【作者】 范丽；

【导师】 马传庚； 陈志武；

【作者基本信息】 安徽医科大学 ， 心脑血管药理学， 2003， 博士

【摘要】 黄蜀葵花总黄酮（Total flavone of Abelmoschl Manihot L medic，TFA）是锦葵科秋葵属植物黄蜀葵的花中提取的有效部位。本室以前的研究初步表明灌胃（intragastric administration，ig）给予TFA对心肌缺血可能有一定的保护作用，但未做系统研究。众所周知，黄酮类化合物口服给药生物利用度较低，影响其药效的发挥，因此，本研究采用静脉注射（intravenous injection，iv）给予TFA的方法，对TFA保护缺血性心肌损伤的作用进行了进一步研究，并重点对TFA药理性预适应（pharmacological preconditioning，PP）作用及其机制进行了较深入探讨，结果如下： 1．TFA延长夹闭气管心肌缺氧小鼠心电消失的时间 采用夹闭气管致小鼠心肌缺氧的模型，观察小鼠尾静脉注射（iv）不同剂量TFA对其心电消失时间的影响。结果发现TFA 32,16,8 mg·kg^-13个剂量组均可使夹闭气管后小鼠心电消失的时间较NS对照组明显延长，最多延长37.88％，差异具有显著性。 2．TFA减轻异丙肾上腺素（Isoproterenol，Iso）诱发的小鼠急性心肌缺血性损伤 连续皮下注射（subcutaneous injection，sc）Iso，可导致小鼠出现急性心肌缺血，并产生典型的缺血心电图（electrocardiogram，ECG）波形。本实验在每次sc Iso前10min尾静脉注射（iv）不同剂量的TFA，连续3天，每天1次，并于第3天注射Iso后立即记录ECG，20min后处死小鼠，迅速取其完整心肌组织，称心肌湿重；110℃干烤8小时后，再称其干重。计算心肌含水量（Myocardial water content，MWC）及心肌指数（Myocardial Index，MI）。结果发现，模型组小鼠MWC较NS安徽医科大学博士学位论文对照组增加1.96%，Ml升高27.04%。TFA 16mg.kg一‘可明显抑制模型组小鼠Mwc的增高，抑制率为65.82%，TEA犯，16 mg·kg一，组对模型组小鼠Ml的升高的抑制率则分别达47，48%和65.82%，差异具有显著性。对模型组小鼠ECG ST段和T波的异常改变，TFA的改善作用显著。 3.TFA减轻离体大鼠心肌缺血再灌注损伤（i schemiarePerfusioninjury，I班） 采用Langendorff离体心脏灌流法建立离体大鼠心肌缺血缺氧再灌注模型，观察到T队100，50，25 mg·L’‘可明显增加缺血再灌注（isehemia:eper九sion，xR）离体大鼠心肌组织匀浆中超氧化物歧化酶（s即eroxide dismutase，SOD）的活力，显著降低丙二醛（malondialdehyde，MDA）生成量，明显减少心肌细胞内乳酸脱氢酶（laetate de抑drogenase，LDH）的漏出;T队100，50 mg·L‘’可明显减少心肌细胞内肌酸磷酸激酶（ereatine phosphokinase，CpK）的漏出;此外，TFA可明显升高心肌组织中一氧化氮合酶（nitrieoxide synthase，Nos）的活力（2 00，25 mg·L一’剂量组）和心肌组织中一氧化氮（nitric。xide，No）含量（一00 mg·L一，剂量组）。 4.TFA增加犬冠脉血流量 采用电磁流量计测定犬冠状动脉左旋支（Left eireumflex braneh of eoronary，LcX）血流量的方法，观察iv TFA对犬Lcx血流量的影响。结果发现，16mg .kg一，TFA在给药后IOmin时，犬LCX的血流量出现显著增高，并一直持续至25min。 5.T队药理性预适应（Pharmacologieal preeonditioning with TFA，TFA一pp）改善家兔心肌IRI 家兔持续静脉推注（smin）不同剂量T队（16，s，4 mg·kg一，），停用10min，再进行IR以模拟缺血预适应（ischemic preeonditioning，Ip）的实验方法，观察家兔LCX经结扎、再灌后，其ECG心律失常发生率、ECGll导联ST段、T波及生化指标的变化情况。结果发现TFA16，8，4 mg·kg一‘对单纯IR组家兔出现的异常EcG改变（ST段抬高、T波高耸等）有较好的改善作用，同时明显降低了心律失常的发生率。生化指标测定结果显示TFA16，8，4 mg·kg一’组不同程度抑制了心肌细胞内乳酸（Lactic Acid，LD）的生成量和LDH的漏出;显著升高血浆中SOD及谷安徽医科大学博士学位论文肤甘肤过氧物酶（glutatnione peroxidase，osH一Px）的活力、明显减少MDA的生成;对血浆中NOS活力的增加和NO生成亦可产生不同程度的促进作用，与IP保护效应相似。 6.T队一PP上调细胞凋亡相关基因bel一2信使核糖核酸（messenger ribonucleic-acid，mRNA）、下调P53 n1RNA的表达 在家兔IP模型上，实验结束时通过采集心肌组织标本，应用原位杂交（ in situhybridization，ISH）分子病理学实验技术检测细胞凋亡相关基因bel一2 mRNA及P53 mRNA的表达情况。结果发现，IR组家兔心肌组织中P53 mRNA分布密度显著增高，呈阳性表达，bel一2 mRNA的表达与之相反。IP组家兔，经短暂缺血预处理后，P53 mRNA分布密度明显降低，表达水平下调;不同剂量的TFA预处理结果与xP组相似，也明显下调P53m胭A的表达（16，8，4 mg·kg一，），同时上调bel一2mRNA的表达水平。 7.TFA一PP下调细胞间粘附分子（intereellular adhesion moleeule一l，ICAM一1）mRNA的表达 同上模型，采用ISH分子病理学实验技术检测心肌ICAM一1 mRNA的表达情况。结果发现，IR组家兔心肌组织中ICAM一1基因mRNA分布密度显著增高，呈阳性表达，而TFA16，8，4mg’k梦3个剂量组经预处理后，显著下调了ICAM一1mRNA?更多还原

