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联合应用莱菔硫烷和利鲁唑对谷氨酸毒性造成的运动神经损伤的保护作用

Protective Effect of Combination of Sulforaphane and Riluzole on Glutamate- Mediated Excitotoxicity

【作者】 常庚

【导师】 李春岩;

【作者基本信息】 河北医科大学 , 神经病学, 2010, 博士

【摘要】 肌萎缩侧索硬化(ALS)是选择性地损伤上、下运动神经元的一种慢性进行性的神经系统变性疾病。临床表现为缓慢进展的四肢肌肉无力,常累及咽喉肌和呼吸肌等。患者多于发病3-5年内死亡。该病目前病因不清,尚无有效治疗。近年来FDA批准的新药利鲁唑临床开始应用,但仅能延长生存期,且价格昂贵、副作用大,一些患者不能耐受,临床并未广泛应用。所以,迫切地需要进一步了解该病的发病机制,寻求新的治疗药物。ALS的发病机制主要集中在以下几个方面:1)Cu/Zn SOD基因突变;2)谷氨酸的兴奋毒作用;3)线粒体功能异常;4)氧化应激;5)小胶质细胞增生等。多数学者认为多种因素相互作用导致了ALS的发病,其中谷氨酸的兴奋性毒作用和氧化应激是ALS的发病过程中最重要的两个致病因素。谷氨酸作为神经系统的一种重要的神经递质,对维持神经细胞的正常生理功能至关重要,但高浓度的谷氨酸则具有神经毒性作用。中枢神经系统维持谷氨酸生理浓度主要依赖于运动神经元和星型胶质细胞的谷氨酸转运体。当谷氨酸转运体功能或结构异常时就会直接造成细胞外谷氨酸浓度的异常增高,激活谷氨酸AMPA受体,引起细胞内钙超载,钙离子大量积聚直接引起线粒体去极化、能量代谢损伤、使产生ATP的电子传递链解耦联,随之大量自由基从线粒体电子传递链中释放,造成严重的氧化应激损伤;同时在运动神经元或反应性增生的胶质细胞又内产生的大量ROS,又会导致星形胶质细胞上谷氨酸转运功能的破坏。多种发病机制在运动神经元损伤的过程中起作用,同时临床试验也证明了,采用单一的治疗策略效果都不理想。因此,联合应用不同机制的药物治疗运动神经元病是未来研究的重点。我们在研究中联合应用力鲁唑和莱菔硫烷治疗运动神经元病,以期取得良好的疗效。利鲁唑是一种经典的抗谷氨酸药物,也是目前公认的可延缓ALS病程的药物。近年来,很多体内和体外的研究证实,利鲁唑不仅能降低细胞外的谷氨酸浓度,而且能有效保护谷氨酸转运体,加强谷氨酸转运体对谷氨酸的摄取。莱菔硫烷( sulforaphane ,SF)广泛存在于十字花科中,可以激活Nrf2/ARE信号通路。而Nrf2/ARE信号通路的激活,可诱导一系列内源性抗氧化酶和抗氧化蛋白上调,协同加强细胞清除ROS/RNS的防御系统,维持细胞内的还原电位。因此,有学者推断此通路有望成为神经变性疾病治疗的新靶点。Nrf2是位于胞浆中一个转录因子,而ARE是存在于多种抗氧化酶和抗氧化蛋白基因的5’侧翼的顺式DNA调节元件,称为抗氧化反应元件。正常情况下,Nrf2被Keap1锚定在胞浆内,Keap1是与actin结合一个的富含半胱氨酸残基的调节蛋白,扮演着分子开关的角色。当Keap1感受到ROS或诱导剂的信号后发生构象改变与Nrf2解离,Nrf2向核内移位与ARE结合后诱导下游靶基因的表达。