臺灣博碩士論文加值系統

Eupafolin是一種類黃酮的物質，經由鴨舌癀Phyla nodiflora. (Verbenaceae) 此中草藥萃取而來。類黃酮通常是用來治療發炎、癌症及一些經細菌感染等的疾病。然而，Eupafolin的抗發炎效果及其作用機轉都尚未十分清楚，因此本篇的主要研究目的是利用老鼠的巨噬細胞 (RAW 264.7) 及小鼠腳掌與肺臟經脂多醣體 (LPS) 刺激發炎產生後，進一步觀察及釐清 Eupafolin 的抗發炎效果及作用機制。首先在in vitro方面，使用RAW 264.7細胞來進行體外實驗；以LPS作為刺激劑，作用於RAW 264.7細胞，來誘發發炎反應的產生，初步結果發現Eupafolin的預先作用能夠有效的降低LPS所誘發的inducible nitric oxide synthase (iNOS) 及cyclooxygenase (COX-2) 表現與NO的產生。藉由西方墨點法及免疫細胞化學染色法的分析，發現Eupafolin 是藉由抑制 LPS 所誘發的MAPKs中的c-Jun NH2-terminal kinase (JNK) 磷酸化的表現，與nuclear factor-kappa B (NF-κB) 及activator protein-1 (AP-1) 這兩種轉錄因子的活化來達到抗發炎的作用。除此之外，在in vivo 實驗方面，經由組織染色的實驗結果也發現，相較於 LPS 單獨注射的刺激組，Eupafolin的預先投予，能有效的降低LPS誘發小鼠後腳掌所產生的發炎症狀-嗜中性球的浸潤現象和腳掌水腫，在肺臟誘發發炎方面，Eupafolin能有效減緩LPS誘發小鼠肺臟所產生的組織發炎、充血和巨噬細胞、嗜中性白血球、淋巴球等發炎細胞聚集的現象。由以上實驗結果推論，Eupafolin可能主要是藉由調降JNK、NF-κB和AP-1等訊息傳遞路徑，抑制發炎因子COX-2、iNOS、NO的表現。綜合上述，此次實驗結果發現Eupafolin 提供了另一種新潁且有效治療發炎相關疾病的藥物選擇。

Eupafolin, a flavonoid isolated from Phyla nodiflora.(Verbenaceae), has been used traditionally to treat disease such as inflammation, cancer and infections by fungi, bacteria, and viruses. However, its mechanism remains unknown. The aim of this study was to evaluate the anti-inflammatory effects and underlying the regulatory mechanisms of the eupafolin on Lipopolysaccharide (LPS)-stimulated RAW 264.7 cells in vitro and mouse paw, lung in vivo. First at all, in in vitro study, eupafolin significantly decreased LPS-induced NO production and expressions of iNOS and COX-2 in RAW 264.7 cells. Furthermore, eupafolin suppressed NF-κB p65 translocation and activator protein-1 (AP-1) activation as well as NH (2)-terminal kinase (JNK) phosphorylation. In in vivo assay, eupafolin reduced the neutrophil infiltration, edema and inflammatory mediators expression in LPS-induced mouse paw and lung. The present results showed that eupafolin exerts its anti-inflammatory effects through the decrease of inflammatory mediators, expression by suppressing pJNK, NF-κB and AP-1 signaling pathways. Taken together, our results indicate that eupafolin may provide a useful therapeutic approach for inflammation-associated diseases.

