【作者】 黄仁彬；

【导师】 刘华钢；

【作者基本信息】 广西医科大学 ， 药理学， 2010， 博士

【摘要】 心肌缺血再灌注损伤(myocardial ischemia reperfusion injury,MIRI)的发生和发展过程是一个多因素相互作用的级联反应,主要的损伤机制有自由基损伤、钙超载、微血管损伤和白细胞的作用等。近年来研究发现,补体、选择素、内皮素和细胞凋亡等均参与了MIRI的病理变化过程。可见,MIRI可能是多分子、多机制、相互影响、相互促进的病理变化。至今MIRI一直是心肌缺血治疗中难以解决的问题。我国中药资源丰富,从中寻找防治MIRI的安全有效药物,特别是对其活性成分进行药效学及作用机制的研究,对于开发我国丰富的中药资源和促进中药现代化具有重要意义。多年来,本课题组一直致力于发掘广西丰富的民族草药资源,筛查副作用小、疗效较好的广西民族草药/活性部位。我们前期开展了大量的基础研究工作,阶段性实验发现:(1)从玉郎伞分离得到的玉郎伞黄酮(YLSF)预处理能明显降低MIRI大鼠血浆中AST、LDH、LDH1和MDA含量,能提高SOD活性,降低心肌梗死范围。提示,YLSF对大鼠心肌MIRI具有显著的保护作用。此外,两种YLS单体对体外培养大鼠乳鼠心肌细胞缺氧/复氧损伤具有保护作用,其机制可能与清除自由基、抑制心肌细胞Ca2+超载有关;进一步研究还发现,YLS两种黄酮单体尚具有抗氧化、耐缺氧、抗凝血以及有较强的清除自由基的作用。(2)从柿叶提取得到的PLF可显著改善MIRI所致的大鼠离休心功能损伤,减少CK、CK-MB、LDH、LDH1的释放和心肌组织MDA的产生,增加SOD、Na+-K+-ATP酶和Ca2+-ATP酶的活性,说明,PLF对大鼠MIRI具有保护作用,其机制可能与提高SOD活性、抗氧自由基、减少脂质过氧化反应有关。而研究PLF对血压的影响,发现PLF对正常大鼠和L-NAME诱导的高血压大鼠的血压和心率均有降低作用。另一研究证明,PLF可能是通过增加内源性舒血管活性物质的释放和减少内源性收缩血管活性物质的释放来调节两者之间的平衡而发挥其抗高血压作用。总之,前期研究表明,YLSF和PLF对大鼠MIRI具有明显的保护作用,其作用机制与抗氧化、清除氧自由基等多个环节有关。为了更进一步地了解YLSF和PLF对大动物(猴、犬)MIRI的影响,完善其临床前研究,我们分别用猴和犬MIRI模型,研究YLSF和PLF对MIRI的保护作用,研究分两部分:第一部分:玉郎伞黄酮对猴心肌缺血再灌注损伤的保护作用及机制研究目的:研究玉郎伞黄酮(YLSF)对猴心肌缺血再灌注损伤(MIRI)的保护作用及其机制。方法:将实验猴随机分为5组,每组4只:假手术(SHAM)组、心肌缺血再灌注损伤(MIRI)模型组、YLS黄酮低剂量组(YLSFL)组、YLS黄酮高剂量(YLSFH)组、地尔硫卓阳性药(DIL)组。缺血前30min经十二指肠注射给药。结扎冠状动脉左前降支60min后,再灌注180min,建立MIRI模型。再灌注结束后测定下列各项指标:(1)利用MPA心功能分析系统观察并纪录在急性缺血和再灌注状态下血流动力学指标(HR、LVSP、Lvedp、+dp/dtmax、-dp/dtmax)的变化。(2)取缺血区的心肌组织,常规制作组织切片、电镜切片,光镜、电镜下观察心肌组织病理结构和超微结构的变化并拍照。(3)从股静脉脉采血,离心,取上层血清,测定AST、LDH、LDH1、CK、CK-MB、SOD、GSH-Px、TNOS、MDA和NEFA;取缺血区心肌组织,按试剂盒说明测定Na+K+-ATPase和Ca2+Mg2+-ATPase活性。(4)取缺血区心肌组织,测定心肌组织凋亡相关蛋白Bcl-2和Bax表达、核因子-κB表达、心肌细胞凋亡、心肌ANT1、Caspase-3 mRNA表达。结果:(1)YLSF对心脏血流动力学的影响:与模型组比较,YLSFH、YLSFL组在再灌注180min时对LVSP、+dp/dtmax有明显的提高作用,而对HR、LVDEP和-dp/dtmax的作用不明显。(2)YLSF对血液生化学指标的影响:YLSF能明显下调各心肌酶、MDA和NEFA含量;提高SOD、GSH-Px、Na+K+-ATPase和Ca2+Mg2+-ATPase活性。(3)YLSF对心肌组织形态学的影响:从组织病理学角度上看,与模型组比较,YLSF给药组心肌受损程度明显减轻。(4)YLSF对相关蛋白Bcl-2和Bax表达、核因子-κB表达的影响:YLSFH、YLSFL组均能明显降低心肌Bax蛋白表达,能显著增加Bcl-2/Bax的比率。YLSFH能明显增加Bcl-2蛋白表达,而YLSFL则对Bcl-2蛋白表达无影响。YLSF给药组NF-κBp65表达降低,阳性细胞百分率下降。(5)YLSF对心肌细胞凋亡的影响:YLSFH组可减轻心肌细胞凋亡程度。(6)YLSF对心肌ANT1、Caspase-3 mRNA表达的影响:与MIRI组比较,YLSFH组、YLSFL组ANT1mRNA表达上调(P<0.05),YLSH组Caspase3的mRNA表达下调(P<0.05)。(7)YLSF对心肌细胞超微结构的影响: YLSF能明显减轻心肌超微结构的破坏。结论:YLSF对猴MIRI具有显著的保护作用,其机制可能与改善心功能、抗脂质过氧化损伤、下调Bax蛋白表达、增加Bcl-2/Bax的比率,下调NF-κBp65表达,减轻心肌细胞凋亡程度,以及上调ANT1mRNA的表达以及通过下调Caspase3 mRNA的表达有关。第二部分:柿叶黄酮对犬心肌缺血再灌注损伤的保护作用及机制研究目的:研究柿叶黄酮(PLF)对犬心肌缺血再灌注损伤(MIRI)的保护作用并对其机制进行初步探讨。方法:选取实验用比格犬随机分为5组,每组6只:假手术组、心肌缺血再灌注损伤(MIRI)组、柿叶黄酮低剂量(PLFL)组、柿叶黄酮高剂量(PLFH)组、地尔硫卓(DIL)组。缺血前30min经十二指肠注射给药。结扎冠状动脉左前降支60min后,再灌注180min,建立MIRI模型。再灌注结束后测定下列各项指标:(1)利用MPA心功能分析系统观察并纪录在急性缺血和再灌注状态下血流动力学指标(HR、LVSP、Lvedp、+dp/dtmax、-dp/dtmax、IT、ET、IT/ET)的变化。(2)取缺血区的心肌组织,常规制作组织切片、电镜切片,光镜、电镜下观察心肌组织病理结构和超微结构的变化并拍照。(3)从股静脉脉采血,离心,取上层血清,按试剂盒说明测定AST、CK、CK-MB、LDH、LDH1,SOD、GSH-Px和TNOS的活性、MDA和NEFA含量;取缺血区心肌组织,按试剂盒说明测定AST、CK、CK-MB、LDH、LDH1,SOD、GSH-Px、TNOS、ATP酶的活性、MDA和NEFA含量。(4)取缺血区心肌组织,免疫组化法测定Bcl-2和Bax蛋白表达。结果:(1)与MIRI组相比,在再灌注180min(r180min)时,PLFH组HR、LVSP、+dp/dtmax、ET显著高于MIRI组(P<0.01或P<0.05),而Lvedp、-dp/dtmax、IT、IT/ET显著低于MIRI组(P<0.01或P<0.05)。(2)光镜下观察,MIRI组心肌纤维排列紊乱、肿胀、部分断裂,甚至出现片状坏死,周围有炎性细胞浸润,红细胞漏出明显。PLFL、PLFH组的损伤明显减轻。电镜下观察,MIRI组的心肌细胞的肌丝、线粒体、闰盘等破坏明显,而PLFL、PLFH组则明显减轻。(3)与MIRI组比较,PLF能显著抑制血清中心肌酶活性和MDA、NEFA含量的提高,抑制血清中SOD、GSH-Px、TNOS活性的降低(P<0.01或P<0.05);PLF还能同时降低组织中MDA和NEFA含量,提高心肌酶、SOD、GSH-Px和ATP酶的活性(P<0.01或P<0.05)。(4)与MIRI组比较, PLFL、PLFH组Bcl-2蛋白表达明显增高(P<0.01或P<0.05),而NF-κBp65、Bax蛋白表达及Bax/Bcl-2的比值明显降低(P<0.01或P<0.05)。结论:柿PLF对MIRI具有明显的保护作用。其抑制可能与抗脂质过氧化损伤、保护ATP酶活性、降低NEFA含量、提高TNOS活性、改善心功能和抑制心肌细胞凋亡有关。更多还原

