慢性低氧低糖培养影响新生大鼠海马神经元α-分泌酶的实验研究

目的探讨慢性低氧低糖培养对新生大鼠海马神经元α-分泌酶活性及表达的影响。方法取新生24h的Wistar大鼠海马原代培养神经元并予以鉴定,培养第七天予以12h、24h、36h低氧低糖培养干预,MTT法检测海马神经元的活力,荧光法检测α、β-分泌酶活性,Western blot检测α-分泌酶蛋白表达水平。结果低氧低糖培养12小时组海马神经元α-分泌酶活性升高(P<0.01),低氧低糖培养24h、36h组α-分泌酶(TACE)活性无明显变化(P>0.05),低氧低糖培养12h、24h、36h组海马神经元β-分泌酶(BACE1)活性升高(P<0.05);低氧低糖培养12h、24h、36h组海马神经元α-分泌酶蛋白表达水平降低(P<0.05)。结论慢性低氧低糖培养降低新生大鼠海马神经元α-分泌酶的表达。     

慢性低氧低糖培养对大鼠海马神经元α-和β-分泌酶活性的影响

目的 探讨慢性低氧低糖培养对大鼠海马神经元α-和β-分泌酶活性的影响.方法 取新生24h的wistar大鼠海马神经元培养,用慢性低氧低糖进行干预,MTT法检测神经元细胞活力,直接荧光法检测α、β-分泌酶活性.结果 低氧低糖组较对照组细胞活力降低,具有显著性差异（P＜0.05）,低氧低糖培养海马神经元12 h的α-分泌酶活性升高（P＜0.0001）,低氧低糖培养海马神经元12、24、36 h的β-分泌酶活性升高（P=0.0032、0.0018、＜0.0001）.结论 慢性低氧低糖培养对海马神经元具有毒性,可能与其升高β-分泌酶活性使具有神经营养作用的sAPPα降低和具有神经毒性作用的Aβ升高有关.     

《中国卒中杂志》2008年第3卷主题词索引

说明:(1)主题词按汉语拼音字母顺序排列;(2)作者、文题后数字依次为年,卷,期号,起止页码。AAdson试验-高凤玲,徐忠宝,李继梅,等.经颅多普勒超声诊断胸廓出口综合征一例.2008,3(2):133-135.阿尔茨海默病-管学能,闫福岭.EGb761对慢性低氧低糖培养的大鼠海马神经元β分泌酶的影响.     

慢性低氧低糖培养对脑微血管内皮细胞RAGE和LRP-1表达的影响

目的：研究慢性低氧低糖对小鼠血脑屏障（BBB）上晚期糖基化终产物受体（RAGE）和低密度脂蛋白受体相关蛋白-1（LRP-1）表达的影响。方法：将小鼠脑微血管内皮细胞系bEnd．3行不同时间的低氧低糖培养后，分别应用RT-PCR和Western blot方法检测细胞RAGE、LRP-1 mRNA和蛋白的表达。结果：与对照组相比，低氧低糖培养24、36和48h组bEnd．3细胞RAGE mRNA和蛋白表达均明显增加（P〈0．05），以48h组最为显著。低氧低糖培养36和48h组LRP-1 mRNA表达明显下降（P〈0．05）；24、36和48h组LRP-1蛋白表达均显著下降（P〈0．05）；48h组LRP-1 mRNA和蛋白表达降为最低。结论：慢性低氧低糖环境下，小鼠BBB上RAGE mRNA表达和蛋白表达水平升高，而LRP-1 mRNA表达和蛋白表达水平下降。     

EGb761对慢性脑缺血大鼠脑组织神经胶质细胞的影响

目的 探讨EGb761(银杏叶提取物)对慢性脑缺血大鼠脑组织神经胶质细胞的影响.方法 将雄性SD大鼠随机分为假手术组、单纯缺血组、EGb761干预组.制备慢性脑缺血大鼠模型,药物干预12周后,免疫组化方法分别检测各组大鼠脑组织中星形胶质细胞和少突胶质细胞的表达.结果 EGb761干预组大鼠的海马、胼胝体、皮质CNPase阳性细胞表达明显高于单纯缺血组,而海马、胼胝体、皮质GFAP阳性细胞表达明显低于单纯缺血组(P<0.05),差异有统计学意义.结论 慢性脑缺血时,EGb761对少突胶质细胞缺血性反应有保护作用,同时减少反应性星形胶质细胞增生.     

