白介素-6对NMDA诱导的小脑神经元放电活动的抑制作用及其机制

目的：观察白介素-6（IL-6）对N-甲基-D-天冬氨酸（NMDA）激发的神经元放电活动的影响及其可能的作用机制。方法：用含IL-6、NMDA和JAK抑制剂ACA90的人工脑脊液（ACSF）灌流小脑脑片,利用离体脑片神经元单位放电细胞外记录技术,记录药物对小脑间位核神经元放电的影响。用Western blot法测定间位核神经元NMDA受体亚单位1（NRI）的磷酸化水平。结果：单独用12．5μmol／L和25μmol／LNMDA灌流,神经元放电频率均较基础放电频率增加;用不同浓度IL-6（50,100,200μg／ml）联合NMDA作用后,神经尤的放电频率出现浓度依赖性地降低;AG490可部分阻断IL-6对NMDA兴奋神经元放电的抑制作用。与单独NMDA处理组比较,用IL-6联合NMDA处理神经元后,神经元的NR1磷酸化水平出现浓度依赖性地降低。AG490可阻断IL-6所致的神经元NR1磷酸化水平的降低。结论：IL-6可抑制NMDA激发的小脑间位核神经元的放电兴奋活动;并同时下调神经元的NR1磷酸化水平。     

鼠尾草酸对Aβ损伤海马神经元的保护作用研究

采用Aβ诱导建立海马神经元损伤模型,CCK-8检测神经元的活性,RT-PCR检测神经元凋亡相关基因Caspase-3 mRNA的表达,探讨了鼠尾草酸对Aβ所致海马神经元损伤的保护作用及其机制。结果显示,浓度在5~10μmol/L时,鼠尾草酸预处理能显著下调Aβ损伤导致的Caspase-3 mRNA表达的升高,增强神经元活力。表明鼠尾草酸预处理可以保护Aβ所致小鼠海马神经元的损伤,其机制可能与鼠尾草酸调控神经元凋亡相关基因caspase-3 mRNA的表达有关。     

MAPKs介导NMDA诱导的大鼠皮层神经元凋亡(英文)

NMDA诱导兴奋毒造成的神经损伤,包括细胞的凋亡和坏死。本研究旨在探讨神经元凋亡在NMDA兴奋毒所致大鼠皮层神经元死亡中的所占比例,并分析了NMDA致神经元凋亡的信号通路机制。通过使用Caspase抑制剂和测定乳酸脱氢酶活性,研究NMDA(100μmol/L,2h)兴奋毒所致的神经元凋亡;并使用MAPKs选择性抑制剂,分别采用Caspase-3活性检测,TUNEL和Annexin V染色方法,进一步观察MAPKs通路中细胞外信号调节激酶(ERK)、c-Jun N-末端激酶(JNK)和p38 MAPK三条不同途径在NMDA所致神经元凋亡中的作用。结果显示:(1)Caspase依赖的凋亡占NMDA所致细胞死亡总数的22.49%;(2)p38 MAPK抑制剂SB203580(10μmol/L)使NMDA诱导的caspase-3活性降低30.43%(P0.05);而ERK抑制剂PD98059(20μmol/L)和JNK抑制剂SP600125(20 μmol/L)不影响caspase-3的活性;(3)SB203580(10μmol/L)使NMDA所致的TUNEL阳性细胞数减少33.10%(P0.05);而PD98059(20μmol/L)或SP600125(20μmol/L)都没有作用;(4)Annexin V染色结果显示,SB203580(10μmol/L)使NMDA所致的早期凋亡细胞减少55.56%(P0.05);SP600125(20μmol/L)使NMDA所致的晚期凋亡/死亡细胞减少67.59%(P0.05);PD98059(20μmol/L)对细胞凋亡/死亡没有明显作用。以上结果表明,NMDA介导的大鼠皮层神经元死亡除坏死外,还包含有一小部分神经元凋亡;p38 MAPK途径,而非JNK和ERK途径,介导了NMDA诱导的神经元凋亡,抑制与此相关的凋亡信号通路可发挥神经保护作用;JNK途径可能介导了NMDA所致的神经元坏死而非凋亡。     

