【作者】 苏静；

【导师】 姜长斌；

【作者基本信息】 大连医科大学 ， 内科学， 2014， 博士

【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。更多还原

【Abstract】 Epilepsy is a common neurological disorder, affecting almost1%of the population and current pharmacological therapies remain insufficient to completely prevent epileptic seizures. Research in traditional medicine represents a promising strategy for developing new antiepileptic drugs and therapeutic approaches.Acori graminei Rhizoma (AGR), the dry rhizomes of Acorus gramineus Solander (Araceae), has been traditionally used in oriental prescriptions to treat epilepsy for hundreds of years. a-Asarone was shown to be the main active anticonvulsive component in AGR Although many studies have shown that a-asarone exerts antiepileptic effects, they are nearly all performed in cell cultures or animal’s attack manifestations. To our knowledge, no study has evaluated the electrophysiological effects of a-asarone in vivo. Previous studies indicate that a-asarone has antiepileptic effects in pentylenetetrazol(PTZ)-induced seizure models. Our findings represent the first attempt at studying the effects of a-asarone with in vivo electrophysiology in PTZ-induced epilepsy in conscious rats. a-Asarone have shown antioxidant effects in many studies both in vivo and vitro. The antiepileptic effect of a-asarone may attribute to its reduction and antioxidant properties. So, the aim of present study was to identify the involvement of nitric oxide (NO) in the antiepileptic effects of a-asarone on PTZ-induced epileptiform activity in rats.Epilepsy is one of the diseases in which NO is regarded as important pathogenetic factor in the mechanisms underlying seizure induction and progression. Whether in the results of animal experiments or clinical studies, it can be seen that NO involved in the development of epilepsy. The role of NO in epilepsy has been examined in a number of in vivo and in vitro studies, however, the obtained results are still contradictory, reporting both pro-and anti-convulsant properties of NO. NO is formed from arginine by the action of three different nitric oxide synthase (NOS) isozymes, two calcium-dependent forms, neuronal (nNOS) and endothelial (eNOS) and one calcium-independent inducible (iNOS). The relationship between NO and epilepsy has been a hotspot. People studied the level of NO in the earlier years, and in the recent years more research focus on detection of changes of NOS. Existing evidences have shown a lack of consistency in detection of NO and NOS level. It is believed that epileptic model, experimental designments, route of administration, attack forms and so on may all affect the results of detection. So there is need for further detailed study.The effects of systemic administration of nitric oxide synthase (NOS) inhibitors, non-selective NG-nitro-L-arginine methyl ester (L-NAME,60mg/kg, i.p.), selective neuronal nitric oxide synthase (nNOS) inhibitor,7-nitroindazole (7-NI,40mg/kg, i.p.), inducible nitric oxide synthase (iNOS) inhibitor, aminoguanidine (AG,100mg/kg, i.p.) and NO precursor, L-arginine (ARG,500mg/kg, i.p.) on the effects of α-asarone were investigated. Considering that NO pathway may be involved in the formation of both models of epilepsy and antiepileptic effect of α-asarone, we designed three experimental programs and NO regulators were administered at different stage. All the results may be helpful to explain the relationship between NO and both PTZ and α-asarone.Experimental programs:1. To compare the characteristics of PTZ-induced epileptiform activity with two doses (50and60mg/kg) of PTZ and choose a suitable dose of PTZ to meet the observation requirements for ECoG analysis (appearance of clonic attack and discharge, appearance of interictal discharge and no less than2h for survival time).To investigate the electrophysiological effects of a-asarone with four different doses (20,40,60and80mg/kg), administered20minutes after PTZ injection.2. An effective dose of L-ARG, AG, L-NAME and7-NI was intraperitoneally administered5min after PTZ application. And animals received the effective dose of a-asarone15min after L-ARG, L-NAME, AG, and7-NI administration.3. Animals received L-NAME,7-NI, AG and LAG15min before a-asarone application. PTZ was administered intraperitoneally20min after a-asarone application.4. An effective dose of L-NAME,7-NI, L-ARG or AG was intraperitoneally administered15min before PTZ application and animals received an effective dose of a-asarone5min after PTZ administration.Results:1. There were no significant differences between the two groups with respect to the following parameters:survival time (observation time=2h), cumulative duration of tonic/clonic seizures, longest duration of a single tonic/clonic seizure, attack times of tonic/clonic seizures and latency of first tonic/clonic