银杏叶提取物EGb761抑制脊髓损伤后的细胞凋亡

目的： 探讨银杏叶提取物EGb761对实验性大鼠脊髓损伤后神经保护的作用及其机制。方法： 成年雄性SD大鼠132 只,体重200~250g,随机分为正常对照组（N组）、损伤组（T组）、甲基强的松龙治疗组（MP组）和 EGb761 治疗组（EGb761组）,每组 33 只。T组、MP组、EGb761组用改良 Allen法以25GCF损伤力度致伤大鼠,建立 T9 脊髓中度损伤模型。术后4h、8h、24h每组随机取3只动物切取损伤区1cm脊髓节段,分别用黄嘌呤氧化酶法和硫代巴比妥酸（TBA）法测定脊髓组织中超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量。分别于术后24h、 3d、 5d、 7d、 14d 处死动物(n=6),快速取T9 节段脊髓,TUNEL法标记细胞凋亡,免疫组化方法检测诱生型一氧化氮合酶（iNOS）的表达。结果：术后4h、8h、24h EGb761治疗组SOD活性及MDA含量与损伤对照组比较均有显著差异（P<0.01）。术后各时相点EGb761治疗组神经细胞凋亡指数和iNOS表达阳性细胞率均低于损伤对照组（P<0.01或P<0.05）。结论：EGb761能抑制脊髓损伤后的脂质过氧化反应,减轻神经细胞的凋亡,其机制可能与抑制iNOS表达有关。     

二十烷酸对大鼠原代皮质神经元APP及BACE1表达的影响

目的 观察二十烷酸对大鼠原代皮质神经元β-淀粉样前体蛋白(APP)及β-分泌酶(BACE1)表达的影响,探讨饱和脂肪酸在阿尔茨海默病发病机制中的作用. 方法 将原代培养的大鼠皮质神经元分为2组:对照组、二十烷酸组.Western blotting法检测神经元APP、BACE1蛋白表达,RT-PCR法分析APP及BACE1 mRNA表达. 结果 与对照组相比,二十烷酸组神经元APP751+770mRNA表达和APP蛋白表达显著升高,BACE1蛋白表达也显著升高,差异有统计学意义(P＜0.05). 结论 饱和脂肪酸增加APP及BACE1表达,可能在阿尔茨海默病发病机制中起重要作用.     

缺氧及复氧对去整合蛋白和金属蛋白水解酶10和淀粉样β分泌酶mRNA水平的影响

目的研究缺氧及复氧损伤对去整合蛋白和金属蛋白水解酶10（ADAM10）和淀粉样β分泌酶（BACE1）mRNA的影响。方法研究分为缺氧12h组，缺氧24h组及各自对照组。缺氧组在缺氧条件下（95％N2，5％CO2）分别培养SH-SYSY细胞12h和24h后复氧，分别于0、12、24h利用逆转录一聚合酶链反应（BT-PCB）方法检测ADAM10、BACE1 mRNA水平；对照组在正常情况下培养，与缺氧组同一时间进行接种和处理。结果缺氧培养12h后复氧各时间点ADAM10 mRNA表达下调（分别较对照组下降19．8％、41．4％、64．6％；P=0．005、0．038、0．001），缺氧培养24h后复氧各时间点ADAM10 mRNA表达下调更明显（分别较对照组下降30．1％、75．9％、86．5％；P=0．009、0．005、0．043）；BACE1 mBNA在缺氧24h后复氧12h和24h表达上调（分别较对照组上调31．5％和35．1％；P=0．028、0．005）。结论缺氧及复氧可以降低ADAM10转录水平和增加BACE1转录水平。     

Egb761对脑慢性低灌注老龄大鼠海马β-分泌酶活性的影响

目的:探讨脑慢性低灌注老龄大鼠海马β-分泌酶活性的改变及银杏叶制剂(Egb761)对其的影响。方法:将大鼠随机分为假手术组、低灌注组和Egb761组,后两组永久性阻断双侧颈总动脉(2VO),Egb761组术后用Egb761灌胃。采用荧光法于术后1、2、4和16周检测双侧海马β-分泌酶的活性。结果:和假手术组相比,2VO组术后各时间点β-分泌酶活性均上调(P<0.05);Egb761组的β-分泌酶活性在用药期间低于2VO组(P<0.05)。结论:脑慢性低灌注可致β-分泌酶活性升高,Egb761可以抑制这种变化。     

