丁苯酞通过PI3K/Akt信号通路对糖尿病小鼠空间学习记忆能力的影响

Effects of butylphthalide on spatial learning and memory ability of diabetic mice through PI3K/Akt signal pathway

目的分析丁苯酞通过磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)信号通路对糖尿病小鼠空间学习记忆能力的影响。方法雄性CB57BL/KsJ-db/db小鼠40只,随机分为模型组与丁苯酞30、60、120 mg/kg组,另选10只CB57BL/KsJ-db/m小鼠为正常对照组(NC);受试组灌胃给药1次/d,模型组和对照组灌胃相应空白溶剂1次/d,共6周。干预6周后观察小鼠一般情况;测量随机血糖和体质量;Morris水迷宫实验和海马长时程增强效应(LTP)诱发实验检测小鼠空间学习记忆能力;光学显微镜观察小鼠海马组织病理学变化;比较各组实时荧光定量PCR、蛋白免疫印迹实验检测海马组织PI3K、Akt、哺乳动物西罗莫司(雷帕霉素)靶蛋白(mTOR)mRNA及蛋白相对表达量。结果药物干预期间丁苯酞30、60、120 mg/kg组小鼠的精神状态和反应灵敏度较糖尿病模型组小鼠改善。与NC组比较,模型组和丁苯酞30、60、120 mg/kg组小鼠的随机血糖和体质量更高(P<0.05)。与模型组比较,丁苯酞组小鼠随机血糖和体质量无明显变化;丁苯酞30、60、120 mg/kg组小鼠Morris水迷宫隐蔽平台实验的逃避潜伏期呈剂量依赖性缩短(P<0.05),探查测试的穿越平台次数、目标象限停留时间百分比呈剂量依赖性增加(P<0.05),海马LTP实验的群峰电位(populatin spike,PS)增幅呈剂量依赖性升高(P<0.05)。光镜下模型组海马CA1区椎体神经元细胞出现不同程度的缺失,细胞形态改变;丁苯酞30、60、120 mg/kg组病理改变程度减轻。与模型组比较,丁苯酞30、60、120 mg/kg组的PI3K mRNA与蛋白相对表达量、磷酸化与非磷酸化的(p)-Akt/Akt、p-mTOR/mTOR蛋白比值均呈剂量依赖性升高(P<0.05)。结论丁苯酞能改善糖尿病小鼠精神状态、反应灵敏度,减轻空间学习记忆能力下降和海马区LTP受损,保护海马CA1区椎体神经元细胞,其机制可能与激活PI3K/Akt信号通路有关。

Objective To analyze the effects of butylphthalide on spatial learning and memory ability of diabetic mice through phosphatidylinositol-3 kinase(PI3K)/protein kinase B(Akt)signal pathway.Methods Forty male CB57BL/KsJ-db/db mice were randomly divided into the model group and 30,60,120 mg/kg butylphthalide groups,while another 10 CB57BL/KsJ-db/m mice were selected as normal control group(NC group).The mice in the test groups were administered the given doses of butylphthalide in oil,while the mice in the model and the NC groups were given the corresponding blank oil,all by gavage once a day for 6 weeks.After 6 weeks of intervention,the general conditions of mice were observed.Blood glucose and body mass were measured.Morris Water maze test and LTP induced test were used to detect the spatial learning and memory ability of mice.The pathological changes in hippocampus were observed by light microscope.The relative expression of PI3K,Akt,mammalian target of sirolimusc(rapamycin)(mTOR)mRNA and protein were detected by RT-qPCR and Western blotting.Results The mental state and response sensitivity of the 30,60 and 120 mg/kg butylphthalide groups were better than those of diabetic mice.Compared with the NC group,the random blood glucose level and body mass were both significantly higher in the model group and the 30,60 and 120 mg/kg butylphthalide groups(P<0.05),and both the random blood glucose level and body mass showed no significant difference between the model group and the three dose butylphthalide test groups(P>0.05).Compared with the model group,the escape latency was significantly shortened(P<0.05),while the number of crossing platform and the percentage of stay time in target quadrant were both significantly increased(both P<0.05),all dose-dependently,in the 30,60 and 120 mg/kg butylphthalide groups in the Morris water maze test.Furthermore,the amplitude of population spike potential(PS)in the hippocampal LTP experiment was also dose-dependently increased in the 30,60 and 120 mg/kg butylphthalide groups than in the model group(P<0.05).Under light microscope,the neurons in the CA1 area of the hippocampus in the model group appeared to be lost to some extent and the the cell morphologies were changed,while the pathological changes were alleviated in the 30,60,120 mg/kg butylphthalide groups.Compared with the model group,the relative expression level of PI3K mRNA and protein as well as the ratio of the phosphorylated and non-phophorylated proteins,(p)-Akt/Akt and p-mTOR/mTOR,were all significantly increased in the 30,60,120 mg/kg butylphthalide groups,all in a dose-dependent manner(P<0.05).Conclusion Butylphthalide could improve the mental state and response sensitivity of diabetic mice,alleviate the reduced spatial learning and memory ability and the damage of LTP in the hippocampus,and protect the neurons in the CA1 area of hippocampus.The possible mechanism is likely related to the activation of PI3K/Akt signaling pathway.