Effect of Xingnaojing Injection(醒脑静注射液)on Hippocampal N-methyl-D-aspartic Acid Receptors of Focal Cerebral Ischemia in Rats

Objective: To observe and elucidate the neuroprotective effect of Xingnaojing (XNJ) injection on hippocampal N-methyl-D-aspartic acid (NMDA) receptors of focal cerebral ischemia in rats. Methods: Cerebral ischemia was established by occluding the middle cerebral artery with an intraluminal suture technique in rats. Neurological deficit score, infarct volume and quantity of NMDA receptors were estimated in all groups and compared. Results: After being treated with XNJ, the score decreased in the initial 6 hours and infarct volume decreased in 24 hours. And within 24 hours, the quantity of NMDA receptors obviously decreased compared with the model group (P<0. 01) It indicated that XNJ could ameliorate neurological behavior of middle cerebral artery occlusion rats and down-regulate the expression of hippocampal NMDA receptors. Conclusion: The neuroprotective effect of XNJ on focal cerebral ischemia is possibly related to down-regulating the expression of NMDA receptors in rats.

Water-soluble lipopolymer delivery of N-methyl-D-aspartic acid receptor 2B siRNA relieves chronic neuropathic pain in rats

Spinal dorsal horn N-Methyl-D-aspartic acid receptor 2B （NR2B） overexpression plays an important role in the production and maintenance of neuropathic pain. Because small interfering RNA （siRNA） can inhibit NR2B expression, siRNA may provide a novel approach to treat neuropathic pain and possibly nerve injury. However, an efficient and safe vector for NR2B siRNA has not been discovered. This study shows that a water soluble lipopolymer （WSLP） comprised of low molecular weight polyethyleneimine （PEI） and cholesterol can deliver siRNA targeting NR2B for the treatment of neuropathic pain. Results show that intrathecal injection of WSLP/siRNA complexes for 3 days inhibit NR2B gene expression with reductions in mRNA and protein levels by 59% and 54%, respectively, compared with control rats （P 〈 0.01）. Injection of WSLP complexed with scrambled siRNA, or PEI with siRNA did not show this inhibitory effect. Moreover, injection of WSLP/siRNA complexes significantly relieved neuropathic pain at 3, 7, 12, and 21 days, while injection of WSLP with scrambled siRNA or PEI with siRNA did not. These results demonstrate that WSLP can efficiently deliver siRNA targeting NR2B in vivo and relieve neuropathic pain.

Comparison of three administration modes for establishing a zebrafish seizure model induced by N-Methyl-D-aspartic acid

BACKGROUND Epilepsy is a complex neurological disorder characterized by recurrent,unprovoked seizures resulting from the sudden abnormal discharge of brain neurons.It leads to transient brain dysfunction,manifested by abnormal physical movements and consciousness.It can occur at any age,affecting approximately 65 million worldwide,one third of which are still estimated to suffer from refractory seizures.There is an urgent need for further establishment of seizure models in animals,which provides an approach to model epilepsy and could be used to identify novel anti-epileptic therapeutics in the future.AIM To compare three administration modes for establishing a seizure model caused by N-Methyl-D-aspartic acid(NMDA)in zebrafish.METHODS Three administration routes of NMDA,including immersion,intravitreal injection and intraperitoneal injection,were compared with regard to their effects on inducing seizure-like behaviors in adult zebrafish.We evaluated neurotoxicity by observing behavioral changes in zebrafish and graded those behaviors with a seizure score.In addition,the protective effects of MK-801(Dizocilpine)and natural active constituent resveratrol against NMDA-induced alterations were studied.RESULTS The three NMDA-administration methods triggered different patterns of the epileptic process in adult zebrafish.Seizure scores were increased after increasing NMDA concentration regardless of the mode of administration.However,the curve of immersion continuously rose to a high plateau(after 50 min),while the curves of intravitreal injection and intraperitoneal injection showed a spike in the early stage(10-20 min)followed by a steady decrease in seizure scores.Furthermore,pretreatment with resveratrol and MK-801 significantly delayed seizure onset time and lowered seizure scores.CONCLUSION By comparing the three methods of administration,intravitreal injection of NMDA was the most suitable for establishing an acute epileptic model in zebrafish.Thus,intraperitoneal injection in zebrafish can be applied to simulate diseases such as epilepsy.In addition,NMDA immersion may be an appropriate method to induce persistent seizures.Moreover,MK-801 and resveratrol showed strong anti-epileptic effects;thus,both of them may be clinically valuable treatments for epilepsy.

