益肾化浊方对快速老化小鼠认知功能与海马神经元形态学的影响及机制
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摘要
目的探讨益肾化浊方对阿尔兹海默病模型快速老化鼠(SAMP)8空间记忆能力、海马神经元形态学的影响及其作用机制。方法将48只9月龄健康雄性SMAP8采用双盲法随机分为SAMP8组、安理申组、益肾化浊方组,每组16只,另取16只SAMR1作为对照组。各组小鼠分别灌胃给予安理申(30 mg/kg)、益肾化浊方(20 g/kg)或等体积0.9%生理盐水,2次/d,连续14 w。分别采用Morris水迷宫检测空间学习记忆能力,尼氏染色观察细胞凋亡情况,电镜观察神经元超微结构,RT-PCR、Western印迹检测β-淀粉样蛋白前体(APP)、早老蛋白(PS1)基因和GSK-3β蛋白表达。结果与对照组比较,SAMP8组游泳总路程、逃避潜伏时间、海马CA1区神经元凋亡数目和APP、PS1基因及GSK-3β蛋白表达量均显著增加(P<0.01),穿越平台次数显著减少(P<0.01),游泳速度显著降低(P<0.01),超微结构损伤明显;与SAMP8组比较,安理申组与益肾化浊方组游泳总路程明显缩短(P<0.01),穿越平台次数明显增加(P<0.01),游泳速度亦显著加快(P<0.01),同时显著减轻神经元超微结构损伤;此外,安理申可显著下调GSK-3β蛋白表达(P<0.01),益肾化浊方可显著减少神经元凋亡数目(P<0.01),同时下调APP、PS1基因表达(P<0.01);组别和训练天数对逃避潜伏时间、总路程、穿越平台次数有明显影响(P<0.01)。结论益肾化浊方可有效提高SAMP8的空间记忆能力,抗神经元凋亡并保护神经元超微结构,其作用机制可能与下调APP、PS1基因和GSK-3β蛋白的表达相关。
Objective To study effects and mechanisms of Yishenhuazhuo recipe( YSHZR) on spatial learning and memory and the hippocampus neurons morphology in the senescence-accelerated prone 8( SAMP8) mice. Methods Forty-eight of 9-month-old SAMP8 mice were randomly allocated to model( SAMP8 mice),Aricept treated and YSHZR treated groups. Meanwhile,sixteen senescence-resistant 1( SAMR1) mice were used as controls( SAMR1 group).Aricept( 30 mg/kg),YSHZR( 20 g/kg) or equal-volume saline( SAMR1 and SAMP8mice) was administered in each mouse for 14 weeks by gastric infusion twice per day.Spatial learning and memory were measured by the Morris water maze.Neuronal apoptosis was quantified by Nissl staining.The mRNA expression of APP and PS1 and the protein expression of GSK-3β in the hippocampus were detected by RT-PCR and Western blot respectively.Results Compared with SAMR1 group,the path length,escape latency,number of neuronal apoptosis,GSK-3β protein and APP,PS1 gene expression in hippocampal CA1 of SAMP8 mice were significantly increased( P<0.01),frequency of entrance was significantly reduced( P < 0. 01),the swim speed was also significantly lowered( P <0.01),and the neuronal ultrastructural damage was very evident; compared with SAMP8 group,the path length in Aricept group and YSHZR group of mice were significantly shorter( P<0.01),the frequency of entrance was significantly increased( P<0.01),swim speed was also significantly faster( P<0.01),and neuronal ultrastructure damage was significantly reduced; moreover,Aricept significantly reduced GSK-3β protein expression( P<0.01),but YSHZR could significantly reduce the number of neuronal apoptosis( P<0.01),and down-regulate the gene expression of APP,PS1( P<0.01); groups and training days had significant effects on escape latency,total distance and the number of cross platform( P<0.01). Conclusions YSHZR plays a role of improving spatial memory,reducing neuronal apoptosis and protecting ultrastructure,which may be related to down-regulating the expression of APP,PS1 gene and GSK-3β protein.
