AMPA受体内化抑制剂GluA2-3Y对慢性脑低灌注模型大鼠认知功能及突触后蛋白表达的影响

Effect of AMPA receptor endocytosis inhibitor GluA2-3Y on cognitive function and postsynaptic protein expression in chronic cerebral hypoperfusion rats

目的探究α-氨基-3-羟基-5-甲基-4-异恶唑-丙酸(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid, AMPA)受体内化抑制剂GluA2-3Y对慢性脑低灌注大鼠认知功能及海马突触后蛋白表达的影响。方法 48只成年雄性SD大鼠按照随机数字表法分为Sham组、2VO组、高剂量GluA2-3Y组和低剂量GluA2-3Y组, 每组12只。采用双侧颈总动脉永久性结扎法(two vessel occlusion, 2VO)构建慢性脑低灌注模型, 其中Sham组行假手术。高剂量GluA2-3Y组和低剂量GluA2-3Y组分别腹腔注射剂量为3 μmol/kg和0.03 μmol/kg的GluA2-3Y, 1次/d, 共2周, 2VO组和Sham组大鼠腹腔注射对照肽。采用Morris水迷宫和新物体识别实验评测大鼠的学习记忆能力, 免疫印迹法评测大鼠海马Akt1、GSK3β(glycogen synthase kinase-3β)、p-GSK3β、GluA2和PSD-95(postsynaptic density-95)的表达, 免疫荧光法评测大鼠海马GluA2和PSD-95的表达。采用SPSS 23.0进行数据分析, 组间比较采用单因素方差分析, Morris水迷宫结果采用重复测量方差分析, 两两比较采用独立样本t检验。结果 (1)重复测量方差分析结果显示, 在Morris水迷宫实验中, 各组大鼠逃避潜伏期的分组与时间的交互作用不显著(F=0.79, P>0.05), 组别主效应与时间主效应均显著(F=24.44, 40.42, 均P<0.05)。在第5天的定位航行检测中, 2VO组大鼠的逃避潜伏期较Sham组长(t=5.87, P<0.05), 低剂量GluA2-3Y组和高剂量GluA2-3Y组大鼠的逃避潜伏期均明显短于2VO组(t=2.20, 3.41, 均P<0.05), 而高剂量GluA2-3Y组与低剂量GluA2-3Y组的逃避潜伏期差异无统计学意义(t=1.37, P>0.05)。2VO组的目标象限停留时间和分辨系数[(14.57±1.40)s, (0.15±0.10)]均明显低于Sham组[(23.71±2.57)s, (0.40±0.06)](t=3.23, 2.24, 均P<0.05), 而高剂量GluA2-3Y组[(20.19±1.53)s]和低剂量GluA2-3Y组[(20.31±2.06)s]的目标象限停留时间均较2VO组长(t=2.71, 2.35, 均P<0.05)。高剂量GluA2-3Y组(0.47±0.10)和低剂量GluA2-3Y组(0.59±0.06)的新物体辨别系数均高于2VO组(t=2.21, 3.94, 均P<0.05)。(2)免疫印迹实验中, 2VO组大鼠海马组织PSD-95和GluA2表达较Sham组明显减少(t=2.31, 2.20, 均P<0.05), 高剂量GluA2-3Y组PSD-95表达(1.026±0.056)较2VO组[(0.760±0.061)]显著增加(t=2.49, P<0.01), 而低剂量GluA2-3Y组PSD-95表达与2VO组比较差异无统计学意义(t=0.96, P>0.05)。低剂量GluA2-3Y组的GluA2表达高于2VO组[(1.130±0.087), (0.766±0.080), t=2.37, P<0.05], 但是高剂量GluA2-3Y组与2VO组间GluA2的表达差异无统计学意义(t=1.06, P>0.05)。(3)免疫荧光结果显示, 与Sham组比较, 2VO组大鼠PSD-95和GluA2的表达均减少(t=4.23, 2.57, 均P<0.05)。与2VO组比较, 高剂量GluA2-3Y组与低剂量GluA2-3Y组的PSD-95和GluA2均显著增加, 差异有统计学意义[PSD-95: (7.757±0.578), (12.057±0.578),t=3.14, 6.96,均P<0.05;GluA2: (9.721±0.950), (16.610±0.950),t=4.56, 9.34,均P<0.05]。(4)免疫印迹结果显示, 各组大鼠海马组织GSK3β的表达差异无统计学意义(F=2.03, 0.30, 4.76, 3.57,均P<0.05)。与Sham组比较, 2VO组的Akt1、p-GSK3β及p-GSK3β/GSK3β百分比均降低, 差异有统计学意义(t=3.00, 2.81, 3.17,均P<0.05)。与2VO组比较, 低剂量GluA2-3Y组和高剂量GluA2-3Y组Akt1、p-GSK3β和p-GSK3β/GSK3β百分比均显著升高, 差异有统计学意义(Akt1:t=2.05, 5.20,均P<0.05;p-GSK3β:t=2.49, 4.15,均P<0.05;p-GSK3β/GSK3β百分比:t=2.30, 2.97, 均P<0.05)。结论 AMPA受体内化抑制剂GluA2-3Y可以改善慢性脑低灌注大鼠的认知损伤, 这可能与增加Akt1、p-GSK3β及突触后蛋白表达有关。

