黄芪总苷对β-淀粉样蛋白和糖皮质激素协同诱导大鼠海马神经元损伤的保护作用及其机理的研究
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摘要
背景中枢神经系统退行性疾病( Progressive neurodegenerative disease)是一类发生在老年前期及老年期的慢性进行性中枢神经系统退行性疾病,严重威胁老龄人身体健康及生活质量,其中以阿尔茨海默病(Alzheimer's disease, AD)较为常见。AD又常被称为老年性痴呆,在临床上,AD患者的症状是以进行性的记忆减退、认知障碍、人格改变等为主要特征的综合症, WHO的调查报告结果表明,65岁以上老人的AD发病率约为5～20%,AD患者的平均存活时间只有5年;脑衰老是多层次,多重性的退行性变化,其病因尚未阐明,国内外也尚无良药来防治该类疾病。因而,研究AD的发病机理及防治方法受到社会的极大关注,也是目前神经药理学等研究的热点之一。
AD的主要病理学特征是在中枢神经系统的皮层、海马等处产生大量的β-淀粉样蛋白(β-Amyloid protein, Aβ),形成Aβ沉积、老年斑(Senile plaque, SP)和神经纤维缠结(Neurofibrilary tangle, NFT),其中Aβ是构成老年斑的主要成份。Aβ是由β-淀粉样前体蛋白(amyloid precursor protein, APP)经β、r-secretase酶裂解生成,Aβ是构成老年斑的主要成份;Aβ在AD患者的神经元中过度生成及其对神经元的细胞毒作用,在AD的发病和进展过程中起关键的作用。糖皮质激素(Glucocorticoids, GCs)是调节机体生长发育、新陈代谢和维持机体功能正常的一类重要激素,也是临床常用的抢救和治疗药物,GCs具有多种生理和药理作用,但长期大剂量应用GCs可对机体免疫和神经等系统造成严重的不良反应。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,仅检索到2篇相关论文。
近年来,国内外都投入了大量人力、物力从事防治中枢神经系统退行性疾病、AD等疾病及其发病机制的研究工作,我国中医中药具有悠久的防治衰老的历史和丰富的临床实践经验,目前,在国内,一些研究单位着重研究了传统延缓衰老的常用中药及其复方的疗效和部分作用机理,取得了一定的进展,但都还基本处于临床前研究阶段,已批准上市的防治AD中药还寥寥无几。黄芪是临床延缓衰老的名药,黄芪总苷(Astragalosides, AST)是从黄芪中提取的有效部位群,我们的前期研究表明, AST不仅具有抗炎、免疫调节和防治局灶性脑缺血和全脑缺血再灌注引起的脑损伤作用,还能明显改善D-半乳糖衰老小鼠和地塞米松(Dexamethasone, DEX)引起的老前期(20 mon)衰老模型小鼠的学习记忆功能,改善环磷酰胺降低的小鼠学习记忆功能;但AST对Aβ和GCs引起的海马神经元损伤是否有保护作用还未见报道。
由于Aβ和GCs分别在AD的发病中均起重要的作用,如Aβ可引起体外培养的海马神经元凋亡和活力下降,向大鼠海马CA1区注射Aβ可引大鼠学习记忆能力下降;小鼠在长期给予GCs时,在引起老前期小鼠海马神经元损伤的同时,还可引起学习记忆能力明显下降,但它们二者之间对神经元是否具有协同的细胞毒性作用仍然不清楚;此外,临床上仍无理想的防治AD的药物和方法。为进一步研究AD的发病机理和寻找有效的防治AD等神经系统退行性疾病的方法和药物,本文拟(1)在体内,研究DEX是否具有协同促进Aβ引起大鼠海马神经元损伤和学习记忆能力下降的作用,然后进一步研究其可能的作用机制。在体外,同样观察DEX协同促进Aβ引起海马神经元损伤,分析其可能的作用机理。(2)进一步探讨AST对A?和DEX诱导大鼠海马神经元损伤的保护作用以及可能的相关分子与基因水平的作用机制。
方法:
第一部分地塞米松协同促进Aβ对大鼠海马神经元毒性及其机理的研究在体内,利用Morris水迷宫和病理组织检查,研究DEX(1 mg/kg/d,sc×7 d)和Aβ(海马CA1区注射、5μg/侧、双侧、单次)联合作用对大鼠学习记忆能力和海马神经元损伤的影响。在体外,取孕第18天Sprague Dawley(SD)大鼠胚胎的海马神经元进行体外原代培养,用MTT(Methyl thiazolyl tetrazolium)方法观察DEX(0.01-10μM)和Aβ25-35(1-40μM)对海马神经元活力的影响,分别找出DEX和Aβ引起海马神经元损伤的亚适浓度;再用MTT和TUNEL(Terminal- Deoxynucleotidyl Transferase Mediated Nick End Labeling)染色法检测DEX(1, 10μM)联合Aβ(1, 5μM)对海马神经元活力和神经元凋亡的影响,同时用激光共聚焦荧光显微镜测定DEX(10μM)联合Aβ25-35(5μM)对海马神经元胞内钙浓度(intracellular calcium, [Ca2+]i)的影响,用western blot分析测定核NF-κB(Nuclear Factor kappa B)、p53和p-tau蛋白含量,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。
第二部分黄芪总苷对A?和DEX协同诱导大鼠海马神经元损伤的保护作用及其机理的研究
采用在大鼠海马CA1区注射A?25-35的方法,建立大鼠AD模型;运用Morris水迷宫方法观察AST对AD模型大鼠学习、记忆能力的影响;利用病理组织学检查,观察AST对AD模型大鼠脑组织病理性损伤的保护作用。在体外,培养胎鼠海马神经元的基础上,观察AST对A?和DEX+A?协同诱导海马神经元损伤的保护作用。采用MTT细胞活力检测法检测海马神经元细胞活力;利用TUNEL法检测细胞凋亡;运用激光共聚焦显微镜(Laser confocal microscopy ,LSCM)检测胞内钙离子浓度变化;用western blot分析测定p-tau蛋白含量;采用RT-PCR法检测P53 mRNA的表达;采用氯化硝基四唑氮蓝(Nitrotetrazolium blue chloride, NBT)法和苯甲酸羟化法观察AST对体外产生氧自由基是否有直接捕获作用。
结果:
第一部分地塞米松协同促进Aβ对大鼠海马神经元毒性及其机理的研究
1. DEX( 5 mg/kg/d, sc×7 d)或向大鼠双侧海马CA1区注射Aβ25-35(5μg/侧)可使大鼠逃避潜伏期和游泳距离有延长趋势,但无统计学意义;DEX( 1 mg/kg/d, sc×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(1/8-3/8)。病理组织学检验结果显示, 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。表明DEX可协同促进Aβ引起大鼠学习记忆能力下降和海马组织病理损伤的作用。
2. DEX(0.01-10μM)对海马神经元细胞活力无明显影响;但A ?25-3(51-40μM)可浓度依赖性地降低海马神经元细胞活力;DEX(1μM, 10μM)预处理海马神经元24 h可明显增强Aβ25-35(1μM, 5μM)引起的海马神经毒性,显著降低海马神经元活力,说明DEX可协同促进A ?引起海马神经元损伤。
3. DEX(1μM, 10μM)不能增加TUNEL染色阳性细胞数(对照组28.3%, DEX30.4%),但DEX(10μM)预处理海马神经元24 h可明显增加Aβ(5μM)诱导的升高的海马神经元凋亡细胞数,表明DEX可协同促进Aβ引起海马神经元凋亡。
4. Aβ25-35(5μM)组彗星拖尾长度明显大于溶剂对照组, DEX(10μM)对彗星拖尾长度无明显影响,但DEX(10μM)可进一步促进Aβ25-35(5μM)引起彗星拖尾长度增长,表明DEX可协同促进Aβ引起海马神经元DNA损伤,进一步证实DEX可协同促进Aβ引起海马神经元凋亡。
