刺五加皂甙对谷氨酸毒性神经元凋亡的保护作用

目的观察神经元在谷氨酸毒性损伤时一氧化氮(NO)的动态变化及其与凋亡的关系,探讨刺五加皂甙(ASS)的有效保护浓度。方法采用谷氨酸(Glu)诱导的皮质神经元凋亡模型。随机分成Glu组、正常对照组及ASS3组;用流式细胞仪检测神经元凋亡率,用硝酸还原酶法测定细胞培养上清液中NO的含量,用MTT法测定神经元存活率并在电镜下观察细胞形态学变化。结果(1)Glu呈剂量和时间依赖性增加神经元培养液中NO含量,ASS能不同程度地减少NO含量;(2)与Glu共培养的神经元,其存活率呈剂量和时间依赖性下降,ASS能增加神经元存活率;(3)经Glu处理的神经元发生凋亡,细胞超微结构呈现凋亡样改变,其凋亡率与正常对照组比较有显著性差异(P<0.01)。ASS能减少Glu毒性神经元凋亡。结论NO介导了Glu毒性神经元凋亡,ASS可能通过抑制NO的释放及其神经毒性作用,拮抗Glu引起的神经元凋亡。     

刺五加皂甙对神经元谷氨酸毒性损伤的保护作用

目的:观察不同谷氨酸浓度和谷氨酸作用不同时间对神经元活性的影响,探讨细胞损伤后刺五加皂甙(ASS)的有效保护浓度。方法:取孕13～15dICR小鼠,无菌条件下对胎鼠大脑皮层神经元进行原代分离培养,建立谷氨酸诱导的皮层神经元损伤模型。用MTT、LDH测定神经元活性,用硝酸还原酶法测定细胞培养上清液中NO的含量,用流式细胞仪检测细胞凋亡率,并在电镜下观察细胞形态学变化。结果:①经谷氨酸处理的神经元,其细胞存活率呈剂量和时间依赖下降、ASS能不同程度提高细胞存活率。②谷氨酸处理组的神经元凋亡率、LDH释放量和NO含量均升高,与正常对照组及ASS组比较有明显差异(P<0.01)。结论:一定浓度的ASS对谷氨酸引起的神经元损伤有保护作用;ASS可能是通过抑制NO的释放和稳定细胞膜,拮抗细胞元损伤。     

一氧化氮在缺氧复氧性神经元凋亡中的作用

目的观察神经元缺氧复氧损伤时NO的动态变化及其与神经元凋亡的关系。方法取孕13~15d ICR小鼠,无菌条件下对胎鼠大脑皮质神经元进行原代分离培养,建立缺氧复氧诱导的皮质神经元凋亡模型。用流式细胞仪检测细胞凋亡率,用硝酸还原酶法测定细胞培养上清液中NO的含量,用MTT测定细胞活性,并在电镜下观察细胞形态学变化。结果经缺氧复氧处理的神经元,随着缺氧时间延长细胞存活率逐渐下降,神经元凋亡率呈时间依赖性升高,NO含量逐渐升高,至缺氧8h复氧24h达到高峰,与正常对照组比较有显著性差异(P<0.01)。细胞超微结构呈现凋亡样改变。结论NO介导了缺氧复氧性神经元凋亡过程。     

Exendin-4对大鼠皮质神经元缺血再灌注损伤后内质网相关性细胞凋亡的影响

目的探讨Exendin-4(Ex-4)对大鼠脑缺血再灌注损伤的保护作用及机制。方法体外培养Spra-gue Dawley(SD)乳鼠皮质神经元并随机分为对照组(正常培养)、模型组(体外模拟缺血再灌注)、治疗组(建模前2h、建模中均给予Ex-4)。免疫荧光染色鉴定神经元纯度,RT-PCR检测GLP-1受体基因表达,ELISA检测神经元细胞浆内cAMP水平,MTT法测定神经元存活率,流式细胞术检测神经元凋亡率,Western-blot免疫印迹法检测葡萄糖调节蛋白78(GRP78)、C/EBP同源蛋白(CHOP)表达。结果大鼠皮质神经元可体外纯化培养。RT-PCR及ELISA检测结果显示皮质神经元存在活性GLP-1受体。体外模拟缺血再灌注后神经元凋亡率明显增加,早期凋亡率及总凋亡率分别为(22.43±1.67)%、(41.87±3.2)%,Ex-4(0.4μg/ml)干预后神经元凋亡率明显下降,早期凋亡率及总凋亡率分别降至(7.53±0.6)%、(13.67±1.77)%,两组比较差异均有统计学意义(t早期凋亡率=14.511、t总凋亡率=13.352,P0.01)。体外模拟缺血再灌注损伤可诱导神经元GRP78、CHOP表达上调,Ex-4干预后神经元GRP78表达进一步增加、CHOP表达反而减少,两组GRP78、CHOP含量比较差异均有统计学意义(tGRP78(%control)=7.103、tCHOP(%control)=7.816,P0.01)。结论 Ex-4对大鼠皮质神经元体外模拟缺血再灌注损伤具有保护作用,其可能机制为抑制损伤后内质网相关性细胞凋亡。     