【Abstract】 The occurance and development of myocardial ischemia reperfusion injury (MIRI) are multiple cascade associated with various factors, for example, free radical injury, calcium overload, microvascular injury and the effects of leucocytes. The most recent researches indicate that comlements, selectins, endothelin and apoptosis are involved in the pathophysiological process of MIRI. Obviously MIRI is a complex pathological process which multiple factors interact and improve each other. MIRI is a troublesome problem of mycardial ischemia for long time.The natural resources of Chinese medicinal materials is very rich in China. It is of great significance to search for the safe and effective herbs for the prevention and treatment of MIRI, particularly for the active components in these herbs and the study of their pharmacodynamics and mechanisms on anti-MIRI, which can help to develop our country’s herbal resources and to make the modernization of Traditional Chinese Medicine.For the last more than ten years, we have gone in for exploring the rich ethnic herbal resources in Guangxi. After screening a large number of herbs, two better effective anti-MIRI active site of the ethnic medical herbs—Yulangsan flavonoids (YLSF) and persimmon leaf flavonoids (PLF) were found. Our preliminary studies showed that: (1) YLSF preconditioning could significantly decrease the activitis of AST, LDH and LDH1, the contents of MDA, and the myocardial infarct size, and increase the activity of SOD. The results indicated that YLSF had a significant protective effect on myocardial ischemia reperfusion injury (MIRI) in rats. In addition, the two flavone morphons had good effects on hypoxia/reoxygenation injury in cultured neonatal rat cardiomyocytes, which was related with scavenging free radicals and inhibiting Ca2+-overloading. The further study showed that the two flavone morphons also had better antioxidation, anti-hypoxia,anticoagulation, and scavenging of free radicals.(2) PLF could obviously recover cardiac function, reduce the releases of CK, CK-MB, LDH, and LDH-1 from rat hearts of MIRI, increase the activities of SOD, Na+-K+-ATPase and Ca2+-ATPase, and decrease the MDA product. It were suggested that PLF may offer myocardial protective effect against MIRI,the mechanism might be related to attenuating free radicals. Other, the study of antihypertensive effect of PLF showed that PLF could decrease blood pressur and heart rat in normal rats and L-NAME induced hypertention in rats, and might adjust the imbalance of cardiovascular active substances by increasing the release of endogenous vasodilators and reducing the release of endogenous vasoconstrictors, resulting in antihypertention.In short, our prior studies indicated that YLSF and PLF had significant protective effects on MIRI in rats, which are related with many kinds of factors,such as antioxidation and scavenging free radicals. To understand further the influence of YLSF and PLF on MIRI in big animal (rhesus monkeys or dogs), and to accomplish their pre-clinical studies, the rhesus monkey or dog model was used to research the effects of YLSF and PLF on MIRI. The experiments contained two parts as the following:Part I Effect and action mechanism of Yulangsan flavonoids on myocardial ischemic reperfusion in rhesus monkeysObjective: To study the effect