丁基苯酞对低糖低氧诱导的大鼠皮层神经细胞损伤的保护作用

为进一步探讨丁基苯肽对神经的保护作用,用原代培养大鼠皮层神经细胞的方法,以LDH和细胞形态等指标,观察了l-丁基苯酞(l-NBP)和d-丁基苯酞(d-NBP)对低糖低氧诱导的大鼠皮层神经细胞损伤的保护作用。结果表明:l-NBP和d-NBP能剂量依赖性地抑制低糖低氧诱导的大鼠皮层神经细胞内LDH的释放,降低细胞死亡率,并能改善受损细胞的形态;此外还能明显减轻低糖低氧诱导的神经细胞内粗面内质网脱颗粒及多聚核糖体解聚。提示:NBP对低糖低氧诱导的大鼠皮层神经细胞损伤有明显保护作用。     

阿米替林对谷氨酸损伤中枢神经细胞的保护作用

目的：研究阿米替林（Ami)对谷氨酸损伤培养大鼠皮层神经细胞的保护作用。方法：分离培养15－18d胎龄的大鼠神经细胞，加入谷氨酸（Glu)，造成神经细胞的损伤，测定乳酸脱氢酶，一氧化氮，丙二醛及超氧化物歧化酶的含量，观察谷氨酸对神经细胞的损伤作用及阿米替林的保护作用。结果：加入谷氨酸后，神经细胞出现明显损伤性变化，死亡率升高，培养上清液中乳酸脱氢酶（LDH），一氧化氮（NO）含量增加，细胞匀浆中丙二醛（MDA）生成增加，超氧化物歧化酶（SOD）含量明显减少，Ami10^-8-10^-6mol/L^-1能不同程度地减轻上述损伤性变化。结论：Ami对谷氨酸所致的神经细胞损伤具有保护作用。其作用机制可能与抗脂质过氧化作用有关。     

银杏叶内酯N对谷氨酸损伤PC12细胞的保护作用

目的 观察银杏叶内酯N(GN)对谷氨酸诱导PC12细胞损伤的保护作用及与银杏叶内酯B(GB)的药效比较.方法 采用谷氨酸诱导的PC12细胞损伤作为脑缺血损伤的体外研究模型,采用MTT比色法和乳酸脱氢酶(LDH)活力检测法,观察GN对谷氨酸致PC12细胞损伤的保护作用.结果 GN能明显改善谷氨酸诱导损伤的PC12细胞的活...     

阿魏酸钠对抗Aβ25-35诱导的鼠神经元凋亡作用研究

目的 探讨阿魏酸钠对β淀粉样蛋白(Aβ25-35)通过谷氨酸诱导的鼠皮层神经元凋亡的影响.方法 培养的皮层神经元分别与谷氨酸(20μmol/L)、Aβ25-35(5μmol/L)、Aβ25-35(5μmol/L)+谷氨酸(20μmol/L)孵育,采用Hoechst 33258荧光染色法分析神经细胞凋亡;然后用谷氨酸( 50μmol/L)诱导神经元凋亡,采用荧光染色法和Westem blot观察阿魏酸钠的保护作用.结果 单独应用Aβ25-35(5μmol/L)和单独加入谷氮酸(20μmol/L)引起的皮层神经元凋亡率与对照组无明显差异;Aβ25-35与皮层神经元共同孵育5天,接着用谷氨酸处理24h,细胞凋亡率从正常的9.2%+1.5%增加到43%±8%:阿魏酸钠能够显著降低谷氨酸诱导的神经细胞凋亡百分比至21%±5%,并对抗谷氨酸引起的Bcl-2蛋白表达的降低.结论 阿魏酸钠能够减弱Aβ25-35提高谷氨酸毒性诱导的鼠皮层神经元凋亡.     

丁基苯酞抑制低氧低糖诱导的大鼠皮质神经细胞凋亡

目的：以原代培养的大鼠胎鼠皮质神经元低氧低糖再复氧为模型,研究丁基苯酞对神经细胞凋亡的抑制作用。方法：用流式细胞术检测DNA含量及凋亡细胞百分率,透射电镜观察细胞形态学变化,DNA琼脂糖凝胶电泳和原位末段标记(TUNEL)检测DNA断裂。 结果：丁基苯酞能减轻细胞核形态的改变,减少DNA断裂和阳性细胞数,使低氧低糖诱导的神经细胞凋亡百分率明显下降,凋亡峰显著降低。 结论：丁基苯酞对低氧低糖诱导的大鼠皮质神经细胞凋亡有抑制作用。     

