丹酚酸B对β-淀粉样蛋白介导原代培养皮层神经元毒性的保护作用

目的　观察一氧化氮 (NO)在谷氨酸 (Glu) ,β 淀粉样蛋白 [β AP(1 40 ) ]引起神经细胞损伤中的作用以及丹酚酸B(SalB)对 β AP(1 40 )引起的神经细胞损伤保护作用。 方法　原代培养大鼠皮层神经元 ,建立了硝普钠(SNP) ,Glu ,β AP(1 40 )等 3种损伤模型 ,用形态学观察、MTT比色法、Griess法分别测定神经元活力 ,培养液中乳酸脱氢酶 (LDH)漏出和NO释放。结果　Glu和 β AP(1 40 )可以引起神经元NO的释放增加 ,造成神经毒性。nNOS在Glu的毒性中起重要作用 ,iNOS可能在Aβ1 4 0 的毒性中起作用。SalB能显著增加细胞活力 ,降低LDH释放率 ,并剂量依赖地减少NO释放。结论　NO介导了Glu和 β AP(1 40 )的毒性 ,SalB可以通过减少NO释放 ,改善 β AP(1 40 )的毒性作用。     

丹酚酸B和银杏叶提取物EGb 761对β-淀粉样蛋白神经毒性抑制作用的比较

目的比较丹酚酸B(Sal B)和银杏叶提取物EGb 761对β-淀粉样蛋白(β-AP)纤维形成及细胞毒作用的影响。方法运用硫黄素T(ThT)荧光法和电子显微镜技术分析Sal B和EGb 761对β-AP_1-40聚集和纤维形成的影响；另将β-AP_25-35预先老化7 d，用MTT法和流式细胞仪检测两种提取物对此老化蛋白造成的PC12细胞毒性的保护作用，用荧光法观察β-AP_25-35作用后细胞内活性氧含量的变化以及两种提取物的作用。结果Sal B和EGb 761都可有效地抑制β-AP_1-40纤维的形成，明显抑制老化的β-AP_25-35对PC12细胞的毒性作用，降低β-AP_25-35造成的细胞内活性氧含量的增加。Sal B的有效剂量远远低于EGb 761。结论Sal B对β-AP神经毒性的抑制作用强于EGb 761。     

谷氨酸诱导体外培养的鸡胚脊髓神经细胞释放NO

用鸡胚脊髓神经细胞的原代培养,测定细胞中亚硝酸盐的含量,研究了谷氨酸(Glu)对原代培养神经细胞中NO的影响。结果表明,谷氨酸作用于原代培养的鸡胚脊髓神经细胞,在诱导神经细胞释放乳酸脱氢酶(LDH)的同时,还可诱导细胞释放一氧化氮(NO)。如先用NO合成酶抑制剂L-NOARG作用细胞,再加入Glu,则发现L-NOARG能降低Glu导致的培养液中LDH活性升高。提示NO可能参与介导Glu的神经毒性作用。     

银杏叶提取物对谷氨酸诱导神经细胞损伤的作用研究

目的：探究银杏叶提取物（EGb）对谷氨酸诱导体外培养神经细胞损伤的保护作用及相关机制,为EGb应用于缺血性卒中提供依据。方法：取孕15～18天SD胎鼠的皮层神经细胞进行原代培养（对照组）,在此基础上,建立谷氨酸诱导神经细胞损伤模型（Glu组）。向Glu组中预先加入4种不同浓度（12．5～100mg·L^-1）EGb作用（研究组）。通过测定神经细胞NO及LDH的释放量来反映神经细胞损伤程度。结果：谷氨酸呈浓度依赖性升高神经细胞NO及LDH释放量;与Glu组相比较,EGb（12．5～100mg·L^-1）能不同程度的抑制NO、LDI-I释放,且EGb浓度为50mg·L^-1时作用最显著（P〈0．01）。结论：谷氨酸通过NO诱导神经细胞损伤,EGb通过抑制NO、LDH释放实现拮抗谷氨酸神经细胞毒性作用。     