and action mechanism of Yulangsan flavonoids (YLSF) on myocardial ischemic reperfusion injury (MIRI) in rhesus monkeys.Methods: Rhesus monkeys were randomly divided into 5 groups: sham operation (SHAM) group, myocardial ischemia reperfusion injury (MIRI) group, YLSF low dose (YLSFL) group, YLSF high dose(YLSFH) group, Diltiazem(DIL) group, and there were 4 animals at each group. Different doses of drug were administered into duodenum 30 min before ligation of the left anterior descending coronary artery (LAD) for 60 min and followed with reperfusion for 180 min to establish MIRI model. Determination of the following indexes: (1) MPA-cardiac function acquisition and analytical system (MPA-CFS) was used to record HR, LVSP, Lvedp, +dp/dtmax, and -dp/dtmax. (2) Myocardial ischemic tissues were taken to make histological section and electron microscopic section S.A. for observing the changes of histopathology and ultrastructure of myocardium under optical microscope or transmission electron microscope respectively, and taking the pictures with camera simultaneously. (3) The blood was collected via the femoral vein, centrifuged, and the upper serum was taken for detecting activities of AST, LDH, LDH1, CK, CK-MB, SOD, GSH-Px, TNOS, MDA and NEFA in each Beagle dog. The myocardial ischemic tissues were taken for determining the activities of ATPase. (4) The myocardial ischemic tissues were taken for determining Bcl-2, Bax and NF-κB protein expressions, cardiac muscle cell apoptosis, ANT1 and Caspase 3 mRNA expressions.Results: (1). Compared with MIRI group, at 180min after reperfusion, YLSFH could significantly increased the LVSP, while could not have effect on HR, LVDEP, +dp/dtmax and -dp/dtmax。(2) Compared with MIRI group, YLSF could significantly inhibited the increasing of myocardial enzyme activity and the content of MDA and NEFA, and increase the activities of SOD, GSH-Px, Na+K+-ATPase and Ca2+Mg2+-ATPase. (3) Histopathological examination confirmed that the degree of myocardial injury in YLSF groups were obviously lessened when compared with model group. (4) The high dosage of YLSF could downregulate the expression of Bax and significantly increase the ratio of Bcl-2/Bax. The high dosage of YLSF could increase the expression of Bcl-2, but the low dosages of YLSF could not have effect on Bcl-2. YLSF could downregulate the expression of NF-κBp65 and the percentage of positive cell. (5) The high dosage of YLSF could lessen the degree of the cardiac muscle cell apoptosis. (6) Compared with MIRI group, expression of ANT1 mRNA increased in YLSFH and YLSFL, expression of Caspase 3 mRNA decreased in YLSFH significantly (P<0.05). (7) YLSF could reduce the injury of the myocardial ultrastructure.Conclusion: Yulangsan flavonoids (YLSF) may offer myocardial protective effects against ischemia reperfusion injury, which may be related to their effects of improving hemodynamics of heart, anti-oxidation, downregulating the expression of Bax protein, increasing the Bcl-2/Bax ratio, downregulating the expression of NF-κBp65, lessening the degree of the cardiac muscle cell apoptosis, and up-regulation of ANT1 mRNA expression and down-regulation of Caspase 3 mRNA expression.Part II The Protective Effect and Mechanisms of Persimmon leaf Flavonid on Myocardial Ischemia Reperfusion Injury in DogsObjective: To study the protective effect of persimmon leaf flavonoid on on myocardial ischemia