6-OHDA诱导PC12细胞凋亡及作用机制

目的 探讨6-羟基多巴胺(6-OHDA)诱导PC12细胞损伤的可能作用机制.方法 不同剂量6-OHDA加入培养的大鼠肾上腺嗜铬细胞瘤细胞(pheochromocytoma cell,PC12)24h后,用四甲基偶氮唑盐法(MTT法)检测细胞的活力,流式细胞仪检测细胞凋亡率以及Bax、Bcl-2的蛋白表达.结果 在加入不同浓度的6-OHDA时PC12细胞活力显著下降,细胞凋亡百分率对照组为8.73±1.09,不同浓度的6-OHDA处理组显著上升,分别为10.97±1.52、25.77±0.95、57.94±1.23,较对照组有显著性差异(P<0.01),并且Bcl-2蛋白表达下降,Bax蛋白表达上升,Bcl-2/Bax比值和正常组比较有显著性差异(P<0.01).结论 6-OHDA能显著诱导PC12细胞损伤,并呈剂量依赖性,其作用机制涉及到促进细胞内bax,以及抑制Bcl-2的表达.     

黄芪甲苷对PC12细胞氧化应激损伤的保护作用

目的 探讨黄芪甲苷对PC12细胞氧化应激损伤的作用。方法 体外培养PC12细胞，6-羟基多巴胺（6-OHDA）作用PC12细胞导致氧化应激损伤。根据细胞作用方法随机分为4组:①对照组，采用完全培养基正常培养；②6-OHDA组，给予终浓度为100 μmol/L的6-OHDA处理24 h；③低、中、高剂量黄芪甲苷组，分别给予终浓度为25、50、100 μmol/L的黄芪甲苷预处理24 h，然后给予终浓度为100 μmol/L 6-OHDA处理24 h；④AG490组，以20 μmol/L AG490（JAK2/STAT3信号通路抑制剂）预处理16 h，加入终浓度为100 μmol/L黄芪甲苷处理24 h，然后再给予终浓度为100 μmol/L 6-OHDA处理24 h。CCK8法检测细胞生存率，流式细胞仪检测细胞凋亡率，酶联免疫吸附实验法检测细胞上清液超氧化物歧化酶（SOD）和丙二醛（MDA）水平，免疫印迹法检测细胞p-JAK2和p-STAT3蛋白表达。结果 6-OHDA作用后，PC12细胞存活率明显降低，细胞凋亡率明显升高，细胞培养液SOD水平明显降低、MDA水平明显升高，细胞p-JAK2和p-STAT3蛋白表达水平明显降低；黄芪甲苷预处理明显逆转6-OHDA的作用，而且呈剂量依赖性；AG490预处理明显逆转黄芪甲苷的作用。结论 6-OHDA作用PC12细胞，可导致氧化应激损伤促使细胞凋亡；黄芪甲苷预处理能激活JAK2/STAT3信号通路，抑制6-OHDA对PC12细胞的损伤，对PC12细胞起保护作用。     

半夏总生物碱对6-OHDA诱导PC12细胞损伤的保护作用及机制

目的探讨半夏总生物碱(TAPT)对6羟基多巴胺(6-OHDA)诱导的大鼠肾上腺嗜铬细胞瘤细胞PC12细胞株损伤的保护作用及其机制。方法通过不同剂量TAPT预处理PC12细胞后,加入6-OHDA诱导氧化应激损伤。采用甲基噻唑基四唑比色法(MTT法)检测PC12细胞活力、天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)活性检测试剂盒测定caspase-3活性、黄嘌呤氧化酶法检测细胞总超氧化物歧化酶(T-SOD)活力,硫代巴比妥法检测丙二醛(MDA)的含量、按Fenton反应原理检测抑制羟自由基(·OH)的能力。结果浓度为100μmol/L的6-OHDA作用PC12细胞24h后,与正常对照组相比,细胞活力明显降低(P<0.05),T-SOD活性和抑制·OH的能力明显下降(P<0.05),而MDA含量和caspase-3活性明显升高(P<0.05);与6-OHDA组相比,TAPT处理组的PC12细胞活力逐渐升高(P<0.05),T-SOD活性和抑制·OH的能力明显上升(P<0.05),而MDA含量和caspase-3活性明显降低(P<0.05)。结论 TAPT对6-OHDA诱导的PC12细胞损伤具有一定的保护作用;提高T-SOD活性和抑制·OH的能力及降低MDA含量和caspase-3活性可能是其作用机制之一。     

