丁基苯酞对帕金森病细胞模型保护作用的初步研究

目的探讨丁基苯酞(dl-3-n-Butylphthalide,NBP)对鱼藤酮诱导的帕金森病细胞模型的保护作用及其机制。方法分别使用终浓度为0.1、1、10、100μM NBP和溶剂二甲基亚砜(DMSO)预处理SH-SY5Y细胞24h后,加入终浓度为200nM的鱼藤酮处理24h建立多巴胺能细胞损伤模型,观察各组细胞形态,采用四甲基偶氮唑盐(MTT)比色法检测细胞活性,流式细胞术检测细胞凋亡率(Annexin V-FITC/PI)、线粒体膜电位(JC-1)、细胞内活性氧水平(DCFH-DA)。结果200nmol/L鱼藤酮处理SH-SY5Y细胞24h能够诱导细胞活性下降和细胞凋亡,NBP预处理后SH-SY5Y细胞存活率明显升高,细胞凋亡率降低,线粒体膜电位显著升高(P0.05),细胞内活性氧水平显著降低(P0.05),且随NBP浓度的增加对SH-SY5Y细胞的保护作用增强。结论NBP对鱼藤酮诱导的SH-SY5Y细胞损伤具有良好的保护作用,线粒体保护可能是其作用机制之一。     

蛋白酶体抑制剂对神经细胞的双重作用

目的探索蛋白酶体抑制剂在不同浓度时对多巴胺能神经元的作用及原因。方法用6-羟多巴(6-OHDA)50uM处理的人SH-SY5Y细胞作为细胞受损伤的模型,加用不同浓度的蛋白酶体抑制剂lactacystin,镜下细胞计数和SRB法测定细胞活力,平行对照组测定蛋白酶体活性。用选择性MEKl/2抑制剂PD98059验证蛋白酶体抑制剂是否通过MAPK途径起作用。结果蛋白酶体抑制剂lactacystin在0.1、0.25、0.5uM浓度时提高细胞存活率,在2、5uM时降低细胞存活率,相应的蛋白酶体活性分别是对照组的83.43%、73.84%、66.14%、24.11%、12.36%,加用PD98059后,0.25、0.5uMlactacystin的保护作用被阻断。结论蛋白酶体抑制剂在低浓度时对多巴胺能神经元有保护作用,高浓度时对多巴胺能神经元有毒性作用,这种不同作用的原因可能与蛋白酶体抑制的程度有关。蛋白酶体抑制剂的保护作用可能通过MAPK途径起作用。     

过量表达野生型和突变型早老素1基因对SH-SY5Y细胞的影响

目的观察转染人野生型和突变型早老素1(Presenilin1,PS1)-EGPF后对SH-SY5Y细胞的影响。方法采用定点突变技术构建含突变PS1基因的质粒及与绿色荧光蛋白共表达载体,转染至SY5Y细胞中,筛选出稳定表达的细胞克隆并鉴定;观察转染后各组细胞之间形态的区别,MTT法测定细胞活力并进行比较。结果稳定表达野生型和突变型PS1的细胞模型构建成功,转染pcDNA3.1/PS1突变质粒的细胞活力明显降低。结论此细胞模型的建立为下一步研究突变型PS1在AD发病机制中的作用奠定了基础。     

通心络对体外培养SH-SY5Y细胞Aβ25-35毒性损伤的保护作用

目的 探讨通心络对体外培养SH-SY5Y细胞Aβ25-35损伤的保护作用。方法采用细胞培养法，以神经母细胞瘤SH～SY5Y细胞系为材料制备Aβ25-35损伤的离体细胞损伤模型。采用细胞形态学观察细胞形态及以MTT法测定细胞存活率。结果 不同浓度通心络作用不同时间其MTT代谢率均高于损伤对照组（P〈0．01），高、中浓度通心络治疗组均高于低浓度治疗组（P〈0．05），而高、中浓度治疗组间差异无统计学意义；相同浓度通心络作用不同时间其MTT代谢率相比较，36、24h组优于12h组（P〈0．05），而24h与36h组间比较差异无统计学意义。结论 通心络可促进神经细胞生存，具有神经细胞保护作用。     

