地黄饮子调节海马神经突触可塑性作用机制研究

Mechanism of Dihuang Drink(地黄饮子)Regulating Synaptic Plasticity in Hippocampus

目的:观察地黄饮子改善阿尔茨海默病(AD)模型小鼠学习记忆能力及对海马突触可塑性的影响,探讨AD治疗机制。方法:选用50只6月龄SAMP8小鼠构建AD动物模型,采用RANDBETWEEN随机函数将其分为模型组、盐酸美金刚组及地黄饮子低、中、高剂量组各10只,选用同龄SAMR1小鼠10只作为正常组。地黄饮子各剂量组及盐酸美金刚组按照给药剂量给予相应药物灌胃,正常组和模型组给予等体积纯水灌胃,连续给药12周。新物体识别、Morris水迷宫、巴恩斯迷宫法分别检测小鼠的学习记忆能力,在体海马场电位实验记录小鼠海马长时程增强(LTP)效应,免疫组化实验检测突触后致密蛋白-95(PSD-95)、突触素(SYN)蛋白水平的变化,蛋白质印迹法检测海马神经元NMDAR1、NMDAR2B、Ca2+在细胞内所结合钙调蛋白依赖性蛋白激酶Ⅱ(CaMKⅡ)蛋白表达情况。结果:与模型组比较,地黄饮子各剂量组小鼠新物体识别系数增加,差异有统计学意义(P<0.001);在目标象限停留时间明显增加,差异均有统计学意义(P<0.05,P<0.01),且平台穿越次数增加,逃避潜伏期缩短;进入逃生箱所需时间减少,但差异无统计学意义(P>0.05),在逃生箱所在象限停留时间明显增加,差异有统计学意义(P<0.05)。在体海马场电位记录实验表明,高频刺激前各组小鼠fEPSPs斜率无明显差异,高频刺激后30 min、60 min地黄饮子各剂量组较模型组斜率增高,差异均有统计学意义(P<0.05)。与模型组比较,地黄饮子各剂量组CA1区海马神经元PSD-95蛋白、SYN蛋白表达水平上升;地黄饮子各剂量组CA3区海马神经元PSD-95蛋白、SYN蛋白表达水平上升。与模型组比较,地黄饮子各剂量组海马神经元CaMKⅡ、NMDAR1、NMDAR2B蛋白表达水平升高。结论:地黄饮子能改善AD模型小鼠的学习记忆能力,机制可能为通过调节海马神经元Ca2+维持神经突触可塑性,。

Objective:To observe the effect of Dihuang Drink(地黄饮子)on improving learning and memory ability and hippocampal synaptic plasticity in model mice with Alzheimer’s disease(AD),and to explore the mechanism of AD treatment.Methods:Total 506-month-old SAMP8 mice were selected to construct AD animal model,and they were divided into model group,memantine hydrochloride group,Dihuang Drink low-dose,medium-dose and high-dose groups by RANDBETWEEN random function,and 10 SAMR1 mice of the same age were selected as normal group.Dihuang Drink groups and memantine hydrochloride group were given corresponding drugs by gavage,and normal group and model group were given equal volume of pure water by gavage for 12 weeks.The learning and memory ability of mice was detected by new object recognition,Morris water maze and Barnes maze respectively.The long-term potentiation(LTP)effect of hippocampus was recorded by field potential experiment.The changes of postsynaptic density protein-95(PSD-95)and synapsin(SYN)protein levels were detected by immunohistochemical experiment.The protein expression of NMDAR1,NMDAR2B and Ca2+-binding calmodulin-dependent protein kinaseⅡ(CaMKII)in hippocampal neurons were detected by Western blotting method.Results:Compared with the model group,the new object recognition coefficient of mice in the Dihuang Drink groups was increased,and the difference was statistically significant(P<0.001);the time of staying in the target quadrant was significantly increased,and the difference was statistically significant(P<0.05,P<0.01),and the number of platform crossing was increased,and the escape latency was decreased;the time required to enter the escape box was reduced,but the difference was not statistically significant(P>0.05),and the time of staying in the quadrant of the escape box was significantly increased,and the difference was statistically significant(P<0.05).The in vivo hippocampal field potential recording experiment showed that there was no significant difference in the slope of fEPSPs in each group of mice before high frequency stimulation,and the slope of the Dihuang Drink groups was significantly higher than that of the model group 30 min and 60 min after high frequency stimulation(P<0.05).Compared with the model group,the expression levels of PSD-95 protein and SYN protein in hippocampal neurons in CA1 region of the Dihuang Drink groups were increased;the expression levels of PSD-95 protein and SYN protein in hippocampal neurons in CA3 region of the Dihuang Drink groups were increased.Compared with the model group,the expression levels of CaMKII,NMDAR1,NMDAR2B protein in hippocampal neurons in the Dihuang Drink groups were increased.Conclusions:Dihuang Drink can improve the learning and memory ability of AD model mice,and the mechanism may be through regulating the Ca2+in hippocampal neurons to maintain synaptic plasticity.