地塞米松促进β-淀粉样蛋白引起大鼠学习记忆能力下降及机理研究
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摘要
阿尔茨海默病(Alzheimer's disease, AD)是一种中枢神经系统退行性疾病,其特征性神经病理变化是老年斑、神经纤维缠结和神经元丢失等,并以海马受累最严重。其病因尚不完全清楚,缺乏有效的治疗措施。因此,积极研究AD的发病机制是临床和基础医学面临的重要课题。β-淀粉样蛋白(Amyloidβ-peptide protein, Aβ)是AD形成和发展的关键因素,Aβ由39～42个氨基酸残基组成,在其多肽序列中,Aβ25-35片断的神经毒性较强。目前研究已证实:Aβ25-35能通过氧化应急增加细胞内Ca2+浓度、影响细胞内信号传导、抑制神经轴突的快速转运机制导致神经元损伤。海马是学习记忆中非常重要的区域,富含大量的糖皮质激素受体(glucocoricoids receptor, GR),是糖皮质激素(glucocorticoids, GCs)作用的主要靶位。研究发现,当人或动物处于强烈的急性应激或长期慢性应激状态时,其学习记忆能力可受到明显的影响。GCs是机体的一类正常激素,具有多种生理和药理作用,近几年的研究结果显示,体内高水平的GCs可以引起海马突触丢失,海马萎缩,加重海马齿状回神经元凋亡。目前,国内外都注重研究Aβ、GCs分别与AD发病之间的关系,但将Aβ与GCs相关联,研究它们之间的相互促进海马神经元细胞毒作用却很少。为此,本研究拟(1)在体内,研究地塞米松(Dexamethasone, DEX)促进Aβ引起大鼠学习记忆能力下降和海马神经元损伤及其可能作用机制;(2)在体外培养大鼠海马神经元的基础上,进一步观察DEX促进Aβ引起海马神经元损伤,研究其可能的作用机制。为探讨AD发病机理提供理论依据。
目的在体内外实验条件下,观察DEX、Aβ及其联合作用对海马神经元的影响,以探讨GCs是否加重Aβ的神经元毒性作用以及机理研究。方法利用大鼠海马CA1区注射Aβ25-35的方法建立大鼠早老性痴呆模型,运用Morris水迷宫和病理组织检查,研究DEX(1 mg/kg/d,5 mg/kg/d)和Aβ(5μg)以及联合作用对大鼠学习记忆能力和海马组织病理的影响。运用免疫组化的方法,检测DEX、Aβ以及DEX+Aβ对p-tau表达的影响。取孕18天Sprague Dawley(SD)大鼠胚胎的海马神经元进行体外原代分离培养,于联合实验中先加入10μM DEX作用24h后,再加入5μM Aβ25-35作用24h观察损伤情况。应用MTT法进行细胞活力分析;用western blot分析测定核NF-κB(Nuclear Factor kappa B)、p53和p-tau蛋白含量,用RT-PCR分析测定p53mRNA的水平,进一步探讨DEX是否通过下调核NF-κB蛋白水平,上调p53和p-tau蛋白水平促进Aβ的神经元毒性。
结果
1.大鼠海马CA1区注射Aβ25-35可诱导大鼠早老性痴呆模型,损伤大鼠的学习记忆能力。大鼠双侧海马CA1区注射5μg Aβ25-35或sc , DEX( 5 mg/kg/d,连续7 d),可使大鼠逃避潜伏期和游泳距离稍延长,但无统计学意义;1 mg/kg/d DEX,sc,连续7 d对大鼠逃避潜伏期和游泳距离无明显影响;DEX和Aβ25-35联合使用后d9,大鼠逃避潜伏期及游泳距离明显延长,并有部分动物死亡。病理检验结果显示, 5 mg/kg/d DEX+ 5μg Aβ25-35组CA1区神经元数目明显减少,排列紊乱,脱失现象明显,核固缩为三角形或多角形,浓染。
2.免疫组化结果显示:溶剂对照组大鼠海马CA1区仅检测到少量tau阳性细胞。DEX(1 mg/kg)组p-tau阳性细胞数量稍增多(p>0.05)。DEX(5 mg/kg /d)、Aβ组大鼠海马神经元中p-tau阳性细胞数量明显增加,与溶剂对照组相比差异有显著性(p<0.05)。与Aβ组比较, DEX(1 mg/kg或5 mg/kg /d )+Aβ组p-tau阳性细胞数量明显增多(p<0.05)。
3.细胞活力检测发现(MTT法),不同浓度DEX(0.01, 0.1, 1, 10μM)对海马神经元活性没有影响(p>0.05), Aβ25-35剂量依赖性地降低海马神经元细胞活力(p<0.05)。DEX(1μM或10μM)预处理海马神经元24 h,可进一步降低Aβ25-35(1μM或5μM)引起的细胞活力下降(p<0.05)
4.在原代培养的海马神经元中加入5μM Aβ25-35后,分别于1 h,2 h,4 h,8 h提取胞浆蛋白和核蛋白,通过western blot分析结果显示:Aβ25-35能时间依赖性的引起核NF-κB p65蛋白水平增加,作用4 h后达到高峰,8 h后略有下降,但仍高于正常。与对照组相比,Aβ25-35组的核NF-κB表达量明显增加(P<0.05),DEX组、DEX+Aβ25-35组变化不明显(p>0.05)。与Aβ组相比,DEX组可下调Aβ25-35引起的上升的海马神经元核NF-κB p65蛋白水平(p<0.05);此外我们进一步研究了DEX与Aβ25-35联合作用对海马神经元胞质IκBα蛋白表达的影响。结果显示,与对照组相比,10μM DEX作用海马神经元时,胞浆IκBα表达量增加(p<0.05),Aβ25-35组表达量降低(P<0.05),DEX+Aβ25-35组变化不明显(p>0.05)。与Aβ组相比,DEX可上调细胞质的IκBα蛋白水平(p<0.05)。
