【作者】 傅秋黎；

【导师】 陈忠；

【作者基本信息】 浙江大学 ， 药理学， 2007， 硕士

【摘要】 阿尔茨海默病（Alzheimer’s disease，AD）是一种以记忆、语言、认知、理解、判断及情感等智能全面低下为特征的神经退行性疾病，以神经元内大量神经原纤维缠结，神经元周围大量β淀粉样蛋白沉积为主要病理特征。几十年有关AD发病机制的研究先后提出了众多假说，而β淀粉样蛋白假说历来备受广大研究者的重视，经过长期大量的研究，逐步成为AD发病机制最重要的假说之一，但其具体作用机制至今未有确切报道。除了Aβ的损伤作用，谷氨酸受体特别是NMDA受体引起的神经兴奋性损伤也被认为是AD重要的发病机制之一。最近，Snyder等发现Aβ引起的神经兴奋性损伤是通过NMDA受体内化进而抑制NMDA受体依赖性LTP及谷氨酸系统而实现的，这一全新的发现揭示了一个新的AD治疗方向。因此，基于β淀粉样蛋白假说的针对AD治疗方法的研究将成为现今及今后研究的热点。组胺作为一种重要的神经递质和神经调质，在中枢神经系统中占有不可替代的位置，参与多种神经系统功能的调节，如情感，运动，压力，学习记忆等。同时组胺也参与了多种神经系统疾病的疾理过程。已有证据表明，在AD中组胺能神经元显示严重的退行性变，同时组胺能神经元胞体所在的TM区有特异的淀粉样蛋白沉积块形成。也有研究表明，在AD中组胺水平和/或组氨酸脱羧酶的活性有明显的下降。种种证据提示，组胺在AD中可能有保护作用，但关于其具体机制的研究至今未有报道。同时一些报道也指出，组胺可以通过直接作用于NMDA受体上的多氨位点或间接激活H₁受体调节NMDA受体的功能，而NMDA受体在AD的病理过程中占有极其重要的位置，说明组胺在AD中可能是通过对NMDA受体调节而发挥作用的。肌肽主要存在于脑，骨骼肌等兴奋性组织，虽然距其发现有近百年的历史，且大量研究表明肌肽具有抗氧化，抗糖基化，抗AGE等大量生物学作用，但其具体的生物学作用机制至今仍不清楚。同时，有研究表明肌肽作为脑内一种重要的神经递质，对多种神经系统疾病如AD具有保护作用。因此，肌肽能否作为神经系统疾病的治疗药物及其具体作用机制的研究成为现近研究的热点。与此同时，一些研究也表明，肌肽能通过组胺酸代谢成组胺，提示肌肽-组胺酸-组胺通路可能是肌肽的保护作用重要机制之一。而本实验室在大鼠点燃癫痫及在PC12细胞上NMDA诱导神经兴奋性毒性研究中也发现，肌肽-组胺酸-组胺是肌肽保护作用占有十分重要的地位。因此本实验的主要目的是检测肌肽对Aβ42诱导的神经兴奋性损伤是否具有保护作用，肌肽-组胺酸-组胺通路是否参与其保护机制，谷氨酸含量，NMDA受体表达及功能在这一过程中是否受到影响，最终阐明肌肽保护作用的具体机制。我们将不同浓度的肌肽或组胺在加入Aβ42前18h加入培养液，观察肌肽或组胺对Aβ42诱导损伤是否具有保护；不同浓度的组胺受体拮抗剂或α-FMH在加入肌肽或组胺前10min加入培养液，观察他们对肌肽或组胺的作用是否具有拮抗作用。Aβ42加入细胞液并孵育24后，用MTT法测定PC12细胞的活性，HPLC测定谷氨酸含量。Clobenpropit在肌肽加入前10min加入培养液，肌肽或clobenpropit作用18h后，用细胞ELISA方法测定细胞膜上NMDA受体的表达量。我们发现Aβ42浓度依赖性诱导细胞损伤。组胺对Aβ42（5μM）诱导细胞损伤具有保护作用，且H₂受体拮抗剂zolantidine能部分逆转组胺的保护作用，但H₁受体拮抗剂diphenhydramine对组胺的保护没有影响。这一结果提示组胺对Aβ42诱导细胞损伤具有保护作用且可能是通过组胺H₂受体。我们同时也发现肌肽在10^-3、5×10^-3，10^-2M浓度时能显著改善Aβ42（5μM）作用24h引起的细胞损伤。肌肽在5mM时的保护作用能被H₃受体拮抗剂thioperamide和clobenpropit所逆转，但不能被H₁受体拮抗剂diphenhydramine，H₂受体拮抗剂zolantidine及HDC酶不可逆的抑制剂α-fluoromethylhistidine（α-FMH）所拮抗。以上结果表明肌肽-组胺酸-组胺通路没有参与肌肽对Aβ42诱导细胞损伤的保护作用机制，虽然我们同时发现组胺单独对Aβ42诱导细胞损伤的保护作用是通过组胺H₂受体。由于组胺H₃受体的特异性，其作为异身受体不仅能调节组胺的合成与释放，而且还能调节其它神经递质的释放。同时鉴于谷氨酸在AD中的重要作用，本实验进一步检测了谷氨酸在胞外的含量。我们发现Aβ42对胞外谷氨酸含量几乎没有影响，肌肽能显著降低胞外谷氨酸含量，而H₃受体拮抗剂clobenpropit和thioperamide能进一步降低由肌肽引起的胞外谷氨酸含量的降低。由于NMDA受体在AD病理过程中的重要地位，本实验进一步检测NMDA受体的表达变化。我们发现肌肽能引起NMDA受体在细胞膜上表达增加，而H₃受体拮抗剂clobenpropit能完全阻断这一现象。以上结果提示了肌肽对Aβ42诱导细胞损伤的保护作用一部分可能是通过对NMDA受体膜上表达调节而引起的。我们同时发现H₃受体拮抗剂thioperamide和clobenpropit也能显著改善Aβ42（5μM）作用24h引起的细胞损伤。Clobenpropit和thioperamide单独能增强胞外谷氨酸含量。同时clobenpropit也能引起NMDA受体在细胞膜上表达的增加。以上结果提示了H₃受体拮抗剂对Aβ42诱导细胞损伤的保护作用可能是通过对胞外谷氨酸含量及NMDA受体膜上表达调节的共同作用而引起的。最后我们还发现NMDA受体完全阻断剂MK801能阻断肌肽和clobenpropit引起的NMDA受体膜上表达增加。提示在本模型中NMDA受体本身对自身在膜上表达也有一定的影响。以上结果表明肌肽对Aβ42诱导细胞损伤的保护作用可能不是通过肌肽-组胺酸-组胺通路，而是通过对NMDA受体调节，谷氨酸及NMDA受体的共同作用而引起的。从而揭示了一个新的AD治疗方向。更多还原

【Abstract】 Alzheimer’s disease （AD） is a progressive neurodegenerative disorder characterized by global cognitive decline involving memory, orientation, judgment, and reasoning, and occurs with pathological hallmarks including widespread neuronal degeneration and neuritic plaques containing amyloid-β peptide （Aβ）. Evidence indicates that Aβ42 is essential for amyloid deposition in the parenchyma and in vessels. In addition to Aβ, enhancement of excitotoxicity by glutamate receptors especially NMDA receptors has also been implicated in the pathogenesis of AD. Among several hypotheses, the "amyloid hypothesis" has firmly taken root, but it currently lacks detail, and certain observations do not fit easily with the simplest version of the hypothesis. Recently, Snyder and colleagues found that Aβ reduces glutamatergic transmission and NMDA receptor-dependent LTP through intemalization of NMDA receptors, which suggests a potential therapeutic direction in AD. Now that therapies based on this idea serve as a basis for the treatment of AD, it is important to clarify these mechanisms.As an important neurotransmitter and neuromodulator, histamine has many actions in the central nervous system, affecting emotion, locomotor activity, stress behavior, learning and memory. Histamine also participates in pathological process of brain diseases. In AD, neurofibrillary tangles occur extensively in the tuberomammillary nucleus, where the cell bodies of histaminergic neurons are located, and histamine levels and/or histidine decarboxylase activity decrease in affected areas of the brain. But the exact role of histamine in AD remains unclear. In addition, several evidences show that histamine causes direct or indirect facilitation of the NMDA receptor, which is one of the most important receptors in AD, through its polyamine modulatory site or as a result of H₁ receptor activation. So regulation of NMDA receptor may be one action of histamine in AD.Carnosine （β-alanyl-histidine） is a major constituent of excitable tissues, brain and skeletal muscles, but more than a century after its isolation, the biological functions of this dipeptide remain enigmatic. Meanwhile, as a putative neurotransmitter in the brain, carnosine has a protective effect in many diseases such as AD, because of its abundant functions such as an anti-oxidant, in anti-glycation, anti-aging. In addition, as a reservoir for histidine, which is a precursor of histamine, there is an association between carnosine and histamine. Our previous work, demonstrating the protective effect of carnosine on kindled seizures in rats and neurotoxicity induced by NMDA, has revealed that the carnosine-histidine-histamine pathway may be one of the most important potential mechanisms in its protective effect. So it is of interest to examine whether the carnosine-histidine-histamine pathway is involved in the protective effect of carnosine in the case of neurotoxicity induced by Aβ42.The present study was designed to investigate whether histamine is involved in the protective effect of carnosine on Aβ42-induced impairment in differentiated PC12 cells. Various concentrations histamine receptor antagonists or α-fluoromethylhistidine （α-FMH） were added 10 min before carnosine or histamine; carnosine, histamine or the H₃ receptor antagonists were added 18 h before Aβ42. Cell viability was determined by 3-（4, 5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay 24 h after application of Aβ42. The amount of glutamate was quantified by HPLC 24 h after application of Aβ42. Clobenpropit was added 10 min before carnosine. Cell surface expression of NMDA receptors was quantified by cell ELISA assays 18 h after application of carnosine or