【作者】 玄承鸾；

【导师】 柳克祥；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 在发展中国家，高血压是心血管疾病的一个重要危险因素。血压的升高可以导致很多心血管事件，如休克、心肌梗死、肾功能衰竭等并发症乃至死亡。预防血压的升高对减少此类心血管事件的发生起着重要作用。血管内皮的收缩因子及舒张因子的失衡是高血压的一个重要致病机制。血管紧张素II通过不同机制使血压升高，主要的机制有收缩阻力血管、刺激醛固酮合成和释放、增加肾小管对钠的重吸收（间接或直接通过醛固酮）、刺激口渴和释放抗利尿激素、提高交感神经的兴奋性。血管紧张素Ⅱ是由血管紧张素Ⅰ在血管紧张素转化酶的作用下，水解产生的多肽（八肽）物质。重要的是，血管紧张素II直接激活血管紧张素1型受体（AT1）和间接地刺激多种生长因子和细胞因子的释放来诱导心肌和血管肥厚、增生。豆类植物作为重要的食物来源，它为饮食提供了复杂碳水化合物，可溶解纤维和基础维生素，并且含有多酚成分，如类黄酮、异黄酮和木酚素。生物活性成分主要为自然多酚，其包括简单多酚，安息香酸衍生物，类黄酮，芪类，单宁类，木酚素类，木素，这些成分的抗氧化能力对身体健康起到了保护作用。扁豆具有比其他豆类更多的抗氧化物质，并且先前的研究已经证实了，生扁豆提取物具有抗氧化、抗高血压的作用。有研究发现，扁豆的热加工过程可以降低扁豆提取物的生物活性成分，但是否同时降低其抗氧化性是本课题的重要研究目的。实验方法：1制备熟扁豆及生扁豆提取物；2大鼠原代心肌细胞培养；3使用可以与ROS结果的荧光探针DHE检测原代培养心肌细胞的自由基水平；4对原代心肌细胞进行免疫组织化学染色，使用Image J软件分析各组中大鼠心肌的增生情况；5测量各组中大鼠心脏的重量，并计算心脏重量与体重比值评价心脏肥大情况；6对大鼠的心脏及肾脏进行HE染色，测量左心室厚度及心肌细胞大小，评价心脏及肾脏血管重构；7对大鼠的心脏及肾脏进行天狼星红染色，评价心脏及肾脏血管周围纤维程度。实验结果：1熟扁豆提取物与生扁豆提取物的抗ROS作用：25μg/ml、50μg/ml、100μg/ml熟扁豆提取物分别可以降低AngII组10.6±1.3%（n=50, p>0.05）、19.8±2.2%(n=50, p<0.05)、37.2±1.3%(n=50, p<0.05)；25μg/ml、50μg/ml、100μg/ml生扁豆提取物分别可以降低16.6±2.7%（n=50, p>0.05）、26.6±3.1%(n=50,p<0.05)、36.5±2.9%(n=50, p<0.05)。2熟扁豆提取物与生扁豆提取物的抗心肌细胞肥大作用：AngII组的大鼠心肌细胞相比，25μg/ml、50μg/ml、100μg/ml熟扁豆提取物分别降低了11.3±2.5%（n=50, p>0.05）、16.0±1.7%(n=50, p<0.05)、27.2±4.9%(n=50, p<0.05)；与AngII组的大鼠心肌细胞相比，25μg/ml、50μg/ml、100μg/ml生扁豆提取物分别可以降低了12.6±4.5%（n=50, p>0.05）、21.2±2.9%(n=50, p<0.05)、28.9±1.2%(n=50,p<0.05)。3熟扁豆提取物与生扁豆提取物的降压作用：AngII200ng/kg/min微量泵皮下注射，明显引起大鼠血压升高，平均动脉压由93.4mmHg±2.0mmHg升高到126.7±2.1mmHg (n=5, P<0.01)。熟扁豆提取物明显减弱了AngII对大鼠血压的影响，Ang II+CLE组大鼠的平均动脉压为105.2±1.8mmHg，明显低于AngII组大鼠的平均动脉压126.7±2.1mmHg （n=5, p<0.05）。同样，生扁豆提取物也明显减弱了AngII对大鼠血压的影响，Ang II+RLE组大鼠的平均动脉压为96.6±1.2mmHg，明显低于AngII组大鼠的平均动脉压126.7±2.1mmHg（n=5, p<0.05）。4熟扁豆提取物与生扁豆提取物抗AngII诱导的心室肥厚：Ang II+CLE组相同切面心室壁厚度为3.51±0.13mm，明显低于AngII组（n=5, p<0.01）；AngII+RLE组相同切面心室壁厚度为3.48±0.11mm，明显低于AngII组（n=5, p<0.01）；AngII+CLE组相同切面心室壁厚度略高于AngII+RLE组，但二者见无明显差别（n=5,p>0.05）。5熟扁豆提取物与生扁豆提取物抗AngII诱导的微血管重构：心脏微血管：AngII+CLE组相同切面小动脉血管壁与血管内腔比值为26.9±1.6%，明显低于AngII组（n=5，p<0.05）；AngII+RLE组相同切面小动脉血管壁与血管内腔比值为25.1±1.3%，明显低于AngII组（n=5, p<0.05）； Ang II+CLE组略高于AngII+RLE组，但二者无统计学差异（n=5, p>0.05）。肾脏微血管：Ang II+CLE组相同切面小动脉血管壁与血管内腔比值为39.6±2.8%，明显低于AngII组（n=5，p<0.05）；AngII+RLE组相同切面小动脉血管壁与血管内腔比值为36.0±1.7%，明显低于AngII组（n=5, p<0.05）； Ang II+CLE组略高于AngII+RLE组，但二者无统计学差异（n=5, p>0.05）。6熟扁豆提取物与生扁豆提取物抗AngII诱导的血管周围纤维化：心脏微血管：Ang II+CLE组相同切面小动脉纤维化比值为29.1±2.6%，明显低于AngII组（n=5，p<0.05）；AngII+RLE组相同切面小动脉纤维化比值为28.2±2.1%，明显低于AngII组（n=5, p<0.05）； Ang II+CLE组略高于AngII+RLE组，但二者无统计学差异（n=5, p>0.05）。肾脏微血管：Ang II+CLE组相同切面小动脉纤维化比值为35.3±2.3%，明显低于AngII组（n=5，p<0.05）；AngII+RLE组相同切面小动脉纤维化比值为33.9±1.7%，明显低于AngII组（n=5, p<0.05）；Ang II+CLE组略高于AngII+RLE组，但二者无统计学差异（n=5, p>0.05）。结论：1.熟扁豆提取物与生扁豆提取物具有降低AngII诱导原代大鼠心肌细胞诱导的ROS;2.熟扁豆提取物与生扁豆提取物具有抗AngII诱导原代大鼠心肌肥大的作用；3.熟扁豆提取物与生扁豆提取物具有降低AngII诱导的大鼠高血压的作用；4.熟扁豆提取物与生扁豆提取物具有抗AngII诱导的心室肥厚的作用；5.熟扁豆提取物与生扁豆提取物具有抗AngII诱导的心脏、肾脏血管重构的作用；6.熟扁豆提取物与生扁豆提取物具有抗AngII诱导的心脏、肾脏血管周围纤维化作用；7.热加工使扁豆提取物的抗氧化作用略有下降，但熟扁豆提取物与生扁豆提取物的抗氧化作用无明显统计学差异。更多还原