5.与对照组相比,10μM DEX作用海马神经元时,p53蛋白水平差异无统计学意义(p>0.05);5μM Aβ组差异有显著性(p<0.05),但与同剂量的Aβ组相比,DEX+Aβ组的p53蛋白表达量变化不明显(p>0.05)。
6.与对照组(0μM Aβ)相比,0.5, 1, 5μM的Aβ作用海马神经元1 h,p-tau表达量明显增加,10μM Aβ作用组,p-tau表达量达到峰值。此外,10μM DEX、5μM Aβ作用海马神经元,其p-tau表达量也明显增加(p<0.05),DEX+Aβ组的p-tau表达量与同剂量的DEX和Aβ相比,差异有显著性(p<0.05)。
7. RT-PCR结果显示: Aβ、DEX+Aβ促进p53 mRNA的水平增加(p<0.05)。DEX+Aβ组的p53 mRNA水平与同剂量的Aβ相比,差异无统计学意义(p>0.05)。
结论
在体内,DEX可加重Aβ引起大鼠学习记忆能力下降,导致大鼠海马CA1区神经元数目明显减少、排列紊乱、脱失现象明显、核固缩为三角形或多角形、浓染,此外,免疫组化结果显示thr231位点磷酸化tau阳性细胞表达明显增加。以上结果提示:海马神经元损伤,学习记忆能力下降可能与神经细胞减少,tau蛋白异常磷酸化增多有关;在体外,DEX下调了Aβ25-35引起的上升的海马神经元核NF-κB蛋白水平,上调了细胞质中的IκBα蛋白水平,上调了Aβ25-35引起的上升的thr231位点磷酸化tau水平。从而提示DEX可能通过促进神经元胞内p-tau蛋白水平的升高、上调IκB蛋白水平或/和抑制核NF-κB蛋白水平来加重神经元毒性作用。此外,DEX+Aβ组的p53蛋白表达和p53 mRNA水平与同剂量的Aβ相比,差异无统计学意义,提示GCs促进Aβ引起海马神经元可能不通过p53途径。因脑内的Aβ随年龄不断增加,糖皮质激素增强Aβ的神经元毒作用可能是GCs诱导老年鼠记忆损伤的机制之一,也可能与AD病因学有关。
Alzheimer's disease(AD) is the major neurodegenerative disorder of the elderly. The main pathological hallmarks of AD is extracellular accumulation of selective insults of neuronal synapsis, neurofibrillary tangle(NFT) and senile plaques (SP) in vulnerable brain regions, composed primarily of aggregatedβ-amyloid peptide(Aβ). The hippocampus, among other areas, is the principal target tissue in the brain for the hormone. The etiology of the more common sporadic cases remains unknown, and up to now, there is no reliable methods to prevent and treat the disease. It is generally considered that Aβplays a pivotal role in the pathogenesis of AD. Aβis a heterogeneous 39-42-amino acid peptide. The hippocampus is closely related to memory and learning impairment which involved much glucocoricoids receptor(GR) , and it is the principal target tissue of which glucocorticoids(GCs) affect. GCs are important adrenal steroids that affect numerous physiological processes in the brain, and widely used in medicine. Previous studies showed that increased plasma levels of DEX can potentiate the loss of hippocampal synapsis and aggravate the apotosis of the dentate gyrus,accompanied with atrophy of hippocampus. Many studies in the world are focusing on the relationship between the GCs, Aβand pathogenesis of AD, but whether GCs could enhance Aβ-inducing hippocampal neuronal injury is not well determined. Therefore, in this study we investigate whether Dexamethasone(DEX) could potentiate Aβ-induced learning and memory impairment and its relative mechanism in SD rats in vivo. On the other hand, we also examine whether DEX could enhance Aβ-induced cell death in hippocampal neurons in vitro, and, if so, what is the underlying mechanism.
