非对称性二甲基精氨酸保护PCI2细胞拮抗谷氨酸兴奋性毒性的损伤作用

目的：探讨一氧化氮合酶（NOS）抑制剂-非对称性二甲基精氨酸（ADMA）对谷氨酸（Glu）兴奋性毒性损伤PCI2细胞的影响及其机制。方法：用不同浓度的谷氨酸处理PCI2细胞，建立谷氨酸兴奋性神经毒性损伤细胞的实验模型；应用四甲基偶氮唑蓝（MTF）比色法检测细胞存活率；乳酸脱氢酶（LDH）释放试验评价细胞的损伤程度；双氢罗丹明123（DHR）染色后流式细胞仪（FCM）检测细胞内活性氧（ROS）水平；应用试剂盒及分光光度计测定NOS活性和NO水平。结果：谷氨酸（1—6mmol／L）处理PC12细胞24h，可呈剂量依赖性地降低PC12细胞的存活率；在谷氨酸作用PCI2细胞前30min给予ADMA，可明显地抑制谷氨酸引起的细胞存活率降低及LDH释放增加，减少谷氨酸引起的细胞内ROS堆积，抑制谷氨酸过度激活NOS和增加NO的生成。结论：ADMA能显著地减弱谷氨酸对PC12细胞的兴奋性毒性损伤作用；其作用机制可能与抑制NOS活性，减少NO生成，进而减轻细胞内ROS的堆积有关。     

樟芝酵素对苯丙胺致PC12细胞毒性损伤的保护作用

目的　探讨樟芝酵素对苯丙胺致PC12细胞损伤的保护作用及其机理。 方法　利用PC12细胞模型,设置对照组,3.5 mmol/L苯丙胺组和3.5 mmol/L苯丙胺+樟芝酵素组。(1)通过显微镜观察细胞形态的变化;(2)通过MTT法检测细胞存活率;(3)通过Western blot分析Caspase-3的变化; (4)通过流式细胞仪检测细胞凋亡和细胞内活性氧的变化。 结果　(1)苯丙胺损伤了PC12细胞的结构,导致细胞凋亡增加,樟芝酵素能够减少细胞的凋亡,提高细胞的存活率;(2)樟芝酵素减少了细胞内ROS的生成;(3)樟芝酵素组procaspase-3（35 kd）蛋白含量比苯丙胺组增多,激活分子caspase-3（17 kd）含量较苯丙胺组减少。 结论　樟芝酵素对苯丙胺致PC12细胞毒性损伤具有保护作用,其机理与抑制细胞线粒体凋亡信号通路以及减少细胞内氧自由基的产生密切相关。     

MPP+对PC12细胞内源性H2S生成的影响

目的： 观察1-甲基4-苯基吡啶离子 (MPP+)对PC12细胞内源性硫化氢（H2S）生成的影响,以探讨MPP+损伤PC12细胞的新机制。方法: RT-PCR方法检测PC12细胞胱硫醚-β-合酶（CBS）mRNA 的表达;亚甲基蓝分光光度计法检测PC12细胞内源性H2S的含量及PC12细胞CBS活性;台盼蓝拒染色法观察PC12细胞的存活率。结果：MPP+ 可以抑制PC12细胞CBS的表达及其活性,减少内源性H2S的生成;MPP+可以明显地降低PC12细胞的存活率;H2S的供体硫氢化钠（NaHS）对MPP+诱导的PC12细胞损伤具有显著的拮抗作用。结论: MPP+能抑制CBS的表达和活性,减少内源性H2S生成,这可能与其损伤PC12细胞的机制有关。     