我们应用谷氨酸转运体抑制剂苏-羟天冬氨酸(THA)诱导的脊髓体外器官型培养谷氨酸兴奋性毒性模型,探讨联合应用莱菔硫烷和利鲁唑对谷氨酸毒性造成的运动神经元损伤的协同保护作用,并研究联合用药的保护机制。研究表明联合应用莱菔硫烷和利鲁唑对谷氨酸毒性造成的运动神经元损伤的有协同保护作用,比单一用药有优势。这对于研究肌萎缩侧索硬化的发病机制提供了坚实的理论依据,并提出了新的联合用药模式。第一部分谷氨酸兴奋性毒性模型的研究目的:探讨建立THA诱导的脊髓器官型培养模型。方法:应用脊髓体外器官型培养模型,随机分成2组:对照组、THA组。用免疫组化法检测运动神经元数目,用多功能酶标仪检测培养基中LDH,MDA和谷氨酸的含量。结果:经过4周培养后,THA组培养液中谷氨酸、LDH和MDA的含量明显高于对照组,运动神经元数目较对照组减少。结论:用THA干预脊髓器官型培养模型后,导致了运动神经元的损伤和培养液中谷氨酸的含量的升高。因此THA诱导的脊髓器官型培养模型是研究慢性谷氨酸毒性的理想模型。第二部分探讨联合应用莱菔硫烷和利鲁唑是否有协同保护作用目的:探讨联合应用莱菔硫烷和利鲁唑是否比单药效果好。方法:应用脊髓体外器官型培养模型,每个实验均随机分成5组:THA组、莱菔硫烷处理组(10μM ,4μM)、利鲁唑处理组(5μM ,2μM)、联合用药组。用免疫组化法检测运动神经元数目,用多功能酶标仪检测培养基中LDH和MDA含量。用流式细胞仪检测线粒体膜电位。结果:经过4周培养后,莱菔硫烷处理组(10μM)、利鲁唑处理组(5μM)和联合用药组运动神经元数目较THA组明显增加,差异显著。在运动神经元数目上,莱菔硫烷处理组(10μM)、利鲁唑处理组(5μM)和联合用药组无显著差别。莱菔硫烷处理组(10μM)、利鲁唑处理组10μM)和联合用药组的培养液中LDH,MDA的含量低于THA组。这三组之间,培养液中LDH、MDA的含量无明显差别。经过4周培养后,莱菔硫烷处理组(4μM)和利鲁唑处理组(2μM)在运动神经元数目和培养液中MDA的含量上,与THA组相比无差异显著。在运动神经元数目上,小剂量联合用药组多于菔硫烷处理组(4μM)、利鲁唑处理组(2μM)和THA组。在培养液中LDH、MDA的含量上,小剂量联合用药组低于莱菔硫烷处理组(4μM)、利鲁唑处理组(2μM)和THA组。结论:联合应用莱菔硫烷和利鲁唑对谷氨酸毒性造成的运动神经元损伤的有协同保护作用,比单一用药有优势。第三部分联合应用莱菔硫烷和利鲁唑对运动神经元损伤的保护机制的探讨目的:探讨应用莱菔硫烷和利鲁唑对运动神经元损伤的保护机制。方法:应用脊髓体外器官型培养模型,随机分成5个组:对照组、THA组、莱菔硫烷处理组、利鲁唑处理组、联合用药组。用Western blot方法检测HO-1、NQO1和Nrf-2蛋白水平的变化,用多功能酶标仪检测培养液中谷氨酸的变化。结果:经过4周培养后,莱菔硫烷处理组和联合用药组能够诱导Ⅱ相酶NQO-1、HO-1、和Nrf-2的表达。利鲁唑组在NQO-1、HO-1、和Nrf-2的表达上与THA组相比无显著差别。利鲁唑组和联合用药组的培养液中谷氨酸的含量较THA组明显降低,莱菔硫烷处理组培养液中谷氨酸的含量与THA组相比无明显差别。结论:联合应用莱菔硫烷和利鲁唑的神经保护机制在于既能诱导Ⅱ相酶的表达,又能降低细胞外液中的谷氨酸。

