本文選題：α細(xì)辛 + 皮層腦電圖��； 參考：《大連醫(yī)科大學(xué)》2014年博士論文

【摘要】：癲癇是一種常見的神經(jīng)疾病,全世界有近1%的人口受此疾病的困擾�，F(xiàn)有的藥物治療手段仍然不能全面有效的阻止癲癇發(fā)作。在傳統(tǒng)醫(yī)學(xué)領(lǐng)域的研究有望為發(fā)展新的抗癲癇藥物和新的抗癲癇治療手段提供有前景的戰(zhàn)略方向。 中醫(yī)認(rèn)為石菖蒲具有豁痰開竅平癲癇的作用,治癲癇單用有效,α細(xì)辛是石菖蒲上述作用的主要活性成分。雖然α細(xì)辛已經(jīng)在大量研究中表現(xiàn)出抗癲癇作用,但這些研究大多集中于研究體外培養(yǎng)的細(xì)胞或是研究動(dòng)物癲癇發(fā)作的行為學(xué)表現(xiàn)。就我們掌握的資料,關(guān)于a細(xì)辛在體的電生理方面的療效評(píng)估尚存在空白。之前的研究已經(jīng)顯示α細(xì)辛在PTZ模型中表現(xiàn)出抗癲癇作用。本研究將首次嘗試采用在體的電生理監(jiān)測技術(shù)評(píng)價(jià)α細(xì)辛在PTZ誘導(dǎo)的大鼠癲癇模型中的抗癲癇作用效果。α細(xì)辛在體內(nèi)或體外研究中都表現(xiàn)出了抗氧化的作用,α細(xì)辛所具備的還原和抗氧化性質(zhì)可能是其在臨床傳統(tǒng)抗癲癇治療中有益的基礎(chǔ)�；诖它c(diǎn),本研究還將探索α細(xì)辛表現(xiàn)出的抗癲癇作用是否有N0通路的參與。 N0在癲癇發(fā)生發(fā)展的細(xì)胞病理過程中起著非常重要而又復(fù)雜的調(diào)節(jié)作用,己成為一個(gè)研究熱點(diǎn)。關(guān)于N0在癲癇中的作用,已經(jīng)進(jìn)行了大量體外和體內(nèi)的研究,不論在動(dòng)物實(shí)驗(yàn)還是臨床研究的結(jié)果中均可以看到N0參與了癲癇的發(fā)生發(fā)展,但是得出的結(jié)論仍然是矛盾的,促癲癇和抗癲癇作用均有報(bào)告。N0由精氨酸在一氧化氮合成酶(Nitrieoxidesthase, NOS)的作用下產(chǎn)生,NOS有三種不同亞型nNOS、iNOS及eNOS。人們對(duì)于N0與癲癇關(guān)系的研究,從最初檢測N0水平的變化,逐漸轉(zhuǎn)移至具體研究哪一種NOS引起的NO變化更有意義�，F(xiàn)有的研究顯示檢測到的N0和NOS水平缺乏一致性表現(xiàn),產(chǎn)生不同的實(shí)驗(yàn)結(jié)果可能與動(dòng)物模型的選擇,實(shí)驗(yàn)設(shè)計(jì)方案,用藥的途徑,發(fā)作形式不同等有關(guān),因此仍需作進(jìn)一步詳盡的研究。 本研究使用四種NO調(diào)節(jié)劑,包括非選擇性一氧化氮合酶(NOS)抑制劑NG-硝基-L-精氨酸甲酯(L-NAME,60mg/kg)、神經(jīng)元型一氧化氮合酶(nNOS)抑制劑7-硝基吲唑(7-NI,40mg/kg)、誘導(dǎo)型一氧化氮合酶(iNOS)抑制劑氨基胍(AG,100mg/kg)和N0前體L-精氨酸(L-ARG,500mg/kg)�？