5. Aβ25-35(5μM)作用海马神经元10 min后,海马神经元胞内[Ca2+]i轻度增加,维持60 min,然后逐渐下降; DEX(10μM)加入培养液中24 h后不能影响海马神经元的[Ca2+]i水平;但DEX(10μM)预处理海马神经元24 h后可进一步提高Aβ25-35(5μM)诱导的[Ca2+]i上升,使海马神经元胞内[Ca2+]i达到较高水平,说明DEX可能通过促进海马神经元胞内钙离子超载促进Aβ引起海马神经元损伤。
6.与对照组(0μM Aβ, 0μM DEX)相比,10μM DEX不能增加海马神经元p53蛋白水平,5μM Aβ25-35组的p53蛋白量明显增加。DEX(10μM)+Aβ25-35(5μM)的p53蛋白表达量与同剂量Aβ25-35组相比,差异没有显著性。
7. 5μM Aβ可时间依赖性地引起海马神经元核NF-κB蛋白表达量增加,与正常对照组相比,10μM DEX可使神经元核NF-κB表达量减少,5μM Aβ25-35能够增加核NF-κB表达量,与同剂量的Aβ(5μM)相比,DEX(10μM )+Aβ25-35(5μM)组的核NF-κB表达量明显下降,说明DEX可能通过抑制海马神经元核NF-κB p65蛋白表达促进Aβ引起海马神经元损伤。此外,与对照组相比,5μM Aβ25-35作用海马神经元4 h后,胞浆IκBα表达量降低,10μM DEX组的胞浆IκBα表达量增加,DEX(10μM)可上调Aβ(5μM)引起的下降的胞浆IκBα表达量,提示DEX可能通过上调海马神经元胞质IκBα蛋白表达、抑制NF-κB p65核转移。
8. Aβ25-35(0-20μM)能够呈剂量依赖性的增加海马神经元p-tau-Thr231蛋白水平,与对照组相比,10μM DEX亦能增加海马神经元p-tau-Thr231的蛋白水平。10μM DEX+5μM Aβ25-35组p-tau-thr231蛋白水平与同剂量DEX和Aβ25-35组相比,可进一步增加p-tau-thr231蛋白水平,提示DEX可能通过促进Aβ25-35引起p-tau-Thr231蛋白表达量增加而促进海马神经元损伤。
第二部分黄芪总苷对A?和DEX协同诱导大鼠海马神经元损伤的保护作用及其机理的研究
1.向大鼠海马CA1区注射A?25-35(10μg/侧,双侧)可制造大鼠AD模型,损伤大鼠的学习记忆能力;AST(20、40、80 mg/kg,ig×7 d)可引起AD模型大鼠升高的逃避潜伏期降低、游泳距离缩短,学习记忆能力恢复,表明AST对A?25-35引起大鼠的学习记忆能力损伤有保护作用。
2.在海马CA1区注射A?25-35组,海马CA1区神经元数目明显减少,排列紊乱,脱失现象明显,部分神经元核消失。AST(40、80mg/kg)能不同程度改善上述神经元损伤,使海马神经元数目明显增加,排列较整齐,核消失现象改善。
3.体外A?25-35(10-40μM)可直接引起海马神经元活力下降。AST(10、20和40μg/ml)对体外A?25-35(10-40μM)和DEX(10μM)+A?25-35(5μM)引起胎鼠海马神经元活力有保护作用,使MTT法A值分别上升3.6-125%和12.3%,表明AST对体外A?25-35和DEX+A?25-35引起的胎鼠海马神经元的损伤有保护作用。
4. A?25-35(10μM)可引起海马神经元发生凋亡。AST(20μg/ml)能显著降低神经元中凋亡细胞的百分率; AST(20μg/ml)亦能明显降低DEX(10μM)+A?25-35(5μM)引起的增高的凋亡细胞百分率。
5. A?25-35(10μM)可诱导海马神经元胞内[Ca2+]i升高;AST(20μg/ml)能使升高的胞内[Ca2+]i降低。AST(20μg/ml)亦能明显降低DEX(10μM)+A?25-35(5μM)引起的增高的海马神经元胞内[Ca2+]i。
6. Aβ25-35(10μM)作用海马神经元1 h后,海马神经元p-tau-Thr231表达量显著升高;AST(10 , 20μg/ml)可显著下调Aβ引起的升高的p-tau-Thr231表达量。AST (10, 20μg/ml)亦能明显降低DEX(10μM)+A?25-35(5μM)引起的增高的海马神经元p-tau-Thr231蛋白表达水平。
7. A?25-35(5μM)可诱导海马神经元p53 mRNA水平升高。加入AST (20μg/ml) 18小时后行RT-PCR,电泳结果显示p53 mRNA条带面积及灰度均降低,积分光密度下降。表明AST可以降低A?25-35诱导增加的海马神经元p53 mRNA水平。
8. AST(5-80μg/ml)可浓度依赖性地抑制黄嘌呤(Xanthine, X)-黄嘌呤氧化酶(Xanthine oxidase, XO)体系和非酶体系引起NBT还原显色反应; AST仅在高浓度(80μg/ml以上)时才对XO活性有抑制作用。AST(0.5-80μg/ml)与甘露醇(·OH捕捉剂)均可浓度依赖性地抑制Fenton反应引起的苯甲酸羟化。
结论:
(1)在体内,DEX具有促进Aβ引起大鼠学习记忆能力下降的作用,导致大鼠海马CA1区神经元数目明显减少、排列紊乱、脱失、浓染和核固缩。表明DEX与Aβ具有协同引起大鼠学习记忆下降和海马组织病理损伤的作用。
(2)AST可逆转A?25-35引起的大鼠学习记忆下降和海马组织病理损伤。
(3)在体外,DEX可明显促进Aβ引起海马神经元活性下降和细胞凋亡率增加,这可能与其具有协同促进Aβ引起海马神经元胞内Ca2+升高、下调升高的核NF-κB蛋白水平,引起tau高度磷酸化有关。另外,DEX对Aβ引起的升高的海马神经元总p53蛋白水平和核p53蛋白水平无影响。
(4)在体外,AST对A?25-35、DEX+A?25-35引起的海马神经元损伤有保护作用, AST能明显降低A?和DEX+A?引起的升高的凋亡细胞百分率,进一步说明AST对A?,DEX协同A?引起的海马神经元损伤有保护作用,这可能与AST能明显降低A?和DEX+A?引起的升高的海马神经元[Ca2+]i、降低增高的海马神经元p-tau-Thr231蛋白表达水平、降低增加的海马神经元p53 mRNA水平以及直接清除超氧阴离子自由基和羟自由基的作用有关。
Background:
Alzheimer's disease(AD) is the major neurodegenerative disorder of the elderly, and is characterized by progressive cognitive deficits such as impairment of memory. One of the pathological hallmarks of Alzheimer's disease is extracellular accumulation of senile plaques composed primarily of aggregated amyloid ?-protein(Aβ). A report from WHO showed that the incidence rate of AD is 5-20% among the people older than sixty five year old. Although mutations in three different genes are known to underlie some cases of the rare, inheritable forms of the disease, the etiology of the more common sporadic cases remains unknown, and up to now, there is no reliable methods to prevent and treat the diesase.Therefore, many researchers focus on studying the machnism and reliable methods to prevent and treat the diesase.
