穿心蓮萃出物及穿心蓮內酯調節 pi 屬麩胱甘肽硫轉移酶表現之研究

Modulation of the expression of pi class of glutathione S-transferase by Andrographis paniculata extracts and andrographolide

張貴婷(Kuei-Ting Chang)

指導教授 ： 陳暉雯 ； 李宗貴

中山醫學大學/健康管理學院/營養學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00132

摘要

目前，世界上有越來越多的人口使用傳統草藥，來作為保健養生及疾病療養之用。穿心蓮，拉丁學名 Andrographis paniculata (Burm. f.) Nees，屬於一種草藥，普遍使用於東南亞國家。穿心蓮具有多種生理功效，例如：抗HIV病毒、抗發炎、抗癌、降低血糖和血壓及抗肝毒性等，穿心蓮內酯是穿心蓮中重要的活性成分之一。過去研究指出，穿心蓮萃出物會增加小鼠肝臟中麩胱甘肽硫轉移酶活性；然而，其中的機轉並不清楚。因此，本實驗利用大鼠初代肝細胞培養模式，探討穿心蓮對誘發 pi 屬麩胱甘肽硫轉移酶 (pi class of glutathione S-transferase, GSTP)的作用。初代肝細胞處理 25 或 50 μg/mL 穿心蓮乙醇及乙酸乙酯萃出物或 10 或 20 μM 穿心蓮內酯 48 小時，觀察 GSTP 的蛋白質、mRNA 及酵素活性的表現情形。結果顯示，穿心蓮萃出物及穿心蓮內酯皆可誘發 GSTP 蛋白質及 mRNA 表現，且呈現劑量關係，而 GSTP 酵素活性也有隨處理劑量增加而上升，在50 μg/mL 穿心蓮萃出物及 20 μM 穿心蓮內酯處理下，GSTP 酵素活性顯著高於控制組 (P < 0.05)。進一步探討 GSTP 基因 promoter 上的 GSTP enhancer element (GPE1 及 GPE2) 在穿心蓮誘發 GSTP 表現過程中所扮演的角色。利用三個含不同長度 GSTP promoter 的報導基因，分別是 pTA-2713 (含 GPE1 及 GPE2)、pTA-2604 (只含 GPE2) 及 pTA-2375 (不含GPE1 及 GPE2)，短暫轉染 (transiently transfect) 至大鼠肝臟細胞株 Clone 9 後，再分別處理穿心蓮萃出物及穿心蓮內酯 24 小時，觀察 GSTP promoter 活化的情形。結果顯示，轉染含有 GPE1 的 pTA-2713 細胞中，穿心蓮萃出物及穿心蓮內酯可以劑量關係誘發 GSTP promoter 活性；但轉染不含 GPE1 的 pTA-2604 或 pTA-2375，則此誘發現象隨即消失，顯示出，GPE1 在穿心蓮誘發 GSTP 表現的過程中是必要的。另外，本實驗也利用 phosphatidylinositol 3-kinase (PI3K)/Akt 抑制劑 -- wortmannin，進一步探討此訊息傳遞路徑是否參與穿心蓮誘發 GSTP 表現。結果顯示，在預處理 wortmannin 後，穿心蓮誘發 GSTP 表現的程度減弱。综合以上實驗結果得知，在大鼠初代肝細胞中，穿心蓮萃出物及穿心蓮內酯皆可誘發 GSTP 表現，且此誘發和GPE1 及 PI3K/Akt 路徑有關。

關鍵字

穿心蓮 ； 穿心蓮內酯 ； pi屬麩胱甘肽硫轉移酶(GSTP) ； GSTP enhancer 1 (GPE1) ； phosphatidylinositol 3-kinase (PI3K)/Akt ； 大鼠初代肝細胞

並列摘要

Andrographis paniculata (Ap) is a commonly used herb for traditional medicine in many Southeast Asian countries. In the present study, we investigated the effect of Ap on the expression of the pi class of glutathione S-transferase (GSTP) in rat primary hepatocytes. Hepatocytes were treated with 25 or 50 μg/mL of ethanol or ethyl acetate extracts of Ap (ApEE or ApEAE) or 10 or 20 μM andrographolide, which is the major active diterpene lactone of Ap, for 48 h. ApEE, ApEAE, and andrographolide dose-dependently induced GSTP protein and mRNA expression. In a GST activity assay, GST activity was significantly higher in cells treated with the maximum concentrations of ApEE, ApEAE, and andrographolide than in control cells (P < 0.05). The pTA-2713 luciferase reporter construct containing GSTP enhancer 1 (GPE1) was transiently transfected into Clone 9 liver cells. Cells treated with ApEE, ApEAE, and andrographolide showed a dose-dependent increase in luciferase activity. GPE1 deletion abolished the induction efficiency of Ap. Also, the induction of GSTP expression by Ap was inhibited by wortmannin, which is an inhibitor of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. These results indicate that ApEE, ApEAE, and andrographolide induce GSTP expression. This induction is likely related to the PI3K/Akt pathway, and GPE1, an enhancer element in GSTP promoter, is essential for the induction.

並列關鍵字

Andrographis paniculata ； Andrographolide ； pi class of glutathione S-transferase (GSTP) ； GSTP enhancer 1 (GPE1) ； rat primary hepatocyte ； phosphatidylinositol 3-kinase (PI3K)/Akt

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