紤]到N0通路可能既參與癲癇模型形成又參與α細(xì)辛抗癲癇作用,故而在藥物干預(yù)和造模的不同階段施與N0調(diào)節(jié)劑,分析不同情況、不同階段三種NOS所起作用,最后綜合分析得出N0與戊四氮癲癇模型及α細(xì)辛抗癲癇作用的關(guān)系。 實(shí)驗(yàn)設(shè)計(jì)分四部分：1、比較兩個(gè)劑量的PTZ(50和60mg/kg)誘發(fā)的癲癇樣腦電活動(dòng),須滿足皮層腦電記錄2小時(shí),根據(jù)比較結(jié)果選擇合適的造模劑量用于α細(xì)辛的療效評(píng)估；選定造模劑量后,在PTZ造模后20分鐘給予α細(xì)辛,觀察四個(gè)不同劑量的α細(xì)辛(20,40,60和80mg/kg)對(duì)PTZ誘發(fā)癲癇放電的作用效果,根據(jù)比較結(jié)果確定α細(xì)辛的劑量。2、在PTZ注入5分鐘后,分別給予四種NO調(diào)節(jié)劑,α細(xì)辛在PTZ給藥20分鐘后腹腔注入,研究NO調(diào)節(jié)劑對(duì)α細(xì)辛抗癲癇作用的影響。3、α細(xì)辛給藥之前15分鐘系統(tǒng)應(yīng)用N0調(diào)節(jié)劑,在α細(xì)辛給藥20分鐘后腹腔注入PTZ,研究NO調(diào)節(jié)劑對(duì)α細(xì)辛預(yù)防癲癇作用的影響。4、造模前15分鐘給予四種NO調(diào)節(jié)劑,PTZ腹腔注入5分鐘后給予α細(xì)辛,研究NO調(diào)節(jié)劑對(duì)癲癇模型及a細(xì)辛抗癲癇作用的影響。 結(jié)果：1、兩個(gè)劑量的PTZ在造模后存活時(shí)間、強(qiáng)直陣攣發(fā)作潛伏期、累計(jì)強(qiáng)直陣攣持續(xù)時(shí)間、單次最長強(qiáng)直陣攣持續(xù)時(shí)間和累計(jì)強(qiáng)直陣攣發(fā)作次數(shù)的比較中均無顯著差異。在陣攣潛伏期的比較中,兩組間有顯著性差異(p=O.001),在PTZ50mg/kg組,陣攣潛伏期為109.3±41.4秒,在PTZ60mg/kg組,陣攣潛伏期為49.0±22.8秒。根據(jù)上述分析結(jié)果,在α細(xì)辛的研究中采用PTZ50mg/kg。α細(xì)辛在60和80mg/kg兩個(gè)劑量時(shí)能顯著減少陣攣放電頻率,60mg/kgα細(xì)辛的抗癲癇作用出現(xiàn)在給藥50分鐘后且持續(xù)不足10分鐘。80mg/kg α細(xì)辛的抗癲癇作用出現(xiàn)在給藥20分鐘后并可持續(xù)達(dá)50分鐘。由此決定,在后續(xù)實(shí)驗(yàn)中α細(xì)辛采用80mg/kg。2、單獨(dú)給予L-NAME或7-NI對(duì)于PTZ模型中的陣攣樣電活動(dòng)和間期放電均無影響,而單獨(dú)使用L-ARG卻能在給藥后最初5分鐘內(nèi)明顯減少間期放電的頻率,AG單獨(dú)給予能明顯增加間期放電的頻率。與α細(xì)辛組相比,L-ARG+α細(xì)辛組中抗陣攣?zhàn)饔锰崆?0分鐘出現(xiàn)。在α細(xì)辛注射15分鐘之前給予L-NAME或7-NI使得a細(xì)辛的抗陣攣?zhàn)饔孟�。L-NAME不僅逆轉(zhuǎn)了a細(xì)辛的抗癲癇作用,甚至使得間期頻率增加：AG也逆轉(zhuǎn)了α細(xì)辛的抗陣攣?zhàn)饔?但AG對(duì)模型中間期放電的不良作用也被α細(xì)辛的作用抵消。