【作者】 张玲；

【导师】 程翼宇；

【作者基本信息】 浙江大学 ， 药理学， 2011， 博士

【摘要】 随着我国人口老龄化进程的加剧,缺血性心脏病的发病率和死亡率均呈现出逐年上升的趋势。药物治疗是缺血性心脏病临床治疗的重要途径之一,特别是对于无法进行手术治疗的冠状动脉弥漫性狭窄病变患者尤为重要。在我国,大量中药方剂和中成药制剂已被用于缺血性心脏病的预防与治疗,但其药效物质基础与作用机制有待进一步研究。治疗性血管新生(Therapeutic Aginogenesis)是近年来提出的缺血性心脏病治疗新途径,其原理是通过促进心肌缺血区域新血管的生成和开放新的侧支循环通路,达到恢复缺血心肌的血流供应、改善患者症状和预后的目的。因此,研究中药方剂促血管新生作用并探讨其作用机制对于缺血性心脏病的治疗具有重要意义。芪参益气方治疗缺血性心脏病临床疗效显著,但抗心肌缺血作用机制还不明确。本论文综合运用多种实验技术手段研究了该方抗急性心肌缺血作用,并探讨其促血管新生的作用及机制；通过体外实验进一步研究了该方中黄芪的主要活性成分——黄芪甲苷对低氧诱导因子-1α(HIF-1α)表达的影响及其促血管新生机制；采用代谢组学技术研究了该方各组分及其配伍对急性心肌缺血大鼠血浆代谢组的影响。主要研究内容如下：1.研究芪参益气方的抗心肌缺血作用并探讨了其促血管新生的作用机制。运用2,3,5,氯化三苯基四氮唑(TTC)染色法、免疫组化(Immunohistochemistry)、荧光定量PCR (Real-time PCR)及蛋白质印迹法(Western blotting)观察了芪参益气方对急性心肌缺血大鼠血管新生的影响。研究发现：(1)芪参益气方显著减小急性心肌缺血大鼠的心肌梗死范围；(2)芪参益气方能够显著增加心肌缺血大鼠缺血交界区的血管数目；(3)芪参益气方能够显著升高缺血心肌中血管内皮生长因子(VEGF)的mRNA和蛋白表达水平；(4)芪参益气方能够显著升高缺血心肌中碱性成纤维细胞生长因子(bFGF)的mRNA和蛋白表达水平；(5)芪参益气方能够显著升高缺血心肌中血小板衍化生长因子B (PDGF-B)的mRNA水平,但对其蛋白表达无明显影响。以上结果提示,芪参益气方可能通过升高VEGF. bFGF的mRNA和蛋白表达水平以及PDGF-B的mRNA水平,促进缺血心肌血管新生,从而发挥抗心肌缺血作用。2.研究该方中君药黄芪的主要活性成分——黄芪甲苷对HIF-1α的影响及其促血管新生机制。分别采用Real-time PCR、Western blotting、免疫荧光、Transwell小室、Matrigel胶及鸡胚尿囊膜实验等方法研究黄芪甲苷对缺氧损伤内皮细胞中HIF-1α表达及其促血管新生机制。研究发现：(1)黄芪甲苷显著提高缺氧损伤后内皮细胞的存活率；(2)黄芪甲苷能够显著升高缺氧损伤后内皮细胞的HIF-1α的蛋白表达水平；(3)黄芪甲苷对HIF-1α的mRNA水平及HIF-1α的蛋白降解几乎无影响；(4)黄芪甲苷能够显著升高内皮细胞中AKT (Ser473)蛋白的磷酸化水平；(5)黄芪甲苷能够促使HIF-1α向细胞核内转移并促进VEGF转录,且P13K抑制剂LY294002能够干预其效果；6)黄芪甲苷能够显著促进内皮细胞迁移、管腔形成及血管新生,且LY294002能够干预其效果。以上结果提示,黄芪甲苷可能通过激活P13K/AKT信号转导通路,促进HIF-1α的蛋白合成并向细胞核内转移,促使VEGF转录增加而发挥促血管新生的作用。3.采用气相色谱-质谱联用技术(GC-MS)分析芪参益气方各组分及其配伍对急性心肌缺血大鼠血浆代谢组的影响。研究发现：与假手术组大鼠相比,急性心肌缺血大鼠血浆中29个内源性代谢物的含量发生改变,其中7个与能量代谢相关和7个与氨基酸代谢相关的内源性代谢物含量均明显增加。对心肌缺血大鼠进行给药后,芪参益气方各组分及其配伍对这14个内源性代谢物含量均有回调作用。提示芪参益气方可能通过调节能量代谢和氨基酸代谢发挥抗急性心肌缺血的作用。本论文研究结果表明,芪参益气方抗心肌缺血的作用机制包括：调节促血管生长因子如VEGF、bFGF、PDGF-B的mRNA和蛋白表达水平,以及调节能量代谢和氨基酸代谢。其君药的主要活性成分——黄芪甲苷主要通过激活PI3K/AKT信号转导通路,促进HIF-1α蛋白合成并促使其向细胞核内转移,升高VEGF的mRNA水平,进而引起内皮细胞迁移、管腔形成,从而促进血管新生。更多还原

