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研究生(外文):Yi-Ching Tseng
論文名稱(外文):Effect of S-Petasin on Corticosterone Secretion in Male Rats
指導教授(外文):Paulus Shyi-Gang Wang, Ph.D.
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硫蜂斗菜素是由Petasites hybridus的根或葉萃取出來且具有生物活性的成份。Petasites hybridus在古希臘歷史中常用來治療胃腸道的和氣喘。此植物體及其萃取物應用在治療使用上已約有兩千年的歷史,然而它是否會對身體的內分泌系統造成影響尚未明朗。我們實驗室曾證實硫蜂斗菜素可抑制大鼠睪丸間隙細胞睪固酮的分泌。至於硫蜂斗菜素對於腎上腺功能的影響還不清楚。因此,本研究論文是以雄性大鼠為實驗動物同時以活體及離體方式探討硫蜂斗菜素對雄鼠皮質固酮分泌的效應及作用機制。活體實驗部分,雄鼠自右頸靜脈植入一 PE-50 軟管,並於 20 小時後,靜注生理鹽水、硫蜂斗菜素、腎上腺皮質促素 (adrenocorticotropin;ACTH)、或腎上腺皮質促素合併硫蜂斗菜素,並於靜注後 0、30、60、120、180及240分鐘抽血,每次 0.5 毫升。血樣取得後離心,分出血漿,以放射免疫法檢測皮質固酮及腎上腺皮質促素之濃度。離體實驗部分,取雄鼠腎上腺皮質組織,以 collagenase 酵解,分離束網層細胞,與不同濃度 (10-6~10-4 M) 之硫蜂斗菜素,伴隨ACTH 、8-Br-cAMP (cAMP的類似物)、forskolin (adenylyl cyclase的活化劑) 或類固醇合成前驅物,包括:25-OH-膽固醇 (P450 side-chain cleaveage, P450scc的受質)、pregnenolone (3-hydroxysteroid dehydrogenase, 3-HSD的受質)、助孕酮 (21-hydroxylase的受質) 及deoxycorticosterone (11-hydroxylase的受質) 一齊培養 1 小時。所得培養液以放射免疫測定法分析皮質固酮濃度。為探討硫蜂斗菜素影響皮質固酮的分泌是否與腎上腺束網狀帶中P450scc或steroidogenic acute regulatory protein (StAR protein) 蛋白質的改變有關,另以西方點墨技術 (Western blot analysis) 分析P450scc及StAR之蛋白質表現。所得結果以變方分析處理,若呈顯著,再以丹肯氏多變域異測驗法進行兩組平均值差異顯著分析,部份結果則以學生式t-測驗進行比較。本研究結果顯示(一)硫蜂斗菜素會抑制大鼠血漿皮質固酮的濃度。(二) 硫蜂斗菜素可直接作用在腎上腺束網狀帶細胞抑制皮質固酮的基礎分泌以及由腎上腺皮質促素刺激之皮質固酮分泌。(三) 硫蜂斗菜素透過抑制adenylyl cyclase的活性抑制皮質固酮的分泌。(四) 硫蜂斗菜素抑制皮質固酮生合成過程中P450scc及11β-hydroxylase的活性。(五) 硫蜂斗菜素經由減少StAR protein的蛋白質表現抑制皮質固酮的分泌。綜合所得的結果顯示硫蜂斗菜素抑制皮質固酮的分泌,其作用機轉包括抑制adenylyl cyclase、P450scc及11β-hydroxylase的活性及減少StAR protein的蛋白質表現。

S-petasin is a bioactive compound isolated from leaves or roots of Petasites hybridus (Engl. Butterbur; German Pestwure) which has been used therapeutically for more than 2000 years. It has been reported that the leaf and root extracts of Petasites hybridus relieve gastrointestinal pain, lung-disease such as asthma and cough, as well as spasms of the urogenital-tract. However, their side effects on the endocrine systems are still not clear. It has been demonstrated that s-petasin inhibits the production of testosterone in rat testicular interstitial cells. Wheather s-petasin affects the adrenal function is unknown. The present study was to explore the effects of s-petasin on the secretion of corticosterone both in vivo and in vitro. A single intravenous injection of s-petasin (10g / kg) decreased both basal and adrenocorticotropin (ACTH)-induced plasma corticosterone concentration in male rats. Administration of s-petasin (3x10-6~10-4M) in vitro caused a reduction of basal and ACTH-stimulated release of corticosterone by the enzymatically dispersed rat zona fasciculata-reticularis (ZFR) cells in a dose-dependent manner. S-petasin decreased corticosterone secretion in response to 8-Br-cAMP (a cAMP analogue, 10-6~10-4M) and forskolin (an adenylyl cyclase activator, 10-6~10-4M). Furthermore, we investigate the effect of s-petasin on steroidogenic enzyme activity by incubation of ZFR cells with steroidogenic precursors (e.g. 25-OH-cholesterol, pregnenolone, progesterone, and deoxycorticosterone, 10-7~10-5M each). We found that s-petasin inhibited corticosterone release induced by 25-OH-cholesterol and deoxycorticosterone. After incubation of rat ZFR cells with s-petasin for 4 h, both basal and ACTH-stimulated StAR protein expressions were decreased. However, the protein expression of P450scc was not altered by the administration of s-petasin. These results suggest that s-petasin inhibits the production of corticosterone in rat zona fasciculata-reticularis cells in part through inhibition of cAMP pathway and reduction of StAR protein expression and the activities of P450scc and 11-hydroxylase during steroidogenesis of corticosterone.

重要名詞中英文對照表 ˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙3
第一章、 文獻回顧˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙8
一、 概述˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙9
二、 腎上腺皮質固酮之特性˙˙˙˙˙˙˙˙˙˙˙˙˙˙10
三、 硫蜂斗菜素˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙26
四、 研究目的與假說˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙32
第二章、 激素及生化物質之檢測與分析˙˙˙˙˙˙˙˙˙33
一、皮質固酮 (corticosterone) 之放射免疫測定˙˙˙34
二、妊烯醇酮 (Pregnenolone) 之放射免疫測定˙˙˙˙35
三、西方墨漬分析 (Western Blot)˙˙˙˙˙˙˙˙˙35
第三章、 硫蜂斗菜素對雄鼠皮質固酮分泌的效應˙˙˙˙˙˙39
一、 緒言˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙40
二、 材料與方法˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙41
三、 結果˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙50
四、 討論˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙˙53

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