钙通道在SIN-1诱导大鼠海马神经元凋亡中的作用

摘要 背景 氯通道参与SIN-1引起的大鼠海马神经元凋亡过程已有报道,而钙通道在这种类型的凋亡中的作用目前未见报道。目的 探讨钙通道在SIN-1诱导大鼠海马神经元凋亡中的作用。方法 方法 离体培养12天的SD大鼠海马神经元,随机分为正常对照组、3-吗啉斯德酮胺(SIN-1)处理组: (SIN-1 1.0 mmol •L-1）、SIN-1处理后加4-4-二异硫氰基苯-2,2’-二磺酸（DIDS,0.1 mmol •L-1）、4-乙酰氨基-4’-异氰酸芪-2,2’-二磺酸(SITS, 1.0 mmol •L-1)、SIN-1处理后加氯化镉（CdCl2）、SIN-1处理后加SITS和CdCl2及SIN-1处理后加DIDS和CdCl2。药物作用时间为18h。DNA荧光染色观察神经元形态及检测凋亡数目的变化,MTT法检测神经元的生存率。结果 SIN-1可以诱导42.32±1.34%的神经元死亡,与正常对照组相比有显著差异（P<0.05）,SIN-1处理的神经元若用氯通道阻断剂SITS或DIDS,生存率分别为87.33±2.03%和83.23±1.01%。SIN-1处理的神经元单独用CdCl2或CdCl2与SITS或DIDS合并使用时,细胞生存率分别为：66.39±2.09%、88.15±1.13%和83.16±2.00%。DNA荧光染色显示,SIN-1处理的神经元若用氯通道阻断剂可以明显减少凋亡百分数,而钙通道阻断剂CdCl2没有明显的抗凋亡作用。结论 氯通道可能参与了SIN-1诱导的大鼠海马神经元凋亡,而钙通道的作用不大。     

Caspase-3在H2O2诱导神经细胞凋亡中的作用

目的研究caspase-3在H2O2诱发神经细胞凋亡中的作用,以探讨氧化应激损伤诱导神经细胞凋亡的机制。方法用海马神经细胞原代培养技术,采用1mmol/L H2O2诱导建立细胞氧化损伤模型,并观察细胞形态学变化,采用TUNEL法检测H2O2诱导大鼠海马神经元凋亡率;采用RT-PCR法检测caspase-3 mRNA的表达。结果形态学观察结果显示模型组比海马组细胞损伤程度较严重;与对照组比较,模型组细胞凋亡率及caspase-3 mRNA的表达显著增高（P〈0.01）。结论本结果提示,caspase-3可能参与了氧化应激损伤诱导的神经细胞凋亡过程。     

刺五加多糖对H2O2诱导的大鼠海马神经元凋亡的保护作用

目的研究刺五加多糖（ASPS）对H2O2诱导的海马神经元凋亡的影响及其机制。方法采用H2O2诱导大鼠海马神经元凋亡。采用末端脱氧核苷酸转移酶介导的dUTP原位切口末端标记法检测细胞凋亡率、免疫组化法检测caspase-3蛋白的表达、逆转录PCR法检测caspase-3 mRNA的表达。结果H2O2作用后,海马神经元凋亡率、caspase-3蛋白和mRNA表达水平均显著增高（P〈0.05）;给予ASPS干预后,均显著下降（P〈0.05）;而且,随ASPS剂量增加,作用效果显著增强（P〈0.05）。结论ASPS具有抑制氧化应激损伤诱导神经细胞凋亡作用,其机制与下调caspase-3 mRNA的表达有关。     

Pinacidil抑制线粒体和死亡受体通路减少大鼠脑缺血再灌注后神经元凋亡(英文)

