清心开窍方对APP/PS1双转基因小鼠学习记忆及海马CA1区Akt、GSK3α、βAPP、Aβ表达的影响
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摘要
目的:探讨清心开窍方对APP/PS1双转基因小鼠学习记忆能力、海马CA1区神经元形态学的改变,以及对Akt、GSK3α、βAPP、Aβ蛋白表达的影响。方法:选取50只3月龄雄性APP/PS1双转基因小鼠,随机分为模型组、多奈哌齐组(1.67 mg·kg~(-1))、清心开窍方高、中、低剂量组(每日给药剂量分别为19、9.5、4.75 g·kg~(-1)),另取10只同背景同性别同月龄的C57BL/6J小鼠作为对照组,对照组、模型组给予等体积生理盐水灌胃,1次/d,连续灌胃12周。采用Morris水迷宫检测小鼠空间学习记忆能力,透射电镜观察小鼠海马CA1区神经元超微结构,尼氏染色法观察小鼠海马CA1区神经元损伤情况,免疫组织化学染色法检测海马CA1区Akt、GSK3α、βAPP、Aβ蛋白的表达情况。结果:与对照组比较,模型组APP/PS1双转基因小鼠逃避潜伏期延长(P<0.01),穿台次数减少,其海马CA1区神经元超微结构出现严重损伤,锥体细胞数量减少,排列紊乱,尼氏体着色变浅,Akt蛋白表达减少(P<0.01),GSK3α、βAPP、Aβ表达增加(P<0.01);与模型组比较,清心开窍方干预组逃避潜伏期均明显缩短(P<0.01),穿台次数增多,同时比较小鼠在原平台停留的时间和路程百分比均明显增加(P<0.05;P<0.01),其神经元结构较完整,核膜清晰,胞浆内核糖体丰富,线粒体数量增多,且嵴结构完整,锥体细胞数量增多,排列紧密,尼氏体着色较深,同时Akt蛋白表达增加(P<0.01;P<0.05),GSK3α、βAPP、Aβ表达减少(P<0.01;P<0.05)。结论:清心开窍方能够改善APP/PS1双转基因小鼠的学习记忆能力,其机制可能与Akt/GSK3α介导的信号通路相关,它能够激活Akt,抑制GSK3α,降低βAPP、Aβ的生成,减少神经元损伤。
Objective: This study was aimed to investigate the changes of spatial cognitive function with Qingxin Kaiqiao Decoction(QKD) in APP/PS1 transgenic mice, the pathological changes of neurons and the expressions of Akt,GSK3α,βAPP and Aβ in hippocampus CA1 region and then explore the possible mechanism of QKD's anti-AD function. Methods: Totally 50 three-month-old male APP/PS1 transgenic mice were used to establish the AD model. They were randomly divided into the model group, the Donepezil group, the QKD high dose group(19 g·kg~(-1)), the QKD middle dose group(9.5 g·kg~(-1)) and the QKD low dose group(4.75 g·kg~(-1)). Besides, 10 three-month-old male C57 BL/6 J mice were selected as the normal control group. The control group and the model group were given equal volume of saline. All mice were gavaged for 12 consecutive weeks. At the end of medication, all mice were tested for their capabilities of spatial navigation and memorization by Morris water maze. The ultrastructure of hippocampus neuronal cells was observed by transmission electron microscopy(TEM)and its nissl body was tested by Nissl staining. Immunohistochemical staining was employed to confirm the expressions of Akt, GSK3α, βAPP and Aβ in hippocampus CA1 region. Results: Compared with the normal group, the escape latency of the APP/PS1 double transgenic mice in the model group were prolonged(P<0.01), while the number of crossing the platform was decreased. In the hippocampal CA1 area, the ultrastructure of neurons were severely damagedand the number of pyramidal cells were decreased while it arranged disorder and stained lightly. The protein expressions of Akt were decreased(P<0.01). GSK3α, βAPPand Aβ were increased(P<0.01). Compared with the model group, the escape latency of QKD groups was significantly shortened(P<0.01), the number of crossing the platform was increasedand the percentage of swimming distancy and swimming time in the target quadrant were significantly increased(P<0.05,P<0.01). Compared with the model group, the neural structure of the QKD groups was mostly healthy, the nuclear membrane was intact, numerous ribosomes and mitochondria were found in cytoplasm, and the mitochondria cristal structure was completed. Results of Nissl staining showed that the number of pyramidal cells in the hippocampal CA1 area was increased and the cells arranged closely and the cytoplasm was in dark blue. The protein expressions of Akt were significantly decreased(P<0.01,P<0.05) and GSK3α, βAPP and Aβ were significantly increased in hipppcampus CA1 region of the QKD groups(P<0.01,P<0.05). Conclusion: These findings suggest that QKD could obviously improve the spatial learning and memory ability of APP/PS1 double transgenic mice. The possible mechanism might be regulating the Akt/GSK3α signaling pathways. QKD could pretect hippocampal neurons by improving the activity of Akt and decreasing GSK3α in mice brain.
