摘要
研究背景：流行病学研究提示：饮用适量红葡萄酒比饮用其他酒精饮料，能更大程度地减少缺血性心脏病的发生率和死亡率，提示红葡萄酒具有心脏保护作用。国外进一步的研究表明，红葡萄酒的某些作用不依赖于酒精，而且白葡萄酒并不完全具备这些作用。已有研究发现，红葡萄酒中的多酚化合物白藜三醇具有抗氧化、抗血小板、抑制平滑肌细胞增殖等多种生物活性，可能就是红葡萄酒心血管保护作用的主要成份，因而近几年受到广泛关注和研究。有关白藜三醇及红葡萄酒对人体的有益作用及其作用机制是当今国际研究的热点课题之一。
红葡萄酒能减少动脉粥样硬化患者的心血管事件发生率，但能否预防实验性动脉粥样硬化的发生，尚无一致的结论；口服白藜三醇是否影响动脉粥样硬化的形成未见研究；国产红葡萄酒是否具有有益作用也未见报道。
目的：本课题拟研究白藜三醇及国产红葡萄酒对：(1)实验性动脉粥样硬化形成的影响；(2)活体动物内皮功能的改善作用；(3)血小板聚集反应及血栓烷合成的影响；(4)主动脉组织PreproET-1、iNOSmRNA表达的影响。通过对上述问题的阐明，以探讨：(1)口服白藜三醇对实验性动脉粥样硬化有无预防作用、实验动物的耐受性以及白藜三醇的作用机制，为白藜三醇能否用于防治动脉粥样硬化性疾病提供依据；(2)在测定国产红葡萄酒中白藜三醇含量的基础上，观察其对实验性动脉粥样硬化形成的影响，为动脉粥样硬化及冠心病的饮食预防提供一个依据和选择。
方法：40只雄性新西兰大白兔，随机分成5组，分别用下
南京医科大学博士学位论文
列饲料喂养：门）普通颗粒状兔饲料，u）高胆固醇饲料，门）
高胆固醇饲料＋红葡萄酒，（4）高胆固醇饲料＋去酒精红葡萄酒，
或门）高胆固醇饲料＋白葱三醇。实验开始前采集静脉血，按要
求进行抗凝、分离血浆并保存，实验结束时重复采血，并进行下
＠列检查：
1．活体血管内皮功能测定：喂养 12周末，使用血管外超声
法测定兔股动脉酬内径、反应性充血后血管内径，计算出血流
＠量介导（内皮依赖性）的血管扩张反应。20分钟后测定硝酸甘
油介导＊ 内皮依赖性）的血管扩张反应；
2．主动脉粥样硬化性病变测定：胸主动脉全长用苏丹IV染
色后照相，扫描后在 Ph成则h叩 6刀下计算斑块面积；等距离将胸
主动脉取 5块标本，切片 H－E染色，用 NYD刁 图象分析系统
计算出平均内膜厚度及内膜冲层比；
3．血标本检测：
门）血脂：使用 OryMPUS AU2400全自动生化分析仪（H
本）测定；
门）血小板聚集：使用二磷酸腺旮为诱导剂，采用h 氏
法测定5分钟最大血小板聚集率；
门）血浆NO：使用硝酸还原酶法，用752紫外分光光度计
读取550一处的光密度，计算NO含量；
门）血浆ETJ和TXB。：用多管自动放免计数仪进行放射
免疫测定。
＠4．NOS、ET－IInRNA表达水平测定：使用 RTPCR技术，
通过与内参照比较，行半定量测定。
＠结呆：
1．正常兔饲料喂养12周，无 1例出现动脉粥样硬化性病变；
高胆固醇饲料喂养的4组均有明显的动脉粥样硬化，单纯高胆固
醇组、RW组、去酒精RW组及白摹三醇组主动脉斑决面积（％）
分别为56．4 L 13．5、41．21 10＊、34二L 12．4和33＊t 19．6（4组
6
南京医科大学博士学位论义
间匕较 ANOVA检验，p—0．of 3x 内膜／中层匕（％）分别为 49．0
】14．5、24．4 ti 0．5、26．8 t 8．4和 13．8 f 5．8（4组间比较 ANOVA
检验，p功刀01h RW组、去酒精 RW组及白暴三醇组均明显低
于单纯高胆固醇组。
＠2．12周后，正常兔饲料喂养者血脂水平无明显改变；高胆
固醇饲料喂养的4组，TG水平不变，TC、LDL七和HDL七在4
周时即明显升高，以TC和LDL七的升高最为显著；12周时高
＠胆固醇饮食的4组之间血脂水平无显著差异；TC及LDL七水平
与AS性斑块面积呈正相关。
3．12周后，单纯高胆固醇饮食组内皮功能明显受损，FMD
（％）低于正常对照组（分别为14．516刀和19．312．8，q检验，
p＝0．002），RW组、去酒精RW组及白暴三醇组内皮功能均有明
显改善（分别为 17 ＊＊1．4、18．8L3．9和 18．9L2．3）。FMD与王动
脉As病变程度呈负相关，与血浆NO水平呈正相关。
4．12周后，正常兔饲料喂养者血浆 NO及 ETq水平均无
明显改变；单纯高胆固醇饮食组血浆NO水平明显降低，而ET八
水平?