目的探讨ATP敏感性钾通道开放剂pinacidil对大鼠脑缺血再灌注后神经元凋亡的保护作用及信号转导机制。方法 100 只Wistar 雄性大鼠随机分为四组:A 组(假手术组)、B组 (缺血组)、C 组 (KATP开放剂处理组)及D组 (KATP开放剂和阻断剂处理组)。用线栓法制备大鼠大脑中动脉缺血(middle cerebral artery occlusion,MCAO)模型,用DNA断端末端标记法(terminal-deoxynucleotidytransferase-mediated dUTP-biotin nick end labeling,TUNEL)检测神经元凋亡,用原位杂交方法检测caspase-3、caspase-8及caspase-9 mRNA的表达。结果 (1) C组12 h、24 h、48 h、72 h 时间点的凋亡细胞数较 B、D 组显著减少(P<0.05 或 P<0.01) ;B 组和 D组之间无显著性差异(P>0.05)。(2) C 组 caspase-3 mRNA 和 caspase-8 mRNA 在各时间点及 caspase-9 mRNA 在 12 h、24 h、48 h、72h 时间点的表达显著少于B组和D组(P<0.01或P<0.05),B组和D组之间无显著性差异(P>0.05)。结论 KATP通道开放剂能显著减少大鼠脑缺血再灌注后的细胞凋亡及caspase-3、caspase-8及caspase-9 mRNA的表达。KATP通道开放剂可能通过抑制线粒体通路和死亡受体通路降低神经元凋亡,保护缺血再灌注损伤后的脑组织。     

GABA_A受体激动剂对AD模型配体门控氯通道的作用

目的观察并分析GABA_A受体激动剂蝇蕈醇对GABA激活的配体门控氯通道的作用,为寻找新的抗阿尔茨海默病(AD)药物提供实验依据。方法 Aβ_(1-42)诱导海马神经细胞作为AD细胞模型;通过膜片钳(Patch clamp)技术在大鼠海马神经元上记录配体门控氯通道瞬时外向电流,采用非特异性氯通道阻断剂(NFA)和无GABA的细胞外液确定该电流成份为瞬时外向氯电流;通过膜片钳技术观察GABA_A受体激动剂蝇蕈醇对于在大鼠海马神经元上记录瞬时外向电流的作用;向AD细胞模型中加入GABA_A受体激动剂,采用MTT观察各组细胞活力。结果加入GABA_A受体激动剂蝇蕈醇后GABA激活氯电流强度增加;加入浓度为1 mmol/L蝇蕈醇的AD模型细胞组存活率为(91.32±3.73)%,略高于未加入蝇蕈醇的模型细胞组,两组对比P0.05。结论 GABA_A受体激动剂蝇蕈醇对于AD细胞模型具有保护作用。     

ATP敏感性钾通道开放剂对大鼠脑缺血再灌注后caspase-12 mRNA及蛋白表达的影响

目的 观察ATP敏感性钾通道(KATP)开放剂对大鼠脑缺血再灌注后caspase-12mRNA和蛋白表达的影响,探讨内质网信号通路是否参与了KATP开放剂对脑缺血后神经元凋亡的抑制机制.方法 200只Wistar雄性大鼠随机分为假手术组,缺血再灌注组,开放剂组及阻断剂组.应用线栓法制备大鼠大脑中动脉缺血再灌注模型,分别应用免疫组化染色、RT-PCR技术检测脑缺血再灌注后各组caspase-12蛋白及mRNA的表达.结果 在缺血再灌注组,开放剂组及阻断剂组,随着缺血再灌注时间的延长,caspase-12mRNA及蛋白的表达逐渐增高,在缺血再灌注后24 h达高峰.开放剂组caspase-12 mRNA及蛋白表达在各时间点均显著少于缺血再灌注组及阻断剂组(P＜0.05或P＜0.01).阻断剂组各时间点与缺血再灌注组相比均无显著性差异(P＞0.05).结论 KATP开放剂可能通过抑制内质网信号通路,减少神经元凋亡,降低脑缺血再灌注损伤.     