麦角甾苷通过PI3K/Akt/GSK3β通路抑制H_2O_2诱导的小鼠胚胎肝细胞凋亡

该文建立了H_2O_2诱导小鼠胚胎肝细胞损伤模型,并探讨了麦角甾苷通过PI3K/Akt/GSK3β通路抑制H_2O_2诱导的胚胎肝细胞凋亡作用机制。CCK-8检测细胞存活率,流式细胞术检测细胞凋亡,Western blot法检测Bcl-x L、Bax、Cyt-c、Akt、p-Akt、GSK3β和p-GSK3β蛋白质水平。结果显示,麦角甾苷可通过降低Bax/Bcl-x L比值、抑制线粒体Cyt-c释放、增加Akt和GSK3β蛋白质磷酸化水平来提高细胞存活率、减少凋亡细胞数量。该研究结果表明,麦角甾苷可通过PI3K/Akt/GSK3β通路调节H_2O_2诱导的小鼠胚胎肝细胞凋亡。     

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.     

APP5肽对糖尿病小鼠学习记忆功能与海马神经元蛋白表达的影响

目的研究APP5肽对糖尿病模型小鼠学习记忆能力及海马神经元蛋白表达的影响。方法用链脲佐菌素诱发小鼠糖尿病模型,应用APP5肽（0.0014 mg/kg）皮下注射治疗,5周后进行Morris水迷宫试验;小鼠脑组织海马做Akt、PI3K、P-CREB、Bcl-2、Bax、CytoC免疫组织化学染色;另一部分鼠脑海马,做Bcl-2、Bax抗体蛋白免疫印记。结果（1）水迷宫试验：糖尿病模型小鼠到达站台游动时间比正常对照组延长（P〈0.01）;而APP5肽皮下注射治疗组较DM组动物分别缩短（P〈0.01）。（2）神经免疫组织化学实验和Western blot：给予APP5肽糖尿病小鼠与对照组小鼠海马组织内神经元表达细胞存活相关蛋白及抗凋亡相关蛋白PI3K、Akt、P-CREB、Bcl-2阳性细胞数相似,明显高于糖尿病小鼠（P〈0.01）;APP5肽给予糖尿病小鼠与对照组小鼠表达凋亡蛋白Bax、cytoC阳性细胞数相似,明显少于糖尿病小鼠（P〈0.01）。Western blot结果相同。结论糖尿病小鼠海马神经元表达细胞存活相关蛋白下降,神经元表达细胞凋亡相关蛋白增加,导致其学习记忆能力下降。APP5肽应用可以使上述蛋白恢复到接近正常,从而改善糖尿病小鼠学习记忆能力。     

阿利克仑抑制血管紧张素原-肾素双转基因高血压小鼠心肌成纤维细胞增殖

本研究旨在观察阿利克仑(aliskiren)在血管紧张素原-肾素(AGT-REN)双转基因高血压(double transgenic hypertension,d TH)小鼠心肌成纤维细胞(cardiac fibroblasts,CFs)增殖中的作用。将培养的AGT-REN d TH小鼠CFs分为高血压组(d TH)和阿利克仑干预组;另培养同品系野生C57B6小鼠CFs作为对照(WT)。采用MTT法观察不同浓度阿利克仑(1×10~(-6)、1×10~(-7)、1×10~(-8)、1×10~(-9) mol/L)对d TH小鼠CFs增殖的影响;选取1×10~(-7) mol/L阿利克仑作用d TH小鼠CFs 24 h,羟脯氨酸试剂盒定量检测细胞胶原含量;Western blot法检测细胞I、III型胶原蛋白表达,观察细胞胶原合成变化;Western blot法检测α-SMA表达,观察细胞转化;DHE检测细胞内活性氧表达,Western blot法检测细胞内NADPH氧化酶蛋白表达,观察细胞内氧化应激反应的改变。结果显示,随年龄增长,AGT-REN d TH组小鼠血压及血浆Ang II水平均呈明显升高趋势,与WT组小鼠相比,CFs增殖明显。1×10~(-6)、1×10~(-7)、1×10~(-8) mol/L阿利克仑均能抑制AGT-REN d TH小鼠CFs增殖。1×10~(-7) mol/L阿利克仑使d TH小鼠CFs表达α-SMA减少,胶原合成及I、III型胶原蛋白表达下降,活性氧表达减少;同时,阿利克仑降低了d TH小鼠CFs内NADPH氧化酶NOX2及NOX4的蛋白表达。阿利克仑抑制AGT-REN d TH小鼠CFs增殖、表型转化及胶原合成,这一过程可能与其抑制细胞内氧化应激反应有关。     

