【摘要】 背景:有实验指出酸性肽可能是通过抑制一氧化氮等毒性化合物的生成而提高阿尔茨海默病模型大鼠的学习记忆能力。目的:建立阿尔茨海默病动物模型,观察给予不同剂量浓度的酸性肽治疗后大鼠脑一氧化氮、一氧化氮合酶与乙酰胆碱酯酶含量的变化。设计:随机对照单一实验。单位:郑州大学基础医学院生物化学与分子生物学教研室。材料:实验于2003-02/07在郑州大学基础医学院生物化学与分子生物学教研室的第二研究室和实验动物房完成。选取雄性SD大鼠100只,用跳台实验除去反应迟钝的动物,共84只大鼠纳入实验,随机分为7组:正常对照组,模型组,生理盐水组,吡拉西坦治疗组,酸性肽15,30,60mg/kg治疗组,12只/组。酸性肽为本课题组从牛脑中分离出的一个新的小分子肽,由三个谷氨酸连成的三肽。方法:除正常对照组外,其余各组大鼠常规饲养1周后,均采用大鼠脑组织立体定位微量注射技术,脑海马注射5μg鹅膏蕈氨酸,以损毁大鼠双侧迈纳特基底核建立阿尔茨海默病模型。正常对照组和模型组不给药,生理盐水组用生理盐水灌胃,吡拉西坦治疗组用0.3g/kg吡拉西坦灌胃,酸性肽15,30,60mg/kg治疗组分别用15,30,60mg/kg酸性肽灌胃,连续20d,1次/d,2mL/次。灌胃期满将大鼠麻醉后断头处死,立即在冰盘中开颅取脑制备组织匀浆。4℃下1000r/min离心10min,取上清液用一氧化氮、一氧化氮合酶和乙酰胆碱酯酶检测试剂盒测定一氧化氮、一氧化氮合酶和乙酰胆碱酯酶的含量。主要观测指标:各组大鼠脑内一氧化氮、一氧化氮合酶和乙酰胆碱酯酶的含量。结果:纳入实验的84只大鼠全部进入结果分析。各组大鼠脑内一氧化氮、一氧化氮合酶和乙酰胆碱酯酶含量的比较:与模型组比较,酸性肽15,30,60mg/kg治疗组一氧化氮含量均明显降低[(1.95±0.20),(1.39±0.10),(1.25±0.07),(1.00±0.04)mmoL/kg,P<0.05];一氧化氮合酶含量均明显降低[(4.53±0.18),(3.39±0.09),(3.10±0.06),(2.97±0.06)μmo1/kg,P<0.05];乙酰胆碱酯酶含量无明显变化[(0.67±0.12),(0.71±0.11),(0.72±0.08),(0.72±0.07)mmo1/L,P>0.05]。结论:酸性肽能够显著降低阿尔茨海默病模型大鼠脑内一氧化氮、一氧化氮合酶的生成,而对乙酰胆碱酯酶的活性并无明显影响。提示酸性肽可能是通过抑制一氧化氮等毒性化合物的生成或毒性作用来提高阿尔茨海默病模型大鼠的学习记忆能力。更多还原

【Abstract】 BACKGROUND: It is pointed in some experiment that acidic peptide improves learning and memory of model rat with Alzheimer disease (AD) by inhibiting the synthesis of toxic compounds of nitric oxide (NO).OBJECTIVE: Animal model with Alzheimer disease was established to observe the changes in the levels of NO, nitric oxide synthase (NOS) and acetylcholinesterase (AChE) treated with acidic peptide of various dose concentration.DESIGN: Randomized control and single experiment.SETTING: Teaching-Research Room of Biochemistry and Molecular Biology of Basic Medical College of Zhengzhou University.MATERIALS: The experiment was performed in 2nd Research Room and Experimental Animal Room of Teaching-Research Room of Biochemistry and Molecular Biology of Basic Medical College of Zhengzhou University. Totally 100 SD male rats were selected and some of them were excluded due to retarded response in step down test. Totally 84 rats were included in the experiment and randomized into 7 groups, named normal control, model group, physiological saline group (PS group), Piracetam group, acidic peptide groups of 15, 30 and 60 mg/kg, 12 rats in each group. Acidic peptide was a new small molecular peptide separated from bovine brain and is tripeptide composed of three glutamic acids.METHODS: Except normal control, in the rest groups, after 1 week routine breeding, cerebral stereotactic microinjection was used to inject 5 μg ibotenic acid in hippocampus of rats to destroy bilateral Meynert’s nucleus basalis to establish AD model. In normal control and model group, no medication was applied. In PS group, physiological saline was used for gastric perfusion. In piracetam group, piracetam of 0.3 g/kg was used for gastric perfusion and in acidic peptide groups of 15, 30 and 60 mg/kg, acidic peptide of 15, 30 and 60 mg/kg was applied for gastric perfusion successively, continuously for 20 days, once per day, 2 mL/time. On the expiration of gastric perfusion, the rats were sacrificed after anesthetized and the brain was collected on ice plate to prepare tissue homogenate. After centrifugated at 1 000 r/minute, 4℃ for 10 minutes, the supernatant was collected to assay the levels of NO, NOS and AChE with NO, NOS and AChE kits successively.MAIN OUTCOME MEASURES: Levels of NO, NOS and AChE in brain of rat in each groupRESULTS: Totally 84 rats were employed in the experiment and all entered result analysis. Comparison of levels of NO, NOS and AChE in rat brain of each group: compared with model group, NO levels in acidic peptide groups of 15, 30 and 60 mg/kg were reduced remarkably (1.95±0.20), (1.39±0.10), (1.25±0.07), (1.00±0.04) mmoL/kg, P < 0.05, NOS levels were reduced remarkably (4.53±0.18), (3.39±0.09), (3.10±0.06), (2.97±0.06) μmo1/kg, P < 0.05 and AChE did not change remarkably (0.67±0.12), (0.71±0.11), (0.72±0.08), (0.72±0.07) mmo1/L, P > 0.05.CONCLUSION: Acidic peptide reduces significantly the synthesis of NO and NOS in brain of AD rat, but it dose not affect AChE activity remarkably. It is suggested that acidic peptide improves learning and memory of rat with Alzheimer disease probably by inhibiting the synthesis of toxic compound of NO or its toxicity.更多还原