【作者】 覃斐章；

【导师】 黄仁彬；

【作者基本信息】 广西医科大学 ， 药理学， 2012， 博士

【摘要】 玉郎伞[Millettia pulchra Kurz var laxior(Dunn) Z. Wei, LYS]是广西壮族的特色药材,具有广泛的心血管药理活性,本课题组对其进行了长期深入的研究,17-甲氧基-7-羟基-苯并呋喃查尔酮(17-methoxyl-7-hydroxy-benzfuranchalcon, YLSC)是从玉郎伞中提取的黄酮类新化合物,其分子式为C18H14O4,分子量为294.3,已授权1项国家发明专利(200710034771.2)。我们体外的研究证实,YLSC具有较强的清除自由基、耐缺氧、抗凝血能力,对H20：及缺氧/复氧所致的心肌细胞损伤有较好的保护作用,其作用机制与抗氧化、抗凝血作用,保护Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性,增加T-SOD、GSH、cNOS、tNOS活性,减少MDA、LDH、iNOS的产生等途径有关。但YLSC对整体动物是否有效,尚不清楚。本实验在前期研究的基础上,研究YLSC对垂体后叶素和缺血再灌注致大鼠心肌缺血的影响,以评价YLSC的药效,为进一步研究YLSC作用机制奠定基础,并为其开发利用提供理论依据。第一部分17-甲氧基-7-羟基-苯并呋喃查尔酮对垂体后叶素致大鼠心肌缺血的保护作用摘要目的：观察17-甲氧基-7-羟基-苯并呋喃查尔酮(YLSC)对垂体后叶素致心肌缺血大鼠的影响。方法：将60只SD大鼠随机分为6组,每组10只：正常对照组,模型对照组,溶媒组,阳性药对照组,YLSC低、高剂量组(2.5,5.0mg·kg-1)。各组尾静脉给予相应药物后,观察垂体后叶素致心肌缺血大鼠Ⅱ导联心电图的变化,伊文思蓝和三苯基氯化四氮唑(TTC)双重染色确定心肌缺血面积,HE染色观察心肌形态学变化,Elisa法检测血清生化标志物MB型肌酸激酶(CK-MB)、肌钙蛋白(cTnl)和肌红蛋白(MyoG)的含量。结果：YLSC能剂量依赖性的减少大鼠15s,30s, lmin,5min的ST段抬高,与模型组比较,差异有显著性(P<0.05或P<0.01)；同时能剂量依赖性的缩小模型组心肌缺血面积(P<0.05或P<0.01),减少心肌标志物CK-MB, cTnl, MyoG的漏出(P<0.05或P<0.01),改善心肌水肿、出血和炎细胞渗出的状况。结论：YLSC对垂体后叶素所致心肌缺血大鼠有明显保护作用。第二部分17-甲氧基-7-羟基-苯并呋喃查尔酮预处理对大鼠离体心脏缺血再灌注损伤的保护作用摘要目的：观察17-甲氧基-7-羟基-苯并呋喃查尔酮(YLSC)预处理对大鼠离体心脏缺血再灌注损伤的影响。方法：将70只SD大鼠随机分为7组,每组10只：正常对照组,缺血再灌注(I/R)组,溶媒组,缺血预处理(IPC)组,阳性组,YLSC低、高剂量组(5.0mg·L-1,10.0mg·L-1)。缺血30min,再灌注30min,记录给药前,再灌注5,10,20,30min的心率(HR)和左室收缩压(LVSP),并检测冠脉流量(CF)和流出液中肌酸激酶(CK)的含量。实验结束后计算心肌梗死面积,并检测心肌组织中一氧化氮(NO),一氧化氮合成酶(NOS),超氧化物歧化酶(SOD),丙二醛(MDA)的含量。结果：YLSC能剂量依赖性的增加I/R组离体心脏再灌注后5,10,20,30min的CF, HR和LVSP (P<0.05或P<0.01),改善心功能低下,同时能缩小缺血再灌注大鼠的心肌梗死面积(P<0.01),减少冠脉流出液中CK的含量(P<0.05或P<0.01)。与I/R组比较,YLSC预处理组MDA生成明显减少,NO, NOS, SOD活性明显升高(P<0.05或P<0.01)。结论：YLSC预处理对大鼠离体心脏缺血再灌注有明显的保护作用,其机制可能与抗氧化,增加NO释放有关。第三部分17-甲氧基-7-羟基-苯并呋喃查尔酮对大鼠心肌缺血再灌注损伤的保护作用摘要目的：观察17-甲氧基-7-羟基-苯并呋喃查尔酮(YLSC)对心肌缺血再灌注损伤大鼠的影响。方法：SD大鼠60只随机分为6组,每组10只：假手术组,模型组,溶媒组,阳性药地尔硫卓组(2.0mg·kg-1), YLSC低剂量组(2.5mg·kg-1)、YLSC高剂量组(5.0mg·kg-1)。缺血30min再灌注60min复制大鼠缺血再灌注(I/R)模型。观察大鼠血流动力学指标如HR, MAP, LVEDP和±dp/dtmax的变化,测定心肌组织中超氧化物歧化酶(SOD)、丙二醛(MDA)、Na+-K+-ATP酶(Na+-K+-ATPase)、Ca2+-ATP酶(Ca2+-ATPase)的含量。TUNEL法检测心肌阳性细胞凋亡率,Western blot法检测凋亡相关蛋白如凋亡信号调节蛋白1(apoptosis signal regulating kinase1, ASK1)、细胞色素c (cytochrome c, cyt c)、Bcl-2、Bax的蛋白表达量,并计算Bcl-2与Bax蛋白表达的比值。结果：与模型组比较,YLSC能剂量依赖性的降低HR、MAP和±dp/dtmax,升高LVEDP (P<0.05或P<0.01); YLSC还能剂量依赖性的减少MDA的释放,增加SOD、 Na+-K+-ATPase和Ca2+-ATPase的活性(P<0.05或P<0.01)。与模型组相比,YLSC能明显降低心肌细胞的凋亡率(P<0.01)；剂量依赖性的减少ASK1、cyt c、Bax蛋白的表达(P<0.05或P<0.01),增加Bcl-2蛋白的表达(P<0.01)；提高Bcl-2/Bax蛋白表达的比值(P<0.01)。结论：YLSC能改善缺血再灌注大鼠的心功能,明显减轻大鼠缺血再灌注诱导的细胞凋亡,机制可能与抗氧化,保护Na+-K+-ATPase和Ca2+-ATPase活性,减少ASK1、cyt c、Bax蛋白表达,增加Bcl-2蛋白表达,升高Bcl-2/Bax比值有关。第四部分17-甲氧基-7-羟基-苯并呋喃查尔酮对乳鼠心肌细胞缺氧／复氧损伤的保护作用摘要目的：观察17-甲氧基-7-羟基-苯并呋喃查尔酮(YLSC)对心肌细胞缺氧/复氧(A/R)损伤的保护作用及其机制。方法：采用差数贴壁法体外分离培养新生乳鼠心肌细胞,缺氧2h复氧4h模拟心肌细胞A/R模型。实验随机分为正常对照组,A/R组,溶媒组,阳性药地尔硫卓组(30μmol·L-1), YLSC低剂量组(75μg·m L-1),YLSC高剂量组(150μg·m L-1).观察心肌细胞形态和搏动频率的变化,采用流式细胞仪检测细胞线粒体膜电位(△Ψm)及细胞内游离钙离子浓度([Ca2+]i)的变化,检测心肌细胞膜Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性,检测心肌细胞L型钙通道的α1c亚单位(Alphac subunit of L-type Ca2+channels,Cav1.2),钠钙交换蛋白(sodium-calcium exchanger,NCX)和肌浆网钙泵-2a (SR calcium ATPase-2a,SERCA2a)mRNA和蛋白表达量的变化。