大鼠戊四氮癫痫模型中一氧化氮的作用及其与α细辛抗癫痫作用的相关性研究

发布时间：2018-05-15 01:13

本文选题：α细辛 + 皮层脑电图　；参考：《大连医科大学》2014年博士论文

【摘要】：癫痫是一种常见的神经疾病,全世界有近1%的人口受此疾病的困扰。现有的药物治疗手段仍然不能全面有效的阻止癫痫发作。在传统医学领域的研究有望为发展新的抗癫痫药物和新的抗癫痫治疗手段提供有前景的战略方向。中医认为石菖蒲具有豁痰开窍平癫痫的作用,治癫痫单用有效,α细辛是石菖蒲上述作用的主要活性成分。虽然α细辛已经在大量研究中表现出抗癫痫作用,但这些研究大多集中于研究体外培养的细胞或是研究动物癫痫发作的行为学表现。就我们掌握的资料,关于a细辛在体的电生理方面的疗效评估尚存在空白。之前的研究已经显示α细辛在PTZ模型中表现出抗癫痫作用。本研究将首次尝试采用在体的电生理监测技术评价α细辛在PTZ诱导的大鼠癫痫模型中的抗癫痫作用效果。α细辛在体内或体外研究中都表现出了抗氧化的作用,α细辛所具备的还原和抗氧化性质可能是其在临床传统抗癫痫治疗中有益的基础。基于此点,本研究还将探索α细辛表现出的抗癫痫作用是否有N0通路的参与。 N0在癫痫发生发展的细胞病理过程中起着非常重要而又复杂的调节作用,己成为一个研究热点。关于N0在癫痫中的作用,已经进行了大量体外和体内的研究,不论在动物实验还是临床研究的结果中均可以看到N0参与了癫痫的发生发展,但是得出的结论仍然是矛盾的,促癫痫和抗癫痫作用均有报告。N0由精氨酸在一氧化氮合成酶(Nitrieoxidesthase, NOS)的作用下产生,NOS有三种不同亚型nNOS、iNOS及eNOS。人们对于N0与癫痫关系的研究,从最初检测N0水平的变化,逐渐转移至具体研究哪一种NOS引起的NO变化更有意义。现有的研究显示检测到的N0和NOS水平缺乏一致性表现,产生不同的实验结果可能与动物模型的选择,实验设计方案,用药的途径,发作形式不同等有关,因此仍需作进一步详尽的研究。本研究使用四种NO调节剂,包括非选择性一氧化氮合酶(NOS)抑制剂NG-硝基-L-精氨酸甲酯(L-NAME,60mg/kg)、神经元型一氧化氮合酶(nNOS)抑制剂7-硝基吲唑(7-NI,40mg/kg)、诱导型一氧化氮合酶(iNOS)抑制剂氨基胍(AG,100mg/kg)和N0前体L-精氨酸(L-ARG,500mg/kg)。考虑到N0通路可能既参与癫痫模型形成又参与α细辛抗癫痫作用,故而在药物干预和造模的不同阶段施与N0调节剂,分析不同情况、不同阶段三种NOS所起作用,最后综合分析得出N0与戊四氮癫痫模型及α细辛抗癫痫作用的关系。实验设计分四部分：1、比较两个剂量的PTZ(50和60mg/kg)诱发的癫痫样脑电活动,须满足皮层脑电记录2小时,根据比较结果选择合适的造模剂量用于α细辛的疗效评估；选定造模剂量后,在PTZ造模后20分钟给予α细辛,观察四个不同剂量的α细辛(20,40,60和80mg/kg)对PTZ诱发癫痫放电的作用效果,根据比较结果确定α细辛的剂量。2、在PTZ注入5分钟后,分别给予四种NO调节剂,α细辛在PTZ给药20分钟后腹腔注入,研究NO调节剂对α细辛抗癫痫作用的影响。3、α细辛给药之前15分钟系统应用N0调节剂,在α细辛给药20分钟后腹腔注入PTZ,研究NO调节剂对α细辛预防癫痫作用的影响。4、造模前15分钟给予四种NO调节剂,PTZ腹腔注入5分钟后给予α细辛,研究NO调节剂对癫痫模型及a细辛抗癫痫作用的影响。结果：1、两个剂量的PTZ在造模后存活时间、强直阵挛发作潜伏期、累计强直阵挛持续时间、单次最长强直阵挛持续时间和累计强直阵挛发作次数的比较中均无显著差异。在阵挛潜伏期的比较中,两组间有显著性差异(p=O.001),在PTZ50mg/kg组,阵挛潜伏期为109.3±41.4秒,在PTZ60mg/kg组,阵挛潜伏期为49.0±22.8秒。根据上述分析结果,在α细辛的研究中采用PTZ50mg/kg。