阿魏酸对阿尔茨海默病转基因小鼠脑内氧化应激和凋亡相关蛋白的影响

本文主要研究阿魏酸对APP/PS1转基因小鼠脑内氧化应激和凋亡相关蛋白的影响。应用Western blot和Real-Time PCR方法检测野生型小鼠、APP/PS1转基因小鼠及不同浓度阿魏酸处理组小鼠脑内凋亡相关蛋白的表达情况,并测定各组小鼠脑内SOD活力和MDA含量。阿魏酸处理后的AD模型小鼠SOD与AD组相比活性增加,MDA含量减少,其中中浓度阿魏酸处理组的效果最为明显;APP/PS1转基因小鼠脑内所检测的凋亡相关蛋白p-JNK、p-C-Jun、Caspase-3、Bax和Bcl-2表达较野生型小鼠明显升高,阿魏酸处理后这些磷酸化蛋白的表达显著减少,并且中浓度阿魏酸处理组的效果最为明显。以上结果表明阿魏酸通过其强大的抗氧化作用降低AD小鼠脑内的氧化应激效应,并且可以降低AD小鼠脑内凋亡相关蛋白的表达,提示阿魏酸在AD的临床治疗方面具有重要的理论意义。     

兔脑皮质静脉闭塞后脑组织Caspase-3活性的变化

目的观察兔脑皮质静脉闭塞后Caspase-3活性的变化。方法采用电凝法制作兔脑皮质引流静脉急性闭塞模型,琼脂糖凝胶电泳、荧光实时定量PCR和Western印迹检测Caspase-3表达。结果脑皮质静脉闭塞后8hCaspase-3活性已升高,24h达高峰,48h明显下降。结论细胞凋亡是脑缺血后脑损伤发生机制,Caspase-3蛋白参与了皮质静脉闭塞后脑缺血后神经元损伤的病理过程。     

雌二醇调控PI3K/Akt通路在大鼠肺泡Ⅱ型上皮细胞缺氧/复氧损伤中的作用

该文探讨了雌二醇在大鼠肺泡Ⅱ型上皮细胞(AECⅡ)缺氧/复氧(H/R)损伤中的作用及其机制。使用AECⅡ构建H/R损伤模型,将AECⅡ细胞随机分为正常对照组(NC组)、缺氧/复氧损伤组(HR组)、不同浓度雌二醇预处理+缺氧/复氧损伤组(E2+HR组)。倒置显微镜观察各组细胞形态学变化; CCK-8法检测各组细胞活力; ELISA法检测细胞培养物上清中IL-6、TNF-α的水平;流式细胞仪检测细胞凋亡情况; Western blot检测Akt、P-Akt、Gsk3β、P-Gsk3β和Caspase-3的表达水平。结果显示,与NC组相比,其余各组细胞活力显著下降, IL-6、TNF-α表达水平显著增加,细胞凋亡水平增加, Akt、P-Akt、P-Gsk3β表达水平降低, Caspase-3表达水平增加;与HR组相比, E2+HR组细胞活力升高, IL-6、TNF-α表达水平降低,细胞凋亡水平降低, Akt、P-Akt、P-Gsk3β蛋白表达水平升高,Caspase-3蛋白表达水平降低。由此提示, PI3K/Akt信号通路参与了大鼠AECⅡ的H/R损伤过程;雌二醇可减轻H/R引起的大鼠AEC...     