Histopathologically, AD is characterized by intraneuronal neurofibrillary tangles, synaptic loss , neuronal death and senile plaques composed primarily of aggregated amyloid ?-protein(Aβ). Aβis a heterogeneous 39-43-amino acid peptide generated by sequential cleavage of amyloid precursor protein by ?-secretase andγ-secretase. It is generally considered that Aβplays a pivotal role in the pathogenesis of AD.
Glucocorticoids(GCs) are important adrenal steroids that affect numerous physiological processes in the brain and body, but long-term elevations of GCs are associated with decreased cognitive performance, attenuated synaptic efficacy and neuronal atrophy. 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.
Astragaloside (AST) is an active compound extracted from the root of astragalus membranaceus, a valuable Chinese Herbs. Our previous studies showed that the AST not only have significant immunomodulatory, anti-inflammatory and anti-stress effect , but also improves the learning and memory impairment induced by cytoxan and dexamethasone in two-month old mice and senescent mice respectively as well as protect against cerebral ischemia- reperfusion injury. However, whether AST could protect hippocampal neuron against the injury induced by Aβor DEX plus Aβwas not elucidated.
Previous studies showed that Aβand GCs all play a important role during the progress of AD. For example, Aβcould induce apoptosis of hippocampal neuron in vitro, injection of A?25–35 into CA1 field of rat hippocampus could damage the brain tissue of rats in vivo, and prolonged exposure to high concentration of DEX induced obvious memory impairment as well as severe histological damage in CA1 field of hippocampus in senescent mice. But synergetic effects of Aβand GCs on hippocampal neuronal injury was not well known. On the other hand, there are no effective methods for prevention and treatment of AD. Therefore, in this study, we first examine whether DEX could potentiate Aβ-induced learning and memory impairment in SD rats in vivo, and, if so, what is the underlying mechanism. Then we want to study the protective effects of AST on hippocampal neuronal injury induced by A? or DEX plus A?, and its relative mechanism.
Methods
PartⅠSynergetic Effect of DEX and A ? on Hippocampal Neurotoxicity and Its Related Mechanisms
Morris water maze test was used to investigate whether DEX could potentiate Aβ-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. Primary hippocampal neurons derived from 18 day embryonic rat were cultured. Cultured cells were treated with 0.01-10μM DEX for 48 h or 1-40μM A?25–35 for 24 h and the suboptimal concentrations of DEX and A?25–35 which producing suboptimal effects on neuronal activity were defined using MTT(3-(4,5-Dimethylthiazol -2-yl)-2,5-Diphenyl Tetrazolium Bromide) assay. Then cultured cells were treated DEX alone at 10μM for 48 h or A?25–35 alone for 24 h in serum-free DMEM, or cultured cells were pretreated with DEX at 10μM for 24 h followed by A?25–35 at 1μM or 5μM for various time in serum-free DMEM. Colorimetric MTT assay and TUNEL(Terminal- Deoxynucleotidyl Transferase Mediated Nick End Labeling) staining were used to investigate the influence of DEX on hippocampal neuronal cell death with Aβ. It was determined the effect of DEX on intracellular calcium ([Ca2+]i) with Aβ25-35 by fluorescence imaging with a confocal laser microscope using fluo-3 acetoxymethylester (AM) as a fluorescent dye. The effects of DEX on Aβ25-35-induced p53 protein, phospho-tau and nuclear factorκB (NF-κB) were analyzed by western blot.
PartⅡNeuroprotective Effects of AST Against Synergistic Hippocampal Neurotoxicity of Aβand DEX in Rat
The AD (Alzheimer,s Disease)model rats were established by injecting Aβ25-35 into the CA1 field of hippocampus. The effect of AST on learning and memory impairment in the model rats were studied by Morris water maze,and the brain protection of AST in model rats were observed through pathomorphologic changes. It were used the methods of MTT assay to investigate the influence of AST on hippocampal neuronal cell death with amyloid ?-protein (A?). The effect of AST on intracellular calcium ([Ca2+]i) with A?25–35 was detected by fluorescence imaging with a confocal laser microscope using fluo-3/acetoxymethylester (AM) as a fluorescent dye, and the effect of AST on hippocampal neuron apoptosis induced by A?25–35 were determined with TUNEL staining. The effects of AST on DEX and Aβ25-35-induced phospho-tau protein were analyzed by western blot. The level of p53 mRNA was obsersed by RT-PCR(Reverse Transcriptase Polymerase Chain Reaction ). The effects of AST on reactive oxygen species(ROS) in vitro were detected by NBT reduction and hydroxylation of benzoic acid.
Results
PartⅠSynergetic Effect of DEX and A ? on Hippocampal Neurotoxicity and Its Related Mechanisms
1. Microinjection of A?25–35 (5μg,each CA1) bilateralis into the CA1 region of adult 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(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. Our results suggest that DEX could potentiate Aβ-induced learning and memory impairment and neuropathological abnormalities.
2. Treatment for 48 h with DEX(0.01-10μM) alone did not cause a significant reduction in MTT compared with vehicle-treated control cultures. Treatment with aggregated A?25–3(51-40μM)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 A?25–35(1 or 5μM), suggesting synergetic hippocampal neurotoxicity of DEX and A?.
3. DEX( 10μM) alone failed to increase the number of cells stained positively for TUNEL in the hippocampal neurons,but pretreatment with DEX(10μM) significantly prompted the A?(5μM) -induced apoptosis in hippocampal neurons. This result suggests that DEX could enhances apoptosis in hippocampal neurons induced by A?.