检测心肌细胞凋亡率和凋亡相关蛋白细胞色素c(cytochrome c,cyt c).Bcl-2.Bax.caspase9和caspase3的表达。结果：A/R组心肌细胞心肌细胞搏动明显减慢,[Ca2+]i显著增加,△Ψ m、Na+-K+-ATP酶、Ca2+、Mg2+-ATP酶活性显著降低,Cavl.2和NCX表达升高,SERCA2a表达降低(与正常对照组比较,P<0.05或P<0.01)。YLSC可呈浓度依赖性显著降低[Ca2+]i,增加△Ψm、Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性,降低Cav1.2和NCX表达(与A/R组比较,P<0.05或P<0.01),对SERCA2a表达无明显影响。与A/R组相比,YLSC明显降低心肌细胞凋亡率,增加心肌Bcl-2/Bax比值,阻止cyt c释放,减少caspase9,caspase3活性(P<0.05或P<0.01)。结论：YLSC可以减轻A/R诱导的心肌细胞损伤,抑制细胞凋亡,这可能与其增强细胞Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶活性,降低Cav1.2和NCX表达,减轻细胞内钙超载和减少凋亡有关。综上所述,YLSC可能是通过清除自由基、减少心肌酶的漏出、有效改善缺血大鼠的血流动力学、减少心肌收缩力、抑制凋亡产生和减少钙超载相关基因蛋白表达等机制而达到抗心肌缺血作用。更多还原

【Abstract】 Yulangsan(YLS), the root of one plant named Millettia pulchra Kurz var laxior (Dunn) Z.Wei, is a traditional Chinese herb used for the treatment of various diseases including hypertension, hyperlipidemia, hyperglycemia and senile dementia. Moreover, our studies have proven that YLS has a significant protective effect against ischemia reperfusion injury in rat hearts and brains in vitro and in vivo. Howerver, until now, the specific active component in YLS was unknown. We have identified eight flavone monomers. After preliminary screening, we determined that YLSC is bioactive. YLSC is a component of flavone whose chemical name is17-methoxyl-7-hydroxyl-benzofuran chalcone. Separation and extraction technology of YLSC has been awarded a national patent(patent number:200710034771.2).In recent studies, YLSC showed the effects of anti-blood coagulation, anti-apoptosis, inhibition of Ca2+overloading, and regulation of oxidation, in particular through its ability to scavenge free radicals. Our research is to observe the effects of YLSC on anti-ischemia and its mechanisms.Part Ⅰ. Protective effect of17-methoxyl-7-hydroxyl-benzofuran chalcone on myocardial ischemia induced by pituitrin in ratsAbstract Objective:To study the cardioprotective effect of17-methoxyl-7-hydroxyl-benzofuran chalcone (YLSC) in rats with myocardial injury induced by pituitrin. Method:60SD rats were randomly divided into six groups:normal group, vehicle group, model control group, positive control group and YLSC groups(2.5,5.0mg.kg-1). Pituitrin(0.75Ukg-1) was injected through sublingual vein to erect model of acute myocardium ischemia. The changes of ST segment on the II road electrocardiogram and contents of creatine kinase-MB(CK-MB), Troponin (cTnI), myoglobin(MyoG) in serum were observed and compared. Meanwhile, myocardium pathological structures were accessed with HE staining. Myocardial ischemia sizes were measured by Evans blue and TTC staining when the experiment was over. Result:Compared with the model control group, YLSC could significantly decrease the height of ST segment at15s,30s, lmin and5min in a dose-dependent manner(P<0.05or P<0.01). It also could reduce myocardial ischemia sizes and contents of CK-MB, cTnI, MyoG in serum (P<0.05or