臺灣博碩士論文加值系統

嗜中性白血球 (neutrophils) 是人體免疫的第一道防線，亦是人體產生發炎反應的重要推手。當嗜中性白血球被活化時，會產生大量的超氧陰離子 (superoxide anion) 並釋放彈性蛋白酶 (elastase) 以毒殺外來的病原菌，但同時也會對周邊的組織產生傷害。目前已知有些發炎疾病與嗜中性白血球的不當調控有關。在中醫理論中，「熱證」是一種發炎反應的表現，其中「熱證」又可以分為慢性的「虛熱證」與急性的「實熱證」。臨床上，兩種「熱證」會選用不同性質的藥物，而黃耆是治療「虛熱證」最具代表性的藥物，但是使用在「實熱證」會擔心引起疾病惡化。本研究使用的黃耆萃取物 (ASWE)，是用傳統的煎煮方法萃取，作為主要實驗藥品，藉以探討 ASWE 是否對於嗜中性白血球的活化有抑制作用。研究發現，ASWE 可以抑制 N-formyl-L-methionyl-L-leucyl-L-phenylalanine 刺激嗜中性白血球活化產生的超氧陰離子、活性氧分子以及釋放彈性蛋白酶的作用。ASWE 不具有直接的細胞毒性。在無細胞的系統中，ASWE 可以清除活性氧分子，但並沒有直接清除超氧陰離子的能力。此外，ASWE 會減少活化態嗜中性白血球 CD11b 的表現並且減少黏連作用 (adhesion)。ASWE 不會影響活化態嗜中性白血球細胞內鈣離子的濃度，也不會影響 Akt、p38 與 JNK 的磷酸化。綜合本研究的結果顯示，ASWE 有潛力減少嗜中性白血球發炎反應造成的氧化傷害。因此，我們建議黃耆可以擴大其適應症：可以應用在嗜中性白血球引起的發炎疾病。

Neutrophils are the first line of defense of human immunity and play an important role in inflammatory reaction. When neutrophils are activated, they release superoxide anion and elastase to destroy pathogens. However, superoxide anion and elastase also damage surrounding tissues. It is well known that some kinds of inflammatory diseases are related to inappropriate regulation of neutrophils. In traditional Chinese medicine theory, “heat pattern” represents inflammatory reaction which can be divided to two types: “vacuity heat pattern” as chronic inflammation and “repletion heat pattern” as acute inflammation. Different medications are used for the two kinds of “heat pattern”. Astragalus is the most representative drug to treat “vacuity heat pattern”. However, using astragalus to treat “repletion heat pattern” is considered to worse the condition. In this study, we used traditional decocting method to make Astragalus mebranaceous water extract (ASWE), and investigated the anti-inflammatory effects in human neutrophils. The results showed ASWE inhibited superoxide anion generation, reactive oxygen species (ROS) production, and elastase release in N-formyl-L-methionyl-L-leucyl-L-phenylalanine activated human neutrophils. ASWE did not have cytotoxic effect in human neutrophils. In cell-free systems, ASWE showed ROS but not superoxide anion scavenging effect. In addition, ASWE repressed CD11b expression and adhesion response in activated human neutrophils. However, ASWE failed to alter Ca2+ mobilization and phosphorylation of Akt, p38 and JNK. This study demonstrates that ASWE may have potential to reduce oxidative damage in neutrophilic inflammatory response. Therefore, we suggest that Astragalus membranaceus may expand its clinical application to treat neutrophil-related inflammatory diseases.

