预缺血通过NMDA受体抑制海马CA1区Bcl-2磷酸化的研究

目的:研究预缺血以及联合给予预缺血和NMDA(N-甲基-D-天冬氨酸)受体抑制剂MK801后对大鼠海马CA1区Bcl-2的磷酸化以及海马CA1区锥体细胞凋亡的影响。方法:采用SD大鼠四动脉结扎全脑缺血及预缺血模型,给药组大鼠在预缺血前1h给予腹腔注射MK801 3mg/kg。用免疫印迹法分析不同处理下大鼠海马CA1区Bcl-2的蛋白表达及其磷酸化水平,焦油紫染色法分析海马CA1区锥体细胞的凋亡情况。结果:脑缺血再灌注组相对于Sham组Bcl-2的磷酸化水平以及海马CA1区锥体细胞的凋亡水平显著增高,预缺血组相对于缺血再灌注组Bcl-2的磷酸化水平以及海马CA1区锥体细胞的凋亡水平显著降低;而预缺血前给予MK801组相对于预缺血组Bcl-2磷酸化水平以及海马CA1区锥体细胞的凋亡水平显著增高;而Bcl-2的蛋白表达水平在以上不同处理条件下均无明显变化。结论:NMDA受体介导了预缺血抑制脑缺血再灌注诱导增加Bcl-2磷酸化以及海马CA1区锥体细胞凋亡。     

皮质酮对原代培养海马神经元的毒性作用及NMDA受体亚基表达的改变

目的研究皮质酮对大鼠海马神经元的毒性作用及NMDA受体亚基表达的影响.方法以体外原代培养的大鼠海马神经元为研究对象,根据影响因素,即给予的不同浓度皮质酮和其它因素分为8个组:对照组、10-7mol/L皮质酮组(简称10-7组)、10-6mol/L皮质酮组(简称10-6组)、10-5mol/L皮质酮组(简称10-5组)、10-6 高糖组、10-5 高糖组、10-6mol/L MK801组和10-5mol/L MK801组,镜下观察不同浓度皮质酮作用下海马神经元形态学的变化,并采用MTT方法测量各组细胞存活率,利用免疫细胞化学结合图象分析对原代培养海马神经元NMDA受体亚基的表达进行观察.结果 10-6、10-5浓度的皮质酮对海马神经元影响较大,细胞存活率较对照组明显降低,但10-6 高糖组、 10-5mol/L 高糖组、10-6mol/L MK801及10-5mol/L MK801 4个组,分别与相同皮质酮浓度处理组比较,细胞存活率显著提高.10-6和10-5组海马神经元上NMDA受体亚基表达较对照组明显降低.10-7mol/L浓度的皮质酮对上述指标影响不大.结论过量的皮质酮对大鼠海马神经元具有损伤作用,NMDA受体参与了此过程,NMDA受体拮抗剂和高浓度葡萄糖可保护海马神经元.     

NMDA受体通道参与大鼠脊髓背角C纤维诱发电位LTP的表达

以往研究表明,激动NMDA受体是引起海马长时程增强(LTP)的必备条件,而LTP的表达主要与AMPA受体的磷酸化及其受体组装到突触后膜有关.但是,近年来有研究表明NMDA受体通道也参与了LTP的表达.为探讨NMDA受体通道是否参与了脊髓背角C纤维诱发电位LTP的表达,诱导LTP后,分别静脉或脊髓局部给予NMDA受体拮抗剂MK801或APV,观察其作用.发现静脉注射非竞争性NMDA受体MK801(0.1mg/kg)对脊髓LTP无影响,注射0.5mg/kg显著抑制LTP,但是当剂量增高到1.0mg/kg时,抑制作用并未进一步增大.脊髓局部给予MK801也能抑制脊髓背角LTP.为验证上述结果,使用了竞争性NMDA受体拮抗剂APⅤ.结果显示,脊髓局部给予50μmol/LAPⅤ对LTP无影响,100μmol/L对LTP有显著的抑制作用,当浓度升至200μmol/L时,抑制作用并未见进一步增强.因此认为,NMDA受体通道部分地参与了脊髓背角C纤维诱发电位LTP的表达.     

