【作者】 徐强；

【导师】 司良毅；

【作者基本信息】 第三军医大学 ， 老年医学， 2010， 博士

【摘要】 研究背景流行病学资料显示,2型糖尿病患者发生动脉粥样硬化及心血管并发症较正常人增加3～4倍,80%的2型糖尿病患者死于心血管疾病。血管内皮功能障碍(endothelial dysfunction,ED)是动脉粥样硬化形成的早期始动环节,并且贯穿于动脉粥样硬化发生发展的全过程。高血糖可通过降低一氧化氮(nitric oxide,NO)生物利用度、增加活性氧(reactive oxygen species,ROS)积聚和抑制内皮依赖性血管舒张功能,导致严重的血管内皮功能障碍。通过干预措施来保护血管内皮功能,对于减少糖尿病患者动脉粥样硬化发病率和降低其致残致死率无疑具有重要的临床意义。白藜芦醇(Resveratrol),化学名为反式-3,5,4’-三羟基二苯乙烯,是一种天然存在于葡萄和红酒、桑椹、花生和虎杖中的多酚类化合物。其独特的抗氧化功效为心血管系统提供了广泛的保护作用。目前越来越多的证据证实白藜芦醇在对抗糖尿病、心血管疾病方面起着积极的作用。从一些细胞和动物层面的研究发现其在血糖控制、血管保护方面的作用可能与激活腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)有关。AMPK是近年发现的调控能量代谢的关键分子,作为细胞能量感受器在维持细胞能量稳态和适应性反应中扮演关键角色。不断积累的研究显示,AMPK不但是细胞能量感受器同时也是功能强大的效应器,通过磷酸化作用调控代谢的多个环节。虽然目前的研究对于阐明如何调节AMPK激活还很不完善,但已经明确AMPK可以通过影响细胞信号转导、代谢和基因表达,增加胰岛素敏感性和减少2型糖尿病的危害。越来越多的证据显示AMPK可能是联系代谢系统和循环系统的纽带。现有的研究已经证实AMPK对内皮型一氧化氮合酶(endothelial nitric oxide synthesis,eNOS)有激活效应,而且AMPK激活后对氧化应激有抑制作用。虽然目前已有关于白藜芦醇对糖尿病相关血管病变有保护作用的报道,但其准确的分子机制并未阐明。研究目地本研究以离体培养的人脐静脉内皮细胞(human umbilical vein endothelial cells ,HUVECs)及小鼠主动脉血管环为研究对象,应用Western blot、血管功能实验等方法,力图证实:1、白藜芦醇可促进培养HUVECs中的eNOS磷酸化激活、NO生成,抑制高糖诱导超氧阴离子增加,最终改善高糖诱导的内皮依赖性血管舒张功能障碍。2、白藜芦醇改善高糖诱导的血管内皮功能障碍的机制可能是通过激活AMPK。研究方法1、离体培养HUVECs,用不同浓度梯度及时间梯度的白藜芦醇干预,Western blot检测培养HUVECs中的eNOS蛋白含量以及磷酸化程度的变化,Griess法检测培养HUVECs中NO生成的变化。同时观察应用eNOS抑制剂L-NAME处理后,白藜芦醇对培养HUVECs中NO生成的影响。2、不同的蛋白酶抑制剂处理培养HUVECs后,Western blot检测白藜芦醇对eNOS蛋白含量及其磷酸化程度的影响;Griess法检测培养HUVECs中NO生成的变化。3、应用Western blot检测白藜芦醇以及高糖对AMPK、乙酰辅酶A羧化酶(acetyl coa carboxylase ,ACC)蛋白含量以及各自磷酸化程度的作用。同时,应用白藜芦醇干预高糖条件下的培养HUVECs,Western blot检测不同时相点AMPK、ACC蛋白含量以及各自磷酸化程度的变化。4、应用Griess法检测高糖对培养HUVECs中超氧阴离子产生的影响。并利用白藜芦醇干预高糖诱导的培养HUVECs,观察超氧阴离子产生的变化。再采用AMPK抑制剂复合物C(compound C,CC)处理,观察白藜芦醇对高糖诱导的培养HUVECs中超氧阴离子产生的变化的影响。5、以小鼠主动脉血管环为研究对象,应用血管功能实验检测不同浓度的白藜芦醇对苯肾上腺素(phenylephrine,PE)预收缩的血管环的舒张作用,并设立去内皮组、L-NAME组、吲哚美辛组和compound C组,对其作用的机制进行探讨。观察高糖对乙酰胆碱(Acetylcholine,Ach)诱导的血管舒张作用的影响,并用白藜芦醇及白藜芦醇加CC干预,观察白藜芦醇对高糖削弱Ach诱导的血管舒张作用的影响并探讨其机制。研究结果1、白藜芦醇孵育60min后,显著增加培养HUVECs中p-eNOS含量,并呈白藜芦醇浓度依赖关系;30μmol/L的白藜芦醇呈时间依赖关系地增加培养HUVECs中p-eNOS含量,但总eNOS无明显变化。同时,白藜芦醇明显增加培养HUVECs中NO生成,然而白藜芦醇这一效应却被L-NAME所抑制。提示白藜芦醇呈浓度和时间依赖地磷酸化激活培养HUVECs中的eNOS,增加衍生自eNOS的NO生成。2、100μmol/L的白藜芦醇显著增加培养HUVECs中p-eNOS含量和NO生成,但这一效应可被AMPK特异性抑制剂compound C所阻止,而PKA、PI3K特异性抑制剂并未影响培养HUVECs中p-eNOS含量和NO生成。提示白藜芦醇通过激活AMPK/eNOS途径促进NO生成。3、白藜芦醇显著增加培养HUVECs中p-AMPK及p-ACC含量,而高糖干预后p-AMPK和p-ACC含量明显下降,同时各组培养HUVECs中AMPKα、ACC含量并无明显变化,提示白藜芦醇可以增加培养HUVECs的AMPK活力,而高糖却抑制AMPK活力。但高糖环境中的培养HUVECs经白藜芦醇30μmol/L处理后,p-AMPK、p-ACC含量明显增加,并且随白藜芦醇处理时间的增加而呈不断增加趋势,AMPKα、ACC含量无明显变化。提示白藜芦醇可恢复被高糖抑制的AMPK活力。4、高糖孵育明显增加培养HUVECs中超氧阴离子含量,而渗透性对照组却无此效应。白藜芦醇可显著减少高糖诱导的超氧阴离子产生增加,但compound C可阻止白藜芦醇对超氧阴离子产生的抑制作用。提示白藜芦醇可通过AMPK途径削弱高糖诱导的培养HUVECs中超氧阴离子产生增加。5、内皮完整的小鼠主动脉血管环经PE预收缩后,白藜芦醇呈浓度依赖地舒张血管环。但经机械性剥脱内皮,加入L-NAME或compound C,白藜芦醇舒张PE预收缩的血管环的作用均被明显削弱,而吲哚美辛却无此作用。这些结果提示白藜芦醇具有直接舒张血管的作用,其作用机制与激活AMPK/eNOS系统有关。6、小鼠主动脉血管环经高糖孵育后可导致Ach诱导的血管舒张反应减弱;加入白藜芦醇后,高糖对Ach诱导的血管舒张反应的损害作用被显著削弱,而compound C则可减弱白藜芦醇这一血管保护效应。提示,白藜芦醇可预防高糖诱导的血管内皮依赖性舒张功能障碍,其机制可能是通过激活AMPK途径。结论1、白藜芦醇可促进培养HUVECs中的eNOS磷酸化激活、NO生成,抑制高糖诱导超氧阴离子产生增加,最终改善高糖诱导的内皮依赖性血管舒张功能受损。2、白藜芦醇改善高糖诱导的血管内皮功能障碍的机制可能是通过激活AMPK。更多还原

