基于αCaMKⅡ-CREB-BDNF信号通路研究调心方对APP/PS1双转基因AD小鼠学习记忆和突触可塑性的影响  

Effect of Tiaoxin Recipe on Memory Learning and Synaptic Plasticity in APP/PS1 Double Transgenic Alzheimer's Disease Mice based onαCaMKⅡ-CREB-BDNF Signal Pathway

作　　者：王晓雯 钱红 沈春娟 李燕军 袁海新 王健 WANG Xiaowen;QIAN Hong;SHEN Chunjuan;LI Yanjun;YUAN Haixin;WANG Jian(The Fifth Rehabilitation Hospital of Shanghai,Shanghai 201699,China;School of Rehabilitation Science,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海市第五康复医院,上海201699 [2]上海中医药大学康复医学院,上海201203

出　　处：《康复学报》2020年第4期299-306,共8页Rehabilitation Medicine

基　　金：上海市自然科学基金项目(13ZR1439700);上海市松江区新一轮医学重点学科建设项目(ZK2019A09)。

摘　　要：目的:观察调心方对APP/PS1双转基因AD小鼠海马CA1区长时程增强(LTP)、海马区α钙/钙调素依赖激酶Ⅱ(αCaMKⅡ)、环磷酸腺苷反应元件结合蛋白(CREB)、脑源性神经营养因子(BDNF)、突触后致密物95(PSD-95)mRNA和蛋白表达的影响,从αCaMKⅡ-CREB-BDNF信号通路的角度探讨调心方改善APP/PS1双转基因AD小鼠学习记忆和突触可塑性的作用机制。方法:选择54只3月龄APP/PS1雄性小鼠,采用随机数字表法分为模型组、调心方组、多奈哌齐组,每组18只,另设同月龄同品系18只C57/BL6雄性野生型小鼠为正常组。参照药理试验中动物与人体间的等效剂量换算标准进行折算,多奈哌齐组和调心方组均按照人的等效剂量(体质量:60 kg)折算出药物剂量。正常组和模型组给予等量的生理盐水,剂量10 mL/(kg·d);多奈哌齐组给予盐酸多奈哌齐灌胃,剂量为0.05 mg/(kg·d),调心方组给予调心方颗粒灌胃,剂量为0.057 g/(kg·d);4组小鼠于3月龄时进行灌胃给药处理,1次/d,连续灌胃3个月。采用电生理技术在离体海马脑片上诱导4组小鼠海马CA1区LTP;采用实时荧光定量PCR(RT-qPCR)法检测4组小鼠海马αCaMKⅡ、CREB、BDNF、PSD-95 mRNA表达情况;采用免疫印迹(WB)法检测4组小鼠海马p-αCaMKⅡ、p-CREB、BDNF、PSD-95蛋白表达情况。结果:①电生理结果:在0.1~0.7 mA刺激强度下,4组小鼠海马I/O曲线斜率无明显差异,差异无统计学意义(P>0.05)。在20~250 ms刺激间隔下,4组小鼠的PPF易化无明显差异,差异无统计学意义(P>0.05)。在第50~60 min稳定期内,与模型组比较,调心方组小鼠诱导的LTP中fEPSP斜率明显提高(P<0.05)。②RT-qPCR结果:与正常组比较,模型组小鼠海马αCaMKⅡ、CREB、BDNF和PSD-95 mRNA表达均明显降低,差异具有统计学意义(P<0.05);与模型组比较,调心方组小鼠海马αCaMKⅡ、CREB、BDNF和PSD-95 mRNA表达均明显增加,差异具有统计学意义(P<0.05)。③Western blot法检测结果:Objective:To observe the effects of Tiaoxin recipe on long-term potentiation(LTP)in hippocampal CA1 region,hippocampal alpha calcium/calmodulin-dependent kinaseⅡ(αCaMKⅡ),cyclic adenosine monophosphate response element binding protein(CREB),brain derived neurotrophic factor(BDNF),postsynaptic density 95(PSD-95)mRNA and protein expression in hippocampal CA1 region of APP/PS1 double transgenic mice.To explore the mechanism of Tiaoxin recipe in improving memory learning and synaptic plasticity in APP/PS1 double transgenic AD mice from the point of view ofαCaMKⅡ-CREB-BDNF signal pathway.Methods:A total of 543-month-old APP/PS1 male mice were randomly divided into the model group,the Tiaoxin recipe group and the Donepezil group according to the method of random number table,with 18 cases in each group.Another 18 C57/BL6 male wild type mice of the same age and the same strain were set as the normal group.According to the conversion standard of equivalent dose between animal and human in pharmacological test,the drug dose was converted according to human equivalent dose(body mass:60 kg)in Donepezil group and Tiaoxin recipe group.The mice in the normal group and the model group were given the same amount of normal saline 10 mL/(kg·d),the Donepezil group was given Donepezil hydrochloride at a dose of 0.05 mg/(kg·d),and the Tiaoxin recipe group was given Tiaoxin recipe granules by gastric perfusion at a dose of 0.057 g/(kg·d).The mice in the four groups were given intragastric administration at the age of three months,once a day for three months,Electrophysiological technique was used to induce LTP in hippocampal CA1 region of four groups of mice in vitro.Real-time fluorescence quantitative PCR(RT-qPCR)was used to detect the expression ofαCaMKⅡ,CREB,BDNF and PSD-95mRNA in hippocampal hippocampus of mice in four groups,and Western blotting(WB)was used to detect the expression of paired p-αCaMKⅡ,p-CREB,BDNF and PSD-95 in hippocampus of four groups of mice.Results:①Electrophysiological results:under the inte

关 键 词：阿尔茨海默病 调心方 突触可塑性 学习记忆 α钙/钙调素依赖激酶Ⅱ 环磷酸腺苷反应元件结合蛋白 脑源性神经营养因子 信号通路 

分 类 号：R285.5[医药卫生—中药学]