Age-dependent effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP): Correlation with monoamine oxidase-B

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration on whole brain dopamine content and monoamine oxidase subtype B (MAO-B) activity were assessed in Swiss-Webster male mice. Groups of mice at six different ages (1,2,4,8,16, and 52 weeks) were treated with either MPTP(12.5 mg/kg) every 2 hours for four subcutaneous injections or with the saline vehicle in the same injection volume. A third group of subjects was untreated and sacrificed at the same ages. The animals were sacrificed 1 week following treatment. The brains were removed and divided into right and left halves. The right brain halves from all subjects of a given age were assayed for MAO-B activity. The left brain halves were assayed for dopamine content using high performance liquid chromatography. The results revealed a significant age-dependent increase in both dopamine content and MAO-B activity in the untreated controls. It was also found that the toxic effect of MPTP as measured by whole brain dopamine depletion was increased at each successive age tested. There was a significant correlation (r = + 0.96) between the baseline levels of MAO-B activity and the degree of lesion induced by MPTP treatment. These results indicate that the increased sensitivity to MPTP reported in aged animals may, in part, be attributable to the increase in MAO-B activity in older animals.     

3-硝基丙酸预处理对多巴胺能神经元保护作用的研究

目的:研究3-硝基丙酸(3-NPA)预处理对MPP+(1-甲基4苯基-吡啶离子)损害的多巴胺能神经元是否具有保护作用。方法:将MPP+加入到培养的多巴胺能神经元SH-SY5Y细胞中制作帕金森病的细胞模型,在加入MPP+(0.25mmol/L)前,分别单次或多次加入3-NPA(0.2mmol/L)形成预处理,应用四氮唑盐(MTT)检测细胞生存率,[3H]DA摄取率测定多巴胺能细胞突触前功能,观察3-NPA预处理对它们的影响。结果:3-NPA预处理后,预处理组细胞生存率分别为71.8％(单次),85.2％(多次),较MPP+组(54.3％)明显提高;[3H]DA摄取率分别为65.8％(单次)80.3％(多次),较MPP+组(50.1％)明显提高,且多次预处理较单次预处理效果更好。单加3-NPA对细胞无影响。结论:3-NPA预处理对多巴胺能神经元有明显的保护作用,多次预处理的保护作用更加显著。     

SH-SY5Y细胞α7神经型尼古丁受体基因沉默对突触相关蛋白的影响

目的研究神经母细胞瘤细胞(SH-SY5Y)α7神经型尼古丁受体基因(α7 nAChR)表达沉默后对细胞突触相关蛋白的影响,探讨α7 nAChR神经保护作用机制及在阿尔茨海默病(Alzheimer disease,AD)的发病机制中的作用。方法用Real-time PCR法和蛋白免疫印迹(Western blot)法分别测定细胞中囊泡相关蛋白(synaptophysin)和突触后膜蛋白(PSD-95)mRNA蛋白表达水平的变化。结果α7 nAChR沉默组的突触相关蛋白PSD-95、SYPmRNA及蛋白表达都有明显的减少。结论沉默SH-SY5Y细胞α7 nAChR水平能够使细胞突触相关蛋白水平减少。这可能提示了α7 nAChR与细胞突触密切相关,并且α7 nAChR对突触有一定的保护作用,进一步说明α7nAChR在阿尔茨海默病的发病中起着重要作用。     

Attenuation of MPTP-induced dopaminergic neurotoxicity by a serotonin uptake blocker

Summary Monoamine oxidase-B (MAO-B) has been determined to be the enzyme responsible for the conversion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into its toxic metabolite 1-methyl-4-phenylpyridine ion (MPP+). Since this enzyme has been localized primarily in astrocytes and serotonergic neurons, it would appear that MPP+ is being produced outside the dopaminergic neurons. To investigate this possibility, the administration of MPTP was preceded by systemically administered fluoxetine. In keeping with its demonstrated ability to inhibit uptake into serotonergic neurons and serotonin uptake into astrocytes, fluoxetine pretreatment resulted in a significant attenuation of MPTP-induced depletions of striatal dopamine and serotonin concentration. These results support the extra-dopaminergic production of MPP+.     