Objective The objectives of this experiments were to explore the mechanisms responsible for dexamethasone enhanced Aβ-induced cell death in hippocampal neurons.
Methods Aβ25–35 was injected into the CA1 field of hippocampus to establish the Alzheimer’s Disease rat model. Morris water maze test was used to investigate whether DEX(1 mg/kg/d,5 mg/kg/d) could potentiate Aβ(5μg, each CA1)-induced learning and memory impairment in SD rats in vivo, and the histopathologic changes in CA1 field of hippocampus was examined under a light microscope. The effect of DEX on Aβ25-35-induced phospho-tau was evalued by immunohistechemical. Primitive hippocampal neurons derived from 18 day embryonic rat were cultured. Cultured cells were treated with DEX alone at 10μM for 48 h or Aβ25-35 alone at 5μM for 24 h, or cultured cells were pretreated with DEX at 10μM for 24 h followed by Aβ25-35 at 5μM for various time, and then the cell viability was measured by colorimetric MTT assay, p53mRNA was assessed using RT-PCR, and the effect of DEX on Aβ25-35-induced nuclear factorκB (NF-κB), phospho-tau and p53 protein was evalued by western blot.
Results
1. Microinjection of Aβ25-35(5μg, each CA1) bilateralis into the CA1 region of SD rats and DEX(5 mg/kg/d, sc×7d)could slightly increase the escape latency and the swim distances in SD rats during training session in the Morris water maze test, but there was no statistic significant compared with vehicle-treated control. DEX(1 mg/kg/d, sc, 7d) did not decreased the escape latency and the swim distances in SD rats during training session in the Morris water maze test, but DEX(1 mg/kg/d ,5 mg/kg/d, sc×7d) could potentiate Aβ(5μg, each CA1)-induced learning and memory impairment in SD rats. Severe histological damage was observed in the CA1 cell fields of the hippocampus in DEX plus Aβ-treated group. These neuropathological changes were characterized by decreased cell number, soma shrinkage and condensation, or nuclear pyknosis.
2. Immunohistechemical results showed that a significant increase of tau(231) positive cells were also detected in DEX(5 mg/kg/d) as well as Aβ、DEX plus Aβtreated group in the hippocampal tissue of rats. The expressions of p-tau in DEX plus Aβtreated group is increased compared with DEX or Aβalone treated group.
