【作者】 朱媛媛；

【导师】 陈忠；

【作者基本信息】 浙江大学 ， 药理学， 2007， 硕士

【摘要】 癫痫是一种严重的慢性中枢神经系统疾病，其特征为反复发作的大脑神经元异常放电，导致暂时性大脑功能失调，临床表现为运动、感觉、意识、植物神经和精神等不同障碍。据WHO提供的数字，目前在世界有约5000万癫痫患者，我国约有800万。癫痫的确切发病机制目前尚未完全阐明。神经元高度的兴奋性、过度的同步化活动、神经元兴奋和抑制之间平衡失调可能是癫痫发作最根本的原因。目前癫痫治疗主要为药物治疗。现有抗癫痫药对脑部无严重病损的患者，尤其是发作较少或发作间歇期脑电图完全正常者，大约80％病人发作可得到控制，但仍有约20％的病人属药物难治性癫痫患者，药物治疗效果较差。而且，长期服用抗癫痫药物如苯妥因钠、卡马西平、丙戊酸钠等还常常伴随着精神、神经、内脏和皮肤系统的不良反应。因此，迫切需要开发新型的安全且有效的抗癫痫药物或者抗癫痫辅助用药。组胺是一种脑内重要的神经递质或神经调质，与睡眠—觉醒周期、食欲、神经内分泌以及学习和记忆等密切相关。已有的研究显示，脑内组胺与癫痫存在着密切联系，可能参与癫痫发作敏感性的调节。本实验组最近的研究发现增加脑内组胺含量（腹腔内注射组氨酸或脑室内注射组胺H₃受体拮抗剂clobenpropit）能明显延缓戊四唑诱导的癫痫形成过程；组氨酸脱羧酶或组胺H₁受体基因敲除小鼠的戊四唑诱导癫痫形成过程较野生型明显加快。而且内源性组胺参与调节癫痫发作的后保护作用过程。以上结果均提示脑内组胺是一种内源性的抗癫痫物质，因此组胺能神经系统的相关药物有望成为新的抗癫痫药物或抗癫痫辅助药物。Carnosine（β-丙氨酰-L-组氨酸）是一种天然二肌肽，是体内组氨酸的重要来源，并可进一步代谢转化为组胺，carnosine可以从外周进入血脑屏障，同时还具有酸碱缓冲、抗氧化、抗衰老、金属鳌合等作用，本实验组最近的实验发现carnosine腹腔内注射可以升高杏仁核的carnosine、组氨酸和组胺含量，显著抑制大鼠杏仁核电点燃癫痫的发作，并且其保护机制可能涉及两条不同的（直接和间接）通路：一是直接激活脑内组胺H₁受体并引起类似于组胺的反应；二是在脑内代谢转变为组胺后再间接激活脑内组胺H₁受体。在此基础上，本课题进一步观察了组氨酸前体物质carnosine在组氨酸脱羧酶基因敲除及其相应野生型小鼠中对戊四唑癫痫的作用，还探讨了carnosine在小鼠杏仁核快速电点燃癫痫模型中的作用。以期通过两种不同的癫痫模型，深入阐明carnosine对癫痫的保护作用。我们的研究发现，carnosine可抑制小鼠戊四唑癫痫的发作过程，其作用机制是通过脑内carnosine-histidine-histamine这条代谢途径完成的。Carnosine也可以抑制小鼠杏仁核快速电点燃模型的发作级别并且增强其发作后保护作用。结果表明，carnosine和组胺一样，是脑内一种重要的内源性抗癫痫物质，并可能与人类癫痫发作后保护作用密切相关。因而，carnosine很有可能在将来可以作为临床治疗癫痫的辅助药物。总之，本研究进一步充实和深化了现有关于carnosine与癫痫关系的研究，并为今后组胺类抗癫痫药物的开发尤其是carnosine的临床应用提供必要的理论与实验基础。1．Carnosine通过组胺能机制对小鼠戊四唑癫痫的保护作用目的：研究carnosine对小鼠戊四唑癫痫发作的作用，并探讨其可能机制。方法：组氨酸脱羧酶基因敲除及相应野生型小鼠腹腔内注射不同剂量carnosine，观察对小鼠急性戊四唑癫痫发作行为及皮层电活动的影响。结果：Carnosine能够显著抑制野生型小鼠癫痫发作的级别、延长肌阵挛发作潜时、升高肌阵挛发作和强直阈值，而且保护作用呈剂量依赖性和时间相关性。而在基因敲除型小鼠中carnosine没有明显的保护作用。我们还发现，在野生型小鼠腹腔内注射组氨酸脱羧酶不可逆性抑制剂α-fluoromethylhistidine（10 mg／kg和20 mg／kg，i.p.），可完全逆转carnosine对小鼠戊四唑癫痫发作的保护作用。此外，carnosine还可显著降低野生型小鼠的皮层电活动。结论：Carnosine可抑制小鼠戊四唑癫痫的发作过程，其作用机制是通过脑内carnosine-histidine-histamine这条代谢途径完成的。本实验也提示carnosine可能是脑内内源性的抗癫痫物质，并且有望成为一种新型的临床抗癫痫药物或抗癫痫辅助药物。2．Carnosine对小鼠杏仁核快速电点燃癫痫的作用目的：研究carnosine对小鼠杏仁核快速电点燃癫痫的作用。方法：小鼠杏仁核快速电点燃，刺激40次，间隔5 min，腹腔内注射500 mg／kg的carnosine，观察对小鼠杏仁核快速电点燃癫痫发作的作用，记录小鼠发作的级别和发作后放电时间。结果：Carnosine可以显著抑制杏仁核快速电点燃癫痫的平均发作级别、大发作的次数，并显著增强癫痫后保护作用。结论：Carnosine对小鼠杏仁核快速电点燃癫痫具有保护作用，其具体作用机制还有待进一步深入探讨。更多还原