低糖诱导人脐静脉内皮细胞一氧化氮与活性氧变化的研究

目的观察低浓度葡萄糖对人脐静脉内皮细胞一氧化氮（NO）与活性氧（ROS）变化的影响。方法以体外培养人脐静脉内皮细胞株HUVEC-12为研究对象,将实验分为正常对照组（5.5mmol/L葡萄糖）、低糖组（2.8mmol/L葡萄糖）、无糖组（0mmol/L葡萄糖）。分别用2.8、0mmol/L葡萄糖干预HUVEC-12细胞,经过1、2、4、12h后,硝酸还原酶法测定NO产量,化学比色法测定一氧化氮合成酶（NOS）活性,Dihydroethidium荧光探针法测定细胞内ROS水平。结果与正常对照组相比,低糖组与无糖组NO水平降低（P〈0.01）,NOS活性下降（P〈0.01）,细胞内ROS水平升高（P〈0.01）,均呈剂量依赖关系。同一浓度组内随着时间的延长,内皮细胞NO水平、NOS活性进一步降低（P〈0.01）,ROS水平进一步升高（P〈0.05）,各指标都具有浓度和时间依赖性。结论低糖可导致内皮细胞功能障碍,NO水平降低,NOS活性下降,其机制可能与低糖导致内皮细胞氧化应激损伤有关。     

细胞外信号调节激酶/miR-133b在甲基苯丙胺致PC12细胞神经毒性的作用及机制

目的 探讨甲基苯丙胺(MA)致神经细胞毒性过程中细胞外信号调节激酶(ERK)和miR-133b的表达变化及调控机制。方法 用MA建立PC12神经细胞损伤模型,采用四甲基偶氮唑盐(MTT)检测细胞活性及镜下形态观察确定MA最佳损伤浓度;应用流式细胞术检测细胞内活性氧（ROS）水平;通过 Western blotting技术测定总ERK1/2和磷酸化ERK1/2 (p-ERK1/2)的表达变化;并应用实时定量聚合酶链反应（Real-time PCR）测定miR-133b的表达变化。为进一步分析ERK/miR133b分子通路的作用关系,经U0126特异阻断ERK通路,检测miR-133b的表达变化。 结果 给予不同浓度的MA,均可导致PC12细胞损伤,其中800μmol/L MA处理后,大部分胞体变圆,神经突起退缩,神经网络消失。MTT结果显示细胞活性明显下降。进一步的细胞毒性机制分析显示,MA处理后,细胞内ROS水平升高,p-ERK表达增高,miR-133b表达降低;并且给予ERK通路抑制剂U0126（10μmol/L）后,miR-133b表达升高,细胞活性增强,胞内ROS水平降低,镜下细胞损伤改善。 结论 MA可通过上调ERK磷酸化抑制miR-133b表达,介导神经元毒性损伤。     

右美沙芬对培养大鼠皮层神经元谷氨酸兴奋毒性和缺氧损伤的保护作用

本文采用16～18d胎龄的大鼠皮层细胞进行分离培养，以培养上清液中乳酸脱氢酶（LDH）活性作为细胞损伤的指标，分别观察了缺氧和过量谷氨酸对皮层神经元的影响以及右美沙芬的保护作用。实验结果表明，外源性谷氨酸（10和50μmol／L）和缺氧（5h）均引起LDH释放增加，而这种作用皆被右美沙芬所抑制。提示右美沙芬对此有明显的保护作用。以上结果说明谷氨酸兴奋毒性与NMDA受体在缺血性脑损伤过程中起着重要的作用。     

NMDA对新生大鼠皮层神经元的兴奋毒作用

目的:建立NMDA诱导原代培养皮层神经元兴奋毒损伤模型,探讨NMDA对NMDA受体过度活化诱导兴奋性神经毒的可能途径。方法:原代培养新生大鼠大脑皮层神经元,通过倒置显微镜形态学观察、细胞活力检测(MTT及LDH释放的检测)及胞内Ca2+的动态测定,探索NMDA诱导毒性作用的适当浓度及时间。通过对ROS、NO检测,分析NMDA诱导毒性作用于线粒体的损伤情况。结果:NMDA(100μmol/L/2 h)引起皮层神经元形态学改变,且引起神经元细胞活力时间和浓度依赖性的下降,由同时伴随LDH释放增加(P<0.05),ROS和NO的生成量明显增加(P<0.05),皮层神经元内Ca2+的快速升高,并维持在高水平。结论:NMDA诱发皮层神经元明显的细胞毒性作用,提示NMDA过度活化NMDA受体后通过神经元膜内Ca2+超载造成ROS和NO的生成量增加,导致皮层神经元产生毒性损伤。     