氯丙嗪对谷氨酸诱导大鼠脑皮质受损神经细胞的保护作用

目的:观察氯丙嗪(CPZ)对谷氨酸(Glu)诱导的大鼠大脑皮质神经细胞兴奋毒性的影响.方法:用倒置显微镜观察神经细胞形态的变化,用Trypan blue染色法观察细胞生存率,测定乳酸脱氢酶(LDH)以定量反映细胞损伤程度.结果:50 μmol•L 1Glu使神经细胞死亡率升高,LDH释放增加351%;1,10 μmol•L 1CPZ能改善神经细胞生长,降低细胞死亡率,减少LDH的释放.结论:CPZ可明显抑制Glu介导的神经毒性作用.     

褪黑激素对β—淀粉样多肽25—35片段所致皮层海马神经细胞毒性损伤的防护作用

目的:观察褪黑激素对β-淀粉样多肽25-35片段(Nβ_(25-35)所致神经细胞毒性作用的影响.方法:用相衬倒置显微镜观察原代培养的神经细胞的形态;用噻唑兰(MTT)法、乳酸脱氢酶(LDH)测定及台盼兰染色观察神经细胞的存活情况.结果:Aβ_(25-35)(20μmol/L)可引起培养的神经细胞数量减少,部分突起消失;台盼兰着色细胞数增加;神经细胞增殖能力降低;培养上清液中LDH释放量增加;褪黑激素(Ⅰ,10μmol/L)可抑制Aβ_(25-35)诱导的上述毒性损伤.结论:Aβ_(25-35)对原代培养的皮质-海马神经细胞有直接的细胞毒作用,褪黑激素对Aβ_(25-35)所致神经细胞毒性损伤具有剂量依赖性的防护作用.     

褪黑素对大脑皮层细胞一氧化氮含量及其神经毒性作用的影响

目的：探讨褪黑素(MT)抗衰老作用与神经细胞NO释放之间的联系。方法：连续给予老年小鼠MT，检测大脑皮层神经细胞NO含量的变化,并用原代培养的大鼠皮层神经元，去血清培养后，观察MT对高钾、谷氨酸（Glu）诱发NO释放及硝普钠（SNP）致神经毒性作用的影响。结果：MT能明显抑制老年小鼠脑内NO含量的增高，并拮抗KCI与Glu诱发的NO释放及SNP引起的神经毒性，对大脑神经元有保护作用。结论：MT抑制大脑皮层NO含量增高，可能是其抗衰老作用的机制之一。     

糖皮质激素促进β-淀粉样蛋白对海马神经元损伤的研究

目的在体外实验条件下,观察糖皮质激素是否促进β-淀粉样蛋白的神经元毒性作用。方法通过LDH释放率法检测细胞活力、TUNEL法测细胞凋亡以及LSCM测胞内Ca²⁺浓度的变化。结果单独的地塞米松(10^-8,10^-7,10^-6mol·L^-1)组或Aβ25-35(0.5μmol·L^-1)组分别作用海马神经元后,细胞活力变化不明显(P>0.05);但与同剂量的DEX、Aβ_25-35组相比,DEX+Aβ_25-35组的细胞活力明显降低(P<0.05),同时胞内Ca²⁺显著上升(P<0.05),神经元的凋亡率增加明显,细胞内出现凋亡的典型形态学特征。结论糖皮质激素可能通过促进Aβ引起海马神经元胞内Ca²⁺升高增加神经元毒性作用。     