头孢曲松对大鼠弥漫性轴索损伤后细胞凋亡的影响

目的探讨头孢曲松对大鼠弥漫性轴索损伤后海马CA1区神经元凋亡的影响。方法选择雄性Wistar大鼠120只,随机分为药物干预组(n=36):制作弥漫性轴索损伤模型,于制作模型后即刻给予头孢曲松200 mg·kg-1。假手术组(n=36):仅行头皮切开缝合处理,给予等量生理盐水腹腔注射。脑损伤组(n=36):制作弥漫性轴索损伤模型,给予等量生理盐水腹腔注射。正常对照组(n=12):不给予任何干预措施。采用免疫组化技术检测干预后12、24和36h时相海马CA1区凋亡蛋白酶激活因子-1(Apaf-1)蛋白的表达水平。结果脑损伤组和药物干预组大鼠海马CA1区Apaf-1蛋白表达水平均于12 h开始增加,24 h达高峰,此后逐渐下降。药物干预组大鼠海马CA1区Apaf-1蛋白表达水平在各个时相点均低于脑损伤组,均差异有统计学意义。结论弥漫性轴索损伤可引起大鼠海马CA1区神经元凋亡,头孢曲松可改善凋亡的发生、发展。     

Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer's disease

The standardized Ginkgo biloba extract EGb 761(definition see editorial) has been shown to produce neuroprotective effects in different in vivo and in vitro models. Since EGb 761 is a complex mixture containing flavonoid glycosides, terpene lactones (non-flavone fraction) and various other constituents, the question arises as to which of these compounds mediates the protective activity of EGb 761. Previous studies have demonstrated that the non-flavone fraction was responsible for the antihypoxic activity of EGb 761. Thus, we examined the neuroprotective and anti-apoptotic ability of the main constituents of the non-flavone fraction, the ginkgolides A, B, C, J and bilobalide. In focal cerebral ischemia models, the administration of bilobalide (5-20 mg/kg, s. c.) 60 min before ischemia dose-dependently reduced the infarct area in mouse brain and the infarct volume in rat brain 2 days after the onset of the injury. 30 minutes of pretreatment with ginkgolide A (50 mg/kg, s. c.) and ginkgolide B (100 mg/kg, s. c.) reduced the infarct area in the mouse model of focal ischemia. In primary cultures of hippocampal neurons and astrocytes from neonatal rats, ginkgolide B (1 microM) and bilobalide (10 microM) protected the neurons against damage caused by glutamate (1 mM, 1 h) as evaluated by trypan blue staining. In addition, bilobalide (0.1 microM) was able to increase the viability of cultured neurons from chick embryo telencepalon when exposed to cyanide (1 mM, 1h). Furthermore, we attempted to find out whether ginkgolides A, B, and J and bilobalide were also able to inhibit neuronal apoptosis (determined by nuclear staining with Hoechst 33 258 and TUNEL-staining). Ginkgolide B (10 microM), ginkgolide J (100 microM) and bilobalide (1 microM) reduced the apoptotic damage induced by serum deprivation (24h) or treatment with staurosporine (200 nM, 24h) in cultured chick embryonic neurons. Bilobalide (100 microM) rescued cultured rat hippocampal neurons from apoptosis caused by serum deprivation (24h), whereas ginkgolide B (100 microM) and bilobalide (100 microM) reduced apoptotic damage induced by staurosporine (300 nM, 24h). Ginkgolide A failed to affect apoptotic damage neither in serum-deprived nor in staurosporine-treated neurons. The results suggest that some of the constituents of the non-flavone fraction of EGb 761 possess neuroprotective and anti-apoptotic capacity, and that bilobalide is the most potent one. In contrast, ginkgolic acids (100-500 microM) induced neuronal death, which showed features of apoptosis as well as of necrosis, but these constituents were removed from EGb 761 below an amount of 0.0005 %. Taking together, there is experimental evidence for a neuroprotective effect of EGb 761 that agrees with clinical studies showing the efficacy of an oral treatment in patients with mild and moderate dementia.     

Preconditioning with Ginkgo biloba (EGb 761®) provides neuroprotection through HO1 and CRMP2

Ginkgo biloba/EGb 761? (EGb 761) is a popular and standardized natural extract used worldwide for the treatment of many ailments. Although EGb 761 is purported to have a plethora of benefits, here, we were interested to study the neuroprotective properties of EGb 761 and its components and determine whether nuclear factor E2 (Nrf2)/heme oxygenase 1 (HO1) induction of the collapsin response mediator protein 2 (CRMP2) pathway contributes to neuroprotection. Mice were pretreated with EGb 761 or one of its constituents (bilobalide, ginkgolide A, ginkgolide B, and terpene free material [TFM]) for 7days and then subjected to transient middle cerebral artery occlusion (tMCAO) and 48 h of reperfusion. All components except TFM significantly reduced infarct volumes and neurologic deficits. Next, we examined the antioxidant and neuritogenic properties of EGb 761 in primary neurons. Compared with vehicle-treated cells, pretreatment with EGb 761 significantly enhanced the survival of neurons exposed to tertiary butylhydroperoxide (t-BuOOH), hydrogen peroxide (H2O2), and N-methyl-D-aspartate (NMDA). Bilobalide and ginkgolide A also protected cells against NMDA-induced excitotoxicity. Immunofluorescence and Western blot analysis showed that EGb 761 pretreatment significantly increased the protein expression levels of Nrf2, HO1, GAPDH, β-actin, CRMP2, and histone H3 during t-BuOOH-induced oxidative stress. These findings suggest that EGb 761 not only has antioxidant activity but also neuritogenic potential. Demonstrating such effects for possible drug discovery may prove beneficial in stroke and ischemic brain injury.     