Rifampicin attenuates the MPTP-induced neurotoxicity in mouse brain

Rifampicin, an antibacterial drug, is highly effective in the treatment of tuberculosis and leprosy. Recently, it has been reported to have neuroprotective effects in in vitro and in vivo models. This study was designed to elucidate its neuroprotective effects against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity (known as an in vivo mouse model of Parkinson's disease). Mice were injected intraperitoneally (i.p.) with MPTP (10 mg/kg) four times at 1-h intervals, and brains were analyzed 3 or 7 days later. Rifampicin at 20 mg/kg (i.p., twice) had protective effects against MPTP-induced neuronal damage (immunohistochemical changes in tyrosine hydroxylase) in both the substantia nigra and striatum. Rifampicin also protected against the MPTP-induced depletions of dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) in the striatum. The maximal concentrations of rifampicin between 30 and 240 min after a single rifampicin injection (20 mg/kg, i.p.) were 2.6 microM (at 30 min) in plasma and 0.77 microM (at 60 min) in striatum. Next, the effects of rifampicin on oxidative stress [lipid peroxidation in mouse brain homogenates and free radical-scavenging activity against diphenyl-p-picrylhydrazyl (DPPH)] were evaluated to clarify the underlying mechanism. At 1 microM or more, rifampicin significantly inhibited both lipid peroxidation in the striatum and free radical production. These findings suggest that in mice, rifampicin can reach brain tissues at concentrations sufficient to attenuate MPTP-induced neurodegeneration in the nigrostriatal dopaminergic neuronal pathway, and that an inhibitory effect against oxidative stress may be partly responsible for its observed neuroprotective effects.     

The investigation of protective effects of glucagon-like peptide-1 (GLP-1) analogue exenatide against glucose and fructose-induced neurotoxicity

Diabetes mellitus (DM) is one of the most common metabolic disorders characterized by hyperglycemia due to insufficiency of insulin and/or insulin resistance. Clinical studies have revealed a higher risk of neurodegenerative disorders such as Alzheimer’s disease or Parkinson’s disease in diabetic patients. Recently, glucagon-like peptide-1 (GLP-1) is an attractive potential treatment modality for various neurodegenerative diseases. In our study, we aimed to investigate whether exenatide, a GLP-1 analogue, has neuroprotective effects against glucose and fructose-induced toxicity in human SH-SY5Y neuroblastoma cell line. Neurotoxicity was induced by incubating SH-SY5Y cells with different doses (25–100?mM) of glucose and fructose for 24, 48 and 72?hours. Following determination of the significant toxic doses of glucose and fructose, the cells were treated with various doses of exenatide (10–250?nM) in the presence or absence of glucose and fructose. Neurotoxicity was evaluated by MTT assay and Hoechst 33258 staining. Caspase-3 activity and the levels of advanced glycation end products (AGEs) were determined in the cytosolic fractions of treated cells. Our results demonstrated that both glucose and fructose treatments decreased cell viability in neuronal cells dose and time-dependently. Glucose and fructose-treated groups showed increased numbers of apoptotic cells, caspase-3 activity and AGEs levels. Treatment of the cells with exenatide significantly prevented cell death. The most prominent effect was observed at 100?nM exenatide-treated cultures. Our results suggest that high doses of glucose and fructose may lead to neurotoxicity, and exenatide may have protective effects against neuronal damage through its anti-apoptotic feature.     