3. Treatment for 48 h with DEX(0.01、0.1、1、10μM) alone did not cause a significant reduction in MTT compared with vehicle-treated control cultures. Treatment with aggregated Aβ25-35 alone decreased cell viability in a concentration-dependent manner. A 24-h preincubation with DEX(1 or 10μM)further decreased the viability of hippocampal neuron induced by Aβ25-35(1 or 5μM).
4. Aβ25-35(5μM) causes a time-dependent increase in the level of nuclear NF-κB p65 proteins, whereas maximal NF-κB p65 was obtained with Aβ25-35 at 4 h. A 24 h preincubation with DEX(10μM) could down-regulate the elevated level of nuclear NF-κB p65 proteins induced by Aβ25-35. Aβ25-35(5μM) alone could decrease the cytoplasmic level of IκBαprotein 24 h after Aβ25-35 was added to the culture. Increased levels of cytoplasmic level of IκBαprotein were observed in hippocampal neurons 24h after incubation with DEX(10μM).
5. Aβ25-35 (5μM) treatment resulted in significant increases in the level of total protein of p53 24 h after Aβwas added to the culture. Treatment with DEX(10μM) alone for 48 h did not increase the levels of total protein of p53. Pretreatment with DEX for 24 h didn’t promote the increased total protein of p53 induced by Aβ25-35..
6. Aβ25-35(0-20μM) dose-dependently induced phosphorylation of tau at Thr-231 in primary hippocampal neurons cocultured for 1 h with Aβ25-35, whereas maximal phosphorylation was obtained with Aβ25-35 at 10μM. Treatment of neurons with DEX in primary culture for 24 h could results in an slight elevation in tau phosphorylation at Thr-231, and pretreated of neuron with DEX at 10μM for 24 h could promote the increased level of phospho-tau at Thr-231 induced by Aβ25-35(5μM).
7. Aβ25-35(5μM) treatment resulted in small but significant increases in the level of total protein protein of p53mRNA 18 h after Aβwas added to the culture. Treatment with DEX(10μM) alone did not increase the levels of total protein of p53mRNA. Pretreatment with DEX for 24 h didn’t promote the increased total protein of p53mRNA induced by Aβ25-35 as well..
Conclusions
In vivo, DEX could potentiate Aβ-induced learning and memory impairment in SD rats. These neuropathological changes were characterized by decreased cell number, soma shrinkage and condensation, or nuclear pyknosis. These results suggested that DEX could enhance Aβ-induced cell death in hippocampal neurons and dysfunction of learing and memory which may involve the decrease of the numbers of neurons and increase of abnormal phosphorylation of tau. In vitro, DEX could potentiate the neurotoxic action of Aβmediated by further increasing the level of phospho-tau at Thr-231, down-regulating the level of nuclear NF-κB protein. Pretreatment of hippocampal neurons with DEX did not influnce p53 proteins level induced by Aβ25-35, which suggested that DEX could enhance Aβ-induced cell death in hippocampal neurons was not possibly carried out by this means. Since Aβand GCs increases in the brain with aging. GCs potentiate the neurotoxic action of Aβmaybe one of the mechanisms responsible for AD.
