板桥党参对激活GSK-3β诱导的AD模型大鼠认知功能障碍的保护作用及其机制
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摘要
目的探索板桥党参(BCP)水煎液对激活糖原合成酶激酶-3β(GSK-3β)诱导的阿尔兹海默病(AD)模型大鼠认知功能障碍的保护作用及其可能机制。方法将50只4月龄♂SD大鼠随机分为假手术组、AD模型组、低剂量板桥党参组、中剂量板桥党参组、高剂量板桥党参组。前2组饮用水灌胃14 d,后3组灌服板桥党参水煎液14 d。灌胃同时进行自主行为测试,d 8开始进行水迷宫训练。训练5 d后,选择能在15 s内找到隐藏平台的治疗组和模型组大鼠进行侧脑室注射渥曼青霉素(Wortmannin,PI3K特异性抑制剂)和GF-109203X(GFX,PKC特异性抑制剂)(浓度均为100μmol·L~(-1))各5μL;假手术组注射2%DMSO 10μL。24 h后进行水迷宫测试。观察各组大鼠侧脑室注射前、后空间学习记忆力是否改变。Western blot和免疫组化检测大鼠脑海马组织Tau磷酸化水平及其相关蛋白激酶GSK-3β表达情况及活性变化。尼氏染色观察神经元尼氏小体变化情况。结果自主行为活动检测结果发现板桥党参能明显减少大鼠活动次数;水迷宫检测结果发现板桥党参能明显改善模型大鼠认知功能障碍;Western blot和免疫组化结果显示模型组海马组织GSK-3β活性升高,并导致Tau蛋白多个位点发生过度磷酸化,而用药后GSK-3β活性降低,Tau蛋白多个位点磷酸化水平下调;尼氏染色结果提示模型组海马神经元内尼氏小体数量减少,而治疗后尼氏小体增多,且呈剂量依赖性。结论板桥党参可有效改善GSK-3β活性升高所诱导的大鼠认知功能障碍,其可能机制与下调GSK-3β活性,进而抑制Tau蛋白过度磷酸化、促进神经元发育有关。
Aim To assess the effects of Banqiao Codonopisis Pilosula( BCP) decoction on learning and memory dysfunction in AD model rats induced by high activity GSK-3β and its possible mechanism. Methods The SD rats( 4 months old,♂) were divided into five groups,namely,sham-operated group( blank group),AD model group,BCP high-dose( 2. 16 g · kg~(-1)·d~(-1)) group,BCP medium-dose( 1. 08 g·kg~(-1)·d~(-1))group,and BCP lower-dose( 0. 54 g · kg~(-1)· d~(-1))group. Treatment group received BCP decoction by gavage once a day for 14 days,while other groups were offered drinking water by gavage once a day for 14 days. The autonomous behavior activities of all rats were observed and recorded after gavage. In the last seven days by gavage,Morris water maze test was used to test the spatial learning and memory ability of the five groups. After five days training,treatment groups and AD model group were injected wortmannin( WT,PI3 K specific inhibitor) and GF~(-1)09203X( GFX,PKC specific inhibitor)( 100 μmol·L~(-1)of each,total volume of 10 μL) into the right lateral ventricle of the rats. The blank group was only injected 2% DMSO.The spatial memory retention was detected by water maze 24 hours after lateral ventricle injection. Then,changes in the spatial learning memory of rats were observed. The level of Tau phosphorylation in SD rat hippocampus and the expression and activity changes of related protein kinase GSK-3β were detected by Western blot and immunohistochemistry. The changes of Nissl bodies in SD rat hippocampus were observed by Nissl's staining. Results After intragastric administration of BCP,the rat autonomous behavior activities in each group all showed a declining trend, and the differences in low-dose and middle-dose groups had statistical significance compared with blank group. The Morris water maze tests showed that the latency navigation of model group was significantly longer than that of blank group( P < 0. 01),while that of the BCP three doses groups was shorter than that of model group( P <0. 05). Compared with the same group,the latency navigation of the three groups after gavage BCP low,middle and high dose was significant shorter than that without gavage( P < 0. 05). Western blot results showed that the activity of GSK-3β in AD model group was up-regulated compared with the blank group.However,BCP inhibited activity of GSK-3β. Western blot and immunohistochemistry results showed the level of Tau phosphorylation in AD model group was increased compared with the blank group in the area of CA3( P < 0. 05). Compared with AD model group,the level of Tau phosphorylation was decreased in treatment group. Nissl' s staining results showed that dendritic spines in AD model group was significantly attenuated compared with the blank group( P < 0. 05). Far more dendritic spines were observed in treatment group than in AD model group. The number of Nissl' s bodies in neuron cells of hippocampus in hippocampal CA3 was obviously larger in treatment groups than in AD model group. These effect of BCP was dose-dependent. Conclusions BCP can prevent the learning and memory dysfunction in AD model rats induced by high activity of GSK-3β. The mechanism may be related to inhibiting GSK-3β activity and then reducing the level of phosphorylation of Tau and improving neural development.
