【作者】 牟艳玲；

【导师】 张岫美；

【作者基本信息】 山东大学 ， 医学基础药理学， 2009， 博士

【摘要】 研究目的急性心肌缺血性损伤（Acute Myrocardial Ischemia,AMI）是严重危害我国中老年人健康的重大疾病,也是最常见的猝死原因之一。心肌缺血后的修复过程及预后与缺血心肌血管的功能状态密切相关,缺血区内是否有血管迅速生成对于心肌缺血后建立良好的侧支循环、改善缺血区血液供应、促进缺血区心肌的存活具有非常重要的意义。介入治疗如经皮冠状动脉腔内成形术（PTCA）和冠状动脉内支架是目前AMI后血运重建的常用方法,但由于介入治疗只适于直径＞2mm的动脉,对直径＜2 mm的动脉和具体负责灌注的微小血管则无能为力,且存在术后再狭窄（25%～50%）问题,以至有相当多的病人（20%～37%）不能达到完全血运重建而影响预后。除了残存血管的重新开放,近年来,诱导血管新生以重建血运挽救濒死的心肌成为治疗AMI所致心肌损伤的一个新策略,这种方法又被称为“治疗性血管新生”。虽然目前对血管新生及治疗性血管新生进行了较多的基础及临床方面的研究,各种血管生长因子及转基因治疗的促进血管新生作用己被认可,但由于多种血管生长因子本身的局限性和应用的烦琐及转基因治疗中尚存在许多问题使其在临床上的应用受到限制,所以需要寻找以诱导血管新生为靶点的更方便、有效、实用的药物来代替生长因子。我们前期的研究结果表明,从葛花中提取分离的异黄酮化合物葛花苷具有明显的耐缺氧活性和抗心肌缺血活性,亦具有明显的改善血液粘度活性及抗血栓形成作用,但是该化合物水溶性较差,使其研究受到一定的限制。因此,我们对葛花苷结构进行修饰,在保留活性基团的基础上,增加水溶性基团,克服该化合物所存在的不足,得到一个新型化合物—甲胺鸢尾素。该化合物水溶性增加,易吸收。小鼠急性毒性实验和体外细胞毒实验表明,该新型化合物毒性较低。我们拟在此基础上深入研究这一新型化合物对缺血心肌的保护作用,从其对缺血心肌心功能、缺血缺氧/再灌注损伤、促血管新生信号通路因子（HIF-1α、VEGF、NO）等多方面深入研究其作用机制,为缺血性心脏病的治疗提供新的药物靶点和创新型药物。研究方法本研究分为两大部分。第一部分采用体内外实验研究甲胺鸢尾素对心肌缺血损伤的保护作用及其机制。第二部分采用体内外实验研究甲胺鸢尾素在急性心肌缺血损伤后促进血管新生的的作用与机制。第一部分:甲胺鸢尾素改善急性心肌缺血损伤的作用及机制研究首先,体内实验观察了新型化合物甲胺鸢尾素对健康小鼠常压耐缺氧存活时间、电刺激大鼠血栓形成时间及对正常大鼠血液粘度的影响,研究甲胺鸢尾素对动物耗氧量及血液流变学的影响;分别采用异丙肾上腺素多次皮下注射和经典的冠状动脉结扎方法建立急性心肌缺血损伤模型,观察甲胺鸢尾素对缺血心肌病理改变、缺血/梗塞面积、心肌酶、心功能、血浆CGRP和ET-1水平等的影响,探讨其对心肌缺血损伤的保护作用。其次,体外实验以原代培养的Wistar乳鼠心肌细胞为研究对象,以高纯氮气（99%）饱和与否建立细胞缺血再灌注损伤（缺氧3h、再灌注6h）模型,以甲胺鸢尾素对损伤心肌细胞形态的变化及细胞存活率的影响、对损伤细胞超氧化物岐化酶（SOD）活性、丙二醛（MDA）含量、乳酸脱氢酶（LDH）的释放量、活性氧生成量、线粒体膜电位、凋亡细胞比率及细胞内钙含量等为观察指标,从细胞水平上探讨甲胺鸢尾素保护心肌细胞缺血损伤的作用机制。第二部分:甲胺鸢尾素在急性心肌缺血损伤后促血管新生的作用与机制研究采用结扎大鼠冠状动脉前降支方法造成急性心肌缺血损伤模型,观察甲胺鸢尾素连续给药30天对缺血心肌梗塞边缘区血管新生的作用、对梗塞边缘区心肌超微结构的影响;分别采用免疫组化、Western blot、实时定量PCR等方法定性、定量的观察甲胺鸢尾素对缺血心肌缺氧诱导因子-1α（HIF-1α）及其调节的下游靶基因血管内皮细胞生长因子（VEGF）、内皮型一氧化氮合酶（eNOS）表达的影响;分别采用ELISA、放免、酶检测法检测甲胺鸢尾素对血清VEGF、血浆降钙素基因相关肽（CGRP）和内皮素（ET-1）水平、血清一氧化氮（NO）含量的影响;观察甲胺鸢尾素对培养的人脐静脉内皮细胞（HUVEC）增殖、迁移和管样结构形成以及缺氧损伤后NO含量的影响,明确甲胺鸢尾素在急性心肌缺血后促进血管新生的作用与机制。结果1.甲胺鸢尾素一次性腹腔注射给药300mg/kg和150mg/kg均能明显增加小鼠常压下耐缺氧存活时间（P＜0.01）;200、100mg/kg剂量可明显降低大鼠血液粘度（P＜0.01）,延长电刺激大鼠颈动脉血栓形成时间（P＜0.05）。2.甲胺鸢尾素200、100mg/kg对异丙肾上腺素所致大鼠心肌缺血损伤具有明显的保护作用,能减轻异丙肾上腺素对心肌缺血的病理性损害,稳定细胞膜,使心肌酶释放减少,与对照组比较差异显著（P＜0.05）。3.甲胺鸢尾素80mg/kg剂量组明显减轻犬急性心肌缺血程度（∑-ST）、缩小缺血范围（N-ST）、缩小心肌梗塞面积;40mg/kg剂量组有降低心肌缺血程度和缩小心肌梗塞范围的趋势,但与模型组比较无明显差异（P＞0.05）。甲胺鸢尾素80mg/kg剂量组给药后2-4h使血清磷酸肌酸激酶同功酶（CK-MB）、LDH释放量明显降低。40mg/kg剂量组给药后4h显著降低LDH释放量（P＜0.05）,对血清谷草转氨酶（AST）、磷酸肌酸激酶（CK）有降低趋势,但与模型组比较无明显差异（P＞0.05）。4.甲胺鸢尾素200mg/kg剂量组可以明显改善大鼠急性心肌梗塞后心肌病理改变;使心肌收缩压、舒张压、左室内压升高,心率减慢（P＜0.01）;血浆CGRP和ET-1水平达到稳态平衡。5.甲胺鸢尾素(10^-7 M,10^-6 M,10^-5 M)能明显提高缺血（3h）再灌注（6h）损伤细胞存活率,提高细胞内SOD活性,明显降低细胞内MDA和ROS生成量、降低凋亡细胞比率,升高线粒体膜电位、明显降低细胞内钙含量,与模型组比较差异显著（P＜0.01）。6.甲胺鸢尾素使急性心肌缺血边缘区心肌新生毛细血管密度增加;明显改善缺血心肌的超微结构。免疫组化和Western Blot结果表明,HIF-1α和VEGF蛋白表达增加;实时定量PCR结果显示HIF-1α基因表达各组未见明显差异,而VEGF、eNOSmRNA表达升高。甲胺鸢尾素200mg/kg使血清VEGF和NO水平提高,与模型组比较显著差异（P＜0.01）。