以電生理方式檢測抗痙攣藥物效果：羅格列酮和天麻苷對NMDA受體促進的體內及體外痙攣模式

Electrophysiological Approaches for Screening Anti-convulsants: Rosiglitazone and Gastrodin For The N-methyl-D-Aspartate Receptor-facilitated Seizures in vivo and in vitro

王緒斌(Shi-Bing Wong)

指導教授 ： 閔明源

國立臺灣大學/生命科學院/生命科學系/博士(2016年)

https://doi.org/10.6342/NTU201610065

摘要

癲癇在孩童時期是一種常見的疾病，導因於腦部皮質的過度放電。不管是局部或泛發性的癲癇，反覆及長時間的發作都曾被證實會對個體的認知能力、情緒控制、學習能力會產生不好的影響。腦皮質的過度放電與皮質神經細胞的突觸傳遞相關。興奮性神經傳導物質的增強，如麩胺酸(glutamate)，抑制性神經傳導物質的減弱，如γ-氨基丁酸(GABA)，均可能造成皮質神經細胞同時間過度興奮而產生抽搐。反覆或長時間的癲癇發作，也會產生許多發炎介質和過氧化物，進而對組織造成二度傷害。因此，在治療上，平衡興奮性和抑制性的動作電位，如增強GABA 或抑制glutamate 的傳遞，以停止癲癇的進行，和減少癲癇所產生的發炎反應和過氧化物，都是目前研究的方向。在本論文中，我們發展出以低鎂溶液誘發海馬迴切片產生癲癇波的體外癲癇模式，另外也使用毛果芸香（Pilocarpine）腹腔注射在活體大鼠引發僵直痙攣性抽搐，這二種癲癇均被認為是經由N-甲基-D-天門冬胺酸受體(N-methyl-D-Aspartase, NMDA)受體激活所誘發出的痙攣。以這些癲癇實驗模式，我們測試了兩種藥物在癲癇上的療效，一為羅格列酮(rosiglitazone)，另一為天麻苷(gastrodin)。
過氧化物增殖激活受體γ（PPARγ）是一種細胞核內的受體。羅格列酮是其中的一種激活劑(agonist)，在毛果芸香誘導的癲癇發作具有神經保護效果。本實驗中發現10μM濃度的羅格列酮可顯著抑制海馬迴CA1神經元癲癇樣放電的頻率和幅度。併以PPARγ拮抗劑GW9662不能阻斷羅格列酮的抗痙攣效果。羅格列酮的能抑制海馬迴CA1-Schaffer collateral的突觸傳遞。通過微小興奮電位突觸電流（mEPSC）分析，羅格列酮顯著抑制突觸前神經傳導物質的釋放。這種現象可以被PPARγ拮抗劑GW9662阻止。此外，羅格列酮可減緩NMDA所造成的興奮性毒性。這個保護作用部分可被高劑量治療GW9662拮抗，表明該此保護效果部分是由PPARγ受體激活導致的。
天麻是一種古老的中國中草藥，是一種有效的抗驚厥藥和止痛藥，並具有抗眩暈，全身癱瘓，癲癇，破傷風和鎮靜的作用。主活性成分被稱為天麻苷，是4-羥基芐醇葡糖苷。天麻可以抑制缺氧，glutamate-，和N-甲基-D-天冬氨酸（NMDA）造成的神經毒性，並減少了癲癇發作的嚴重程度。 本實驗中發現25μM天麻苷可顯著抑制NMDA在海馬迴CA3造成的神經傷害。但天麻苷反而會加速腹腔注射毛果芸香的痙攣作用（P = 0.051）。天麻苷也未能抑制誘發低鎂誘發的癲癇樣放電。對於海馬迴突觸傳遞，天麻苷對興奮性突觸後電位（EPSP）沒有顯著效果。但高劑量天麻苷(200μM)可減少海馬迴雙刺激的促進效果（P = 0.002），暗示著高劑量天麻苷可能會影響細胞內鈣離子的存量。
結論上，羅格列酮可通過抑制突觸前神經傳導物質的釋放，來抑制NMDA受體所傳導的的癲癇樣放電。羅格列酮通過激活PPARγ來保護海馬切片抵抗NMDA的興奮性毒性。我們認為，羅格列酮有潛力可用來治療顳葉癲癇患者。另一種化合物天麻苷減緩癲癇的發生。天麻苷對神經突觸傳遞沒有影響。這些結果表明天麻苷無法以抑制NMDA受體傳遞的癲癇發生。但天麻苷仍表現出NMDA毒性的保護作用。因此，天麻苷對中風和癲癇患者的仍存潛在好處。

