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研究生:王前喬
研究生(外文):Wang Chien Chiao
論文名稱:無患子和安息香活性成分抑制嗜中性白血球功能的機轉探討
論文名稱(外文):Inhibition of human neutrophil functions by Sapindus mukorossi and Styrax benzoin
指導教授:黃聰龍黃聰龍引用關係
指導教授(外文):Hwang Tsong Long
學位類別:碩士
校院名稱:長庚大學
系所名稱:天然藥物研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2005
畢業學年度:93
語文別:中文
論文頁數:108
中文關鍵詞:嗜中性白血球無患子安息香
外文關鍵詞:human neutrophilSapindus mukorossiStyrax benzoin
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中文摘要
人類嗜中性白血球產生的活性氧類及顆粒蛋白酶和發炎性疾病有關如:類風濕關節炎、腎絲球腎炎、皮膚病、局部缺血性再灌流損傷、心肌梗塞、慢性肺阻塞疾病和氣喘。抑制嗜中性白血球過度或不適當活化被認為可以改善這些發炎性疾病。然而,在臨床實驗中只有少數有效的藥物能直接調控嗜中性白血球的發炎反應。本論文中主要探討結構為heteragenin-3-O-(3,4-O-di-acetyl-β-D-arabinopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (SM),來自無患子科的天然物(第一單元)與dammarenolic acid (SB)來自安息香的天然物(第二單元)對人類嗜中性白血球產生發炎物質的影響與作用機轉。

第一單元: SM 能以濃度相關性的方式抑制fMLP活化的人類嗜中性白血球 O2.- 的生成和elastase的釋放。此抑制活性不是由細胞毒性而來,因為以SM處置細胞時,並不會引起致乳酸去氫酶釋放出來。SM不具有抗氧化作用和直接清除O2.- 的能力,並且排除是由cAMP路徑而來。在NADPH oxidase活性實驗中,SM明顯地抑制NADPH oxidase組合而不直接改變其活性。SM具有相關性的方式抑制fMLP活化的人類嗜中性白血球所造成的P47PHOX轉移到細胞膜的現象。另一方面,SM會抑制由fMLP刺激活化人類嗜中性白血球所引起的鈣離子濃度增加和p38的磷酸化,但未影響ERK1/2的磷酸化。以上結果表示SM抑制嗜中性白血球呼吸爆炸是經由直接影響NADPH oxidase的組合過程。另外,抑制Ca2+ 與p38磷酸化也共同參與調控抑制O2.-生成和elastase的釋出作用。

第二單元:SB以濃度相關的方式抑制fMLP刺激嗜中性白血球所造成的O2.-生成和elastase釋放現象。此抑制活性不是由細胞毒性而來,因為嗜中性白血球以SB處置時,並不會導致乳酸去氫酶釋放出來。SB 不具有直接清除O2.-及抗氧化的能力,也不會改變NADPH oxidase的活性。SB會抑制由fMLP刺激人類嗜中性白血球所引起的ERK1/2和p38磷酸化。另外,fMLP誘導的鈣離子變化會被SB抑制,但鈣離子ATPase抑制劑thapsigargin的作用則不會被SB抑制。另一方面,SB不會活化AC/cAMP/PDE的訊息路徑,而PKA抑制劑也無法逆轉SB造成的抑制現象。總結以上結果,SB抑制fMLP誘導人類嗜中性白血球產生O2.- 和elastase的作用可能是與ERK1/2及p38磷酸化的減少及阻斷鈣離子流動有關。
English abstract
Reactive oxygen species and granule proteases produced by neutrophils contribute to the pathogenesis of inflammatory diseases, such as rheumatoid arthritis, glomerulonephritis, dermatoses, ischemia-reperfusion injury, myocardial infarction, chronic obstructive pulmonary disease, and asthma. Suppression of the extensive or inappropriate activation of neutrophils has been proposed to ameliorate these inflammatory diseases. There are, in spite of this, only a few currently available agents that directly modulate neutrophil pro-inflammatory responses in clinical practice. In the current study, the anti-inflammatory effects of heteragenin-3-O-(3,4-O-di-acetyl-β-D-arabinopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranoside (SM) isolated from Sapindus mukorossii (Part I) and dammarenolic acid (SB) isolated from Styrax benzoin (Part II) were determined in human neutrophils.
Part Ι: SM was demonstrated to inhibit the generation of O2.- and the release of elastase in formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-activated human neutrophils in a concentration-dependent fashion. This inhibitory effect was not due to cytoxicity because culturing with SM did not cause lactate dehydrogenase release. SM did not have antioxidant and superoxide anion-scavenging ability, and it failed to alter the cAMP-dependent pathways. SM significantly inhibited the assembly of NADPH oxidase, but not directly altered the activity of NADPH oxidase, in a reconstituted system. Furthermore, SM decreased the membrane-associated p47phox in fMLP-stimulated human neutrophils in a concentration-dependent fashion, suggesting that the translocation of p47phox mediates the inhibition of O2.- release. On the other hand, SM inhibited calcium mobilization and p38 but not ERK1/2 phosphorylation in fMLP-activated human neutrophils. In summary, these results indicate that the inhibition of O2.- generation by SM is through a direct effect on NADPH oxidase assembly. In addition, the inhibition of Ca2+ mobilization and p38 MAPK phosphorylation is also associated with an decrease in the generation of O2.- and the release of elastase.
PartⅡ: SB concentration-dependently inhibited fMLP-induced O2.- generation and elastase release in human neutrophils. This inhibitory effect was not due to cytoxicity because culturing with SB did not cause lactate dehydrogenase release. SB did not display antioxidant and O2.--scavenging ability, as well as it failed to alter the subcellular NADPH oxidase activity. SB inhibited the phosphorylation of p38 and ERK1/2 MAPK in fMLP-activated human neutrophils. Moreover, fMLP- but not calcium ATPase inhibitor thapsigargin-induced calcium mobilization was inhibited by SB. In contrast, SB did not alter the AC/cAMP/PDE pathways, and the protein kinase A inhibitor failed to reverse the SB-caused inhibition. In summary, these data indicate that the inhibition of O2.- generation and elastase release by SB in fMLP-activated human neutrophils can probably be attributed to the decrease of p38 and ERK1/2 MAPK phosphorylation and the blockade of calcium mobilization.
目 錄
指導教授推薦書
口試委員會審定書
博碩士指本論文著作授權書……………………………………………………….. Ⅰ
誌謝………………………………………………………………………………….. Ⅱ
目錄……………………………………………………………………………… 1
縮寫表……………………………………………………………………………… 2
中文摘要…………………………………………………………………………. 5
英文摘要………………………………………………………………………… 7
緒論……………………………………………………………………………… 9
實驗材料與方法………………………………………………………………….. 22
Part Ⅰ: 無患子活性成分SM抑制嗜中性白血球機轉之探討………………… 31
結果………………………………………………………………………… 32
討論……………………………………………………………………… 37
圖表……………………………………………………………………… 40
Part Ⅱ: 安息香活性成分SB抑制嗜中性白血球機轉之探討………………… 60
結果……………………………………………………………………………. 61
討論……………………………………………………………………………. 65
圖表…………………………………………………………………………… 67
參考文獻…………………………………………………………………….. 84
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