【Abstract】 With the gradually increasing of the aging population, the morbidity and mortality of ischemic heart disease is vastly increasd in recent years. Drug therapy is one of the most important ways in the clinical therapeutics of ischemic heart disease, especially for patients of coronary artery stenosis, which can not be treated by surgery. In china, a large number of traditional Chinese medicines were used for the prevention and treatment of ischemic heart disease, but the active componets and mechanisms of action of many traditional Chinese medicines are still unclear. Therapeutic angiogenesis is a recently proposed approach for the treatment of ischemic heart disease. It is belived that angiogenesis in ischemic region of myocardium may lead to the construction of new collateral circulation and resume blood supply. Therefore, studies on therapeutic angiogenesis induced by traditional Chinese medicine may play important roles in the treatment of ischemic heart disease.QI-SHEN-YI-QI showed a significant effect in treatment of ischemic heart disease, but the mechanisms were not clear. In this study, experimental techniques of pharmacology were used to study the angiogenic mechanisms of QI-SHEN-YI-QI. In vitro experiments were performed to study the accumulation of hypoxia-inducible factor-1α(HIF-1α) and angioenesis by astragaloside IV. Then metabolomics technique was used to study the effect of QI-SHEN-YI-QI on regulation of endogenous metabolites in rat plasma. The main contents of dissertation include:1. This study aims to determine the possible cardioprotective effects of QI-SHEN-YI-QI and its angiogenic mechanism. Male Sprague-Dauley rats were subjected to ligation of left anterior descending artery (LAD). Triphenyltetrazolium chloride (TTC) staining, immunohistochemistry, real-time PCR and western blotting were used to study the agiogenic effect of QI-SHEN-YI-QI. The results showed that QI-SHEN-YI-QI significantly reduced infarct size and increased density of vessel. The mRNA levels of VEGF, bFGF and PDGF-B were increased in QI-SHEN-YI-QI treated group compared to myocardial infarct group. Western blotting results further demonstrated that protein expression of VEGF and bFGF were also notably raised in QI-SHEN-YI-QI treated group. Our study demonstrated that therapeutic angiogenesis induced by QSYQ may lead to neovascularization and conferring myocardial protection by increaing expression of angiogenic factors.2. This study aims to determine the HIF-la accumulation and angiogenic effect of astragaloside IV and its underlying mechanisms. MTT assay, western blotting, real-time PCR, immunofluorescence, transwell, matrigel and chick chorioallantoic membrane (CAM) assays were used to study the effect of astragaloside IV on accumulation of HIF-1αand angiogenesis. The results showed that astragaloside IV significantly improved cell viability after hypoxia and stimulated HIF-1αaccumulation during hypoxia. Mechanism studies revealed that astragaloside IV did not affect the degradation of HIF-1αprotein or the mRNA level of HIF-1α. In contrast, astragaloside IV apparently activated the PI3K/AKT pathway, which regulates HIF-la protein synthesis. Moreover, HIF-1αwas translocated to nuclear and mRNA level of VEGF was significantly increased in astragaloside IV treated group. Astragaloside IV also stimulated cell migration, increased tube formation and promoted angiogenesis in chick chorioallantoic membrane assay. All angiogenic effects of astragaloside IV were reversed by PI3K inhibitor LY LY294002. Our data suggested that astragaloside IV was a novel regulator of HIF-1αand angiogenesis through the PI3K/AKT pathway in HUVECs exposed to hypoxia.3. GC-MS was employed to profiling the effect of QI-SHEN-YI-QI on endogenous metabolites in myocardial infarcted rats. The results showed that seven metabolites related to energy metabolism and seven metabolites related to metabolism of amino acids were significantly increased in the plasma of myocardial infracted rats. However, administration of QI-SHEN-YI-QI regulated those metabolites in energy and amino acids metabolism to the normal level. It suggested that QI-SHEN-YI-QI may exert its protective effect against myocardial ischemia through regulation of energy and amino acids metabolism.Taken together, QI-SHEN-YI-QI promoted angiogenesis by collaborative effect of VEGF, bFGF, PDGF-B and protected against myocardial ischemia through regulation of energy and amino acids metabolism. Astragaloside IV improved cell viability during hypoxia, stimulated HIF-1αaccumulation by P13K/AKT pathway, and increased mRNA level of VEGF, which may contribute the angiogenesis effect during hypoxia.更多还原