【作者】 许银燕；

【导师】 李胜男；

【作者基本信息】 南京医科大学 ， 药理学， 2007， 硕士

【摘要】 系统性高血压(Systemic hypertension)是一种复杂的病理生理状态，其主要原因是由机体长期处于高血压所导致，它是引发中风、缺血性心脏病、外周血管性疾病和进行性肾损害的主要危险因素。肾素一血管紧张素系统(renin-angiotensin system，RAS)活动的亢进在原发性高血压的发展中起了关键性作用，有机构估计，在促成各种类型高血压的因素中，RAS至少占了百分之十到百分之三十的比例。因此众多研究已经直接面向RAS及其与控制血压之间的关系。阻断RAS会降低血压，减轻高血压的病理生理影响，同时编码RAS的一些基因同高血压具有相关性。因此可以断定RAS是调节正常血压和原发性高血压病理生理状况的基本要素。Genistein是一种异黄酮类的植物雌激素，与17β-estradiol结构相似。它是天然植物雌激素，可以选择性与雌激素受体结合。Genistein可以预防心血管疾病因而近几年来受到广泛的关注。它可以降低胆固醇浓度，降低血压，增加高密度胆固醇，抑制血管平滑肌增殖，增加动脉顺应性和抗氧化作用。因此，食用植物雌激素可能是通过这些作用来降低心血管疾病发病率。血管紧张素转换酶(angiotensin-converting enzyme，ACE)是羧基末端二肽基肽链端解酶，在RAS中发挥了重要作用。ACE是存在于肉皮细胞细胞膜的(胞)外酶，调控血管紧张度和渗透性，将血管紧张素Ⅰ(angiotensinⅠ，AngⅠ)转化成血管紧张素Ⅱ(angiotensinⅡ，AngⅡ)并降解缓激肽(bradykinin，BK)。血管紧张素转换酶抑制剂(angiotensin-converting enzyme inhibitor，ACEI)已经在治疗大部分心血管疾病的临床试验中取得成功。本研究探讨了genistein对于大鼠ACE活性及ACE mRNA表达的影响，进一步拓展对genistein血压调节作用机制的认识。本研究内容包括离体和在体两部分实验，概述如下：第一部分Genistein对Sprague-Dawley(SD)大鼠主动脉肉皮细胞(rat aortic endothelial cells，RAECs)ACE水平及ACE mRNA表达的影响RAECs随机分成以下几组：(1)对照组；(2)genistein 10μM，50μM，100μM and 200μM四个不同浓度组；(3)17β-estradiol 100μM；(4)captopril 1 mM；(5)tamoxifen 100μM；(6)genistein 100μM+ta-moxifen 100μM；(7)17β-estradiol 100μM+tamoxifen 100μM；(8)PD98059 10μM和(9)genistein 100μM+PD98059 10μM。孵育48 h。在以上(6，7，9)组的细胞先给予tamoxifen或PD98059一小时后给予genistein或17β-estradiol处理。免疫荧光及逆转录酶-多聚酶链式反应(reverse transcript polymerase chain reaction，RT-PCR)方法用于检测ACE水平及ACE mRNA表达情况。Genistein浓度依赖性的降低RAECs的ACE水平。Genistein对RAECs的ACE mRNA表达的影响与免疫荧光结果极为相似。此外，genistein对RAECs的ACE水平的影响被证明是通过雌激素受体(estrogen receptor)及细胞外信号调节激酶1／2(extracellular signal-regulated kinase 1／2，ERK 1／2)通路来发挥作用的。第二部分Genistein对Sprague-Dawley(SD)大鼠ACE水平及ACE mRNA表达的影响大鼠被随机分成以下几组：(1)对照组；(2) genistein(2.5 mg／kg／day，10 mg／kg／day，25 mg／kg／day，50 mg／kg／day)四个浓度组；(3)17β-estradiol(0.5 mg／kg／day)；(4) captopril(8 mg／kg／day)；(5) tamoxifen(5 mg／kg／day)；(6) genistein(25 mg／kg／day)+tamoxifen(5 mg／kg／day)；(7) 17β-estradiol(0.5 mg／kg／day)+tamoxifen(5 mg／kg／day)；(8)PD98059(4 mg／kg／day)；(9) genistein(25 mg／kg／day)+PD98059(4 mg／kg／day)；(10) 17β-estradiol(0.5 mg／kg／day)+PD98059(4 mg／kg／day)。在以上相关组中先注射tamoxifen或PD98059十分钟后注射genistein或17β-estradiol。药物均采取尾静脉注射方式，连续注射两天，第二天给药一小时后从大鼠胸主动脉收集血样测血清ACE水平。同时，取胸主动脉测定组织ACE水平及组织ACE mRNA表达情况。注射不同浓度genistein可以剂量依赖性的降低血清ACE水平。在组织ACE水平及组织ACE mRNA表达情况与其类似。实验进一步证明，注射genistein对大鼠ACE的影响是通过雌激素受体及ERK1／2通路发挥作用的。综上所述，在体试验及离体试验均显示，genistein可以剂量依赖性的降低ACE的水平，该影响是通过雌激素受体及激活ERK1／2通路发挥作用的。我们的研究结果拓展了对于genistein在心血管方面的作用机制的理解，对今后心血管疾病的治疗具有潜在价值，同时给那些患有雌激素替代疗法禁忌症及希望自然疗法的妇女带来了希望。更多还原

