鱼藤酮对大鼠中脑神经元多巴胺转运、储存和代谢的影响

目的探讨鱼藤酮对多巴胺(DA)神经元选择性毒性作用的可能机制。方法大鼠背部sc鱼藤酮(1.0或1.5mg.kg-1.d-1)28d,观察大鼠的外观和行为变化。然后麻醉处死大鼠,取中脑黑质组织。分别用免疫组织化学法、Western蛋白印迹法和RT-PCR法检测鱼藤酮对大鼠中脑黑质DA神经元突触囊泡单胺转运体(VMAT2)表达的影响;以卞胺为底物进行比色,检测组织单胺氧化酶(MAO)的活性;高效液相色谱-荧光检测器流速梯度法测定黑质组织内DA及其代谢产物3,4-二羟基苯乙酸(DOPAC)和高香草酸(HVA)的含量。结果注射鱼藤酮28d后,大鼠发生外观和行为改变,出现类帕金森病症候群特征;中脑黑质DA神经元VMAT2蛋白免疫深染的细胞数量减少;鱼藤酮1.0和1.5mg.kg-1组VMAT2蛋白和基因表达与正常对照组比较明显降低,MAO活性分别从对照组的(23.6±7.6)升高至(37.8±5.0)和(40.5±4.3)kU.h-1.g-1蛋白,均具有统计学意义;实验组大鼠中脑黑质组织内DA及其代谢产物DOPAC和HVA含量明显减少。结论鱼藤酮可抑制大鼠中脑黑质DA神经元VMAT2的表达,增强DA代谢相关酶MAO的活性,进而影响DA的转运、储存和代谢,这可能是鱼藤酮DA神经元选择性毒性作用机制之一。     

黄芩对实验性帕金森模型大鼠的保护作用

目的:研究黄芩对鱼藤酮所致帕金森(PD)模型大鼠的保护作用。方法:Wistar大鼠每日颈、背部皮下注射鱼藤酮葵花油乳化液制备PD大鼠模型;阳性组及不同剂量药物组在每日注射鱼藤酮之前1小时灌胃给药。ELISA法检测大鼠脑硫氧还蛋白(Trx);荧光探针法检测纹状体活性氧(ROS);分光光度法检测纹状体谷胱甘肽(GSH)、黑质丙二醛(MDA)含量。结果:造模后模型组大鼠出现一定程度的行为学变化,模型组Trx含量明显低于空白组、阳性组和低剂量组,药物可以一定程度上抑制氧化应激反应。结论:黄芩可以提高帕金森大鼠脑内Trx的表达,对鱼藤酮所致帕金森模型大鼠具有保护作用,这种作用可能与Trx相关。     

α-硫辛酸对帕金森病大鼠脑内黑质多巴胺能神经元保护机制研究

目的 探讨α-硫辛酸(LA)对帕金森病(PD)大鼠模型脑内黑质多巴胺能神经元的保护机制.方法 60只3月龄雄性Wistar大鼠随机分为对照组、实验组和药物干预组,每组20只.对照组大鼠给予背部皮下注射葵花油,实验组和药物干预组背部皮下注射鱼藤酮制备PD模型,药物干预组大鼠同时给予LA腹腔注射;采用Western blot检测大鼠中脑多巴胺能神经元中酪氨酸羟化酶(TH)、转录因子NF-E2相关因子-抗氧化反应元件(Nrf2-ARE)和血红素氧合酶1(HO-1)的表达变化;采用分光光度法检测大鼠脑内纹状体中丙二醛(MDA)和还原型谷胱甘肽(GSH)含量变化.结果 实验组大鼠中脑TH蛋白表达比对照组明显降低(P<0.05);药物干预组中脑TH蛋白在黑质比鱼藤酮组明显增高(P<0.05),但是较对照组仍有明显减少(P<0.05).实验组大鼠Nrf2和HO-1蛋白表达比对照组明显降低(P<0.05);药物干预组Nrf2和HO-1蛋白表达比实验组明显增高(P<0.05),但是较对照组仍有明显降低(P<0.05).与对照组相比,实验组大鼠纹状体组织中脂质代谢产物MDA含量明显增加(P<0.01),药物干预后纹状体中MDA含量明显减少(P<0.05),但较对照组仍明显增高(P<0.05).与对照组相比,实验组大鼠GSH的含量明显减少(P<0.01),药物干预后GSH明显增加(P<0.05),但较对照组仍显著降低(P<0.05).结论 LA能激活Nrf2-ARE信号通路对PD大鼠模型中脑多巴胺能神经元起到有效的神经保护作用,改善PD样症状.     