4. A?25–35(5μM) could significantly increased the length of the whole comet in alkaline single-cell gel electrophoresis. DEX( 10μM) did not influence the the length of the whole comet, but the length of the whole comet in DEX ( 10μM) plus A?25–35(5μM) treated group was markedly longer than that in A?25–35 alone treated group. This result futher suggest that DEX increase the vulnerability of the hippocampal neuron to A?.
5. Treatment with A? 25-35(5μM) alone induced a slight increase in [Ca2+]i in neurons 10 min later. [Ca2+]i then declined but remained higher than basal level until analyzed over 60 min. DEX(10μM ) alone did not affect [Ca2+]i in hippocampal neurons 24 h after DEX was added to the culture, but pre-incubation of neurons with DEX (10μM) markedly increased the A? 25-35(5μM) -triggered elevation in [Ca2+]i. These results imply that DEX could potentiate the neurotoxic action of A? mediated by increasing the level of intracellular Ca2+.
6. A?25–35(5μM) causes a time-dependent increase in the level of nuclear NF-κB p65 proteins. A 24 h preincubation with DEX (10μM)could down-regulate the elevated level of nuclear NF-κB p65 proteins induced by A?25–35, suggesting that DEX increase the vulnerability of the hippocampal neurons to A? mediated by down-regulating the level of nuclear NF-κB proteins. A?25–35(5μM) alone could decrease the cytoplasmic level of IκBαprotein 4 h after A?25–35 was added to the culture. Increased levels of cytoplasmic IκBαprotein were observed in hippocampal neurons 28 h after incubation with DEX(10μM), and pretreatment of hippocampal neurons with DEX(10μM) for 24 h could slow the disappearance of cytoplasmic IκBαprotein mediated by A?25–35, suggesting that DEX down-regulation of the elevated level of nuclear NF-κB p65 proteins induced by A? might be associated with increased rate of IκBαprotein synthesis.
7. A? 25–35 (5μM) treatment resulted in small but significant increases in the level of total protein and nuclear protein of p53 24 h after A? was added to the culture. Treatment with DEX (10μM) alone for 48 h did not increase the levels of total protein and nuclear protein of p53. Pretreatment with DEX for 24 h didn’t promote the increased total protein and nuclear protein of p53 induced by A?25–35..
8. 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(10μM). DEX treatment of neurons in primary culture for 24 h could results in an slight elevation in tau phosphorylation at Thr-231, and pretreated with DEX at 10μM for 24 h could promote the increased level of phospho-tau at Thr-231 induced by Aβ25-35(5μM). These results suggest that DEX increasing the vulnerability of the hippocampal neurons to Aβwas mediated by promoting Aβ-induced tau phosphorylation at Thr-231.
PartⅡNeuroprotective Effects of AST Against Synergistic Hippocampal Neurotoxicity of Aβand DEX in Rat
1. AD was modeled by microinjection of A?25–35 (10μg/CA1)bilateralis into the CA1 region of adult rats under stereotaxic guidance. AST(20, 40, 80mg/kg, ig×7 d) could decreased the escape latency and the swim distances of AD model rats, suggesting that AST could improve the ability of learning and memory in AD model rats in the Morris water maze test.
2. Injection of A?25–35 (10μg/CA1) into CA1 field of rat hippocampus could damage the brain tissue of rats in vivo and the neuropathological changes were characterized by decreased cell number, soma shrinkage and condensation, or nuclear pyknosis. AST( 40 and 80mg/kg )improved histopathologic change in the CA1 field of rats hippocampus.
3. AST(10μg/ml, 20μg/ml and 40μg/ml) could protect hippocampal neurons against A?25-35(10-40μM)-induced hippocampal neuronal injury in vitro. AST(10μg/ml, 20μg/ml and 40μg/ml)could also protect hippocampal neurons against DEX(10μM) plus A?25-35(5μM)- induced hippocampal neuronal injury. These results suggest that AST could protect hippocampal neuron against synergistic neurotoxicity of Aβand DEX.
4. It was used the method of TUNEL staining to investigate the influence of AST on hippocampal neuronal cell apoptosis induced by A?25-35 or DEX plus A?25-35 . Results showed that AST (20μg/ml) could significantly inhibited the apoptosis of the hippocampal neurons induced by A?25-35(10μM) or DEX(10μM) plus A?25-35(5μM).
5. Treatment with A? 25-35(10μM) alone induced a increase in intracellular calcium( [Ca2+]i) in neurons 10 min after A? was added into the culture. [Ca2+]i then declined but remained higher than basal level until analyzed over 60 min. AST (20μg/ml) could inhibit the increased levels of the [Ca2+]i induced by A?25–35. Pre-incubation of neurons with DEX(10μM) markedly increased the A? 25-35(5μM )-triggered elevation in [Ca2+]i , and AST (20μg/ml) could also inhibit the increased levels of the [Ca2+]i induced by DEX(10μM) plus A?25-35(5μM).
6. Aβ25-35(10μM) could increase the level of phospho-tau at Thr-231 in primary hippocampal neurons. Pretreatment with DEX at 10μM for 24 h could further promote the increased level of phospho-tau at Thr-231 induced by 5μM Aβ25-35. AST( 10, 20μg/ml) could significantly inhibit the level of phospho-tau at Thr-231 induced by Aβ25-35(10μM) or DEX(10μM) plus Aβ25-35(5μM).
7. The level of p53 mRNA increased 18 h after A?25-35(5μM) was added to the cultured. AST( 20μg/ml) could significantly inhibit the level of p53 mRNA 18 h after A?25-35 was added to the culture.
8. AST(5- 80μg/ml) could inhibit NBT reduction induced by both xanthine-xanthine oxidase and non-enzyme generated superoxide anions ( ), but the inhibitory effect of this compound on activity of xanthine oxidase was obtained only at high concentration (more than 80μg/ml). It was also found that AST(0.5-80μg/ml) could dose-dependantly inhibit the hydroxylation of benzoic acid induced by Fenton reaction generated . OH.
Conclusions
(1) In vivo, DEX could potentiate Aβ-induced learning and memory impairment and pathological damage in CA1 field of hippocampus in SD rats.
(2) AST could protect against A?-induced learning and memory impairment. Injection of A? into CA1 field of rat hippocampus could damage the brain tissue of rats in vivo, and AST could improve histopathologic change in the CA1 field of SD rats.
(3) in vitro, DEX could potentiate the neurotoxic action of A? and could enhances apoptosis in hippocampal neurons induced by A?, which might be related to its effects in increasing the A?-triggered elevation in [Ca2+]i, down-regulating the elevated level of nuclear NF-κB p65 proteins, slowing the disappearance of cytoplasmic IκBαprotein as well as promoting the increased level of phospho-tau at Thr-231 with Aβ. In contrary, DEX didn’t promote the increased levels of total protein and nuclear protein of p53 induced by A?.