目的在体内外实验条件下,观察DEX、Aβ及其联合作用对海马神经元的影响,以探讨GCs是否加重Aβ的神经元毒性作用以及机理研究。方法利用大鼠海马CA1区注射Aβ25-35的方法建立大鼠早老性痴呆模型,运用Morris水迷宫和病理组织检查,研究DEX(1 mg/kg/d,5 mg/kg/d)和Aβ(5μg)以及联合作用对大鼠学习记忆能力和海马组织病理的影响。运用免疫组化的方法,检测DEX、Aβ以及DEX+Aβ对p-tau表达的影响。取孕18天Sprague Dawley(SD)大鼠胚胎的海马神经元进行体外原代分离培养,于联合实验中先加入10μM DEX作用24h后,再加入5μM Aβ25-35作用24h观察损伤情况。应用MTT法进行细胞活力分析;用western blot分析测定核NF-κB(Nuclear Factor kappa B)、p53和p-tau蛋白含量,用RT-PCR分析测定p53mRNA的水平,进一步探讨DEX是否通过下调核NF-κB蛋白水平,上调p53和p-tau蛋白水平促进Aβ的神经元毒性。
结果
1.大鼠海马CA1区注射Aβ25-35可诱导大鼠早老性痴呆模型,损伤大鼠的学习记忆能力。大鼠双侧海马CA1区注射5μg Aβ25-35或sc , DEX( 5 mg/kg/d,连续7 d),可使大鼠逃避潜伏期和游泳距离稍延长,但无统计学意义;1 mg/kg/d DEX,sc,连续7 d对大鼠逃避潜伏期和游泳距离无明显影响;DEX和Aβ25-35联合使用后d9,大鼠逃避潜伏期及游泳距离明显延长,并有部分动物死亡。病理检验结果显示, 5 mg/kg/d DEX+ 5μg Aβ25-35组CA1区神经元数目明显减少,排列紊乱,脱失现象明显,核固缩为三角形或多角形,浓染。
2.免疫组化结果显示:溶剂对照组大鼠海马CA1区仅检测到少量tau阳性细胞。DEX(1 mg/kg)组p-tau阳性细胞数量稍增多(p>0.05)。DEX(5 mg/kg /d)、Aβ组大鼠海马神经元中p-tau阳性细胞数量明显增加,与溶剂对照组相比差异有显著性(p<0.05)。与Aβ组比较, DEX(1 mg/kg或5 mg/kg /d )+Aβ组p-tau阳性细胞数量明显增多(p<0.05)。
3.细胞活力检测发现(MTT法),不同浓度DEX(0.01, 0.1, 1, 10μM)对海马神经元活性没有影响(p>0.05), Aβ25-35剂量依赖性地降低海马神经元细胞活力(p<0.05)。DEX(1μM或10μM)预处理海马神经元24 h,可进一步降低Aβ25-35(1μM或5μM)引起的细胞活力下降(p<0.05)
4.在原代培养的海马神经元中加入5μM Aβ25-35后,分别于1 h,2 h,4 h,8 h提取胞浆蛋白和核蛋白,通过western blot分析结果显示:Aβ25-35能时间依赖性的引起核NF-κB p65蛋白水平增加,作用4 h后达到高峰,8 h后略有下降,但仍高于正常。与对照组相比,Aβ25-35组的核NF-κB表达量明显增加(P<0.05),DEX组、DEX+Aβ25-35组变化不明显(p>0.05)。与Aβ组相比,DEX组可下调Aβ25-35引起的上升的海马神经元核NF-κB p65蛋白水平(p<0.05);此外我们进一步研究了DEX与Aβ25-35联合作用对海马神经元胞质IκBα蛋白表达的影响。结果显示,与对照组相比,10μM DEX作用海马神经元时,胞浆IκBα表达量增加(p<0.05),Aβ25-35组表达量降低(P<0.05),DEX+Aβ25-35组变化不明显(p>0.05)。与Aβ组相比,DEX可上调细胞质的IκBα蛋白水平(p<0.05)。
5.与对照组相比,10μM DEX作用海马神经元时,p53蛋白水平差异无统计学意义(p>0.05);5μM Aβ组差异有显著性(p<0.05),但与同剂量的Aβ组相比,DEX+Aβ组的p53蛋白表达量变化不明显(p>0.05)。
6.与对照组(0μM Aβ)相比,0.5, 1, 5μM的Aβ作用海马神经元1 h,p-tau表达量明显增加,10μM Aβ作用组,p-tau表达量达到峰值。此外,10μM DEX、5μM Aβ作用海马神经元,其p-tau表达量也明显增加(p<0.05),DEX+Aβ组的p-tau表达量与同剂量的DEX和Aβ相比,差异有显著性(p<0.05)。
7. RT-PCR结果显示: Aβ、DEX+Aβ促进p53 mRNA的水平增加(p<0.05)。DEX+Aβ组的p53 mRNA水平与同剂量的Aβ相比,差异无统计学意义(p>0.05)。
结论
在体内,DEX可加重Aβ引起大鼠学习记忆能力下降,导致大鼠海马CA1区神经元数目明显减少、排列紊乱、脱失现象明显、核固缩为三角形或多角形、浓染,此外,免疫组化结果显示thr231位点磷酸化tau阳性细胞表达明显增加。以上结果提示:海马神经元损伤,学习记忆能力下降可能与神经细胞减少,tau蛋白异常磷酸化增多有关;在体外,DEX下调了Aβ25-35引起的上升的海马神经元核NF-κB蛋白水平,上调了细胞质中的IκBα蛋白水平,上调了Aβ25-35引起的上升的thr231位点磷酸化tau水平。从而提示DEX可能通过促进神经元胞内p-tau蛋白水平的升高、上调IκB蛋白水平或/和抑制核NF-κB蛋白水平来加重神经元毒性作用。此外,DEX+Aβ组的p53蛋白表达和p53 mRNA水平与同剂量的Aβ相比,差异无统计学意义,提示GCs促进Aβ引起海马神经元可能不通过p53途径。因脑内的Aβ随年龄不断增加,糖皮质激素增强Aβ的神经元毒作用可能是GCs诱导老年鼠记忆损伤的机制之一,也可能与AD病因学有关。