槲皮苷对过氧化氢诱导PC12细胞损伤的保护作用

目的探讨槲皮苷对H2O2所致的PC12细胞凋亡的保护作用及机制。方法 PC12细胞培养后,MTT检测细胞存活率的方法进行H2O2损伤模型的摸索和槲皮苷药物浓度的筛选,将PC12细胞分为对照组、模型组和不同剂量槲皮苷组。用400μmol H2O2刺激PC12神经元细胞使其发生凋亡复制阿尔茨海默病(AD)模型,MTT法检测PC12细胞存活率、硫辛酰胺脱氢酶催化的INT显色反应检测乳酸脱氢酶(LDH)释放量和DAPI荧光核染色观察细胞凋亡形态学改变,Western blot方法检测Cytc和caspase-3表达的变化。结果 400μmol H2O2诱导PC12细胞损伤明显,与模型组比较,槲皮苷组PC12细胞存活率显著提高(P<0.01),凋亡率显著下降(P<0.01),LDH释放量和凋亡相关蛋白Cytc和caspase-3的表达显著减少(P<0.01)。结论槲皮苷可抑制H2O2诱导的PC12细胞凋亡,其机制可能与抑制细胞凋亡线粒体途径中凋亡相关蛋白Cytc和caspase-3的表达有关。     

西洛他唑对大鼠皮层细胞糖氧剥离损伤的保护作用

目的探讨磷酸二酯酶抑制剂西洛他唑对糖氧剥离后大鼠皮层细胞培养的影响及作用机制。方法原代混合培养大鼠皮层细胞,建立糖氧剥离的细胞损伤模型模拟细胞"缺血损伤",然后进行干预。测定细胞培养上清中乳酸脱氢酶(LDH)、丙二醛(MDA)、谷胱甘肽过氧化物酶(GSH-Px)、神经元型一氧化氮合酶(nNOS)及诱导型一氧化氮合酶(i NOS)的含量;测定一氧化氮(NO)的分泌水平;测定细胞内环磷酸腺苷(cAMP)水平及四唑盐(MTT)比色试验测定细胞活力。结果西洛他唑组及依达拉奉组与糖氧剥离模型组比较,LDH、MDA漏出量显著减少(P均0.05),GSH-Px释放量明显升高(P均0.05),nNOS、i NOS的水平及NO的分泌量显著下降(P均0.05),细胞内cAMP水平明显升高(P均0.05);细胞存活率显著提高(P均0.05);西洛他唑与依达拉奉组比较,LDH、MDA漏出量及GSH-Px的释放量无差别,nNOS、i NOS和NO的水平明显降低(P均0.05),细胞内cAMP水平显著升高(P0.05);细胞存活率明显提高(P0.05)。结论西洛他唑对培养大鼠皮层细胞在糖氧剥离损伤中具有保护作用,其作用机制可能通过抗氧化、降低nNOS及i NOS的水平从而降低NO的分泌、升高细胞内cAMP水平来实现的。     

托吡酯对大鼠脑缺血再灌注损伤的神经保护作用

目的探讨托吡酯(TPM)对大鼠脑缺血再灌注损伤的神经保护作用及其机制。方法将健康30只雄性SD大鼠随机分为假手术组、缺血再灌注组和TPM干预组。用线栓法建立大鼠脑缺血再灌注模型,TPM干预组给予TPM80mg/kg腹腔注射,2次。缺血再灌注24h时进行神经功能评分、TTC染色法测量梗死体积、高效液相色谱分析法测定脑组织谷氨酸(Glu)及γ-氨基丁酸(GABA)的含量;免疫组化法检测GABAA受体阳性表达。结果(1)与缺血再灌注组比较,TPM干预组神经功能评分明显增高(P<0.01),脑梗死体积减少(P<0.05);(2)TPM干预组缺血侧脑皮质Glu含量显著低于缺血再灌注组(P<0.01),与假手术组比较差异无显著性;GABA含量显著高于假手术组和缺血再灌注组(均P<0.01);(3)TPM干预组缺血侧脑皮质GABAA受体阳性细胞数显著高于缺血再灌注组(P<0.01)。结论TPM对脑缺血再灌注损伤有神经保护作用,其机制可能为TPM降低兴奋性递质Glu水平、增加抑制性递质GABA的释放及GABAA受体的表达。     