对培养的HUVEC细胞,甲胺鸢尾素预处理可使培养的HUVEC细胞增殖加快（S期和G2期细胞增多）、迁移能力增强、管样结构形成数目增多。缺血缺氧损伤模型组内皮细胞NO含量及NOS活性明显低于正常对照组（P＜0.01）,而预先用甲胺鸢尾素(10^-7 M,10^-6 M,10^-5M)处理的各组内皮细胞内NO含量及NOS活性则明显高于模型组,显著差异（P＜0.01）。结论1.甲胺鸢尾素能明显增加耐缺氧小鼠存活时间、延长电刺激大鼠血栓形成时间、降低大鼠血液粘度;减轻异丙肾上腺素对心肌的病理性损害、减少心肌酶的释放;明显减轻冠脉结扎犬心肌缺血/梗塞面积;明显改善急性心肌梗塞后大鼠心功能,具有明显的心肌缺血保护作用,其机制可能与改善受损心肌供血、供氧,稳定细胞膜,从而调节能量代谢、改善血液流变作用有关。2.甲胺鸢尾素可提高缺血再灌注损伤细胞存活率、提高氧自由基清除能力、降低细胞活性氧水平、升高线粒体膜电位。其机制可能与增强机体抗氧化酶活性、降低缺氧再给氧产生的活性氧水平,从而改善线粒体功能、降低细胞内钙超载有关。3.缺血缺氧损伤心肌HIF-1α基因表达未见明显变化、蛋白表达增加,VEGF基因和蛋白表达均明显增加,NOS活性增强、NO合成增加,说明在我们的实验条件中缺血缺氧并不影响HIF-1α基因表达,但可减少其降解,增加其活性,启动HIF-1α-VEGF-NO信号通路的转导,代偿性起到心肌保护作用。但是随着缺血时间的延长,代偿作用减弱。因此,维持HIF-1α—VEGF—NO信号通路的转导是治疗缺血性心脏病的靶点之一。4.甲胺鸢尾素能明显增加心肌缺血边缘区微血管形成,可使HIF-1α、VEGF表达均维持在较高水平,血清VEGF、NO水平升高,说明其可以促进HIF-1α—VEGF—NO这一信号通路的转导,从而改善缺血心肌微循环和氧的供需平衡,起到“药物搭桥”治疗心肌缺血的作用。5.甲胺鸢尾素可以促进体外培养的HUVEC细胞增殖、迁移和管样结构形成,增加内皮细胞NOS合成分泌NO的能力,这可能是其发挥改善微循环、增加缺血区血供,从而产生“治疗性血管新生”的机制之一。本研究的意义与创新点心肌缺血性损伤是严重危害人类健康的重要疾病,研究防治此类疾病的药物并探讨其机制具有重要的理论意义和现实意义。本研究明确了新型化合物甲胺鸢尾素对缺血心肌的保护作用及其机制,为其防治缺血性心脏病的研究开发提供理论基础。主要创新点:1.对葛花苷进行结构修饰,得到有良好药理活性的具有自主知识产权的新型化合物—甲胺鸢尾素,申报国家专利号:200610068403.5。2.对心肌缺血模型中血浆CGRP和ET-1水平进行动态监测,明确心肌缺血发生发展的病理生理基础:明确了促进HIF-1α—VEGF—NO信号通路的转导是治疗缺血性心脏病的靶点之一。3.阐明了甲胺鸢尾素可以通过稳定细胞膜、改善微循环、降低钙超载、减轻线粒体损伤,促进HIF-1a—VEGF—NO这一信号通路的转导,从而提高促血管新生因子（HIF-1α、VEGF、eNOS）的表达,发挥心肌缺血的保护作用。4.本课题研究为中药黄酮类提取物的研究提供思路。更多还原

【Abstract】 Objective:Acute myocardial ischemia（AMI） is one of the worst diseases harming the health of middle and elder people in our country,which is also one of the commonest reasons leading to sudden death.The reparative process and prognosis of acute myocardial infarction is correlated to functional status of vassular in myocardial tissue.Therefore,forming and opening of coronary artery collateral circulation, ameliorating blood supply and myocardial survival of infarction zone by rapid angiogenesis or revascularization is extremely considerable significance.Nowadays, interventional therapy such as percutaneous tranluminal coronary angioplasty（PTCA） and intracoronary stent are the commonly used methods of revascularization after AMI.Interventional therapy is only suitable for arterial diameter above 2 mm, however,which is no use for capillary and arterial diameter below 2 mm.Furthermore, interventional therapy exist re-narrow（25%～50%） resulting in considerable acute patient who can not achieve completely revascularization and influence prognosis. Recently,besides opening of coronary artery collateral circulation,inducing angiogenesis to reestablish revascularization have developed to a new strategy for curing myocardial ischemia after AMI.The new strategy is also called "therapeutic angiogenesis".There are major foundation and clinical research related to angiogenesis