(4) AST could protect against A? or DEX plus A?-induced hippocampal neuronal injury , which might be related to its down-regulating the increased levels of the [Ca2+]i and phospho-tau at Thr-231, inhibiting the level of p53 mRNA induced by Aβor DEX plus Aβand scavenging and . OH generated by xanthine-xanthine oxidase system, non-enzyme system and Fenton reaction.
AD的主要病理学特征是在中枢神经系统的皮层、海马等处产生大量的β-淀粉样蛋白(β-Amyloid protein, Aβ),形成Aβ沉积、老年斑(Senile plaque, SP)和神经纤维缠结(Neurofibrilary tangle, NFT),其中Aβ是构成老年斑的主要成份。Aβ是由β-淀粉样前体蛋白(amyloid precursor protein, APP)经β、r-secretase酶裂解生成,Aβ是构成老年斑的主要成份;Aβ在AD患者的神经元中过度生成及其对神经元的细胞毒作用,在AD的发病和进展过程中起关键的作用。糖皮质激素(Glucocorticoids, GCs)是调节机体生长发育、新陈代谢和维持机体功能正常的一类重要激素,也是临床常用的抢救和治疗药物,GCs具有多种生理和药理作用,但长期大剂量应用GCs可对机体免疫和神经等系统造成严重的不良反应。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,仅检索到2篇相关论文。
近年来,国内外都投入了大量人力、物力从事防治中枢神经系统退行性疾病、AD等疾病及其发病机制的研究工作,我国中医中药具有悠久的防治衰老的历史和丰富的临床实践经验,目前,在国内,一些研究单位着重研究了传统延缓衰老的常用中药及其复方的疗效和部分作用机理,取得了一定的进展,但都还基本处于临床前研究阶段,已批准上市的防治AD中药还寥寥无几。黄芪是临床延缓衰老的名药,黄芪总苷(Astragalosides, AST)是从黄芪中提取的有效部位群,我们的前期研究表明, AST不仅具有抗炎、免疫调节和防治局灶性脑缺血和全脑缺血再灌注引起的脑损伤作用,还能明显改善D-半乳糖衰老小鼠和地塞米松(Dexamethasone, DEX)引起的老前期(20 mon)衰老模型小鼠的学习记忆功能,改善环磷酰胺降低的小鼠学习记忆功能;但AST对Aβ和GCs引起的海马神经元损伤是否有保护作用还未见报道。
由于Aβ和GCs分别在AD的发病中均起重要的作用,如Aβ可引起体外培养的海马神经元凋亡和活力下降,向大鼠海马CA1区注射Aβ可引大鼠学习记忆能力下降;小鼠在长期给予GCs时,在引起老前期小鼠海马神经元损伤的同时,还可引起学习记忆能力明显下降,但它们二者之间对神经元是否具有协同的细胞毒性作用仍然不清楚;此外,临床上仍无理想的防治AD的药物和方法。为进一步研究AD的发病机理和寻找有效的防治AD等神经系统退行性疾病的方法和药物,本文拟(1)在体内,研究DEX是否具有协同促进Aβ引起大鼠海马神经元损伤和学习记忆能力下降的作用,然后进一步研究其可能的作用机制。在体外,同样观察DEX协同促进Aβ引起海马神经元损伤,分析其可能的作用机理。(2)进一步探讨AST对A?和DEX诱导大鼠海马神经元损伤的保护作用以及可能的相关分子与基因水平的作用机制。
方法:
第一部分地塞米松协同促进Aβ对大鼠海马神经元毒性及其机理的研究在体内,利用Morris水迷宫和病理组织检查,研究DEX(1 mg/kg/d,sc×7 d)和Aβ(海马CA1区注射、5μg/侧、双侧、单次)联合作用对大鼠学习记忆能力和海马神经元损伤的影响。在体外,取孕第18天Sprague Dawley(SD)大鼠胚胎的海马神经元进行体外原代培养,用MTT(Methyl thiazolyl tetrazolium)方法观察DEX(0.01-10μM)和Aβ25-35(1-40μM)对海马神经元活力的影响,分别找出DEX和Aβ引起海马神经元损伤的亚适浓度;再用MTT和TUNEL(Terminal- Deoxynucleotidyl Transferase Mediated Nick End Labeling)染色法检测DEX(1, 10μM)联合Aβ(1, 5μM)对海马神经元活力和神经元凋亡的影响,同时用激光共聚焦荧光显微镜测定DEX(10μM)联合Aβ25-35(5μM)对海马神经元胞内钙浓度(intracellular calcium, [Ca2+]i)的影响,用western blot分析测定核NF-κB(Nuclear Factor kappa B)、p53和p-tau蛋白含量,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。
第二部分黄芪总苷对A?和DEX协同诱导大鼠海马神经元损伤的保护作用及其机理的研究
采用在大鼠海马CA1区注射A?25-35的方法,建立大鼠AD模型;运用Morris水迷宫方法观察AST对AD模型大鼠学习、记忆能力的影响;利用病理组织学检查,观察AST对AD模型大鼠脑组织病理性损伤的保护作用。在体外,培养胎鼠海马神经元的基础上,观察AST对A?和DEX+A?协同诱导海马神经元损伤的保护作用。采用MTT细胞活力检测法检测海马神经元细胞活力;利用TUNEL法检测细胞凋亡;运用激光共聚焦显微镜(Laser confocal microscopy ,LSCM)检测胞内钙离子浓度变化;用western blot分析测定p-tau蛋白含量;采用RT-PCR法检测P53 mRNA的表达;采用氯化硝基四唑氮蓝(Nitrotetrazolium blue chloride, NBT)法和苯甲酸羟化法观察AST对体外产生氧自由基是否有直接捕获作用。
结果:
第一部分地塞米松协同促进Aβ对大鼠海马神经元毒性及其机理的研究
1. DEX( 5 mg/kg/d, sc×7 d)或向大鼠双侧海马CA1区注射Aβ25-35(5μg/侧)可使大鼠逃避潜伏期和游泳距离有延长趋势,但无统计学意义;DEX( 1 mg/kg/d, sc×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(1/8-3/8)。病理组织学检验结果显示, 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。表明DEX可协同促进Aβ引起大鼠学习记忆能力下降和海马组织病理损伤的作用。
2. DEX(0.01-10μM)对海马神经元细胞活力无明显影响;但A ?25-3(51-40μM)可浓度依赖性地降低海马神经元细胞活力;DEX(1μM, 10μM)预处理海马神经元24 h可明显增强Aβ25-35(1μM, 5μM)引起的海马神经毒性,显著降低海马神经元活力,说明DEX可协同促进A ?引起海马神经元损伤。
3. DEX(1μM, 10μM)不能增加TUNEL染色阳性细胞数(对照组28.3%, DEX30.4%),但DEX(10μM)预处理海马神经元24 h可明显增加Aβ(5μM)诱导的升高的海马神经元凋亡细胞数,表明DEX可协同促进Aβ引起海马神经元凋亡。
4. Aβ25-35(5μM)组彗星拖尾长度明显大于溶剂对照组, DEX(10μM)对彗星拖尾长度无明显影响,但DEX(10μM)可进一步促进Aβ25-35(5μM)引起彗星拖尾长度增长,表明DEX可协同促进Aβ引起海马神经元DNA损伤,进一步证实DEX可协同促进Aβ引起海马神经元凋亡。