clobenpropit.We found that Aβ42 caused a concentration-dependent reduction of viability in PC12 cells and pretreatment with histamine ameliorated Aβ42-induced impairment. This amelioration was significantly reversed by the H₂ receptor antagonist zolantidine, but not reversed by the H₁ receptor antagonist diphenhydramine. These results indicated that histamine attenuates Aβ42- induced neurotoxicity through H₂ receptor.Pretreatment with carnosine also ameliorated Aβ42-induced impairment. This amelioration was reversed by the H3 receptor antagonists thioperamide and clobenpropit, but neither the H₁ receptor antagonist diphenhydramine nor the H₂ receptor antagonist zolantidine. Further, α-FMH, an irreversible inhibitor of histidine decarboxylase, also had no effect. These results indicated that the carnosine-histidine-histamine pathway may not be involved in the protection of carnosine on Aβ42- induced neurotoxicity, although histamine alone attenuated Aβ42-induced neurotoxicity through H₂ receptor.Since the H₃ receptor, which is a heteroreceptor, not only provides negative feedback to restrict histamine synthesis and release, but also regulates the release of other transmitters such as glutamate and GABA, and also because of the importance of glutamate in AD, we examine the glutamate release, hoping its regulation could go with the cell survival. We found Aβ42 did not affect the glutamate release. In the presence of Aβ42, carnosine significantly decreased glutamate release, which was further reduced by clobenpropit and thioperamide. Then we examine whether NMDA receptor is involved in the protective effect of carnosine on Aβ42-induced neurotoxicity because of the important role of NMDA receptor in AD. And we found that carnosine increased the cell surface expression of NMDA receptor, which was abolished by clobenpropit. These results implicated that regulation of NMDA receptor trafficking may be involved in the protective effect of carnosine on Aβ42-inducedneurotoxicity.In addition, we also found that the H₃ receptor antagonists clobenpropit and thioperamide significantly ameliorated Aβ42-induced impairment. Both of them in the present of Aβ42 increased glutamate release. And clobenpropit increased the cell surface expression of NMDA receptor. It seems that regulation of both glutamate release and NMDA receptor trafficking are worked in the protective effect of carnosine on Aβ42-induced neurotoxicity.Finally, we found that MK801, a complete antagonist of NMDA receptor, abolished increasing cell surface expression of NMDA receptor afforded by carnosine or clobenpropit. These data indicated that NMDA receptor activity is necessary for surface expression of NMDA receptors under these conditions.In conclusion, the results indicate that the mechanism by which carnosine attenuates Aβ42- induced neurotoxicity is independent of the carnosine-histidine-histamine pathway, but may act through regulation of NMDA receptor trafficking, which could be abolished by the H₃ receptor antagonist. Although the mechanism by which histamine attenuates Aβ42- induced neurotoxicity is through H₂ receptors.更多还原