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…     

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。     

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。     

线粒体钙单向转运体在匹鲁卡品致痫大鼠海马神经损伤中的作用

目的研究线粒体钙单向转运体(mitochondrial calcium uniporter,MCU)在氯化锂-匹鲁卡品(pilocarpine,PILO)癫痫大鼠模型神经损伤中的作用。方法将84只雄性SD大鼠随机分成空白对照(CON)组、PILO组(2 h、8 h、24 h和72 h 4个亚组)、Ru360组和精胺(Spermine)组。观察各组大鼠行为学改变,并荧光染色法测定海马组织线粒体Ca~(2+)浓度,TUNEL染色检测海马CA3区神经元凋亡,并采用Western Blot法检测凋亡相关蛋白Bax、Bcl-2、Cyt C和caspase-3表达变化。结果 (1)与CON组比较,PILO组海马神经元线粒体Ca~(2+)浓度明显升高,凋亡明显增加(P0.05);与CON组比较,PILO组海马Bax表达水平、线粒体Cyt C释放及caspase-3激活明显增多,而Bcl-2表达水平明显减少(P0.05)。(2)与PILO组比较,Ru360组海马神经元线粒体Ca~(2+)浓度明显降低,凋亡明显减少(P0.05);与PILO组比较,Ru360组海马Bax表达水平、线粒体Cyt C释放及caspase-3激活明显减少,而Bcl-2表达水平明显增多(P0.05)。(3)与PILO组比较,Spermine组海马神经元线粒体Ca~(2+)浓度明显升高,凋亡明显增加(P0.05);与PILO组比较,Spermine组海马Bax表达水平、线粒体Cyt C释放及caspase-3激活明显增多,而Bcl-2表达水平明显减少(P0.05)。结论 MCU在PILO癫痫大鼠海马神经损伤中发挥重要作用,抑制MCU可发挥对海马神经元凋亡的保护作用,其机制可能是通过抑制线粒体介导的细胞凋亡途径。     

Role of chloride channels in nitric oxide-induced rat hippocampal neuronal apoptosis In vitro

BACKGROUND:Chloride channels participate in non-neuronal apoptosis.However,it remains unclear whether chloride channels are involved in ischemic neuronal apoptosis.OBJECTIVE:To explore the effects of 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS),two chloride channel blockers,on the hippocampal neuronal apoptosis induced by 3-morpholinosydnonimine (SIN-1) based on the nitric oxide toxicity theory of neuronal apoptosis following ischemic brain injury.DESIGN,TIME AND SETTING:Comparative observation and in vitro experiments were performed at the laboratory of Zhuhai Campus of Zunyi Medical College from January to May 2009.MATERIALS:SIN-1,SITS,and DIDS were purchased from Sigma,USA.METHODS:Hippocampal neurons from Sprague-Dawley rats,aged 1 day,were cultured In vitro for 12 days and randomly assigned to control,SIN-1,or chloride channel blocker groups.SIN-1 group neurons were induced by SIN-1 for 18 hours to establish a model of ischemic neuronal apoptosis.Neurons in chloride channel blocker groups were treated with SITS or DIDS plus SIN-1 for 18 hours.The controls were cultured in DMEM/Ham's F12 complete medium alone.MAIN OUTCOME MEASURES:The apoptotic neurons and nuclear appearance were detected by Hoechst 33258 fluorescence staining; neuronal viability was quantitatively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis.Caspase-3 activity was analyzed by Western blot.RESULTS:SIN-1 (1 mmol/L) dramatically induced apoptosis (50%-60%).SITS and DIDS inhibited nitric oxide-induced neuronal injury in a dose-dependent manner,suppressed caspase-3 activation,reduced neuronal apoptosis,and improved neuronal survival.CONCLUSION:Chloride channel blockers can protect against neuronal injury induced by NO.Chloride channels might be involved in neuronal apoptosis following cerebral ischemia.     

ClC-3 chloride channel in hippocampal neuronal apoptosis

Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area fol- lowing ischemic brain injury. In this study, an apoptotic model in rat hippocampal neurons was es- tablished by 0.5 mmol/L 3-morpholinosyndnomine （SIN-l）, a nitric oxide donor. The models were then cultured with 0.1 mmol/L of 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS; the chloride channel blocker）for 18 hours. Neuronal survival was detected using the 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining. Western blot analysis and immunochemilumi- nescence staining were applied to determine the changes of activated caspase-3 and CIC-3 channel proteins. Real-time PCR was used to detect the mRNA expression of CIC-3. The results showed that SIN-1 reduced the neuronal survival rate, induced neuronal apoptosis, and promoted CIC-3 chloride channel protein and mRNA expression in the apoptotic neurons. DIDS reversed the effect of SIN-I. Our findings indicate that the increased activities of the CIC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide.     