硫化氢对抗过氧化氢对PC12细胞的损伤作用

目的探讨硫化氢(hydrogen sulfide,H2S)对抗过氧化氢(hydrogen peroxide,H2O2)对PC12细胞的损伤作用及有关机制。方法应用H2O2在PC12细胞建立氧化应激损伤的实验模型;应用甲氮甲唑蓝(MTT)法检测细胞存活率,碘化丙啶(PI)染色流式细胞技术(FCM)检测细胞凋亡率,罗丹明123(Rhodamine 123,Rh123)染色FCM检测细胞线粒体膜电位(mitochondrial membrane potential,MMP),双氢罗丹明123染色FCM检测细胞内活性氧(reactive oxygen species,ROS)的含量。应用硫化氢钠(sodium hydrosulfide,NaHS)作为H2S的供体。结果200μmol和400μmolH2O2作用PC12细胞24h均使细胞的存活率明显降低及凋亡率显著增加,200μmolH2O2引起PC12细胞的MMP明显降低及ROS生成显著增多。当NaHS与H2O2(200或400μmol/L)共同作用于PC12细胞时,NaHS(100～400μmol/L)浓度依赖性的阻断H2O2引起PC12细胞的存活率降低及细胞凋亡率增加。400μmolNaHS明显地阻断200μmolH2O2引起PC12细胞的MMP降低及ROS增多。结论H2S能明显地保护PC12细胞对抗H2O2引起的损伤,阻断MMP降低及ROS生成可能是H2S的细胞保护机制之一。     

硫化氢对缺氧诱导的大鼠皮层神经元损伤的保护作用

 目的:探讨硫化氢(H 2S)对缺氧诱导的皮层神经元损伤的影响及作用机制。方法:将SD大鼠皮层神经元在2% O 2、5% CO 2、93 % N 2、37 ℃培养箱培养24 h,建立细胞缺氧模型。以硫氢化钠（NaHS）作为H 2S的供体,应用CCK-8分析细胞活性;采用荧光探针DCFH-DA检测神经元活性氧(ROS)含量;用Rh123染色测定线粒体膜电位(MMP);采用乳酸脱氢酶(LDH)试剂盒分析神经元LDH释放率,反映神经元的损伤情况。结果:(1)缺氧引起神经元ROS含量和LDH释放率升高,NaHS预处理可抑制缺氧所致神经元ROS含量和LDH释放率的升高;(2)缺氧降低神经元MMP和细胞活性,NaHS和活性氧清除剂NAC预处理均显著抑制缺氧所致神经元MMP和细胞活性的降低。结论:缺氧增加神经元ROS含量,降低神经元MMP和细胞活性,而H 2S通过其抗氧化作用,减轻缺氧所致神经元的损伤。     

一氧化氮合酶抑制物在胃黏膜损伤中的作用与机制

目的:研究一氧化氮合酶抑制物非对称性二甲基精氨酸(ADMA)在不同因素诱发的胃黏膜损伤中的作用,并初步探讨其机制。方法:用乙醇、吲哚美辛、应激诱发大鼠胃黏膜损伤模型,检测胃黏膜溃疡指数(UI),一氧化氮(NO)和ADMA水平,以及二甲基精氨酸-二甲胺水解酶(DDAH)的活性;检测幽门螺旋杆菌(Hp)处理胃黏膜上皮细胞(GES-1)细胞培养液中NO,ADMA和TNF-α水平,以及细胞中DDAH活性。结果:乙醇、吲哚美辛、应激诱发大鼠胃黏膜损伤的同时ADMA水平显著升高以及DDAH活性下降;Hp处理GES-1细胞24 h后,ADMA和TNF-α水平显著升高,DDAH活性下降;外源性ADMA也能显著升高GES-1细胞TNF-α水平。结论:ADMA是促进胃黏膜损伤的重要因子,除抑制NO生成外,还具有直接致炎作用。     

