【作者】 沈耀；

【导师】 陈忠；

【作者基本信息】 浙江大学 ， 药理学， 2009， 博士

【摘要】 组胺是一种体内广泛存在的神经递质和神经调质。它在许多中枢神经活动中起着重要的调节作用,如觉醒-睡眠、痛觉、神经内分泌调节、饮水摄食调节、体温调节、运动以及学习记忆等。同时有研究表明组胺可能参与了脑缺血的过程,它对缺血过程中由于NMDA受体过度激活而引起的神经元损伤具有保护作用。大鼠脑缺血后给予组胺的前体物质组氨酸,能够减轻大鼠纹状体部位神经元的损伤。相反,α-fluoromethylhistidine（α-FMH）,一种不可逆的组氨酸脱羧酶的抑制剂,能显著性地增加大鼠海马CA1区锥体神经元的坏死。课题组的前期研究发现组胺在原代培养的皮层神经元上具有明显的抗兴奋性损伤的神经保护作用。然而,组胺本身是一种炎症介质,可能会引起一些炎症反应;同时它也不能通透血脑屏障,这些都限制了组胺在临床上成药的可能性。因此,寻找一些既具有组胺样的神经保护功能,又能避免组胺的不良作用的药物,对临床上防治脑缺血具有重要的意义。肌肽（β-丙氨酸-L-组氨酸）在一个世纪前就被前苏联学者Gulewitsch和Amiradzibi从牛肉提取物中检测到。肌肽在生物体内的分布比较广泛,如骨骼肌、脑、心脏、肾脏等。肌肽具有广泛的生物学作用,如抗氧化、清除自由基、调节生理pH值、抗炎症、抗糖基化,在嗅球区还可能起着神经递质的功能。但是迄今为止,肌肽在脑内的生理功能还是不很清楚。另一方面,有报道肌肽能在肌肽酶和组氨酸脱羧酶的作用下代谢生成组胺,同时也能通过组胺的H₁受体起到一个神经保护作用。鉴于肌肽和组胺之间的这种关系,我们首次提出在脑内是否存在肌肽-组氨酸-组胺这样一条代谢通路?肌肽能否通过这样一条代谢通路从而代替组胺,成为一个在临床上治疗脑缺血的有效药物?所以本课题探讨了肌肽对NMDA诱导的分化的PC12细胞兴奋性损伤以及组氨酸脱羧酶基因敲除小鼠的永久性局灶性脑缺血的作用,试图阐明肌肽的保护作用及其与组胺的相关性。1肌肽对NMDA诱导的分化的PC12细胞兴奋性损伤的保护作用及其机制MTT实验结果显示肌肽预处理能显著降低由NMDA诱导PC12细胞的死亡率;Hoechst 33342和PI的核双染实验同时也显示了肌肽能明显降低NMDA诱导的PC12细胞的凋亡和坏死。肌肽预处理能显著降低胞外谷氨酸的含量,同时能提高PC12细胞内组氨酸脱羧酶的活力,显著提高胞内胞外肌肽、组氨酸、组胺的含量。进一步研究发现,肌肽的这种保护作用能被对组氨酸脱羧酶的选择性且不可逆的抑制剂α-FMH所逆转;同时这种保护作用也能够被组胺H₁受体拮抗剂吡拉明（pyrilamine）和组胺H₃受体拮抗剂thioperamide所逆转。Thioperamide逆转肌肽的保护作用可能与它能逆转肌肽对谷氨酸的抑制作用相关。因此,研究提示在PC12细胞内存在肌肽-组氨酸-组胺这条代谢通路,肌肽对NMDA诱导的PC12细胞损伤的保护机制主要是通过肌肽-组氨酸-组胺这条代谢通路和H₁/H₃受体介导的,此外还可能与其抑制谷氨酸水平的作用有关。本研究显示肌肽可能是一种潜在的抗兴奋性损伤的药物。2肌肽对小鼠永久性局灶性脑缺血的保护作用及其机制在NGF诱导分化的PC12细胞兴奋性损伤模型上我们发现了肌肽的保护作用通过了肌肽-组氨酸-组胺这条代谢通路。那么,肌肽及这条代谢通路在整体动物的脑缺血模型中是否也有作用?为了进一步验证肌肽及其代谢通路的作用,我们利用组氨酸脱羧酶基因敲除（缺乏组胺的合成酶,即阻断组氨酸-组胺代谢通路,长期缺乏组胺）小鼠及它的野生型小鼠来观察肌肽在永久性局灶性脑缺血中的作用及这条代谢通路在其中的作用。于术前30 min腹腔内给与肌肽750 mg/kg均能使组氨酸脱羧酶基因敲除小鼠和它的野生型小鼠的神经功能得到改善,梗死体积减少;肌肽的这种保护作用在两种小鼠上没有统计学差异。肌肽能使小鼠脑内谷氨酸的水平降低,并能维持小鼠缺血后脑内谷氨酸转运体GLT-1的表达。在原代培养的皮层星形胶质细胞进行体外缺糖缺氧（oxygen-glucose deprivation,OGD）模拟在体缺血的实验模型中同样证实了肌肽对谷氨酸释放（或再摄取）及其GLT-1的表达具有调节作用。另外,在星形胶质细胞上肌肽能抑制OGD导致的线粒体内活性氧自由基（ROS）的大量生成和线粒体膜电势的丧失。肌肽还能降低鱼藤酮诱导的星形胶质细胞线粒体内ROS生成,抑制GLT-1的表达下调。我们的结果表明无论组氨酸脱羧酶基因敲除与否都不影响肌肽对小鼠永久性局灶性脑缺血的保护作用。因此,肌肽对小鼠在体脑缺血的保护作用可能不是通过组胺,而是与它能有效地调节星形胶质细胞上的谷氨酸转运体GLT-1和降低谷氨酸水平的作用相关。总之,本课题发现了肌肽-组氨酸-组胺这条代谢通路的存在,并且在分化的PC12细胞的兴奋性损伤模型上起到保护作用。肌肽对小鼠的在体永久性局灶性脑缺血具有神经保护功能,但是没有涉及到肌肽-组氨酸-组胺这条代谢通路,而是通过了有效的调节星形胶质细胞上GLT-1的表达,降低缺血后脑内的兴奋性损伤,从而起到脑保护作用。更多还原