Objective: This study was aimed to investigate the changes of spatial cognitive function with Qingxin Kaiqiao Decoction(QKD) in APP/PS1 transgenic mice, the pathological changes of neurons and the expressions of Akt,GSK3α,βAPP and Aβ in hippocampus CA1 region and then explore the possible mechanism of QKD's anti-AD function. Methods: Totally 50 three-month-old male APP/PS1 transgenic mice were used to establish the AD model. They were randomly divided into the model group, the Donepezil group, the QKD high dose group(19 g·kg~(-1)), the QKD middle dose group(9.5 g·kg~(-1)) and the QKD low dose group(4.75 g·kg~(-1)). Besides, 10 three-month-old male C57 BL/6 J mice were selected as the normal control group. The control group and the model group were given equal volume of saline. All mice were gavaged for 12 consecutive weeks. At the end of medication, all mice were tested for their capabilities of spatial navigation and memorization by Morris water maze. The ultrastructure of hippocampus neuronal cells was observed by transmission electron microscopy(TEM)and its nissl body was tested by Nissl staining. Immunohistochemical staining was employed to confirm the expressions of Akt, GSK3α, βAPP and Aβ in hippocampus CA1 region. Results: Compared with the normal group, the escape latency of the APP/PS1 double transgenic mice in the model group were prolonged(P<0.01), while the number of crossing the platform was decreased. In the hippocampal CA1 area, the ultrastructure of neurons were severely damagedand the number of pyramidal cells were decreased while it arranged disorder and stained lightly. The protein expressions of Akt were decreased(P<0.01). GSK3α, βAPPand Aβ were increased(P<0.01). Compared with the model group, the escape latency of QKD groups was significantly shortened(P<0.01), the number of crossing the platform was increasedand the percentage of swimming distancy and swimming time in the target quadrant were significantly increased(P<0.05,P<0.01). Compared with the model group, the neural structure of the QKD groups was mostly healthy, the nuclear membrane was intact, numerous ribosomes and mitochondria were found in cytoplasm, and the mitochondria cristal structure was completed. Results of Nissl staining showed that the number of pyramidal cells in the hippocampal CA1 area was increased and the cells arranged closely and the cytoplasm was in dark blue. The protein expressions of Akt were significantly decreased(P<0.01,P<0.05) and GSK3α, βAPP and Aβ were significantly increased in hipppcampus CA1 region of the QKD groups(P<0.01,P<0.05). Conclusion: These findings suggest that QKD could obviously improve the spatial learning and memory ability of APP/PS1 double transgenic mice. The possible mechanism might be regulating the Akt/GSK3α signaling pathways. QKD could pretect hippocampal neurons by improving the activity of Akt and decreasing GSK3α in mice brain.
引文
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[4] 徐冬梅,王逸如,胡海燕,等.清心开窍方有效成分对AD大鼠海马区IL-1β、GFAP及Aβ表达的影响[J].中华中医药学刊,2015,35(3):595-598.
[5] 王逸如,张益慧,陈志裕,等.清心开窍方有效成分对AD大鼠学习记忆能力及其海马区Bax、Bcl-2、Caspase-3和βAPP表达的影响[J].中国中西医结合杂志,2017(2):189-197.
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[11] Wang ZX,Tan L,Liu J,et al.The Essential Role of Soluble Aβ Oligomers in Alzheimer’s Disease[J].Mol Neurobiol,2016,53(3):1905-1924.
[12] Selkoe D,Mandelkow E,Holtzman D.Deciphering Alzheimer Disease[J].Csh Perspect Med,2012,2(1):a011460.
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[14] Kim B,Feldman EL.Insulin resistance in the nervous system[J].Trends Endocrin Met,2012,23(3):133-141.
[15] Stanley M,Macauley SL,Holtzman DM.Changes in insulin and insulin signaling in Alzheimer’s disease:cause or consequence?[J].J Exp Med,2016,213(8):1375-1385.
[16] O’ NC.PI3-kinase/Akt/mTOR signaling:impaired on/off switches in aging,cognitive decline and Alzheimer’s disease.[J].Exp Gerontol,2013,48(7):647-653.
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[18] Linda Adlerz,Sofia Holback,GerdMulthaup,et al.IGF-l-induced processing of the amyloid precursor protein family is mediated by different signaling pathways[J].JBiol Chem.2007,282(14):10203-10209.
[19] Phiel CJ,Wilson CA,Lee VM,et al.GSK-3alpha regulates production of Alzheimer’s disease amyloid-beta peptides[J].Nature,2003,423(6938):435.