7,8-二羟基-4-甲基香豆素对谷氨酸诱导的PC12细胞损伤的神经保护作用

目的探讨7,8-二羟基-4-甲基香豆素(Dhmc)对谷氨酸诱导的PC12细胞损伤的神经保护作用。方法采用谷氨酸诱导PC12细胞损伤凋亡模型,给予Dhmc处理后,MTT法检测细胞增殖;化学检测法测定乳酸脱氢酶(LDH)漏出量、细胞上清液中丙二醛(MDA)含量及超氧化物歧化酶(SOD)水平;采用Annexin-V-FITC双染法检测细胞凋亡率;采用荧光法检测活性氧(ROS)的生成;采用流式细胞仪检测线粒体膜电位(MMP);Western blot法检测Bcl-2、Bax、Cyt C、cleaved caspase-3蛋白表达。结果 Dhmc可显著降低由谷氨酸诱导的细胞损伤,提高了细胞存活率,减少LDH漏出量,降低MDA含量,增加SOD活性,抑制ROS的生成,维持线粒体膜电位水平,下调Bcl-2/Bax比率,减少Cyt C的释放,降低Caspase-3活性。结论 Dhmc具有减轻由谷氨酸诱导的PC12细胞损伤的作用,其机制可能与抗氧化应激和减轻线粒体损伤有关。     

姜黄素对皮层神经元氧化损伤的保护作用

目的　观察姜黄素对第三丁基过氧化氢 (tert butylhy droperoxide,t BHP ,tBHP)诱导的大鼠皮层神经元氧化损伤的影响 ,探讨可能的机制。方法　培养胚胎鼠皮层神经元 ,MTT法测定细胞活力 ,DNA断裂评价细胞凋亡 ,流式细胞术测定线粒体膜电位和细胞内活性氧水平 ,分光光度法测定细胞内谷胱甘肽 (GSH)水平 ,Westernblot法测定Bcl 2和Bax蛋白和胞浆细胞色素C以及活化型半胱氨酸天冬氨酸蛋白酶 3(caspase 3)和多聚 (ADP 核糖 )聚合酶 [poly (ADP ribose)poly merase,PARP]水平。结果　姜黄素 (2 5～ 2 0 μmol·L-1)可有效减少tBHP对神经元的氧化损伤和tBHP引起的细胞内GSH水平降低 ,降低细胞内的活性氧水平 ,增加线粒体膜电位和细胞内GSH以及Bcl 2蛋白水平 ,减少线粒体内细胞色素C向胞浆释放和Bax蛋白表达水平 ,最终明显减少cas pase 3和PARP活化和tBHP引起的神经元凋亡。结论　姜黄素可减弱tBHP对原代皮层神经元的氧化损伤作用 ,其作用可能与降低细胞内的活性氧水平 ,保护线粒体功能有关     

低频经颅磁刺激对颞叶癫(癇)海马超微结构的影响

目的:研究低频经颅磁刺激(rTMS)对颞叶癫(癇)大鼠海马超微结构的影响.方法:用清洁级Wistar大鼠制备氯化锂-匹罗卡品诱导的颞叶癫(癇)大鼠模型,随机分为对照组和rTMS组,每组5只,对rTMS组大鼠进行rTMS治疗.利用电子显微镜从神经元、神经纤维、突触、胶质细胞和毛细血管5个方面观察颞叶癫(癇)对海马超微结构的病理损伤和rTMS对颞叶癫(癇)海马区病理损伤的作用.结果:颞叶癫(癇)对海马区超微结构具有明显病理损伤,包括神经元出现凋亡和坏死,核浓缩,染色质聚集,线粒体嵴断裂,神经细胞和胶质细胞突起明显水肿,神经纤维局部髓鞘降解,轴浆内微管、微丝排列紊乱,血管内皮细胞肿胀,胶质细胞损伤.rTMS后海马超微结构明显改善,包括脑组织水肿减轻,凋亡和损伤细胞减少,突触和神经毡病变改善和血管水肿减轻.结论:rTMS可改善颞叶癫(癇)对海马超微结构的病理损伤,具有神经保护作用.     