Alzheimer's disease(AD) is the major neurodegenerative disorder of the elderly. The main pathological hallmarks of AD is extracellular accumulation of selective insults of neuronal synapsis, neurofibrillary tangle(NFT) and senile plaques (SP) in vulnerable brain regions, composed primarily of aggregatedβ-amyloid peptide(Aβ). The hippocampus, among other areas, is the principal target tissue in the brain for the hormone. The etiology of the more common sporadic cases remains unknown, and up to now, there is no reliable methods to prevent and treat the disease. It is generally considered that Aβplays a pivotal role in the pathogenesis of AD. Aβis a heterogeneous 39-42-amino acid peptide. The hippocampus is closely related to memory and learning impairment which involved much glucocoricoids receptor(GR) , and it is the principal target tissue of which glucocorticoids(GCs) affect. GCs are important adrenal steroids that affect numerous physiological processes in the brain, and widely used in medicine. Previous studies showed that increased plasma levels of DEX can potentiate the loss of hippocampal synapsis and aggravate the apotosis of the dentate gyrus,accompanied with atrophy of hippocampus. Many studies in the world are focusing on the relationship between the GCs, Aβand pathogenesis of AD, but whether GCs could enhance Aβ-inducing hippocampal neuronal injury is not well determined. Therefore, in this study we investigate whether Dexamethasone(DEX) could potentiate Aβ-induced learning and memory impairment and its relative mechanism in SD rats in vivo. On the other hand, we also examine whether DEX could enhance Aβ-induced cell death in hippocampal neurons in vitro, and, if so, what is the underlying mechanism.
Objective The objectives of this experiments were to explore the mechanisms responsible for dexamethasone enhanced Aβ-induced cell death in hippocampal neurons.
Methods Aβ25–35 was injected into the CA1 field of hippocampus to establish the Alzheimer’s Disease rat model. Morris water maze test was used to investigate whether DEX(1 mg/kg/d,5 mg/kg/d) could potentiate Aβ(5μg, each CA1)-induced learning and memory impairment in SD rats in vivo, and the histopathologic changes in CA1 field of hippocampus was examined under a light microscope. The effect of DEX on Aβ25-35-induced phospho-tau was evalued by immunohistechemical. Primitive hippocampal neurons derived from 18 day embryonic rat were cultured. Cultured cells were treated with DEX alone at 10μM for 48 h or Aβ25-35 alone at 5μM for 24 h, or cultured cells were pretreated with DEX at 10μM for 24 h followed by Aβ25-35 at 5μM for various time, and then the cell viability was measured by colorimetric MTT assay, p53mRNA was assessed using RT-PCR, and the effect of DEX on Aβ25-35-induced nuclear factorκB (NF-κB), phospho-tau and p53 protein was evalued by western blot.