Objective To study effects and mechanisms of Yishenhuazhuo recipe( YSHZR) on spatial learning and memory and the hippocampus neurons morphology in the senescence-accelerated prone 8( SAMP8) mice. Methods Forty-eight of 9-month-old SAMP8 mice were randomly allocated to model( SAMP8 mice),Aricept treated and YSHZR treated groups. Meanwhile,sixteen senescence-resistant 1( SAMR1) mice were used as controls( SAMR1 group).Aricept( 30 mg/kg),YSHZR( 20 g/kg) or equal-volume saline( SAMR1 and SAMP8mice) was administered in each mouse for 14 weeks by gastric infusion twice per day.Spatial learning and memory were measured by the Morris water maze.Neuronal apoptosis was quantified by Nissl staining.The mRNA expression of APP and PS1 and the protein expression of GSK-3β in the hippocampus were detected by RT-PCR and Western blot respectively.Results Compared with SAMR1 group,the path length,escape latency,number of neuronal apoptosis,GSK-3β protein and APP,PS1 gene expression in hippocampal CA1 of SAMP8 mice were significantly increased( P<0.01),frequency of entrance was significantly reduced( P < 0. 01),the swim speed was also significantly lowered( P <0.01),and the neuronal ultrastructural damage was very evident; compared with SAMP8 group,the path length in Aricept group and YSHZR group of mice were significantly shorter( P<0.01),the frequency of entrance was significantly increased( P<0.01),swim speed was also significantly faster( P<0.01),and neuronal ultrastructure damage was significantly reduced; moreover,Aricept significantly reduced GSK-3β protein expression( P<0.01),but YSHZR could significantly reduce the number of neuronal apoptosis( P<0.01),and down-regulate the gene expression of APP,PS1( P<0.01); groups and training days had significant effects on escape latency,total distance and the number of cross platform( P<0.01). Conclusions YSHZR plays a role of improving spatial memory,reducing neuronal apoptosis and protecting ultrastructure,which may be related to down-regulating the expression of APP,PS1 gene and GSK-3β protein.
引文
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2 Mario Nizzari,Stefano Thellung,Alessandro Corsaro,et al.Neurodegeneration in Alzheimer disease:Role of amyloid precursor protein and presenilin1 intracellular signaling[J].J Toxicol,2012;doi:10.1155/2012/187297.
3傅凯丽,林翠茹,张玉莲,等.“益肾化浊方”治疗轻度阿尔茨海默病15例临床研究[J].江苏中医药,2012;44(8):28-9.
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6刘金凤,聂坤,栗振杰,等.Morris水迷宫测评方法的改进及对快速老化痴呆小鼠SAMP8认知功能的再评价[J].中国中医急症,2013;22(1):5-7,22.
7郭振球.老年期痴呆的证治学研究[J].中医药研究,1991;1:16.
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10李延峥,李林,张兰.快速老化小鼠SAMP8在阿尔茨海默病研究中的应用[J].中国康复理论与实践,2012;18(12):1119-22.
11 Morris R.Developments of a water maze procedure for studying spatial,learning in the rat[J].J Neurosci Methods,1984;11(1):47-60.
12 Nowakowska E,Kus K,Bobkiewicz-Kozlowska T.Role of neuropeptides in antidepressant and memory improving effects of venlafaxine[J].Pol JPharmacol,2002;54(6):605-13.
13 Frisone DF,Frye CA,Zimmerberg B.Social isolation stress during the third week of life has age-dependent effects on spatial learning in rats[J].Behav Brain Res,2002;128(2):153-60.
14 Shanmugam G,Polavarapu PL,Láng E,et al.Conformational analysis of amyloid precursor protein fragment containing amino acids 667-676,and the effect of D-Asp and iso-Asp substitution at Asp672 residue[J].JStruct Biol,2012;177(3):621-9.
15 Torreilles F,Touchon J.Pathogenic theories and intrathecal analysis of the sporadic from of Alzheimers'disease[J].Prog Neurobiol,2002;66(3):191-203.
16 Deshpande A,Mina E,Glabe C,et al.Different conformations of amyloidbeta induce neurotoxicity by distinct mechanisms in human cortical neurons[J].J Neurosci,2006;26(22):6011-8.
17 Guo Q,Fu W,Sopher BL,et al.Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock in mice[J].Nat Med,1999;5(1):101-6.
18 Duff K,Eckman C,Zehr C,et al.Increased amyloid-beta 42(43)in brains of mice expressing mutant presenilin-1[J].Nature,1996;383(6602):710-3.
19 Biernat J,Gustke N,Drewes G,et al.Phosphorylation of Ser262 strongly reduces binding of tau to microtubules:distinction between PHF-like immunoreactivity and microtubule binding[J].Neuron,1993;11(1):153-63.
20 Sengupta A,Kabat J,Novak M,et al.Phosphorylation of tau at both Thr231 and Ser262 is required for maximal inhibition of its binding to microtubules[J].Arch Biochem Biophys,1998;357(2):299-309.
21 Abraha A,Ghoshal N,Gamblin TC,et al.C-term inal inhibition of tau assembly in vitro and in Alzheimer’s disease[J].J Cell Sci,2000;113(Pt21):3737-45.
