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背景:以往的很多研究表明高同型半胱氨酸血症通过增强氧化应激诱导动脉粥样硬化,而银杏叶提取物可以清除氧自由基。目的:观察在同型半胱氨酸诱导细胞黏附分子表达中活性氧基团和核因子κB的作用及银杏叶提取物对这一过程的影响。设计:随机对照动物实验。单位:华中科技大学同济医学院附属协和医院神经内科。材料:选用健康雄性家兔24只,6月龄。蛋氨酸(Sigma公司);银杏叶提取物(贵州益佰制药股份有限公司提供,粉剂)。方法:实验于2003-02/2004-03在华中科技大学同济医学院附属协和医院神经内科实验室完成。①适应性喂养2周后,按随机摸球法分为3组:模型组(12只):按80mg/(kg·d)剂量皮下注射蛋氨酸;银杏叶提取物组(8只):皮下注射蛋氨酸前1h予喂饲(与食物混合)银杏叶提取物50mg/(kg·d);对照组(4只):注射与蛋白氨酸等剂量的生理盐水。连续用药7周。②光镜和透射电镜下观察动脉组织学变化;比色法(721型分光光度计)测定活性氧水平;免疫组织化学方法检测动脉内皮细胞黏附分子和核因子κB的表达。高效液相色谱法测定血浆同型半胱氨酸浓度。主要观察指标:各组动物动脉组织学变化、活性氧水平,以及动脉内皮细胞黏附分子和核因子κB的表达。结果:家兔24只均进入结果分析。①动脉内皮细胞活性氧水平:模型组给药结束时活性氧水平明显增高(2.92±0.20,2.48±0.26,P<0.05),银杏叶提取物组和对照组给药结束时分别为2.41±0.23和2.43±0.20,与给药前比较,差异不明显(2.31±0.27,2.47±0.32,P>0.05)。②动脉组织学变化:模型组颈总动脉动脉组织表现为早期动脉粥样硬化形态(内皮细胞脱落,平滑肌细胞排列紊乱);对照组和银杏叶提取物组动脉壁结构基本正常。③动脉内皮细胞细胞黏附分子和核因子κB表达:模型组细胞黏附分子和核因子κB表达明显高于对照组(P<0.05),银杏叶提取物组与对照组相近(P>0.05)。④血浆同型半胱氨酸浓度:给药7周后,模型组与银杏叶提取物组血浆同型半胱氨酸浓度分别为(25.01±6.80),(26.71±2.36)μmol/L,高于对照组[(16.85±1.64)μmol/L,P<0.05]。结论:同型半胱氨酸可诱导家兔主动脉上皮细胞表达细胞黏附分子,主要由氧化应激作用激活核因子κB而介导。银杏叶提取物通过抑制活性氧水平和核因子κB活性而抑制细胞黏附分子的表达。
BACKGROUND: Many previous studies have shown that hyperhomocysteinemia induces atherosclerosis by enhancing oxidative stress, whereas Ginkgo biloba extract can scavenge oxygen free radicals. Objective: To observe the role of reactive oxygen species and nuclear factor kappa B in the expression of cell adhesion molecules induced by homocysteine and the effect of Ginkgo biloba extract on this process. Design: Randomized controlled animal experiments. Unit: Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. MATERIALS: Twenty-four healthy male rabbits were selected and were 6 months old. Methionine (Sigma); Ginkgo biloba extract (provided by Guizhou Yijun Pharmaceutical Co., Ltd., powder). METHODS: The experiment was performed at the Department of Neurology, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology from November 2003 to March 2004. 1 After 2 weeks of adaptive feeding, they were randomly divided into 3 groups according to the random ball method: Model group (12): subcutaneous injection of methionine at 80 mg/(kg·d); Ginkgo biloba extract group (8): subcutaneous injection Methionine was fed 1 h before (mixed with food) Ginkgo biloba extract 50 mg/(kg·d), and the control group (4): injected with a dose of normal saline such as amino acid. Continuous medication for 7 weeks. 2 The histological changes of the arteries were observed under light and transmission electron microscopes. The level of reactive oxygen species was measured by colorimetric method (721 spectrophotometer). The expression of adhesion molecules and nuclear factor kappa B in arterial endothelial cells were detected by immunohistochemistry. The plasma homocysteine concentration was determined by high performance liquid chromatography. MAIN OUTCOME MEASURES: Histological changes, reactive oxygen species, and the expression of arterial endothelial cell adhesion molecules and nuclear factor kappa B in all animals. RESULTS: Twenty-four rabbits were involved in the result analysis. 1 Reactive oxygen species in arterial endothelial cells: The level of reactive oxygen species was significantly higher in the model group at the end of the administration (2.92±0.20, 2.48±0.26, P<0.05), and 2.41±0.23 at the end of the administration of the Ginkgo biloba extract group and control group, respectively. And 2.43±0.20, compared with pre-dose, the difference was not significant (2.31±0.27, 2.47±0.32, P>0.05). 2 Arterial histological changes: Arterial tissue of common carotid arteries in the model group showed early atherosclerotic morphology (endothelial cell shedding, smooth muscle cell arrangement disorder); the arterial wall structure of the control group and Ginkgo biloba extract group was basically normal. 3The expression of cell adhesion molecule and nuclear factor-κB in artery endothelial cells: The expression of cell adhesion molecules and nuclear factor-κB in the model group was significantly higher than that in the control group (P<0.05). The Ginkgo biloba extract group was similar to the control group (P>0.05). 4 Plasma homocysteine concentration: After 7 weeks of administration, plasma homocysteine concentrations in the model group and the Ginkgo biloba extract group were (25.01±6.80) and (26.71±2.36) μmol/L, respectively, which were higher than the control group. Group [(16.85±1.64) μmol/L, P<0.05]. CONCLUSION: Homocysteine can induce the expression of cell adhesion molecules in rabbit aortic epithelial cells, and is mainly mediated by oxidative stress that activates nuclear factor kappa B. Ginkgo biloba extract inhibits the expression of cell adhesion molecules by inhibiting reactive oxygen species and nuclear factor kappa B activity.