Aim To assess the effects of Banqiao Codonopisis Pilosula( BCP) decoction on learning and memory dysfunction in AD model rats induced by high activity GSK-3β and its possible mechanism. Methods The SD rats( 4 months old,♂) were divided into five groups,namely,sham-operated group( blank group),AD model group,BCP high-dose( 2. 16 g · kg~(-1)·d~(-1)) group,BCP medium-dose( 1. 08 g·kg~(-1)·d~(-1))group,and BCP lower-dose( 0. 54 g · kg~(-1)· d~(-1))group. Treatment group received BCP decoction by gavage once a day for 14 days,while other groups were offered drinking water by gavage once a day for 14 days. The autonomous behavior activities of all rats were observed and recorded after gavage. In the last seven days by gavage,Morris water maze test was used to test the spatial learning and memory ability of the five groups. After five days training,treatment groups and AD model group were injected wortmannin( WT,PI3 K specific inhibitor) and GF~(-1)09203X( GFX,PKC specific inhibitor)( 100 μmol·L~(-1)of each,total volume of 10 μL) into the right lateral ventricle of the rats. The blank group was only injected 2% DMSO.The spatial memory retention was detected by water maze 24 hours after lateral ventricle injection. Then,changes in the spatial learning memory of rats were observed. The level of Tau phosphorylation in SD rat hippocampus and the expression and activity changes of related protein kinase GSK-3β were detected by Western blot and immunohistochemistry. The changes of Nissl bodies in SD rat hippocampus were observed by Nissl's staining. Results After intragastric administration of BCP,the rat autonomous behavior activities in each group all showed a declining trend, and the differences in low-dose and middle-dose groups had statistical significance compared with blank group. The Morris water maze tests showed that the latency navigation of model group was significantly longer than that of blank group( P < 0. 01),while that of the BCP three doses groups was shorter than that of model group( P <0. 05). Compared with the same group,the latency navigation of the three groups after gavage BCP low,middle and high dose was significant shorter than that without gavage( P < 0. 05). Western blot results showed that the activity of GSK-3β in AD model group was up-regulated compared with the blank group.However,BCP inhibited activity of GSK-3β. Western blot and immunohistochemistry results showed the level of Tau phosphorylation in AD model group was increased compared with the blank group in the area of CA3( P < 0. 05). Compared with AD model group,the level of Tau phosphorylation was decreased in treatment group. Nissl' s staining results showed that dendritic spines in AD model group was significantly attenuated compared with the blank group( P < 0. 05). Far more dendritic spines were observed in treatment group than in AD model group. The number of Nissl' s bodies in neuron cells of hippocampus in hippocampal CA3 was obviously larger in treatment groups than in AD model group. These effect of BCP was dose-dependent. Conclusions BCP can prevent the learning and memory dysfunction in AD model rats induced by high activity of GSK-3β. The mechanism may be related to inhibiting GSK-3β activity and then reducing the level of phosphorylation of Tau and improving neural development.
引文
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[14]Maqbool M,Mobashir M,Hoda N.Pivotal role of glycogen synthase kinase-3:a therapeutic target for Alzheimer's disease[J].Eur J Med Chem,2016,107(7):63-81.
[15]Shim S S,Stutzmann G E.Inhibition of glycogen synthase kinase-3:an emerging target in the treatment of traumatic brain injury[J].J Neurotrauma,2016,33(23):2065-76.
[16]陈地灵,陈鹏,林励,等.巴戟天低聚糖对Aβ25-35致拟痴呆模型大鼠学习记忆障碍的影响[J].中国药理学通报,2013,29(2):271-6.Chen D L,Chen P,Lin L,et al.Effect of oligosaccharides from Morinda Officinalis onβ-amyloid-induced learning and memory dysfunction in rats[J].Chin Pharmacol Bull,2013,29(2):271-6.
[17]李林,张兰.中药治疗阿尔茨海默病的作用特点[J].生物化学与生物物理进展,2012,39(8):816-28.Li L,Zhang L.Action characteristics of traditional Chinese medicine in treatment of Alzheimer's disease[J].Prog Biochem Biophys,2012,39(8):816-28.
[18]Medina M,Avila J.New insights into the role of glycogen synthase kinase-3 in Alzheimer's disease[J].Expert Opin Ther Targets,2014,18(1):69-77.
[19]Ma T.GSK3 in Alzheimer's disease:mind the isoforms[J].J Alzheimers Dis,2014,39(4):707-10.
[20]King M K,Pardo M,Cheng Y,et al.Glycogen synthase kinase-3inhibitors:rescuers of cognitive impairments[J].Pharmacol Ther,2014,141(1):1-12.
[21]张雄,李昱.姜黄素通过抑制GSK-3β的活性防治AD的体外研究[J].中国药理学通报,2009,25(11):1507-12.Zhang X,Li Y.Curcum in inhibits the activity of GSK-3βto prevent AD in vitro[J].Chin Pharmacol Bull,2009,25(11):1507-12.
[22]Alonso Adel C,Mederlyova A,Novak M,et al.Promotion of hyperphosphorylation by frontotemporal dementia Tau mutation[J].J Biol Chem,2004,279(33):34878-81.
