AMPK是一種能量調控的酵素，在細胞能量的調節上扮演了很重要的角色，尤其是生物體面臨能量缺乏的壓力的時候，如：細胞內ATP量的減少或AMP量的增加時，AMPK便會被活化而調控一連串的訊息傳遞途徑，以保護生物體免於代謝失衡之疾病。從分子生物學的觀點來看，AMPK的活化主要和其α-subunit上的Thr172磷酸化有關。除此之外，近幾年來在細胞及動物實驗的研究發現AMPK也可能參與了調控發炎反應的訊息傳遞途徑。在這份研究裡，利用AMPK的活化劑-AICAR，我們探討了AMPK在LPS誘發的發炎反應中所扮演的角色。實驗証實了AICAR可以藉由減弱iNOS和COX-2的基因表現，來抑制LPS對巨噬細胞及微小神經膠細胞刺激而誘發的NO和PGE2生成。再者，AICAR亦能抑制LPS誘導的NFKB, AP-1, CREB及C/EBPβ的活化，減弱這些轉錄因子與基因啟動子的結合。相反的，AICAR卻不能抑制LPS誘導的IKK磷酸化，IKB降解或p65進入細胞核。我們亦証實了AICAR可以直接抑制NFKB, CREB及C/EBPβ對基因啟動子的結合。儘管AICAR可以促進AMPK的磷酸化，但其對LPS誘發的發炎反應的抑制作用卻與其活化AMPK無關。証據是因5''-iodotubercidin可藉由抑制adenosine kinase而抑制AICAR代謝成ZMP，使得AICAR無法活化 AMPK，但卻無法使AICAR對LPS的抑制作用產生逆轉。然而，在缺乏LPS刺激的情況下，AICAR本身在巨噬細胞、微小神經膠細胞和人類臍靜脈內皮細胞卻可微弱地增加iNOS及/或COX-2的表現。此作用可被5''-iodotubercidin逆轉，且可能與AICAR活化IKK, PKC, p38, ERK及Akt相關，故我們推測AMPK可能參與了各種細胞生理功能調控的訊息傳遞途徑，深具進一步研究的價值。簡而言之，我們的研究証實了AICAR對iNOS和COX-2的基因表現可藉由AMPK的參與或不參與而具有雙面的調控能力，顯示了AICAR可能在未來發炎相關疾病的治療具有研發價值。

AMP-activated protein kinase (AMPK), an energy-sensing enzyme that is activated in response to cellular stress, is a critical signaling molecule for the regulation of energy homeostasis and might play a part in protecting the body from metabolic diseases. AMPK is activated by the cellular stresses causing ATP depletion. At the molecular level, AMPK activation is associated with Thr172 phosphorylation on the catalytic α subunit by AMPK kinase (AMPKK). Besides, AMPK might be a novel anti-inflammatory signaling pathway. Recent observations in cultured cells as well as in the animal model suggest the regulatory role of AMPK signaling pathway in inflammatory process, and 5-aminoimidazole-4-carboxamide riboside (AICAR), an AMPK activator, of therapeutic value in treating inflammatory diseases. In this study, we examined the possible role of AMPK in the lipopolysaccharide (LPS)-induced inflammatory process in cultured cells and demonstrated that AICAR inhibits LPS-induced NO and PGE2 production in RAW264.7 macrophages and BV-2 microglia through downregulation of iNOS and COX-2 gene transcription. Moreover, AICAR could block LPS-mediated NFKB, AP-1, CREB and C/EBPβ activation. Conversely, AICAR fails to inhibit LPS-mediated IKK phosphorylation, IKB degradation and p65 nuclear translocation. In vitro direct addition of AICAR in EMSA assays reveals the interruption of NFKB, CREB and C/EBP binding to specific DNA element. Despite the ability of AICAR to enhance AMPK phosphorylation, these effects of AICAR are not associated with AMPK, as 5’-iodotubercidin, an inhibitor of adenosine kinase which blocks AICAR conversion to AMPK activator, fails to change AICAR action. Notably in the absence of LPS challenge we also observed a weak stimulatory effect of AICAR on iNOS and COX-2 protein expression in macrophages, microglia and human umbilical vein endothelial cells (HUVEC). This action of AICAR can be blocked by 5’-iodotubercidin, and might result from the activation of IKK, PKC, p38, ERK and/or Akt. These results imply AMPK might be involved in the upstream regulation of multiple signaling pathways, and in turn exerts diverse cellular functions which open a new research interest. Taken together, our current study documents the dual roles of AICAR on iNOS and COX-2 gene transcription through different action mechanisms, and suggests AICAR may be of therapeutic value in treating inflammatory diseases.

Abbreviations-----------------------------------------------------------------------------1

AMPK活化劑-AICAR對iNOS及COX-2基因表現的調節作用
AMPK-dependent and -independent action mechanisms of AICAR in the regulation of iNOS and COX-2 gene expressions--------------------------------3

Abstract------------------------------------------------------------------------------------4

Chinese abstract--------------------------------------------------------------------------6

Introduction-------------------------------------------------------------------------------7

Materials and methods-----------------------------------------------------------------16

Results------------------------------------------------------------------------------------22

Discussion-------------------------------------------------------------------------------34

Reference--------------------------------------------------------------------------------41

Figure-------------------------------------------------------------------------------------59

Appendix---------------------------------------------------------------------------------88

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