预热激对6-OHDA诱导SH-SY5Y细胞神经毒性损伤的保护作用

目的探讨预热激对6-羟多巴胺(6-OHDA)诱导人神经母细胞瘤细胞(SH-SY5Y)神经毒性损伤的保护作用及其机制。方法对实验组SH-SY5Y细胞进行预热激(42±0.5)℃处理,再通过6-OHDA对其进行神经毒性诱导,未行预热激处理细胞作为对照组。逆转录酶-聚合酶链式反应(RT-PCR)检测不同热激时间(15 min、30 min、60 min)SH-SY5Y细胞内葡萄糖调节蛋白(GRP78、GRP94)的表达水平;显微镜下观察细胞形态学变化,并用四甲基偶氮唑蓝(MTT)比色法检测SH-SY5Y细胞活性情况。结果 6-OHDA对SH-SY5Y细胞的神经毒性损伤呈剂量依赖性及时间依赖性关系,实验组SH-SY5Y细胞较对照组所受的神经毒性损伤轻(P<0.05),且实验组间差异也具有统计学意义(P<0.05);SH-SY5Y细胞中GRP78和GRP94 mRNA表达水平与热应激时间正相关(P<0.05)。结论预热激可降低6-OHDA诱导SH-SY5Y细胞的神经毒性损伤作用,且热激过程中SH-SY5Y细胞中GRP78和GRP94表达上调。     

MPP+ induced apoptotic cell death in SH-SY5Y neuroblastoma cells: An electron microscope study

PD is a common, late-onset neurodegenerative disorder that results in part from the gradual loss of dopaminergic neurons in the substantia nigra pars compacta. The neurotoxin MPTP can induce PD-like clinical symptomatology and neuropathological destruction and, thus, has been used as a PD model. The human neuroblastoma cell line SH-SY5Y possesses many of the qualities of human neurons and, as such, has served as a model for them. Apoptosis is the mode of cell death induced in SH-SY5Y cells by MPTP, and this was confirmed with nick end labeling and bisbenzimide staining. Transmission electron microscopic analysis of the ultrastructural changes occurring in neurotoxin exposed SH-SY5Ys revealed many morphological characteristics consistent with apoptosis. These changes included plasmalemmal blebbing, altered cytosolic density, nuclear condensation and fragmentation, pronounced vacuole formation, ribosomal dispersion, and the disappearance of the golgi complex, microtubules, and smooth endoplasmic reticulum. Limited amounts of rough endoplasmic reticulum and mitochondria exhibited normal morphology throughout the apoptotic changes but then were disrupted during secondary necrotic changes. The in vitro induction of apoptosis by a parkinsonism neurotoxin might be reflective of the mechanisms of in vivo nigral degeneration occurring during PD. J. Neurosci. Res. 48:226–237, 1997. © 1997 Wiley-Liss, Inc.     

Endogenous urinary monoamine oxidase inhibitor excretion in Parkinson's disease and other neurological disorders

Summary In this study, urinary output of both neutral (tribulin) and basic monoamine oxidase inhibitory activity was measured in parkinsonian patients, other neurological patients and controls. No significant differences in output were found between these different groups. In general, tribulin output rose with age, in parallel with known changes in monoamine oxidase B activity.     

Neuroprotective effect of thalidomide on MPTP-induced toxicity

Thalidomide is a sedative with unique pharmacological properties; studies on epilepsy and brain ischemia have shown intense neuroprotective effects. We analyzed the effect of thalidomide treatment on the neurotoxicity caused by the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahidropyridine (MPTP) in mice. Thalidomide was administered at two times; before and after the exposure to MPTP. In both circumstances thalidomide improved the neurotoxicity induced by MPTP as seen by a significant raise of the striatal contents of dopamine and simultaneous decrease of monoamine-oxidase-B (MAO-B). These results indicate that in the experimental model of Parkinson's disease the administration of thalidomide improves the functional damage on the nigrostriatal cell substratum as seen by the production of dopamine. This neuroprotective effect seems to be mediated by inhibition of excitotoxicity. Our results suggest that thalidomide could be investigated as potential adjuvant therapy for Parkinson's disease.     