5. Aβ25-35(5μM)作用海马神经元10 min后,海马神经元胞内[Ca2+]i轻度增加,维持60 min,然后逐渐下降; DEX(10μM)加入培养液中24 h后不能影响海马神经元的[Ca2+]i水平;但DEX(10μM)预处理海马神经元24 h后可进一步提高Aβ25-35(5μM)诱导的[Ca2+]i上升,使海马神经元胞内[Ca2+]i达到较高水平,说明DEX可能通过促进海马神经元胞内钙离子超载促进Aβ引起海马神经元损伤。
6.与对照组(0μM Aβ, 0μM DEX)相比,10μM DEX不能增加海马神经元p53蛋白水平,5μM Aβ25-35组的p53蛋白量明显增加。DEX(10μM)+Aβ25-35(5μM)的p53蛋白表达量与同剂量Aβ25-35组相比,差异没有显著性。
7. 5μM Aβ可时间依赖性地引起海马神经元核NF-κB蛋白表达量增加,与正常对照组相比,10μM DEX可使神经元核NF-κB表达量减少,5μM Aβ25-35能够增加核NF-κB表达量,与同剂量的Aβ(5μM)相比,DEX(10μM )+Aβ25-35(5μM)组的核NF-κB表达量明显下降,说明DEX可能通过抑制海马神经元核NF-κB p65蛋白表达促进Aβ引起海马神经元损伤。此外,与对照组相比,5μM Aβ25-35作用海马神经元4 h后,胞浆IκBα表达量降低,10μM DEX组的胞浆IκBα表达量增加,DEX(10μM)可上调Aβ(5μM)引起的下降的胞浆IκBα表达量,提示DEX可能通过上调海马神经元胞质IκBα蛋白表达、抑制NF-κB p65核转移。
8. Aβ25-35(0-20μM)能够呈剂量依赖性的增加海马神经元p-tau-Thr231蛋白水平,与对照组相比,10μM DEX亦能增加海马神经元p-tau-Thr231的蛋白水平。10μM DEX+5μM Aβ25-35组p-tau-thr231蛋白水平与同剂量DEX和Aβ25-35组相比,可进一步增加p-tau-thr231蛋白水平,提示DEX可能通过促进Aβ25-35引起p-tau-Thr231蛋白表达量增加而促进海马神经元损伤。
第二部分黄芪总苷对A?和DEX协同诱导大鼠海马神经元损伤的保护作用及其机理的研究
1.向大鼠海马CA1区注射A?25-35(10μg/侧,双侧)可制造大鼠AD模型,损伤大鼠的学习记忆能力;AST(20、40、80 mg/kg,ig×7 d)可引起AD模型大鼠升高的逃避潜伏期降低、游泳距离缩短,学习记忆能力恢复,表明AST对A?25-35引起大鼠的学习记忆能力损伤有保护作用。
2.在海马CA1区注射A?25-35组,海马CA1区神经元数目明显减少,排列紊乱,脱失现象明显,部分神经元核消失。AST(40、80mg/kg)能不同程度改善上述神经元损伤,使海马神经元数目明显增加,排列较整齐,核消失现象改善。
3.体外A?25-35(10-40μM)可直接引起海马神经元活力下降。AST(10、20和40μg/ml)对体外A?25-35(10-40μM)和DEX(10μM)+A?25-35(5μM)引起胎鼠海马神经元活力有保护作用,使MTT法A值分别上升3.6-125%和12.3%,表明AST对体外A?25-35和DEX+A?25-35引起的胎鼠海马神经元的损伤有保护作用。
4. A?25-35(10μM)可引起海马神经元发生凋亡。AST(20μg/ml)能显著降低神经元中凋亡细胞的百分率; AST(20μg/ml)亦能明显降低DEX(10μM)+A?25-35(5μM)引起的增高的凋亡细胞百分率。
5. A?25-35(10μM)可诱导海马神经元胞内[Ca2+]i升高;AST(20μg/ml)能使升高的胞内[Ca2+]i降低。AST(20μg/ml)亦能明显降低DEX(10μM)+A?25-35(5μM)引起的增高的海马神经元胞内[Ca2+]i。
6. Aβ25-35(10μM)作用海马神经元1 h后,海马神经元p-tau-Thr231表达量显著升高;AST(10 , 20μg/ml)可显著下调Aβ引起的升高的p-tau-Thr231表达量。AST (10, 20μg/ml)亦能明显降低DEX(10μM)+A?25-35(5μM)引起的增高的海马神经元p-tau-Thr231蛋白表达水平。
7. A?25-35(5μM)可诱导海马神经元p53 mRNA水平升高。加入AST (20μg/ml) 18小时后行RT-PCR,电泳结果显示p53 mRNA条带面积及灰度均降低,积分光密度下降。表明AST可以降低A?25-35诱导增加的海马神经元p53 mRNA水平。
8. AST(5-80μg/ml)可浓度依赖性地抑制黄嘌呤(Xanthine, X)-黄嘌呤氧化酶(Xanthine oxidase, XO)体系和非酶体系引起NBT还原显色反应; AST仅在高浓度(80μg/ml以上)时才对XO活性有抑制作用。AST(0.5-80μg/ml)与甘露醇(·OH捕捉剂)均可浓度依赖性地抑制Fenton反应引起的苯甲酸羟化。
结论:
(1)在体内,DEX具有促进Aβ引起大鼠学习记忆能力下降的作用,导致大鼠海马CA1区神经元数目明显减少、排列紊乱、脱失、浓染和核固缩。表明DEX与Aβ具有协同引起大鼠学习记忆下降和海马组织病理损伤的作用。
(2)AST可逆转A?25-35引起的大鼠学习记忆下降和海马组织病理损伤。
(3)在体外,DEX可明显促进Aβ引起海马神经元活性下降和细胞凋亡率增加,这可能与其具有协同促进Aβ引起海马神经元胞内Ca2+升高、下调升高的核NF-κB蛋白水平,引起tau高度磷酸化有关。另外,DEX对Aβ引起的升高的海马神经元总p53蛋白水平和核p53蛋白水平无影响。
(4)在体外,AST对A?25-35、DEX+A?25-35引起的海马神经元损伤有保护作用, AST能明显降低A?和DEX+A?引起的升高的凋亡细胞百分率,进一步说明AST对A?,DEX协同A?引起的海马神经元损伤有保护作用,这可能与AST能明显降低A?和DEX+A?引起的升高的海马神经元[Ca2+]i、降低增高的海马神经元p-tau-Thr231蛋白表达水平、降低增加的海马神经元p53 mRNA水平以及直接清除超氧阴离子自由基和羟自由基的作用有关。
Background:
Alzheimer's disease(AD) is the major neurodegenerative disorder of the elderly, and is characterized by progressive cognitive deficits such as impairment of memory. One of the pathological hallmarks of Alzheimer's disease is extracellular accumulation of senile plaques composed primarily of aggregated amyloid ?-protein(Aβ). A report from WHO showed that the incidence rate of AD is 5-20% among the people older than sixty five year old. Although mutations in three different genes are known to underlie some cases of the rare, inheritable forms of the disease, the etiology of the more common sporadic cases remains unknown, and up to now, there is no reliable methods to prevent and treat the diesase.Therefore, many researchers focus on studying the machnism and reliable methods to prevent and treat the diesase.
