黃芩苷及漢黃芩素對睡眠調節影響之探討

Abstract

Baicalin及Wogonin為中國傳統草藥黃芩 (Scrutellaria baicalensis Georgi) 之成分，傳統常使用在消炎、腸胃不適、抗過敏、解熱及鎮靜，近年來研究指出其有抗菌、抗病毒、抗腫瘤、抗發燒 (antipyretic)、神經保護、及解焦慮等作用。近年來，已有許多黃芩成分相關機制的研究，有些報告指出，Baicalin能夠有效的清除多種自由基，且Baicalin及Wogonin能藉由抗氧化作用幫助神經細胞抵抗氧化壓力的傷害，而Baicalin及Wogonin也能抑制微膠細胞 (microglia cells) 活化，使NF-κB的活化量下降，降低cytokines及chemokines分泌量及RNA的表現量。也有報告指出，Baicalin及Wogonin是GABAA受體的擬效劑 (agonists)，能夠與受體上的BZ結合位 (Benzodiazepine binding site) 結合，因其沒有鎮靜及認知缺損的副作用，因此受到了醫學界的重視。由於在正常睡眠的調控機制上，IL-1β、TNF-α及GABA皆與睡眠的內分泌調控作用密不可分，由其是在Non-rapid eye movement sleep (NREMS) 方面，但Baicalin及Wogonin對睡眠的影響至今尚未有任何相關研究報告。本實驗的目的是觀察黃芩主成分Baicalin與Wogonin對大鼠正常睡眠的影響，進一步探討其內在作用機制，並尋求在睡眠醫學上可能的應用方式。 在我們的研究結果中，發現Baicalin及Wogonin對正常睡眠具有兩個階段的作用，第一個階段是打藥後的第1~2個小時，使NREMS下降，我們稱之為初期 (initiate phase) 的作用。第二個階段是在第7~12個小時，使NREMS大幅度上升的作用，稱之為晚期 (late phase) 的作用。而initiate phase的作用，主要是在Light-onset的實驗結果中出現。在Late phase的作用，則是在Dark-onset的實驗結果中較容易看到，於Light-onset的實驗中則較不明顯。因此，我們假設在Light-onset的作用是與亮期時腦中的IL-1β或TNF-α的量或作用程度被改變所導致。而在同時打入IL-1β及Baicalin之後的NREMS，與單獨打入IL-1β的NREMS比較，有明顯下降的情形，Wogonin亦有相同的結果。而在IL-1β的ELISA結果中，我們發現，在打入Baiclin之後的第二個小時，下視丘 (hypothalamus)、海馬迴 (hippocampus) 及皮質 (cortex) 處的IL-1β的量都沒有明顯改變。而關於晚期的作用，我們假設其有可能是經由GABAA受體的作用或是經由某些機制使GABA的分泌量所造成。我們觀察在Dark-onset先打入Baicalin或Wogonin，待其作用至第七個小時，再給予GABAA及GABAB的拮抗劑 (antagonists)。之後的結果發現，wogonin的NREMS上升作用，有被GABAA的拮抗劑所抵銷。 在此研究中，結果中證明了初期的作用極有可能是藉由IL-1β受體受到Baicalin及Wogonin的阻抗所造成的。而GABA組的實驗結果說明了，Wogonin的晚期作用可能是藉由增加GABA的分泌量，而使NREMS有增加的結果。

Baicalin and wogonin are active compounds originated from the root of traditional Chinese herb Scrutellaria baicalensis Georgi. They have been used in anti-inflammation, anti-bacteria, anti-hypertension, anti-allergy and sedative actions since ancient China. Baicalin and wogonin possess abilities to decrease the expression of pro-inflammatory cytokines and NF-κB activity. It has been described that baicalin and wogonin are both the free radical scavengers and exhibit anti-inflammatory activity. It has also been demonstrated that the anxiolytic-like effect of baicalin is mediated by GABAA receptors. The activity of GABAergic sleep-active neurons is related to sleep regulation. In the homeostatic sleep regulation, cytokines, especially IL-1β and TNF-α, plays an important role in the regulation of slow wave sleep (SWS). However, there is a lack of evidence linking baicalin and wogonin to the sleep regulation. The purpose of this study is investigating the underlying mechanism of baicalin and wogonin on spontaneous sleep. We respectively i.c.v. injected baicalin and wogonin into brain and EEG-defined sleep-wake activities were collected. Our results indicated that both baicalin and wogonin exhibited a biphasic effect on spontaneous sleep, a initiate phase and a late phase of sleep regulation. In the initiate phase, the time spent in SWS during hours 1~2 of the light period were decreased from 63.19 ± 3.62 %, obtained after vehicle (DMSO), to 47.73 ± 6.88 % acquired after administration of baicalin 20 mins prior to light onset. Besides, the time spent in SWS were increased during hours 8~12 of dark period from 17.03 ± 1.86 %, obtained after DMSO, to 26.6 ± 2.63 % acquired after injection of baicalin prior to dark onset. Wogonin also exhibited a similar sleep-wake regulation as that of baicalin. When we administrated Baicalin+IL-1β，we investigated the increasing of NREMS of IL-1β disappeared. When wogonin+IL-1β was given, the effects on NREMS was the same. And the IL-1β ELISA result demonstrated the IL-1β concentration of hypothalamus, hippocampus and cortex was not changed after administration of baicalin. After we injected wogonin at dark-onset and injected GABAA antagonist, bicuculline, or GABAB antagonist, 2-hydroxysaclofen, during the middle of dark period (7th hour), the increasing of NREMS of wogonin’s effect decreased The observation elucidated that both wogonin and baicalin exhibited dual responses to regulate sleep-wake activity; an initiate phase to suppress SWS during the light period and a late phase to enhance SWS during the dark period. We demonstrated that the anti-inflammatory effect of wogonin and baicalin may be involved in the regulation of the initial phase. they may be interfere the function of IL-1β receptor or blocked it. And wogonin may be somehow have some mechanism to interfere the secretion of GABA in the brain of the late phase response.