seizure. There was a significant difference in the latency of the first clonic seizure between the two groups (p＝0.001). The latencies were109.3±41.4sec (PTZ50mg/kg group) and49.0±22.8sec (PTZ60mg/kg group), respectively. We decided to use50mg/kg PTZ in the following experiment. a-Asarone significantly decreased the clonic frequency at doses of60and80mg/kg. The antiepileptic effects of the60mg/kg dose lasted no more than10min. The antiepileptic effects of the80mg/kg dose lasted50min. We decided to use80mg/kg a-asarone in the following experiment.2. Administration of L-NAME or7-NI5min after PTZ injection did not influence either the clonic activity or interictal discharge in both the frequency and amplitude. Administration of L-ARG significant decreased the mean frequency of interictal discharge in the first5min. The mean frequency of interictal discharge was significantly increased after AG administration, and the effect lasted for20min. a-Asarone significantly decreased the mean frequency of clonic activity and the significant effects appeared20min after a-asarone injection and lasted for50min. The administration of L-NAME reversed the anticlonic activity of a-asarone and induced significant increasement of interictal discharge. Administration of7-NI reversed the anticlonic effect of a-asarone without affecting clonic amplitude. The anticlonic effects appeared10min earlier in the L-ARG+a-asarone group compared with a-asarone group alone and lasted for60min. The administration of AG reversed the beneficial effect ofa-asarone on clonic activity, whereas the detrimental effect of AG on intericatal discharge was inhibited in aminoguanidine+a-asarone group.3. a-Asarone significantly decreased the mean frequency of clonic activity and the significant effects lasted for30min. L-NAME reversed the anticlonic activity of a-asarone and a significant increasement of clonic activity was induced70min after PTZ injection in L-NAME+a-asarone+PTZ group. Administration of7-N1also reversed the anticlonic activity of α-asarone. In the AG+α-asarone+PTZ group, similarly the mean frequency of clonic activity was decreased significantly compared to that of clonic activity in a-asarone+PTZ group. In the L-ARG+α-asarone+PTZ group, the mean frequency of clonic activity was temporally increased in the10min after PTZ injection and the anticlonic activity of a-asarone was reversed, whereas the mean frequency of interictal discharge significantly decreased in the50min after PTZ injection and lasted for20min compared with PTZ group.4. Administration of7-NI and AG significantly increased the mean frequency of clonic activity. The significant effect of7-NI occurred earlier than that of AG. Administration of L-NAME and L-ARG did not influence either clonic activity or interictal discharge. a-Asarone significantly decreased the mean frequency of clonic activity and the significant effects lasted for30min. The pro-convulsant effect of7-NI was reversed in7-NI+PTZ+α-asarone group. The pro-convulsant effect of AG was reversed in AG+PTZ+α-asarone group. In the case of using L-NAME, the antiepileptic effect of a-asarone was reversed. In the case of using L-ARG the antiepileptic effect of α-asarone was also reversed.Conclusion:Our results confirm that α-asarone, at the80mg/kg dose, significantly decreased the mean frequency of clonic epileptiform activity. All the three NOS isoforms were activated in PTZ-induced epilepsy, and NOS play an important key role in the genesis and spreading of epileptiform hyperactivity but not NO. Different NOS exhibits different effect and NO is only the mediator in realization of NOS’s effects. NO itself has neither pro-nor anti-epileptic property. NO produced by iNOS and nNOS exhibited protective effect in PTZ model. NO produced by eNOS exhibited pro-epileptic effect in PTZ model. NO may start different pathway by acting on different receptors or sites, ultimately showed similar or opposite effect. The results indicated that NOSs were activated at different stage of PTZ modeling. eNOS was activated within5min, while iNOS and nNOS were activated later, and nNOS was activated earlier than nNOS, but the action of iNOS lasted longer than that of nNOS.We could comment that both nNOS/NO and eNOS/NO pathways involved in the anticonvulsant effect of a-asarone. It can be concluded that a-asarone promoted NO synthesis and exerted anticonvulsant effects by acting on nNOS. On the other hand, a-asarone inhibited NO synthesis and exerted anticonvulsant effects by acting on eNOS. Both promoting and inhibiting synthesis of NO involved in the antiepileptic mechanism of a-asarone. It showed that eNOS and nNOS were influenced at different conditions, and this may due to the experimental programs. While iNOS was unrelated to the inhibition of a-asarone on PTZ induced epileptiform activity.更多还原