P<0.01). Furthermore,these damages induced by pituitrin including myocardium edema, hemorrhage and exudation of inflammatory cells were improved in YLSC treated groups. Conclusion:YLSC has obvious protective effect against pituitrin induced myocardial injury in rats.Part Ⅱ.17-methoxyl-7-hydroxyl-benzofuran chalcone participating in pharmacological preconditioning against cardiac ischemia reperfusion injury in isolated rat heartsAbstract Objective:To investigate whether17-methoxyl-7-hydroxyl-benzofuran chalcone (YLSC) participated in pharmacological preconditioning against cardiac ischemia reperfusion injury in isolated rat hearts. Methods:70SD rats were randomly divided into seven groups:normal group, ischemia reperfusion(I/R) group,vehicle group, ischema preconditioning(IPC) group, positive control group and YLSC groups (5.0mg·L-1,10.0mg·L-1). Isolated rat hearts were subjected to ischemia by stopping perfusion for30min followed by reperfusion for30min in Langendorff perfusion system. The coronary arterial flow(CF), heart rate(HR) and left ventricular developed pressure(LVSP) were recorded. The activity of creatine kinase (CK) in the coronary effluent fluid was measured. The infarction sizes were measured with TTC dying method. Contents of NO, NOS, SOD and MDA were measured when the experiment was over. Results:Compared with the I/R group, YLSC could significantly increase CF, HR and LVSP at5,10,20,30min after reperfusion in a dose-dependent manner(P<0.05or P<0.01).YLSC preconditioning could improve the recovery of cardiac function, decrease the activiy of CK, and minimize the size of myocardial infarction(P<0.05or P<0.01). It also could increase activity of NO, NOS and SOD, reduce content of MDA compared with the I/R group(P<0.05or P<0.01). Conclusion:YLSC has obvious protective effect against ischemia reperfusion injury in the isolated rat hearts, the mechanism may involve in stimulation of NO release and inhibition of lipid peroxidation.Part Ⅲ. Protective effects of17-methoxyl-7-hydroxyl-benzofuran chalcone on rats with myocardial ischemia reperfusion injuryAbstract Purpose:To study the cardioprotective effect of7-methoxyl-7-hydroxyl-benzofuran chalcone (YLSC) on rats with myocardial ischemia reperfusion injury. Methods:60SD rats were randomly divided into six groups: Sham group, Model group, Vehicle group, Dilthiazem(Dil) group, YLSCL group(2.5mg/kg) and YLSCH group(5.0mg/kg). Myocardial ischemia reperfusion injury model was established through occluding left anterior descending branch of coronary artery for30min and removing ligation later to reperfuse for1h. The changes of hemodynamic, including HR, MAP, LVEDP and±dp/dtmax were recorded. Contects of SOD, MDA, Na+-K+-ATPase and Ca2+-ATPase were detected when the experiment was over. Myocardium apoptosis rate was measured by TUNEL.Expressions of apoptosis signal regulating kinase1(ASK1),cytochrome c (cyt c), Bcl-2and Bax protein were tested by western blot.Meanwhile, Bcl-2/Bax ratio