Amyloid precursor protein metabolism is regulated toward alpha-secretase pathway by Ginkgo biloba extracts

Clinical trials report that Ginkgo biloba extracts (e.g., EGb761) reduce cognitive symptoms in age-associated memory impairment and dementia, including Alzheimer disease (AD). However, the mechanisms behind their neuroprotective ability remain to be fully established. In this study, the effect of EGb761 on the amyloid precursor protein (APP) metabolism has been investigated by both in vitro and in vivo models. To this aim, alpha-secretase, the enzyme regulating the non-amyloidogenic processing of APP and the release of alphaAPPs, the alpha-secretase metabolite, were studied in superfusates of hippocampal slices after EGb761 incubation, and in hippocampi and cortices of EGb761-treated rats. PKC translocation state was evaluated as well. EGb761 increases alphaAPPs release through a PKC-independent manner. This effect is not accompanied by a modification of either APP forms or alpha-secretase expression. Moreover, EGb761 influence on alphaAPPs release was strictly dependent on treatment dosage. Our findings suggest that the benefit of EGb761 reported by previous clinical studies is underscored by a specific biological mechanism of this compound on APP metabolism, directly affecting the release of the non-amyloidogenic metabolite. Additional research will be needed to clearly define the effective clinical relevance, thus considering EGb761 as a possible supplementary treatment in dementing diseases.     

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.     

Neuroprotective effects of bilobalide, a component of the Ginkgo biloba extract (EGb 761), in gerbil global brain ischemia.

The neuroprotective effect of Ginkgo biloba extract (EGb 761) against ischemic injury has been demonstrated in animal models. In this study, we compared the protective effect of bilobalide, a purified terpene lactone from EGb 761, and EGb 761 against ischemic injury. We measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global forebrain ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In addition, both bilobalide and EGb 761 protected against ischemia-induced reductions in COX III mRNA in CA1 neurons prior to their death, at 1 day of reperfusion. These results suggest that oral administration of bilobalide and EGb 761 protect against ischemia-induced neuron death and reductions in mitochondrial gene expression.     

The Ginkgo biloba extract (EGb 761) protects hippocampal neurons against cell death induced by beta-amyloid

Substantial evidence suggests that the accumulation of beta-amyloid (Abeta)-derived peptides, and to a lesser extent free radicals, may contribute to the aetiology and/or progression of Alzheimer's disease (AD). Ginkgo biloba extract (EGb 761) is a well-defined plant extract containing two major groups of constituents, i.e. flavonoids and terpenoids. It is viewed as a polyvalent agent with a possible therapeutic use in the treatment of neurodegenerative diseases of multifactorial origin, e.g. AD. We have investigated here the potential effectiveness of EGb 761 against toxicity induced by (Abeta)-derived peptides (Abeta25-35, Abeta1-40 and Abeta1-42) on hippocampal primary cultured cells, this area being severely affected in AD. A co-treatment with EGb 761 concentration-dependently (10-100 microg/mL) protected hippocampal neurons against toxicity induced by Abeta fragments, with a maximal and complete protection at the highest concentration tested. Similar, albeit less potent protective effects were seen with the flavonoid fraction of the extract (CP 205), while the terpenes were ineffective. Most interestingly, EGb 761 (100 microg/mL) was even able to protect (up to 8 h) hippocampal cells from a pre-exposure to Abeta25-35 and Abeta1-40. EGb 761 was also able to both protect and rescue hippocampal cells from toxicity induced by H2O2 (50-150 microM), a major peroxide possibly involved in mediating Abeta toxicity. Moreover, EGb 761 (10-100 microg/mL), and to a lesser extent CP 205 (10-50 microg/mL), completely blocked Abeta-induced events, e.g. reactive oxygen species accumulation and apoptosis. These results suggest that the neuroprotective effects of EGb 761 are partly associated with its antioxidant properties and highlight its possible effectiveness in neurodegenerative diseases, e.g. AD via the inhibition of Abeta-induced toxicity and cell death.     