9-(4-乙氧羰基苯氧基)-6,7-二甲氧基-1,2,3,4-四氢吖啶盐酸盐抑制自由基诱发鼠皮层神经元毒性及脑缺血损伤

目的考察9-(4-乙氧羰基苯氧基)-6,7-二甲氧基-1,2,3,4-四氢吖啶盐酸盐(EDT)对自由基致原代培养大鼠皮层神经毒及小鼠脑缺血损伤的影响。方法原代培养的鼠皮层神经细胞,用H₂O₂致自由基损伤模型,测定细胞内丙二醛(MDA)及超氧化物歧化酶(SOD)活性。结扎单侧颈总动脉及迷走神经造成小鼠慢性脑缺血模型,用跳台法研究EDT对记忆障碍的影响。同时,检测了大脑皮层形态学变化,脑匀浆内MDA,NO含量及SOD活力。结果在原代培养神经元,0.01～3 μmol·L^-1 EDT浓度依赖地抑制H₂O₂诱发的MDA生成及SOD活力降低。在小鼠脑缺血模型,EDT 2.5,5和10 mg·kg^-1 ig 5 d可显著改善脑缺血小鼠的记忆障碍,对抗脑内NO释放及MDA生成,增加SOD活力。结论EDT能有效对抗自由基诱发的神经元毒性及脑缺血损伤。     

五味子酚对氧化低密度脂蛋白引起的血管内皮细胞及神经细胞损伤的保护作用

目的：氧化低密度脂蛋白（ox-LDL）对血管内皮细胞及神经细胞均有毒性作用，与动脉粥样硬化，神经退行性疾病的发展密切相关。本文探讨了具有很强抗氧化作用的五味子有效成分五味子酚（Sal）对ox-LDI。引起的牛主动脉内皮细胞及神经细胞NGl08-15的损伤和凋亡的保护作用及其初步的机制。方法：以细胞形态，MTT法，LDH释放检测ox-LDL对细胞的损伤及Sal的作用；DNA电泳，核染色质荧光染色观察细胞的凋亡，流式细胞术检测凋亡细胞的百分率；自由基（ROS）荧光探针DCF检测细胞内ROS变化，Western-blot法检测线粒体细胞色素C的释放。结果：ox-LDL可引起内皮细胞和NGl08-15细胞的损伤、凋亡，细胞形态发生明显变化，存活率降低，胞内LDH泄漏增加，出现核染色质聚集、DNA梯状条带，流式细胞术检测出现G1亚峰，凋亡细胞百分率显著增加，并可刺激细胞内ROS产生，促进NGl08-15细胞线粒体细胞色素C的释放增加。提前加入Sal对上述细胞损伤具有明显的保护作用，并能显著抑制细胞的凋亡，减少细胞内ROS产生，降低细胞色素C的表达。结论：Sal能抑制ox-LDL引起的血管内皮细胞及神经细胞的毒性损伤、凋亡，其机制可能与减少胞内自由基、抑制线粒体细胞色素C的释放有关。     

藻酸双酯钠对培养神经细胞谷氨酸兴奋毒性的保护作用

目的 :观察藻酸双酯钠 (PSS)对培养神经细胞谷氨酸兴奋毒性的保护作用。方法 :体外培养大鼠胚胎皮层神经细胞 ,加入谷氨酸观察谷氨酸对神经细胞的兴奋毒性及PSS的保护作用。结果 :PSS 5 0 ,1 0 0 ,1 5 0mg·L-1能显著减少细胞死亡 ,降低乳酸脱氢酶(LDH)漏出量 ,一氧化氮 (NO)含量及丙二醛 (MDA)生成 ,提高超氧歧化酶 (SOD)活性。结论 :PSS对培养神经细胞谷氨酸兴奋毒性具有显著的保护作用 ,其机制可能与PSS抗脂质过氧化作用有关。     