Histopathologically, AD is characterized by intraneuronal neurofibrillary tangles, synaptic loss , neuronal death and senile plaques composed primarily of aggregated amyloid ?-protein(Aβ). Aβis a heterogeneous 39-43-amino acid peptide generated by sequential cleavage of amyloid precursor protein by ?-secretase andγ-secretase. It is generally considered that Aβplays a pivotal role in the pathogenesis of AD.
Glucocorticoids(GCs) are important adrenal steroids that affect numerous physiological processes in the brain and body, but long-term elevations of GCs are associated with decreased cognitive performance, attenuated synaptic efficacy and neuronal atrophy. 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.
Astragaloside (AST) is an active compound extracted from the root of astragalus membranaceus, a valuable Chinese Herbs. Our previous studies showed that the AST not only have significant immunomodulatory, anti-inflammatory and anti-stress effect , but also improves the learning and memory impairment induced by cytoxan and dexamethasone in two-month old mice and senescent mice respectively as well as protect against cerebral ischemia- reperfusion injury. However, whether AST could protect hippocampal neuron against the injury induced by Aβor DEX plus Aβwas not elucidated.
Previous studies showed that Aβand GCs all play a important role during the progress of AD. For example, Aβcould induce apoptosis of hippocampal neuron in vitro, injection of A?25–35 into CA1 field of rat hippocampus could damage the brain tissue of rats in vivo, and prolonged exposure to high concentration of DEX induced obvious memory impairment as well as severe histological damage in CA1 field of hippocampus in senescent mice. But synergetic effects of Aβand GCs on hippocampal neuronal injury was not well known. On the other hand, there are no effective methods for prevention and treatment of AD. Therefore, in this study, we first examine whether DEX could potentiate Aβ-induced learning and memory impairment in SD rats in vivo, and, if so, what is the underlying mechanism. Then we want to study the protective effects of AST on hippocampal neuronal injury induced by A? or DEX plus A?, and its relative mechanism.
Methods
PartⅠSynergetic Effect of DEX and A ? on Hippocampal Neurotoxicity and Its Related Mechanisms
Morris water maze test was used to investigate whether DEX could potentiate Aβ-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. Primary hippocampal neurons derived from 18 day embryonic rat were cultured. Cultured cells were treated with 0.01-10μM DEX for 48 h or 1-40μM A?25–35 for 24 h and the suboptimal concentrations of DEX and A?25–35 which producing suboptimal effects on neuronal activity were defined using MTT(3-(4,5-Dimethylthiazol -2-yl)-2,5-Diphenyl Tetrazolium Bromide) assay. Then cultured cells were treated DEX alone at 10μM for 48 h or A?25–35 alone for 24 h in serum-free DMEM, or cultured cells were pretreated with DEX at 10μM for 24 h followed by A?25–35 at 1μM or 5μM for various time in serum-free DMEM. Colorimetric MTT assay and TUNEL(Terminal- Deoxynucleotidyl Transferase Mediated Nick End Labeling) staining were used to investigate the influence of DEX on hippocampal neuronal cell death with Aβ. It was determined the effect of DEX on intracellular calcium ([Ca2+]i) with Aβ25-35 by fluorescence imaging with a confocal laser microscope using fluo-3 acetoxymethylester (AM) as a fluorescent dye. The effects of DEX on Aβ25-35-induced p53 protein, phospho-tau and nuclear factorκB (NF-κB) were analyzed by western blot.
PartⅡNeuroprotective Effects of AST Against Synergistic Hippocampal Neurotoxicity of Aβand DEX in Rat
The AD (Alzheimer,s Disease)model rats were established by injecting Aβ25-35 into the CA1 field of hippocampus. The effect of AST on learning and memory impairment in the model rats were studied by Morris water maze,and the brain protection of AST in model rats were observed through pathomorphologic changes. It were used the methods of MTT assay to investigate the influence of AST on hippocampal neuronal cell death with amyloid ?-protein (A?). The effect of AST on intracellular calcium ([Ca2+]i) with A?25–35 was detected by fluorescence imaging with a confocal laser microscope using fluo-3/acetoxymethylester (AM) as a fluorescent dye, and the effect of AST on hippocampal neuron apoptosis induced by A?25–35 were determined with TUNEL staining. The effects of AST on DEX and Aβ25-35-induced phospho-tau protein were analyzed by western blot. The level of p53 mRNA was obsersed by RT-PCR(Reverse Transcriptase Polymerase Chain Reaction ). The effects of AST on reactive oxygen species(ROS) in vitro were detected by NBT reduction and hydroxylation of benzoic acid.
Results
PartⅠSynergetic Effect of DEX and A ? on Hippocampal Neurotoxicity and Its Related Mechanisms
1. Microinjection of A?25–35 (5μg,each CA1) bilateralis into the CA1 region of adult 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(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. Our results suggest that DEX could potentiate Aβ-induced learning and memory impairment and neuropathological abnormalities.
2. Treatment for 48 h with DEX(0.01-10μM) alone did not cause a significant reduction in MTT compared with vehicle-treated control cultures. Treatment with aggregated A?25–3(51-40μM)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 A?25–35(1 or 5μM), suggesting synergetic hippocampal neurotoxicity of DEX and A?.
3. DEX( 10μM) alone failed to increase the number of cells stained positively for TUNEL in the hippocampal neurons,but pretreatment with DEX(10μM) significantly prompted the A?(5μM) -induced apoptosis in hippocampal neurons. This result suggests that DEX could enhances apoptosis in hippocampal neurons induced by A?.
4. A?25–35(5μM) could significantly increased the length of the whole comet in alkaline single-cell gel electrophoresis. DEX( 10μM) did not influence the the length of the whole comet, but the length of the whole comet in DEX ( 10μM) plus A?25–35(5μM) treated group was markedly longer than that in A?25–35 alone treated group. This result futher suggest that DEX increase the vulnerability of the hippocampal neuron to A?.