3、在PTZ造模20分鐘前給予α細(xì)辛能明顯抑制陣攣發(fā)作的頻率,持續(xù)可達(dá)30分鐘。α細(xì)辛之前給予L-NAME或7-NI抑制了α細(xì)辛的抗癲癇作用,并且在應(yīng)用L-NAME時(shí)觀察到陣攣頻率顯著增加,這種促癲癇的效果出現(xiàn)在PTZ注入70分鐘后。L-ARG也抑制了α細(xì)辛的抗癲癇作用,并且呈現(xiàn)出雙相作用,在造模初期增加陣攣發(fā)作頻率而在后期卻減少間期放電頻率。AG對(duì)α細(xì)辛的作用無影響。4、7-NI和AG都能明顯增加PTZ誘發(fā)的陣攣發(fā)作,并且7-NI的作用要早于AG的作用。L-NAME和L-ARG對(duì)PTZ誘發(fā)的陣攣樣放電和間期放電均無明顯作用。PTZ后5分鐘給予α細(xì)辛能顯著減少陣攣的平均發(fā)作頻率而對(duì)間期放電無作用,這種抗癲癇效果出現(xiàn)在PTZ注入50分鐘后并持續(xù)30分鐘。α細(xì)辛能夠逆轉(zhuǎn)7-NI和AG在PTZ模型中的促驚厥作用。在L-NAME和L-ARG存在的條件下,α細(xì)辛的抗癲癇作用被抵消。 結(jié)論：在PTZ模型中,三種NOS均被激活,但不同NOS合成的N0發(fā)揮的作用不同,eNOS合成的NO發(fā)揮促癲癇作用,nNOS和iNOS合成的N0具有抗癲癇作用,這似乎說明N0并未直接參與癲癇發(fā)生過程,其本身不具有確定的促癲癇或是抗癲癇性能,N0通過作用于不同的受體或位點(diǎn)而啟動(dòng)不同的機(jī)制,最終表現(xiàn)出相似或相反的作用效果。實(shí)驗(yàn)結(jié)果提示不同NOS在PTZ造模后不同階段被活化,造模5分鐘內(nèi)eNOS迅速被激活,造模5分鐘后nNOS和iNOS逐漸被活化,且nNOS的活化早于iNOS出現(xiàn),但iNOS的活化持續(xù)時(shí)間更長。在癲癇中各種不同亞型的NOS相互制約、相互協(xié)調(diào),維持著使機(jī)體最小損傷的微妙平衡。 在PTZ造模5分鐘后給予α細(xì)辛?xí)r,α細(xì)辛可能是通過抑制eNOS合成N0而起到抗癲癇作用；在PTZ造模20分鐘后給予α細(xì)辛?xí)r,nNOS及其產(chǎn)生的NO參與了a細(xì)辛在大鼠PTZ癲癇模型中的抗癲癇作用,α細(xì)辛可能是通過誘導(dǎo)nNOS合成NO而起到抗癲癇作用：iNOS未參與α細(xì)辛抗癲癇的作用機(jī)制。在α細(xì)辛的抗癲癇作用中,同時(shí)有促NO合成和抑制N0合成的作用。α細(xì)辛的抗癲癇作用涉及兩種不同NOS/NO機(jī)制,且未發(fā)現(xiàn)兩種機(jī)制同時(shí)起作用的證據(jù),有理由相信α細(xì)辛誘導(dǎo)nNOS或是抑制eNOS跟不同的實(shí)驗(yàn)設(shè)計(jì)方案有關(guān),這提示NOS的活動(dòng)可能存在程序性啟動(dòng)和相互調(diào)節(jié)的情況。
[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.

【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.1

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