Targeting harmful effects of non-excitatory amino acids as an alternative therapeutic strategy to reduce ischemic damage

The involvement of the excitatory amino acids glutamate and aspartate in ce rebral ischemia and excitotoxicity is well-documented.Nevertheless,the role of non-excitatory amino acids in brain damage following a stroke or brain trauma remains largely understudied.The release of amino acids by necrotic cells in the ischemic core may contribute to the expansion of the penumbra.Our findings indicated that the reversible loss of field excitato ry postsynaptic potentials caused by transient hypoxia became irreversible when exposed to a mixture of just four non-excitatory amino acids(L-alanine,glycine,L-glutamine,and L-serine)at their plasma concentrations.These amino acids induce swelling in the somas of neurons and astrocytes during hypoxia,along with permanent dendritic damage mediated by N-methyl-D-aspartate receptors.Blocking N-methyl-D-aspartate receptors prevented neuronal damage in the presence of these amino acids during hypoxia.It is likely that astroglial swelling caused by the accumulation of these amino acids via the alanine-serine-cysteine transporter 2 exchanger and system N transporters activates volume-regulated anion channels,leading to the release of excitotoxins and subsequent neuronal damage through N-methyl-D-aspartate receptor activation.Thus,previously unrecognized mechanisms involving non-excitatory amino acids may contribute to the progression and expansion of brain injury in neurological emergencies such as stroke and traumatic brain injury.Understanding these pathways co uld highlight new therapeutic targets to mitigate brain injury.

Kainic Acid, NMDA and Bicuculline Induce Elevation in Concentrations of Glutathione and Amino Acids <i>in Vivo</i>: Biomarkers for Seizure Predisposition?

The present study was carried out to investigate the effect of NMDA, bicuculline and kainic acid (KA) on the extracellular concentration of glutathione, phosphoethanolamine (PEA) and taurine in rat hippocampus in vivo. Rats were implanted with intrahippocampal microelectrodes perfused with free-glucose Krebs-Ringer solution and allowed to recover for about 2 h. After assaying baseline concentrations of amino acids, NMDA or bicuculline was administered intrahippocampally, whereas KA was given systemically. Either treatment resulted in significant high extracellular concentrations of glutathione, but only NMDA or KA resulted in high concentrations of PEA and taurine. Interestingly, the increase in glutathione concentration due to KA was followed by a delayed increase of glutamate and PEA. Our results demonstrated that increased efflux of glutathione, a common consequence of different neuroexcitotoxic agents, occurs in vivo. Given that the agents used in the present study were also convulsunts, the implication of the findings on seizure predisposition was also considered.

去氢表雄酮对抗NMDA神经毒性与抑制细胞内Ca^(2+)升高有关(英文)

去氢表雄酮(Dehydroepiandrosterone,DHEA)是一种神经甾体物质,虽然已经发现对中枢神经系统有保护作用,但其分子机制尚未阐明.从胚胎小鼠(E15～16)大脑皮层分离神经元,离体培养10d,用MTT法检测DHEA对抗NMDA神经毒性的作用.用荧光染料Fluo＿3处理后,激光共聚焦显微镜分析胞内Ca2+浓度的变化,研究了DHEA神经保护作用与胞内Ca2+浓度的相关性.结果发现:(1)NMDA的剂量、时间依赖方式降低神经元的活性,(2)DHEA预处理可以明显改善NMDA诱导的形态学变化,且剂量依赖性预防NMDA的毒性,(3)1mmoL/LNMDA诱导细胞内游离Ca2+荧光浓度的迅速升高,而DHEA可以阻断这种变化.以上数据表明,DHEA可以保护神经元,对抗NMDA的神经毒性作用,其机制可能与抑制胞内Ca2+升高有关.

脊髓EAAs及NMDA受体在神经病理性疼痛及针刺镇痛中的作用研究进展

神经病理性疼痛以自发性疼痛、痛觉过敏、痛觉超敏为特征,其发病机制目前还不十分清楚。脊髓背角神经元的中枢敏化在神经病理性疼痛的产生和维持中发挥了重要的作用。而脊髓兴奋性氨基酸及其NMDA受体介导了脊髓中枢敏化现象,参与了自发性疼痛、痛觉过敏、痛觉超敏的形成,在神经病理性疼痛的形成和维持中具有重要作用。针刺可通过降低脊髓兴奋性氨基酸的含量及NMDA受体的表达,抑制中枢敏感化的产生,减缓痛觉过敏的形成从而达到镇痛的效应。