5. Treatment with A? 25-35(5μM) alone induced a slight increase in [Ca2+]i in neurons 10 min later. [Ca2+]i then declined but remained higher than basal level until analyzed over 60 min. DEX(10μM ) alone did not affect [Ca2+]i in hippocampal neurons 24 h after DEX was added to the culture, but pre-incubation of neurons with DEX (10μM) markedly increased the A? 25-35(5μM) -triggered elevation in [Ca2+]i. These results imply that DEX could potentiate the neurotoxic action of A? mediated by increasing the level of intracellular Ca2+.
6. A?25–35(5μM) causes a time-dependent increase in the level of nuclear NF-κB p65 proteins. A 24 h preincubation with DEX (10μM)could down-regulate the elevated level of nuclear NF-κB p65 proteins induced by A?25–35, suggesting that DEX increase the vulnerability of the hippocampal neurons to A? mediated by down-regulating the level of nuclear NF-κB proteins. A?25–35(5μM) alone could decrease the cytoplasmic level of IκBαprotein 4 h after A?25–35 was added to the culture. Increased levels of cytoplasmic IκBαprotein were observed in hippocampal neurons 28 h after incubation with DEX(10μM), and pretreatment of hippocampal neurons with DEX(10μM) for 24 h could slow the disappearance of cytoplasmic IκBαprotein mediated by A?25–35, suggesting that DEX down-regulation of the elevated level of nuclear NF-κB p65 proteins induced by A? might be associated with increased rate of IκBαprotein synthesis.
7. A? 25–35 (5μM) treatment resulted in small but significant increases in the level of total protein and nuclear protein of p53 24 h after A? was added to the culture. Treatment with DEX (10μM) alone for 48 h did not increase the levels of total protein and nuclear protein of p53. Pretreatment with DEX for 24 h didn’t promote the increased total protein and nuclear protein of p53 induced by A?25–35..
8. 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(10μM). DEX treatment of neurons in primary culture for 24 h could results in an slight elevation in tau phosphorylation at Thr-231, and pretreated with DEX at 10μM for 24 h could promote the increased level of phospho-tau at Thr-231 induced by Aβ25-35(5μM). These results suggest that DEX increasing the vulnerability of the hippocampal neurons to Aβwas mediated by promoting Aβ-induced tau phosphorylation at Thr-231.
PartⅡNeuroprotective Effects of AST Against Synergistic Hippocampal Neurotoxicity of Aβand DEX in Rat
1. AD was modeled by microinjection of A?25–35 (10μg/CA1)bilateralis into the CA1 region of adult rats under stereotaxic guidance. AST(20, 40, 80mg/kg, ig×7 d) could decreased the escape latency and the swim distances of AD model rats, suggesting that AST could improve the ability of learning and memory in AD model rats in the Morris water maze test.
2. Injection of A?25–35 (10μg/CA1) into CA1 field of rat hippocampus could damage the brain tissue of rats in vivo and the neuropathological changes were characterized by decreased cell number, soma shrinkage and condensation, or nuclear pyknosis. AST( 40 and 80mg/kg )improved histopathologic change in the CA1 field of rats hippocampus.
3. AST(10μg/ml, 20μg/ml and 40μg/ml) could protect hippocampal neurons against A?25-35(10-40μM)-induced hippocampal neuronal injury in vitro. AST(10μg/ml, 20μg/ml and 40μg/ml)could also protect hippocampal neurons against DEX(10μM) plus A?25-35(5μM)- induced hippocampal neuronal injury. These results suggest that AST could protect hippocampal neuron against synergistic neurotoxicity of Aβand DEX.
4. It was used the method of TUNEL staining to investigate the influence of AST on hippocampal neuronal cell apoptosis induced by A?25-35 or DEX plus A?25-35 . Results showed that AST (20μg/ml) could significantly inhibited the apoptosis of the hippocampal neurons induced by A?25-35(10μM) or DEX(10μM) plus A?25-35(5μM).
5. Treatment with A? 25-35(10μM) alone induced a increase in intracellular calcium( [Ca2+]i) in neurons 10 min after A? was added into the culture. [Ca2+]i then declined but remained higher than basal level until analyzed over 60 min. AST (20μg/ml) could inhibit the increased levels of the [Ca2+]i induced by A?25–35. Pre-incubation of neurons with DEX(10μM) markedly increased the A? 25-35(5μM )-triggered elevation in [Ca2+]i , and AST (20μg/ml) could also inhibit the increased levels of the [Ca2+]i induced by DEX(10μM) plus A?25-35(5μM).
6. Aβ25-35(10μM) could increase the level of phospho-tau at Thr-231 in primary hippocampal neurons. Pretreatment with DEX at 10μM for 24 h could further promote the increased level of phospho-tau at Thr-231 induced by 5μM Aβ25-35. AST( 10, 20μg/ml) could significantly inhibit the level of phospho-tau at Thr-231 induced by Aβ25-35(10μM) or DEX(10μM) plus Aβ25-35(5μM).
7. The level of p53 mRNA increased 18 h after A?25-35(5μM) was added to the cultured. AST( 20μg/ml) could significantly inhibit the level of p53 mRNA 18 h after A?25-35 was added to the culture.
8. AST(5- 80μg/ml) could inhibit NBT reduction induced by both xanthine-xanthine oxidase and non-enzyme generated superoxide anions ( ), but the inhibitory effect of this compound on activity of xanthine oxidase was obtained only at high concentration (more than 80μg/ml). It was also found that AST(0.5-80μg/ml) could dose-dependantly inhibit the hydroxylation of benzoic acid induced by Fenton reaction generated . OH.
Conclusions
(1) In vivo, DEX could potentiate Aβ-induced learning and memory impairment and pathological damage in CA1 field of hippocampus in SD rats.
(2) AST could protect against A?-induced learning and memory impairment. Injection of A? into CA1 field of rat hippocampus could damage the brain tissue of rats in vivo, and AST could improve histopathologic change in the CA1 field of SD rats.
(3) in vitro, DEX could potentiate the neurotoxic action of A? and could enhances apoptosis in hippocampal neurons induced by A?, which might be related to its effects in increasing the A?-triggered elevation in [Ca2+]i, down-regulating the elevated level of nuclear NF-κB p65 proteins, slowing the disappearance of cytoplasmic IκBαprotein as well as promoting the increased level of phospho-tau at Thr-231 with Aβ. In contrary, DEX didn’t promote the increased levels of total protein and nuclear protein of p53 induced by A?.
(4) AST could protect against A? or DEX plus A?-induced hippocampal neuronal injury , which might be related to its down-regulating the increased levels of the [Ca2+]i and phospho-tau at Thr-231, inhibiting the level of p53 mRNA induced by Aβor DEX plus Aβand scavenging and . OH generated by xanthine-xanthine oxidase system, non-enzyme system and Fenton reaction.
引文
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