褐藻多糖硫酸酯对6-羟基多巴胺所致MN9D细胞损伤的保护作用及其机制

目的 以6-羟基多巴胺(6-OHDA)为工具药,建立帕金森病(PD)的细胞模型,观察褐藻多糖硫酸酯(Fuc)对细胞模型的保护作用并初步探讨其作用机制.方法 用不同浓度(12.5、25、50、100、200 μmol/L)的6-0HDA处理MN9D细胞24 h,挑选出合适浓度50 μmol/L.再以50μmol/L 6-OHDA处理MN9D细胞不同时间(6、12、24及48 h),建立细胞损伤模型,挑选出合适时间24h.用0.01、0.1、1.0mg/mL Fuc预孵育MN9D细胞1 h后加入50μmol/L 6-OHDA共同作用24h,以探讨Fuc的保护作用.MTT法检测细胞存活率,生化法测定乳酸脱氢酶(LDH)释放量,二氯荧光索乙二酯(DCF-DA)染色法检测细胞内的氧化应激水平.结果 随着6-OHDA浓度增加或作用时间延长,MN9D细胞MTT值逐渐降低.50 μmol/L6-OHDA处理细胞24 h,MTT值明显下降,LDH释放量增加.而0.1、1.0mg/mL的Fuc预孵育1 h可明显减轻MN9D细胞的损伤,提高MTT值并降低LDH释放量,细胞形态学改变与生化实验结果一致.50 μmol/L6-OHDA作用8h可明显升高MN9D细胞内的氧化应激水平,而1.0mg/mL的Fuc预处理1 h可以拮抗6-OHDA引起的细胞内氧化应激水平增高.结论 Fuc可以有效的拮抗6-OHDA对MN9D细胞的损伤作用,其作用机制可能与抗氧化活性有关.     

17β-雌二醇对6-OH多巴胺损伤的多巴胺能神经细胞的影响

目的 研究17β-雌二醇(17β-E2)对6-羟基多巴胺(6-OHDA)所致的黑质多巴胺(DA)能神经细胞损伤的影响,探讨17β-E2是否具有神经保护作用.方法 新生2～3 d的雄性SD大鼠行常规中脑脑片培养,6-OHDA(200 μmol/L)诱导损伤后分为实验对照组和17β-E2组,实验对照组继续常规培养,17β-E2组又分为1.0× 10-8 mol/L、1.0×10-9 mol/L和1.0×1 0-10 mol/L 3个亚组,分别按上述浓度添加17β-E2进行干预.Western blotting方法 检测每组细胞干预15min、30 min、60 min后酪氨酸羟化酶(TH)的表达.结果 1.0×10-9 mol/L 173-E2组TH的表达最高,与实验对照组比较差异有统计学意义(P＜0.05);增加或减少17β-E2的浓度均不能上调TH的表达;3个不同时间点中,17β-E2作用15min时,TH的表达最高,延长作用时间并不能增加TH的表达量.结论 适当浓度(1.0× 10-9 mol/L)17β-E2作用一定时间(15 min)可以对受损的DA能神经细胞起到保护作用.     