關鍵字

顳葉癲癇 ； 羅格列酮 ； 天麻苷 ； 神經保護 ； N-甲基-D-天冬氨酸

並列摘要

Epilepsy is a common among childhood. Seizures are induced by synchronous discharges from cerebral cortex. Repetitive or prolonged seizures, either focal or generalized- origin, are proved to impair the individuals' cognition, emotion control and learning ability. The synchronous discharges of cerebral cortex based on imbalanced synaptic transmission, either enhanced excitatory neurotransmitter release, such as glutamate, or declined inhibitory neurotransmitter release, such as GABA. Repetitive or prolonged seizures may release many inflammatory cytokines and peroxides, and further lead to tissue injury. Thus, researches for seizure management focus on rebalancing of synaptic transmission, and reducing inflammatory or peroxide responses. In this dissertation, we introduced in vivo and in vitro seizure models to evaluate anti-convulsive properties for different chemicals. In vivo we applied pilocarpine intraperitoneally toward adult rats to induce generalized-tonic-clonic seizures. In vitro we incubated Mg2+-free medium toward acute hippocampal slices to provoke epileptiform discharges. Both of these were N-methyl-D-Aspartase receptor facilitated seizures. Two compounds were included in this dissertation, including rosiglitazone and gastrodin.
Peroxisomal proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor whose agonist, rosiglitazone has a neuroprotective effect to hippocampal neurons in pilocarpine-induced seizures. Application of 10μM rosiglitazone significantly suppressed amplitude and frequency of epileptiform discharges in CA1 neurons. Pretreatment with the PPARγ antagonist GW9662 did not block the effect of rosiglitazone on suppressing discharge frequency, but reverse the effect on suppressing discharge amplitude. Application of rosiglitazone suppressed synaptic transmission in the CA1-Schaffer collateral pathway. By miniature excitatory-potential synaptic current (mEPSC) analysis, rosiglitazone significantly suppressed presynaptic neurotransmitter release. This phenomenon can be reversed by pretreating PPARγ antagonist GW9662. Also, rosiglitazone protected cultured hippocampal slices from NMDA-induced excitotoxicity. The protective effect of 10μM rosiglitazone was partially antagonized by concomitant high dose GW9662 treatment, indicating that this effect is partially mediated by PPARγ receptors.
Tian ma (Gastrodia elata) is an ancient Chinese herbal medicine that has been suggested to be effective as an anticonvulsant and analgesic, and to have sedative effects against vertigo, general paralysis, epilepsy, and tetanus. The primary active ingredient isolated from G. elata has been termed gastrodin, which is the glucoside of 4-hydroxybenzyl alcohol. Gastrodin can abolish hypoxia-, glutamate-, and N-methyl-D-aspartate (NMDA) receptor-induced toxicity in primary cultures of rat cortical neurons, and reduces seizure severity in seizure-sensitive gerbils. Application of 25 μM gastrodin significantly suppressed NMDA excitotoxicity in CA3 hippocampus but not in CA1 hippocampus and dentate gyrus. Intraventricular gastrodin accelerated seizure onset for 12 min after intraperitoneal pilocarpine injection (P = 0.051). Three of five rats (60%) in the gastrodin group and three of four (75%) in the dimethyl sulfoxide (DMSO) group died within 3 days after status epilepticus. Gastrodin also failed to inhibit epileptiform discharges in hippocampal slices induced by Mg2+-free medium. The frequencies of the spontaneous epileptiform discharges were similar under gastrodin 25 μM, gastrodin 200 μM, and DMSO treatments. For the evaluation of gastrodin on synaptic transmission, application of DMSO, gastrodin 25 μM, or gastrodin 200 μM had no significant effect on excitatory postsynaptic potential (EPSP) slopes. Gastrodin 200 μM decreased paired-pulse facilitation from 1.23 ± 0.04 to 1.12 ± 0.04 (P = 0.002).
In conclusion, rosiglitazone suppressed NMDA receptor-mediated epileptiform discharges by inhibition of presynaptic neurotransmitter release. Rosiglitazone protected hippocampal slice from NMDA excitotoxicity partially by PPARγ activation. We suggest that rosiglitazone could be a potential agent to treat patients with TLE. Another compound gastrodin failed to suppress in vivo and in vitro seizures in our study. Gastrodin showed no effect on hippocampal Schaffer collateral EPSP. These findings suggest that gastrodin does not interact with ionotropic glutamate receptors to inhibit NMDAR-facilitated seizures. But gastrodin showed protective effects against NMDA toxicity on cultured hippocampal slices. Nevertheless, gastrodin is still a potential neuroprotective agent against NMDA excitotoxicity, with potential benefits for stroke and patients with epilepsy.

並列關鍵字

gastrodin ； neuroprotection ； NMDA receptor ； rosiglitazone ； temporal lobe seizure

參考文獻

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