[2]Sagare A P,Bell R D,Zlokovic B V.Neurovascular defects and faulty amyloid-βvascular clearance in Alzheimer's disease[J].J Alzheimers Dis,2013,33(Suppl 1):S87-100.
[3]Wang J Z,Liu F.Microtubule-associated protein tau in development,degeneration and protection of neurons[J].Prog Neurobiol,2008,85(2):148-75.
[4]Mandelkow E M,Mandelkow E.Biochemistry and cell biology of tau protein in neurofibrillary degeneration[J].Cold Spring Harb Perspect Med,2012,2(7):a006247.
[5]Luo H B,Xia Y Y,Shu X J,et al.SUMOylation at K340 inhibits tau degradation through deregulating its phosphorylation and ubiquitination[J].Proc Natl Acad Sci USA,2014,111(46):16586-91.
[6]Bukar M M,Al-Hilaly Y K,Serpell L C.Nuclear tau and its potential role in Alzheimer's disease[J].Biomolecules,2016,6(1):9.
[7]Khan S S,Bloom G S.Tau:the center of a signaling nexus in Alzheimer's disease[J].Front Neurosci,2016,10(1):31.
[8]Wang J,Wang X,Liu R,et al.In vitro analysis of tau phosphorylation sites and its biological activity[J].Chin Med Sci J,2002,17(1):13-6.
[9]Liu S J,Zhang A H,Li H L,et al.Overactivation of glycogen synthase kinase-3 by inhibition of phosphoinositol-3 kinase and protein kinase C leads to hyperphosphorylation of tau and impairment of spatial memory[J].J Neurochem,2003,87(6):1333-44.
[10]黄丽亚,叶嗣颖.党参注射液上调抗氧化酶表达作用的实验研究[J].中国老年学杂志,2006,26(1):70-1.Huang L Y,Ye S Y.Study on the effect of Codonopsis pilosula injection increase the expression of antioxidant enzymes[J].Chin J Gerontol,2006,26(1):70-1.
[11]朱永红,吕盼,欧晓群.板桥党参的研究现状[J].亚太传统医药,2012,8(3):189-90.Zhu Y H,Lyu P,Ou X Q.Research status of Banqiao Codonopisis Pilosula[J].Asia-Pac Tradit Med,2012,8(3):189-90.
[12]姜霞.L-肉毒碱在Alzheimer样Tau蛋白过度磷酸化和空间记忆障碍中的保护作用[D].武汉:华中科技大学,2008.Jiang X.Effect of L-Carnitine on Alzheimer-like tau hyperphosphorylation and spatial memory retention deficits[D].Wuhan:Huazhong University of Science and Technology,2008.
[13]姜霞.空间学习对AD模型鼠海马突触可塑性及神经发生的影响[D].武汉:华中科技大学,2012.Jiang X.Roles of spatial learning in synaptic plasticity and neurogenesis in Alzheimer's disease models[D].Wuhan:Huazhong University of Science and Technology,2012.
[14]Maqbool M,Mobashir M,Hoda N.Pivotal role of glycogen synthase kinase-3:a therapeutic target for Alzheimer's disease[J].Eur J Med Chem,2016,107(7):63-81.
[15]Shim S S,Stutzmann G E.Inhibition of glycogen synthase kinase-3:an emerging target in the treatment of traumatic brain injury[J].J Neurotrauma,2016,33(23):2065-76.
[16]陈地灵,陈鹏,林励,等.巴戟天低聚糖对Aβ25-35致拟痴呆模型大鼠学习记忆障碍的影响[J].中国药理学通报,2013,29(2):271-6.Chen D L,Chen P,Lin L,et al.Effect of oligosaccharides from Morinda Officinalis onβ-amyloid-induced learning and memory dysfunction in rats[J].Chin Pharmacol Bull,2013,29(2):271-6.
[17]李林,张兰.中药治疗阿尔茨海默病的作用特点[J].生物化学与生物物理进展,2012,39(8):816-28.Li L,Zhang L.Action characteristics of traditional Chinese medicine in treatment of Alzheimer's disease[J].Prog Biochem Biophys,2012,39(8):816-28.
[18]Medina M,Avila J.New insights into the role of glycogen synthase kinase-3 in Alzheimer's disease[J].Expert Opin Ther Targets,2014,18(1):69-77.
[19]Ma T.GSK3 in Alzheimer's disease:mind the isoforms[J].J Alzheimers Dis,2014,39(4):707-10.
[20]King M K,Pardo M,Cheng Y,et al.Glycogen synthase kinase-3inhibitors:rescuers of cognitive impairments[J].Pharmacol Ther,2014,141(1):1-12.
[21]张雄,李昱.姜黄素通过抑制GSK-3β的活性防治AD的体外研究[J].中国药理学通报,2009,25(11):1507-12.Zhang X,Li Y.Curcum in inhibits the activity of GSK-3βto prevent AD in vitro[J].Chin Pharmacol Bull,2009,25(11):1507-12.
[22]Alonso Adel C,Mederlyova A,Novak M,et al.Promotion of hyperphosphorylation by frontotemporal dementia Tau mutation[J].J Biol Chem,2004,279(33):34878-81.