茶多酚对D-半乳糖与Aβ25～35诱导脑神经细胞凋亡的保护效应

背景:有研究表明茶多酚对β淀粉样蛋白、6-羟多巴和氧化性物质诱致海马神经细胞的神经毒性具有保护作用,临床初试表明其对老年性痴呆高危人群认知功能减退具有一定的防治作用,但其作用靶点及作用机制仍是研究的热点。目的:观察茶多酚干预D-半乳糖与Aβ25～35诱致小鼠脑神经细胞凋亡作用。设计:随机对照动物实验。单位:暨南大学药学院药理教研室。材料:实验于2004-09/2005-01在暨南大学医学实验中心完成。选用90只2月龄健康昆明种小鼠,雌雄各半,体质量26～28g,由广东省医学实验动物中心提供。茶多酚(浙江东方茶业科技公司,批号:20040203);D-半乳糖(上海试剂二厂,批号:20030708);Aβ25～35(Sigma,批号:13/01/2004);维生素E(上海信谊药厂,批号:20030708)。方法:实验干预:按体质量将小鼠随机分为6组:假手术组(n=17),模型组(n=16),维生素E组(n=16),茶多酚低剂量组(n=13),茶多酚中剂量组(n=14)、茶多酚高剂量组(n=14)。除假手术组外,小鼠均皮下注射D-半乳糖,120mg/(kg·d),连续给药至第12周,并在第7周时,于侧脑室缓慢注射4nmolAβ25～35。假手术组小鼠侧脑室内注射等容积的人工脑脊液。各组药物处理于造模后第1周开始,假手术组和模型组给予蒸馏水,维生素E组给予100mg/kg维生素E,茶多酚低、中、高剂量组分别给予100,250,625mg/kg,给药方式均为灌胃,容量均为10mL/kg,连续给药12周。实验评估:给药后各组分别采用LW-Ⅱ型水迷宫仪测定小鼠学习记忆情况;分别取动物脑、肝脏组织和血清测定超氧化物歧化酶活性、丙二醛含量;Fura-2/AM负载法测定小鼠红细胞内和脑神经元细胞浆钙离子浓度;流式细胞仪检测各组小鼠脑神经细胞凋亡。主要观察指标:①小鼠学习记忆情况。②血清、肝脏和脑组织内超氧化物歧化酶活性、丙二醛含量。③各组红细胞内和脑神经元细胞浆内钙离子水平变化。④小鼠脑神经细胞凋亡情况。结果:纳入大鼠90只均进入结果分析。①药物处理12周后,茶多酚中、高剂量组和维生素E组的小鼠游出迷宫时间短于模型组,进入迷宫盲端的错误次数较模型组减少,差异均有统计学意义(P<0.05～0.01)。②茶多酚中、高剂量组超氧化物歧化酶活性较模型组有所增高,茶多酚高剂量组丙二醛含量较模型组有所降低,差异有统计学意义(P<0.05～0.01)。③茶多酚中、高剂量组和维生素E组红细胞内和脑神经元细胞浆钙离子浓度均低于模型组,差异有统计学意义(P<0.05～0.01)。④茶多酚各剂量组神经细胞凋亡率均低于模型组,差异有显著性意义(P<0.05)。结论:茶多酚具有抑制D-半乳糖与Aβ25～35诱致脑神经细胞凋亡作用,并明显改善摸型小鼠学习记忆能力,其作用可能与提高全身性抗氧化能力,改善氧化应激损伤引起的细胞内钙超载有关。

Protective effects of tea polyphenols on cerebral nerve cell apoptosis induced by D-galactose and beta-amyloid peptide 25-35

