3-正丁基苯酞对慢性酒精中毒模型小鼠情绪记忆改善作用及机制

Effect and mechanism of DL-3-n-butylphthalide on emotional memory in mice with chronic alcoholism

目的探讨3-正丁基苯酞(DL-3-n-Butylphthalide,NBP)对慢性酒精中毒模型小鼠的情绪记忆的改善作用及可能机制。方法24只C57/BL6成年雄性小鼠,随机分为对照组(CON组,n=8),模型组(AT组,n=8),治疗组(AT+NBP组,n=8)。AT组及AT+NBP组小鼠采用酒精灌胃方法建立慢性酒精中毒小鼠模型。AT+NBP组在酒精造模期间每天一次给予40 mg/kg的NBP灌胃,AT组和CON组给予等剂量的玉米油灌胃,连续14 d。采用旷场实验评估小鼠焦虑样行为,悬尾实验评估抑郁样行为,Morris水迷宫和新物识别评估小鼠记忆能力,Tunel染色评估神经元凋亡数量。原代培养小鼠神经元,在细胞水平上给予酒精和NBP干预,采用荧光钙成像技术检测神经元内的钙离子浓度变化。采用SPSS 17.0进行描述性分析,t检验和方差分析。结果AT+NBP组小鼠在旷场中间区域探索时间较AT组长[(50.68±7.82)s,(38.50±13.93)s;t=-2.16,P<0.05)];空间记忆测试中,AT+NBP组在目标象限探索时间较AT组长[(28.02±7.13)s,(20.98±5.58)s;t=-2.20,P<0.05];AT+NBP组在短记忆测试中2 h时其认知系数RI(0.83±0.08)比AT组(0.68±0.10)高(t=-3.13,P<0.05)。与CON组比,AT组小鼠前额叶皮层神经元凋亡增加[(17.33±2.51)个,(115.67±6.50)个;t=-24.41,P<0.001],AT+NBP组较AT组减少[(45.00±5.57)个](t=14.29,P<0.001);AT组海马齿状回神经元凋亡[(13.75±4.79)个]也较AT+NBP组少[(5.75±3.30)个](t=2.75,P<0.05);神经细胞内钙离子浓度检测结果显示,3种浓度的酒精(100 mmol/L、200 mmol/L、300 mmol/L)均导致神经细胞内相对荧光单位(RFU)△F/F显著增加[(1.43±0.32),(2.31±1.39),(1.21±0.73);t=-7.67,-2.85,-2.86,均P<0.05],与之相对应地使用NBP干预的3组神经细胞内RFU变化相对稳定[(-0.04±0.01),(-0.03±0.01),(-0.04±0.02);t=7.96,2.96,2.92,均P<0.05]。结论3-正丁基苯酞可以改善慢性酒精中毒模型小鼠的学习记忆能力,这可能与抑制中毒模型小鼠神经元凋亡、影响神经元细胞内钙离子稳态有关。

Objective To investigate the improvement and possible mechanism of dl-3-n-butylphthalide(NBP)on the emotional memory of chronic alcoholism model mice.Methods Twenty-four adult male C57/BL6 mice were randomly divided into control group(CON,n=8),model group(AT,n=8),treatment group(AT+NBP,n=8).Mice in AT group and AT+NBP group were administrated with alcohol to establish chronic alcoholism model.In the AT+NBP group,the mice was administrated with NBP(40 mg/kg)by gavage once a day for 14 days during the alcohol modeling period.Ang the mice in AT group and CON group was given the same dose of corn oil by gavage.Open field test was used to evaluate anxiety-like behavior,tail suspension test to evaluate depression-like behavior,Morris water maze and new object recognition to evaluate memory ability,and TUNEL staining to evaluate the number of neuron apoptosis.The primary cultured neurons were interfered by alcohol and NBP at the cell level,and the calcium concentration in the neurons was detected by fluorescence calcium imaging.Descriptive analysis,t-test and one-way ANOVA were processed by SPSS 17.0.Results The results of open field test showed that the exploration time of AT+NBP group was longer than that of AT group((50.68±7.82)s,(38.50±13.93)s;t=-2.16,P<0.05)).In the spatial memory test,the target quadrant exploration time of AT+NBP group was longer than that of AT group((28.02±7.13)s,(20.98±5.58)s;t=-2.20,P<0.05).In short memory test,the cognitive coefficient RI(0.83±0.08)of AT+NBP group was higher than that of AT group(0.68±0.10)(t=-3.13,P<0.05).Compared with CON group,the number of neuron apoptosis in prefrontal cortex in AT group increased((17.33±2.51),(115.67±6.50);t=-24.41,P<0.001),and that in AT+NBP group decreased compared with AT group((45.00±5.57))(t=14.29,P<0.001).The number of apoptosis neurons in the dentate gyrus of the hippocampus in AT group(13.75±4.79)was also less than that in the AT+NBP group(5.75±3.30)(t=2.75,P<0.05).Calcium concentration in nerve cells was detected that the three concentrations of alcohol(100 mmol/L,200 mmol/L and 300 mmol/L)led to a significant increase in the RFU within the nerve cells(△F/F)((1.43±0.32),(2.31±1.39),(1.21±0.73);t=-7.67,-2.85,-2.86,all P<0.05).In comparison,the changes of RFU in the three groups treated with NBP treatment were relatively stable((0.04±0.01),(-0.03±0.01),(-0.04±0.02);t=7.96,2.96,2.92,all P<0.05).Conclusion The 3-n-Butylphthalide can improve the learning and memory ability of chronic alcoholism model mice,which may be related with the inhibition of neuron apoptosis and the influence of intracellular calcium homeostasis.