多巴胺对鱼藤酮细胞毒性介导作用研究

目的探讨多巴胺在鱼藤酮细胞毒性损伤中的作用。方法采用鱼藤酮(1μmol/L)处理PC12细胞,噻唑蓝比色法检测细胞活性,流式细胞术检测DHR123荧光强度;并采用多巴胺耗竭剂-利血平预处理3小时,观察上述指标。结果 1μmol/L鱼藤酮处理PC12细胞24小时,导致细胞活力较正常组显著下降(P<0.05),吸光度为0.730±0.01(正常组为1.112±0.025);利血平(1μmol/L和5μmol/L)预处理后再给予鱼藤酮,孵育24小时后,吸光度分别为0.945±0.02和1.06±0.03,细胞活力较鱼藤酮组显著升高(P<0.05)。鱼藤酮处理24小时,过氧化物水平升高至正常组的281%,而利血平(1μmol/L和5μmol/L)预处理后,分别降至正常组的248%和232%,与鱼藤酮组比较有显著差异(P<0.05)。结论多巴胺介导了鱼藤酮对多巴胺能细胞的毒性作用。     

尿酸减轻6-羟基多巴胺对PC12细胞的毒性作用

目的：评价尿酸减轻6．羟基多巴胺（6-OHDA）对PC12细胞的毒性作用。方法：应用PCI2细胞制作帕金森细胞模型,分为对照组、尿酸组、6-OHDA组、尿酸＋6-OHDA组。采用MTT测定各组PC12细胞活性,免疫荧光法观察各组PCI2细胞caspase-3激活情况,流式细胞术检测各组PC12细胞凋亡率。尿酸100～400μmol·L^-1不影响PCI2细胞生存率,尿酸100～400μmol·L-1可显著提高6-OHDA50gmol-L。作用6、12和24h造成的PCI2细胞生存率的下降（P〈0．01）;尿酸能减少6-OHDA导致的PCI2细胞caspase-3激活,降低6-OHDA导致的凋亡率（P〈0．05）。结论：尿酸具有减轻6-OHDA对PC12细胞的毒性作用。     

6-羟基多巴胺诱导的PC12细胞的凋亡和谷氨酸的释放不依赖蛋白激酶C

目的:了解6-羟基多巴胺(6-OHDA)诱导的PC12细胞凋亡及谷氨酸(Glu)的释放与蛋白激酶C(PKC)的关系,为探讨6-OHDA的毒性作用机制及诱导Glu释放的信号传递途径提供实验依据。方法:培养PC12细胞,以特定浓度6-OHDA刺激24h,用缺口末端标记法(TUNEL)测定细胞凋亡,用噻唑蓝(MTT)法检测细胞存活率,用高效液相色谱法(HPLC)检测Glu的释放量。提前1h加入蛋白激酶C抑制剂HA-100,观察细胞活力和Glu水平的变化。结果:HA-100对6-OHDA诱导的PC12细胞外Glu水平无明显影响,且不能减少6-OHDA诱导的细胞凋亡。结论:6-羟基多巴胺诱导的PC12细胞凋亡和谷氨酸的释放是PKC非依耐性的。     

雌二醇对6-羟基多巴胺引起的大鼠嗜铬细胞瘤细胞损伤的影响

目的:研究雌激素对6-羟基多巴胺(6-OHDA)引起的大鼠嗜铬细胞瘤细胞(PC12)损伤的影响。方法:采用台盼蓝染料排除法和检测细胞乳酸脱氢酶(LDH)漏出量水平,估测PC12细胞的损伤程度。结果:同型异构体17β雌二醇和17α雌二醇均可明显减轻6-OHDA引起的PC12细胞存活率的降低,雌激素受体拮抗剂他莫昔芬对17β雌二醇和17α雌二醇的细胞保护作用无明显影响。结论:雌激素可能是通过非受体依赖的抗氧化机制对6-OHDA引起的PC12细胞损伤起保护作用。     

