【作者】 蒲云飞；

【导师】 祝之明；

【作者基本信息】 第三军医大学 ， 内科学， 2013， 博士

【摘要】 背景：我国正迈入老龄化社会，高血压是最常见的老年慢性病之一，也是心脑血管病最主要的危险因素。近百年来人们围绕盐与血压的关系进行研究，基本上确定了盐是高血压的重要易患因素。但在人群内个体间对盐负荷或限盐却呈现不同的血压反应，即存在盐敏感性问题。肾脏生理功能异常是诱发及维持高血压的重要因素，而高血压又可成为慢性肾损害的病因，两者相互影响。近年来研究发现，血管内皮氧化应激参与高血压、肾脏受损、老龄化血管功能障碍等多种心肾血管疾病的发生发展。其中，内皮功能损伤和高血压特别是盐敏感性高血压的发病机制密切相关。流行病学研究表明，多种膳食因素可影响血压和血管功能，在过去的十余年中，通过减少钠盐的摄入、增加钾盐的摄入和增加以蔬菜和水果为基础的“清淡饮食”的摄入，可有效的降低血压和改善血管功能。其中，膳食植物多酚广泛分布于各种水果、蔬菜、谷物中，其对健康的影响受到人们越来越多的关注。芹菜素和姜黄素是两种重要的植物多酚，既往研究发现芹菜素和姜黄素具有降压、扩血管、抗氧化等作用，但具体机制尚不清楚。研究发现激活过氧化物酶体增殖物激活受体δ(peroxisome proliferator-activated receptor δ，PPARδ)产生的非基因作用可通过磷酯酰肌醇-3-激酶/丝氨酸-苏氨酸激酶/内皮型一氧化氮合酶(phosphatidylinositol-3-kinase/serine-threonine kinase/endothelial nitric oxide synthase，PI3K/Akt-eNOS)信号通路改善内皮依赖性血管功能障碍，而芹菜素可激活PPARγ发挥抗炎作用；研究表明，姜黄素可以调节老龄小鼠的血管内皮功能障碍相关的5’-AMP激活的蛋白激酶(5’-AMP-activated protein kinase，AMPK)信号通路。因此我们推测，芹菜素可能通过上调PPARδ激活PI3K/Akt-eNOS，促进内皮细胞一氧化氮(nitric oxide，NO)的生成，改善血管功能、预防盐敏感性高血压和保护肾脏；姜黄素可通过激活AMPK、上调解偶联蛋白2(uncoupling protein2，UCP2)，减少活性氧簇(oxygen species，ROS)的产生，改善老龄化相关的脑血管功能障碍。目的：为了验证上述假设，本研究分为三部分进行。第一部分研究膳食芹菜素对醋酸脱氧皮质酮-盐(deoxycorticosterone acetate-salt，DOCA-盐)高血压大鼠是否具有降压作用和改善内皮依赖性血管舒张功能作用，芹菜素对DOCA-盐高血压大鼠24小时尿蛋白、肾小球硬化的影响。第二部分观察芹菜素对DOCA-盐高血压大鼠肾内动脉内皮ROS和NO水平、PPARδ-PI3K/Akt-eNOS通路信号分子蛋白表达的影响，应用芹菜素与SD大鼠肾内动脉孵育12小时，观察芹菜素是否通过PPARδ-PI3K/Akt-eNOS发挥改善血管功能和抗氧化应激作用。第三部分通过体内和体外实验，明确姜黄素是否通过激活AMPK/UCP2信号通路减少老龄鼠的脑血管ROS的产生和增加NO的生成，从而改善老化脑血管功能障碍。材料与方法：整个研究由在体实验和离体实验两部分组成。在体试验采用了多种动物模型。首先以DOCA-盐高血压大鼠作为盐敏感性高血压模型，按照是否注射DOCA造高血压模型和是否喂食芹菜素饲料4周，分为对照组、芹菜素组、DOCA组、DOCA+芹菜素组。选取24个月的SD大鼠、UCP-/-小鼠和对照鼠作为老龄鼠，分别喂食普通饲料和姜黄素饲料，选取6个月龄大鼠喂食普通饲料被用来作为青龄大鼠对照组。离体实验选以SD大鼠肾内动脉、SD大鼠和UCP2-/-小鼠脑动脉以及培养的内皮细胞为研究对象。1．无创鼠尾血压仪测大鼠鼠尾血压、多通道生理检测仪记录大鼠平均颈动脉压。2．组织冰冻切片HE染色观察大鼠肾脏病理结构变化。3．小动物代谢笼收集大鼠24小时尿液，γ放射免疫法检测尿蛋白含量，全自动生化检测仪检测尿电解质。4．小血管张力测定技术检测芹菜素急性刺激后或长期干预后大鼠肾内动脉舒张功能；姜黄素急性刺激后或长期干预后鼠脑基底动脉舒张功能。5．荧光显像技术检测芹菜素长期干预后对大鼠肾内动脉ROS和NO水平的影响；姜黄素长期干预后对老龄鼠脑动脉ROS和NO水平影响。6．蛋白免疫印迹法检测芹菜素对大鼠肾内动脉NO合成相关信号分子(PPARδ、p-Akt、Akt、eNOS、p-eNOS)的蛋白表达；姜黄素对老龄鼠脑动脉和内皮细胞ROS产生相关通路(AMPK、UCP2)的蛋白水平。结果：1．膳食芹菜素降低DOCA-盐高血压大鼠鼠尾血压和平均颈动脉压，改善DOCA-盐高血压大鼠肾内动脉内皮依赖性舒张功能，减轻DOCA-盐高血压大鼠蛋白尿、肾小球硬化程度。2．膳食芹菜素降低DOCA-盐高血压大鼠肾内动脉ROS水平、增加NO合成和磷酸化eNOS蛋白表达。3．芹菜素呈浓度依赖地内皮依赖性舒张大鼠肾内动脉，去除内皮、应用eNOS抑制剂、PPARδ抑制剂或PI3K抑制剂明显削弱血管的舒张反应。4．膳食芹菜素可显著上调DOCA-盐高血压大鼠肾内动脉PPARδ、磷酸化Akt蛋白表达；芹菜素孵育SD大鼠肾内动脉12小时可显著上调大鼠肾内动脉PPARδ、磷酸化Akt蛋白表达，加入PPARδ抑制剂或PI3K抑制剂可消除芹菜素对PPARδ、磷酸化Akt和磷酸化eNOS影响。5．膳食姜黄素干预改善老龄大鼠脑基底动脉内皮依赖性血管舒张功能，减少老龄大鼠脑血管ROS产生和增加eNOS磷酸化水平；膳食姜黄素通过UCP2依赖的方式减少老龄小鼠脑血管ROS产生和改善内皮依赖性血管舒张功能。6．膳食姜黄素上调老龄大鼠脑血管AMPK和UCP2蛋白表达；姜黄素与老龄大鼠脑血管或动脉内皮细胞孵育12小时后可上调内皮细胞AMPK、UCP2和磷酸化eNOS蛋白表达，改善血管内皮依赖性血管舒张功能或降低内皮细胞ROS和增加NO水平，AMPK抑制剂和UCP2抑制剂可分别显著减弱姜黄素的作用。结论：1．芹菜素降低DOCA-盐高血压大鼠血压、改善内皮依赖性血管舒张功能、防止肾脏损伤。2．芹菜素上调DOCA-盐高血压大鼠血管PPARδ并通过PI3K/Akt-eNOS介导增加NO合成，减轻血管氧化应激。3．姜黄素降低老年鼠脑血管内皮ROS的产生、增加NO生成，改善内皮依赖性血管舒张功能，其作用依赖AMPK激活介导的UCP2上调。更多还原