阿司匹林对炎症条件下人血管内皮细胞一氧化氮的产生及诱导型一氧化氮合酶mRNA表达的影响

目的:探讨炎症时阿司匹林(AS)对内皮细胞一氧化氮(NO)的产生及诱导型一氧化氮合酶(iNOS)基因表达的抑制作用。方法:Griess法测上清液NO^-₂/NO^-₃水平、黄递酶法测NOS活性、常规生化法测乳酸脱氢酶(LDH)、丙二醛(MDA)浓度,染料排除法测细胞活力,RT-PCR技术分析iNOSmRNA水平。结果:白介素(IL)-1β、肿瘤坏死因子(TNF)-α、γ-干扰素(INF)联用脂多糖(LPS)诱导后上清液中NO^-₂/NO^-₃由(4.27±0.75)μmol/L增加到(9.35±1.25)μmol/L,对内皮细胞造成明显的损伤。但3mmol/LAS组NO生成及NOS活性明显降低,LDH释放率及MDA浓度下降,细胞存活率上升,与NO诱导组相比差异显著。并随AS剂量的增加对NO的抑制及对细胞的保护作用更加明显,但AS对生理水平的NO没有抑制作用。同时发现10mmol/L浓度以下AS对iNOSmRNA表达水平没有影响;但10-20mmol/L的AS则可在转录水平上抑制iNOSmRNA的表达。并观察到水杨酸钠及消炎痛不具有抑制NO产生的作用。结论:AS具有明显抑制IL-1β、TNF-α、γ-INF及LPS诱导NO生成的作用,从而保护血管内皮细胞避免炎症时高浓度NO的损伤。     

同型半胱氨酸硫内酯损伤血管内皮细胞的机制研究

目的: 在体外培养的内皮细胞中探讨同型半胱氨酸硫内酯(HTL)致血管内皮细胞损伤作用及其机制。方法: 体外培养的人脐静脉内皮细胞,与不同浓度的HTL孵育,用ELISA检测内皮细胞中肿瘤坏死因子α(TNF-α)和可溶性细胞间黏附分子(sICAM)-1的浓度,荧光显微镜检测活性氧簇(ROS)的含量、核转录因子κB(NF-κB)的激活及IκBα蛋白表达,同时检测内皮细胞活力、乳酸脱氢酶(LDH)漏出量与一氧化氮(NO)水平。结果: HTL(1 mmol/L)孵育内皮细胞3 h后显著增加细胞 ROS含量,刺激NF-κB转入胞核而活化,孵育细胞24 h后明显升高细胞上清液中sICAM-1和TNF-α浓度;降低细胞活力和NO的水平,增加LDH的漏出量。抗氧化剂NAC、NADPH氧化酶抑制剂Apocynin和NF-κB抑制剂PDTC可显著抑制HTL所致ROS含量的增加以及NF-κB激活,降低HTL刺激的培养液中sICAM-1和TNF-α的浓度,增加培养液中NO水平。结论: HTL诱导的血管内皮细胞功能损伤的机制可能与诱导氧化应激以及NF-κB活化有关。     

Glutamate-induced free radical formation in rat brain synaptosomes is not dependent on intrasynaptosomal mitochondria membrane potential