莱菔硫烷对帕金森病大鼠脑内多巴胺能神经元的保护作用及机制

目的 观察莱菔硫烷(sulforaphane,SF)对帕金森(PD)病大鼠模型脑内黑质多巴胺能神经元的保护作用.方法 健康成年雄性Wistar大鼠背部皮下注射鱼藤酮制备PD大鼠模型,药物治疗组同时给予大鼠腹腔注射EGCG.采用分光光度法检测大鼠脑内丙二醛(MDA)和还原型谷胱甘肽(GSH),免疫细胞化学和免疫印记检测大鼠中脑黑质及纹状体中络氨酸羟化酶(TH)的表达变化.结果 Western blot结果显示试验组大鼠脑内中脑和纹状体中TH蛋白表达都比对照组有明显降低(P<0.05);莱菔硫烷药物干预后TH蛋白在中脑和纹状体比试验组明显增高(P<0.05),但是较对照组仍有明显减少(P<0.05).实验组大鼠在给予鱼藤酮背部皮下注射后导致大鼠脑内纹状体中脂质代谢产物MDA含量比对照组明显增加(P<0.01).莱菔硫烷药物干预组大鼠脑内纹状体中MDA明显减少(P<0.05),但较对照组仍明显增加(P<0.05).同时我们发现鱼藤酮背部皮下注射可以导致实验组大鼠脑内纹状体中GSH的含量比对照组明显减少(P<0.01);莱菔硫烷药物干预后大鼠脑内纹状体中GSH含量较实验组明显增加(P<0.05),但较对照组仍明显减少(P<0.05).结论 氧化应激在PD发病中起着非常重要的作用,抗氧化治疗能有效减轻大鼠脑内多巴胺能神经元损伤情况,同时改善PD样症状,为PD的治疗提供了新的靶点.     

毛钩藤碱对6-羟基多巴胺诱导的帕金森病模型大鼠神经保护作用及机制研究

目的 探讨毛钩藤碱对帕金森病(Parkinson′s disease, PD)模型大鼠的神经保护作用及机制。方法 采用右侧中脑黑质致密部注射6-羟基多巴胺(6-hydroxydopamine, 6-OHDA)制备大鼠PD模型,将建模成功的PD大鼠随机分为模型组、美多芭组、毛钩藤碱低剂量和高剂量组,同时设置正常对照组,每组12只。给予相应药物灌胃干预,每天1次,连续1个月。采用旋转试验、转棒试验和Morris水迷宫试验检测大鼠行为学变化;HE染色法观察大鼠右侧中脑黑质病理学变化;高效液相色谱法(HPLC)检测大鼠右侧纹状体中多巴胺(dopamine, DA)水平;免疫组化法检测大鼠右侧中脑黑质中多巴胺能神经元标志物酪氨酸羟化酶(tyrosine hydroxylase, TH)表达;ELISA法检测大鼠右侧中脑黑质IL-6、IL-1β和TNF-α水平;Western blot检测大鼠右侧中脑黑质中NF-κB p65、NF-κB抑制因子α(IκBα)、p-IκBα等蛋白表达水平。结果 毛钩藤碱能显著改善PD大鼠行为学障碍及中脑黑质损伤,升高纹状体DA含量及中脑黑质中TH阳性神经元数量和Iκ...     