α细辛在60和80mg/kg两个剂量时能显著减少阵挛放电频率,60mg/kgα细辛的抗癫痫作用出现在给药50分钟后且持续不足10分钟。80mg/kg α细辛的抗癫痫作用出现在给药20分钟后并可持续达50分钟。由此决定,在后续实验中α细辛采用80mg/kg。2、单独给予L-NAME或7-NI对于PTZ模型中的阵挛样电活动和间期放电均无影响,而单独使用L-ARG却能在给药后最初5分钟内明显减少间期放电的频率,AG单独给予能明显增加间期放电的频率。与α细辛组相比,L-ARG+α细辛组中抗阵挛作用提前10分钟出现。在α细辛注射15分钟之前给予L-NAME或7-NI使得a细辛的抗阵挛作用消失。L-NAME不仅逆转了a细辛的抗癫痫作用,甚至使得间期频率增加：AG也逆转了α细辛的抗阵挛作用,但AG对模型中间期放电的不良作用也被α细辛的作用抵消。3、在PTZ造模20分钟前给予α细辛能明显抑制阵挛发作的频率,持续可达30分钟。α细辛之前给予L-NAME或7-NI抑制了α细辛的抗癫痫作用,并且在应用L-NAME时观察到阵挛频率显著增加,这种促癫痫的效果出现在PTZ注入70分钟后。L-ARG也抑制了α细辛的抗癫痫作用,并且呈现出双相作用,在造模初期增加阵挛发作频率而在后期却减少间期放电频率。AG对α细辛的作用无影响。4、7-NI和AG都能明显增加PTZ诱发的阵挛发作,并且7-NI的作用要早于AG的作用。L-NAME和L-ARG对PTZ诱发的阵挛样放电和间期放电均无明显作用。PTZ后5分钟给予α细辛能显著减少阵挛的平均发作频率而对间期放电无作用,这种抗癫痫效果出现在PTZ注入50分钟后并持续30分钟。α细辛能够逆转7-NI和AG在PTZ模型中的促惊厥作用。在L-NAME和L-ARG存在的条件下,α细辛的抗癫痫作用被抵消。结论：在PTZ模型中,三种NOS均被激活,但不同NOS合成的N0发挥的作用不同,eNOS合成的NO发挥促癫痫作用,nNOS和iNOS合成的N0具有抗癫痫作用,这似乎说明N0并未直接参与癫痫发生过程,其本身不具有确定的促癫痫或是抗癫痫性能,N0通过作用于不同的受体或位点而启动不同的机制,最终表现出相似或相反的作用效果。实验结果提示不同NOS在PTZ造模后不同阶段被活化,造模5分钟内eNOS迅速被激活,造模5分钟后nNOS和iNOS逐渐被活化,且nNOS的活化早于iNOS出现,但iNOS的活化持续时间更长。在癫痫中各种不同亚型的NOS相互制约、相互协调,维持着使机体最小损伤的微妙平衡。在PTZ造模5分钟后给予α细辛时,α细辛可能是通过抑制eNOS合成N0而起到抗癫痫作用；在PTZ造模20分钟后给予α细辛时,nNOS及其产生的NO参与了a细辛在大鼠PTZ癫痫模型中的抗癫痫作用,α细辛可能是通过诱导nNOS合成NO而起到抗癫痫作用：iNOS未参与α细辛抗癫痫的作用机制。在α细辛的抗癫痫作用中,同时有促NO合成和抑制N0合成的作用。α细辛的抗癫痫作用涉及两种不同NOS/NO机制,且未发现两种机制同时起作用的证据,有理由相信α细辛诱导nNOS或是抑制eNOS跟不同的实验设计方案有关,这提示NOS的活动可能存在程序性启动和相互调节的情况。
[Abstract]:Epilepsy is a common neurological disease, and nearly 1% of the world's population is plagued by the disease. The existing drug therapy is still unable to effectively prevent seizures. The research in the traditional medicine field is expected to provide a promising strategic direction for the development of new antiepileptic drugs and new antiepileptic hand segments.