Glutamate induces reactive oxygen species formation (ROS) in neurons. Free radicals can potentially be synthesized by NADPH oxidase or mitochondria. The primary source of ROS origin has yet to be identified. In addition, pro-oxidant action of glutamate receptors on neuronal presynaptic terminals is still not characterized. We investigated the influence of glutamate and agonists of its ionotropic receptors on ROS formation detected by fluorescent dye DCFDA in rat brain synaptosomes. Glutamate in concentration 10 and 100μM led to an increase of probe fluorescence pointing to free radical accumulation. This effect was mimicked by 100μM of NMDA or 100μM of kainate. Glutamate-induced ROS formation was sensitive to NMDA inhibitors MK-801 (10μM), NO synthase (NOS) inhibitor l-NAME (100μM) and NADPH oxidase inhibitors DPI (30μM) and not affected by mitochondrial uncoupler CCCP (10μM) and mitochondrial toxins rotenone (10μM)+oligomycin (5μg/ml). We also showed that 100μM of glutamate leads to a decrease of intrasynaptosomal mitochondrial potential monitored by fluorescent dye Rhodamine-123. Hence, the depolarization of intrasynaptosomal mitochondria is not a primary cause of glutamate-induced ROS formation in neuronal presynaptic terminals. Activation of NMDA receptors might be responsible for a certain part of glutamate pro-oxidant action. Most likely, sources of glutamate-induced ROS formation in neuronal presynaptic terminals are NADPH oxidase and NOS activation.     

Protective effect of 7-difluoromethoxy-5,4'-Di-hydroxyl isoflavone against the damage induced by glutamate in PC12 cells

7-difluoromethoxy-5,4'-Di-hydroxyl isoflavone (dFGEN), prepared by the difluoromethylation of genistein, is an active chemical entity. In this study, our main purpose was to investigate whether dFGEN had an effect on glutamate-induced apoptosis in cultured PC12 cells. The PC12 cells were treated with different glutamate concentrations for 24 h in vitro. The PC12 cells impaired by glutamate were used as the cell model of excitability. Cells were incubated for 30 min with genistein, dFGEN, vitamin E, and exposed to 10 mM glutamate for 24 h. Cell morphology was observed by light microscopy. The growth and proliferation of PC12 cells were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Cell apoptosis was determined by flow cytome-try (FCM) with propidium iodide (PI) staining. The activity of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and the content of malondialdelyde (MDA) were measured by kits, respectively. Acridine orange (AO) staining was used to detect characteristics of cell apoptosis. When PC12 cells were incubated with glutamate for 24 h, cells appeared to have significant changes in shape. The cellular viability was reduced and the apoptotic rate was increased. The levels of LDH and the content of MDA were increased. The activity of SOD was decreased. After PC12 cells were pretreated with dFGEN, dFGEN significantly improved cell morphology, cell growth and proliferation, suppressed apoptosis of cells, reduced the release of LDH, improving SOD activity and decreased MDA content in a concentration-dependent manner. AO staining displayed that apoptosis was decreased. These results suggested that dFGEN has a protective effect against glutamate-induced damage in PC12 cells. dFGEN seemed to have a better protective effect than the lead compound genistein in a concentration-dependent manner. The mechanism of protective effect of dFGEN may be mainly related to its antioxidative activity.     

Asymmetric dimethylarginine induces oxidative and nitrosative stress in murine lung epithelial cells

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by epithelial and inflammatory cells are key mediators of the chronic airway inflammation of asthma. Low L-arginine levels can result in the uncoupling of nitric oxide synthase (NOS) leading to production of both ROS and RNS. Asymmetric dimethylarginine (ADMA) is a competitive endogenous inhibitor of all NOS isoforms and has been demonstrated to inhibit NO formation and increase oxidative stress in vascular endothelial and smooth muscle cells. The effect of ADMA on inducible NOS (iNOS) activity in epithelial cells has not been explored. In this study, we investigated whether addition of exogenous ADMA alters the generation of NO and superoxide anion (O2-), leading to peroxynitrite (ONOO-) formation in a mouse epithelial cell line. In stimulated LA-4 cells, ADMA dose-dependently inhibited nitrite accumulation after 24 h of treatment. In addition, ADMA concentrations as low as 10 microM induced rapid increases in O2- production as measured by dihydroethidium oxidation. Furthermore, using dihydrorhodamine to monitor ONOO- formation, ADMA caused a dose-dependent increase in ONOO- after treatment for 24 h. Similar effects of ADMA were seen using purified iNOS protein in a cell-free system. Together, these data indicate that elevated ADMA may contribute to the production of ROS and RNS in airway inflammation.     