NMDA受体介导脑缺血/再灌注诱导GluR6 巯基亚硝基化的研究

目的:研究脑缺血再灌注以及联合给予脑缺血和NMDA(N-甲基-D-天冬氨酸)受体抑制剂MK801对大鼠海马CA1区Glu R6巯基亚硝基化以及海马CA1区锥体细胞凋亡的影响。方法:采用四动脉结扎法构建大鼠全脑缺血再灌注模型,给予SD大鼠腹腔注射NMDA受体特异性抑制剂MK801(3 mg/kg)。主要运用'生物素转化法'(Biotin-Switch method)、SDS-PAGE、免疫印迹、焦油紫染色等方法对Glu R6的巯基亚硝基化(S-亚硝基化)、蛋白表达水平以及海马CA1区锥体细胞的凋亡水平进行研究。结果:脑缺血/再灌注显著促进Glu R6的巯基亚硝基化以及海马CA1区锥体细胞的凋亡,给予NMDA受体特异性抑制剂MK801能够显著抑制脑缺血/复灌诱导增加的Glu R6的S-亚硝基化以及海马CA1区锥体细胞的凋亡。结论:脑缺血/再灌注早期NMDA受体介导了Glu R6的巯基亚硝基化以及海马CA1区锥体细胞的凋亡,从而为临床治疗缺血再灌注脑损伤提供了理论依据。     

Sertraline联合Aripiprazole可显著上调大鼠脑部的CREB和BDNF水平

为了探讨SSRI联合抗精神病药物对脑源性神经营养因子(brain derived neurotrophic factor, BDNF)-cAMP反应元件结合蛋白(cAMP response element binding, CREB)信号通路的影响,本研究将SD大鼠随机分成5组,每组10只,各组大鼠分别腹腔注射阿立哌唑(5 mg·kg-1·d-1,阿立哌唑组)、舍曲林(5 mg·kg-1·d-1,舍曲林组)、阿立哌唑+舍曲林(5 mg·kg-1·d-1+5 mg·kg-1·d-1,联合组),奥氮平(5 mg·kg-1·d-1,奥氮平组)和不含药物的溶液(对照组),连续注射3周。研究显示,联合组显著增加大鼠的海马区BDNF平均荧光强度和蛋白水平,但在其他组未观察到对BDNF水平的影响。另外,不同组处理对额皮质中的BDNF水平没有影响。联合组显著增加了海马和额皮质的CREB磷酸化,而单独药物处理对CREB磷酸化无影响。联合组显著增加大鼠的海马和额皮质中CREB和TrkB (BDNF受体)的mRNA表达水平,以及AKT的磷酸化。综上所述,舍曲林联合抗精神病药(阿立哌唑)可显著上调大鼠脑部的CREB和BDNF水平,并且参与调节BDNF-CREB-AKT信号通路及相关分子。     

针刺百会穴对产后抑郁小鼠行为学改变和海马区NMDAR相关蛋白表达的影响

摘要 目的：研究针刺百会穴对产后抑郁小鼠行为学改变和海马区N-甲基-D-天冬氨酸受体（NMDAR）相关蛋白表达的影响。方法：30只C57BL/6母鼠被随机分为对照组、模型组和治疗组,每组30只。模型组和治疗组小鼠在妊娠期间通过皮下注射地塞米松磷酸钠建立产后抑郁小鼠模型,对照组小鼠皮下注射等量的生理盐水作为对照。治疗组小鼠分娩后通过针刺百会穴治疗14天,模型组和对照组小鼠不进行治疗。比较三组小鼠24 h食物消耗量和体重,黑白箱实验中白箱停留时间和黑白箱穿梭次数,以及强迫游泳实验不动状态时间和悬尾实验中悬尾不动时间。同时,通过免疫印记法检测三组小鼠海马去NMDA受体（NR2A和NR2B）、cAMP 结合蛋白(CREB) 和钙调蛋白激酶II(CaMKII) 蛋白表达水平。结果：治疗前,产后抑郁小鼠24 h食物消耗量、体重、白箱停留时间、黑白箱穿梭次数以及海马区CREB蛋白表达水平均显著低于对照组小鼠（P<0.05）,而游泳不同状态时间、悬尾不动时间和海马区NR2A、NR2B、cAMP蛋白表达水平均显著高于对照组小鼠（P<0.05）。治疗后,针刺百会穴治疗组小鼠24 h食物消耗量、体重、白箱停留时间和黑白箱穿梭次数以及海马区CREB蛋白表达水平均显著高于模型组小鼠（P<0.05）,而游泳不同状态时间、悬尾不动时间和海马区NR2A、NR2B、cAMP蛋白表达水平均显著低于模型组小鼠（P<0.05）。结论：针刺百会穴可以显著改善产后抑郁小鼠行为学情况,提高其运动能力,其可能与影响产后抑郁小鼠NMDAR相关蛋白表达有关。     

