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標題: | 苦藍盤萃取物在神經/精神疾病動物模式之行為研究 Behavioral studies of KLP-1 in animal models of neurological and psychiatric disorders |
作者: | Yu-Hsiang Liao 廖御翔 |
指導教授: | 邱麗珠(Lih-Chu Chiou) |
關鍵字: | 苦藍盤,高活動力,刻版化攀爬行為,動物模式,小腦,妥瑞氏症,精神分裂症, KLP-1,hyperlocomotion,stereotypy climbing,cerebellum,tourette syndrome,schizophrenia, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | 我們在一則臨床案例報告發現,一位患有頑固性抽動的小女孩,發現在其服用苦藍盤汁液的萃取物1小時後,她的抽動症狀獲得有效緩解。因此,我們從苦藍盤葉的乙醇萃取物中,鑑定並分離出活性成分,為一種純化合物,KLP-1。利用在動物上模擬妥瑞氏症(Tourette syndrome)及精神病分裂症的疾病模式,我們發現KLP-1能顯著地改善由甲基安非他命(methamphetamine)及NMDA通道阻斷劑所引起的前脈衝抑制不良(PPI);而且也能改善在小鼠上慢性給予天使塵(phencyclindine)來誘導社交行為退縮的現象,為在動物上模擬精神分裂症的負向症狀模式。結合學研究還表明KLP-1在GABAA受體(GABAARs)及兒茶酚臨位甲基轉移酶(COMT)有著微莫耳濃度(μM)的親和力。先前的研究還指出,KLP-1是一個GABAARs的正向調控者,其中包括在小腦顆粒細胞中獨有表現的α6次單元(α6GABAARs)。
在這項研究中,我們進而對KLP-1 進行了全面的行為研究,影響各種行為模型小鼠的運動功能,其中包括甲基安非他命(促多巴胺釋放激素)或阿樸嗎啡(多巴胺第2受體促進劑)所引起的活動力增加現象,以及後者所誘使的刻版化攀爬行為。我們還研究了抑制COMT是否對KLP-1的效果有貢獻。要做到這一點,使用α6GABAAR的正向調控者Ro15-4513作為正向對照以及不影響α6GABAAR的diazepam作為負向對照,同時,一個非競爭α6GABAAR的拮抗劑furosemide也被使用。此外,COMT的選擇性抑制劑OR-486被用來作為COMT抑制效果的正向對照。 第一部分的動物行為模式實驗,我們發現不論是腹腔注射或小腦埋管給藥KLP-1,皆會修復甲基安非他命(促多巴胺分泌素)所造成活動力增加的現象。且小腦內注射α6GABAA接受器的活化劑Ro15-4513,也可修復甲基安非他命所造成高活動力的現象。另外,相較於KLP-1與Ro15-4513,diazepam無法作用於α6GABAAR。在小腦內注射diazepam後,並無法修復甲基安非他命所造成活動力增加的現象。另外也使用對於α6GABAA接受器有高選擇性的阻斷劑,furosemide,其可有效的抑制KLP-1與Ro15-4513所造成修復甲基安非他命活動力增加的效果。另外,我們也用了典型的COMT抑制劑OR-486,其並不對甲基安非他命所造成高活動力的現象造成影響。這些結果顯示KLP-1修復甲基安非他命所造成活動力增加的現象歸因於其在小腦顆粒細胞的α6GABAA接受器的正向調控效果,並非歸因於COMT抑制的效果。 第二部分的動物行為模式實驗,我們發現KLP-1是不對阿樸嗎啡(多巴胺第2受體促進劑)所造成活動力增加及刻板化的攀爬行為有影響。而市售的抗精神疾病藥物haloperidol,能顯著地改善阿樸嗎啡所誘導的刻板化攀爬行為。相對於KLP-1,diazepam在修復阿樸嗎啡所造成活動力增加情形的劑量下,也觀察到有嚴重的鎮靜、抗焦慮(在升高十字迷津測試下)和活動力削弱的情形。另外,在KLP-1能有效的改善甲基安非他命所誘導高活動力的劑量下,則是發現不影響自發性探索行為、肢體運動的協調性(在滾輪試驗測試下)、也不造成任何鎮靜或抗焦慮的作用。所以KLP-1的作用形式是不同於diazepam的,例如在KLP-1扮演正向調控效果及在diazepam俱敏感性GABAA受體α1-3,5次單元(被指出與抗焦慮的效果相關),結果指出對KLP-1能修復高活動力行為是不具有貢獻的。 總結,在KLP-1有效地修復由大量多巴胺所誘導活動力增加的劑量下,同時是沒有任何副作用的,例如破壞自發性探索行為、肢體運動協調性、運動力削弱或鎮靜。而其修復高活動力的現象可能藉由在小腦顆粒細胞中正向調控GABAA受體,增加小腦的調控,進而抑制紋狀體(striatal)的多巴胺系統。KLP-1的這些作用,加上其能修復前脈衝抑制不良的缺陷以及社交行為退縮的特質,顯示KLP-1很有潛力發展成治療精神分裂症及抽動症狀的藥物。特別值得一提KLP-1在改善社交行為退縮現象上,與精神分裂症病患的負向症狀是很類似的,負向症狀是現今抗精神病藥物無法有效治療,而亟待解決的臨床問題。在KLP-1進行基因毒性試驗(GLP TOX test)後,能進而支持藥物發展安全性的問題。 In a pediatric patient with intractable motor tics, we previously found that her motor tics was subsided within 1 hour after taking the leaf extract of Clerodendron inerme (CI) Gaertn. Therefore, we have tried to identify the active constituent(s) from the ethanol extract of CI leaves and isolated a pure compound, KLP-1. In animal models mimicking Tourette syndrome (TS), a spectrum of tic disorders, and schizophrenia, we found that KLP-1 significantly rescued the disruption of prepulse inhibition (PPI) induced by methamphetamine and NMDA channel blockers, as well as improved the social withdrawal like behaviors in mice induced by chronical treatement with phencyclindine, an animal model mimicking the negative syndrome of schizophrenia. Binding studies also demonstrated KLP-1 with micromolar affinity to GABAA receptors (GABAARs) and catechol-O-methyltransferase (COMT). A previous study also demonstrated that KLP-1 is a positive modulator of GABAARs, including the subtype of α6 subunit-containing GABAARs (α6GABAARs), which is exclusively expressed in the cerebellar granule cells. In this study, we further conducted a comprehensive study examining effects of KLP-1 on motor functions of mice by various behavioral models, including the hyperlocomotion induced by methamphetamine (a dopamine releaser) or by apomorphine (a D2 receptor agonist), and stereotypy climbing behaviors induced by apomorphine. We also examined the relative contribution of activation of α6GABAARs and COMT inhibition in the effects of KLP-1. To serve the purpose, we used Ro15-4513, a positive modulator of α6GABAAR as a positive controls, and diazepam that does not affect α6GABAAR as a negative control, and furosemide, a non-competitive α6GABAAR antagonist. Besides, OR-486, a COMT selective inhibitor, was used as a positive control for COMT inhibition. First, we found that either intraperitoneal (i.p.) or intra-cerebellar (i.cb.) microinjection of KLP-1 significantly normalized the hyperlocomotion induced by methamphetamine, a dopamine releaser. Ro15-4513 given by i.cb. microinjection also effectively normalized methamphetamine-induced hyperlocomotion. However, i.cb. diazepam, which is insensitive to α6GABAAR, did not affect methamphetamine-induced hyperlocomotion. Moreover, i.cb. furosemide, which selectively antagonizes α6GABAARs, significantly antagonized the normalizing effect induced by i.cb. KLP-1 and Ro15-4513 on methamphetamine-induced hyperlocomotion. On the other hand, OR-486, a typical COMT inhibitor, did not affect methamphetamine-induced hyperlocomotion. These results suggest that the inhibitory effect of KLP-1 on methamphetamine-induced hyperlocomotion is mediated by positively modulating α6GABAARs in cerebellar granule cells, but not by COMT inhibition. Second, we found that KLP-1 (i.p.) did not affect hyperlocomotion and stereotypy climbing behaviors induced by apomorphine, a D2 receptor agonist. Haloperidol, a commercially available antipsychotic, significantly reduced apomorphine-induced stereotypy climbing behaviors. Unlike KLP-1, diazepam reduced apomorphine-induced hyperlocomotion. This effect was observed at doses of diazepam with severe sedative, anxiolytic (revealed by the elevated plus maze test) and motor weakness activity. In contrast, KLP-1 at doses effectively normalized methamphetamine-induced hyperolocomotion did not affect spontaneous motor activity and motor coordination in the rotarod test, but did not display any significant sedation or anxiolytic activity. Therefore, the action mode of KLP-1 is different from that of diazepam, i.e. the positive modulatory effect of KLP-1 on diazepam-sensitive GABAARs, the α1-3,5 subunit-containing GABAARs, dose not contribute to KLP-1-induced normalization of hyperlocomotion. In summary, KLP-1 at the doses effectively normalizing hyperlocomotion induced by hyper-dopaminergic tone did not have side effects, such as impairment of spontaneous motor activity, motor coordination, causing motor weakeness or sedation. Its reduction of hyperlocomotion might be attributed to a positive modulation of GABAARs in cerebellar granule cells, leading to enhancing cerebellar inhibitory control on the striatal dopaminergic activity. In combination with its effectiveness in rescuing PPI disruptions and in improving social withdrawal behaviors, KLP-1 is a potential compound for treating schizophrenia or tic disorders. Characteristically, KLP-1-induced improvement of social withdrwal behaviors, which resembles the negative syndrome of schizophrenia patients, is especially noteworthy since schizophrenia negative syndrome is resistant to current antipsychotic drugs and is an unmet medical need in clinical practice. The absence of genotoxicity of KLP-1 in a GLP TOX test further supports its safety issue in terms of drug development. |
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