牛樟芝(Antrodia cinnamomea)為台灣傳統上藥用真菌，只生長在台灣獨有的牛樟樹上。牛樟芝傳統上被用於治療腹瀉、嘔吐、食物中毒，近年來研究證實具有多種生理活性，包括抗氧化和抗腫瘤。本實驗以脂多醣(lipopolysaccharides, LPS)刺激小鼠巨噬細胞RAW 264.7產生急性發炎模式，探討牛樟芝萃取物之抗發炎活性，和其作用機制。結果顯示牛樟芝子實體分離物ME能夠顯著抑制LPS誘發細胞內自由基、一氧化氮( nitric oxide, NO)、前列腺素E2(prostaglandin E2, PGE2)與細胞激素(cytokines)產生，且能夠降低一氧化氮合成酶(inducible nitric oxide, iNOS)與第二型環氧酵素(cyclooxygenase 2, COX-2)之蛋白質表現量。已知nuclear factor-κB (NF-κB)和mitogen-activated protein kinases (MAPK)路徑在發炎反應中扮演重要腳色，結果顯示ME能夠減少NF-κB反應路徑中IKK磷酸化、IκB裂解與NF-κB進入細胞。另外一方面ME能夠減少MAPK反應路徑中ERK、p38與JNK磷酸化。綜合以上實驗結果，我們發現牛樟芝分離物ME可以有效抑制促發炎物質的生成，且可能透過抑制NF-κB和MAPK的訊息傳遞路徑降低發炎反應。

Antrodia cinnamomea ( AC ) is a traditional medicinal mushroom which grows only on the old trunk cavity of the endemic species Cinnamomum kanehirae ( Bull camphor tree )of Taiwan. On folk remedies, AC has been used for the treatment of diarrhea, vomiting and food poisoning etc. Recent studies have demonstrated a variety of physiological activities, including antioxidant and anticancer. In the present study, the lipopolysaccharide(LPS) induced mouse macrophage RAW 264.7 acute inflammation model was used to evaluate the anti-inflammatory activity of AC extract, named as “ME”, and its mechanism of action. The results showed that ME could significantly inhibit LPS-induced inflammatory on intracellular radical production, nitric oxide(NO), prostaglandin E2(PGE2) and cytokines, and also showed the capability to inhibit the synthesis of nitric oxide enzymes (iNOS) and cyclooxygenase 2 (COX-2) protein expression. As we all know the nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) play important roles in inflammation. The result showed that ME could inhibit NF-κB activation through the prevention of inhibitor IKK phosphorylation, IκBα degradation and NF-κB nuclear translocation. In addition, ME also inhibited MAPK activation through the prevention of inhibitor ERK, p38, JNK phosphorylation. Based on the above results, we conclude that the AC isolates ME should be through both the inhibition of NF-κB and MAPK pathway to suppress the production of pre-inflammatory substances.