二苯乙烯苷对帕金森病模型大鼠行为学及多巴胺能神经元的影响

［摘要］　目的：观察何首乌主要有效成分二苯乙烯苷（TSG）对帕金森病（PD）模型大鼠行为学及脑内黑质多巴胺能神经元的影响。方法：采用6-羟基多巴胺（6-OHDA）单侧脑内黑质致密部（SNC）和中脑腹侧被盖区（VTA）两点注射法制备PD大鼠模型后，灌胃给予TSG低、中、高剂量 (50，100，200 mg&#8226;kg-1)，qd，连续5周。实验同时设正常对照组和美多芭阳性对照组（125 mg&#8226;kg-1），每组10只。用旋转行为检测及Rotarod试验观察各组大鼠的行为学变化，用免疫组化法观察大鼠脑黑质酪氨酸羟化酶(TH)阳性神经元的表达，用高效液相色谱-电化学法（HPLC-ECD）测定大鼠脑黑质多巴胺（DA）及3,4-二羟基苯乙酸（DOPAC）含量。结果：行为学检测结果表明，模型组大鼠旋转次数及运动协调性均低于正常对照组(P<0.01)，而TSG与美多芭给药可改善大鼠旋转行为及运动协调性。TH免疫组化检测结果显示，TSG处理组脑黑质部位损毁较轻，有较多残存的TH阳性细胞；而模型组黑质大部损毁，几乎无残存的TH阳性细胞；从HPLC-ECD测定结果可以看出，与正常对照组比较，模型组DA及DOPAC含量下降显著(P<0.05)，与模型组相比，TSG处理组脑内DA及DOPAC含量明显增加(P<0.05)。结论：TSG可改善PD大鼠的行为学改变，增加黑质-纹状体多巴胺及其代谢物含量并提高黑质多巴胺能神经元的残存率，提示TSG对PD可能具有一定的神经保护作用。     

多巴胺转运蛋白研究进展

多巴胺转运蛋白(dopaminetransporter，DAT)，位于中枢多巴胺能神经元末梢，是一种膜蛋白，属于Na+／Cl-依赖性转运体基因家族。其生理作用为将突触间隙内已发挥生理效应的多巴胺(DA)重新摄入突触前膜，以备再次利用，同时终止神经细胞间的信息传递。由于分子生物学技术的应用，对DAT的作用有了深一层的认识。DAT已成为调节DA神经系统功能的重要环节。近年来的研究表明，DA能神经毒素MPP+是通过DAT进入神经元的，且单光子发射计算机断层扫描（SPECT）显示早期帕金森病（PD）病人的DAT水平明显降低。因此，对DAT深入广泛的研…     

人参皂苷Rg1可能通过抗氧化作用来保护帕金森病鼠黑质神经元

研究人参皂苷Rgl对1—甲基—4—苯基—1，2，3，6—四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,MPTP)诱导的小鼠黑质神经元凋亡的抗氧化保护作用。方法：采用MPTP制备帕金森病(PD)小鼠模型，经人参皂苷Rg1预处理后，用尼氏(Nissl)染色和TH组化染色观察黑质神经元的存活情况，通过活化型Caspase3的免疫组化染色和TUNEL染色了解黑质神经元的凋亡情况，并用生化技术对黑质区域谷胱甘肽（GSH)浓度及超氧化物歧化酶(SOD)活力进行检测。结果：人参皂苷Rg1预处理能提高黑质区域GSH的浓度及降低SOD活力，相应地减少PD鼠黑质致密带Nissl阳性神经元和TH阳性神经元的脱失现象，使活化型Caspase3表达阳性细胞减少，降低黑质神经元TUNEL染色的阳性率。结论：人参皂苷Rg1可能对MPTP诱导的小鼠黑质神经元凋亡有抗氧化保护作用。     