【Abstract】 Total flavone of Abelmoschl Manihot L medic (TFA) was extracted from the flower of Abelmoschus, which has been preliminary assessed to be of protection against myocardial ischemia given via intragastric administration (ig) route. It is known that the absorption of flavonoides is poor when given intragastricly. The low bioavailability would affect their validity. The advanced study on the protective effect of TFA against ischemic myocardium injury and its pharmacological pre-conditioning mechanisms through intravenous injection (iv) was performed in this paper:1 Prolongating effect of TFA on ECG survival time in trachea clamping miceOn the myocardial anoxia model induced by clamping trachea in mice, it is found that the ECG survival time were much longer in the groups pretreated with TFA (32,16, 8 mg kg-1) than that in NS control group. The maximal elongation rate of ECG survival time reached a height of 37.88%.2 Protective effect of TFA on the acute myocardial ischemia injury in mice induced by isoproterenolAcute myocardial ischemia episodes and typical ischemia ECG developed after subcutaneous injection (sc) of Iso continuously. The animals were pretreated with TFA at doses of 32, 16 or 8 mg kg-1 administrated intravenously at 10 minutes before giving Iso once a day for 3 days. On the third day, ECG was recorded at the end of sc Iso. 20minutes later, the mice were killed, the wet weights of myocardium were weighted immediately. Their dry weights were weighted after 8 hours baking at 110C. The results showed that the myocardial water content (MWC) and the myocardial index (MI)in model group were 1.96% and 27.04% higher than NS control. TFA (16mg kg-1) can significantly inhibit the increase of MWC of model group. Its inhibition rate was 65.82%. TFA (32, 16mg kg-1) can also remarkably inhibit the increase of MI of model group and the inhibition rates were 47.48% and 65.82% respectively. Moreover, TFA showed remarkedly the amelioration effect on the abnormal changes of ECG vs the model control.3 Protective effect of TFA on myocardial ischemia reperfusion injury (IRI) in rat Langendorff heartsUsing the isolated perfused rat heart as a model set up by Langendorff, it was found that TFA( 100,50, 25mg L-1) can obviously increase the activity of superoxide dismutase (SOD) in myocardium homogenates, significantly reduce the lipid peroxidation product, malondialdehyde (MDA) , decrease the transudation of creatine phosphokinase(CPK) and lactate dehydrogenase (LDH) from cardiomyocyte. And also, TFA remarkedly raised nitric oxide (NO) level and the activity of NO synthase( NOS).4 TFA increases the blood flow volume of left circumflex branch of coronary (LCX) in dogsIt was found that TFA (16mg kg-1) can significantly increase the LCX blood flow in dogs determined by the electromagnetic flow meter at lOminute after administration of TFA and the effect lasted foe over 15 minutes.5 Protective effect of TFA-PP on IRI in rabbitsBefore the 30 minutes ischemia and the followed 60 minutes reperfusion, the rabbit hearts were preconditioned with 5 minutes ischemia and 10 minutes reperfusion so as to elicit cardioprotection of ischemic preconditioning (IP). In order to mimic IP, before ischemia reperfusion (IR), different doses of TFA were perfused intra-venously for 5 minutes, then followed a 10 minutes break. The results showed that TFA (16, 8, 4mg kg-1) could ameliorate the abnormal changes of ECG (ST segment and T wave), decrease the incidence rate of cardiac arrhythmia. And also, TFA at the samedoses significantly reduce the content of lactic acid (LD) in myocardial homogenate, decrease the transudation of lactate dehydrogenase (LDH) from myocardial cells, enhance the activities of SOD and glutathione peroxidase (GSH-PX), decrease MDA level in plasma. Meanwhile, TFA could also reinforce the activity of NOS, increase the content of NO in plasma. Such effective changes of biochemical markers in TFA groups are about the same as IP group.6 Effect of TFA on the expression of the ap更多还原