褪黑激素对大脑皮层谷氨酸释放及其神经毒的拮抗作用

AIM: To observe the effects of melatonin (Mel) on glutamate (Glu) release from the cortical synaptosomes in old mice and on neurotoxicity induced by KCl, Glu in cultured cortical cells of fetal rat and to explore the antiaging mechanism of Mel. METHODS: Glu release by the synaptosomes in old mouse cerebral cortex was detected in a spectrofluorophotometer. The neuronal viability in primary cultures from rat cerebral cortex was assessed using MTT stain and lactate dehydrogenase (LDH) efflux in the bathing medium. RESULTS: Mel inhibited the K+ (30 mmol.L-1)-induced Glu release from synaptosomes either in calcium dependent or independent conditions [control (10.6 +/- 1.1), (9.2 +/- 0.7) mumol.g-1 (protein); Mel 0.1 mumol.L-1 (6.5 +/- 0.9), (7.5 +/- 0.6) mumol.g-1 (protein), respectively, P < 0.01 vs control group), increased MTT activity (control 0.67 +/- 0.04, 0.81 +/- 0.03; Mel 0.1 mumol.L-1 0.715 +/- 0.023, 0.925 +/- 0.027, P < 0.01 vs control group] and decreased LDH efflux (control 0.400 +/- 0.016, 0.379 +/- 0.016; Mel 0.1 mumol.L-1 0.345 +/- 0.021, 0.340 +/- 0.012, respectively, P < 0.01 vs control group), therefore, protected the neuronal viability against KCl and Glu-induced injury. CONCLUSION: The inhibitory effect of Mel on Glu release from cortical synaptosome and the protective effect of Mel on cortical neurons against neurotoxicity are its antiaging mechanisms.     

L—焦谷氨酸对抗从氨酸钠诱发的大鼠皮层神经元损伤

目的：在大鼠皮层神经元研究Ｌ－吡咯烷酮羧酸（Ｌ－ＰＧＡ）对谷氨酸钠（Ｇｌｕ）诱发神经毒性的拮抗作用。方法：原代培养的皮层神经元取自１６ｄ龄的胎鼠,与Ｇｌｕ作用３０分钟,２４后测定神经元的存活及增益昌质中亚硝酸盐的浓度;以Ｆｕｒａ ２－ＡＭｏ ｘｌｅｑｍｗ 〖Ｃａ＾２＋〗;荧光探针,,ＡＲ－ＣＭ－ＭＩＣ阳离子测定系统测定〖Ｃａ＾２＋〗ｉ。结果：Ｌ－ＰＧＡ１０－８０βｍｏｌ．Ｌ＾－１浓度依赖地抑制Ｇ     

Serofendic acid prevents acute glutamate neurotoxicity in cultured cortical neurons

We have previously reported that a novel neuroprotective substance named serofendic acid was purified and isolated from ether extract of fetal calf serum. In the present study, we investigated the effect of serofendic acid on acute neurotoxicity induced by L-glutamate (Glu) using primary cultures of rat cortical neurons. Exposure of cortical cultures to Glu for 1 h caused a marked decrease in cell viability, as determined by trypan blue exclusion. This acute Glu neurotoxicity was prevented by N-methyl-D-aspartate (NMDA) receptor antagonists, extracellular Ca(2+) removal, nitric oxide (NO) synthase inhibitor and NO scavenger. Serofendic acid prevented acute Glu neurotoxicity in a concentration-dependent manner. Acute neurotoxicity was induced by ionomycin, a Ca(2+) ionophore, and S-nitroso-L-cysteine, an NO donor. Serofendic acid also prevented both ionomycin- and S-nitroso-L-cysteine-induced neurotoxicity. Moreover, the protective effect of serofendic acid on acute Glu neurotoxicity was not affected by cycloheximide, a protein synthesis inhibitor, and actinomycin D, an RNA synthesis inhibitor. These results indicate that serofendic acid protects cultured cortical neurons from acute Glu neurotoxicity by reducing the cytotoxic action of NO and de novo protein synthesis is not required for this neuroprotection.     