人参皂苷Rg1通过激活Nrf2/xCT/GPX4通路抑制神经元铁死亡改善缺血性脑卒中损伤北大核心CSCD

目的研究人参皂苷Rg1对缺血性脑卒中后神经元铁死亡的抑制作用及其机制。方法细胞水平建立HT22细胞氧糖剥夺/复氧(oxygen glucose deprivation/reoxygenation,OGD/R)模型,CCK-8法检测Rg1对OGD/R损伤后HT22细胞活力的影响,试剂盒检测细胞铁死亡标志物GSH/GSSG、SOD、MDA和Fe 2+含量的影响;Western blot检测细胞GPX4、xCT和Nrf2蛋白的变化;ML385干预Nrf2验证其在Rg1调节OGD/R引起的细胞铁死亡中的作用。动物水平建立大鼠短暂性大脑中动脉阻塞(middle cerebral artery occlusion,MCAO)再灌注模型,分为假手术组、模型组、Rg1治疗组(40 mg·kg^(-1))和Fer-1铁死亡抑制剂组(0.2 mg·kg^(-1))。缺血90 min后拔栓并给药,再灌注24 h后进行Zea Longa和Garcia Test评分评估神经受损程度;透射电镜观察皮质神经元线粒体形态变化;Western blot检测GPX4和xCT蛋白水平。结果Rg1能明显提高OGD/R后HT22细胞存活率,上调GSH/GSSG比值,恢复SOD活性,降低MDA和Fe 2+含量,并促进GPX4,xCT和Nrf2蛋白表达,而ML385干预明显抑制了Rg1对OGD/R后HT22细胞存活率及GPX4、xCT蛋白的上调作用。在体动物实验结果表明,Rg1可上调MCAO大鼠皮质组织GPX4和xCT的表达,缓解神经元线粒体的形态损伤,改善神经功能。结论人参皂苷Rg1可通过促进Nrf2/xCT/GPX4通路抑制缺血性脑卒中神经元铁死亡。     

二价金属离子转运体对铁、铅转运作用的研究现状

二价金属离子转运体1（divalent metal transporter 1，DMT1）又称自然抵抗相关的巨噬细胞蛋白2（natural resistance-associated macrophage protein 2，Nramp2），是一种质子依赖性金属离子转运蛋白。肠道的DMT1是已知唯一的肠腔内摄取非血红素铁的跨膜转运体，其功能主要是介导上皮细胞对铁的吸收及参与铁从内吞小体移位到胞浆的过程。     

Glutathione,iron and Parkinson's disease

Parkinson's disease (PD) is a progressive neurodegenerative disease involving neurodegeneration of dopaminergic neurons of the substantia nigra (SN), a part of the midbrain. Oxidative stress has been implicated to play a major role in the neuronal cell death associated with PD. Importantly, there is a drastic depletion in cytoplasmic levels of the thiol tripeptide glutathione within the SN of PD patients. Glutathione (GSH) exhibits several functions in the brain chiefly acting as an antioxidant and a redox regulator. GSH depletion has been shown to affect mitochondrial function probably via selective inhibition of mitochondrial complex I activity. An important biochemical feature of neurodegeneration during PD is the presence of abnormal protein aggregates present as intracytoplasmic inclusions called Lewy bodies. Oxidative damage via GSH depletion might also accelerate the build-up of defective proteins leading to cell death of SN dopaminergic neurons by impairing the ubiquitin-proteasome pathway of protein degradation. Replenishment of normal glutathione levels within the brain may hold an important key to therapeutics for PD. Several reports have suggested that iron accumulation in the SN patients might also contribute to oxidative stress during PD.     

Alteration of Striatal Tetrahydrobiopterin in Iron-Induced Unilateral Model of Parkinson's Disease

It has been suggested that transition metal ions such as iron can produce an oxidative injuries to nigrostriatal dopaminergic neurons, like Parkinson''s disease (PD) and subsequent compensative increase of tetrahydrobiopterin (BH₄) during the disease progression induces the aggravation of dopaminergic neurodegeneration in striatum. It had been established that the direct administration of BH₄ into neuron would induce the neuronal toxicity in vitro. To elucidate a role of BH₄ in pathogenesis in the PD in vivo, we assessed the changes of dopamine (DA) and BH₄ at striatum in unilateral intranigral iron infused PD rat model. The ipsistriatal DA and BH₄ levels were significantly increased at 0.5 to 1 d and were continually depleting during 2 to 7 d after intranigral iron infusion. The turnover rate of BH₄ was higher than that of DA in early phase. However, the expression level of GTP-cyclohydrolase I mRNA in striatum was steadily increased after iron administration. These results suggest that the accumulation of intranigral iron leads to generation of oxidative stress which damage to dopaminergic neurons and causes increased release of BH₄ in the dopaminergic neuron. The degenerating dopaminergic neurons decrease the synthesis and release of both BH4 and DA in vivo that are relevance to the progression of PD. Based on these data, we propose that the increase of BH₄ can deteriorate the disease progression in early phase of PD, and the inhibition of BH4 increase could be a strategy for PD treatment.     