Results
1. Microinjection of Aβ25-35(5μg, each CA1) bilateralis into the CA1 region of SD rats and DEX(5 mg/kg/d, sc×7d)could slightly increase the escape latency and the swim distances in SD rats during training session in the Morris water maze test, but there was no statistic significant compared with vehicle-treated control. DEX(1 mg/kg/d, sc, 7d) did not decreased the escape latency and the swim distances in SD rats during training session in the Morris water maze test, but DEX(1 mg/kg/d ,5 mg/kg/d, sc×7d) could potentiate Aβ(5μg, each CA1)-induced learning and memory impairment in SD rats. Severe histological damage was observed in the CA1 cell fields of the hippocampus in DEX plus Aβ-treated group. These neuropathological changes were characterized by decreased cell number, soma shrinkage and condensation, or nuclear pyknosis.
2. Immunohistechemical results showed that a significant increase of tau(231) positive cells were also detected in DEX(5 mg/kg/d) as well as Aβ、DEX plus Aβtreated group in the hippocampal tissue of rats. The expressions of p-tau in DEX plus Aβtreated group is increased compared with DEX or Aβalone treated group.
3. Treatment for 48 h with DEX(0.01、0.1、1、10μM) alone did not cause a significant reduction in MTT compared with vehicle-treated control cultures. Treatment with aggregated Aβ25-35 alone decreased cell viability in a concentration-dependent manner. A 24-h preincubation with DEX(1 or 10μM)further decreased the viability of hippocampal neuron induced by Aβ25-35(1 or 5μM).
4. Aβ25-35(5μM) causes a time-dependent increase in the level of nuclear NF-κB p65 proteins, whereas maximal NF-κB p65 was obtained with Aβ25-35 at 4 h. A 24 h preincubation with DEX(10μM) could down-regulate the elevated level of nuclear NF-κB p65 proteins induced by Aβ25-35. Aβ25-35(5μM) alone could decrease the cytoplasmic level of IκBαprotein 24 h after Aβ25-35 was added to the culture. Increased levels of cytoplasmic level of IκBαprotein were observed in hippocampal neurons 24h after incubation with DEX(10μM).
5. Aβ25-35 (5μM) treatment resulted in significant increases in the level of total protein of p53 24 h after Aβwas added to the culture. Treatment with DEX(10μM) alone for 48 h did not increase the levels of total protein of p53. Pretreatment with DEX for 24 h didn’t promote the increased total protein of p53 induced by Aβ25-35..
6. Aβ25-35(0-20μM) dose-dependently induced phosphorylation of tau at Thr-231 in primary hippocampal neurons cocultured for 1 h with Aβ25-35, whereas maximal phosphorylation was obtained with Aβ25-35 at 10μM. Treatment of neurons with DEX in primary culture for 24 h could results in an slight elevation in tau phosphorylation at Thr-231, and pretreated of neuron with DEX at 10μM for 24 h could promote the increased level of phospho-tau at Thr-231 induced by Aβ25-35(5μM).
7. Aβ25-35(5μM) treatment resulted in small but significant increases in the level of total protein protein of p53mRNA 18 h after Aβwas added to the culture. Treatment with DEX(10μM) alone did not increase the levels of total protein of p53mRNA. Pretreatment with DEX for 24 h didn’t promote the increased total protein of p53mRNA induced by Aβ25-35 as well..
Conclusions
In vivo, DEX could potentiate Aβ-induced learning and memory impairment in SD rats. These neuropathological changes were characterized by decreased cell number, soma shrinkage and condensation, or nuclear pyknosis. These results suggested that DEX could enhance Aβ-induced cell death in hippocampal neurons and dysfunction of learing and memory which may involve the decrease of the numbers of neurons and increase of abnormal phosphorylation of tau. In vitro, DEX could potentiate the neurotoxic action of Aβmediated by further increasing the level of phospho-tau at Thr-231, down-regulating the level of nuclear NF-κB protein. Pretreatment of hippocampal neurons with DEX did not influnce p53 proteins level induced by Aβ25-35, which suggested that DEX could enhance Aβ-induced cell death in hippocampal neurons was not possibly carried out by this means. Since Aβand GCs increases in the brain with aging. GCs potentiate the neurotoxic action of Aβmaybe one of the mechanisms responsible for AD.
引文
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