以脂多醣體刺激之巨噬細胞模式探討丁香醇與異丁香醇衍生物之抗發炎活性及機轉

本研究實驗的藥物是不同的eugenol 和isoeugenol 衍生物，像是 eugenolol、isoeugenolol、isoeugenodilol 和glyceryl-isoeugenol 已被本實驗室合成並研究。之前的研究發現這些衍生物有血管舒張、抗氧化、支氣管擴張、β–blocker 或?恁Vblocker 等作用。許多研究顯示 isoeugenol 和eugenol 展現了抗發炎的作用，然而這些衍生物的抗發炎活性仍未被探討，因此本研究的目的為評估這些衍生物的抗發炎功效並進而探討其機轉。在我們的實驗中，我們發現eugenol 的衍生物— eugenolol 能夠有效的抑制脂多醣體(lipopolysaccharide, LPS)刺激的老鼠巨噬細胞株RAW264.7 中環氧化酶(cyclooxygenase-2, COX-2)、誘導型一氧化氮合成酶(inducible nitric oxide synthase, iNOS)表現和一氧化氮(nitric oxide, NO) 、腫瘤壞死因子-??(tumor necrosis factor-???z?nTNF?{??)、介白素-1??(interleukin-1???z?nIL-1??)釋放量，且作用比 eugenol 更為顯著。我們也發現了eugenolol 能藉著阻斷Akt、inhibitor κB (IκB)?恁Bextracellular signal-regulated kinase 1/2 (ERK1/2) 磷酸化來減少LPS 誘導的nuclear factor-?羠 (NF-?羠)次單元p65 的核轉移以及 NF-?羠的DNA 結合活性，作用比eugenol 更強。而在isoeugenol 衍生物方面，我們發現glyceryl-isoeugenol 的抑制COX-2、iNOS、TNF?{?? 以及NO 表現活性是所有isoeugenol 衍生物中最強的，而此作用主要是藉著阻斷MAPK、Akt、I?羠?捘C酸化以及接續的p65 核轉移和NF-?羠的DNA 結合活性。除此之外，我們也探討了其他轉錄因子，像是 activator protein-1(AP-1) 、cAMP-response element-binding protein (CREB)、hypoxia-induced factor (HIF)是否參與衍生物的抗發炎作用。我們發現eugenolol 和glyceryl-isoeugenol 能夠抑制LPS 誘導的 HIF-1α 表現和AP-1-DNA 結合活性。總結來說，eugenolol 和 glyceryl-isoeugenol能夠藉著抑制MAPKs及Akt/I?羠?捘C酸化來阻斷下游的NF-?羠、AP-1 DNA 結合能力和HIF-1?捖J白表現，進而達到抗發炎的效果。

關鍵字

巨噬細胞；脂多醣體；丁香醇；異丁香醇

並列摘要

During the past decade, various eugenol and isoeugenol derivatives such as eugenolol, isoeugenolol, isoeugenodilol and glyceryl-isoeugenol have been synthesized and investigated in our laboratory. Our previous studies have demonstrated that the agents possess vasodilatory, antioxidant, tracheal relaxant, β–adrenoceptor blocking, or α–adrenoceptor blocking properties. Several studies have demonstrated isoeugenol and eugenol exert anti-inflammatory actions. However, the anti-inflammatory action of these derivatives is still not determined. Therefore we evaluate the anti-inflammatory effects of these derivatives and study the mechanisms. In this study, we demonstrated that eugenol derivative, eugenolol, exhibited stronger inhibition effects on cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor-?n???n?vTNF?{??), interleukin -1???n(IL-1???w as well as NO production than those of eugenol in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. We also found that eugenolol reduced LPS-induced nuclear translocation of nuclear factor-?羠 (NF-?羠) subunit p65 and the DNA binding activity of NF-?羠 more potently than eugenol by blocking phosphorylation of Akt and inhibitor κB (IκB)?? as well as the subsequent degradation of IκB???|?nWe also revealed that eugenolol repress the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) but not c-Jun NH2-terminal kinase (JNK) and p-38. Besides, the anti-inflammatory effects of isoeugenol derivatives are also investigated, and we discovered that glyceryl-isoeugenol is the strongest COX-2, iNOS and TNF-?? inhibitor of these derivatives by preventing the Akt and I?羠?? phosphorylation and the following nuclear translocation of p65. Moreover, glyceryl-isoeugenol can repress all three MAPKs. Aside from this, we also studied other transcription factor such as activator protein-1(AP-1), cAMP-response element-binding protein (CREB), hypoxia-induced factor (HIF), and found that eugenolol and glyceryl-isoeugenol inhibited the LPS-induced HIF-1α expression and AP-1 binding activity. Taken together, eugenolol and glyceryl-isoeugenol can suppress iNOS and COX-2 expression by blocking MAPKs-mediated pathways with the attendant activation of AP-1, HIF-1?? and CREB, and also by preventing the phosphorylation of Akt and I?羠?? and the subsequent p65 nuclear translocation as well as the NF-?羠 binding activity.