大豆苷元对结扎大鼠冠状动脉所致损伤的保护作用(一)
详细内容
作者：叶和杨,胡志苹,周莉,黄志华,曾靖
【关键词】 大豆
Protective effect of Daidzein on myocardial ischemia injury in rats
【Abstract】 AIM: To observe the protective effect of Daidzein on rats whose branch of left anterior descending coronary arteries were ligated. METHODS: Rats ischemia injury model was established by ligating the branch of left anterior descending coronary artery for a durative course of 120 min. Thirtytwo male rats were randomly divided into 4 groups: sham operation group, model group, highdose (5.0 mg/kg) Daidzein group and lowdose （2.5 mg/kg） Daidzein group. The two Daidzein groups were injected with Daidzein of 1 mL/100 g through sublingual vein 10 min before ischemia injury and saline was given in sham operation group and model group. The leadⅡ ECG was recorded before myocardial ischemia injury, 15 min after ischemia and 30, 60, 90 and 120 min after injection with Daidzein. The ∑ST of lead Ⅱ ECG at each moment mentioned above was analyzed and the Jpoint and Tware of ECG in acute myocardial ischemia model were observed to eva luate the protective effect of Daidzein. The activity of creatine phosphokinase (CK) and lactate dehydrogenase (LDH) in serum, the contents of malondialdehyde (MDA) and superoxide dismutase (SOD) in myocardial tissue were observed to eva luate the protective effect of Daidzein. The myocardial infarct size was measured by TTC staining of cardiac muscle cells in another 24 rats (divided into 3 groups: model group, Daidzein 5.0 mg/kg group, Daidzein 2.5 mg/kg group) with myocardial ischemia injury induced by the same methods. RESULTS: pared with those in model group, the area of acute myocardial infarction declined, the value of ∑ST reduced and the maximum values of Jpoint and Tware of ECG declined in the 2 Daidzein groups. The activity of LDH and CK in the 2 Daidzein groups was lower than that in the model group. The content of SOD was significantly higher and MDA was markedly lower in the Daidzein groups than those in the model group. All the changes mentioned above showed some dosedependent effect. CONCLUSION: Daidzein has obvious protective effect on myocardium against ischemia injury caused by ligating the branch of left anterior descending coronary artery.
【Keywords】 Daidzein; myocardial ischemia; coronary artery ligation; serum enzyme; free radicals
【摘要】 目的：观察大豆苷元对结扎大鼠左冠状动脉前降支损伤的保护作用. 方法：取32只雄性Wister大鼠，随机分为4组，每组8只，分别为假手术组、缺血模型组、大豆苷元高、低剂量组（5.0,2.5 mg/kg）. 于结扎冠状动脉左前降支前10 min经舌下静脉注入大豆苷元溶液，缺血模型组及假手术组经舌下静脉注入等体积生理盐水. 结扎冠状动脉左前降支使心肌缺血并持续120 min建立缺血模型. 记录缺血前、缺血15 min，给药后30，60，90和120 min Ⅱ导联ECG，统计各标测点Ⅱ导联ECG的ST段偏移∑ST、统计各组大鼠心电图J点和T波上升的最大变化值，评价心肌缺血程度. 实验结束后心内取血测定血清LDH、CK值；测定心肌组织MDA和SOD值. 另用24只Wister大鼠分缺血模型组、大豆苷元高、低剂量组（5.0,2.5 mg/kg）组，同上述动物模型制作方法，取其心肌作TTC染色测心肌梗死面积. 结果：大豆苷元组与缺血模型组比较，心肌梗死面积明显缩小，LDH, CK值降低，MDA值减小，SOD值升高，心电图∑ST值减少，J点和T波的上升的最大值明显降低，且呈一定剂量依赖性. 结论：大豆苷元对大鼠结扎冠状动脉的缺血损伤具有保护作用.