Anion channel blockers attenuate delayed neuronal cell death induced by transient forebrain ischemia

Chloride efflux is known to be involved in the progression of apoptosis in various cell types. We have recently shown that the volume-sensitive outwardly rectifying (VSOR) anion channel serves as the pathway for apoptotic chloride efflux in some cells. In the present study, we tested the neuroprotective effects of drugs that can block the VSOR anion channel, on delayed neuronal death (DND) induced by transient forebrain ischemia. The functional expression of the VSOR anion channel was first examined in hippocampal neurons in both primary culture and hippocampal slice preparations, by the whole-cell patch-clamp technique. We then tested the channel's sensitivity to an anion channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and a tyrosine kinase blocker, genistein. By histological examinations and cytochrome c release assessments, the protective effects of these drugs on the DND of hippocampal CA1 neurons in mice subjected to transient ischemia were examined. Drugs were administered via the jugular vein prior to ischemic treatment and into the peritoneal cavity after reperfusion. Hippocampal neurons were found to express the volume-sensitive Cl(-) channel, which exhibits outward rectification and is sensitive to DIDS and genistein. Administration of DIDS or genistein reduced cytochrome c release and the number of damaged neurons in the CA1 region after transient forebrain ischemia. This fact suggests that the DND induction mechanism involves the activity of the VSOR anion channel and that this channel may provide a therapeutic target for the treatment of stroke.     

Disparity between ionic mediators of volume regulation and apoptosis in N1E 115 mouse neuroblastoma cells

Cellular volume loss or shrinkage is a ubiquitous feature of apoptosis and thus may contribute to this form of degeneration. Chloride (Cl(-)) and potassium (K(+)) efflux has been shown to participate in volume regulation and several recent reports have implicated K(+) efflux in apoptotic neuronal death. Here pharmacological inhibitors of various K(+) and Cl(-) channels and transporters were used to decipher the relationship between cellular volume regulation and apoptosis. Following exposure to a hypotonic media, cells swell but over time gradually recover, returning to their original cell volume in a process known as regulatory volume decrease (RVD). RVD in N1E 115 neuroblastoma cells was monitored using time-lapse videomicroscopy, cell size and DNA degradation were followed using flow cytometry and fragmented apoptotic nuclei were visualized using Hoechst staining. RVD was blocked by high K(+), TEA and 4-AP (K(+) channel blockers), DIDS and niflumic acid but not SITS (Cl(-) channel blockers), ethacrynic acid (Cl(-) pump blocker), bumetanide (Na(+)/K(+)/Cl(-) cotransporter blocker) and furosemide (K(+)/Cl(-) cotransport blocker). In contrast, only DIDS and SITS (blockers of the Cl(-)/HCO(3) exchanger) inhibited apoptosis, suggesting that a common mechanistic link between RVD and apoptosis is the Cl(-)/HCO(3) exchanger. Thus, this study does not support the notion that K(+) channels are universal anti-apoptotic targets. Instead, the Cl(-)/HCO(3) exchanger may prove to be a viable target of therapeutic intervention for treating pathological apoptosis and neurodegeneration.     

NGF对烧伤大鼠血清引起纹状体神经元细胞毒性的影响

观察NGF对烧伤大鼠血清引起神经毒性的影响。初步探讨NGF对烧伤后神经元损伤的保护作用及其机制,测定烧伤大鼠纹状体组织NO和LDH含量;给予原代培养纹状体神经元不同浓度NCF24h后,加入不同浓度烧伤大鼠血清,测定细胞存活率及培养液中NO含量,大鼠烧伤后,纹状体组织NO和LDH含量明显升,烧伤大鼠血清可引起卢培养的纹状体神经元存活率下降,培养液中NO含量升高,NGF能降低纹状体组织中NO和LDH的含量,提高培养的纹状体神经元的存活率,减少培养液中NO含量,其作用呈剂量依赖性,NGF对神经元存活率的影响与NO含量呈显著负相关,NGF对烧伤大鼠血甭引起的纹状体神经元损伤有保护作用。其作用机制可能是通过抑制NO的神经毒性。     