【Abstract】 Epilepsy is a serious chronic neurological disease, which is characterized by recurrent abnormal discharge of cerebral neurons and transient disfunction of brain. The clinical symptoms include a variety of abnormities in motor, sense, conscious state, vegetative nervous and psychology. According to WHO, 50 million persons worldwide, and 8 million in China have suffered from the diseases. The exact pathogenesis of epilepsy remains largely unclear. Excessive excitability, over-synchronized discharges and the imbalance between excitation and inhibition of cerebral neurons may contribute to seizures. With the currently available antiepileptic drugs, the disease in about 80% patients who have no serious injuries in the brain, especially those with less frequency of seizures and normal interictal EEGs, are well controlled. By contrast, about 20% epileptic patients can’t get effective therapies using those drugs. This epilepsy is called drug-resistance or intractable epilepsy. Moreover, long application of those antiepileptic drugs often results in some adverse responses in psychological, neurological, splanchnic and skin system. Thus, developing new safe and effective antiepileptic drugs is urgently expected.Histamine, a biogenic amine, is an important neurotransmitter or neuromodulator in the mammalian central nervous system. Brain histaminergic system seems to be involved in various physiological and behavioral functions including sleep-wake cycles, appetite control, neuroendocrine, learning and memory through its specific receptors. The studies from others and our laboratory have alsoreported that brain histamine may be involved in the epileptogenesis and regulate seizure susceptibility as an endogenous anticonvulsant substance. Recently, our group further found that increasing brain histamine levels （e.g. i.p. injection of histidine; i.c.v. injection of histamine or H₃ receptor antagonist clobenpropit） markedly delays pentylenetetrazol-induced seizure development, while knock out of histamine H₁ receptor or histidine decarboxylase genes accelerates pentylenetetrazol-induced seizure development in mice. These evidences indicate that histaminergic system exerts an antiepileptic effect in the brain. Moreover, it is proposed that certain histaminergic drags may be used as new anticonvulsants in clinic in future.Carnosine （β-alanyl-1-histidine） is a naturally occurring dipeptide, it serves as a reservoir for histidine, which is a precursor of histamine, and can easily enter the central nervous system from the periphery. It has many putative roles such as anti-inflammatory agent, free radical scavenger, and protein glycosylation inhibitor. Our recent studies have showed that it can significantly inhibit amygdaloid kindled seizures in rats, and it simultaneously induces a significant increase in carnosine, histidine and histamine levels in the amygdala. The mechanisms of the protective effect of carnosine may involve at least two different （direct and indirect） pathways: pathway I, directly acting at histamine H₁-receptors and provoking a histamine-like response; pathway II, indirectly activating histamine Hi-receptors after metabolic transformation into histamine. In the present study, we used both histidine decarboxylase-deficient （HDC-KO） and wild-type （WT） mice to further elucidate the possible role of carnosine in pentylenetetrazol （PTZ）-induced seizures, which is an animal model of human absence epilepsy and myoclonic, generalized tonic-clonic seizures. We also derived a fast electrical amygdala kindling model in WT mice to test the effects of carnosine. We expect to elucidate the relations between carnosine and epilepsy in the two different epilepsy models. It has been found that carnosine can protect against PTZ-induced seizures and its action is mainly through the carnosine-histidine-histamine metabolic pathway. Additionally, in the fast electrical amygdala kindling model, carnosine inhibit the seizure grades and induced a significant enhance of postictal seizure protection in WT mice. In conclusion, ourstudy provides more evidences that carnosine has a significant anticonvulsant effect on seizures in mice, we develops the knowledge about the relations between carnosine and epilepsy, provides necessary theories for the development of histaminergic antiepileptic drugs, especially the medical use of carnosine in future.1. Mechanism of carnosine on PTZ-induced seizures in miceIn the present study, we used both histidine decarboxylase-deficient （HDC-KO） mice and wild-type （WT） mice to elucidate the possible role of carnosine in pentylenetetrazol （PTZ）-induced seizures, In the acute PTZ challenge study, PTZ （75 mg/kg） was injected intraperitoneally （i.p.） to induce seizures. Carnosine （200, 500 or 1000 mg/kg, i.p.） significantly decreased seizure stage, and prolonged the latency for myoclonic jerks in WT mice in a dose-dependent manner. The effects of carnosine （500 mg/kg） were time-dependent and reached a peak at 1 h. However, it had no significant effect on HDC-KO mice. Carnosine （500 mg/kg） also significantly elevated the thresholds in WT mice but not HDC-KO mice following intravenous （tail vein） administration of PTZ. We also found that α-fluoromethylhistidine substantially reversed the protective effects of carnosine in WT mice. In addition, carnosine pretreatment reduced the cortical EEG activity induced by PTZ （75 mg/kg, i.p.）. These results indicate that carnosine can protect against PTZ-induced seizures and its action is mainly through the carnosine—histidine-histamine metabolic pathway. This suggests that carnosine may be an endogenous anticonvulsant factor in the brain and may be used as a new antiepileptic drug in the future.2. The effect of carnosine on fast electrical amygdala kindling in miceTo test the effect of carnosine in fast electrical amygdala kindling in mice, we derived a fast electrical amygdala kindling model （40 stimulations, 5 min intervals）. We found that an injection of carnosine （500 mg/kg） significantly decreased seizure stage, afterdischarge duration, generalized seizure, and induced a significant enhance of postictal seizure protection in WT mice. The study suggests that carnosine can protect against the fast electrical amygdala kindling. It may be an endogenous anticonvulsant factor in the brain and could be used as a new antiepileptic drug in the future.更多还原