【Abstract】 BackgroundEpidemiologic data indicate that the risk of developing atherosclerosis and cardiovascular complications is 3-fold to 4-fold higher for patients with type 2 diabetes compared to the general population, and that 80 percents of diabetic patients dies of cardiovascular diseases. Endothelial dysfunction (ED) plays a key role in the initial stage of atherosclerosis, and is involved in the whole process of atherosclerotic lesions. Hyperglycemic can increase the production of reactive oxygen species (ROS) and decrease the nitric oxide (NO) bioavailability, and subsequently induce the vascular ED. The strategies for the protection of vascular endothelial function may reduce the risk of atherosclerosis and its disability rate and mortality in patients with diabetes.Resveratrol, trans-3, 4’, 5-trihydroxyestilbene, is a kind of natural polyphenols produced by several plants, such as grape, mulberry, peanut and giant knotweed rhizome. The unique anti-oxidative effect of resveratrol provides a comprehensive protective role in cardiovascular system. Recently, increasing evidences suggest that resveratrol plays a beneficial effect against diabetes and cardiovascular diseases. Some cellular and animal studies suggest that the beneficial effects of resveratrol on plasma glucose control and vascular protection require AMP-activated protein kinase (AMPK) activity.AMPK, a recently indentified key molecule regulating energy metabolism, plays a pivotal role in cellular energy homeostasis and adaptation reaction, as a sensor of cellular energy status. Accumulative studies demonstrate that AMPK is not only an energy sensor but also a powerful effecter, which is involved in several metabolic processes via its phosphorylating action. Although the regulatory mechanism of AMPK activation is not completely elucidated, activation of AMPK can enhance the insulin sensitivity and reduce the complications of type 2 diabetes mellitus through regulating cellular signal transduction, metabolism and gene expression. Increasing evidences suggest that AMPK may be a linker between energy metabolism and circulatory diseases. Recent studies have demonstrated that AMPK can activate endothelial nitric oxide synthesis (eNOS), and consequently inhibit oxidative stress. Although it is reported that resveratrol can protect from cardiovascular complications of diabetes, the underlying precise molecular mechanisms have not been clarified.ObjectivesThe present study was to test the hypotheses: 1) resveratrol can promote the phosphorylation and activation of eNOS, inhibit hyperglycemia-induced overproduction of superoxide anion in cultured human umbilical vein endothelial cells (HUVECs), and finally improve hyperglycemia-induced impairment of endothelium-dependent vasodilatation; 2) the protection effect of resveratrol on the improvement of endothelial dysfunction is mediated by the activation of AMPK.Methods1. After the cultured HUVECs were treated with varying concentrations of resveratrol for different duration time, the expression and phosphorylation of eNOS were measured by Western blotting and the production of NO was detected by Griess assay. Additionally, L-NAME was used to test the involvement of eNOS enzyme in the effect of resveratrol on the production of NO.2. After the cultured HUVECs were incubated with inhibitors of several kinases, the expression and phosphorylation of eNOS were measured by Western blotting and the production of NO was detected by Griess assay.3. Test the effect of resveratrol on the expression and phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) using Western blotting assay. In addition, test the effect of resveratrol on hyperglycemia-induced changes in the expression and phosphorylation of AMPK and ACC by Western blotting.4. Test the effect of high glucose on the production of superoxide anion by Griess assay and the inhibitory effect of resveratrol on hyperglycemia-induced overproduction of superoxide, and investigate the involvement of AMPK in the protective action of resveratrol using AMPK inhibitor compound C (CC). 