二苯乙烯苷对脑缺血啮齿动物脑NMDA受体及细胞内钙离子的影响

目的　研究二苯乙烯苷对脑缺血再灌注啮齿动物脑组织N 甲基 D 天 (门 )冬氨酸 (NMDA)受体结合力的影响 ,同时观察二苯乙烯苷对神经细胞内钙离子浓度的影响 ,初步探讨二苯乙烯苷对缺血再灌注动物的脑保护作用的机制。方法　麻醉后夹闭沙土鼠和小鼠双侧颈总动脉 10min后 ,小心撤去动脉夹制作缺血再灌注动物模型 :(1)沙土鼠再灌注 7d后处死 ,取前脑进行NMDA受体结合力实验 ;(2 )小鼠再灌注 15min后迅速断头取脑 ,切成脑片 ,加入Fluo 3/AM负载后 ,在激光扫描共聚焦显微镜上进行光切 ,观察不同层面皮层和海马区细胞内游离钙的荧光强度。结果　模型组的NMDA受体结合力比假手术组明显升高 ,而二苯乙烯苷治疗组可以降低模型动物的NMDA受体结合力 ;脑片的光切结果显示 ,脑缺血再灌注模型组神经细胞内游离钙离子浓度比假手术组明显升高 ;与模型组相比 ,二苯乙烯苷治疗组的细胞内游离钙离子浓度降低。结论　二苯乙烯苷能够抑制啮齿动物脑缺血再灌注所导致的脑组织NMDA受体结合力及神经细胞内钙离子浓度的升高 ,减轻钙超载导致的脑组织损伤 ,可能具有脑保护作用     

何首乌中芪合酶基因的克隆、鉴定及其功能分析

2,3,5,4’-四羟基二苯乙烯-2-O-β-D-葡萄糖苷(2,3,5,4’-tetrahydroxy-stilbene-2-O-β-D-glucoside,TSG),简称二苯乙烯苷,是我国传统大宗中药材何首乌特有的药效成分,具有抗氧化、清除自由基等功效。二苯乙烯苷属于芪类次生代谢     

HPLC法测定降脂方中2,3,5,4’-四羟基二苯乙烯苷-2-O-β-D-葡萄糖苷的含量

目的建立降脂方中何首乌的有效成分2,3,5,4’-四羟基二苯乙烯苷-2-O-β-D-葡萄糖苷的测定方法。方法改进药典法,采用高效液相色谱法对降脂方中主药何首乌的主要成分2,3,5,4’-四羟基二苯乙烯苷-2-O-β-D-葡萄糖苷进行含量测定,色谱柱为Hypersil CN(10μm,4.6mm×150mm);流动相:乙腈-水(18∶82);检测波长:320nm;柱温:25℃;流速:1mL/min,并与药典法进行比较。结果 2,3,5,4’-四羟基二苯乙烯苷-2-O-β-D-葡萄糖苷在20.48~102.4μg/mL线性关系良好(r=0.999975),理论塔板数3207,分离度4.08,平均回收率100.76%,RSD为0.402%。结论改进法的结果与药典法相当,但相对于药典法,其流动相有机溶剂使用更少,环境污染少,对供试品的分离度更好,含量测定更准确,适用于降脂方中22,3,5,4’-四羟基二苯乙烯苷-2-O-β-D-葡萄糖苷的含量测定。     

超高效液相色谱法测定安乐片中甘草苷和2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷含量

目的 用UPLC法测定安乐片中甘草苷和2,3,5,4-四羟基二苯乙烯-2-O-β-D-葡萄糖苷含量.方法 色谱柱为Waters BEH C18 (50 mm×2.1 mm×1.7 μm),流动相为乙腈-水(10:90),流速为0.2 mL·min-1,检测波长为280 nm,柱温 20 ℃.结果 甘草苷在133.25～2665 ng与峰面积线性关系良好(r=0.9998),2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷在127.25～2545 ng与峰面积线性关系良好(r=0.9997),其平均回收率(n=6)分别为99.3% (RSD为1.6%)和96.6% (RSD为1.7%).结论 本法灵敏、准确、专属性强,能简便有效的控制安乐片中甘草苷和2,3,5,4'-四羟基二苯乙烯-2-O-β-D-葡萄糖苷的含量.     