【Abstract】 Background:Hypertension is the most common chronic diseases associated with aging, but also themost important risk factor for heart and vascular disease. Over the past century, around therelationship between salt and blood pressure, it is confirmed that salt is the important riskfactor of hypertensive. However, salt loading or salt restriction leads to a different bloodpressure response in the crowd, that is salt-sensitive. Abnormal renal physiology is animportant factor to induce and maintain high blood pressure, and high blood pressure can be acause of chronic renal damage, also, which both affect each other. Recent studies have foundthat the vascular endothelium oxidative stress involved in high blood pressure, kidney damage,the aging vascular dysfunction and a variety of cardiovascular disease development.Hypertension, especially salt-sensitive hypertension, is closely related to the pathogenesis ofendothelial dysfunction. Epidemiological studies have shown that a variety of dietary factorsaffect blood pressure and vascular function. In the past ten years, reducing sodium intake andincreaseing potassium salt intake, vegetable and fruit dietary can effectively lower bloodpressure and improve blood vessel function. The plant polyphenols widely distributed in avariety of fruits, vegetables and grains, and its impact on health attentioned by the peoplemore and more. Apigenin and curcumin are two important plant polyphenols. Previous studiesfound that apigenin and curcumin with effects of reduceing blood pressure, vasodilation,anti-oxidation, but the exact mechanisms of those are unclear. Recent studies found thatactivation of peroxisome proliferator-activated receptor δ (PPARδ) generating non-geneticeffects through phosphatidylinositol-3-kinase/serine-threonine kinase/endothelial nitric oxidesynthase (PI3K/Akt-eNOS) pathway improve endothelial function. Apigenin activatingPPARγ plays anti-inflammatory role. Studies also have shown that curcumin can adjust theaging mouse endothelial dysfunction related to5’-AMP-activated protein kinase (AMPK)signaling pathway. Therefore, we hypothesized that apigenin can activate PI3K/Akt-eNOS through PPARδ, promote the endothelial synthesis and release of nitric oxide (NO), improveblood vessel function, prevention of salt-sensitive high blood pressure and protect the kidneys.Curcumin can reduce reactive oxygen species (ROS) generation through AMPK-Uncouplingprotein2(UCP2) pathway and improve the aging cerebrovascular dysfunction.Objective:In order to verify the above assumptions, the present study was divided into three parts.First, we monitored blood pressure and vascular diastolic function of deoxycorticosteroneacetate-salt (DOCA-salt) hypertensive rats, to investigate the effect by apigenin ofanti-salt-sensitive hypertension and improving vascular