重复高压氧预处理对氧糖剥夺神经元保护作用的研究

目的研究重复高压氧预处理(HBO-PC)对新生鼠脑皮层神经元缺血损伤的影响。方法建立体外培养皮层神经元氧糖剥夺损伤模型,分为对照组(CON)和HBO-PC组(0.35 MPa,2 h)。通过形态学观察、甲基噻唑基四唑(MTT)比色微量分析、细胞凋亡检测和乳酸脱氢酶(LDH)测定等方法观察氧糖剥夺处理所导致的神经元损伤以及HBO-PC对这种损伤的影响。结果氧糖剥夺后24 h对照组与HBO组相比,细胞活性下降、光密度值明显降低、多数神经元出现凋亡、细胞LDH释放量明显增多(P<0.05)。结论氧糖剥夺可导致神经元出现损伤,而HBO-PC对这种损伤具有保护作用。     

亚低温对缺血性神经元凋亡后凋亡诱导因子表达的影响

目的研究亚低温对凋亡诱导因子(AIF)介导的缺血性神经元凋亡通路的影响。方法建立离体大鼠皮质神经元凋亡模型。皮质神经元原代培养,分为常温组(37℃)和亚低温组(33℃),在剥夺血清后的8h、16h和24h,测定神经元生存率;应用免疫细胞化学的方法检测微管相关蛋白-2(MAP-2);通过Western Blotting方法测定神经元的细胞核和细胞浆中AIF的含量变化。结果缺血24h组,神经元生存率分别为42.34%(37℃)和65.76% (33℃),差异有统计学意义(P<0.05)。随着缺血时间的延长,MAP-2的表达逐渐减低,在缺血16h和24h,两组间差异有统计学意义(P<0.05)。细胞核中AIF的含量逐渐增加,而细胞浆中AIF的含量逐渐减少,在缺血16h和24h,与常温组相比,低温组细胞核AIF的含量显著性减低(P<0.05)。结论亚低温能促进缺血后神经元的存活,减少MAP-2的降解。亚低温干预了AIF介导的细胞凋亡途径,减少了缺血性损伤导致的AIF核位移。     

L-NMMA和L-Arg对体外培养海马神经元缺血缺氧损伤调控作用及其机制的研究

近年来研究发现一氧化氮 (NO)在缺血缺氧机制中具有重要作用。 NO与细胞凋亡的关系仍存在着较大分歧 ,实验证明 NO既可诱导凋亡又可抑制凋亡。本研究利用体外培养海马神经元 ,通过去除培养液中糖和氧气模拟缺血缺氧状态 ,以细胞形态学、细胞存活率、乳酸脱氢酶变化描述细胞损伤程度 ,观察 NO合成前体物左旋精氨酸 (L - Arg)、NO合成酶 (NOS)抑制剂 N单甲基 - L -精氨酸 (L - NMMA)对缺血缺氧损伤体外培养海马神经元的影响 ,并初步探讨了两者与细胞凋亡的关系。1　材料与方法采用新生的 Wistar大鼠进行海马神经元体外培养 :分离海…     

Differential effects of natural polyphenols on neuronal survival in primary cultured central neurons against glutamate- and glucose deprivation-induced neuronal death

Neuronal injury in the central nervous system following ischemic insult is believed to result from glutamate toxicity and glucose deprivation. In this study, polyphenols isolated from Scutellaria baicalensis Georgi, including baicalin, baicalein, and wogonin, were investigated for their neuroprotective effects against glutamate/NMDA (Glu/NMDA) stimulation and glucose deprivation in primary cultured rat brain neurons. Cell death was accessed by lactate dehydrogenase (LDH) release assay for necrosis, and mitochondrial activity was accessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction activity assay. It was found that both baicalin and baicalein decreased LDH release of the cultured neurons after 24 h treatment, whereas wogonin profoundly increased LDH release after 2 h treatment and resulted in neuronal death after 24 h. Glu/NMDA treatment profoundly increased LDH release and moderately decreased MTT reduction activity in an NMDA receptor-dependent manner. Both baicalin and baicalein significantly reduced Glu/NMDA-increased LDH release, in which baicalein is much more potent than baicalin. Glu/NMDA-increased intracellular calcium was also significantly attenuated by baicalin and baicalein. Baicalin and baicalein did not affect glutamate receptor binding activity, but baicalein did moderately decrease Glu/NMDA-induced nitric oxide (NO) production. In the glucose deprivation (GD) study, baicalein but not baicalin showed significant protective effects on the GD-increased LDH release, without affecting the GD-induced NO production, in cultured rat brain neurons. These results suggest that baicalein is the most effective compound among three polyphenols tested in preventing neurotoxicity induced by both glutamate and GD, whereas baicalin was only effective in preventing glutamate toxicity. Wogonin might have a neurotoxic effect on the brain.     