超高效液相色谱法测定安乐片中甘草苷和2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷含量

目的用UPLC法测定安乐片中甘草苷和2,3,5,4-四羟基二苯乙烯-2-O-β-D-葡萄糖苷含量。方法色谱柱为Waters BEHC18(50 mm×2.1 mm×1.7μm),流动相为乙腈-水(10∶90),流速为0.2 mL.min-1,检测波长为280 nm,柱温20℃。结果甘草苷在133.25~2 665 ng与峰面积线性关系良好(r=0.999 8),2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷在127.25~2 545 ng与峰面积线性关系良好(r=0.9997),其平均回收率(n=6)分别为99.3%(RSD为1.6%)和96.6%(RSD为1.7%)。结论本法灵敏、准确、专属性强,能简便有效的控制安乐片中甘草苷和2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷的含量。     

HPLC法测定扶正颗粒中2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷的含量

目的对扶正颗粒中的主要有效成分2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷,建立含量测定方法。方法采用高效液相色谱法,测定本制剂中二苯乙烯苷的含量。结果 2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷的含量测定线性范围在0.026～0.130mg·mL~(-1),平均回收率为99.32%。结论建立的方法准确、稳定、重现性好,可用于该制剂的质量控制。     

何首乌中芪合酶基因的克隆、鉴定及其功能分析

2,3,5,4'-四,羟基二苯乙烯-2-O-β-D-葡萄糖苷(2,3,5,4'-tetrahydroxy-stilbene-2-O-β-D-glucoside,TSG),简称二苯乙烯苷,是我国传统大宗中药材何首乌特有的药效成分,具有抗氧化、清除自由基等功效.二苯乙烯苷属于芪类次生代谢产物,现有研究证实,以白藜芦醇为代表的三羟基芪类化合物是通过苯丙氨酸代谢途径,由芪合酶(stilbene synthase,STS)催化底物丙二酰-CoA和香豆酰-CoA合成[2].何首乌中含有的二苯乙烯苷为四羟基芪类物质,其生物合成是否经相同的途径催化合成至今还未见报道,芪合酶作为这一代谢途径的关键酶,成为解答这一问题的关键.     

HPLC法测定复方首乌补液中二苯乙烯苷的含量

目的:建立测定复方首乌补液中二苯乙烯苷(2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷)含量的高效液相色谱法。方法:hypersil ODS柱,流动相为乙腈:水(30:70);流速1.0mL/min,检测波长320nm。结果:2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷线性范围为25-400mg/mL,r=0.999 5;平均回收率为99.4%。结论:本方法分离效果好,方法简便、快速,适用于该制剂的含量测定。     

HPLC测定安尔眠胶囊中2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷

目的建立安尔眠胶囊中2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷（C20H2209）的含量测定方法。方法采用高效液相色谱（HPLC）法,以Dionex Acclaim 120C18色谱柱（150 mm×4.6 mm,5μm）为分离柱,以乙腈-1%甲酸溶液（23：77）为流动相,体积流量1.0 mL/min,检测波长320 nm,柱温25℃。结果 2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷在0.010~0.200μg呈现良好的线性关系（r=0.999 6）,平均回收率为97.35%,RSD为2.07%。结论该方法准确可靠,适用于安尔眠胶囊中2,3,5,4′-四羟基二苯乙烯-2-O-β-D-葡萄糖苷的含量测定。     