[20] Parr Callum,Carzaniga Raffaela,Gentleman Steve M,et al.Glycogen synthase kinase 3 inhibition promotes lysosomal biogenesis and autophagic degradation of the amyloid-β precursor protein[J] .Mol Cell Biol,2012,32(21):4410-4418.
[21] 张介宾.景岳全书[M].上海:第二军医大学出版社,2006:685.
[22] Wengenack TM,Whelan S,Curran GL,et al.Quantitative histological analysis of amyloid deposition in Alzheimer’s double transgenic mouse brain[J].Neuroscience,2000,101(4):939-944.
[2] 胡海燕,汤家铭,朱未名,等.清心开窍方对Aβ大鼠痴呆模型学习记忆能力和胆碱能系统的影响[J].中华中医药学刊,2008,26(8):1804-1806.
[3] 胡海燕,朱未名.清心开窍方对AD小鼠脑组织NO、AchE含量及海马CA1区神经细胞形态学改变的实验研究[J].福建中医药,2007,38(1):40-41.
[4] 徐冬梅,王逸如,胡海燕,等.清心开窍方有效成分对AD大鼠海马区IL-1β、GFAP及Aβ表达的影响[J].中华中医药学刊,2015,35(3):595-598.
[5] 王逸如,张益慧,陈志裕,等.清心开窍方有效成分对AD大鼠学习记忆能力及其海马区Bax、Bcl-2、Caspase-3和βAPP表达的影响[J].中国中西医结合杂志,2017(2):189-197.
[6] Mao DD,Yang WY,Li Y,et al.Effect of Qingxin Kaiqiao Fang on Hippocampus mRNA Expression of the Inflammation-Related Genes IL-1β,GFAP,and Aβ in an Alzheimer’s Disease Rat Model[J].Evid-Based Compl Alt,2018,2018:9267653.
[7] 李仪奎.中药药理实验方法学[M].上海:上海科学技术出版社,1991:36.
[8] Mattson MP.Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives[J].Physiol Rev,1997,77:1081-1132.
[9] Calderon-Garcidue?±As A L,Duyckaerts C.Alzheimer disease[J].Handb Clin Neurol,2017(13):325-337.
[10] Yan SD,Fu J,Soto C,et al.An intracellular protein that binds amyloid-beta peptide and mediates neurotoxicity in Alzheimer’s disease[J].Nature,1997,389(6652):689-695.
[11] Wang ZX,Tan L,Liu J,et al.The Essential Role of Soluble Aβ Oligomers in Alzheimer’s Disease[J].Mol Neurobiol,2016,53(3):1905-1924.
[12] Selkoe D,Mandelkow E,Holtzman D.Deciphering Alzheimer Disease[J].Csh Perspect Med,2012,2(1):a011460.
[13] Steen E,Terry BM,Rivera EJ,et al.Impaired insulin and insulin-like growth factor expression and signaling mechanisms in Alzheimer’s disease-is this type 3 diabetes?[J].J Alzheimers Dis,2005,7(1):63-80.
[14] Kim B,Feldman EL.Insulin resistance in the nervous system[J].Trends Endocrin Met,2012,23(3):133-141.
[15] Stanley M,Macauley SL,Holtzman DM.Changes in insulin and insulin signaling in Alzheimer’s disease:cause or consequence?[J].J Exp Med,2016,213(8):1375-1385.
[16] O’ NC.PI3-kinase/Akt/mTOR signaling:impaired on/off switches in aging,cognitive decline and Alzheimer’s disease.[J].Exp Gerontol,2013,48(7):647-653.
[17] Woodgett JR.A common denominator linking glycogen metabolism,nuclear oncogenes and development[J].Trends Biochem Sci,1991,16(5):177-181.
[18] Linda Adlerz,Sofia Holback,GerdMulthaup,et al.IGF-l-induced processing of the amyloid precursor protein family is mediated by different signaling pathways[J].JBiol Chem.2007,282(14):10203-10209.
[19] Phiel CJ,Wilson CA,Lee VM,et al.GSK-3alpha regulates production of Alzheimer’s disease amyloid-beta peptides[J].Nature,2003,423(6938):435.
[20] Parr Callum,Carzaniga Raffaela,Gentleman Steve M,et al.Glycogen synthase kinase 3 inhibition promotes lysosomal biogenesis and autophagic degradation of the amyloid-β precursor protein[J] .Mol Cell Biol,2012,32(21):4410-4418.
[21] 张介宾.景岳全书[M].上海:第二军医大学出版社,2006:685.
[22] Wengenack TM,Whelan S,Curran GL,et al.Quantitative histological analysis of amyloid deposition in Alzheimer’s double transgenic mouse brain[J].Neuroscience,2000,101(4):939-944.
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