二甲双胍改善慢性脑低灌注大鼠认知功能障碍的作用和机制

Roles and mechanisms of metformin in improving cognitive dysfunction induced by chronic cerebral hypoperfusion in rats

目的:探讨二甲双胍(metformin)改善慢性脑低灌注大鼠认知功能障碍的作用和机制。方法:82只3~4月龄SPF级雄性SD大鼠按照随机数字表法分为假手术对照组(Con组,n=15)、假手术二甲双胍给药组(Met组,n=20)、双侧颈总动脉结扎(2-vessel occlusion,2VO)组(2VO组,n=22)、双侧颈总动脉结扎+二甲双胍给药组(2VO+Met组,n=25),采用双侧颈总动脉结扎法建立慢性脑低灌注模型,假手术组只分离血管,不接扎。建模后按照每天100 mg/kg剂量连续4周给予二甲双胍溶液饮用水。二甲双胍干预4周后,Morris水迷宫实验检测大鼠的空间认知功能,在体电生理技术检测大鼠的长时程增强,酶联免疫吸附实验(ELISA)检测海马组织的炎性因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白介素-1β(interleukin-1β,IL-1β)和白介素-6(interleukin-6,IL-6)浓度水平,同时采用高尔基染色观察海马神经元树突棘的密度,透射电镜观察海马神经元的突触结构,尤其是囊泡密度。采用SPSS 16.0进行统计分析,水迷宫7 d重复学习训练的逃避潜伏期数据采用重复测量方差分析,其余数据多组间比较采用单因素方差分析,进一步两两比较采用Dunnett's t检验。结果:Morris水迷宫结果显示,在7 d的学习训练中,4组大鼠逃避潜伏期的时间和组别交互作用不显著(F=0.93,P>0.05),但是时间主效应(F=25.90,P<0.05)和组别主效应(F=13.20,P<0.05)显著;在第3~7天的平台位置学习中,2VO组大鼠逃避潜伏期显著长于Con组和2VO+Met组(均P<0.05)。休息1 d后检测大鼠短期记忆,结果显示:2VO组大鼠逃避潜伏期显著长于Con组和2VO+Met组(P<0.01),同时2VO大鼠的平台区域滞留时间和穿梭次数均少于Con组(P<0.01)和2VO+Met组(P<0.01)。电生理结果显示:高频刺激后,2VO组相对兴奋性突触后场电位斜率[(1.29±0.09)]显著低于Con组[(2.07±0.09)]和2VO+Met组[(1.69±0.08)](P<0.01)。ELISA结果显示:2VO组海马组织TNF-α含量水平显著高于Con组和2VO+Met组;2VO组海马组织IL-1β和IL-6含量水平显著高于Con组和2VO+Met组。2VO组海马神经元树突棘密度显著低于Con组和2VO+Met组。2VO组海马神经元不成熟树突棘密度和比例显著高于Con组和2VO+Met组。2VO组海马CA1区神经元的突触囊泡密度[(230.29±19.44)个/μm^(2)]显著低于Con组[(414.52±13.17)个/μm^(2)]和2VO+Met组[(313.19±12.42)个/μm^(2)](均P<0.05)。结论:二甲双胍能够降低慢性脑低灌注海马组织神经炎性反应,同时能够改善突触可塑性和认知功能障碍,在治疗血管性认知功能障碍方面可能具有潜在的应用价值。

Objective To explore the roles and mechanisms of metformin in the improvement of cognitive dysfunction induced by chronic cerebral hypoperfusion in rats.Methods Total 82 SD male rats(SPF grade)aged 3-4 months were randomly divided into four groups:sham operation control group(Con group,n=15),sham operation with metformin treatment group(Met group,n=20),2-vessel occlusion group(2VO group,n=22),and 2-vessel occlusion with metformin administration group(2VO+Met group,n=25).The chronic cerebral hypoperfusion model was established by bilateral common carotid artery ligation,and the carotid arteries of rats in Con group and Met group were only separated without ligation.After 2VO operation,rats in 2VO+Met group and Met group were given metformin solution in drinking water at a dose of 100 mg/kg per day for 4 weeks.After 4-week continuous intervention with metformin,Morris water maze was performed to test the spatial cognitive function of the rats,in vivo electrophysiological technology was used to detect the long-term potential of the rats,and enzyme-linked immunosorbent assay(ELISA)was used to detect the concentrations of inflammatory factor tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)in the hippocampus.The density of dendritic spines of hippocampal neurons was observed by Golgi staining,and the synaptic structure of hippocampal neurons,especially the vesicle density,was observed by transmission electron microscopy.SPSS 16.0 software was used for statistical analysis.Repetitive measurement ANOVA was used for the escape latency data of 7 days repeated learning training in water maze.One-way ANOVA was used for the comparison of other data among multiple groups,and Dunnett's t test was used for further pairwise comparison.Results Morris water maze results showed that during 7 days of learning training,the time and group interaction for escape latency was not significant in the 4 groups of rats(F=0.93,P>0.05),but the time main effect(F=25.90,P<0.05)and group main effect(F=13.20,P<0.05)were significant.Morris water maze test showed that from the 3rd to 7th day,the escape latencies in 2VO group were significantly longer than those in Con group and 2VO+Met group(all P<0.05).The short-term memory of rats was detected after 1 day of rest.The results showed that the escape latency in 2VO group was significantly longer than that in Con group and 2VO+Met group(P<0.01).The retention time and crossing times in the platform area of 2VO rats were less than those in Con group and 2VO+Met group(P<0.01).Electrophysiological results showed that the relative field excitatory postsynaptic potential slope of 2VO group(1.29±0.09)was significantly lower than that in Con group(2.07±0.09)and 2VO+Met group(1.69±0.08)(P<0.01).ELISA results showed that TNF-αlevel in hippocampal tissue of 2VO group was significantly higher than that in Con group and 2VO+Met group;IL-1βand IL-6 levels in hippocampal tissue of 2VO group were significantly higher than those in Con group and 2VO+Met group.Density of dendritic spines in hippocampal neurons of 2VO group was significantly lower than that in Con group and 2VO+Met group.The density and proportion of immature dendritic spines in hippocampal neurons of 2VO group were significantly higher than those in Con group and 2VO+Met group.Synaptic vesicle density of neurons in CA1 area of hippocampus in 2VO group((230.29±19.44)vescicles/μm^(2))was significantly lower than that in the Con group((414.52±13.17)vescicles/μm^(2))and 2VO+Met group((313.19±12.42)vescicles/μm^(2)).Conclusion Metformin can reduce neuroinflammation of hippocampus with chronic cerebral hypoperfusion and improve synaptic plasticity and cognitive dysfunction.It may have potential application value in the treatment of vascular cognitive dysfunction.