石杉碱甲对血管性痴呆小鼠海马NMDA受体及mRNA表达的影响

目的:观测石杉碱甲对血管性痴呆小鼠海马神经元N-甲基D-天门冬氨酸(NMDA)受体免疫组化和原位杂交变化的影响,探讨石杉碱甲干预作用的机制.方法:双侧颈总动脉线结,反复缺血-再灌注法制备模型,药物组用石杉碱甲溶液灌胃,跳台试验和水迷宫试验观测其行为学改变,采用免疫组化和原位杂交技术观测小鼠海马神经元NMDA受体及其mRNA的表达变化.结果:在跳台试验中,模型组小鼠学习阶段的反应时间延长、错误次数增加,记忆阶段的潜伏时间缩短、错误次数增加;水迷宫试验中,其学习阶段的游全程时间延长和错误次数增加,记忆阶段的游全程时间延长和错误次数增加(与假手术组相比,P＜0.01).石杉碱甲明显改善了模型小鼠上述学习、记忆成绩(P＜0.01).同时,药物组海马的NMDA受体及其mRNA表达也较模型组的表达明显增高(P＜0.01),且海马两者的表达在药物组与假手术组间差异无显著性.结论:石杉碱甲改善血管性痴呆小鼠学习、记忆成绩与其恢复海马低水平的NMDA受体及其mRNA有关.     

Effects of developmental lead exposure on inhibitory avoidance learning and glutamate receptors in rats

Chronic lead (Pb) exposure during development is known to produce learning deficits. AMPA and NMDA receptors have been shown to participate in the synaptic mechanisms involved in certain forms of learning and memory. We investigated whether the effects of Pb on AMPA and NMDA receptors are associated with Pb-induced impairment in learning and memory. Rats were exposed to 0.2% lead acetate at different developmental stages including a maternally exposed group (including gestation and lactation period), a postweaning exposed group, and a continuously exposed group. Lead treatment impaired learning acquisition, but not memory retention in step-down avoidance learning task in all treatment groups. In parallel with the behavioral data, autoradiographic analyses of brain sections indicated that the [³H]AMPA binding was decreased in the CA1 and dentate gyrus of the hippocampus and entorhinal cortex in all three Pb-exposed groups. However, an increase in [³H]MK801 binding was only observed in CA1 of the hippocampus in the continuously Pb-exposed rats. The findings suggest that alterations in AMPA receptor may contribute to the Pb-induced deficits in learning acquisition of inhibitory avoidance.     

ZDY102对拟痴呆大鼠脑M受体的影响

目的　观察知母活性成分ZMS的异构体ZDY10 2对拟痴呆大鼠脑M受体及学习记忆功能的影响。方法　大鼠单侧脑基底核部位定位注射 β 淀粉样肽 (2 5～ 35 )和鹅蕈膏酸造成拟痴呆模型 ,设立不同剂量ZDY10 2治疗组、模型组和对照组 (注射生理盐水 ) ,分别用Y 迷宫测定学习记忆能力及放射配基结合分析法测定注射同侧脑内M胆碱受体密度。结果　拟痴呆模型大鼠学习和记忆成绩和脑M受体密度明显低于对照组。连续两个月喂服ZDY10 2的模型大鼠学习和记忆能力显著好于对照组 ,脑M受体密度也显著提高 ,而且脑M受体密度与动物的学习记忆能力呈显著正相关。结论　ZDY10 2能明显改善拟痴呆大鼠的学习记忆能力 ,同时能显著提高脑内M受体密度 ,从两者的显著正相关来看 ,提高脑M受体密度可能是ZDY10 2改善拟痴呆模型学习记忆的重要机制。和以往对知母活性成分的研究结果相比较可以看出 ,2 5位甲基的立体异构对上述效果无明显影响     

Visual recognition memory is related to basic expression level of NMDA receptor NR1／NR2B subtype in hippocampus and striatum of rats