l—S.R—蝙蝠葛苏林碱通过减少一氧化氮的产生保护培养的海马神经元对 …

AIM: To explore mechanisms of l-S.R-daurisoline (DS)-mediated protection of cultured hippocampal neurons from sodium glutamate (Glu) cytotoxicity. METHODS: Cultured neurons obtained from rat hippocampus were used to examine the protective effect of DS against Glu neurotoxicity. Cell viability was estimated using trypan blue dye exclusion method and [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay. Release of nitric oxide (NO) from the hippocampus was assayed using rat thoracic aorta in vitro. RESULTS: DS 0.01-10 mumol.L-1 concentration-dependently inhibited Glu cytotoxicity and increased cell viability with 50% prevention of cell death 2.8 mumol.L-1 (95% confidence limit 1.2-5.9 mumol.L-1). This protection was mostly attenuated by L-arginine (Arg) 1 mmol.L-1. DS 0.01-10 mumol.L-1 did not prevent sodium nitropusside (SNP) 500 mumol.L-1-induced cytotoxicity. DS 10 mumol.L-1 blocked Glu-elicited relaxation of the endothelium-denued rat aortic rings contracted by norepinephrine (NE) 10 mumol.L-1 in the presence of hippocampal tissue, but did not affect that induced by SNP. This indicated that DS inhibited Glu-triggered NO generation but did not prevent the effects of NO. CONCLUSION: DS prevented neurons from Glu neurotoxicity by inhibiting Glu-triggered NO generation.     

The role of NO and B-50 in neurotoxicity of excitatory amino acids

To investigate the direct evidence for the role which nitric oxide (NO) plays in the neurotoxicity of excitatory amino acids, we evaluated NO level by Greiss testing solution when glutamate (Glu) and kainate (KA) induced neuronal degeneration in primary cortical cultures. Glutamate-induced neurotoxicity was accompanied by a rise in NO. 5 mM hemoglobin (Hb) led to a decrease of NO content and prevented excitotoxicity induced by 1 mM glutamate. 1 mM L-arginine (L-Arg) reversed the effect of hemoglobin by raising the NO level. No change in NO content was found in KA-induced neurotoxicity, which was not affected by L-Arg, Hb or L-Arg + Hb. It is suggested that NO plays an important role in glutamate-, but not KA-induced neurotoxicity in primary cortical cultures. We also investigated the effects of glutamate on a growth-associated protein, B-50. The B-50 level declined significantly 24 h after exposure to 100 microM glutamate for 30 min and then recovered 2 days later. The effect of glutamate on B-50 was concentration-dependent. This indicates that B-50 might be involved in both glutamate neurotoxicity and the following neuronal repair process.     

Nitric oxide-mediated effect of nipradilol, an alpha- and beta-adrenergic blocker, on glutamate neurotoxicity in rat cortical cultures

Nipradilol (3,4-dihydro-8-(2-hydroxy-3-isopropylamino)propoxy-3-nitroxy-2H-1-benzopyran) is used clinically as an anti-glaucoma ophthalmic solution in Japan, and was recently reported to suppress N-methyl-d-aspartate-induced retinal damage in rats. Here we investigated cytotoxic and cytoprotective actions of nipradilol on primary cultures of rat cortical neurons. Treatment of cortical cultures with a high concentration (500 microM) of nipradilol significantly reduced cell viability, increased lactate dehydrogenase (LDH) release and nitrite concentration in culture medium, whereas desnitro-nipradilol (3,4-dihydro-8-(2-hydroxy-3-isopropylamino)propoxy-3-hydroxy-2H-1-benzopyran) had no significant effects. Nipradilol-induced neuronal damage was inhibited by S-hexylglutathione, a glutathione S-transferase inhibitor, and FeTPPS (5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III) chloride), a peroxynitrite decomposition catalyst. On the other hand, relatively low concentrations (10-100 microM) of nipradilol but not desnitro-nipradilol prevented neuronal cell death induced by 24 h application of 100 microM glutamate. Importantly, neuroprotective concentration (100 microM) of nipradilol suppressed glutamate-induced elevation of intracellular Ca2+ concentrations, but had no effect on intracellular cyclic GMP levels. Hence, nipradilol can protect cultured cortical neurons against glutamate neurotoxicity via cyclic GMP-independent mechanisms, and nitric oxide (NO) released from the nitoroxy moiety of nipradilol may mediate neuroprotective effect through the modulation of NMDA receptor function.