NMDA对培养的大鼠腺垂体细胞分泌生长激素影响的研究

应用细胞培养、结合放射免疫测定法探讨了 N-甲基 -D-天冬氨酸 (NMDA)对培养的大鼠腺垂体细胞分泌生长激素 (GH)的影响。结果表明 :在腺垂体细胞培养中 ,试验组 (分别添加1 0 - 8、1 0 - 6 、1 0 - 4M NMDA)培养液中的 GH比对照组分别提高了 67.92 % ,87.46% ,1 0 0 .67% ,差异显著 (P <0 .0 5)。试验表明 NM-DA能直接刺激离体腺垂体细胞分泌 GH。

雌激素和Glu-NMDA受体通路与学习记忆相关性的研究进展

雌激素(estrogen,E)是维持女性第二性征最主要的甾体物质,其在神经系统也发挥广泛的药理作用,如抗脑缺血再灌注损伤减少梗死面积,抗自由基形成,调节兴奋性氨基酸释放等。雌激素自身作为一种抗氧化剂能够通过调节体内氧化还原平衡发挥神经保护作用。另外,雌激素在神经系统药理作用的发挥与其受体密不可分,经典的雌激素受体(estrogen receptor,ER)主要位于细胞核内,包括ER-α和ER-β两种亚型,二者均可在大脑皮质和海马表达:卵巢切除大鼠学习记忆能力降低的同时,ER-α在大脑皮质区和海马区表达也显著减少;ER-β基因敲除能够严重影响学习记忆行为、长时程增强(long term potentiation,LTP)和突触强度,该受体可能参与了高级脑功能的调节。N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)受体作为与学习记忆密切相关的受体,与雌激素受体在海马区存在共表达。雌激素可能通过膜相关的雌激素受体快速激活ERK1/2信号转导通路,进一步诱导NMDA受体NR2B亚基磷酸化,激活NMDA受体,三者均会影响突触可塑性。本文就雌激素及其受体和Glu-NMDA受体通路与学习记忆的相关性进行综述。

豹皮菌中2－Amino－3－（1，2－dicarboxyethylthio）propanoic Acid非对映异构体的分离和鉴定

从豹皮菌中的新鲜子实体的乙醇提取物中分离得到一种ＮＭＤＡ受体格抗剂，经理化常数和波谱数据的测定鉴定为２－Ａｍｉｎｏ－３－（１，２－ｄｉｃａｒｂｏｘｙｅｔｈｙｌｔｈｉｏ）ｐｒｏｐａｎｏｉｃａｃｉｄ非对映异构体的混合物，该化合物是首次从豹皮菌中得到，经进一步分离，得到两种非对映体异构体。

非NMDA受体拮抗剂对大鼠牙髓伤害性刺激诱导的延髓Fos表达

目的 研究兴奋性氨基酸非 NMDA受体在牙髓伤害性信息传递中的作用 ,为揭示牙痛的产生机制打下基础 .方法 将实验动物随机分为 4组 .第 1组单纯给予机械穿髓刺激 ;第 2组预先侧脑室注射非 NMDA受体拮抗剂 CNQX,再给予相同的穿髓刺激 ;第 3组只行 CNQX侧脑室注射 ,不予穿髓刺激 ;第 4组为正常对照组 .动物存活相同时间后取延髓做 Fos蛋白免疫组化反应 ,观察 3组动物 Fos蛋白表达变化 .结果 第 2组动物延髓 Fos免疫反应阳性细胞的数量与第 1组比较没有显著性差异 .第 3组只有很少 Fos阳性细胞出现 .结论 兴奋性氨基酸非 NMDA受体在机械穿髓致痛动物模型中很可能不参与介导牙髓伤害性信息的中枢传递 .

酸性环境抑制视网膜水平细胞NMDA受体活动

目的:研究酸性环境对NMDA受体的影响;方法:在分离的视网膜水平细胞上,施加不同pH值的含有NMDA和Glycine的Ringer液,测量NMDA受体电流;结果:低pH值对NMDA受体活动有抑制作用,半效抑制效应的pH值为6.6;结论:低pH值条件下,NMDA受体受到抑制,提示缺血等病理条件下的酸性环境抑制NMDA受体,起到保护作用。

N-methyl-D-aspartic acid receptor 1 （NMDAR1） aggravates secondary inflammatory damage induced by hemin-NLRP3 pathway after intracerebral hemorrhage