AIM: To examine the basic expression levels of N-methyl-D-aspartate (NMDA) receptor NR1 and NR2B subunits in six brain regions of Sprague-Dawley (SD) rats with different visual recognition memory. METHODS: Rats were tested by a novel-object-recognition model and grouped into the high and the low visual recognition memory groups. The expression levels of NR1 and NR2B subunits in the cortex, hippocampus, striatum, amygdala, diencephalon, and olfactory bulb were measured by semiquantitative immunoblotting. RESULTS: The NR1 and NR2B subunit protein levels in the hippocampus of the high visual recognition memory group were 35.9% (P<0.01) and 53.4% (P<0.05) higher respectively than those in the low group. In addition, the NR2B level in the striatum in the high visual recognition memory group was 25.0% (P<0.05) higher than that in the low one. However, no significant difference was found in the levels of the subunits between the two groups in other brain regions. CONCLUSION: The visual recognition memory in rats is related to the basic expression level of NMDA receptor NR1/NR2B subtype in the hippocampus and striatum.     

Hippocampal NMDA receptors and anxiety: At the interface between cognition and emotion

David De Wied had a fundamental interest in the brain and behaviour, with a particular interest in the interface between cognition and emotion, and how impairments at this interface could underlie human psychopathology. The NMDA subtype of glutamate receptor is an important mediator of synaptic plasticity and plays a central role in the neurobiological mechanisms of emotionality, as well as learning and memory. NMDA receptor antagonists affect various aspects of emotionality including fear, anxiety and depression, as well as impairing certain forms of learning and memory. The hippocampus is a key brain structure, implicated in both cognition and emotion. Lesion studies in animals have suggested that dorsal and ventral sub-regions of the hippocampus are differentially involved in dissociable aspects of hippocampus-dependent behaviour. Cytotoxic lesions of the dorsal hippocampus (septal pole) in rodents impair spatial learning but have no effect on anxiety, whereas ventral hippocampal lesions reduce anxiety but are without effect on spatial memory. This role for the ventral hippocampus in anxiety is distinct from the role of the amygdala in other aspects of emotional processing, such as fear conditioning. Recent studies with genetically modified mice have shown that NR1 NMDA receptor subunit deletion, specifically from the granule cells of the dentate gyrus, not only impairs short-term spatial memory but also reduces anxiety. This suggests that NMDA receptors in ventral hippocampus may be a key locus supporting the anxiolytic effects of NMDA receptor antagonists. These data support Gray's neuropsychological account of hippocampal function.     

Memantine improves memory for taste-avoidance learning in day-old chicks exposed to isolation stress

Activation of NMDA receptors by glutamate is particularly important in the initial stages of memory consolidation. Memantine, a noncompetitive NMDA receptor antagonist, ameliorates memory impairment under certain circumstances, despite blocking the activation of NMDA receptors. The present experiments tested the hypothesis that memantine can improve memory deficits induced by isolation stress in day-old chicks (Gallus gallus domesticus) trained in a one-trial taste-avoidance task. Three experiments assessed the effects of memantine at different concentrations and in combination with isolation stress. The results of Experiment 1 indicate that, under normal, non-stressed conditions, memory in control animals is strong and 15.0 mM memantine impairs memory, similar to that seen in many studies of the effects of NMDA receptor antagonists on learning. However, the results of Experiments 2 and 3 showed that, when chicks were exposed to isolation stress during the pre-training period, memory formation for saline-injected control animals was impaired and 5.0 mM memantine significantly improved memory in an inverted U-shaped dose response function. The current results extend the findings that memantine can ameliorate memory impairment and supports the hypothesis that memantine, despite its action to reduce NMDA receptor activity, can facilitate normalized memory acquisition.     