中文摘要…………………………………………………………………I
Abstract.…………………………………………………….…………II
目錄……………………………………………………………………III
表目錄 …………………………………………………………………IX
圖目錄…………………………………………………………………XIV
第一章 前言 ……………………………………………………………1
第二章 文獻回顧 ………………………………………………………3
一、 牛樟芝簡介 …………………………………………………3
1. 生長特徵 ……………………………………….…………3
2. 分類法與命名……………………………………….………4
3. 牛樟芝功效…………………………………….……………5
4. 活性成分………………………………………….…………6
4.1 皮膚修復 ………………………………….….…..…10
4.2 保護肝臟………………………………….….………11
4.3 抗發炎………………………………………..………11
4.4 抗腫瘤………………………………………..………11
二、 發炎活性評估……………………………………..…....…13
1. 巨噬細胞(Macrophage) ………………………..………13
2. 巨噬細胞和Lipopolysaccharide(LPS) ………..….……13
3. 細胞激素(cytokines) ……………………………..……15
4. inducible NOS(iNOS)及Nitric oxide(NO)……….….…15
5. Cyclooxygenase-2(COX-2)和Prostaglandin E2(PGE2)……..……17
6. NF-κB路徑.……………………………………..………17
7. PI3K/AKT路徑…………………………………….……19
8. MAPK路徑…………………………………...…….……19
9. Activating protein-1 (AP-1) ……………………….……21
第三章 研究目的…………………………………….……………...…23
第四章 實驗架構……………………………………….……...………25
第五章 材料方法………………………………………….…...………27
一、 牛樟芝子實體萃取……………………………...….……27
1. 索氏萃取法 .……………………………….…...………27
2. 管住層析法 .……………………………….…...………27
3. HPLC分析 .……………………………………………28
二、 小鼠巨噬細胞珠RAW 264.7……………………………28
1. DMEM細胞培養液配置.………………………………28
2. 細胞型態觀察 .…………………………………………29
3. 冷凍細胞解凍及活化 .…………………………………29
4. 細胞繼代級分盤 .………………………………………30
5. 細胞凍管保存 .…………………………………………31
三、 細胞存活率測試 ………………………………………31
四、 巨噬細胞測量一氧化氮的生成 ………………………32
五、 巨噬細胞內ROS檢測…………………………………33
六、 巨噬細胞測量PGE2的生成 .…………………………34
七、 反轉錄酵素聚合反應 …………………………………35
1. RNA 萃取………………………………………………35
2. RNA 定量………………………………………………36
3. 反轉錄酵素反應 .………………………………………36
4. 聚合連鎖反應 .…………………………………………37
八、 西方墨點法 ……………………………………………38
1. 細胞蛋白質萃取 .………………………………………38
2. 細胞核蛋白質的萃取 …………………….……………38
3. 蛋白質定量 ………………………………….…………39
4. 蛋白質電泳 ……………………….……………………39
5. 蛋白質轉漬 ……………………….……………………40
6. 免疫化學呈色 …………………….……………………41
九、 統計分析 ………………………………………………41
第六章 實驗結果………………………………………………………43
一、 牛樟芝子實體之萃取與存純化………………....…43
1. 牛樟芝子實體萃取.. …………………………………43
2. 管住層析 …………………………….………………43
二、 細胞存活率測試(MTT assay) ………….…….………44
1. PBS溶劑對RAW 264.7 細胞株之存活率影響....…44
2. 牛樟芝子實體管柱分離萃取物 (ME、FK1~FK6 )對RAW 264.7細胞株之存活率影響 ……….…....……44
3. 脂多醣LPS對RAW 264.7細胞株之存活率影響....44
三、 抗氧化測試 ……………………………….……...…45
1. 細胞內自由基之檢測 …………………….…………45
四、 發炎反應之相關物質測試 …………….……………45
1. ME對LPS 誘發RAW 264.7細胞株細胞激素之基因表現………………………………………45
1.1. IL-1β之基因表現.………………….…….……45
1.2. IL-6之基因表現……………………..…….…..46
1.3. TNF-α之基因表現 ...........................................46
2. ME對LPS 誘發RAW 264.7細胞株生成NO之影響……………………………………………………….46
3. ME對LPS 誘發RAW 264.7細胞株生成PGE2之影響……………………………………………………….47
4. ME對LPS 誘發RAW 264.7細胞株發炎之蛋白表現……………………………………………………….47
4.1. iNOS 蛋白表現…………………………..……47
4.2. COX-2蛋白表現………………….……………48
五、 發炎反應之訊息傳遞探討……………………..……48
1. 探討NF-κB之蛋白之表現……………………..…..…48
1.1. p-P65蛋白表現…………………………...……48
1.2. IκBα蛋白表現………………………………....48
1.3. p-IKK蛋白表現………………………...…..…49
2. 探討PI3K/AKT之蛋白之表現………………...…..…49
3. 探討AP-1之蛋白之表現………………………...……50
3.1. p-c-Fos蛋白表現………………………...…….50
3.2. p-c-Jun蛋白表現………………………...…….50
4. 探討MAPK之蛋白之表現…………………......…….50
4.1. p-p38蛋白表現………………………......…….50
4.2. p-ERK蛋白表現……………………......…..…51
4.3. p-JNK蛋白表現…………………….......…..…51
第七章 討論………………………………………………………….73
第八章 結論………………………………………………………….79
第九章 未來展望…………………………….………………………81
第十章 參考文獻…………………………….………………………83

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