心肌营养素-1对超氧化损伤神经元的保护作用

目的在体外培养神经元凋亡损伤模型,观察重组腺病毒-心肌营养素1(Adv-CT1)对损伤神经元存活的保护作用,了解CT-1对神经元的作用和机制,为神经损伤提供新的治疗措施。方法诱导大鼠神经干细胞(NSCs)分化为神经元,建立超氧化诱导神经元凋亡损伤模型,以Adv-CT1转染神经元,应用免疫组化、流式细胞仪凋亡检测等技术,观察CT-1对神经元生长存活的作用以及CT-1和caspase-3基因在损伤神经元表达的变化。结果分离培养NSCs,应用无血清小剂量碱性成纤维细胞生长因子(bFGF)神经元培养基诱导NSCs定向分化培养神经元;在神经元凋亡模型中转染Adv-CT1,免疫组化显示神经元中CT-1表达增高(P<0.05,或P<0.01),caspase-3表达降低(P<0.05);流式细胞检测显示CT-1可减少损伤神经元凋亡比例(P<0.01,或P<0.05),促进细胞存活。结论Adv-CT1转染到凋亡神经元后,CT-1表达增加,caspase-3表达降低,提示Adv-CT1对损伤神经元有保护作用,是CT-1通过减少神经元凋亡基因caspase-3表达,抑制凋亡发生,从而促进神经元存活。     

Differential vulnerability of immature murine neurons to oxygen-glucose deprivation

In vivo studies support selective neuronal vulnerability to hypoxia-ischemia (HI) in the developing brain. Since differences in intrinsic properties of neurons might be responsible, pure cultures containing immature neurons (6-8 days in vitro) isolated from mouse cortex and hippocampus, regions chosen for their marked vulnerability to oxidative stress, were studied under in vitro ischemic conditions-oxygen-glucose deprivation (OGD). Twenty-four hours of reoxygenation after 2.5 h of OGD induced significantly greater cell death in hippocampal than in cortical neurons (67.8% vs. 33.4%, P = 0.0068). The expression of neuronal nitric oxide synthase (nNOS) protein, production of nitric oxide (NO), and reactive oxygen species (ROS), as well as glutathione peroxidase (GPx) activity and intracellular levels of reduced glutathione (GSH), were measured as indicators of oxidative stress. Hippocampal neurons had markedly higher nNOS expression than cortical neurons by 24 h of reoxygenation, which coincided with an increase in NO production, and significantly greater ROS accumulation. GPx activity declined significantly in hippocampal but not in cortical neurons at 4 and 24 h after OGD. The decrease in GSH level in hippocampal neurons correlated with the decline of GPx activity. Our data suggest that developing hippocampal neurons are more sensitive to OGD than cortical neurons. This finding supports our in vivo studies showing that mouse hippocampus is more vulnerable than cortex after neonatal HI. An imbalance between excess prooxidant production (increased nNOS expression, and NO and ROS production) and insufficient antioxidant defenses created by reduced GPx activity and GSH levels may, in part, explain the higher susceptibility to OGD of immature hippocampal neurons.     

Transient ADC change precedes persistent neuronal death in hypoxic-ischemic model in immature rats

A brief ischemia causes delayed neuronal death (DND) in some areas vulnerable to ischemia. Additionally, it causes a transient reduction in the apparent diffusion coefficients (ADCs) obtained from diffusion-weighted magnetic resonance imaging (DWI), which is a powerful tool to detect ischemic changes in the brain at a very early stage. The present study examined long-term histopathological changes in the hippocampal neurons up to 30 days after a very mild hypoxic-ischemic (HI) insult in immature rats. Three-week-old male rats were subjected to 15- and 30-min HI insults (15-min HI and 30-min HI) and serial DWI was performed. Only animals whose ADC reduction pattern was transient were examined histopathologically. ADCs decreased significantly during the insult, and the ADC values of 30-min HI group were significantly lower than those of 15-min HI group. Ischemic neuronal changes were observed up to 30 days after the insult in 30-min HI group, although ADCs in the chronic stage were within the normal range. In addition, neuron density in 30-min HI group was significantly lower in the chronic stage (on days 14 and 30) than in 15-min HI group. A very mild hypoxia-ischemia followed by a transient ADC reduction causes persistent neuronal death, which can be predicted by measuring ADCs during the acute insult.     