Objective： Inflammation plays a critical role in secondary brain damage after intracerebral hemorrhage （ICH）. However, the mechanisms of inflammatory injury following ICH are still unclear, particularly the involvement of NLRP3 inflammasome, which are crucial to sterile inflammatory responses. In this study, we aim to test the hypothesis that NLRP3 signaling pathway takes a vital position in ICH-induced sec- ondary inflammatory damage and detect the role of N-methyl-D-aspartic acid receptor 1 （NMDARI） in this progress. Methods： ICH was induced in mice by microinjection of heroin into the striatum. The protein levels of NMDAR1, NMDAR1 phosphorylation, NLRP3 and IL-113 were measured by Western blot. The binding of NMDARI to NLRP3 was detected by immunoprecipitation. Results： The expression of NMDARI, NMDAR1 phosphorylation, NLRP3 and IL-I ~ were rapidly increased after ICH. Heroin treatment enhanced NMDAR1 expression and NMDAR1 phosphorylation, as well in cultured microglial cells treated by hemin. Hemin up-regulated NLRP3 and IL-I]3 level, which was reversed by MK801 （NMDAR antagonist） in vitro. Hemin also promoted the binding of NMDAR1 to NLRP3. Conclusion： Our findings suggest that NMDARI plays a pivotal role in hemin-induced NLRP3-mediated inflammatory damage through synergistic activation.

Basic roles of key molecules connected with NMDAR signaling pathway on regulating learning and memory and synaptic plasticity

With key roles in essential brain functions ranging from the long-term potentiation(LTP) to synaptic plasticity,the N-methyl-D-aspartic acid receptor(NMDAR) can be considered as one of the fundamental glutamate receptors in the central nervous system.The role of NMDA R was first identified in synaptic plasticity and has been extensively studied.Some molecules,such as Ca^(2+),postsynaptic density 95(PSD-95),calcium/calmodulin-dependent protein kinase II(Ca MK II),protein kinase A(PKA),mitogen-activated protein kinase(MAPK) and cyclic adenosine monophosphate(c AMP) responsive element binding protein(CREB),are of special importance in learning and memory.This review mainly focused on the new research of key molecules connected with learning and memory,which played important roles in the NMDAR signaling pathway.

钩吻素己对NMDA受体的抑制作用

为了揭示钩吻素己对N-甲基-D-天冬氨酸(NMDA)受体活性的抑制作用,将钩吻素己、喹啉酸、钩吻素己+喹啉酸、地佐环平+喹啉酸分别作用于小鼠神经母瘤细胞(Neuro-2a),运用噻唑蓝(MTT)试验方法检测细胞活力度;采用5-乙炔基-2′-脱氧胞苷(EdU)细胞增殖试验方法检测细胞增殖;应用Western Blot试验方法研究钩吻素己对NMDA受体的抑制作用效果。结果显示,钩吻素己拮抗喹啉酸对Neuro-2a的细胞毒性,提高细胞的增殖能力(P<0.05),并且钩吻素己通过调控N-甲基-D-天冬氨酸ε2(NMDAε2)蛋白降低N-甲基-D-天冬氨酸ζ1(NMDAζ1)蛋白活性(P<0.05)。本试验结果表明钩吻素己能够有效抑制NMDA受体蛋白活性。

NMDA通过Wnt/β-catenin通路促进大鼠神经元中神经连接蛋白3表达

目的:探讨N-甲基-D-天冬氨酸(NMDA)对大鼠神经元来源的细胞系H19-7细胞中神经连接蛋白3(NLGN3)表达的影响及分子机制。方法:分别利用NMDA、NMDA受体(NMDAR)特异性拮抗剂D-AP5和β-catenin特异性siRNA处理大鼠海马神经元细胞系H19-7细胞,通过Western Blot和real time RT-PCR检测β-连环蛋白(β-catenin)、NLGN3及c-Myc的表达变化。结果:NMDA刺激后H19-7细胞中β-catenin蛋白上调,与此同时NLGN3和c-Myc表达增加;使用D-AP5阻断NMDAR以及特异性siRNA沉默β-catenin均能抑制NLGN3的表达。结论:NMDA能够通过Wnt/β-catenin通路促进大鼠神经元中NLGN3表达。

Excitatory amino acid on secondary spinal cord injury: effect anmechanism

Sprague-Dawley rats were subjected to traumatic spinal cord injury using Allen's method. Regional concentrations of excitatory amino acid (EAA) were assessed by high-performance liquid chromatography, and regional water and ion content were measured by the atomic absorption spectrophotometry at various times after intrathecal administration of different dosage of 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP),a specific antagonist of N-methyl-D-aspartame (NMDA) receptor,to rat at 48 h posttrauma.The results showed that concentrations of glutamate and aspartate were elevated rapidly at 15, 30 min and were correlated positively with the severity of injury. In the injured segment there was a significant increase in water, Na+ ,Ca++ content and a decrease in K+ , Mg++. Whereas in CPP treated groups they had a statistically significant return, which was related to the dosage and time of administration. This effect of CPP suggests that ion and water content were related to overactivation of NMDA receptor. Excessive high levels of EAA may be involved in secondary tissue damage following spinal cord injury.