葛根素对谷氨酸所致大鼠原代神经细胞损伤的保护作用

目的从膜脂流动性等观察葛根素对谷氨酸所致大鼠原代神经细胞损伤的保护作用。方法建立大鼠原代神经细胞谷氨酸损伤模型,以1,6-二苯基-1,3,5-己三烯(DPH)为探针,用荧光偏振法测定荧光偏振度(P)值和微粘度(η),研究细胞膜脂流动性的改变;MTT(四甲基偶氮唑盐)法、培养介质乳酸脱氢酶(LDH)活力测定观察葛根素的保护作用。结果葛根素组荧光偏振度和微粘度低于谷氨酸损伤组,与尼莫地平作用相当,且呈现剂量依赖关系。葛根素组原代神经细胞LDH的释放减少,吸光度增加,但葛根素对正常细胞增殖无影响。结论葛根素对谷氨酸所致的大鼠原代神经细胞损伤具有保护作用,其保护作用可能与葛根素改善神经细胞膜脂流动性有关。     

头孢曲松神经保护作用及其与胶质细胞谷氨酸转运体1表达的关系

目的:研究头孢曲松对神经细胞的保护作用及其机制。方法:培养大鼠脑皮层神经细胞,加头孢曲松保护和谷氨酸损伤,光镜及四氮唑蓝(MTT)比色法观察细胞改变,逆转录-聚合酶链反应(RT- PCR)检测GLT1 mRNA、EAAC1 mRNA表达变化;激光共聚焦显微镜(LSCM)实时观测加或不加二氢红藻氨酸(DHK)后谷氨酸诱导钙超载时细胞内Ca~(2+)荧光强度变化。结果:头孢曲松组较对照组神经细胞损伤减轻,GLT1 mRNA表达增加(0.435±s 0.006,P<0.01);细胞内Ca~(2+)荧光强度在钙超载后上升缓慢但恢复迅速,加入DHK后,与对照组变化基本相同。结论:头孢曲松通过上调GLT1 mRNA表达,减轻谷氨酸细胞毒性和内钙超载,对神经细胞产生保护作用。     

The role of mitochondrial function in glutamate-dependent metabolism in neuronal cells

Glutamate is an important neurotransmitter in neurons and glial cells and it is one of the keys to the neuron-glial interaction in the brain. Glutamate transmission is strongly dependent on calcium homeostasis and on mitochondrial function. In the present work we presented several aspects related to the role of mitochondria in glutamate signaling and in brain diseases. We focused on glutamateinduced calcium signaling and its relation to the organelle dysfunction with cell death processes. In addition, we have discussed how alterations in this pathway may lead or aggravate a variety of neurodegenerative diseases. We compiled information on how mitochondria can influence cell fate during glutamate stimulation and calcium signaling. These organelles play a pivotal role in neuron and glial exchange, in synaptic plasticity and several pathological conditions related to Aging, Alzheimer's, Parkinson's and Huntington's diseases. We have also presented autophagy as a mechanism activated during mitochondrial dysfunction which may function as a protective mechanism during injury. Furthermore, some new perspectives and approaches to treat these neurodegenerative diseases are offered and evaluated.     

谷氨酸转运体靶点药物的抗脑缺血作用研究进展

兴奋性氨基酸毒性是脑缺血损伤的主要机制之一。缺血期间谷氨酸的大量累积会导致神经元细胞、星形胶质细胞等神经细胞发生兴奋性毒性损伤,因此对缺血期间谷氨酸水平的调控一直是脑缺血防治药物研究的重点。近年来研究表明,通过上调星形胶质细胞上谷氨酸转运体GLAST(EAAT1)和GLT-1(EAAT2)的表达或活性,增加缺血时谷氨酸的摄取,维持突触间隙内谷氨酸的正常浓度,从而降低兴奋性毒性,减轻缺血性脑损伤。一些化合物如β-内酰胺类抗生素、尿酸、甲状腺激素、雌激素、山楂酸等已在体内或体外实验中被证实对谷氨酸转运体的调节作用,对抗谷氨酸毒性,发挥神经保护作用。研究和开发以星形胶质细胞谷氨酸转运体为作用靶点的药物,为缺血性脑损伤的预防和治疗提供了一条新的途径。     

β—内啡肽增强谷氨酸神经毒性

探讨β－内啡肽对谷氨酸单钠诱导的神经毒性的影响。方法：形态学观察、神经元面积图象分析、线粒体膜蛋白结合钙和单细胞内游离钙浓度测定。结果：β－内啡肽在０．５－５．０ｍｇ·ｋｇ＾－１范围内以剂量依赖方式加剧谷氨酸单钠诱导的下丘脑弓状核神经元损伤，谷氨酸单钠诱导的线粒体膜蛋白结合钙增多可以被β－内啡肽２ｇ·Ｌ＾－１加强，谷氨到单钠诱导的单细胞内钙浓度升高也被β－内啡肽２ｇ·Ｌ＾－１从３２０±８４提高至５     

Effects of ticlopidine, a new platelet antiaggregating agent, and its analogues on mitochondrial metabolism. Oxidative phosphorylation, protein synthesis and DNA polymerase activity