Mitochondrial involvement in nitric oxide-induced cellular death in cortical neurons in culture

Nitric oxide (NO) is an unstable molecule with physiological and pathological properties. In brain, NO acts as a modulator of neurotransmission as well as a protector against neuronal death from several death stimuli. However, beside this protector effect, high NO concentrations produce neuronal death by a mechanism in which the caspase pathway is implicated. In this work, we demonstrate that in cortical neurons the NO toxicity is mediated by mitochondrial dysfunction. SNAP, an NO donor, induces apoptosis in these cells because it 1) increases the p53 and 2) induces cytochrome c release and activation of caspase-9 and caspase-3. SNAP also induces necrosis, through 1) breakdown of the mitochondrial membrane potential, 2) ATP decrease, 3) ROS formation, and 4) LDH and ATP release, indicative of oxidative stress and death by necrosis. To sum up, in cortical neurons, high NO concentrations produced cellular death by both an apoptotic and a necrotic mechanism in which the mitochondria are implicated.     

Transgenic expression of human FGF-1 protects against hypoxic-ischemic injury in perinatal brain by intervening at caspase-XIAP signaling cascades

Perinatal hypoxia-ischemia (HI) is a major cause of neurological disability and mortality in infant and children. In the present study, we explored the neuroprotective efficacy of FGF-1 in a rat model of perinatal HI. Carotid ligation combined with hypoxia caused marked infarctions in the ipsilateral cerebral hemisphere with significant loss of ipsilateral striatal, cortical and hippocampal volumes. Morphological analyses revealed both apoptotic and necrotic form of neuronal death determined by Nissl histology, dark-field microscopy and TUNEL staining. HI induced a marked increase in activated caspase-9, caspase-3 and PARP cleavage at 12 h to 7 days after HI in brain areas displaying TUNEL (+) cells. In addition, expression of the anti-apoptotic protein X-linked inhibitor of apoptosis (XIAP) was decreased under similar conditions of HI. Expression of human FGF-1 in brain significantly reduced the extent of both apoptotic and necrotic injury caused by HI. FGF-1 attenuated the HI-induced increase in activated caspase-3, caspase-9 and cleaved PARP protein levels and markedly blocked the HI-induced decrease in XIAP expression under the conditions at which FGF-1 showed significant neuroprotection. These findings demonstrate that FGF-1 prevents the onset of both apoptotic and necrotic death in neurons otherwise "destined to die" following hypoxic-ischemic injury by intervening at the level of caspase-signaling cascades and by restoring prosurvival protein XIAP expression in central neurons.     

Endothelin B receptor deficiency augments neuronal damage upon exposure to hypoxia-ischemia in vivo

The role of functional endothelin-B (ETB)-receptors on neuronal survival upon hypoxia-ischemia (HI) has been investigated in 14-day-old ETB-receptor-deficient spotting lethal (sl/sl) and wildtype (+/+) rats. Carotid ligation followed by exposure to 8% oxygen for 2 h produced distinct cortical and hippocampal neuronal damage. Damage severity 24 h after HI was mild to intermediate in +/+ rats whereas large cortical infarcts and profound apoptosis of the hippocampus evolved in sl/sl rats. The number of apoptotic cells in the dentate 24 h after HI amounted to 30 +/- 7 cells/0.1 mm(2) in sl/sl compared to 9 +/- 3 cells/0.1 mm(2) in wildtype rats (mean +/- S.E.M., n=10-11, P=0.0093). In-vitro hypoxia (15 h) resulted in a comparable increase in cell death in primary pure neuronal hippocampal cultures from both groups (49.8 +/- 1.6% in sl/sl, 51.4 +/- 0.9% in +/+, mean +/- S.E.M., n=5, P=0.0560). To conclude, absence of functional ETB receptors is associated with an increased susceptibility to HI in-vivo, which is not intrinsic to neurons. Antagonism of ETB receptors seems not to be desirable in ischemic stroke.