香茹草乙醇萃取物抗發炎機制之探討

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Title:	香茹草乙醇萃取物抗發炎機制之探討 Anti-inflammatory Mechanism of Ethanol Extract of Hsiang Ju
Authors:	王麗淑 Li-Su Wang
Contributors:	吳明娟嘉南藥理科技大學：生物科技研究所
Keywords:	香茹巨噬細胞誘導型一氧化氮誘導型環氧化酶細胞激素 Glossogyne tenuifolia nitric oxide (NO) inducible nitric oxide synthase(iNOS) cytokine NF-kappa B luteolin-7-glucoside oleanolic acid
Date:	2003
Issue Date:	2008-10-08 15:45:28 (UTC+8)
Abstract:	香茹(Glossogyne tenuifolia, GT)是澎湖島的原生植物，民間將其作為退熱、解毒、抗發炎之用藥，但目前並無與抗發炎作用機轉及有效成分相關的文獻報導。當巨噬細胞受到內毒素脂多醣LPS（lipopolysaccharide）或免疫刺激（Interferon-gamma, IFN-）時，會產生各種發炎調節因子，誘發一系列的發炎反應。本研究主要以LPS刺激誘導的老鼠巨噬細胞株RAW264.7做為反應模式，探討香茹的乙醇萃取物(GT)對於促發炎的調節因子的釋放及相關調控基因的影響。此論文第一部分主要探討GT對LPS刺激誘導的老鼠巨噬細胞株RAW264.7的亞硝酸鹽產生的抑制的效果。結果顯示GT (0.05-0.25 mg/ml)對一氧化氮產生的抑制作用，隨濃度的增加遞增。利用西方吸漬法及RT-PCR分析誘導性一氧化氮合成酶(iNOS)的基因表達時發現，GT對iNOS 基因表達的抑制，亦隨濃度的增加遞增。另外利用添加actinomycin D 抑制RNA生合成時發現，GT並不會影響iNOS mRNA的穩定度。因此GT主要是藉調控iNOS的轉錄來抑制一氧化氮合成酶的表達，進而抑制一氧化氮的生合成。此外，GT與nuclear factor-B(NF-B)活化的抑制劑pyrrolidine dithiocarbonate (PDTC) 或NOS的抑制劑aminoguanidine(AG)共同作用在活化的RAW264.7時，可協同抑制一氧化氮的產生。在此論文的第二部分主要探討香茹草的乙醇萃取物(GT)對於花生四烯酸的代謝終產物(PGE2)的抑制。由結果中發現，雖然GT能夠抑制PGE2的產生，但對於COX-2 mRNA及蛋白質的表達皆無明顯抑制的效果，因此推論GT可能抑制了COX-1途徑或是影響了COX酵素的活性或是影響了PGE2合成酵素的生合成等。論文的第三部分則是探討GT對一級發炎調節因子的釋放的影響。在實驗結果中顯示GT能夠顯著的抑制受活化的巨噬細胞產生TNF-, IL-1 IL-6, IL-12等促發炎細胞激素。抑制效果隨GT劑量的增加而遞增。論文的第四部分是探討GT對發炎基因共同的轉錄因子NF-B的活化的影響。利用西方吸漬法發現GT能夠顯著的抑制細胞質裡的IB的磷酸化及進一步的水解。利用轉染含NF-B responsive element及luciferase reporter gene的質體進入RAW264.7時發現，GT可顯著抑制LPS誘發的NF-B的活化。在論文的最後一部分進一步探討香茹主要成份oleanolic acid及luteolin-7-glucoside，對nitrite、細胞激素及NF-B活化的抑制效果。我們發現此這些物質皆能抑制受LPS活化的巨噬細胞的NO, TNF-, IL-6, IL-12的產生及NF-B 的活化。然而此三種成份與PDTC及aminoguanidine無協同抑制NO的產生。因此，本研究的主要貢獻為確立香茹草乙醇萃取物之抗發炎分子藥理機轉，並確定luteolin-7-glycoside及 oleanolic acid為其抗發炎之指標成份。 Glossogyne tenuifolia (Hsiang-Ju) is a traditional anti-pyretic herb in Chinese medicine; however, no information is available to describe its action. The objective of this research is to elucidate the molecular pharmacological activity and the effective components in the ethanol extract of Glossogyne tenuifolia (GT). We found that GT had potent anti-inflammatory effects on LPS-activated murine macrophages, RAW264.7. We found that GT (0.05-0.25mg/ml) produced a significant and concentration-dependent decreases in NO production in a endotoxin-stimulated RAW264.7 macrophage. Western blot analysis and RT-PCR assay revealed that GT inhibited the inducible nitric oxide synthase (iNOS) expression in dose-dependent manners. The mRNA stability analysis further suggested that the inhibition of iNOS expression likely by acting at transcriptional rather than post-transcriptional level. Therefore, GT down-regulates LPS-induced iNOS expression primarily by blocking its transcription. In combination with pyrrolidine dithiocarbonate (PDTC), which is an inhibitor for nuclear factor-B activation, or aminoguanidine, which is a competitive NOS inhibitor, GT exerted dose-dependent super-inhibition effect on NO production. Moreover, GT exhibited dose-dependent inhibition on the LPS-stimulated release of arachidonic metabolite and proinflammatory cytokines, such as prostaglandin E2 (PGE2), TNF-, IL-1, IL-6, and IL-12. GT also blocked the phosphorylation and disappearance of IB from cytosolic fraction. In addition, GT reduced LPS-induced luciferase activity in macrophages transfected with a vector coding for the luciferase reporter gene under the control of NF-B cis-acting elements. These results suggested that GT attenuated proinflammatory mediator synthesis on activated macrophages through NF-B dependent pathway. The active components of GT are identified as oleanolic acid and luteolin-7-glucoside. Both of these compounds exhibited dose-dependent inhibition on nitric oxide (NO), TNF-, IL-6 and IL-12 release and NF-B activation. Cooperative inhibition effect on NO production could not be found, when activated RAW264.7 cells were treated with these compounds in combination with PDTC or aminoguanidine. In conclusion, GT and its active ingradients elicit anti-inflammatory effects by down-regulating iNOS transcription and proinflammatory cytokine release through inhibition of NF-KappaB activation.
Relation:	校內校外均不公開
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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