and therapeutic angiogenesis,and a number of vascular growth factors and transgenic therapy have been approved to promote angiogenesis.However,the local and tedious application of vascular growth factors,and transgenic therapy existing many problems,all that confine its clinical application.Therefore,we need search more convenient,effect and pragmatic drugs to replace growth factors.Our previous research showed that kakklide extracting from Puerariae Flos could counteract hypoxia and myocardial ischemia significantly,and also significantly counteract thrombosis and improve blood viscosity.We modified the structure of the compound owing to its worse water-solubility,and obtained a new compound—methylamine irisolidone.The toxicity testing of mice and cells showed that this new compound had less toxicity.On the contrary,the new compound could increase the survival time of standing hypoxia in mouse.Accordingly,on the basis of our previous study,we want to identify the protection and mechanisms in myocardial ischemia of methylamine irisolidone,including the improvement of microcirculation,apoptosis and angiogenesis of ischemic myocardium.,which may offer a new strategy and medicine for curing myocardial ischemia.Methods:There are two parts of our study.The first part researched the effects and mechanism of methyalmine irisolidone on acute myocardial infarction and myocardial cells cultured under ischemia and hypoxia.The second part investigated the mechanism of methylamine irisolidone on angiogenesis induced by acute myocardial infarction.Part 1:In vivo experiment,the survival test of hypoxic mice,and electrical stimulation of thrombosis model in rats were used to evaluate the pharmacy activity of methylamine irisolidone.Isoprenaline and coronary artery ligation were used to induce the acute myocardial infarction model,several experiments including the degree of myocardial ischemia（∑-ST）,the range of myocardial ischemia（N-ST） and the infarct area,myocardial zymogram（AST,LDH,CK,CK-MB）,heart function,the serum levels of CGRP and ET-1 were investigated.In vivro experiment,we investigated the effect of methylamine irisolidone on hypoxia injury in cultured rat cardial myocytes.Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 3 hours and subsequently reoxygenated for 6 hour.Myocytes injury was determined by the release of lactate dehydrogenase （LDH）,and the activity of SOD,MDA and ROS levels,the change of mitochondrial membrane potential（MMP）,myocardial cells cytosolic Ca²⁺ content were measured to investigate the effects and mechanism of methylamine irisolidone.Part 2:Coronary artery ligation was used to induce the acute myocardial ischemia model.After 30 d