NMDA受体的活化调节原代皮层神经元的Wnt/β-catenin信号通路

经典的Wnt/β-catenin信号通路在中枢神经系统突触形成和功能中发挥重要的调节作用。作为兴奋性神经递质的谷氨酸,与其受体结合,参与许多信号调节活动。为了探讨NMDA受体活化对Wnt/β-catenin信号通路的作用,该文利用18 d的C57小鼠胚胎培养皮层神经元（离体10 d）,用10μmol/L谷氨酸钠（monosodium glutamate,MSG）和50μmol/L N-甲基-D-天冬氨酸（NMDA）处理细胞,通过蛋白免疫印迹技术或者细胞免疫荧光染色分析Wnt/β-catenin信号通路关键成员。结果发现,NMDA受体的活化能使GSK-3β的Ser9位磷酸化水平增加,活性被抑制,胞浆内β-catenin蛋白降解减少,入核增加,激活下游基因表达。这些结果提示,NMDA受体激活能够上调Wnt/β-catenin信号通路。     

Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3

Activated protein C (APC), a serine protease with anticoagulant and anti-inflammatory activities, exerts direct cytoprotective effects on endothelium via endothelial protein C receptor-dependent activation of protease activated receptor 1 (PAR1). Here, we report that APC protects mouse cortical neurons from two divergent inducers of apoptosis, N-methyl-D-aspartate (NMDA) and staurosporine. APC blocked several steps in NMDA-induced apoptosis downstream to nitric oxide, i.e., caspase-3 activation, nuclear translocation of apoptosis-inducing factor (AIF), and induction of p53, and prevented staurosporine-induced apoptosis by blocking caspase-8 activation upstream of caspase-3 activation and AIF nuclear translocation. Intracerebral APC infusion dose dependently reduced NMDA excitotoxicity in mice. By using different anti-PARs antibodies and mice with single PAR1, PAR3, or PAR4 deletion, we demonstrated that direct neuronal protective effects of APC in vitro and in vivo require PAR1 and PAR3. Thus, PAR1 and PAR3 mediate anti-apoptotic signaling by APC in neurons, which may suggest novel treatments for neurodegenerative disorders.     

选择性代谢性谷氨酸受体5激动剂CHPG对创伤性神经元损伤的保护作用研究

目的：研究选择性代谢性谷氨酸受体5激动剂2-氯-4羟苯基甘氨酸（CHPG）对创伤性神经元损伤的保护作用,并初步探讨其保护机制。方法：大鼠皮层神经元原代培养10天后,采用机械划伤的方法建立损伤模型,采用乳酸脱氢酶（LDH）测定和Hoechst 33342染色观察CHPG对神经元的保护作用。结果：①CHPG显著降低损伤后LDH的释放和神经元凋亡。②与对照组相比,CHPG增加了ERK与Akt的磷酸化水平。③使用ERK抑制剂PD98059或者Akt抑制剂LY294002都可以部分逆转CHPG的保护作用。结论：CHPG可以减轻创伤性神经元损伤,这种保护作用可能是由ERK和Akt信号通路介导的。     