【关键词】 大豆苷元；心肌缺血；冠脉结扎；血清酶；自由基
0引言
大豆苷元（Daidzein，DD）系葛根中的主要成份，葛根具有解肌退热、生津透疹、升阳止泻的功效〔1〕，其化学成分复杂. 主要有效成分为葛根素和大豆苷元，现已能人工合成. 葛根素的临床应用非常广泛，对心脑血管疾病〔1-2〕、视网膜疾病〔3〕等具有良好疗效；大豆苷元也具有抗心律失常〔4〕、抑制胆囊收缩〔5〕、耐缺氧〔6〕、影响免疫器官〔7〕等作用. 我们通过实验观察大豆苷元对心肌缺血损伤的保护作用.
1材料和方法
1.1材料健康雄性Wister大鼠，体质量(220±20)g，由江西省实验动物中心提供（许可证号0219712，动物合格证号JXA2004118，清洁级）. 大豆苷元实验样品由沈阳药科大学植化教研室邱峰博士提供,纯度为97%. 实验前用N二甲基甲酰胺溶解DD、双蒸水稀释至所需浓度(3∶7)；溶剂对照液含相等量的N二甲基甲酰胺. 红四氮唑，2,3,5triphenylterazolium chloride（TTC）,中国医药集团上海化学试剂公司；甲醛溶液，南昌洪都试剂化工厂（批号040202）；其他试剂均为国产分析纯. 超氧化物歧化酶(SOD)，丙二醛(MDA)，GSH，乳酸脱氢酶(LDH)，磷酸肌酸激酶(CK)试剂盒均购自南京建成生物工程研究所. Mettler Toledo AG135型光电天平，made in Switzerland；中佳KLC2046型低速冷冻离心机；721分光光度计，上海精密科学仪器有限公司；XDH3心电图机；DH140动物人工呼吸机，浙江医科大学医学仪器厂；电热恒温浴锅，湖北黄石医疗器械厂； JNA型天平（上海第二天平仪器厂）.
1.2方法〔8-9〕取32只雄性Wister大鼠，随机分为假手术组、缺血模型组、大豆苷元高剂量组（5.0 mg/kg）、大豆苷元低剂量组（2.5 mg/kg), n=8. 用100 g/L水合氯醛(3 mL／kg)腹腔麻醉后，气管插管. 左侧第3~4肋间开胸，剪开心包，暴露心脏，于肺动脉园锥与左心房之中间找出冠状动脉左前降支，并用圆形无创缝合针6／0丝线置于左冠状动脉前降支起始部下2 mm处备用. 除假手术组仅穿线不结扎外，其余各组均以0号线立即结扎. 结扎冠状动脉，以心电图Ⅱ导联ST段抬高0.1 mV或T波高耸、心肌颜色变暗红色为结扎成功的标志，记录结扎前，结扎15 min，给药后30，60，90，120 min的Ⅱ导联心电图. 于结扎冠状动脉左前降支前10 min经舌下静脉注入大豆苷元溶液，缺血模型组及假手术组经舌下静脉注入等体积生理盐水. 结扎120 min后实验结束，左心室心内穿刺取血，分离血清置-20℃冰箱保存侍测，测定血清LDH、CK值；然后迅速取出心脏，并测定心肌组织MDA和SOD值. 操作步骤按南京建成生物工程研究所试剂盒说明书进行. 统计各标测点Ⅱ导联心电图的ST段偏移∑ST、统计各组大鼠心电图J点和T波上升的最大变化值，评价心肌缺血程度. 另取24只Wister大鼠分缺血模型组、大豆苷元高、低剂量组（5.0,2.5 mg/kg）组(n=8)，同上述动物模型制作方法进行实验，取心肌作TTC染色测心肌梗死面积. 实验结束后取出心脏，用冰生理盐水洗净称质量. 将左心室均匀地横切成5份，用滤纸吸去表面水份，然后放入1.25 /L的TTC 37℃环境中染色15 min，非梗塞区不变色，梗塞区呈灰白色. 置于40 g/L的甲醛溶液中固定24 h，将梗塞区心肌剪下，用光电天平称其质量，以梗塞区心肌湿质量占全心肌湿质量的百分比表示心肌梗塞的范围. 评价大豆苷元组（高、低剂量）对大鼠心肌结扎冠状动脉损伤的保护作用.