【Abstract】 Histamine is one of the most widely distributed neurotransmitter or neuromodulator in the central nervous system and controls a variety of neurobiological functions and behavioral responses including sleep-wake cycle,nociception,water consumption,food,motor activity and learning and memory.Histamine is involved in ischemic pathogenesis,and has a protective effect on the delayed neuronal death mediated by N-methyl-D-aspartate（NMDA） receptors.Postischemic loading with histidine,a precursor of histamine,decreases the amount of neuronal damage in the rat striatum.Histamine depletion withα-fluoromethylhistidine（α-FMH）,an irreversible inhibitor of histidine decarboxylase（HDC）,significantly increases the number of necrotic pyramidal cells in hippocampal CA1 region in rats subjected to cerebral ischemia.Histamine also protects against NMDA-induced necrosis in cultured cortical neurons.However,histamine itself cannot cross the blood-brain barrier and it is involved in brain inflammation.Therefore,specific histamine related compounds which have histamine-like effect but without the side-effect may have the potential clinical uses in preventing and treating cerebral ischemia.Carnosine（β-alanyl-L-histidine）,first identified by Gulewitsch and Amiradzibi from Liebig’s meat extract nearly a century ago and it occurs in innervated tissues including the animal and human brain and can easily enter the central nervous system from the periphery.There are many theories about its biological functions,such as anti-inflammatory agent,free radical scavenger,and protein glycosylation inhibitor,and may serve as a neurotransmitter in the olfactory bulb.However,so far,the physiological functions of carnosine in the brain remain obscure,and a unifying concept has not yet emerged.On the other hand,previous studies have suggested that carnosine can be metabolically transformed into histamine by carnosinase and histidine decarboxylase enzyme that exist in the brain and exerts its protective action through histamine H₁ receptor.Given the relationship between carnosine and histamine,carnosine is proposed as a new histaminergic drug that can replace histamine and avoid inflammation in clinical therapeutics.Therefore,our present studies concentrate on the effects of carnosine and the carnosine-histidine-histamine metabolic pathway on NMDA induced neurotoxicity in differentiated PC12 cells and focal cerebral ischemia in histidine decarboxylase knock-out（HDC-KO） mice.1 Neuroprotective effect of carnosine on NMDA-induced neurotoxicity in differentiated PC12 cellsPretreatment with carnosine increased the viability and decreased the number of apoptotic and necrotic cells in NMDA induced injury in differentiated PC12 cells measured by MTT and Hoechst 33342 and propidium iodide（PI） double staining assays. Carnosine also can reduce the extracellular glutamate level and increase HDC activity and the intracellular and extracellular contents of carnosine,histidine and histamine detected by high-performance liquid chromatography（HPLC）.The protection by carnosine was reversed byα-fluoromethylhistidine,a selective and irreversible inhibitor of histidine decarboxylase（HDC）.Pyrilamine and thioperamide,selective central histamine H₁ and H₃ antagonists also significantly reversed the protection of carnosine. Further,the inhibition of glutamate release by carnosine was reversed by thioperamide. Therefore,the protective mechanism of carnosine may not only involve the carnosine-histidine-histamine pathway,but also H1/H3 receptors and the effective inhibition of glutamate release.This study indicates that carnosine may be an endogenous protective factor and calls for its further study as a new antiexcitotoxic agent.2 Role of carnosine in permanent focal cerebral ischemia in HDC-KO mice Since camosine can protect against excitotoxicity in differentiated PC12 cells through a histaminergic pathway,in this study,we used HDC-KO and the corresponding wild type（WT） mice to elucidate whether histaminergic or other mechanisms are involved in.the effects of carnosine on permanent middle cerebral artery occlusion（pMCAO）.The results showed that carnosine significantly improved neurological function and decreased infarct size both in HDC-KO and its WT mice,to the same extent.Carnosine decreased the glutamate levels,and preserved the expression of glutamate transporter-1（GLT-1） but not glutamate/aspartate transporter（GLAST） in astrocytes exposed to ischemia in vivo and in vitro.It suppressed the dissipation of mitochondrial membrane potentil（△Ψm） and generation of mitochondrial ROS induced by OGD in astrocytes.Furthermore,carnosine also decreased the mitochondrial ROS level and reversed the decrease of GLT-1 induced by rotenone,an inhibitor of mitochondrial complex I.Our study suggests that carnosine is neuroprotective in pMCAO in HDC-KO and WT mice and its mechanism of action may not be mediated by the histaminergic pathway,but by the effective regulation the expression of GLT-1 in astrocytes due to improving the moitochondrial function.In conclusion,our study demonstrates that there exists carnosine-histidine-histamine pathway in differentiated PC12 cells and it plays a protective roles in NMDA induced excitotoxicity.Carnosine exerts a neuroprotective effect on pMCAO induced injuries in HDC-KO and its WT mice and its action may not involve the histaminergic pathway,but through reducing glutamate excitotoxicity by reversing the GLT-1 decrease in astrocytes.更多还原