Mitoapocynin Treatment Protects Against Neuroinflammation and Dopaminergic Neurodegeneration in a Preclinical Animal Model of Parkinson’s Disease

Mitochondrial dysfunction, oxidative stress and neuroinflammation have been implicated as key mediators contributing to the progressive degeneration of dopaminergic neurons in Parkinson’s disease (PD). Currently, we lack a pharmacological agent that can intervene in all key pathological mechanisms, which would offer better neuroprotective efficacy than a compound that targets a single degenerative mechanism. Herein, we investigated whether mito-apocynin (Mito-Apo), a newly-synthesized and orally available derivative of apocynin that targets mitochondria, protects against oxidative damage, glial-mediated inflammation and nigrostriatal neurodegeneration in cellular and animal models of PD. Mito-Apo treatment in primary mesencephalic cultures significantly attenuated the 1-methyl-4-phenylpyridinium (MPP⁺)-induced loss of tyrosine hydroxylase (TH)-positive neuronal cells and neurites. Mito-Apo also diminished MPP⁺-induced increases in glial cell activation and inducible nitric oxide synthase (iNOS) expression. Additionally, Mito-Apo decreased nitrotyrosine (3-NT) and 4-hydroxynonenol (4-HNE) levels in primary mesencephalic cultures. Importantly, we assessed the neuroprotective property of Mito-Apo in the MPTP mouse model of PD, wherein it restored the behavioral performance of MPTP-treated mice. Immunohistological analysis of nigral dopaminergic neurons and monoamine measurement further confirmed the neuroprotective effect of Mito-Apo against MPTP-induced nigrostriatal dopaminergic neuronal loss. Mito-Apo showed excellent brain bioavailability and also markedly attenuated MPTP-induced oxidative markers in the substantia nigra (SN). Furthermore, oral administration of Mito-Apo significantly suppressed MPTP-induced glial cell activation, upregulation of proinflammatory cytokines, iNOS and gp91phox in IBA1-positive cells of SN. Collectively, these results demonstrate that the novel mitochondria-targeted compound Mito-Apo exhibits profound neuroprotective effects in cellular and pre-clinical animal models of PD by attenuating oxidative damage and neuroinflammatory processes.     

Iron-binding characteristics of neuromelanin of the human substantia nigra

The vulnerability of the dopaminergic neurons of the substantia nigra (SN) in Parkinson's disease has been related to the presence of the pigment neuromelanin (NM) in these neurons. It is hypothesised that NM may act as an endogenous storage molecule for iron, an interaction suggested to influence free radical production. The current study quantified and characterised the interaction between NM and iron. Iron-binding studies demonstrated that both NM and synthetically-produced dopamine melanin contain equivalent numbers of high and low-affinity binding sites for iron but that the affinity of NM for iron is higher than that of synthetic melanin. Quantification of the total iron content in iron-loaded NM and synthetic melanin demonstrated that the iron-binding capacity of NM is 10-fold greater than that of the model melanin. This data was in agreement with the larger iron cluster size demonstrated by M?ssbauer spectroscopy in the native pigment compared with the synthetic melanin. These findings are consistent with the hypothesis that NM may act as an endogenous iron-binding molecule in dopaminergic neurons of the SN in the human brain. The interaction between NM and iron has implications for disorders such as Parkinson's disease where an increase in iron in the SN is associated with increased indices of oxidative stress.     