神经生长因子对大鼠脑缺血再灌注海马神经细胞损伤的影响

目的 :观察神经生长因子 (NGF)对脑缺血再灌注后海马CA1区神经细胞损伤的影响。方法 :采用大鼠脑缺血再灌注模型 ,在光镜和透射电镜下观察NGF治疗组和缺血再灌组动物脑缺血 3 0min再灌注 2 4h和 72h时海马组织学及超微结构的改变。结果 :在缺血 3 0min再灌注 2 4h和 72h时 ,NGF治疗组海马CA1区神经细胞的结构损伤与缺血再灌组比较显著减轻 ;NGF可以减轻海马迟发性神经细胞死亡 (DND)性损伤。结论 :NGF对缺血再灌注所致的神经细胞损伤可能具有一定的保护作用     

Mercury compounds disrupt neuronal glutamate transport in cultured mouse cerebellar granule cells

Cerebellar granule cells are targeted selectively by mercury compounds in vivo. Despite the affinity of mercury for thiol groups present in all cells, the molecular determinant(s) of selective cerebellar degeneration remain to be elucidated fully. We studied the effect of mercury compounds on neuronal glutamate transport in primary cultures of mouse cerebellar granule cells. Immunoblots probed with an antibody against the excitatory amino acid transporter (EAAT) neuronal glutamate transporter, EAAT3, revealed the presence of a specific band in control and mercury-treated cultures. Micromolar concentrations of both methylmercury and mercuric chloride increased the release of endogenous glutamate, inhibited glutamate uptake, reduced mitochondrial activity, and decreased ATP levels. All these effects were completely prevented by the nonpermeant reducing agent Tris-(2-carboxyethyl)phosphine (TCEP). Reduction of mitochondrial activity by mercuric chloride, but not by methylmercury, was inhibited significantly by 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) and by reduced extracellular Cl- ion concentration. In addition, DIDS and low extracellular Cl- completely inhibited the release of glutamate induced by mercuric chloride, and produced a partial although significant reduction of that induced by methylmercury. We suggest that a direct inhibition of glutamate uptake triggers an imbalance in cell homeostasis, leading to neuronal failure and Cl(-)-regulated cellular glutamate efflux. Our results demonstrate that neuronal glutamate transport is a novel target to be taken into account when assessing mercury-induced neurotoxicity.     

Aerobic exercise combined with huwentoxin-I mitigates chronic cerebral ischemia injury

Ca2+ channel blockers have been shown to protect neurons from ischemia, and aerobic exercise has significant protective effects on a variety of chronic diseases. The present study injected huwentoxin-I (HWTX-I), a spider peptide toxin that blocks Ca2+ channels, into the caudal vein of a chronic cerebral ischemia mouse model, once every 2 days, for a total of 15 injections. During this time, a subgroup of mice was subjected to treadmill exercise for 5 weeks. Results showed amelioration of cortical injury and improved neurological function in mice with chronic cerebral ischemia in the HWTX-I + aerobic exercise group. The combined effects of HWTX I and exercise were superior to HWTX-I or aerobic exercise alone. HWTX-I effectively activated the Notch signal transduction pathway in brain tissue. Aerobic exercise up-regulated synaptophysin mRNA expression. These results demonstrated that aerobic exercise, in combination with HWTX-I, effectively relieved neuronal injury induced by chronic cerebral ischemia via the Notch signaling pathway and promoting synaptic regeneration.     

Voltage-gated sodium channel blockers for the treatment of neuropathic pain

Pain serves a crucial physiological function, warning the body of impending or actual tissue damage, preventing further damage and aiding the healing process. Neuropathic pain, resulting from nervous system injury or dysfunction, can be a serious medical problem and especially difficult to treat. Although sodium channel blockers are clinically useful for treating pain, they often provide only partial relief and adverse effects associated with nonspecific actions can limit their use. Research on the roles of sodium channels in neuronal excitability and pain shows that specific sodium channel isoforms are crucial determinants of nociception and neuropathic pain, indicating that it should be possible to develop sodium channel blockers with lower toxicity and enhanced efficacy for treating neuropathic pain.