Objective To investigate the effect of GluA2-3Y which is an inhibitor of AMPA(α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid)receptor internalization on cognitive function and hippocampal postsynaptic protein expression in rats with chronic cerebral hypoperfusion.Methods Forty-eight adult male SD rats were randomly divided into Sham group,2VO group,high-dose GluA2-3Y group and low-dose GluA2-3Y group according to random number table,with 12 rats in each group.The chronic cerebral hypoperfusion model of rat was established by two vessel occlusion(2VO)while the Sham operation was performed in rats of Sham group.The rats in high dose GluA2-3Y group and low dose GluA2-3Y group were intraperitoneal injected with 3μmol/kg and 0.03μmol/kg GluA2-3Y respectively once a day for 2 weeks.Rats in 2VO group and Sham group were intraperitoneally injected with control peptide.Morris water maze test and new object recognition test were performed to evaluate the learning and memory ability of rats,and Western blot was used to evaluate the expression of Akt1、GSK3β、p-GSK3β、GluA2 and PSD-95 in rat hippocampus.The expressions of GluA2 and PSD-95 in rat hippocampus were evaluated by immunofluorescence.SPSS 23.0 software was used for data analysis.The comparison between multiple groups was analyzed by one-way ANOVA and repeated measurement ANOVA was used to analyze Morris water maze results.And independent-samples t-test was used for pairwise comparisons.Results(1)In Morris water maze trials,the results of repeated measurement ANOVA showed that the interaction between group and time of escape latency of rats in each group was not significant(F=0.79,P>0.05),and the group main effect and time main effect were significant(F=24.44,40.42,both P<0.05).On the 5th day of navigation trials,the escape latency of rats in 2VO group was longer than that in sham group(t=5.87,P<0.05).The escape latency of rats in low dose GluA2-3Y group and high dose GluA2-3Y group were significantly shorter than that in 2VO group(t=2.20,3.41,both P<0.05),but there was no significant difference between low dose GluA2-3Y group and high dose GluA2-3Y group(t=1.37,P>0.05).The target quadrant residence time and resolution coefficient((14.57±1.40)s,(0.15±0.10))in 2VO group were significantly lower than those in Sham group((23.71±2.57)s,(0.40±0.06))(t=3.23,2.24,both P<0.05),while the target quadrant residence time in high dose GluA2-3Y group((20.19±1.53)s)and low dose GluA2-3Y group((20.31±2.06)s)were longer than that in 2VO group(t=2.71,2.35,both P<0.05).The discrimination coefficients in high dose GluA2-3Y group(0.47±0.10)and low dose GluA2-3Y group(0.59±0.06)were higher than that of 2VO group(t=2.21,3.94,both P<0.05).(2)The Western blot results showed that the expression of PSD-95 and GluA2 in hippocampus of rats in 2VO group were significantly lower than those in Sham group(t=2.31,2.20,both P<0.05),and the expression of PSD-95 in high dose GluA2-3Y group(1.026±0.056)was significantly higher than that in 2VO group((0.760±0.061),t=2.49,P<0.05),while there was no significant difference between low-dose GluA2-3Y group and 2VO group(t=0.96,P>0.05).The expression of GluA2 in low-dose GluA2-3Y group was higher than that in 2VO Group((1.130±0.087),(0.766±0.080),t=2.37,P<0.05),but there was no significant difference between high-dose GluA2-3Y group and 2VO group(t=1.06,P>0.05).(3)Immunofluorescence showed that compared with Sham group,the expression of PSD-95 and GluA2 in 2VO group decreased(t=4.23,2.57,P<0.05).Compared with 2VO group,the expression of PSD-95 and GluA2 in high dose GluA2-3Y group and low dose GluA2-3Y group increased significantly,and the differences were statistically significant(PSD-95:(7.757±0.578),(12.057±0.578),t=3.14,6.96,both P<0.05;(9.721±0.950),(16.610±0.950),t=4.56,9.34,both P<0.05).(4)The results of Western blot showed that the expression GSK3βin hippocampus of rats in each group were not statistically different(F=2.03,P>0.05).There were significant differences in the expression of Akt1,p-GSK3βand the percentage of p-GSK3β/GSK3βin hippocampus of rats in each group(F=8.30,4.76,3.57,all P<0.05).Compared with Sham group,the levels of Akt1,p-GSK3βand the percentage of p-GSK3β/GSK3βin 2VO group were significantly lower(t=3.00,2.81,3.17,all P<0.05).Compared with 2VO group,the levels of Akt1,p-GSK3βand p-GSK3β/GSK3βpercentage in low dose GluA2-3Y group and high-dose GluA2-3Y group were significantly higher(Akt1:t=2.05,5.20,both P<0.05;p-GSK3β:t=2.49,4.15,both P<0.05;p-GSK3β/GSK3βpercentage:t=2.30,2.97,both P<0.05).Conclusion GluA2-3Y,an AMPA receptor internalization inhibitor,can alleviate the cognitive impairment in rats with chronic cerebral hypoperfusion,which may be related to the increased expression of Akt1,p-GSK3βand postsynaptic proteins.