【Abstract】 Systemic hypertension is a complex pathophysiological state that isprimarily manifested as chronic high blood pressure and is a major riskfactor for stroke, ischemic heart disease, peripheral vascular disease, andprogressive renal damage. It is well established that a hyperactiverenin-angiotensin system (RAS) plays a key role in the development andmaintenance of human primary hypertension and, by some estimates,contributes to at least 10% to 30% of all cases of hypertension. It is notsurprising that tremendous efforts have been directed toward the under-standing of the RAS and its involvement in the control of blood pressure.The interruption of the RAS attenuates high blood pressure and manypathophysiological aspects of hypertension. Also, some genes encodingmembers of the RAS are associated with hypertension. Thus, it is rea-sonable to conclude that the RAS is essential in the normal regulation ofblood pressure and pathophysiology of systemic hypertension.Genistein is a principal isoflavone found in soy phytoestrogen, and ithas structural similarity with 17β-estradiol. It is a naturally occurringplant estrogen with selective estrogen receptor modulator properties. Genistein has received a great deal of attention over the last few yearsbecause of its potentially preventive roles against cardiovascular diseases.It decreases cholesterol concentration, lowers blood pressure and in-creases HDL cholesterol. Genistein inhibits vascular smooth muscle cellproliferation, improves arterial compliance and it has an antioxidant ac-tion. Therefore, dietary phytoestrogen may contribute to a low incidenceof heart disease through these actions.Angiotensin-converting enzyme (ACE) is a carboxyl-terminaldipeptidyl exopeptidase that plays a pivotal role in the RAS. ACE is anecto-enzyme anchored in plasma membrane of endothelial cells control-ling vascular tone and permeability by converting angiotensinⅠ(AngⅠ)into angiotensinⅡ(AngⅡ) and by degrading bradykinin (BK). Angio-tensin-converting enzyme inhibitors (ACEI) have been an increasinglyand impressively successful approach for a wide range of patients withvascular disease.The objective of present study is to investigate the relationship be-tween genistein and ACE in rats.Present study can be divided into two parts, in vitro and in vivo.PartⅠEffects of genistein on ACE activity and ACE expression inrat aortic endothelial cells (RAECs)RAECs were randomly divided and subjected to different treatmentsfor 48 h as follows: (1) control; (2) genistein at four concentrations (10 μM, 50μM, 100μM and 200μM); (3) 17β-estradiol 100μM; (4) capto-pril 1 mM; (5) tamoxifen 100μM; (6) genistein 100μM + tamoxifen 100μM; (7) 17β-estradiol 100μM + tamoxifen 100μM; (8) PD98059 10μM;and (9) genistein 100μM + PD98059 10μM. In relevant groups above (6,7, 9), cells were pretreated with tamoxifen or PD98059 for 60 min beforegenistein or 17β-estradiol treatment. ACE expression was assessed by theimmunofluorescence and the reverse transcriptase-polymerase chain re-action (RT-PCR) assay.Genistein decreased ACE activity on RAECs in a concentra-tion-dependent manner. The effects of genistein on ACE mRNA expres-sion in RAECs were highly consistent with the data in the immunofluo-rescence analysis. Furthermore, our data showed that genistein exertedthe effect on ACE activity and ACE expression by activating the estrogenreceptor and extracellular signal-regulated kinase 1/2 (ERK1/2) pathway.PartⅡEffects of genistein on ACE activity and ACE expression inSprague-Dawley (SD) ratsSD rats were randomly divided into the following groups: (1) control;(2) genistein at four concentrations (2.5 mg/kg/day, 10 mg/kg/day, 25mg/kg/day, and 50 mg/kg/day); (3) 17β-estradiol (0.5 mg/kg/day); (4)captopril (8 mg/kg/day); (5) tamoxifen (5 mg/kg/day); (6) genistein (25mg/kg/day) + tamoxifen (5 mg/kg/day); (7) 17β-estradiol (0.5 mg/kg/day)+ tamoxifen (5 mg/kg/day); (8) PD98059 (4 mg/kg/day); (9) genistein (25 mg/kg/day) + PD98059 (4 mg/kg/day); (10) 17β-estradiol (0.5 mg/kg/day)+ PD98059 (4 mg/kg/day). In relevant groups above, genistein or17β-estradiol was administered 10min after pretreatment with tamoxifenor PD98059. All drugs were administered intravenously via tail vein bybolus. One hour after the administration on day two, blood samples wereobtained from the thoracic aortas for the determination of serum ACE ac-tivity. Meanwhile, thoracic aortas were harvested to analyze tissue ACEactivity and tissue ACE mRNA expression.Administration of genistein induced a dose-related decrease in serumACE activity. Similar dose-related decreases were detected in ACE activ-ity in the aorta and tissue ACE mRNA expression with genistein. Our re-sults also proved that genistein functioned via activation of estrogen re-ceptor and ERK1/2 pathway.In conclusion, results of the present study indicated that genisteindose-dependently decreased ACE levels in rats both in vivo and in vitro.The effect was mediated by the estrogen receptor and subsequent activa-tion of the ERK1/2 pathway. This finding expands our knowledge of themechanisms of genistein and may further support the potential use ofgenistein as a therapeutic agent in cardiovascular events. It may provide apromising approach in women with contraindications to the use of con-ventional estrogen replacement or those wanting a natural alternative aswell.更多还原