administration of methylamine irisolidone,the gene and protein expression of HIF-1α,VEGF,eNOS in ischemia border zones were detected and the serum level of VEGF,CGRP,ET-1,NO and NOS activity were measured by ELISA,radioimmunity,nitroreductase methods,respectively.At the same time,the multiplication and migration and tube structure formation of HUVECs were detected.Results:1.Methylamine irisolidone administration markedly prolonged the living time of the hypoxia experimetal mice at the doses of 300mg and 150mg/kg,reduced the formation time of carotid artery thrombosis,decreased blood viscosity shear rate.2.For in vivo experiments,compared with model group,pretreatment with methylamine irisolidone at dose of 200 and 100mg/kg could improve the myocardiac injury induced by isoprenaline,and decrease the release of cardiac creatase in rats.3.Methylamine irisolidone（80 mg/kg,i.v） could reduce the myocardial infarct areas in dogs with myocardial infarction.The serum lactate dehydrogenase（LDH） activity and MB isoenzyme of creatine kinase（CK-MB） were suppressed by methylamine irisolidone after 2 and 4 h of administration.Methylamine irisolidone （80 mg/kg） can lessen the degree of myocardial ischemia obviously,manifestly reduce the range of myocardial ischemia（N-ST） in contrast with the myocardial ischemia control group.Myocardial infarct area displayed by the N-BT staining is roughly similar to the results measured by the epicardial electrogram.Methylamine irisolidone （80 mg/kg） exhibited obvious effects of lessening injury in myocardial ischemia,and the infarct area is conspicuously decreased as compared with the ischemia control group.4.Methylamine irisolidone could improve the heart function of rats in AMI injury,increased the systolic pressure and diastolic pressure,decreased the heart rate. Methylamine irisolidone could increase the level of calcitonin gene related protein （CGRP） and decrease the level of endothelin-1（ET-1） in the blood plasma and achieve the dynamic balance.5.The cell viability of cardiomyocytes treated with methylamine irisolidone during H/R injury significantly increased compared with methylamine irisolidone-untreated cells.Similar results were shown by LDH release.Methylamine irisolidone enhanced the mitochondrial membrane potential and decrease intracelluar calcium associated with the attenuated reactive oxygen species（ROS） generation,reduced levels of malondialdehyde（MDA） and increased activity of superoxide dismutase （SOD） after H/R in a dose-dependent manner.6.Methylamine irisolidone induced angiogenesis in the ischemic myocardium, myocardial vessel density was significantly increased in ischemic zones.The gene expression or activation of