"Smart fats", healthy brain and function of lipid-sensing NMDA receptors

NMDA receptor channels play a significant role in learning and memory and their dysfunction can cause neuronal cell death leading to dementia. Research had shown that lipids change the risk for dementia, especially some omega-3 lipids appear to lower Alz-heimer’s risk, yet only limited research exists on the modulation of NMDA receptor channels by lipids. Here we review recent literature concerning molecular determinants that influence the NMDA receptor channel gating via membrane lipids and fatty acids with profound significance for understanding how altered NMDA signalling leads to neuronal cell death linked to age-related dementia’s. Future discovery of lipid-like modulators of NMDA receptor function offer the potential for the development of new bioceu-ticals and affordable nutritional supplements to combat neuronal degeneration as well as to promote well being and healthy aging.

Inhibition of N-methyl-D-aspartate-activated Current by Bis(7)-tacrine in HEK-293 Cells Expressing NR1/NR2A or NR1/NR2B Receptors

In normal rat forebrain, the NR1/NR2A and NR1/NR2B dimmers are the main constitutional forms of NMDA receptors. The present study was carried out to determine the functional properties of the heteromeric NMDA receptor subunits and their inhibition by bis(7)-tacrine (B7T). Rat NR1, NR2A and NR2B cDNAs were transfected into human embryonic kidney 293 cells (HEK-293).The inhibition of NMDA-activated currents by B7T was detected in HEK-293 cell expressing NR1/NR2A or NR1/NR2B receptors by using whole-cell patch-clamp techniques. The results showed that in HEK-293 cells expressing NR1/NR2A receptor, 1μmol/L B7T inhibited 30μmol/L NMDA- and 1000μmol/L NMDA-activated steady-state currents by 46% and 40%, respectively (P>0.05; n=5), suggesting that the inhibition of B7T on NR1/NR2A receptor doesn’t depend on NMDA concentration, which is consistent with a non-competitive mechanism of inhibition. But for the NR1/NR2B receptor, 1μmol/L B7T inhibited 30μmol/L NMDA- and 1000 μmol/L NMDA-activated steady-state currents by 61% and 13%, re-spectively (P<0.05; n=6), showing that B7T appears to be competitive with NMDA. In addition, simultaneous application of 1μmol/L B7T and 1000μmol/L NMDA produced a moderate inhibition of peak NMDA-activated current, followed by a gradual decline of the current to a steady state. However, the gradual onset of inhibition produced by B7T applied simultaneously with NMDA was eliminated when B7T was given 5s before NMDA. These results suggested that B7T inhibition of NMDA current mediated by NR1/NR2B receptor was slow onset, and it did not depend on the presence of the agonist. With holding potentials ranging from -50 to +50 mV, the B7T inhibition rate of NMDA currents didn’t change significantly, and neither did the reversal potential. We are led to conclude that the NR1/NR2B recombinant receptor can serve as a very useful model for studying the molecular mechanism of NMDA receptor inhibition by B7T.

STAT3 ameliorates truncated tau-induced cognitive deficits

Proteolytic cleavage of tau by asparagine endopeptidase(AEP)creates tau-N368 fragments,which may drive the pathophysiology associated with synaptic dysfunction and memory deterioration in the brain of Alzheimer’s disease patients.Nonetheless,the molecular mechanisms of truncated tau-induced cognitive deficits remain unclear.Evidence suggests that signal transduction and activator of transcription-3(STAT3)is associated with modulating synaptic plasticity,cell apoptosis,and cognitive function.Using luciferase reporter assays,electrophoretic mobility shift assays,western blotting,and immunofluorescence,we found that human tau-N368 accumulation inhibited STAT3 activity by suppressing STAT3 translocation into the nucleus.Overexpression of STAT3 improved tau-N368-induced synaptic deficits and reduced neuronal loss,thereby improving the cognitive deficits in tau-N368 mice.Moreover,in tau-N368 mice,activation of STAT3 increased N-methyl-D-aspartic acid receptor levels,decreased Bcl-2 levels,reversed synaptic damage and neuronal loss,and thereby alleviated cognitive deficits caused by tau-N368.Taken together,STAT3 plays a critical role in truncated tau-related neuropathological changes.This indicates a new mechanism behind the effect of tau-N368 on synapses and memory deficits.STAT3 can be used as a new molecular target to treat tau-N368-induced protein pathology.