function. Analysising24-hour urinaryalbumin and glomerulosclerosis of DOCA-salt hypertensive rats, to explore effect by apigeninprotecting against renal damage of salt-sensitive hypertensive. Second, comparing ROS andNO production and PPARδ-PI3K/Akt-eNOS pathway protein expression of arteries fromDOCA-salt hypertensive rats. Third, we provide in vivo and in vitro experimental evidencethat curcumin reduces ROS production and increases NO production, thereby rescuingcerebrovascular endothelium dysfunction through the AMPK/UCP2pathway in agingrodents.Materials and Methods:1. Non-invasive rat tail blood pressure, carotid artery mean arterial blood pressure ofDOCA-salt hypertensive rats.2. HE staining of kidney sections of DOCA-salt hypertensive rats.3.24-hour urine urine protein analysising of DOCA-salt hypertensive rats.4. Determination renal interlobular artery function after acute stimulation or long-terminterventions by apigenin. Cerebral basilar artery function changes by curcumin acutestimulation or long-term intervention.5. Fluorescence imaging of ROS and NO levels of DOCA-salt hypertensive rats renalartery. ROS and NO basilar arteries from aging rats after long-term curcumin intervention.6. Western blot detection of NO synthesis signaling proteins expression (PPARδ, Akt,eNOS, phospho-eNOS) in renal artery by apigenin treatment. Protein Level of ROS pathway(AMPK, UCP2) in brain basilar artery and endothelial cells treated by curcuminResults:1. Dietary apigenin reduced blood pressure, improved artery endothelium-dependent vasodilation, prevented proteinuria and glomerular sclerosis in DOCA-salt hypertensive rats.2. Dietary apigenin reduced artery ROS levels, increased NO production andphosphorylation of eNOS protein expression in DOCA-salt hypertensive rats.3. Dietary apigenin can significantly improve the DOCA-salt hypertensive rats renalartery endothelium-dependent vasodilation, and apigenin showed a concentration-dependentmanner vasodilation of rat renal artery, which dependented with PPARδ-PI3K/Akt-eNOSpathway.4. Impaired cerebrovascular endothelial function and increased ROS in aging rats can beimproved by chronic dietary curcumin through a UCP2-dependent pathway.5. Curcumin mediated UCP2up-regulation involves the activation of the AMPK in thecerebrovascular endothelium.6. Activation of the AMPK/UCP2pathway by curcumin antagonizes the production ofsuperoxide anions and prevents NO reduction in endothelial cells.Conclusions:1. Apigenin reduced salt-sensitive hypertension, improved endothelium-dependentvascular function and prevented against renal damage.2. Apigenin activated vascular PPARδ and increased NO production throughPI3K/Akt-eNOS pathway.3. Curcumin improves aging-related cerebrovascular dysfunction through theAMPK/UCP2pathway.更多还原