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論文名稱(外文):cAMP/ICER-associated inhibition of andrographolide-induced pi class of glutathione S-transferase expression in rat primary hepatocyte
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穿心蓮內酯是一種bicyclic diterpenoid lactone,主要分佈於穿心蓮葉子中,它在中國傳統中藥應用上,大多用於治療感染性疾病或發炎疾病。本實驗室先前研究發現大鼠初代肝細胞處理穿心蓮乙醇萃出物、乙酸乙酯萃出物或穿心蓮內酯均可顯著誘發GSTP蛋白質和mRNA表現且呈劑量關係,且此誘發作用也可能與PI3K/Akt pathway有關,但是,其它調控GSTP表現的機轉仍不清楚。本實驗利用MAPKs抑制劑 (PD98059、SP600125、SB203580)、PI3K抑制劑 (wortmannin)、adenylate cyclase活化劑 (forskolin)或cAMP類似物 (dibutyryl cAMP, 8-(4-chlorophenylthio)-cAMP和8-Bromo cAMP) 預處理大鼠初代肝細胞發現PI3K抑制劑 (wortmannin)、adenylate cyclase活化劑 (forskolin) 或cAMP類似物 (dibutyryl cAMP, 8-Bromo cAMP 和8-(4-chlorophenylthio)-cAMP) 可有效抑制穿心蓮內酯誘發GSTP 蛋白質及mRNA表現。由結果推論cAMP/PKA路徑在穿心蓮內酯誘發GSTP表現可能是扮演一個負向調控角色。另外,細胞預處理7.5 uM H89 (PKA 抑制劑),發現可部份阻斷cAMP類似物抑制穿心蓮誘發GSTP表現,但無法阻斷 5 uM forskolin之抑制作用;而大鼠初代肝細胞處理40 uM 穿心蓮內酯或25 uM forskolin或是兩者共同處理發現均可增加CREB 磷酸化,而ICER mRNA表現卻只有在25 uM forskolin或是兩者共同處理下有顯著上升,且在共同處理組出現加乘作用。Electromobility gel shift assay (EMSA) 結果顯示穿心蓮內酯處理1hr或穿心蓮內酯與forskolin共同處理1 hr或3 hr均會增加轉錄因子與cAMP response element (CRE) 結合能力。這些結果顯示cAMP/CREB/ICER在穿心蓮內酯誘發GSTP 表現過程中扮演負向調控角色。

Andrographolide is a bicyclic diterpenoid lactone isolated from the leaves of Andrographis paniculata. Andrographis paniculata, one of Chinese herbs, is used for the treatment of viral infections and inflammatory diseases. We have previously reported that rat primary hepatocytes treated with 40 μM andrographolide for 48 hr resulted in the induction of the expression of pi class of glutathione S-transferase (GSTP) protein and mRNA; however, the mechanism was not completely clarified. In the present study, we found that the induction of the expression of GSTP protein and mRNA by Ap was inhibited by wortmannin, forskolin (adenylate cyclase activator), dibutyryl cAMP, 8-Bromo cAMP and 8-(4-chlorophenylthio)-cAMP (cAMP analogs), but not by MAP kinase inhibitors including PD98059, SP600125 and SB203580. Moreover, hepatocytes pretreated with 7.5 uM H89 (PKA inhibitor) partially blocked the inhibitory effect of cAMP analogs. Also, we investigated the effect of andrographolide, forskolin, or both on the expression of phosphorylated CREB protein and ICER mRNA in rat primary hepatocytes. Hepatocytes treated with 25 uM forskolin, 40 uM andrographolide, or both increased the expression of phosphorylated CREB; however, forskolin is necessary for the ICER mRNA expression. Electromobility gel shift assay (EMSA) showed that the DNA binding activity of cAMP-response element (CRE) was increased by 40 uM andrographolide treatment after 1hr and 25 uM forskolin treatment after 1hr and 3hr in the presence of 40 uM andrographolide compared with the control. Taken together, these results suggest that cAMP/CREB/ICER pathway plays a down-regulatory role in the induction of GSTP by andrographolide.

第一章 文獻回顧
1-1 穿心蓮介紹...................6
1-2 穿心蓮與癌症..................7
1-3 穿心蓮與保肝..................8
1-4 穿心蓮與慢性發炎疾病..............9
二、 pi屬麩胱甘肽硫轉移酶 (pi class of glutathione S-transferase, GSTP)
2-1 生物轉換酵素系統 (biotransformation enzyme system) ...10
2-2 麩胱甘肽硫轉移酶 (glutathione S-transferases) 簡介....11
2-3 GSTP介紹...................12
三、3’,5’- cyclic adenosine monophosphate/protein kinase A/ cAMP-responsive element binding protein (cAMP/PKA/CREB) 簡介及調控機轉
3-1 cAMP / PKA...................15
3-2 PKA / CREB...................16
3-3 ICER......................17
第二章 研究目的....................19
第三章 實驗材料與方法.................20
第四章 實驗結果與圖表.................32
第五章 討論......................47
第六章 結論......................52
第七章 未來方向....................53

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