Agonism of Peroxisome Proliferator Receptor-Gamma may have Therapeutic Potential for Neuroinflammation and Parkinson's Disease

Evidence suggests inflammation, mitochondria dysfunction, and oxidative stress play major roles in Parkinson's disease (PD), where the primary pathology is the significant loss of dopaminergic neurons in the substantia nigra (SN). Current methods used to treat PD focus mainly on replacing dopamine in the nigrostriatal system. However, with time these methods fail and worsen the symptoms of the disease. This implies there is more to the treatment of PD than just restoring dopamine or the dopaminergic neurons, and that a broader spectrum of factors must be changed in order to restore environmental homeostasis. Pharmacological agents that can protect against progressive neuronal degeneration, increase the level of dopamine in the nigrostriatal system, or restore the dopaminergic system offer various avenues for the treatment of PD. Drugs that reduce inflammation, restore mitochondrial function, or scavenge free radicals have also been shown to offer neuroprotection in various animal models of PD. The activation of peroxisome proliferator receptor- gamma (PPAR-gamma ) has been associated with altering insulin sensitivity, increasing dopamine, inhibiting inflammation, altering mitochondrial bioenergetics, and reducing oxidative stress - a variety of factors that are altered in PD. Therefore, PPAR-gamma activation may offer a new clinically relevant treatment approach to neuroinflammation and PD related neurodegeneration. This review will summarize the current understanding of the role of PPAR-gamma agonists in neuroinflammation and discuss their potential for the treatment of PD.     

DJ-1 mediates paraquat-induced dopaminergic neuronal cell death

There are two causes of Parkinson's disease (PD): environmental insults and genetic mutations of PD-associated genes. Environmental insults and genetic mutations lead to mitochondrial dysfunction, and a combination of mitochondrial dysfunction and increased oxidative stress in dopaminergic neurons is thought to contribute to the pathogenesis of PD. Among the PD-associated genes, DJ-1 acts as a redox sensor for oxidative stress and has been also proposed to maintain mitochondrial complex I activity.To understand molecular functions of DJ-1 in the cell, we have generated DJ-1 null cells from the DJ-1(−/−) mouse embryos. Using these null cells, we investigated the susceptibility to an environmental toxin, paraquat, which is known to inhibit mitochondrial complex I. Interestingly, we found that DJ-1 null cells showed a resistance to paraquat-induced apoptosis, including reduced poly (ADP-ribose) polymerase and procaspase-3. Also DJ-1 null cells generated less superoxide than SN4741 cells by paraquat treatment. Consistent with the reduced paraquat sensitivity, DJ-1 null cells showed reduced complex I activity, which was partially rescued by ectopic DJ-I expression. In summary, our results suggest that DJ-1 is critical to maintain mitochondrial complex I and complex I could be a key target in interaction of paraquat toxicity and DJ-1 for giving rise to PD.     

Cryptotanshinone protects against oxidative stress in the paraquat-induced Parkinson's disease model

Parkinson's disease (PD) is a common neurodegenerative disorder associated with striatal dopaminergic neuronal loss in the Substantia nigra. Oxidative stress plays a significant role in several neurodegenerative diseases. Paraquat (PQ) is considered a potential neurotoxin that affects the brain leading to the death of dopaminergic neurons mimicking the PD phenotype. Various scientific reports have proven that cryptotanshinone possesses antioxidant and anti-inflammatory properties. We hypothesized that cryptotanshinone could extend its neuroprotective activity by exerting antioxidant effects. This study was designed to evaluate the effects of cryptotanshinone in both cellular and animal models of PQ-induced PD. Annexin V-PI double staining and immunoblotting were used to detect apoptosis and oxidative stress proteins, respectively. Reactive oxygen species kits were used to evaluate oxidative stress in cells. For in vivo studies, 18 B6 mice were divided into three groups. The rotarod data revealed the motor function and immunostaining showed the survival of TH+ neurons in SNpc region. Our study showed that cryptotanshinone attenuated paraquat-induced oxidative stress by upregulating anti-oxidant markers in vitro, and restored behavioral deficits and survival of dopaminergic neurons in vivo, demonstrating its therapeutic potential.     

帕金森病与氧自由基

该文对近年来在帕金森病与氧自由基关系方面的研究工作做了回顾 ,许多事实表明 ,氧自由基在帕金森病的发病过程中起着重要作用 ,帕金森病患者机体处于氧应激状态 ,帕金森病病变部位的选择性决定了其氧自由基产生的特点 ,其它有关帕金森病的致病学说均与氧自由基有着密切关系。