Ginkgo biloba extract EGb 761® in the context of current developments in the diagnosis and treatment of age-related cognitive decline and Alzheimer's disease: a research perspective

In June 2011 a two-day expert meeting "The Ageing Brain" took place in Amsterdam, The Netherlands. The main aim was to discuss the available preclinical and clinical data on Ginkgo biloba special extract EGb 761? in the context of current developments in the diagnosis and treatment of age-related cognitive decline and Alzheimer's disease. 19 dementia experts covering the disciplines bio- and neurochemistry, gerontology, neurology, pharmacology, and psychiatry from Australia, Asia, Europe and North America reviewed available preclinical and clinical data for EGb 761? and identified core topics for future research. Based on a wide range of preclinical effects demonstrated for Ginkgo biloba, EGb 761? can be conceptualized as a multi-target compound with activity on distinct pathophysiological pathways in Alzheimer's disease (AD) and age-related cognitive decline. While symptomatic efficacy in dementia and mild cognitive impairment (MCI) has been demonstrated, interpretation of data from dementia prevention trials is complicated by important methodological issues. Bridging pre-clinical research and clinical research as well as deciding on suitable study designs for future trials with EGb 761? remain important questions. The participants of the "Ageing Brain" meeting on Ginkgo biloba special extract EGb 761? concluded that there is plenty of promising data, both pre-clinical and clinical, to consider future research with the compound targeting cognitive impairment in old age as a worthwhile activity.     

A ginkgo biloba extract promotes proliferation of endogenous neural stem cells in vascular dementia rats

The ginkgo biloba extract EGb761 improves memory loss and cognitive impairments in patients with senile dementia. It also promotes proliferation of neural stem cells in the subventricular zone in Parkinson’s disease model mice and in the hippocampal zone of young epileptic rats. However, it remains unclear whether EGb761 enhances proliferation of endogenous neural stem cells in the brain of rats with vascular dementia. In this study, a vascular dementia model was established by repeatedly clipping and reperfusing the bilateral common carotid arteries of rats in combination with an intraperitoneal injection of a sodium nitroprusside solution. Seven days after establishing the model, rats were intragastrically given EGb761 at 50 mg/kg per day. Learning and memory abilities were assessed using the Morris water maze and proliferation of endogenous neural stem cells in the subventricular zone and dentate gyrus were labeled by 5-bromo-2-deoxyuridine immunofluorescence in all rats at 15 days, and 1, 2, and 4 months after model establishment. The escape latencies in Morris water maze tests of rats with vascular dementia after EGb761 treatment were significantly shorter than the model group. Immunofluorescence staining showed that the number and proliferation of 5-bromo-2-deoxyuridine-positive cells in the subventricular zone and dentate gyrus of the EGb761-treated group were significantly higher than in the model group. These experimental findings suggest that EGb761 enhances proliferation of neural stem cells in the subventricular zone and dentate gyrus, and significantly improves learning and memory in rats with vascular dementia.     

Neuroprotective effects of bilobalide, a component of Ginkgo biloba extract (EGb 761) in global brain ischemia and in excitotoxicity-induced neuronal death

In this study, we compared the protective effect of bilobalide, a purified terpene lactone component of ginkgo biloba extract EGb 761, (definition see editorial) and EGb 761 against ischemic injury and against glutamate-induced excitotoxic neuronal death. In ischemic injury, we measured neuronal loss and the levels of mitochondrial DNA (mtDNA)-encoded cytochrome oxidase (COX) subunit III mRNA in vulnerable hippocampal regions of gerbils. At 7 days of reperfusion after 5 min of transient global ischemia, a significant increase in neuronal death and a significant decrease in COX III mRNA were observed in the hippocampal CA1 neurons. Oral administration of EGb 761 at 25, 50 and 100 mg/kg/day and bilobalide at 3 and 6 mg/kg/day for 7 days before ischemia progressively protected CA1 neurons from death and from ischemia-induced reductions in COX III mRNA. In rat cerebellar neuronal cultures, addition of bilobalide or EGb 761 protected in a dose-dependent manner against glutamate-induced excitotoxic neuronal death (effective concentration [EC (50)] = 5 microg/ml (12 microM) for bilobalide and 100 microg/ml for EGb 761. These results suggest that both EGb 761 and bilobalide are protective against ischemia-induced neuronal death in vivo and glutamate-induced neuronal death in vitro by synergistic mechanisms involving anti-excitotoxicity, inhibition of free radical generation, scavenging of reactive oxygen species, and regulation of mitochondrial gene expression.