知母皂苷元及其异构体对老年大鼠学习记忆和脑内M_1受体密度的影响

目的　观察知母皂苷元 (ZMS)及其异构体 (ZMR)对老年大鼠学习记忆和脑内M1受体密度的作用。方法　选择 2 4mon龄SD老年大鼠 ,将动物分为老年对照组、ZMS组和ZMR组 ,并以 3～ 4mon龄青年大鼠作为正常对照 ,用迷宫法测定学习记忆能力 ,采用放射配基结合分析法测定脑内M1受体密度。结果　用药组大鼠连续口服ZMS和ZMR 4 0d后 ,与老年对照组比较 ,其学习记忆能力明显增强 ,脑内M1受体密度升高。结论　知母皂苷元及其异构体对老年性痴呆的胆碱能系统功能渐进性退化有一定的预防和治疗作用     

知母皂苷元对拟痴呆大鼠β-淀粉样肽沉积及胆碱能系统功能的影响

目的　观察知母皂苷元 (ZMS)对拟痴呆大鼠模型脑内 β 淀粉样肽沉积及胆碱能系统功能的作用。 方法　单侧基底核内联合注射 β 淀粉样肽2 5-3 5片段 (β AP2 5-3 5)和兴奋性氨基酸建立大鼠拟痴呆模型 ,然后将模型动物分为假手术组 37、模型组和ZMS组 ,采用免疫组织化学方法和图像分析法测定β AP2 5-3 5沉积斑块的面积。用避暗法及跳台法测定动物的学习记忆功能 ,用放射配基结合分析法分别测定脑胆碱乙酰转移酶 (ChAT)活性和M受体密度。结果　一次性脑内联合注射 β AP +IBO后 ,模型大鼠脑内有明显的 β AP斑块沉积 ,而模型大鼠喂服ZMS 6 0d后 ,能有效地减少β AP沉积的面积 ,同时ZMS能明显改善模型动物的学习记忆功能 ,并提高模型动物脑内ChAT活性和M受体密度。结论　ZMS可能对脑内 β AP的沉积有一定的清除作用 ,并对AD低下的胆碱能系统功能有一定的改善和治疗作用     

电针相关井穴治疗血管性痴呆记忆障碍的实验研究

目的观察电针相关井穴(少冲、中冲、涌泉和少商)对血管性痴呆(VD)大鼠学习记忆和脑及血清一氧化氮(NO)水平的影响。方法104只大鼠随机分为假手术组、模型组、井穴组和西药组。参照改良Pulsinelli4血管闭塞法(4-VO)获得VD大鼠模型,记录治疗前后跳台法行为学测试潜伏期和3min内错误次数;采用硝酸还原酶法检测治疗后大鼠脑组织及血清中NO含量。结果治疗前假手术组潜伏期较长,错误次数较少,和其余各组比较均有非常显著性差异(P<0.01);治疗后井穴组和模型组比较,潜伏期显著延长(P<0.01),错误次数显著减少(P<0.05),脑组织(P<0.05)和血清(P<0.01)NO含量明显降低。井穴组和西药组在潜伏期、错误次数和NO水平上的比较均无显著性差异(P>0.05)。结论电针相关井穴能显著改善VD大鼠的记忆障碍,降低脑组织及血清中NO含量。对脑缺血损伤后NO的过度产生和相关神经毒性的抑制可能是其作用机制之一。     

NMDA受体2A亚单位mRNA和蛋白质在大鼠海马前脑缺血再灌注损伤中的变化

目的观察分析大鼠前脑缺血再灌注早期,NMDA受体2A亚单位mRNA和蛋白质表达的改变,探讨两者在缺血性脑损伤中的变化和相互关系。方法选择SD大鼠30只,随机为分正常对照组（NC组）6只,假手术对照组（SC组）12只,缺血再藻注组（IR组）12只,大鼠脑8μm厚石蜡切片,原位杂交染色,TUNEL染色,焦油紫染色。图像和镜下分析。结果（1）NR2A mRNA在海马的CA1区缺血24h后,显著高于NC组水平,是SC组的141．5％（P〈0．01）。（2）NR2A蛋白质水平在海马的CA1区缺血复灌48h后,显著下降,是SC组的59．3％（P〈0．01）。结论缺血性脑损伤早期中,NMDA受体2A亚单位mRNA和蛋白质表达并不呈现一致变化,提示缺血过程中2A亚单位的转录水平未受影响,而翻译水平受到了干扰。