Rottlerin通过抑制PKCδ磷酸化减轻6羟基多巴胺对多巴胺能细胞的毒性作用

目的观察蛋白激酶C6亚型（PKCδ）磷酸化在6羟基多巴胺（6-OHDA）引起的多巴胺能神经细胞死亡过程中的作用,探讨帕金森病中神经元缺失的分子机制。方法体外培养大鼠嗜铬细胞瘤细胞系PC12细胞,观察预先加入的PKC抑制剂（bisindolylmaleimide I,Go6976及Rottlerin）和激动剂佛波酯对6-OHDA毒性作用的影响,噻唑蓝比色法检测细胞存活率,免疫印迹法观察磷酸化PKC8的表达。结果PKC8抑制剂Rottlerin（2μmol／L）可抑制PKC8的磷酸化,减轻6-OHDA引起的细胞死亡,细胞存活率上升至69．6±2．63％（P〈0．05）。PKCα抑制剂bisindolylmaleimide I和钙依赖性PKC抑制剂Go6976对6-OHDA的毒性作用及PKCδ磷酸化均无显著影响,而PKC8激活剂佛波酯（100nmol／L）能提高PKC8磷酸化水平,加重6-OHDA的损害作用,使细胞存活率下降至单用6-OHDA组水平的49．8±5．06％（P〈0．001）。结论Rottlerin能抑制PKCδ的磷酸化,进而减轻6-OHDA对多巴胺能神经细胞死亡的诱导作用,说明PKCδ505位点丝氨酸的磷酸化是6-OHDA发挥毒性作用的关键,提示PKCδ在帕金森病病人神经元缺失中起重要作用。     

Cerebrospinal fluid monoamine and monoamine metabolite concentrations in melancholia

Cerebrospinal fluid levels of norepinephrine and six monoamine metabolites were measured in 28 medication-free depressed patients. Patients with a major depressive episode with melancholia (n = 15) had significantly lower levels of the three dopamine metabolites: homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and conjugated dihydroxyphenylacetic (CONJDOPAC), when compared with a combined group of patients with a major depressive episode or dysthymic disorder (n = 13). In patients with major depressive episode with melancholia, levels of HVA and of the serotonin metabolite 5-hydroxyindoleacetic acid significantly correlated with the severity of depression. In the total group of 28 depressed patients, cerebrospinal fluid (CSF) levels of norepinephrine significantly correlated with symptoms of anxiety. In both patients with major depressive episode and major depressive episode with melancholia, those who were non-suppressors on the dexamethasone suppression test had significantly higher CSF levels of the norepinephrine metabolite 3-methoxy-4-hydroxyphenylglycol compared to those who were suppressors.     

Cannabinoids and monoamine neurotransmission with focus on monoamine oxidase

Progress in understanding the mechanisms of action of cannabinoids was made after discovery of cannabinoid receptors and finding their endogenous ligands. New findings are obtained using both endogenous cannabinoids and plant or synthetic cannabinoids. Activation of cannabinoid receptors on synaptic terminals results in regulation of ion channels, neurotransmitter release and synaptic plasticity. Neuromodulation of synapses by cannabinoids is proving to have a wide range of functional effects, making them potential targets as medical preparations in a variety of illnesses, including some neurodegenerative and mental disorders. Brain monoamines are involved in many of the same processes affected by neuropsychiatric disorders and by different psychotropic drugs, including cannabinoids. Basic information is summarized in the paper about mechanisms of action of cannabinoids on monoaminergic systems, with a view to inhibition of monoamine oxidase.     

Effect of chronic nicotine administration on monoamine and monoamine metabolite concentrations in rat brain

The effects on rat brain tissue monoamine and monoamine metabolite concentrations of chronic nicotine administration at two doses (3 and 12 mg/kg/day) using constant infusion were studied. After 21 days of treatment, tissue concentrations of dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT), and several metabolites in striatum, hypothalamus, and frontal cortex were determined by high performance liquid chromatography with electrochemical detection. Compared with a control group, nicotine treatment significantly decreased NE in frontal cortex but not in other regions. The concentration of 5HT also was decreased in frontal cortex but increased in the hypothalamus at the higher dose of nicotine. The 5HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) was not significantly altered in any region. The 5HT index (5-HIAA/5-HT) was significantly decreased in the hypothalamus and increased in frontal cortex at the higher dose. Concentrations of DA and the metabolite homovanillic acid (HVA) were not significantly altered by nicotine. Nevertheless, significant decreases in the DA metabolite dihydroxyphenyl-acetic acid (DOPAC) were observed in both striatum and hypothalamus. Moreover, the DA index [(DOPAC + HVA)/DA] was significantly decreased in all three brain regions. In contrast to other studies using acute dose and in vitro perfusion paradigms that have reported increased CNS catecholamine release stimulated by nicotine, chronic administration appears to be associated with decreased catecholamine turnover in some brain regions.     

