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  • 學位論文

苯妥英鈉和多酚對神經傳遞的影響

Effects of Phenytoin and Polyphenols on Neurotransmission

指導教授 : 潘建源

摘要


癲癇是一種神經過度興奮性症狀,而苯妥英鈉是一種常見的抗癲癇藥物,主要的作用是抑制鈉離子通道的活性。然而過去研究,對於苯妥英鈉 (phenytoin) 如何透過神經突觸的傳遞,來調節緩解癲癇症狀的機制仍不甚了解。在此研究中,使用初級培養的大鼠胚胎神經細胞進行實驗,發現苯妥英鈉以濃度依賴性的方式,抑制內流鈉離子的電流,其半抑制濃度(IC50)約為16.8 M。在處理苯妥英鈉濃度100 M時,對鈉電流的抑制作用與給予的刺激頻率有使用依賴性的正比關係;且顯著降低神經細胞的動作電位頻率。利用雷射光裂解技術於局部釋放興奮性神經傳導物質--麩胺酸 (Glutamate),並測量受刺激的目標神經及其相鄰連接的神經細胞胞內鈣離子濃度的變化,以探討神經傳導的變化。在給予強度光裂解刺激下,苯妥英鈉能顯著抑制目標神經和鄰近細胞被誘發的細胞內鈣離子濃度的上升,但此明顯的抑制作用在給予中度光裂解刺激時,則會被減弱。進一步以第一型γ-氨基丁酸 (GABA)受體拮抗劑印防己毒素 (picrotoxin) 處理神經細胞,以增加自發興奮性突觸後電流 (sEPSC) 的發生。而苯妥英鈉對> 100 pA的自發興奮性突觸後電流,有較為顯著的抑制效果,但對< 100 pA的突觸後電流,則僅有輕微影響。在人類胚胎腎細胞 (HEK293) 中,表現第一型電壓調控依賴性鈉離子通道 (Nav1.1),苯妥英鈉能抑制通道電流,並延長了鈉離子通道從失活態恢復到可再開啟的時間,但不影響通道激活和失活的特性。這些結果顯示,苯妥英鈉藉由延長鈉離子通道恢復所需時間,限制了鈉離子通道的可用性,以降低神經傳遞的效率,以舒緩因癲癇發作造成神經過度興奮的高頻神經活動。 綠茶萃取物對於人體具有許多健康的益處,其中表兒茶素沒食子酸鹽 ((-)-Epicatechin-3-gallate,ECG) 及表沒食子兒茶素沒食子酸酯 (Epigallocatechin-3-gallate,EGCG) 是綠茶多酚的主要化合物;一般多針對其抗氧化能力、對神經退化性疾病和癌症的相關保護作用進行討論,但是對於抗焦慮及神經鎮靜作用的機制仍不甚了解。在本研究中,前處理一小時綠茶多酚的初級培養大鼠胚胎神經細胞,其鈉離子內流電流、麩胺酸受體電流及動作電位頻率都會受到抑制。分別刺激離子型或代謝型麩胺酸亞受體,所誘發的細胞內鈣離子變化,也都會受到綠茶多酚前處理的抑制,因此對整體的神經活性產生鎮靜的作用。

並列摘要


Phenytoin is an effective antiepileptic drug (AED) that inhibits Na+ channel activities; however, how phenytoin modulates synaptic transmission to soothe epileptic symptoms is not clear. To characterize the effects of phenytoin on neurotransmission, we used cultured embryonic cortical neurons to study the electrophysical properties before and after phenytoin treatment. Phenytoin inhibited the inward Na+ current in a dose-dependent manner with an IC50 of 16.8 μM, and at 100 μM, the inhibitory effect of phenytoin on the Na+ current was proportional to the stimulation frequency applied. In cultured neurons, phenytoin significantly decreased the action potential (AP) firing rate. To study the effect of phenytoin in neurotransmission, we measured the intracellular calcium concentration ([Ca2+]i) responses from stimulated target neurons and their neighboring neurons. Phenytoin significantly suppressed the [Ca2+]i responses evoked by strong stimulations in the target and neighboring neurons, and exerted a decreased inhibitory effect under moderate stimulation. Picrotoxin, a GABAA receptor antagonist, enhanced the recorded spontaneous excitatory postsynaptic current (sEPSC) activities. After picrotoxin-induced enhancement, phenytoin had a more pronounced effect on the suppression of the hyper-exciting sEPSC (> 100 pA), but it only mildly inhibited the averaged EPSC. Furthermore, phenytoin inhibited the Nav1.1 currents expressed in HEK293T cells and prolonged the recovery time from inactivation without affecting the voltage-dependent activation and inactivation properties. Our results demonstrate that phenytoin suppresses the efficacy of neurotransmission especially for the high-frequency stimulation by lengthening the Na+ channel recovery time from inactivation which limits Na+ channel availability and can potentially alleviate epileptic activity. The (−)-epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG) are the major compounds of green tea polyphenols. It has been extensively studied for their neuroprotective effects on neurodegenerative diseases and cancer, but the effects on neurotransmission are unclear. In this study, we used calcium imaging and patch-clamp techniques to determine the effects of ECG and EGCG on regulating the neurotransmission among cultured embryonic cortical neurons. The results indicated that ECG and EGCG at 10 μM inhibit the voltage-gated Na+ currents, reduce the N-methyl-D-aspartate (NMDA)-evoked currents, and furthermore decrease the AP firing rate. By monitoring the [Ca2+]i, both ECG and EGCG inhibited the [Ca2+]i responses via activating the α-amino-3-hydroxy-5-methyl-4-isoxazole-proprionate (AMPA) receptors or the metabotropic glutamate receptors. Overall, ECG and EGCG suppress the neurological activities and synaptic transmission resulting in sedation.

參考文獻


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