雌激素快速影响大鼠发育期海马NMDA受体活性的机制

为了探讨雌激素对发育期大鼠海马NMDA受体活性的快速影响,对出生后18d的雄性大鼠进行苯甲酸雌二醇皮下注射,1h后用WesternBlot检测海马NMDA受体NR1和NR2B亚基、雌激素β受体、ERK1/2蛋白的表达,以及NR2B和ERK1/2的磷酸化水平;并通过海马内给予雌激素受体拮抗剂ICI182,780和MEK1/2抑制剂U0126预处理,进一步分析苯甲酸雌二醇影响NR2B和ERK1/2磷酸化的作用机制。结果显示,苯甲酸雌二醇不影响NR1、NR2B、ERβ和ERK1/2的表达,但能快速增强NR2B和ERK1/2的磷酸化水平。雌激素受体拮抗剂ICI182,780和MEK1/2抑制剂U0126均能明显抑制苯甲酸雌二醇诱导的NR2B和ERK1/2磷酸化水平的增加。以上结果提示,雌激素可能通过雌激素受体的非基因组机制激活ERK/MAPK信号转导通路,快速诱导NMDA受体NR2B亚基磷酸化,激活NMDA受体。     

海马NMDA受体与神经肽Y在慢性应激性抑郁发生中的作用及其关系

本文旨在探讨N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid,NMDA)受体与神经肽Y(neuropeptide Y,NPY)在慢性应激抑郁发生中的作用与关系。建立慢性不可预见性温和应激(chronic unpredictable mild stress,CUMS)抑郁模型,海马单侧分别微量注射非竞争性NMDA受体拮抗剂MK-801、NPY-Y1受体阻断剂GR231118和NMDA后,利用体重测量及糖水偏爱测试、强迫游泳及敞箱实验等方法观察动物行为变化,运用免疫组织化学方法检测海马CA3区和齿状回(dentate gyrus,DG)内NPY的表达。结果显示,CUMS组大鼠表现出抑郁样行为变化,海马NPY表达显著降低;海马微量注射NMDA或NPY-Y1受体阻断剂GR231118,动物行为学表现均与CUMS组相同,注射NMDA可使NPY表达显著降低;海马微量注射MK-801能明显改善应激引起的抑郁样行为表现,并使海马NPY表达增加。联合注射GR231118与MK-801后,GR231118可以显著减弱MK-801的抗抑郁样行为的效应。以上结果表明,CUMS可能使谷氨酸(glutamic acid,Glu)过量释放,NMDA受体过度激活,抑制NPY表达,导致抑郁发生。NPY抗抑郁作用主要是通过NPY-Y1受体实现。     

罗布麻叶总黄酮提取物促进小鼠抗抑郁基因CREB、BDNF的表达

罗布麻是中国药典收录的传统中药,本研究提取它的主要有效成分罗布麻总黄酮,采用经典抗抑郁评价模型小鼠强迫游泳实验对罗布麻叶总黄酮进行抗抑郁活性评价.试验中给小鼠罗布麻叶总黄酮提取物25 mg/kg、50 mg/kg和100 mg/kg后,观察罗布麻叶总黄酮对小鼠强迫游泳不动时间的影响,结果显示罗布麻总黄酮提取物能显著缩短小鼠强迫游泳不动时间(P<0.05),药效与氟西汀相似,表明罗布麻具有明显的抗抑郁活性.在此基础上,应用皮质酮损伤的PC12细胞模型,采用荧光相对定量RT-PCR法检测罗布麻对皮质酮损伤的PC12细胞中脑内脑源性神经营养因子(BDNF)和环磷酸腺苷反应元件结合蛋白(CREB)表达水平的影响.结果表明,皮质酮处理后PC12细胞中BDNF、CREB基因的表达量最低,罗布麻处理后,表达量显著增加,较处理前增加了近十倍,且呈剂量依赖性.本研究结果提示罗布麻抗抑郁机制可能是通过(AC-cAMP-CREB)信号通路促进BDNF、CREB的基因表达而发挥的.     