The effects of ticlopidine and six of its analogues on mitochondrial functions were studied in isolated rat liver mitochondria. The influence of ticlopidine and each of the following analogues: PCR 5325, PCR 4099, PCR 3787, PCR 2362, PCR 4499 and PCR 0665 was evaluated by determining their interaction with three major mitochondrial activities. (A) Oxidative phosphorylation, measured by oxypolarography, was assayed in the presence of glutamate or succinate as source of energy, and both State 4 and State 3 were recorded. Ticlopidine, at 20 micrograms/ml, slightly increased glutamate State 4, whereas it was without effect on that of succinate. At higher concentration (40 micrograms/ml), ticlopidine caused 40-45% inhibition of State 4 with both substrates. All the other analogues tested at either 20 or 40 micrograms/ml were virtually without effect on the respiration. However, at 20 micrograms/ml, ticlopidine and some of its analogues inhibited mitochondrial State 3, while under similar conditions other analogues had little or no effect on this state. (B) Mitochondrial protein synthesis, measured by [14C]-L-leucine incorporation, was not affected significantly by any of these drugs. Whereas chloramphenicol at 10 micrograms/ml caused 80% inhibition, ticlopidine and its analogues in concentrations inhibitory to State 3 did not inhibit mitochondrial protein synthesis. (C) Mitochondrial DNA polymerase activity, determined by [3H] thymidine 5'-triphosphate incorporation, was not inhibited by these drugs. We conclude that, while ticlopidine and analogues have little or no effect on either mitochondrial protein synthesis or mitochondrial DNA polymerase activity, ticlopidine and some of its analogues are inhibitory of the energy conserving mechanism in mitochondria.     

Inhibition of mouse liver respiration by Chelidonium majus isoquinoline alkaloids

The alkaloids from Chelidonium majus L. which had a significant inhibitory effect in mitochondrial respiration were those which contain a positive charge due to a quaternary nitrogen atom, i.e., chelerythrine, sanguinarine, berberine and coptisine, both with malate+glutamate or with succinate as substrates. When malate+glutamate was used as substrate, chelerythrine and berberine, which contain methoxy groups, were particularly more active, since they had a strong effect even at low concentrations. In submitochondrial particles, berberine and coptisine had a marked inhibitory effect on NADH dehydrogenase activity but practically no effect on succinate dehydrogenase activity, whereas chelerythrine and sanguinarine inhibited more strongly succinate dehydrogenase than NADH dehydrogenase, which is in agreement with the results found for mitochondrial respiration. Protopine and allocryptopine, which did not inhibit mitochondrial respiration, strongly inhibited NADH dehydrogenase in submitochondrial particles, but had no effect on succinate dehydrogenase activity.     

Effect of glutamate receptor ligands on mitochondrial membrane potential in rat dissociated cerebellar cells

The effect of three different glutamate receptor ligands on mitochondrial membrane potential has been studied in rat pup dissociated cerebellar cells by measuring rhodamine 123 fluorescence. L-glutamate, NMDA (N-methyl-D-aspartate) and kainate (from 10^–8 to 10^–3 M) decreased in a concentration-dependent manner the mitochondrial membrane potential with EC₅₀ values of 6.7±1.7, 3.8±0.5, and 37.4±14 M, respectively.Dizocilpine ((+)MK 801) was able to inhibit the NMDA- and L-glutamate-induced decrease in rhodamine 123 fluorescence, while kainate-induced fluorescence-decreases were unaffected. However, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) totally prevented the effect of kainate on mitochondrial membrane potential, but failed to block the L-glutamate effect. It is concluded that, in our cell preparation, L-glutamate exerts its action mainly through NMDA-subtype receptors, and that Ca²⁺ and Na⁺ entry through ionotropic glutamate receptors could be responsible for an impairment of mitochondrial membrane potential.     

Effect of methylglyoxal bis(guanylhydrazone) on hepatic, heart and skeletal muscle mitochondrial carnitine palmitoyltransferase and beta-oxidation of fatty acids