【Abstract】 Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative disorders characterized by the progressive and selective death of upper and lower motor neurons. The disease is characterized by progressive musele weakness and the patient’s death is usually caused by respiratoy failure,occurring 3-5 years after the first appearance of symptoms. The cause of this process is mostly unknown and there has been no effective treatment. Riluzole, which was the only drug currently registered for ALS, prolonged survival by only approximately three months but its serious makes it impossible to use extensively.Several mechanisms for the pathogenesis of ALS have been proposed: 1)mutations of the copper/zinc superoxide dismutase (Cu/Zn SOD) gene;2)glutamate excitotoxicity;3)mitochondrial dysfunction;4)oxidative stress;5)Immunity and inflammation,et al. It has been demonstrated that several mechanisms have been proposed to work together in contributing to the disease. Excitotoxicity and oxidative stress are the important pathogenic factors. Glutamate is a major excitatory neurotransmitter, which play important role in maintaining the neuron’s function. the level of extracellular or synaptic cleft glutamate is tightly controlled by glutamate transporters, . Glutamate excitotoxicity is thought to result mainly from excess Ca2+ entry into neurons triggered by over-stimulation of postsynaptic glutamate receptor, and a large rise in intracellular Ca2+ in these cells causes rapid mitochondrial Ca2+ overload, resulting in mitochondrial damage and mitochondrial generation of reactive oxygen species (ROS). Mitochondrial damage may lead to necrosis and apoptosis, as a result of potential loss of ATP synthesis and release to cytoplasm of apoptogenic factors. Increased ROS levels may cause oxidative damage within motor neurons and also cause oxidative damage and disruption of glutamate transport in surrounding astrocytes, the latter resulting from the release of ROS from injured motor neurons.Riluzole is the only drug proven to slow the disease process in humans and has anti-excitotoxic properties. A growing body of work shows that riluzole has neuroprotective properties in some animal models and in cell cultures. riluzole can reduce glutamate release and enhance the activity of the glutamate transporters.Sulforaphane (SF), which is present in broccoli, can activate the Nrf2-ARE signaling, which functions as one of the most important anti-oxidant defense mechanisms by inducing and up-regulating many cytoprotective and antioxidant phase II enzymes. Therefore, the Nrf2-ARE signaling has been recognized as the novel pharamacological target for ALS treatment. Nrf2 is a nuclear transcription factor in cytoplasm,while antioxidant response element (ARE) , which is present in the promoter regions of many antioxidant genes. Nrf2 is sequestered in the cytoplasm by interacting with two molecules of Keap1. Oxidative stress and other inducers can result in a conformational change that renders Keap1 unable to bind to Nrf2, thereby inducing translocation of Nrf2 to the nucleus and binds to the antioxidant response element (ARE) .In our research, we use the organotypic spinal cord cultures interfered with Threohydroxyaspartate (THA) as our research model. The objective of the current study is to investigate whether the combination of SF and riluzole is superior to either one used alone. Our results suggest that the combination of sulforaphane and riluzone was more effective than each drug used alone in the protective action against glutamate-mediated excitotoxicity. It represents a novel approach in potential combination therapy for ALS.Part I Study the model of glutamate excitotoxicityObjective: To study the model of THA-induced spinal cord organotypic culture.Methods: The SD rat pups spinal cord organotypic cultures were divided into two groups at random: control and 100μmol/L THA group. The number of motor neurons was assessed by immunohistochemistry and the level of LDH、MDA and glutamate in the medium was assayed with ELIASA.Results: At the end of the 3-week THA treatment, we found that the motor neurons number in the group treated with THA was less than the control group and the level of LDH、MDA and glutamate in the medium in THA group is higher than that in the control group.Conclusions: Threohydroxyaspartate (THA) causes glutamate excitotoxicity in motor neurons in organotypic culture of rat spinal cord. So the model is an ideal model for studying chronic glutamate-induced excitotoxicity.PartⅡThe objective of the current study is to investigate whether the combination of SF and riluzole is superior to either one used aloneObjective: To investigate whether SF can act together with riluzole in protecting motor neurons against glutamate-induced excitotoxicity in organotypic spinal cord cultures.Results: In riluzole (5μM) , SF (10μM) and the combination treatment groups, the number of motor neuron is higher than that in THA group, and there was no significant difference among the three groups. In riluzole (5μM) , SF (10μM) and the combination treatment groups, the level of MDA、LDH in the medium is lower than that in THA group, and there was no significant difference among the three groups.There was no difference among the SF (4μM) treatment group、riluzole (2μM) treatment group and the THA group, as measured by the number of motor neuron, medium MDA, and LDH level. In the combination treatment group, the number of motor neuron was significantly higher than that in the THA group, the SF(4μM) group and the riluzole(2μM) group. In the combination treatment group, the level of LDH and MDA was significantly lower than that in the THA group, the SF(4μM) group and the riluzole(2μM) group.Conclusions: The combination treatment is more effective than each drug used alone. PartⅢInvestigate the neuroprotective mechanisms of the combinationObjective: To investigate the neuroprotective mechanisms of the combination of sulforaphane with riluzole.Methods: The SD rat pups spinal cord organotypic cultures were divided into five groups at random: control, the THA group, the SF treatment group,. the riluzole treatment group and the combination treatment group. the explants are harvested for measurement of expression of Nrf2, NADPH: quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1) by immunoblotting analysis. The level of glutamate was assayed with ELIASA.Results: At the end of the treatment, Nrf2 accumulated and the expression of two Phase II enzymes, including HO-1 and NQO1, increased in the SF treatment group and the combination treatment group. In the SF treatment group and the combination treatment group, the level of glutamate in the medium is lower than that in the THA treatment group.Conclusions: combination of SF and riluzole can stimulate the expression of phase II enzymes and reduces glutamate accumulation in the extracellular space.Conclusions: The neuroprotective mechanisms of the combination ar is that combination treatment can stimulate the expression of phase II enzymes and reduces glutamate accumulation in the extracellular space.

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