卡维地洛对氧自由基诱导的内皮细胞内源性一氧化氮合酶抑制物代谢的影响

目的： 观察卡维地洛对氧自由基（OFR）培养的人脐静脉内皮细胞（HUVECs）二甲精氨酸-二甲赖氨酸水解酶 (DDAH)活性及表达的影响，以探讨卡维地洛对不对称二甲精氨酸（ADMA）代谢机制的影响。 方法： 采用改良的Jaffe法培养原代人脐静脉内皮细胞（HUVECs），取生长良好的3-6代HUVECs用于实验，分为①空白对照组：加DMEM培养液；②OFR组:加入OFR（0.01 mmol/L,0.1 mmol/L）；③OFR+卡维地洛组: 同时加入0.1 mmol/L OFR及卡维地洛（10 μmol/L）共孵24 h后, 检测上清液中一氧化氮（NO）、内皮素（ET）、ADMA含量、L-胍氨酸(L-cit)浓度及一氧化氮合酶（NOS）活性。用Western blotting法测定细胞裂解液中二甲基精氨酸-二甲基赖氨酸水解酶 (DDAH)的蛋白表达。 结果： OFR条件培养下,内皮细胞的代谢产物ADMA、ET的量均高于空白对照组,而NO的量及NOS的活性少于空白对照组;反映DDAH酶活性的L-cit浓度显著降低,且有浓度依赖性，而DDAH的表达无明显变化。卡维地洛干预组的ADMA、ET的量均低于OFR组，NOS活性及NO、L-cit浓度明显高于OFR组。 结论： OFR培养下，内皮损伤ADMA的增加与DDAH的活性减弱有关，而与DDAH的表达无关。卡维地洛通过增加DDAH活性促进ADMA代谢，使NOS活性增加，抑制OFR对内皮功能的损伤。     

谷氨酸诱导海马神经元凋亡涉及线粒体释放细胞色素C

目的：建立体外谷氨酸诱导神经元兴奋损伤模型，探索其凋亡发生是否通过线粒体信号转导途径介导的细胞色素C（Cyt C）释放而实现，为今后干预性使用神经保护剂提供依据。方法:分离及培养新生Wistar大鼠海马神经元，选用合适谷氨酸浓度建立神经元损伤模型；利用LDH测定及流式细胞仪Annexin V/PI双染色法检测谷氨酸暴露后不同时点神经元凋亡及坏死的动态改变；采用Western blotting法检测caspase-3活性及线粒体内和胞浆内Cyt C水平动态变化。结果:谷氨酸诱导神经元损伤呈明显浓度及时间依赖性，50 μmol/L浓度可使LDH释放量明显增加 (18.4%,P<0.05)，暴露后6 h凋亡率显著增加；凋亡发生前，神经元caspase-3活性已明显增高（3 h），6 h达高峰；线粒体Cyt C释放发生在caspase-3增高前，30 min时胞浆内Cyt C水平即明显增加（P<0.05），3 h胞浆内Cyt C水平超过线粒体内，而线粒体内Cyt C水平进行性减少。结论:50 μmol/L谷氨酸可诱导海马神经元凋亡，凋亡机制可能是通过损伤线粒体膜，使Cyt C易位释放入胞浆激活caspase级联反应而致。     

Blockade of nitric oxide formation does not prevent glutamate-induced neurotoxicity in neuronal cultures from rat hippocampus.