Methylglyoxal bis(guanylhydrazone) (MGBG) is an antileukemic agent and a structural polyamine analogue which inhibits S-adenosyl methionine decarboxylase. However, MGBG also produces profound mitochondrial structural damage and inhibition of fatty acid oxidation. Carnitine palmitoyltransferase-A (CPT-A) is located on the outer surface of the inner mitochondrial membrane and is the putative rate-controlling enzyme for mitochondrial long-chain fatty acid oxidation. The present experiments were designed to determine if MGBG inhibits CPT-A. Liver, heart and skeletal muscle mitochondria were isolated from rats following 24 hr of starvation. Measuring the reaction in the direction of palmitoylcarnitine plus CoA formation from palmitoyl-CoA plus carnitine ("forward reaction"), MGBG was competitive with l-carnitine. The MGBG CPT-A Ki values were (mM): liver, 5.0 +/- 0.6 (N = 15); heart 3.2 +/- 1.2 (N = 3); and skeletal muscle, 2.8 +/- 1.0 (N = 3). Lysis of hepatic mitochondria with Triton X-100 yielded a Ki of 4.0 +/- 2.0, which was not significantly different from intact mitochondria or inverted vesicles (4.9 mM). Purified hepatic CPT had a Ki of 4.2 mM. MGBG did not inhibit purified CPT in the "reverse reaction" (palmitoyl-CoA plus carnitine formation from palmitoylcarnitine plus CoA). Spermine and spermidine, which are structurally similar to MGBG, did not inhibit either CPT activity or acid-soluble product formation from 1-[14C]palmitoyl-CoA. MGBG inhibited mitochondrial state 3 oxidation rates of palmitoyl-CoA and palmitoylcarnitine, as well as of glutamate. However, the fatty acid substrates were considerably more sensitive than glutamate to MGBG inhibition. MGBG also increased hepatic mitochondrial aggregation which was reversed by l-carnitine. Fluorescence polarization, using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe, indicated that MGBG increased membrane rigidity in a dose-dependent manner. This effect was not altered by l-carnitine. MGBG also inhibited purified pigeon breast carnitine acetyltransferase (CAT; Ki = 1.6 mM). While MGBG appeared to be competitive with l-carnitine for both CPT and CAT, MGBG also exhibits a number of effects which may be mediated through membrane interaction and which are not reversed by carnitine.     

Basal metallothionein-I/II protects against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures

N-Methyl-D-aspartate (NMDA) receptor overactivation-mediated oxidative stress has been proposed to contribute to brain injury. Metallothionein-I/II (MT-I/II), a member of cysteine-rich metalloproteins, has been found to express in the central nervous system primarily in cortical tissues and be upregulated following brain injury. To address the role of MT-I/II on NMDA-mediated oxidative injury, we established primary cortical neuron/astrocyte cultures from neonatal MT-I/II deficient (MT?/?) and wild type (MT+/+) mice to test whether basal MT-I/II protects cortical cultures against NMDA-mediated injury. We found that MT-I/II expression was increased by NMDA in MT+/+ cultures but was not detectable in MT?/? cultures. NMDA concentration-dependently induced oxidative injury in both MT+/+ and MT?/? cultures as evidenced by decrease of cell viability, increases of lipid peroxidation and DNA damage. However, these toxic effects were greater in MT?/? than MT+/+ cultures. NMDA significantly increased reactive oxygen species (ROS) generation and disrupted mitochondrial membrane potential in neurons in MT+/+ cultures, and these effects were exaggerated in MT?/? cultures. Our findings clearly show that basal MT-I/II provides protection against NMDA-mediated oxidative injury in cortical neuron/astrocyte cultures, and suggest that the protective effects are possibly associated with inhibition of ROS generation and preservation of mitochondrial membrane potential.     

四嗪二甲酰胺抑制肝癌细胞株HepG2增殖并诱导细胞凋亡的研究

目的观察四嗪二甲酰胺(ZGDHu-1)体外抑制肝癌细胞株HepG2增殖并诱导细胞凋亡作用。方法将不同浓度的ZGDHu-1与HepG2细胞在体外培养,用台盼蓝染色、MTT法、5′-溴-2′脱氧尿苷(B rdu)-ELISA法观察ZGDHu-1对HepG2细胞增殖的抑制作用;用细胞形态学、DNA凝胶电泳、DNA含量及细胞周期分析、Annexin-V/PI双标记、Ho-echst33258荧光染色和ELISA法测定DNA片段等技术检测细胞凋亡。结果ZGDHu-1能抑制HepG2细胞增殖和活力,呈现作用时间和剂量的量效关系。HepG2细胞与ZG-DHu-1作用后,大部分细胞阻滞于G2-M期;出现典型的细胞形态改变,DNA片断化,亚G1峰检出并增加,Annexin V+/PI-表达升高,细胞内DNA片段含量增加,Hoechst33258荧光染色后出现凋亡细胞的特征性改变等均证实ZGDHu-1能诱导HepG2细胞凋亡。结论ZGDHu-1能抑制HepG2细胞增殖,并可诱导其细胞凋亡。