22 Haase C,Stieler JT,Arendt T,et al.Pseudophosphorylation of tau protein alters its ability for self-aggregation[J].J Neurochem,2004;88(6):1509-20.
23 Cai Z,Zhao Y,Zhao B.Roles of glycogen synthase kinase 3 in Alzheimer's disease[J].Curr Alzheimer Res,2012;9(7):864-79.
2 Mario Nizzari,Stefano Thellung,Alessandro Corsaro,et al.Neurodegeneration in Alzheimer disease:Role of amyloid precursor protein and presenilin1 intracellular signaling[J].J Toxicol,2012;doi:10.1155/2012/187297.
3傅凯丽,林翠茹,张玉莲,等.“益肾化浊方”治疗轻度阿尔茨海默病15例临床研究[J].江苏中医药,2012;44(8):28-9.
4张琳琳,周震,李程,等.益肾化浊方对SAMP8小鼠NEI网络相关指标的影响[J].世界中医药,2014;9(6):689-92.
5王凯,张琳琳,宋宛珊,等.益肾化浊方对阿尔茨海默病模型大鼠海马区BDNF及其受体Trk B蛋白表达的影响[J].中国实验方剂学杂志,2015;21(5):111-4.
6刘金凤,聂坤,栗振杰,等.Morris水迷宫测评方法的改进及对快速老化痴呆小鼠SAMP8认知功能的再评价[J].中国中医急症,2013;22(1):5-7,22.
7郭振球.老年期痴呆的证治学研究[J].中医药研究,1991;1:16.
8傅仁杰,罗社文.中医药治疗老年性呆病的临床研究述评[J].北京中医药大学学报,1994;17(3):2.
9 Del Valle J,Duran-Vilaregut J,Manich G,et al.Early amyloid accumulation in the hippocampus of SAMP8 mice[J].J Alzheimers Dis,2010;19(4):1303-15.
10李延峥,李林,张兰.快速老化小鼠SAMP8在阿尔茨海默病研究中的应用[J].中国康复理论与实践,2012;18(12):1119-22.
11 Morris R.Developments of a water maze procedure for studying spatial,learning in the rat[J].J Neurosci Methods,1984;11(1):47-60.
12 Nowakowska E,Kus K,Bobkiewicz-Kozlowska T.Role of neuropeptides in antidepressant and memory improving effects of venlafaxine[J].Pol JPharmacol,2002;54(6):605-13.
13 Frisone DF,Frye CA,Zimmerberg B.Social isolation stress during the third week of life has age-dependent effects on spatial learning in rats[J].Behav Brain Res,2002;128(2):153-60.
14 Shanmugam G,Polavarapu PL,Láng E,et al.Conformational analysis of amyloid precursor protein fragment containing amino acids 667-676,and the effect of D-Asp and iso-Asp substitution at Asp672 residue[J].JStruct Biol,2012;177(3):621-9.
15 Torreilles F,Touchon J.Pathogenic theories and intrathecal analysis of the sporadic from of Alzheimers'disease[J].Prog Neurobiol,2002;66(3):191-203.
16 Deshpande A,Mina E,Glabe C,et al.Different conformations of amyloidbeta induce neurotoxicity by distinct mechanisms in human cortical neurons[J].J Neurosci,2006;26(22):6011-8.
17 Guo Q,Fu W,Sopher BL,et al.Increased vulnerability of hippocampal neurons to excitotoxic necrosis in presenilin-1 mutant knock in mice[J].Nat Med,1999;5(1):101-6.
18 Duff K,Eckman C,Zehr C,et al.Increased amyloid-beta 42(43)in brains of mice expressing mutant presenilin-1[J].Nature,1996;383(6602):710-3.
19 Biernat J,Gustke N,Drewes G,et al.Phosphorylation of Ser262 strongly reduces binding of tau to microtubules:distinction between PHF-like immunoreactivity and microtubule binding[J].Neuron,1993;11(1):153-63.
20 Sengupta A,Kabat J,Novak M,et al.Phosphorylation of tau at both Thr231 and Ser262 is required for maximal inhibition of its binding to microtubules[J].Arch Biochem Biophys,1998;357(2):299-309.
21 Abraha A,Ghoshal N,Gamblin TC,et al.C-term inal inhibition of tau assembly in vitro and in Alzheimer’s disease[J].J Cell Sci,2000;113(Pt21):3737-45.
22 Haase C,Stieler JT,Arendt T,et al.Pseudophosphorylation of tau protein alters its ability for self-aggregation[J].J Neurochem,2004;88(6):1509-20.
23 Cai Z,Zhao Y,Zhao B.Roles of glycogen synthase kinase 3 in Alzheimer's disease[J].Curr Alzheimer Res,2012;9(7):864-79.