糖尿病大鼠前额叶部位淀粉样蛋白水平与自噬水平变化的关系研究
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摘要
目的观察糖尿病大鼠前额叶部位淀粉样蛋白(β-amyloid,Aβ)表达与自噬水平变化的关系。方法90只雄性SD大鼠随机分为正常组(n=45)和糖尿病组(n=45)。建立STZ诱导的糖尿病大鼠模型,分别在成模第8、10、12周时,从两组随机取出15只大鼠行Morris水迷宫测试大鼠的学习与记忆能力,电镜检测大鼠前额叶神经元细胞的超微结构,qRT-PCR检测大鼠前额叶APP、自噬相关蛋白Beclin-1、LC3Ⅰ、LC3ⅡmRNA的表达情况,Western blot和免疫组化检测大鼠前额叶Aβ、Beclin-1、LC3Ⅰ、LC3Ⅱ和p62蛋白的表达情况。结果与正常组大鼠相比,糖尿病组大鼠在成模第8周Morris水迷宫测试成绩下降,且随着糖尿病大鼠病程的延长,认知功能下降明显(P<0.05)。与正常组大鼠相比,糖尿病大鼠在成模第8周时前额叶神经元细胞的超微结构改变,线粒体肿胀,到第12周时神经元细胞核碎裂,染色质固缩凝集。第8周糖尿病大鼠前额叶神经元细胞Aβ沉积明显高于正常组大鼠(P<0.05),且随着糖尿病大鼠成模时间的延长,Aβ沉积增加明显,呈时间依赖性(P<0.05)。糖尿病大鼠在成模第8周时,前额叶神经元细胞Beclin-1蛋白及mRNA表达增加(P<0.05),LC3Ⅱ/Ⅰ蛋白及mRNA的比值升高(P<0.05),到第12周时这种差异最为明显。p62表达两组间差异无统计学意义(P>0.05)。第8～12周,随着时间延长,Aβ表达水平与Beclin-1、LC3Ⅱ/Ⅰ表达呈正相关(P<0.01)。结论糖尿病大鼠自成模第8周开始出现认知功能的下降,这与前额叶神经元细胞自噬水平改变、自噬流不通、Aβ清除障碍有关。
Objective To study the correlation between the levels ofβ-amyloid(Aβ)and autophagy in the prefrontal lobe of diabetic rats.Methods A total of 90 male Sprague-Dawley rats were divided randomly into the control group(n=45)and diabetic model(DM)group(n=45).The streptozocin(STZ)-induced diabetic rat models were established.15 rats were taken randomly from each group to measure their learning and memory ability by Morris water maze test at the 8 th,10 th and 12 th week respectively.Meanwhile,the electron microscopy was used to examine the ultrastructure of their prefrontal lobe neurons,the qRT-PCR assay was adopted to detect the expression levels of APP,Beclin-1,LC3Ⅰ and LC3Ⅱ in the prefrontal lobe,and the Western blot and immunohistochemistry staining methods were used to detect the expression levels of Aβ,Beclin-1,LC3Ⅰ,LC3Ⅱand p62 in the prefrontal lobe.Results The escape latency in the Morris water maze test and the course of disease were significantly longer and the cognitive function was significantly lower at the 8 th week in the DM group than in the control group(P<0.05).Compared with the control group,the ultrastructure of the rats' prefrontal neurons in the DM group changed and the mitochondria swelled at the 8 th week.At the 12 th week,the nuclei of neurons fragmented and the chromatin condensed and agglutinated.The Aβdeposition in the prefrontal lobe increased more significantly at the 8 th week in the DM group than in the control group(P<0.05).With the prolongation of time,the Aβdeposition in the DM group increased significantly in a time-dependent manner(P<0.05).The expression levels of Beclin-1 and mRNA in the prefrontal lobe neurons increased significantly(P<0.05)and the levels of LC3Ⅱ/Ⅰ and mRNA increased significantly(P<0.05)at the 8 th week in the DM group(P<0.05),and the differences were the greatest at the 12 th week.There was no significant difference in the p62 expression level between the two groups(P>0.05).The Aβexpression level was correlated positively with the expression levels of Beclin-1 and LC3Ⅱ/Ⅰ respectively(P<0.01)with the time going from the 8 th week to the 12 th week.Conclusion The cognitive function of the diabetic rats began to decline at the 8 th week after the model establishment,which may be correlated with the changes of the autophagy level,the blocked autophagy flow and the impaired clearance of Aβin in the prefrontal lobe neurons.
Objective To study the correlation between the levels ofβ-amyloid(Aβ)and autophagy in the prefrontal lobe of diabetic rats.Methods A total of 90 male Sprague-Dawley rats were divided randomly into the control group(n=45)and diabetic model(DM)group(n=45).The streptozocin(STZ)-induced diabetic rat models were established.15 rats were taken randomly from each group to measure their learning and memory ability by Morris water maze test at the 8 th,10 th and 12 th week respectively.Meanwhile,the electron microscopy was used to examine the ultrastructure of their prefrontal lobe neurons,the qRT-PCR assay was adopted to detect the expression levels of APP,Beclin-1,LC3Ⅰ and LC3Ⅱ in the prefrontal lobe,and the Western blot and immunohistochemistry staining methods were used to detect the expression levels of Aβ,Beclin-1,LC3Ⅰ,LC3Ⅱand p62 in the prefrontal lobe.Results The escape latency in the Morris water maze test and the course of disease were significantly longer and the cognitive function was significantly lower at the 8 th week in the DM group than in the control group(P<0.05).Compared with the control group,the ultrastructure of the rats' prefrontal neurons in the DM group changed and the mitochondria swelled at the 8 th week.At the 12 th week,the nuclei of neurons fragmented and the chromatin condensed and agglutinated.The Aβdeposition in the prefrontal lobe increased more significantly at the 8 th week in the DM group than in the control group(P<0.05).With the prolongation of time,the Aβdeposition in the DM group increased significantly in a time-dependent manner(P<0.05).The expression levels of Beclin-1 and mRNA in the prefrontal lobe neurons increased significantly(P<0.05)and the levels of LC3Ⅱ/Ⅰ and mRNA increased significantly(P<0.05)at the 8 th week in the DM group(P<0.05),and the differences were the greatest at the 12 th week.There was no significant difference in the p62 expression level between the two groups(P>0.05).The Aβexpression level was correlated positively with the expression levels of Beclin-1 and LC3Ⅱ/Ⅰ respectively(P<0.01)with the time going from the 8 th week to the 12 th week.Conclusion The cognitive function of the diabetic rats began to decline at the 8 th week after the model establishment,which may be correlated with the changes of the autophagy level,the blocked autophagy flow and the impaired clearance of Aβin in the prefrontal lobe neurons.
引文
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[4]Funderburk S F,Marcellino B K,Yue Z.Cell"self-eating"(autophagy)mechanism in Alzheimer's disease[J].Mt Sinai JMed,2010,77(1):59-68.
[5]Gallagher L,Williamson L,Chan E.Advances in autophagy regulatory mechanisms[J].Cells,2016,5(2):24.
[6]Nilsson P,Loganathan K,Sekiguchi M,et al.Aβsecretion and plaque formation depend on autophagy[J].Cell Rep,2013,5(1):61-69.
[7]Chen L,Gong S,Shan L D,et al.Effects of exercise on neurogenesis in the dentate gyrus and ability of learning and memory after hippocampus lesion in adult rats[J].Neurosci Bull,2006,22(1):1-6.