梓醇对Aβ损伤SH-SY5Y细胞BDNF表达的影响及机制研究

摘要 目的：探讨梓醇对β-淀粉样肽（β-amyloid, Aβ）损伤SH-SY5Y细胞脑源性神经营养因子（brain-derived neurotrophic factor, BDNF）表达的影响及调节BDNF表达的机制。方法：以全反式维甲酸诱导分化的神经母细胞瘤细胞SH-SY5Y为研究对象，研究梓醇对Aβ 损伤细胞的作用。采用MTT实验测定细胞存活率，ELISA测定BDNF的含量，Western blot检测转录因子cAMP反应元件结合蛋白（cAMP response element binding protein, CREB）及其活化形式磷酸化CREB（pCREB）表达量，RT-PCR测定BDNF mRNA及转录子的表达水平；用RNA干扰的方法阻断CREB表达后，用RT-PCR测定BDNF mRNA表达量的变化。结果：梓醇预保护提高Aβ损伤SH-SY5Y细胞的存活率，显著增加细胞培养上清中BDNF含量和胞内BDNF mRNA水平，促进BDNF转录子IV及其关键调节转录因子pCREB的表达，干扰CREB表达后，梓醇上调BDNF mRNA表达的作用部分消失。结论：梓醇可能通过上调CREB磷酸化促进BDNF的表达，从而发挥神经保护作用。     

Reactive Transformation and Increased BDNF Signaling by Hippocampal Astrocytes in Response to MK-801

MK-801, also known as dizocilpine, is a noncompetitive N-methyl-D-aspartic acid (NMDA) receptor antagonist that induces schizophrenia-like symptoms. While astrocytes have been implicated in the pathophysiology of psychiatric disorders, including schizophrenia, astrocytic responses to MK-801 and their significance to schizotypic symptoms are unclear. Changes in the expression levels of glial fibrillary acid protein (GFAP), a marker of astrocyte activation in response to a variety of pathogenic stimuli, were examined in the hippocampus of rats treated with the repeated MK-801 injection (0.5 mg/10ml/kg body weight for 6 days) and in primary cultured hippocampal astrocytes incubated with MK-801 (5 or 20 μM for 24 h). Moreover, the expression levels of BDNF and its receptors TrkB and p75 were examined in MK-801-treated astrocyte cultures. MK-801 treatment enhanced GFAP expression in the rat hippocampus and also increased the levels of GFAP protein and mRNA in hippocampal astrocytes in vitro. Treatment of cultured hippocampal astrocytes with MK-801 enhanced protein and mRNA levels of BDNF, TrkB, and p75. Collectively, our results suggest that hippocampal astrocytes may contribute to the pathophysiology of schizophrenia symptoms associated with NMDA receptor hypofunction by reactive transformation and altered BDNF signaling.     

Increased levels of messenger RNAs for neurotrophic factors in the brain during kindling epileptogenesis.

Kindling, induced by repeated subconvulsive electrical or chemical stimulations leads to progressive and permanent amplification of seizure activity, culminating in generalized seizures. We report that kindling induced by electrical stimulation in the ventral hippocampus leads to a marked and transient increase in mRNA for NGF and BDNF in the dentate gyrus, the parietal cortex, and the piriform cortex. BDNF mRNA increased also in the pyramidal layer of hippocampus and in the amygdaloid complex. No change was seen in the level of HDNF/NT-3 mRNA. The increased expression of NGF and BDNF mRNAs was not influenced by pretreatment with the NMDA receptor antagonist MK801, but was partially blocked by the quisqualate, AMPA receptor antagonist NBQX. The presumed subsequent increase of the trophic factors themselves may be important for kindling-associated plasticity in specific neuronal systems in the hippocampus, which could promote hyperexcitability and contribute to the development of epileptic syndromes.     

The effect of postnatal manganese exposure on the NMDA receptor signaling pathway in rat hippocampus

Overexposure to manganese (Mn) is associated with neurological disorders in children. Evidence indicated that N‐methyl‐d ‐aspartate (NMDA) receptor signaling pathway was critical for neurobehavioral function. However, whether NMDA receptor signaling pathway contributes to Mn‐induced neurotoxicity remains unknown. In this study, newborn Sprague–Dawley rats were randomly assigned to four groups exposed to 0, 10, 20, and 30 mg/kg of Mn²⁺ by intraperitoneal injection (n = 10/group: five males and five females). After 3 weeks of Mn exposure, messenger RNA (mRNA) and protein expression of NMDA receptor subunits (NR1, NR2A, and NR2B), cAMP‐response element binding protein (CREB), and brain‐derived neurotrophic factor (BDNF) in hippocampus were measured by real‐time quantitative RT‐PCR and Western blot. In Mn‐exposed rats, decreased mRNA and protein expression of NR1, NR2A, and NR2B, CREB, and BDNF was observed. The results imply that downregulated NMDA receptor signaling pathway may be of vital importance in the neuropathological process of Mn‐induced neurotoxicity.     