reperfusion injury in dogs and its related mechanisms.Methods: Healthy Beagle dogs were picked out and randomly divided into 4 groups with 6 dogs in each group: sham operation (SHAM) group, myocardial ischemia reperfusion injury (MIRI) group, PLF low dose (PLFL) group, PLF high (PLFH) dose group,positive drug (DIL)group. Different doses of drug were administered into duodenum 30 min before ligation of the left anterior descending coronary artery (LAD) for 60 min and followed with reperfusion for 180 min to establish MIRI model. Determine the following indexes: 1. MPA-cardiac function acquisition and analytical system (MPA-CFS) was used to record HR, LVSP, Lvedp、+dp/dtmax, -dp/dtmax, IT, ET and IT/ET. 2. Myocardial ischemic tissues were taken to make histological section and electron microscopic section S.A. for observing the changes of histopathology and ultrastructure of myocardium under optical microscope and transmission electron microscope respectively, and taking the pictures with camera simultaneously. 3.The blood was collected via the femoral vein, centrifuged, and the upper serum was taken for detecting activities of AST, CK, CK-MB, LDH, LDH1, SOD, GSH-Px and TNOS, and content of MDA and NEFA in each Beagle dog. The myocardial ischemic tissues were taken for determining the activities of AST, CK, CK-MB, LDH, LDH1, SOD, GSH-Px, TNOS and ATPase, and content of MDA and NEFA. 4. The myocardial ischemic tissues were taken for determining Bcl-2 and Bax protein expressions by immunohistochemical method.Results: 1. Compared with MIRI group, at 180min after reperfusion, HR, LVSP, +dp/dtmax and ET in PLFH group increased significantly (P<0.01 or P<0.05), while the Lvedp, -dp/dtmax, IT, IT/ET decreased significantly (P<0.01 or P<0.05). 2. Myocardial fibers were in disorder condition under optical microscope in MIRI group, in which cellular edema, break or necrosis, infiltration of inflammatory cells, or obvious leakage of red blood cells could be observed. The above injuries could be significantly alleviated in PLFL and PLFH group. The obvious damages of myocardial fibers, mitochondria and intercalated disks could be observed in MIRI group under electronic microscope, but these damages were attenuated significantly in PLFL and PLFH group. 3. Compared with MIRI group, persimmon leaf flavonoid could inhibited the increasing of myocardial enzyme activity and the content of MDA in the serum significantly (P<0.01), and inhibited the decreasing in the activities of SOD, GSH-Px and TNOS in the serum significantly (P<0.01 or P<0.05). Persimmon leaf flavonoid also could decrease the content of MDA and NEFA in the myocardial ischemic tissues significantly (P<0.01 or P<0.05), and increase the myocardial enzyme activity and the activities of SOD, GSH-Px and ATPase in the myocardial ischemic tissues(P<0.01 or P<0.05). 4. Compared with the MIRI group, the expression of Bc1-2 protein increased significantly in PLFL and PLFH group (P> 0.05), the expresion of NF-κBp65 protein,Bax protein and the ratio of Bax protein to Bc1-2 decreased significantly (P <0.05),Conclusion: Persimmon leaf extracts have the protective effects on myocardial ischemical reperfusion injury in dogs. The mechanism may be related to anti-oxidation, protect the activity of ATPase, decrease the content of NEFA, increase the activity of TNOS, improve hemodynamics of heart and inhibit the cardiocyte apoptosis.更多还原