5. Test the vasodilatation action of resveratrol in phenylephrine constricted mouse aortic rings treated with or without endothelium denudation, L-NAME, indomethacin and CC. Test the effect of resveratrol on acetylcholine (Ach)-induced vasodilatation in mouse aortic rings with or with out incubation of high glucose.Results1. Treatment with resveratrol for 60 min dose-dependently increased the phosphorylation of eNOS in cultured HUVECs. Additionally, resveratrol (30μM) time-dependently increased the phosphorylation of eNOS in cultured HUVECs. Moreover, resveratrol significantly increased the production of NO, which can be attenuated by the incubation of L-NAME, indicating the resveratrol can increase the NO bioavailability through time- and dose-dependently activating eNOS.2. Resveratrol (100μM) significantly increased the production of NO in cultured HUVECs, which can be attenuated by CC (a specific inhibitor of AMPK) but not the inhibitors of PKA and PI3K, indicating that resveratrol-induced eNOS activation and NO production were mediated by AMPK activation.3. Resveratrol significantly enhanced the phosphorylation of AMPK and ACC but not the total expression of AMPK and ACC, while treatment of high glucose remarkably reduced the phosphorylation of AMPK and ACC, which can by reversed by the treatment of resveratrol (30μM), indicating that resveratrol can protect from hyperglycemia-induced reduction in the activation of AMPK through activating ACC.4. Resveratrol significantly inhibited hyperglycemia-induced overproduction of superoxide anion, which can be attenuated by the incubation of CC, indicating that AMPK activation is required in resveratrol-induced reduction in superoxide production.5. Resveratrol dose-dependently dilated the phenylephrine-constricted mouse aortic rings with intact endothelium, which can be partially attenuated by endothelium denudation, L-NAME and CC, but unaffected by indomethacin, indicating that resveratrol-induced vasodilatation is required AMPK/eNOS pathway.6. High glucose but not isotonic mannitol significantly impaired Ach-induced vasodilatation, which can be patially reversed by resveratrol. However, treatment with CC can significantly block the beneficial effect of resveratrol, indicating that resveratrol can protect the aortae from hyperglycemia-induced endothelial dysfunction through activating AMPK.Conclusions1. Resveratrol can promote the phosphorylation and activation of eNOS, increase the NO production, inhibit hyperglycemia-induced overproduction of superoxide anion in cultured human umbilical vein endothelial cells (HUVECs), and finally improve hyperglycemia-induced impairment of endothelium-dependent vasodilatation.2. The protection effect of resveratrol on the improvement of endothelial dysfunction is mediated by the activation of AMPK.更多还原