. The TGF-β1 participates in the early period of ventricular remodeling and myocardial fibrosis. The ventricular remodeling is closely correlated with myocardial fibrosis. The study shows that the TGF-β1 expression on the myocardium significantly increased in M than TH group after infarction, the TGF-β1 expression was significantly lower （p<0.01） after tongxinluo treatment, which indicates that TGF-β1 plays key role in ventricular remodeling and myocardial fibrosis. Preventing and treating the ventricular remodeling and myocardial fibrosis with tongxinluo is effective. 3 The effects of tongxinluo on TNF-αexpression after infarction in ratTo observe changes of TNF-αprotein expression in the myocardium after infarction in the rats, 80 rats were randomly divided into two groups: sham operation group and coronary artery ligation group. Sham operation group was fed with 0.9 % NaCl for 4 weeks （n=10, after 4 weeks, n=8）, and killed after 4 weeks; 70 rats underwent the left descending coronary artery ligation, 38 surviving rats were randomly assigned to one of the following groups: AMI model group （M group, the rats were fed with 0.9 % NaCl for 4 weeks, n=9）, high dose Tongxinluo group (TH group, 1.0 g kg^-1 d^-1, n=10), Captopril group (C group, 15 mg kg^-1 d^-1, n=10); low dose Tongxinluo group (TL group, 0.05 g kg^-1 d^-1, n=9). Latter three of all the groups were fed with the medicine for 4 weeks. At the end of the experiment, the rat hearts were fixed and pathologically analyzed. Morphological characteristics were evaluated with HE and Masson staining. The protein expression of TNF-αon the myocardium was detected by immunochemistry and Western blot.Compared to sham operation rats, the TNF-αexpression was enhanced in M group. In comparison with the M group, TNF-αexpression was significantly lower （p<0.01） in the TH group and C group, but TNF-αexpression was not lower in the TL group.TNF-αis not expressed or expressed in low level in normal myocardium, AMI increased expression of TNF-αand resulted in ventricular remodeling triggered by myocardium injury and ischemia. Meanwhile, TNF-αwas closely correlated with left ventricular function. The activation of TNF-αcan affect left ventricular function. The tongxinluo has the inhibitory effects on activation of TNF-α. The left ventricular function was improved after treatment with tongxinluo.Conclusion:1 The tongxinluo reduces the activation of TNF-αand TNF-αexpression. The NF-κB expression in the myocardium of TH group and C group is lower than M group. The phenomenon suggests that NF-κB plays an important role in ventricular remodeling after myocardial infarction. The prevention of ventricular remodeling with tongxinluo after infarction is an effective method .2 Nuclear factor NF-κB is a proinflammatory transcription factor. The proinflammatory cytokines are implicated in the pathogenesis of ventricular remodeling. The NF-κB is activated by myocardium injury and ischemia. NF-κB has some correlation with ventricular remodeling.3 The inhibition of NF-κB expression is based on the anti-oxidative action of tongxinluo.4 The TGF-β1 participates in the early period of ventricular remodeling and myocardial fibrosis. Preventing and treating the myocardial fibrosis with tongxinluo is effective.5 TNF-αhas closely correlation with left ventricular function. The activation of TNF-αaffects left ventricular function. The tongxinluo has the inhibitory effects on activation of TNF-α. The left ventricular function is improved after treatment with tongxinluo.6 The proinflammatory cytokines triggered by myocardial infarction has close correlation with ventricular remodeling.更多还原