【作者】 毕静；

【导师】 安利佳； 姜波；

【作者基本信息】 大连理工大学 ， 生物化工， 2012， 博士

【摘要】 本研究以帕金森(PD)和脑缺血损伤模型为例,采用原代皮层纯星形胶质细胞培养体系、皮层或中脑纯神经元培养体系以及中脑神经元-星形胶质细胞混合培养体系作为研究对象,探讨梓醇和PBEF在神经退行性疾病中的保护作用和机理,主要内容如下：1.梓醇的神经保护作用梓醇在纯星形胶质细胞培养体系中的神经保护作用。一方面,采用H202诱导的星形胶质细胞氧化应激损伤,结果发现,梓醇能够明显提高细胞活力,减少细胞内ROS的形成。此外,梓醇还通过增强谷胱甘肽还原酶(GR)、谷胱甘肽过氧化物酶(GSH-Px)等谷胱甘肽代谢循环中关键酶的活性以及降低氧化型谷胱甘肽(GSSG)在总谷胱甘肽(GSx)中的比例,从而抑制H202诱导的氧化应激损伤。但是,梓醇对过氧化氢酶(CAT)活性的增强作用却并不明显,说明梓醇在此损伤模型中潜在的保护机制主要是增强谷胱甘肽代谢循环效率以及抑制ROS生成。另一方面,采用脂多糖(LPS)与干扰素-Y(IFN-Y)共同刺激星形胶质细胞诱导炎症反应,结果表明,梓醇显著抑制了一氧化氮(NO)和ROS的生成,削弱了诱导型一氧化氮合酶(iNOS)的活性。此外,梓醇还明显下调了相关炎症基因iNOS、环氧化酶-2(COX-2)和Toll样受体-4(TLR4)的表达。进一步研究发现,梓醇的抗炎作用是通过调节核转录因子-kB(NF-KB)的激活以控制下游炎症因子的表达与释放,从而抑制星形胶质细胞激活诱发的炎症反应,最终实现神经保护的目的。梓醇在中脑纯神经元培养体系中的神经保护作用。利用鱼藤酮诱导中脑神经元损伤以模拟PD的发生,通过细胞形态学、免疫组化以及流式细胞分析,发现梓醇能够有效地抑制鱼藤酮诱导的中脑神经元凋亡或坏死。更进一步,发现梓醇是通过调控细胞外调节蛋白激酶(ERK)以抑制iNOS表达与NO释放,从而使神经元免于变性死亡的命运。梓醇在中脑神经元-星形胶质细胞混合培养体系中的神经保护作用。1-甲基-4-苯基-1,2,3,6-四氢吡啶(MPTP)已经是众所周知的临床以及生化水平上的神经毒素,可以模拟PD的发生。其主要作用机理是：MPTP先被星形胶质细胞内的单胺氧化酶-B(MAO-B)转化成有毒性的1-甲基-4-苯基吡啶离子(MPP+),然后被神经元选择性吸收,并且损伤神经元的线粒体造成功能紊乱,而最终导致神经元变性死亡。本研究中,利用MPTP分别处理中脑神经元-星形胶质细胞混合培养体系,以进一步探索梓醇的神经保护作用及其潜在的保护机制。结果发现,梓醇预处理明显削弱了神经元变性死亡的趋势,其保护机制涉及对线粒体功能的改善,主要体现在：增强了线粒体复合物I活性,阻止了线粒体膜电位(MMP)的损失,抑制了ROS的产生与Ca2+超载以及降低了线粒体通透性转运孔(MPTp)的开放度。2. PBEF的神经保护作用前B细胞克隆增强因子(PBEF)是烟酰胺(NAM)转化成烟酰胺腺嘌呤二核苷酸(NAD+)过程中的限速酶,在NAD+生物合成补救途径过程中起到重要作用。最近以小鼠为对象的研究表明,PBEF主要在野生型(+／+)以及半敲除型(+／-)小鼠的神经元中表达。采用光栓法建造脑缺血模型,发现与PBEF+/+相比,PBEF+/-小鼠更容易受到损伤而导致更大的缺血面积。本研究以原代小鼠皮层神经元为实验对象,采用氧糖剥夺模型(OGD)以及谷氨酸神经兴奋性毒性体外模拟脑缺血缺氧损伤,继续探索PBEF在脑缺血缺氧损伤中的神经保护作用。结果表明,作为PBEF的底物与产物,NAM和NAD+在OGD以及谷氨酸诱导的神经损伤模型中表现出较好的神经保护作用。利用PBEF特异性抑制FK866展开进一步研究,发现FK866的处理会加剧OGD诱导的神经损伤,表现在降低的细胞活力和NAD+水平上；而且,在谷氨酸诱导的神经兴奋性毒性损伤中,野生型PBEF过表达的神经元表现出更强的生命力,提示作为NAD+合成的限速酶,PBEF在脑缺血缺氧损伤模型中扮演着至关重要的角色。通过对线粒体功能的检测与分析,发现外源NAD+与NAM的补充明显削弱了OGD与FK866引起的线粒体生物合成障碍。此外,野生型PBEF过表达的神经元能够在一定程度上抑制谷氨酸诱导的MMP损失,提示PBEF的神经保护作用一部分是通过对线粒体功能的调节和优化实现的。更多还原