This study examined the role of nitric oxide (NO) in glutamate-induced, N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in rat hippocampal neuronal cultures grown under serum-free conditions. Formation of cGMP was used as an indirect measure of NO formation. Neuronal cell degeneration was monitored by measuring the release of lactate dehydrogenase (LDH). Neuronal cells showed a 4-fold increase in cGMP formation and release of LDH upon exposure to 30 microM glutamate. cGMP formation was fully inhibited by 1 microM nitro-arginine (N-Arg), 100 microM hemoglobin or 1 microM MK-801. In the presence of 1 microM MK-801, glutamate induced neither cGMP formation nor neuronal cell degeneration. However, when NO formation was inhibited by means of 100 microM N-Arg, glutamate still induced neurotoxicity. Therefore, in serum-free hippocampal cultures glutamate neurotoxicity occurs notwithstanding complete inhibition of the NO-synthase enzyme by N-Arg. Our data provide evidence that NO, synthesized upon glutamate exposure, has not a primary toxic action in pure hippocampal neuronal cultures.     

Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis

Glutamate toxicity on PC12 cells is mediated by oxidative stress as a consequence of the inhibition of a cystine uptake system with depletion of GSH. In this study we report that glutamate decreases PC12 cell viability, inhibiting the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This decrease was prevented by the antioxidants vitamin E, idebenone and L-deprenyl, which were also shown to be effective in reducing the accumulation of reactive oxygen species (ROS) in cells exposed to glutamate, decreasing the fluorescence of 2',7'-dichlorofluorescein (DCF). Incubation of PC12 cells with high glutamate concentrations induced mitochondrial dysfunction, leading to the loss of mitochondrial transmembrane potential, evaluated as a decrease in rhodamine 123 (Rh123) retention by mitochondria, and to the decrease of intracellular ATP levels. The mitochondrial dysfunction, induced by glutamate, can be involved in the observed increase of [Ca2+]i. The elevation of [Ca2+]i occurred after GSH depletion, suggesting that oxidative stress is involved in the disturbances of intracellular calcium homeostasis. In conclusion, our data indicate that glutamate, at concentrations which block cystine uptake in PC12 cells leading to GSH depletion and inducing oxidative stress, increases ROS accumulation and decreases cell survival by a mechanism involving mitochondrial dysfunction and impairment of Ca2+ homeostasis.     

炎性细胞因子及脂多糖诱导产生的一氧化氮对人血管内皮细胞的损伤作用

目的:探讨炎症过程诱生的一氧化氮(NO)对内皮细胞的损伤及其作用机制。方法:黄递酶法及Griess法检测可诱导性NO合酶(iNOS)活性、NO₂^-/NO₃^-水平,RT-PCR技术分析iNOSmRNA的表达;同时观察NO产生后对内皮细胞的损伤作用。结果:IL-1β2×10⁵U/L、TNF-α5×10⁵U/L、γ-INF2×10⁵U/L联用LPS(10mg/L)可诱导出高浓度NOS合成及NO产生,比单用这些细胞因子或LPS诱导的量高两倍多,iNOSmRNA的表达水平也显著增加;同时MDA及LDH释放率明显增加,细胞存活率下降,并伴随细胞受损的形态学改变。而单用上述细胞因子或LPS,以及降低剂量或缩短处理时间,其诱生的NOS及NO与正常对照相比P＞0.05;但MDA及LDH释放率仍增加明显。使用2倍剂量的这些炎性细胞因子或延长处理时间到48h,与标准剂量及24h组相比NOS和NO的增加虽不显著,但细胞生长受限更明显。NOS抑制剂能阻止NO的产生及其对细胞的损伤作用。结论:炎性细胞因子及脂多糖可激活iNOS大量表达诱生高浓度的NO,对内皮细胞具有明显的氧化损伤作用。