臭椿酮诱导人黑色素瘤A375细胞凋亡及其机制研究

为研究臭椿酮(Ailanthone,AIL)诱导人黑色素瘤A375细胞凋亡的作用及作用机制,以人黑色素瘤A375细胞为研究对象,采用MTT法测定AIL对人黑色素瘤A375细胞生长增殖的抑制作用。用倒置相差显微镜观察AIL对A375细胞形态的影响,用荧光倒置显微镜观察Hoechst33258染色后AIL对A375细胞核的影响,用AnnexinV-FITC/PI双染法检测AIL诱导A375细胞凋亡的作用,用分光光度法检测caspase-3和caspase-9的活性,Westernblot检测p-PI3Kβ(Ser1070),PI3Kβ,p-Akt(Ser473)和Akt蛋白表达水平的变化,接着用PI3K抑制剂LY294002进行干预,进一步验证AIL对PI3K/Akt信号通路及细胞凋亡的影响。实验结果表明,AIL能够明显抑制A375细胞增殖,使A375细胞数目变少、附着力和透光性减弱,AIL能够诱导A375细胞凋亡,使其细胞核染色质发生固缩并呈现高亮,且使A375细胞早期及晚期凋亡率均增加,AIL作用后能够使caspase-3和caspase-9活性增加,AIL能够抑制PI3K和Akt蛋白磷酸化,从而使PI3K/Akt信号通路失活。较AIL单独作用,AIL和LY294002共同作用后对PI3K和Akt蛋白磷酸化的抑制作用增强且诱导凋亡作用增加,进一步说明AIL通过失活PI3K/Akt信号通路来诱导A375细胞凋亡。     

Neuroprotective effect of the endogenous neural peptide apelin in cultured mouse cortical neurons

The adipocytokine apelin and its G protein-coupled APJ receptor were initially isolated from a bovine stomach and have been detected in the brain and cardiovascular system. Recent studies suggest that apelin can protect cardiomyocytes from ischemic injury. Here, we investigated the effect of apelin on apoptosis in mouse primary cultures of cortical neurons. Exposure of the cortical cultures to a serum-free medium for 24 h induced nuclear fragmentation and apoptotic death; apelin-13 (1.0-5.0 nM) markedly prevented the neuronal apoptosis. Apelin neuroprotective effects were mediated by multiple mechanisms. Apelin-13 reduced serum deprivation (SD)-induced ROS generation, mitochondria depolarization, cytochrome c release and activation of caspase-3. Apelin-13 prevented SD-induced changes in phosphorylation status of Akt and ERK1/2. In addition, apelin-13 attenuated NMDA-induced intracellular Ca²⁺ accumulation. These results indicate that apelin is an endogenous neuroprotective adipocytokine that may block apoptosis and excitotoxic death via cellular and molecular mechanisms. It is suggested that apelins may be further explored as a potential neuroprotective reagent for ischemia-induced brain damage.     

β-淀粉样肽诱导小胶质细胞培养上清致海马神经元损伤模型的建立

目的：建立β淀粉样肽（Aβ1-40）诱导激活小胶质细胞的上清致海马神经元损伤的细胞模型,并初步研究神经元损伤的机制。方法：用不同浓度的可溶性Aβ1-40诱导激活小胶质细胞,光镜下观察不同时间点的细胞形态,ELISA检测其分泌的肿瘤坏死因子仪;用激活后的小胶质细胞条件培养基刺激海马神经元,光镜下观察细胞形态,Western blot检测刺激后海马神经元内诱导型一氧化氮合酶（iNOS）和硝基酪氨酸（NT）的表达水平,ELISA检测海马神经元内胱冬蛋白酶-3（caspase-3）活性来评价神经元的损伤程度。结果：终浓度为10μmol／L的Aβ1-40与小胶质细胞孵育24h后,取上清液加到培养的海马神经元,孵育24-72h,海马神经元较对照组形态有明显变化;经Western blot检测,神经元内iNOS、NT表达明显增加;ELISA检测神经元内caspase-3活性明显增高。结论：小胶质细胞被Aβ1-40激活后,其释放物有明显的致神经元损伤效应,表明建立了神经元损伤模型。     

Neuroprotective Effects of Hydroxysafflor Yellow A Against Excitotoxic Neuronal Death Partially Through Down-Regulation of NR2B-Containing NMDA Receptors