Effect of NMDA-receptor ligands on neocortical and hippocampal EEG activity of rat brain.

Neocortex and hippocampus play important role in motor activity, neuronal plasticity and learning and memory mechanisms. Electroencephalographic (EEG) activity of neocortex and hippocampus of rat following NMDA-receptor agonist, N-methyl-D-aspartate (NMDA), 0.25-2 nmol in 10 microliters, ICV and noncompetitive NMDA-receptor antagonists, MK 801 (0.025-0.1 mg/kg, ip) and ketamine (10-50 mg/kg, ip) at OH, 1/2H, 4H, 8H and 24H was recorded. The electrodes were implanted stereotaxically in hippocampus and neocortex respectively. NMDA (0.25 and 1 nmol) showed longer lasting decrease in amplitude in hippocampus and in frequency in cortical neurons while 2 nmol produced epileptogenic neurotoxicity. Opposite effect i.e. increase in amplitude in both, hippocampus and neocortex was observed with MK 801 and ketamine and these agents also showed longer lasting influence. Administration of MK 801 (0.05 mg/kg) and ketamine (50 mg/kg) prior to NMDA 2 nmol protected 40% animals from NMDA-induced neurotoxicity and blockade of NMDA-induced long term influence. The EEG effect of NMDA agonist and NMDA-induced neurotoxicity at higher dose and its modification by NMDA-antagonist, MK 801 and ketamine suggest that beside NMDA agonists (NMDA), its antagonists may, also affect long lasting changes in hippocampus and cortex. These antagonists reverse NMDA-mediated long term influence in these brain areas.     

The effects of chronic alcoholization on the expression of brain-derived neurotrophic factor and its receptors in the brains of mice genetically predisposed to depressive-like behavior

Brain-derived neurotropic factor (BDNF) plays an important role in mechanisms of depression. Precursor protein of this factor (proBDNF) can initiate apoptosis in the brain, while the mature form of BDNF is involved in neurogenesis. It is known that chronic alcoholization leads to the activation of apoptotic processes, neurodegeneration, brain injury, and cognitive dysfunction. In this work, we have studied the influence of long-term ethanol exposure on the proBDNF and BDNF protein levels, as well as on the expression of genes that encode these proteins in the brain structures of ASC mice with genetic predisposition to depressive-like behavior and in mice from parental nondepressive CBA strain. It was shown that chronic alcoholization results in a reduction of the BDNF level in the hippocampus and an increase in the amount of TrkB and p75 receptors in the frontal cortex of nondepressive CBA mice. At the same time, the long-term alcoholization of depressive ASC mice results in an increase of the proBDNF level in the frontal cortex and a reduction in the p75 protein level in the hippocampus. It has also been shown that, in depressive ASC mice, proBDNF and BDNF levels are significantly lower in the hippocampus and the frontal cortex compared with nondepressive CBA strain. However, no significant differences in the expression of genes encoding the studied proteins were observed. Thus, changes in the expression patterns of proBDNF, BDNF, and their receptors under the influence of alcoholization in the depressive ASC strain and nondepressive CBA strain mice are different.     

Different pathways for iNOS-mediated toxicity in vitro dependent on neuronal maturation and NMDA receptor expression

Co-localization of activated microglia and damaged neurones seen in brain injury suggests microglia-induced neurodegeneration. Activated microglia release two potential neurotoxins, excitatory amino acids and nitric oxide (NO), but their contribution to mechanisms of injury is poorly understood. Using co-cultures of rat microglia and embryonic cortical neurones, we show that inducible NO synthase (iNOS)-derived NO aloneis responsible for neuronal death from interferon gamma (IFNgamma) +lipopolysaccharide (LPS)-activated microglia. Neurones remain sensitive to NO irrespective of maturation state but, whereas blocking NMDA receptor activation with MK801 has no effect on NO-mediated toxicity to immature neurones, MK801 rescues 60-70% of neurones matured in culture for 12 days. Neuronal expression of NMDA receptors increases with maturation in culture, accounting for increased susceptibility to excitotoxins seen in more mature cultures. We show that MK801 delays the death of more mature neurones caused by the NO-donor DETA/NO indicating that NO elicits an excitotoxic mechanism, most likely through neuronal glutamate release. Thus, similar concentrations of nitric oxide cause neuronal death by two distinct mechanisms: NO acts directly upon immature neurones but indirectly, via NMDA receptors, on more mature neurones. Our results therefore extend existing evidence for NO-mediated toxicity and show a complex interaction between inflammatory and excitotoxic mechanisms of injury in mature neurones.