Acorus calamus has the effect of opening the phlegm to open the orifices and treating epilepsy, and it is effective in treating epilepsy alone. Alpha asari is the main active component of the above effects of Acorus calamus. Although alpha asari has shown antiepileptic effects in a large number of studies, most of these studies focus on the study of cells in vitro or the behavioral study of epileptic seizures in animals. Now. As far as we know, there is still a gap in the evaluation of the electrophysiological effects of a Asarum in the body. Previous studies have shown that alpha asari showed antiepileptic effect in the PTZ model. This study will be the first attempt to evaluate the antiepileptic activity of alpha asari in the rat model of PTZ induced epilepsy by using the electrophysiological monitoring technique in vivo. The effect of alpha Asarum has shown antioxidation in both in vivo and in vitro studies, and the reductive and antioxidant properties of alpha Asarum may be a useful basis for its clinical traditional antiepileptic therapy. Based on this, this study will also explore the involvement of the N0 pathway in the antiepileptic use of alpha Asarum.
N0 plays a very important and complex regulatory role in the pathological process of the development of epilepsy, and it has become a research hotspot. The role of N0 in epilepsy has been studied in vitro and in vivo. Both in animal experiments and in the results of clinical research, we can see that N0 is involved in the development of epilepsy. The conclusion is still contradictory. Epilepsy and antiepileptic effects are reported in the report that.N0 is produced by arginine under the action of Nitrieoxidesthase (NOS). There are three different subtypes of nNOS, iNOS and eNOS. in the study of the relationship between N0 and epilepsy. From the initial detection of the change of N0 level, it is gradually transferred to a specific study. Which NOS induced NO changes are more meaningful. Existing studies show that the detected N0 and NOS levels are not consistent, and the results may be related to the selection of animal models, the design scheme, the way of drug use, the different form of the attack, and so on, so further detailed study is needed.
Four kinds of NO regulators, including non selective nitric oxide synthase (NOS) inhibitor NG- nitro -L- arginine methyl ester (L-NAME, 60mg/kg), neuronal nitric oxide synthase (nNOS) inhibitor 7- nitro indazole (7-NI, 40mg/kg), inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (iNOS) and precursor arginine, were used in this study. /kg). Considering that the N0 pathway may not only participate in the formation of epileptic model but also participate in the antiepileptic effect of alpha asarone, it is applied with N0 regulator at different stages of drug intervention and modeling, analyzing the roles of three kinds of NOS in different situations and different stages. Finally, the relationship between the epileptic model of N0 and amyl four nitrogen and the antiepileptic effect of alpha Asarum is analyzed.
The experimental design is divided into four parts: 1, comparing two doses of PTZ (50 and 60mg/kg) induced epileptic EEG activity, which must meet the cortical EEG for 2 hours. According to the comparison results, the appropriate dosage of the model dose is selected for the evaluation of the effect of alpha asari; after the dosage of the model, alpha Asarum is given 20 minutes after the model of PTZ, and four different doses of alpha are observed. The effect of Asarum (20,40,60 and 80mg/kg) on PTZ induced epileptic discharge was determined according to the comparison results, and the dose.2 of alpha Asarum was determined. After 5 minutes of PTZ injection, four NO regulators were given respectively. Alpha asari was injected intraperitoneally after PTZ Administration for 20 minutes, and the effect of NO regulator on the antiepileptic effect of alpha Asarum was studied, and the system should be 15 minutes before the administration of alpha Asarum. The effect of NO regulator on the effect of alpha Asarum on the prevention of epilepsy was studied with N0 regulator 20 minutes after the administration of alpha Asarum. Four kinds of NO modifiers were given 15 minutes before the model, and 5 minutes after PTZ intraperitoneal injection was given to the alpha Asarum. The effects of NO conditioner on epilepsy model and the antiepileptic effect of a Asarum were studied.