Hydroxysafflor yellow A (HSYA) is a component of the flower Carthamus tinctorius L. that elicits neuroprotective effects in vivo and in vitro. The purpose of this study was to investigate pharmacological properties of HSYA on neurotoxicity of glutamate in primary cultured rat cortical neurons along with its possible mechanism of action. After challenge with N-methyl-d-aspartate (NMDA, 100 μM) for 30 min, loss of cell viability and excessive apoptotic cell death were observed in cultured cortical neurons. However, the excitotoxic neuronal death was attenuated markedly by HSYA treatment. Western blot analysis revealed that HSYA decreased expression of Bax and rescued the balance of pro-and anti-apoptotic proteins. In addition, HSYA significantly reversed up-regulation of NR2B-containing NMDA receptors by exposure to NMDA, while it did not affect the expression of NR2A-containing NMDA receptors. These finding suggest that HSYA protects cortical neurons, at least partially, from inhibiting the expression NR2B-containing NMDA receptors and by regulating Bcl-2 family.     

Ketamine Affects the Neurogenesis of Rat Fetal Neural Stem Progenitor Cells via the PI3K/Akt‐p27 Signaling Pathway

Ketamine is widely used as an anesthetic, analgesic, or sedative in pediatric patients. We reported that ketamine alters the normal neurogenesis of rat fetal neural stem progenitor cells (NSPCs) in the developing brain, but the underlying mechanisms remain unknown. The PI3K‐PKB/Akt (phosphatidylinositide 3‐kinase/protein kinase B) signaling pathway plays many important roles in cell survival, apoptosis, and proliferation. We hypothesized that PI3K‐PKB/Akt signaling may be involved in ketamine‐altered neurogenesis of cultured NSPCs in vitro. NSPCs were isolated from Sprague‐Dawley rat fetuses on gestational day 17. 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation, Ki67 staining, and differentiation tests were utilized to identify primary cultured NSPCs. Immunofluorescent staining was used to detect Akt expression, whereas Western blots measured phosphorylated Akt and p27 expression in NSPCs exposed to different treatments. We report that cultured NSPCs had properties of neurogenesis: proliferation and neural differentiation. PKB/Akt was expressed in cultured rat fetal cortical NSPCs. Ketamine inhibited the phosphorylation of Akt and further enhanced p27 expression in cultured NSPCs. All ketamine‐induced PI3K/Akt signaling changes could be recovered by N‐methyl‐d ‐aspartate (NMDA) receptor agonist, NMDA. These data suggest that the inhibition of PI3K/Akt‐p27 signaling may be involved in ketamine‐induced neurotoxicity in the developing brain, whereas excitatory NMDA receptor activation may reverse these effects     

Phosphorylation of neurofilament subunit NF-M is regulated by activation of NMDA receptors and modulates cytoskeleton stability and neuronal shape

The cytoskeleton is essential for the structural organization of neurons and is influenced during development by excitatory stimuli such as activation of glutamate receptors. In particular, NMDA receptors are known to modulate the function of several cytoskeletal proteins and to influence cell morphology, but the underlying molecular and cellular mechanisms remain unclear. Here, we characterized the neurofilament subunit NF-M in cultures of developing mouse cortical neurons chronically exposed to NMDA receptor antagonists. Western blots analysis showed that treatment of cortical neurons with MK801 or AP5 shifted the size of NF-M towards higher molecular weights. Dephosphorylation assay revealed that this increased size of NF-M observed after chronic exposure to NMDA receptor antagonists was due to phosphorylation. Neurons treated with cyclosporin, an inhibitor of the Ca(2+)-dependent phosphatase calcineurin, also showed increased levels of phosphorylated NF-M. Moreover, analysis of neurofilament stability revealed that the phosphorylation of NF-M, resulting from NMDA receptor inhibition, enhanced the solubility of NF-M. Finally, cortical neurons cultured in the presence of the NMDA receptor antagonists MK801 and AP5 grew longer neurites. Together, these data indicate that a blockade of NMDA receptors during development of cortical neurons increases the phosphorylation state and the solubility of NF-M, thereby favoring neurite outgrowth. This also underlines that dynamics of the neurofilament and microtubule cytoskeleton is fundamental for growth processes.