Ebselen (DR3305) ameliorates delayed cerebral vasospasm in a canine two-hemorrhage model

Abstract

Ebselen, a seleno-organic compound which inhibits arachidonic acid lipoxygenase activity and exerts glutathione peroxidase-like activity, ameliorated delayed cerebral vasospasm in a canine two-hemorrhage model Twenty-five dogs were exposed to subarachnoid h~morrhage and divided into two groups. In the Ebselen-treated group (6 dogs), 50 mg kg- ¹ of Ebselen was administered twice a days for 7 days. The other 19 dogs without administration of Ebselen were used as a control group. In the Ebselen-treated group, the basilar artery on Day 7 after subarachnoid hemorrhage was constricted to 68.1 ± 6.4% (mean ± 50, n = 6) of the angiographic diameter on Day 0, before subarachnoid hemorrhage. This percentage was significantly larger than the 41.3 ± 4.6% (n = 19) in the control group. The basilar artery segment obtained on Day 7 in the Ebselen-treated group produced 3.32 ± 1.82 nmol of 5-hydroxyeico~atetraenoicacid/mg protein/ 5 minutes (n = 5), significantly less than the 6.73 ± 0.95 (n = 3) produced by the artery in the control group. Thus, administration of Ebselen suppressed arachidonate 5-lipoxygenase activation and had a beneficial effect on angiographically detected delayed vasospasm. [Neural Res 1997; 19: 563-565]     

Method for measuring neurotoxicity of aggregating polypeptides with the MTT assay on differentiated neuroblastoma cells

Reliable in vitro assays are essential for study of the effects of neurotoxic compounds such as beta-amyloid peptides (Abeta). The MTT assay has been used in cultures of different cells, e.g. SH-SY5Y neuroblastoma cells, for the quantitative measurement of Abeta toxicity. In our laboratory differentiated SH-SY5Y cells were used in the MTT assay. Cell differentiation with 10 microM all-trans-retinoic acid resulted in a constant cell number. The cells possess highly developed neurites and exhibit high sensitivity against Abeta. Owing to the constant cell number in differentiated SH-SY5Y cultures the decrease of the redox activity is directly proportional to the neurotoxicity of the substances, no correction is needed. The results of the MTT assay of Abeta peptides on differentiated SH-SY5Y cells displayed a good correlation also with the in vivo results. The present experiments reveal an effective assay for the study of potentially neurotoxic compounds.     

Superoxide dismutase 1 (SOD1) and cadmium: A three models approach to the comprehension of its neurotoxic effects

Cadmium (Cd) is a widespread toxic environmental contaminant, released by anthropogenic activities. It interferes with essential metal ions homeostasis and affects protein structures and functions by substituting zinc, copper and iron. In this study, the effect of cadmium on SOD1, a CuZn metalloenzyme catalyzing superoxide conversion into hydrogen peroxide, has been investigated in three different biological models. We first evaluated the effects of cadmium combined with copper and/or zinc on the recombinant GST-SOD1, expressed in E. coli BL21. The enzyme activity and expression were investigated in the presence of fixed copper and/or zinc doses with different cadmium concentrations, in the cellular medium. Cadmium caused a dose-dependent reduction in SOD1 activity, while the expression remains constant. Similar results were obtained in the cellular model represented by the human SH-SY5Y neuronal cell line. After cadmium treatment for 24 and 48 h, SOD1 enzymatic activity decreased in a dose- and time-dependent way, while the protein expression remained constant. Finally, a 16 h cadmium treatment caused a 25 % reduction of CuZn-SOD activity without affecting the protein expression in the Caenorhabditis elegans model. Taken together our results show an inhibitory effect of cadmium on SOD1 enzymatic activity, without affecting the protein expression, in all the biological models used, suggesting that cadmium can displace zinc from the enzyme catalytic site.     