【Abstract】 Hypertension is an important risk factor of cardiovascular disease in somedeveloping countries. Elevation of blood pressure is a risk factor for adversecardiovascular outcomes, including stroke, myocardial infarction, renal failure anddeath. Prevention of increased blood pressure therefore plays a crucial role in areduction of those outcomes. Broke balance between relaxing and contracting factorsin the endothelium of blood vessels is an important pathogenic mechanism ofhypertension. Increased pro-oxidant and decreased antioxidant activities have beenshown to be some of the mechanisms of the pathogenesis of hypertension.Ang II increases blood pressue by various mechanisms, including constrictingresistance vessels, stimulating aldosterone synthesis and release and renal tubularsodium reaborption, stimulating thirst and release of antidiuretic hormone, andenhacing sympathetic outflow from the brain. Importantly, Ang II induces cardiac andvascular cell hypertrophy and hyperplasia directly by activating the Ang II type1receptor and indirectly by stimulating relase of several growth factors and cytokines.Legumes are an important source of foods, which supply the diet with solublefibers, complex carbohydrates, essential vitamins, metals, and polyphenols such asflavonoids, isoflavones and lignans. Natural polyphenols, including simple phenols,phenylpropanoids, benzoic acid derivatives, flavonoids, tannins, stilbenes, and lignins,exert their beneficial health effects by their antioxidant activities. Lentil has morenatural polyphenols than other type of legumes. Some studies have shown lentilextract has the function of anti-oxidant and anti-hypertension.Someone reported that cook process could reduce the natural polyphenols oflentil. However, whether the cooked lentil extract still have anti-oxidant function isthe object of our study. Methods:1Preparation of cooked lentil extracts and raw lentil extracts.2Primary rat cardiomyocytes.3Measurement of intracellular ROS generation of cardiomyocytes with DHE.4Immunocytochemistry and measurement of cell surface area with Image Jsoftware.5Assessed cardiac hypertrophy by heart weight-to-body weight ratio (HW/BW).6Assessment of left ventricle hypertrophy and arterial remodeling in heart andkidney with HE staining.7Assessment of perivascular fibrosis in heart and kidney with Sirius red staining.Results:1Effects of cooked lentil extracts and raw lentil extract on Ang II-induced cellularROS stress in cultured cardiomyocytes: pretreatment of cardiomyocytes with cookedlentil extracts significantly attenuated Ang II-induced increase of in intracellular ROSlevel by10.6±1.3%（n=50, p>0.05）、19.8±2.2%(n=50, p<0.05)、37.2±1.3%(n=50,p<0.05) in25μ g/ml、50μ g/ml、100μ g/ml respectively; pretreatment ofcardiomyocytes with raw lentil extracts significantly attenuated Ang II-inducedincrease of in intracellular ROS level by16.6±2.7%（n=50, p>0.05）、26.6±3.1%(n=50, p<0.05)、36.5±2.9%(n=50, p<0.05) in25μg/ml、50μg/ml、100μg/ml respectively.2Effects of cooked lentil extracts and raw lentil extract on Ang II-inducedcardiomyocyte hypertrophy: The Ang II-induced cardiomyocyte hypertrophy wasattenuated by11.3±2.5%（n=50, p>0.05）、16.0±1.7%(n=50, p<0.05)、27.2±4.9%(n=50, p<0.05) after treatment with cooked lentil extracts at doses of25μg/ml、50μg/ml and100μg/ml; The Ang II-induced cardiomyocyte hypertrophy wasattenuated by12.6±4.5%（n=50, p>0.05）、21.2±2.9%(n=50, p<0.05)、28.9±1.2%(n=50, p<0.05) at doses of25μg/ml、50μg/ml and100μg/ml.3Cooked