vascular endothelial growth factor（VEGF）,and endothelial nitric oxide synthase（eNOS） which correlated with angiogenesis were also induced by pretreated with methylamine irisolidone.On the other hand, methylamine irisolidone could improve the ultrastructural organization of ischemic myocardial zones,myofibrillae well-arranged,mitochondria structure integrated, nuclear chromatin well-distributed and a few blood capillary appeared in mesenchymal.For cultured serial subcultivation HUVECs,pretreated with methylamine irisolidone could speed up the endothelial cell proliferation（cells in S and G2 period increased）,cell migrate and tube structure formation.Conclutions:1.The present study demonstrated that methylamine irisolidone could lessen the degree of myocardial ischemia obviously（∑-ST） and the range of myocardial ischemia（N-ST） and the infarct area in acute myocardial ischemia dogs,and could improve the myocardial injury induced by isoprenaline.These results suggested that methylamine irisolidone exert the protective effects on myocardial ischemia injury, which may be due to its function of inhibiting LDH and CK-MB releasing,stabilizing myocardial cell membrane and improving myocardial microcirculation and metabolism.2.The present study demonstrated that methylamine irisolidone could directly protect cardiomyocyte against hypoxia injury,primarily as a result of reduction of the intracellular Ca²⁺ overload coincident with an attenuation of ROS generation and ROS-mediated lipid peroxidation which may contribute to the preservation of mitochondrion function and antioxidant against hypoxic injury.3.Under ischemic and hypoxic connditions,there is no change of HIF-1αmRNA expression,however,HIF-1αprotein expression enhanced.At the same time,the activity of NOS increased and NO level enhanced.That is to say,reduce HIF-1αdegradation,improve its activity,priming HIF-1α—VEGF—NO signal pathway,can induce the compensation protective effects.But with time extended,the compensation is attenuated,the concentration of VEGF and NO were decreased markedly.So, improving the HIF-1α—VEGF—NO transduction is one of the targets of treatment of ischemic heart diseases.4.Under ischemic and hypoxic connditions,pretreated with methylamine irisolidone could improve the formation of capillary in ischemic zone.It is indicated that improve the transduction of HIF-1α—VEGF—NO sinal pathway could induce "drug bridge" effect for treating ischemic disease.5.For cultured serial subcultivation HUVECs,pretreated with methylamine irisolidone could speed up the endothelial cell proliferation,cell migrate and tube structure formation,which may be one of the mechanisms of its therapeutic angiogenesis.更多还原