【Abstract】 In this study, the protective effects and mechanisms of catalpol and PBEF on neurodegenerative disease were investigated with Parkinson’s disease (PD) and Cerebral ischemia models in cortical astrocyte-enriched cultures, cortical or mesencephalic neuron-enriched cultures and mesencephalic neuron-astrocyte cultures, the primary coverage as follows:1. Neuroprotective Effects of CatalpolNeuroprotective effects of catalpol in astrocytes primary cultures. On one hand, we treated astrocytes with H2O2to induce oxidant stress. The results showed that catalpol could significantly increase the cell viability and reduce the intracellular ROS formation. Furthermore, catalpol attenuated H2O2-induced oxidative stress via preventing the decrease in the activities of antioxidant enzymes in glutathione redox cycling such as glutathione reductase (GR), glutathione peroxidase (GSH-Px), and glutathione content. However, the catalase (CAT) activity did not appear to be elevated by catalpol adequately. Together, the main mechanism underlying the protective effects of catalpol in H2O2-injured astrocytes might be related to the maintenance of glutathione metabolism balance and the decrease of ROS formation. On the other hand, astrocytes were pretreated with catalpol prior to lipopolysaccharide (LPS) plus interferon-γ (IFN-γ) stimulation which induced a inflammatory response. Biochemical analyses showed that nitric oxide (NO), ROS production and the inducible nitric oxide synthase (iNOS) activity were significantly reduced by catalpol. The data at transcriptional level also demonstrated that catalpol potently attenuated gene expressions which involved in inflammation, such as iNOS, cyclooxygenase-2(COX-2) and toll-like receptor4(TLR4). In addition, our exploration further revealed that the suppressive action of catalpol on inflammation was mediated via inhibiting nuclear factor-KB (NF-κB) activation. Collectively, these results adequately suggested that catalpol could exert inhibitory effects on inflammatory reaction in astrocytes and the inactivation of NF-κB could be the major determinant for its anti-inflammatory mechanism.Neuroprotective effects of catalpol in mesencephalic neurons. In current study, we stimulated primary mesencephalic neurons by rotenone to simulate PD occurring, and the results indicated that catalpol inhibited primary mesencephalic neurons from apoptosis by morphological assay, immunocytochemistry and flow cytometric evaluation. Moreover, the Extracellular regulated protein kinases (ERK) signaling pathway plays an important role in NO-mediated degeneration of neuron.Neuroprotective effects of catalpol in mesencephalic neuron-astrocyte cultures. MPTP is well known to produce clinical, biochemical and neurochemical changes similar to those which occur in PD. Furthermore, the accumulated evidence suggests that MPP+, conversed by monoamine oxidase type B (MAO-B) in astrocytes principally, is the active metabolite of MPTP and the major cause to PD associated with mitochondrial dysfunction. In this study, we treated mesencephalic neuron-astrocyte with MPTP respectively to investigate the neuroprotective effects of catalpol and the underlying protective mechanisms. Our results showed that pre-treatment with catalpol prior to MPTP treatment attenuated mitochondrial dysfunction by reversing the activity of mitochondrial complex I, mitochondrial membrane potential (MMP), intracellular Ca+level, and ROS accumulation as well as mitochondrial permeability transition pore (MPTp) opening.2. Neuroprotective Effects of PBEFPre-B-Cell Colony-Enhancing Factor (PBEF) is a rate-limiting enzyme to convert nicotinamide to nicotinamide mononucleotide (NMN) in the salvage pathway of nicotinamide adenine dinucleotide (NAD+) biosynthesis. Previously we found PBEF is exclusively expressed in neurons in mouse brain and heterozygous PBEF knockout (Pbef+/-) mice have larger ischemic lesion than wild type mice using photothrombosis-induced ischemia model. For mechanistic study of neuronal protective role of PBEF, we used in vitro oxygen-glucose deprivation (OGD) and glutamate excitotoxicity models of primary cultured neurons. Our results show that the treatments of neurons with nicotinamide and NAD+, the substrate and product of PBEF respectively, reduce neuronal death after OGD and glutamate excitotoxicity. Neurons with treatment of FK866, a PBEF inhibitor, have reduced cell viability and NAD+level after OGD as compared with neurons without treatment. Furthermore, overexpression of PBEF reduced glutamate excitotoxicity. We further tested whether PBEF affect mitochondrial function and biogenesis. Inhibition of PBEF reduces mitochondrial biogenesis, while addition of NAD+and NAM increase mitochondrial biogenesis. We further show overexpression of PBEF in neurons reduces MMP depolarization following glutamate stimulation using fluorescent live cell imaging. We conclude that PBEF exerts neuroprotection in ischemia through its enzymatic activity for NAD+production that can ameliorate mitochondrial dysfunction.更多还原