Background: Epidemiological studies have showed that moderate consumption of red wine has more beneficial effects than other alcohol beverages in decreasing the mobidity and mortality of coronary heart disease. These findings suggest that red wine posses cardioprotective effects. Recently, the further studies in abroad showed that many effects of red wine are alcohol independent and white wine did not have all those effects. It has been discovered that resveratrol, a polyphenol compound existed in red wine, has many biological activities including antioxidation, antiplatelet and supression of smooth muscle cell proliferation. It may be the primary biological compound existed in red wine and has been studing extensively and increasingly. Studies on the benificial actions of resveratrol or red wine and their mechanism is a hot-spot topic.
Red wine could decrease the mobidity of cardiovascular event in patients with atherosclerosis, but the results about whether red wine can prevent atherosclerosis are inconclusive. There are few reports about the effect of resveratrol taken orally on experimental atherosclerosis and we also haven't found the reports about the benificial effects of red wine manufactured in China on human health.
Objective: In this study, we will observe the effects of resveratrol or Chinese red wine on: ( 1 ) experimental atherosclerosis in rabbit; (2 ) the endothelial function in experimental atherosclerotic rabbits; ( 3 ) platelet aggregation in experimental atherosclerotic rabbits in vivo
and the synthesis of TXB2; ( 4 ) PreproET-1 , iNOS mRNA expression in aortic tissue. With these results, we wish to clarify the preventive effects of resveratrol taken orally on experimental atherosclerosis and its safety and mechanisms and to provide evidence for the practical use of resveratrol. At the same time, we also measure the concentrations of resveratrol in some Chinese red wine and to explore the effects of Chinese red wine on experimental atherosclerosis.
Methods: 40 New Zealand male rabbits, divided into 5 groups randomly, were fed with forages as follow respectively: ( 1) normal forage, ( 2 ) high-cholesterol forage (containing cholesterol 1.5%), (3 ) high-cholesterol forage with red wine, (4) high-cholesterol forage with de-alcohol red wine, or( 5 ) high-cholesterol forage with resveratrol. Venous bloods were drawn before experiment and at the end of experiment. Bloods were mixed with various anticoagulants according to different measurement requirement. Plasma was obtained after centrifugation and stored at -20℃or-70℃.
1.Measurement of endothelial function in rabbits in vivo: femoral artery diameters were measured both at rest and during reactive hyperemia in two-dimension (2-D) images to calculate the flow-mediated vasodilation (FMD). After a 20 min rest period, 2-D images of the femoral artery were again obtained at baseline and 5 min after sublingual administration of nitroglycerin.
2. Atherosclerotic lesion quantification: immediately upon excision, thoracic aortas were fixed in formaldehyde and then immersed in Sudan IV solution. The whole specimen was photographed and plaque area was quantitated using Photoshop 6.0. 5 cross sections (0.4cm-thick) of the aortas were cut in equal appearing interval and subjected to regular histological handling. All slides of specimen were examined using NYD-1000 image analysis
system and intimal and medial layer thicknesses were measured.
3. Blood specimen measurement:
( 1 ) Lipid: Triglycerides, total and HDL cholesterol were measured by the enzymatic method using OLYMPUS AU2400 biochemical analysis apparatus.
(2) Platelet aggregation: ADP-induced platelet aggregation was measured using Bern's method.
( 3 ) NO: Plasma NO concentration was measured with nitrate reductase method. The reaction product was quantified on the basis of absorbance at 550 nm.
(4) ET-1 and TXB2: Plasma ET-1 and TXB2 were measured with radioimmunoassay method..
4. iNOS, ET-1 mRNA expression level: demi-quantitative determ
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