臺灣博碩士論文加值系統

台灣八角金盤是台灣特有種的常綠灌木植物，於傳統中草藥中，其葉可治療感冒咳嗽、風濕關節炎、腫瘤以及瘡疹，而樹皮用於袪風、止痛、行血。這些功效有一部分與抗發炎有關，但其有效成分不明。最近已有研究將台灣八角金盤葉子與樹枝部位進行萃取，並且分離出九種三萜類化合物，3α-hydroxyolean-11, 13(18)-dien-28-oic acid (HODA)為其中含量較高的三萜類分子之一。在本研究中，我們研究HODA，以及其乙醯基化學修飾的衍生物 (Ac-HODA)的抗發炎效果。分析兩個化合物的細胞毒性，發現HODA沒有明顯毒性，但是Ac-HODA卻可選擇性地抑制小鼠腫瘤細胞株RAW264.7巨噬細胞以及人類肝癌細胞HepG2與人類子宮頸癌細胞HeLa的生長，而對小鼠正常肝細胞FL83B則無明顯毒性。進行DNA fragmentation初步測試發現Ac-HODA可能是導致RAW264.7產生細胞凋亡，所以使用FL83B細胞作為模型，研究兩個化合物的抗發炎效果。結果，HODA與Ac-HODA可抑制TNF-a (tumor necrosis factor-a)所誘導的inducible nitric oxide synthase的表現、NF-kB (nuclear factor-kappa B) 轉位至細胞核、IKK (inhibitor of NF-kB kinase)的活化、以及IkB (inhibitor of NF-kB)的磷酸化，而Ac-HODA的效果比HODA更明顯。另外，HODA可抑制TNF-a所誘導的JNK (c-Jun N-terminal kinase), ERK1/2 (extracellular signal-regulated kinase 1/2)的活化，對p38則無明顯抑制效果。但是Ac-HODA卻會誘導JNK、ERK、p38的活化。在小鼠實驗中也發現HODA與Ac-HODA皆可有效抑制皮膚發炎。發炎反應可分為「起始期」與「解除期」兩個階段。在發炎解除期中一些因子如TGF-b1 (transforming growth factor-b1)、IL-10 (interleukin-10)以及脂氧化酶 (lipoxygenase, LOX)家族所催化產生的一些脂質類分子等，會被表現而促進發炎解除。我們發現HODA與Ac-HODA隨著時間的增加會抑制15-LOX (15-lipoxygenase)的表現，卻增加了5-LOX (5-lipoxygenase)的表現。所以，本研究的結果顯示台灣八角金盤的天然物HODA以及其衍生物Ac-HODA具有抗發炎的效果，而其分子機制與IKK/NF-kB pathway的抑制有關。研究並證實於HODA的3a-OH上進行化學修飾會影響其活性。另外，這些化合物是否促進發炎解除仍須更進一步的分析。

Fatsia polycarpa is an evergreen shrub plant endemic to Taiwan. Its foliage is used in traditional medicine to treat cold, coughing, and rheumatoid arthritis; its twig is used in pain relief and improving blood circulation. However, the effective constituents in its extract are not clear. Previously, nine oleanane-type triterpenoids were isolated from the leaves and twigs of Fatsia polycarpa. Among them, 3α-hydroxylean-11,13 (18)-dien-28-oic acid (HODA) is one of the abundant triterpenes. In this study, the anti-inflammatory effects of HODA and its acetylated derivative, Ac-HODA, were characterized. HODA did not display obvious cytotoxicity, whereas Ac-HODA selectively inhibited the growth of the macrophage line RAW264.7 and the tumor lines HepG2 and HeLa cell, yet did not affect the survival of FL83B cells, a normal liver cell line. DNA fragmentation assay showed that Ac-HODA might induce apoptosis of RAW264.7 cells. Thus, FL83B cells were used as a model to study the anti-inflammatory effects of these compounds. Consequently, HODA and Ac-HODA both inhibited TNF-a-induced expression of inducible nitric oxide synthase, the translocation of nuclear factor kappa B (NF-kB), the activation of inhibitor of NF-kB kinase (IKK), and the phosphorylation of inhibitor of NF-κB (IκB), and Ac-HODA was more effective than HODA. In addition, HODA inhibited the activation of c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase 1/2 (ERK1/2), yet Ac-HODA promoted the activation of JNK, ERK1/2, and p38. Animal tests using mouse ear edema as a model confirmed that both of HODA and Ac-HODA could ameliorate skin inflammation. Inflammation can be divided into “initiation phase” and “resolution phase”. The resolution phase involves the production of factors such as transforming growth factor-b1 (TGF-b1), interleukin-10 (IL-10), and lipid mediators produced by lipoxygenase (LOX). HODA and Ac-HODA inhibited the expression of 15-LOX, yet promoted those of 5-LOX. In summary, results from this study suggest that HODA and its derivative, Ac-HODA, possess anti-inflammatory activities, and the underlying mechanism likely involves the inhibition of IKK / NF-kB pathway. The data also confirmed that chemical modification on the 3a-OH of HODA affected the activity of the molecule. Whether HODA and Ac-HODA promote the resolution of inflammation needs further investigation.

中文摘要
Abstract
誌謝
目錄
圖表目錄
第1章 前言
1.1 研究背景
1.2 研究動機與架構
1.3 研究成果的重要性
第2章 文獻回顧
2.1 發炎反應
2.2發炎反應的訊號傳遞路徑
2.2.1 NF-κB路徑
2.2.2 MAPK路徑
2.2.3 發炎解除的分子機制
2.3 腫瘤壞死因子-α (Tumour necrosis factor-α, TNF-α)
2.4 一氧化氮 (NO)
2.5 抗發炎研究常用的細胞模型
2.6 抗發炎研究常用的動物模型
第3章 材料與方法
3.1材料
3.1.1細胞株
3.1.2 細胞培養基
3.1.3實驗藥品
3.1.4 試劑及緩衝溶液
3.2 實驗儀器
3.3 實驗方法
3.3.1 細胞培養
3.3.2 細胞存活率分析 (MTT assay)
3.3.3 蛋白質的粗萃取
3.3.4 蛋白質萃取之核質分離
3.3.5 發炎解除路徑之分子機制5-LOX與15-LOX路徑鑑定
3.3.6西方墨點法分析 (Western blot analysis)
3.3.7老鼠耳朵水腫模式
3.8實驗數據統計分析
第4章 結果
4.1分析HODA與Ac-HODA的細胞毒性
4.2 HODA和Ac-HODA的抗發炎效果
4.3 HODA和Ac-HODA抗發炎的分子機制
4.3.1 IKK/NF-κB路徑的抑制
4.3.3 對MAPKs的影響
4.4 分析HODA和Ac-HODA是否有促進「發炎解除」的效果
4.5 HODA和Ac-HODA的動物實驗
第5章 討論
第6章 結論
參考文獻
作者簡介

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