Results: 1, the survival time of the two doses of PTZ, the latent period of tonic clonic seizure, the cumulative tonic clonic duration, the single most long tonic clonic duration and the number of cumulative tonic clonic seizures were not significantly different. There were significant differences between the two groups in the clononic latency (p=O.001), in the group PTZ50mg/kg, The clonus incubation period was 109.3 + 41.4 seconds, in group PTZ60mg/kg, the clonus incubation period was 49 + 22.8 seconds. According to the above analysis, the clonic discharge frequency was significantly reduced by PTZ50mg/kg. alpha Asarum at two doses of 60 and 80mg/kg, and the antiepileptic effect of 60mg/kg alpha Asarum occurred after 50 minutes and lasted less than 10 minutes. The antiepileptic effect of clock.80mg/kg alpha Asarum lasted for 20 minutes and lasted for up to 50 minutes. Thus, it was determined that alpha asari was used in subsequent experiments with 80mg/kg.2, and L-NAME or 7-NI alone had no effect on the clonus like electrical activity and interval discharge in the PTZ model, but the use of L-ARG alone was significantly reduced in the first 5 minutes after the administration. Compared with alpha asari group, the resistance to clonoclonus in the L-ARG+ alpha Asarum group was 10 minutes earlier than that in the alpha Asarum group. The anti clonoclonic effect of a Asarum to L-NAME or 7-NI before the alpha Asarum injection was 10 minutes earlier than that in the alpha Asarum group. The anti clononus effect of a Asarum.L-NAME did not only reverse the antiepileptic effect of a asarum, and even made it possible to reverse the anti epileptic effect of a Asarum. AG also reversed the anti clonus effect of alpha asari, but the adverse effect of AG on the middle stage of the model was also counteracted by alpha asari, which could inhibit the frequency of clonic seizures for up to 30 minutes before 20 minutes of PTZ model. L-NAME or 7-NI inhibited the antiepileptic effect of alpha asari before alpha Asarum. A significant increase in the clononic frequency was observed at the time of application of L-NAME. The effect of the epilepsy showed that.L-ARG also inhibited the antiepileptic effect of alpha Asarum after 70 minutes of PTZ injection, and showed a biphasic effect, which increased the frequency of clonic seizures at the early stage of the model and decreased the effect of the interval discharge frequency.AG on alpha Asarum at the later stage. Both.4,7-NI and AG can significantly increase the clonic seizure induced by PTZ, and the effect of 7-NI is earlier than that of AG,.L-NAME and L-ARG have no significant effect on the average seizure frequency of the clonus induced by PTZ induced clonic discharge and interval discharge, 5 minutes after.PTZ, but no effect on interphase discharge. Now PTZ is injected 50 minutes and lasts for 30 minutes. Alpha Asarum can reverse the effect of 7-NI and AG on the convulsion in the PTZ model. In the presence of L-NAME and L-ARG, the antiepileptic effect of alpha Asarum is counteracted.
Conclusion: in the PTZ model, three kinds of NOS were activated, but different NOS synthesized N0 played different roles, eNOS synthesized NO played an epileptic effect. NNOS and iNOS synthesized N0 had antiepileptic effect. This seemed to indicate that N0 did not directly participate in the process of epilepsy, which itself did not have certain epilepsy or anti epileptic properties, N0 through the action. Different mechanisms were initiated at different receptors or sites, and the results showed similar or opposite effects. The results showed that different NOS were activated at different stages after PTZ modeling, eNOS was activated rapidly in 5 minutes, and nNOS and iNOS were activated gradually after 5 minutes of modeling, and the activation of nNOS was earlier than iNOS, but the activation of iNOS was sustained. The NOS of various subtypes in epilepsy are mutually restricted and coordinated, maintaining a delicate balance that minimize the damage of the body.
When alpha asari was given 5 minutes after the PTZ model, alpha asari might play an antiepileptic effect by inhibiting eNOS to synthesize N0. When PTZ was given to alpha asari after 20 minutes, nNOS and its produced NO were involved in the antiepileptic effect of a Asarum in the rat PTZ epileptic model, and alpha Asarum may be antiepileptic by inducing nNOS to synthesize NO. INOS does not participate in the mechanism of alpha Asarum antiepileptic action. In the anti epileptic effect of alpha asarum, it also has the effect of promoting NO synthesis and inhibiting the synthesis of N0. The antiepileptic effect of alpha Asarum involves two different NOS/NO mechanisms, and there is no evidence of the simultaneous action of the two mechanisms, and there is reason to believe that alpha Asarum induces nNOS or inhibits eNOS following different realities. The design plan is relevant, which indicates that there may be procedural startup and mutual adjustment in NOS activities.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1

【参考文献】