lentil extracts and raw lentil extracts attenuated Ang II-induced increase inBP: Chronic subcutaneous infusion of Ang II (200ng/kg/min) significantly increasedMAP from93.4±2.0to126.7±2.1mmHg (n=5, p<0.01); The pressor effect of Ang IIwas significantly attenuated by preeatment with cooked lentil extracts (BP:126.7± 2.1mmHg in Ang II group and105.2±1.8mmHg in Ang II+CLE group respectively,n=5, p<0.05) or raw lentil extracts (BP:126.7±2.1mmHg in Ang II group and96.6±1.2mmHg in Ang II+CLE group respectively, n=5, p<0.05).4Effects of CLE and RLE on cardiac remodeling induced by Ang II: Oraladministration of CLE significantly attenuated Ang II-induced increases in leftventricular wall thickness from4.00±0.11mm to3.51±0.13mm (n=5, p<0.01); RLEalso significantly attenuated Ang II-induced increases in left ventricular wall thicknessfrom4.00±0.11mm to3.48±0.11mm (n=5, p<0.01). The left ventricular wallthichness of rats receiving RLE is smaller than CLE, but there is no significantdifference between two groups.5Effects of CLE and RLE on peripheral vascular remodeling: Oral administrationof CLE significantly attenuated increase in small arterial media/lumen ratio (26.9±1.6%vs31.5±1.4%, n=5, p<0.05), and RLE also significantly attenuated increase insmall arterial media/lumen ratio (25.1±1.3%vs31.5±1.4%, n=5, p<0.05) in ratheart; Oral administration of CLE significantly attenuated increase in small arterialmedia/lumen ratio (39.6±2.8%vs51.7±1.9%, n=5, p<0.05), and RLE alsosignificantly attenuated increase in small arterial media/lumen ratio (36.0±1.7%vs51.7±1.9%, n=5, p<0.05) in rat kidney. The peripheral vascular remodeling of ratsreceiving RLE is feebler than CLE, but there is no significant difference between twogroups.6Effects of CLE and RLE on perivascular fibrosis induced by Ang II: Oraladministration of CLE significantly attenuated increase in perivascular fibrosis (29.1±2.6%vs37.1±3.0%, n=5, p<0.05), and RLE also significantly attenuated increasein perivascular fibrosis (25.1±1.3%vs37.1±3.0%, n=5, p<0.05) in rat heart; Oraladministration of CLE significantly attenuated increase in perivascular fibrosis (35.3±2.3%vs43.9±2.9%, n=5, p<0.05), and RLE also significantly attenuated increasein perivascular fibrosis (33.9±1.7%vs43.9±2.9%, n=5, p<0.05) in rat kidney. Theperivascular fibrosis of rats receiving RLE is feebler than CLE, but there is nosignificant difference between two groups.Conclusion: 1Oral administration of CLE or RLE significantly attenuated Ang II-induced ROSgeneration of cardiomyocytes;2Oral administration of CLE or RLE significantly attenuated Ang II-inducedcaridomyocytes hypertrophy;3Oral administration of CLE or RLE significantly attenuated Ang II-inducedhypertension;4Oral administration of CLE or RLE significantly attenuated Ang II-induced leftventricle hypertrophy;5Oral administration of CLE or RLE significantly attenuated Ang II-induced heartand kidney small arterial remodeling;6Oral administration of CLE or RLE significantly attenuated Ang II-induced heartand kidney perivascular fibrosis;7The cook process attenuated the effects of antihypertensive and antioxidant insome degree, but the CLE has similar antihypertensive and antioxidant effect as RLE.更多还原