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論文口試委員審定書
致謝.....iii
中文摘要.....iv
Abstract...v
目錄.....vi
縮寫對照表...x
表目錄....xii
圖目錄....xiii
附圖目錄.....xv
第一章 緒論.1
1.1.　研究動機.....1
1.2.　人類嗜中性白血球的活化.3
1.3.　受活化嗜中性白血球引起發炎反應的機制......4
1.3.1.　呼吸爆破作用 (Respiratory Burst).......5
1.3.2.　去顆粒化作用 (Degranulation)...6
1.3.3.　嗜中性白血球細胞外網狀結構 (Neutrophil extracellular traps) ...7
1.4.　嗜中性白血球在免疫反應的訊號傳遞路徑......8
第二章 實驗材料與方法.....16
2.1.實驗材料........16
2.1.1.實驗藥品來源.....16
2.1.2.實驗藥品黃耆的藥材來源.17
2.1.3.黃耆水萃取物的製備方法與產率.......17
2.1.4.黃耆水萃取物高效能液相層析 (HPLC).......18
2.1.5.黃耆水萃取物中黃耆甲苷定量分析 (LC MS/MS)18
2.2.實驗方法........21
2.2.1.人類嗜中性白血球製備......21
2.2.2.細胞存活率 (Cell viability)......21
2.2.3.超氧自由基 (Superoxide anion, O2·–) 釋放之測定22
2.2.4.彈性蛋白酶 (Elastase) 釋放之測定......24
2.2.5.細胞毒性測試 Lactate dehydrogenase (LDH).25
2.2.6.直接清除自由基能力之測定......26
2.2.7.細胞表面 CD11b 表現測定.29
2.2.8.細胞黏附作用 (adhesion) 測試....30
2.2.9.細胞移行作用 (migration) 測試....30
2.2.10.細胞內鈣離子濃度（[Ca2+]i）測定..31
2.2.11.西方墨點法 (Western Blotting).....32
2.2.12.統計方法...33
第三章 實驗結果 34
3.1.黃耆水萃取物 (ASWE) 顯著抑制 fMLF 活化後的人類嗜中性白血球生成超氧陰離子與釋放彈性蛋白酶。......34
3.2.黃耆水萃取物 (ASWE) 抑制嗜中性白血球的活性並非經由細胞毒性而來。.....34
3.3.黃耆水萃取物 (ASWE) 能夠顯著抑制 fMLF 活化後人類嗜中性白血球細胞內及細胞外超氧自由基的生成。..35
3.4.黃耆水萃取物 (ASWE) 具有選擇性清除 AAPH 氧屬自由基的能力。..36
3.5.黃耆水萃取物 (ASWE) 顯著抑制 MMK1 活化後的人類嗜中性白血球生成超氧陰離子與釋放彈性蛋白酶。......39
3.6.黃耆水萃取物 (ASWE) 不影響 PMA 及 m-3M3FBS 活化後的人類嗜中性白血球生成超氧陰離子與釋放彈性蛋白酶。......40
3.7.黃耆水萃取物 (ASWE) 顯著抑制 PAF、LTB4及IL-8 活化後的人類嗜中性白血球釋放彈性蛋白酶的能力。......41
3.8.黃耆水萃取物 (ASWE) 減少 fMLF 活化後人類嗜中性白血球細胞表面 CD11b 的表現量。.41
3.9.黃耆水萃取物 (ASWE) 減少 fMLF 活化後人類嗜中性白血球的黏附作用 (adhesion)。.42
3.10.黃耆水萃取物 (ASWE) 不影響被活化的人類嗜中性白血球細胞內 Ca2+ 的濃度變化。.....42
3.11.黃耆水萃取物 (ASWE) 會輕微抑制 ERK 的磷酸化，但不影響 Akt、p38、JNK 的磷酸化。.43
第四章 討論 ........44
第五章 圖表........53
附圖..............86
參考文獻...............90

表目錄
Table 1. ASWE inhibits superoxide anion generation and elastase release in different stimulants activated human neutrophils.....53

圖目錄
Figure 1. Astragaloside IV and ASWE chromatogram by LC MS/MS......54
Figure 2. ASWE inhibits superoxide anion generation and elastase release in fMLF activated human neutrophils...56
Figure 3. ASWE does not induce LDH release in human neutrophils......58
Figure 4. ASWE inhibits ROS generation in activated human neutrophils in luminol-enhanced chemiluminescence assay........61
Figure 5. ASWE inhibits intracellular ROS generation in fMLF activated human neutrophils........ ......63
Figure 6. ASWE does not have superoxide anion scavenging ability in a cell-free xanthine/xanthine oxidase system........65
Figure 7. ASWE does not affect uric acid production in a cell-free xanthine/xanthine oxidase system...66
Figure 8. ASWE does not have reactive nitrogen species scavenging ability in cell-free assays...68
Figure 9. Oxygen radical absorbance capacity of ASWE in a cell-free system.71
Figure 10. ASWE inhibits superoxide anion generation and elastase release in MMK1 activated human neutrophils...73
Figure 11. ASWE does not inhibit superoxide anion generation and elastase release in m-3M3FBS activated human neutrophils......75
Figure 12. ASWE does not inhibit superoxide anion generation in PMA activated human neutrophils........ .......76
Figure 13. ASWE inhibits elastase release in PAF, LTB4 or IL-8 activated human neutrophils........ .......78
Figure 14. ASWE inhibits CD11b expression in fMLF activated human neutrophils...80
Figure 15. ASWE represses adhesion of fMLF activated human neutrophils to bEnd 3.1 cells........82
Figure 16. ASWE fails to affect intracellular Ca2+ mobilization in fMLF activated human neutrophils.......83
Figure 17. The effects of ASWE on phosphorylation of Akt and MAPKs in fMLF activated human neutrophils.......85

附圖目錄
附圖.......86
附圖1. Neutrophil recruitment to sites of inflammation..86
附圖2. Structure and activation of NADPH oxidase......87
附圖3. Neutrophil degranulation87
附圖4. Formation of neutrophil extracellular traps........88
附圖5. Signaling pathways of GPCRs involved in inflammation.....88
附圖6. The ERK, p38, and JNK MAP kinase cascades89
附圖7. Astragalus membranaceus sample.89

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