was calculated. Results: Compared with the Model group, YLSC could decrease HR, MAP and±dp/dtmax, increase LVEDP, inhibit a increase of MDA, and increase the activity of SOD, Na+-K+-ATPase and Ca2+-ATPase in a dose-dependent manner (P<0.05or P<0.01).Compared with that in the Model group, apoptosis rate was significantly decreased in YLSC groups(P<0.01).Expression levels of ASK1, cyt c and Bax protein were lower in YLSC groups than that in Model group (P<0.05or P<0.01), while the level of Bcl-2protein and Bcl-2/Bax ratio were higher in YLSC groups than that in Model group(P<0.01), respectively. Conclusion: YLSC plays a protective role in rats with myocardial ischemia reperfusion injury. The mechanism may involve in anti-apoptosis, inhibting effect on lipid peroxidation, protecting activity of Na+-K+-ATPase and Ca2+-ATPase, decreasing ASK1, cyt c, Bax protein expression, increasing Bax protein expression, and augmenting Bcl-2/Bax ratio in myocardium of the rat.Part Ⅳ. The protective effects of YLSC on cultured cardiomyocytes in anoxia/reoxygenation injuryAbstract Objective:To study protective effects and mechanisms of17-methoxyl-7-hydroxyl-benzofuran chalcone(YLSC) on damage induced by anoxia/reoxygenation(A/R) in myocardial cell. Methods:Primary cultured neonate rat myocardial cells were pretreated with2h anoxia following4h reoxygenation. They were randomly assigned to six groups:control group, A/R group, vehicle group, Diltiazem (Dil) group(30μmol·L-1), YLSL group(75μg·mL-1), YLSH group(150μg·mL-1). At the end of experiment, myocardial cells beating rate was calculated, intracellular Ca2+([Ca2+];) and mitochondrial membrane potential (ΔΨm) were determined by flow cytometric analysis. Besides, Na+-K+-ATPase and Ca2+-Mg2+-ATPase, Alphac subunit of T-type Ca2+channels(Cav1.2), Sodium-calcium exchanger (NCX) and SR calcium ATPase-2a(SERCA2a) expressions were also evaluated in each group. Results: Compared with the A/R group, the beating rate,[Ca2+]; and ΔΨm of YLSC groups was significantly increased(P<0.05or P<0.01). Compared with the A/R group, Cav1.2and NCX mRNA and protein expressions were significantly decreased(P<0.05or P<0.01), but SERCA2a mRNA and protein expression were not changed. Compared with the A/R group, treatment with YLSC significantly decreased apoptosis(P<0.01). Furthermore, YLSC inhibited cyt c release from the mitochondria and prevented caspase9/3activation(P<0.05. or P<0.01). Conclusion:YLSC exerts significantly cardioprotective effects against A/R in cultured myocardial cells. The protection of calcium overload and anti-apoptosis may be involved in the mechanism of YLSC protection.In summary, the present results suggest that YLSC is effective in anti-ischemia. As a possible mechanism, YLSC can scavenge free radicals, reduce the contents of myocardium kinase, regulate hemodynamic, ruduce myocardial contractility, inhibit apoptosis, inhibit calcium overload, and inhibit the expressions of calcium overload-related gene.更多还原