目錄
口試委員會審定書 i
致謝 …… ii
中文摘要 …… iii
英文摘要 …… iv
目錄 …… v
圖目錄 …… viii
壹、 緒論 …… 1
一、引言 1
二、發炎的成因 1
三、巨噬細胞與發炎之關係 2
四、內毒素與發炎之關係 2
五、NO、iNOS、COX-2表現與發炎之關係 3
六、Mitogen Activated Protein Kinases、Akt 與發炎之關係 4
七、NF-κB、AP-1與發炎之關係 5
八、Eupafolin之藥理性質與應用 6
九、研究動機 6
貳、 實驗材料 7
叁、 實驗方法 14
一、細胞實驗 (in vitro) 14
1.細胞培養 (Cell culture) 14
2.細胞活性分析 (MTT assay) 14
3. 西方墨點分析法 (Western blotting). 14
4. 免疫細胞螢光染色法 (Immunocytochemistry; ICC) 17
5. Cytokine Array 17
6. 一氧化氮檢測試驗 (NO test; Griess assay) 18
7. 動物實驗 18
8. 蘇木紫- 伊紅染色 (Hematoxylin-eosin staining) 20
9. 支氣管肺泡灌洗液 (bronchoalveolar lavage fluid; BALF) 21
10. 流式細胞儀分析 (Flow cytometry analysis) 21
11. 免疫組織化學染色 (Immunohistochemistry; IHC) 22
肆、 實驗結果 25
一、細胞實驗 (in vitro study) 25
1. 利用細胞活性分析法觀察LPS對RAW 264.7之影響 25
2. 利用細胞活性分析法觀察eupafolin對RAW 264.7細胞之影響. 25
3. 利用西方墨點法分析LPS對RAW 264.7細胞的COX-2之表現 25
4. Eupafolin藉由調控RAW 264.7細胞中發炎相關蛋白COX-2之表現達到抑制經LPS誘發發炎的作用 26
5. Eupafolin藉由調控RAW 264.7細胞發炎相關蛋白iNOS和NO之表現達到抑制經LPS誘發發炎的作用 26
6. Eupafolin藉由調控RAW 264.7細胞發炎相關cytokines和chemokines之表現達到抑制經LPS誘發發炎的作用 27
7. 在RAW 264.7細胞中eupafolin透過抑制MAPKs及Akt活化達到抑制LPS經誘發發炎的作用 28
8. Eupafolin在經LPS誘發發炎狀況的RAW 264.7細胞中抑制磷酸化MAPKs蛋白質表現 28
9. Eupafolin能透過抑制AP-1和NF-κB進入RAW 264.7細胞核內，達到抗發炎效果 29
二、動物實驗 (in vivo study) 30
1. Eupafolin減緩老鼠腳掌組織經LPS刺激所誘發的發炎反應…………… 30
2. Eupafolin藉由調控LPS誘發之巨噬細胞聚集所產生發炎相關蛋白COX-2、iNOS之表現達到抑制老鼠腳掌發炎的作用………. 31
3. Eupafolin減緩老鼠肺臟組織經LPS刺激所誘發的發炎反應 31
4. Eupafolin調控受LPS刺激的老鼠支氣管肺泡灌洗液中的發炎細胞表達 31
5. Eupafolin藉由調控LPS誘發之巨噬細胞聚集所產生發炎相關蛋白COX-2、iNOS之表現達到抑制老鼠肺臟發炎的作用 32
伍、 討論 -----------------------------------------------------------------------------------------33
陸、 參考文獻------------------------------------------------------------------------------------37
柒、 附圖------------------------------------------------------------------------------------------42


圖目錄
圖. 1 Eupafolin的結構式 42
圖. 2 利用細胞活性分析法觀察LPS對RAW 264.7細胞之影響 43
圖. 3 利用細胞活性分析法觀察eupafolin對RAW 264.7細胞之影響 44
圖. 4 LPS對RAW 264.7細胞中COX-2之表現 45
圖. 5 Eupafolin藉由調控RAW 264.7細胞發炎相關蛋白COX-2之表現達到抑制經LPS誘發發炎的作用 46
圖. 6 以免疫細胞螢光染色法觀察eupafolin及LPS 對COX-2 之表現影響 47
圖. 7 Eupafolin藉由調控RAW 264.7細胞中發炎相關蛋白iNOS和NO之表現達到抑制經LPS誘發發炎的作用 48
圖. 8 Eupafolin 藉由調控RAW 264.7細胞發炎相關cytokines和chemokines之表達抑制經LPS誘發發炎的作用 50
圖. 9 在RAW 264.7細胞中LPS經活化MAPKs及Akt訊息傳遞路徑誘發發炎的作用 51
圖. 10 Eupafolin在經LPS誘發發炎狀況的RAW 264.7細胞中抑制磷酸化 MAPKs蛋白質表現 52
圖. 11 Eupafolin在經LPS誘發發炎狀況的RAW 264.7中抑制磷酸化MAPKs蛋白質表現 53
圖. 12 Eupafolin能透過抑制NF-κB進入RAW 264.7細胞核內， 達到抗發炎效果 54
圖. 13 Eupafolin能透過抑制NF-κB位移進入RAW 264.7細胞核內，達到抗發炎效果 55
圖. 14 Eupafolin能透過抑制AP-1進入RAW 264.7細胞核內，達到抗發炎效果 56
圖. 15 Eupafolin減緩老鼠腳掌組織經LPS刺激所誘發的發炎反應 57
圖. 16 Eupafolin藉由調控LPS誘發之巨噬細胞聚集所產生發炎相關蛋白COX-2、iNOS之表現達到抑制老鼠腳掌發炎的作用 58
圖. 17 Eupafolin減緩老鼠肺臟組織經LPS刺激所誘發的發炎反應 59
圖. 18 Eupafolin調控受LPS刺激的老鼠支氣管肺泡灌洗液中的發炎細胞 表達 60
圖. 19 Eupafolin藉由調控老鼠肺臟發炎相關蛋白COX-2之表現達到抑制經LPS誘發發炎的作用 62
圖. 20 Eupafolin藉由調控老鼠肺臟發炎相關蛋白iNOS之表現達到抑制經LPS誘發發炎的作用 63
圖. 21 細胞模式圖 64

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