酸性肽对阿尔茨海默病模型大鼠脑内N-甲基-D-天冬氨酸受体、神经生长因子和β淀粉样蛋白水平的调节作用(英文)

Regulatory effects of acidic peptide on the levels of N-methyl-D-aspartate receptor,nerve growth factor and beta-amyloid in the brain of rats with Alzheimer disease

背景:一些研究已经证实酸性肽对痴呆模型大鼠有良好的治疗作用,但其发挥作用的途径尚需探讨。目的:观察酸性肽能否抑制阿尔茨海默病大鼠脑内N-甲基-D-天冬氨酸受体和淀粉样β蛋白的产生以及促进神经生长因子的产生和分泌。设计:随机对照动物实验。单位:郑州大学医学院生物化学与分子生物学教研室。材料:实验于2005-03/2006-05在郑州大学生物活性肽研究所第一实验室和郑州大学基础医学院细胞培养中心完成。选取10周龄的健康且经Y-迷宫测定无痴呆症状的雄性SD大鼠70只,单纯随机分为7组,正常对照组、模型组、生理盐水组、谷氨酸0.3g/kg组、酸性肽15,30,60mg/kg组,每组10只。方法:正常对照组不干预,其他6组用鹅膏蕈氨酸损毁大鼠双侧迈内特基底核,建立阿尔茨海默病病的动物模型,造模后谷氨酸0.3g/kg组灌胃谷氨酸0.3g/kg,酸性肽15,30,60mg/kg组灌胃相应剂量酸性肽(自制),生理盐水组灌胃等量生理盐水,1次/d,2mL/次,连续20d。主要观察指标:①灌胃期满后各组大鼠即做Y-迷宫实验以检测大鼠的学习记忆能力,以正确反应次数为指标。②学习记忆测试结束后,各组大鼠断头取脑,免疫组化染色,再用BiosensDigitalImagingSystem灰度扫描仪扫描以检测大鼠脑内神经生长因子、N-甲基-D-天冬氨酸受体、淀粉样β蛋白的水平。结果:70只大鼠全部进入结果分析。①Y-迷宫实验中正确反应次数:其他6组均低于正常组(P<0.01);酸性肽30,60mg/kg组高于模型组、生理盐水组、谷氨酸0.3g/kg组及酸性肽15mg/kg组(P<0.01)。②基底前脑神经生长因子灰度值:模型组、生理盐水组、谷氨酸0.3g/kg组及酸性肽15mg/kg组低于正常对照组(69.60±2.41,69.62±1.46,69.62±1.46,69.73±1.87,80.77±2.72,P<0.01),酸性肽30,60mg/kg组与正常对照组比较差异不显著(79.39±2.23,80.20±1.7,P>0.05),但高于其他几组。③大脑皮质N-甲基-D-天冬氨酸受体和淀粉样β蛋白表达的灰度值:模型组、生理盐水组、谷氨酸0.3g/kg组及酸性肽15mg/kg组高于正常对照组(N-甲基-D-天冬氨酸受体:81.01±1.38,81.31±2.06,81.37±1.39,79.38±1.23,69.50±1.04;淀粉样β蛋白:74.26±1.39,74.89±8.66,74.88±1.46,74.16±2.48,67.40±3.06,P<0.01),酸性肽30,60mg/kg组与正常对照组比较差异不显著(N-甲基-D-天冬氨酸受体:70.55±2.89,69.78±1.24;淀粉样β蛋白:67.66±2.25,67.58±2.21,P>0.05),但低于其他几组。结论:30,60mg/kg的酸性肽能明显改善阿尔茨海默病模型大鼠的学习记忆能力,其治疗作用途径可能是通过上调基底前脑中的神经生长因子水平,下调大脑皮质中N-甲基-D-天冬氨酸受体和β淀粉样蛋白的水平实现的。

BACKGROUND： It has been confirmed that acidic peptide has good therapeutic effect on rat models of Alzheimer disease, but the mechanism still needs further exploration. OBJECTIVE： To observe whether acidic peptide can inhibit the production of N-methyl-D-aspartate receptor （NMDAR） and beta-amyloid （β-amylord） in brain, and accelerate the production and excretion of nerve growth factor （NGF） in rats with Alzheimer disease. DESIGN： A randomized controlled animal experiment. SETTING： Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Zhengzhou University. MATERIALS： The experiments were finished in the first laboratory of Institute of Bioactive Peptide, Zhengzhou University and the Cellular Culture Center, School of Basic Medical Sciences of Zhengzhou University from March 2005 to May 2006. Seventy 10-week-old healthy male SD rats without dementia symptoms were randomly divided into 7 groups with 10 rats in each group： normal control group, model group, saline group, glutamic acid 0.3 g/kg group, acidic peptide 15, 30 and 60 mg/kg groups. METHODS： Except the normal control group, the rats in the other 6 groups were induced into models of Alzheimer disease by damaging bilateral nucleus basalis of Meynert with ibotenic acid, and then intragastric administration of glutamic acid （0.3 g/kg） was given in the glutamic acid 0.3 g/kg group, acidic peptide of corresponding dosages in the acidic peptide 15, 30 and 60 mg/kg groups, and isovolume saline in the saline group respectively, 2 mL for each time, once a day for 20 days continuously. MAIN OUTCOME MEASURES： ① The learning ability of the rats was detected with Y-maze test immediately after the end of intragastric administration, and the times of correct responses were recorded. ② After the end of learning and memory test, the head was cut rapidly to remove brain, treated with immunohistochemical staining, and gray value was scanned with Bioscns Digital Imaging System to determine the contents of NMDAR, NGF and 13-amyloid in the brain of rats. RESULTS： All the 70 rats were involved in the analysis of results. ① Times of correct responses in the Y-maze test were lower in the other 6 groups than in the normal control group （P 〈 0.01）, but higher in the acidic pcptidc 30 and 60 mg/kg groups than in the model group, saline group, glutamic acid 0.3 g/kg group and acidic peptide 15 mg/kg group （P 〈 0.01）. ② The gray values of NGF in basal forebrain in the model group, saline group, glutamic acid 0.3 g/kg group and acidic peptide 15 mg/kg group were lower than that in the normal control group （69.60±2.41, 69.62±1.46, 69.62±1.46, 69.73±1.87, 80.77±2.72, P 〈 0.01）; There were no significant differences between the acidic peptide 30 and 60 mg/kg groups （79.39±2.23, 80.20±1.7, P 〉 0.05）, which were higher than the other groups. ③ The gray values of NMDAR and β-amyloid in cerebral cortex in the model group, saline group, glutamic acid 0.3 g/kg group and acidic peptide 15 mg/kg group were lower than those in the normal control group （NMDAR： 81.01 ±1.38, 81.31±2.06, 81.37±1.39, 79.38±1.23, 69.50±1.04; 13-amyloid： 74.26±1.39, 74.89±8.66, 74.88±1.46, 74.16±2.48, 67.40±3.06, P 〈 0.01）, and There were no significant differences between the acidic peptide 30 and 60 mg/kg groups （P 〉 0.05）, which were lower than the other groups. CONCLUSION： Acidic peptide of 30 and 60 mg/kg can obviously ameliorate the learning and memory abilities in rat models of Alzheimer disease, which may be realized mainly through up-regulating the NGF content in basal forebrain and down-regulating the NMDAR and β-amyloid contents in cerebral cortex.