Chronic monoamine oxidase-B inhibitor treatment blocks monoamine oxidase-A enzyme activity

Patients with Parkinson’s disease receive selective irreversible monoamine oxidase (MAO)-B inhibitors, but their effects on MAO-A activity are not known during long-term application. We determined MAO-A inhibition in plasma samples from patients with MAO-B inhibitor intake or without MAO-B inhibitor treatment and from healthy controls. We detected a 70 % reduction of MAO-A activity in patients with MAO-B inhibitor therapy in comparison to the other groups. Our results suggest that treatment with MAO-B inhibitor may also influence MAO-A activity in vivo, when administered daily.     

Immunocytochemical localization of serotonin, monoamine oxidase and assessment of monoamine oxidase activity in human dental pulp

In the present study serotonin (5-hydroxytryptamine, 5-HT) and monoamine oxidase (MAO) were both found localized in the blood vessel walls of human dental pulp. Our discovery of MAO activity in human dental pulp suggests a functional relationship between serotonin and MAO in this region.     

Platelet monoamine oxidase activity and psychopathy

Platelet monoamine oxidase (MAO) activity appears to be correlated to certain diagnostic subgroups and symptom patterns. A group of criminal offenders hospitalized for forensic psychiatric assessment were studied. Male nurses and construction workers were used as controls. Patients who were diagnosed as psychopaths according to the criteria of Cleckley had significantly lower platelet MAO activity than the control group of construction workers.     

Cerebrospinal fluid monoamine metabolites and suicide

Prospective studies of the serotonergic system and suicide report that low 5-hydroxyindolacetic acid (5-HIAA) in the cerebrospinal fluid (CSF) and a history of attempted suicide predict suicide risk. Low CSF homovanillic acid (HVA) is reported to be associated with past and future lethality of suicide attempts but not with suicide. The interrelationships between monoamine metabolites, violent method, suicide intent and lethality of suicidal behaviour are complex. We hypothesized that CSF 5-HIAA and HVA levels are related to suicide intent, violence and lethality of suicidal behaviour. Fifteen male suicide attempters admitted to a psychiatric ward at the Karolinska University Hospital and eight healthy male volunteers were submitted to lumbar puncture and CSF 5-HIAA and HVA were assayed. Suicide intent with the Beck Suicide Intent Scale (SIS), lethality and violence of suicidal behaviour were assessed. All patients were followed up for causes of death. Six suicides and one fatal accident were identified with death certificates. Mean CSF 5-HIAA but not CSF HVA differed between suicides and survivors. Violent suicides had higher suicide intent and CSF 5-HIAA than non-violent suicides. In violent suicides, CSF 5-HIAA levels were negatively correlated with SIS. Greater suicide intent may be associated with greater aggressive intent and predicts a violent suicide method.     

Ischemia-induced seizures and cortical monoamine levels

Seizure activity as a component of the ischemic process possibly responsible for monoamine changes described in the gerbil stroke model was the subject of this study. Abnormal motor activity suggestive of seizures developed one to three hours after unilateral ligation of the common carotid artery in approximately 50% of gerbils that exhibited signs of stroke. Reduction of cortical levels of dopamine and norepinephrine was observed only when seizures occurred in association with stroke. The levels of 5-hydroxytryptamine were reduced bilaterally in animals with and without signs of stroke and were reduced further in animals with stroke plus seizures. Further study is needed to establish whether the catecholamine changes associated with ischemia-induced seizures are primary and causative or secondary to seizure activity itself. In the ischemic brain, 5-hydroxytryptamine metabolism appears disordered independent of seizure activity. Seizure activity must be taken into account when the mechanisms of disordered monoamine metabolism are being examined in the gerbil stroke model.