BACKGROUND: Some researches demonstrate that tea polyphenols (TP) has protective effects on neurotoxicity of hippocampal nerve cells induced byβ-amyloid peptide (Aβ), 6-hydroxydopamine (6-OHDA) and oxidative substances. In addition, clinical preliminary examination indicates that TP plays a certain preventive and therapeutic effects on the reduction of recognition function in high-risk population with Alzheimer disease (AD); however, its target and mechanism are still hot topics.OBJECTIVE: To observe the interfering effects of TP on cerebral nerve cell apoptosis induced by D-galactose and Aβ25～35 in mice.DESIGN: Randomized controlled animal study.SETTING: Department of Pharmacology, Pharmacological College of Jinan University.MATERIALS: The experiment was carried out in the Experimental Center of Jinan University from September 2004 to January 2005. A total of 90 healthy Kumning mice, aged 2 months, each gender in half, weighing 26-28 g, were provided by Guangdong Provincial Medical Laboratory Animal Center. Tea polyphenols was provided by Zhejiang Oriental Tea Science and Technology Corporation (batch number: 20040203); D-galactose by Shanghai Number 2 Reagent Plant (batch number: 20030708); Aβ25～35 by Sigma (batch number: 13/01/2004); vitamin E (Vit-E) by Shanghai Xinyi Pharmaceutical Factory (batch number: 20030708).METHODS: Experimental interference: Mice based on body mass were randomly divided into 6 groups: sham operation group (n =17), model group (n =16), vitamin E group (n =16), low-dose (n =13), moderate-dose (n =14) and high-dose (n =14) tea polyphenols groups. In above-mentioned animals, except those in the sham operation group, all were given 120 mg/(kg·d) D-galactose for 12 consecutive weeks, and Aβ25～35 (4 nmol) was slowly injected into the lateral cerebral ventricle. In sham operation group, the same volume of artificial cerebral spinal fluid (CSF) was internally injected into lateral ventricle. Drug treatment began at the first week. Mice in the sham operation group and model group were given distilled water, and the animals in other groups were given the above-mentioned drugs (100 mg/kg Vit-E, 100, 250 and 625 mg/kg TP), respectively. The volume of perfusion was 10 ml/kg, and the treatment lasted for 12 consecutive weeks. Experimental evaluation: After administration, LW-Ⅱ water maze was used to measure learning and memory condition; brain, liver tissues and serum were obtained to measure activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA); Fura-2/AM loading method was used to measure Ca2+ concentration in erythrocytes and neurons; flow cytometer was used to detect cerebral nerve cell apoptosis.MAIN OUTCOME MEASURES: Cl) Learning and memory ability; (2) SOD activity and MDA content in serum, liver and brain tissues; (3) Ca2+ concentration in erythrocytes and neurons;flow cytometer was used to cerebral nerve cell apoptosis.MAIN OUTCOME MEASURES：①Learning and memory ability;②SOD activity and MDA content in serum,liver and brain tissues；③Ca2+ concentration in erythrocytes and neurons；④cerebral nerve cell apoptosis．RESULTS：All 90 mice were involved in the final analysis．①At 12 weeks after administration,time to swim out of the water maze in the moderete-dose and high-dose TP groups and Vit-E group was shorter than that in the model group,and numbers of errors in passing the blind alleys in the water maze was reduced as compared with those in the model group,and there was significant difference(P＜0．05-0．01)．②SOD activities in the moderate-dose and high-dose TP groups were increased as compared with that in the model group, but MDA content in the high-dose TP group was decreased as compared with that in the model group．There was significant difference (P＜0．05-0．01)．③Ca2+ concentration in erythrocytes and neurons in the modemte-dose and high-dose TP groups and Vit-E group was lower than that in the model group, and there was significant difference(P＜0．05-0．01)．④The rates of brain neurons apoptosis in treatment groups with different doses of TP were 12．6％，18．6％, and 24．1％ respectively, exhibiting significant difference as compared with the mice in sham operation group(P＜0．05-0．01) CONCLUSION：TP can inhibit cerebral nerve cell apoptosis induced by D-galactose and Aβ25～35 and improve learning and memory ability in model mice．The effects may be related to its action of raising general anti-oxidative ability and improvement of intrecellular Ca overload induced by oxidative stress injury．