川芎嗪对MPTP所致小鼠多巴胺能神经元损伤的保护作用及机制

目的 探讨川芎嗪对MPTP所致小鼠多巴胺能神经元损伤的保护作用及可能机制.方法 C57BL/6J雄性小鼠32只,随机分为4组:生理盐水对照组(NS+NS)、生理盐水组(NS+MPTP)、川芎嗪高剂量组(LT50+MPTP)、川芎嗪低剂量组(LT20+MPTP),每组8只动物.分别采用HPLC法检测纹状体中DA的含量,免疫组化检测黑质中TH阳性细胞数,荧光显色法检测黑质SOD活力、GSH含量.结果 LT50+MPTP组纹状体DA含量、黑质DA神经元数量、黑质SOD活力、黑质GSH含量较NS+MPTP组显著增高(P<0.01).结论 川芎嗪对对MPTP所致的小鼠多巴胺能神经元损伤具有保护作用,其保护机制可能与其调节调节小鼠黑质中的SOD、GSH含量有关.

Abstract：

Objective To investigate the neuroprotective effects and mechanisms of ligustrazine on the MPTP-in-duced dopaminergic neurodegeneration in a mouse model of PD. Methods Male C57BL/6J mice were randomly divided into following four treatment groups ( n = 8/group) : ( 1 ) the saline control group ( NS + NS), mice pretreated with saline followed by saline treatment;(2) the mice pre-treated with saline followed by saline treatment (NS + MPTP) ; (3) the mice pretreated with 50mg/kg of ligustrazine followed by MPTP treatment (LT50 + MPTP) ;(4) the mice pretreated with 20mg/kg of ligustrazine followed by MPTP ( LT20 + MPTP). HPLC,immunohistochemistry and fluorimetry were used. Results The residual DA contents, TH-IR positive cells, SOD activity and GSH content in the mice pretreated with 50mg/kg ligustrazine ( LT50 + MPTP) were significantly higher than those of the saline-pretreatd mice ( NS + MPTP) ( P < 0.01 ).Conclusions Ligustrazine ameliorated MPTP-induced dopaminergic neurodegeneration in mice. The neuroprotective effect of ligustrazine may be associated with their strong antioxidant capacity in vivo.     

C-terminal mechano growth factor protects dopamine neurons: A novel peptide that induces heme oxygenase-1

To assess potential efficacy of mechano growth factor (MGF) for chronic neurodegenerative disorders, we studied whether MGF protects dopamine (DA) neurons subjected to neurotoxic stress. We show that a short 24-amino acid C-terminal peptide of MGF (MGF24) upregulates heme oxygenase-1 (HO-1) expression and protects SH-SY5Y cells against apoptosis and cell loss induced by three DA cell-specific neurotoxins: 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenylpyridinium (MPP⁺), and rotenone. MGF24 maintains the mitochondrial membrane potential and blocks the release of mitochondrial apoptotic-inducing factor into the cytoplasm induced by 6-OHDA, MPP⁺, and rotenone. Chemical inhibition of HO-1 with zinc protoporphyrin-IX prevents neuroprotection by MGF24 against the three neurotoxins. MGF24 does not activate Akt signaling nor does Akt inhibition block MGF24 protection of SH-SY5Y cells. In 6-OHDA-lesioned rats, central or peripheral MGF24 administration protects against the development of contralateral forelimb under-utilization, reduces ipsilateral nigral DA cell body loss, and attenuates tyrosine hydroxylase fiber loss in the ipsilateral striatum, independent of IGF-1 receptor activation. Peripheral MGF24 administration upregulates HO-1 expression in striatal and midbrain tissue. This report is the first to demonstrate that a small peptide, MGF24, upregulates HO-1, an important cell defense mediator, and protects DA cells, suggesting new strategies for neuroprotection in Parkinson's disease.     

A link between monoamine oxidase-A and apoptosis in serum deprived human SH-SY5Y neuroblastoma cells

Summary Increased monoamine oxidase (MAO) activity was recently shown to accompany apoptotic cell death of various neuronal cells following growth factor deprivation. Here we show that in serum deprived SH-SY5Y cells, MAO-A mRNA levels and catalytic activities are increased, linked with activation of the apoptotic executioner caspase-3. Importantly, specific inhibition of MAO-A activity resulted in loss of apoptotic cell morphology. Our study indicates that MAO catalytic activity is involved in apoptotic signalling in response to serum withdrawal in neuronal cells.