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研究生:蔡萱亭
研究生(外文):Shan-Tin Tsai
論文名稱:中藥黃芩及化合物Wogonin在人類肝癌細胞中的抗發炎機制之探討
論文名稱(外文):Mechanistic Studies of Anti-inflammatory Activity of Scutellariae radix and Wogonin in Human Hepatoma Cells
指導教授:葉小帆葉小帆引用關係
指導教授(外文):Sheau-Farn Yeh
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:中文
論文頁數:78
中文關鍵詞:間白素趨化細胞素
外文關鍵詞:IL-1βcc-chemokinesNF-κB
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IL-1β及CC-chemokines皆為促發炎的細胞激素,在許多慢性發炎疾病當中扮演重要的角色。而CC-chemokines為 IL-1β 的下游基因,受到 IL-1β 的刺激而表現,進而促使免疫細胞移動至受感染的位置,產生免疫發炎反應。本實驗室過去研究已知中草藥黃芩及其成份 wogonin 有抑制B型肝炎病毒的作用,在此研究中,利用 IL-1β 在肝癌細胞株Huh7中誘發CC-chemokines MIP-1β 之篩選模式,來研究中藥-黃芩及黃芩中的化合物 wogonin 是否具有抗發炎活性。本論文主要探討黃芩如何能夠有效抑制 IL-1β 所活化之 MIP-1β 的基因表現, 及其抑制調控此發炎相關基因的轉錄因子 c-jun 和 NF-κB 的作用機制。
為了瞭解黃芩及 wogonin 抑制人類肝癌細胞 Huh7 中 IL-1β 所活MIP-1β表現作用的分子機制。透過處理不同的MAPK及NF-κB 抑制劑時,發現IL-1β是透過活化JNK路徑及NF-κB路徑而促使MIP-1β基因表現;進一步利用西方墨點法研究wogonin及黃芩對於IL-1β所活化之訊息傳遞路徑的影響,發現wogonin與黃芩的作用相同,不影響IL-1β所活化之c-jun蛋白質表現及c-jun蛋白質之磷酸化作用,但是會影響IL-1β所活化之p38蛋白質磷酸化作用及抑制以IL-1β刺激和大量表現p65時所誘發之NF-κB啟動子活性。
雖然p38路徑並非調控MIP-1β表現的因子,但p38路徑與NF-κB也皆為掌控發炎基因之重要因子。
為了釐清wogonin及黃芩是如何調控NF-κB的活性,針對(1) NF-κB的進核 (2) NF-κB的轉錄活性 (3) NF-κB的DNA結合活性做進一步的探討。利用免疫螢光染色及以細胞核萃取物以西方墨點法分析p65在細胞當中的分佈情形,發現wogonin及黃芩並不影響IL-1β所活化之NF-κB進核步驟;使用one hybrid assay實驗分析,判別wogonin及黃芩對於p65轉錄活性的影響,在細胞株中轉染含有p65 transactivation domain及Gal4 DNA binding domain的融合蛋白,利用Gal4 response element 的活性,得知wogonin及黃芩會抑制p65之轉錄活性;並利用在凝膠電泳遷移實驗分析,在大量表現p65時wogonin及黃芩同樣都會抑制p65結合在MIP-1β啟動子上,因此當p65大量表現時,並無法回復wogonin及黃芩對MIP-1β啟動子及訊息核醣核酸的抑制作用。
在本研究中,探討黃芩及其中有效成份wogonin抗發炎活性的作用機制,得知黃芩及其成份wogonin能透過抑制NF-κB的轉錄活性及DNA結合的活性而降低IL-1β下游的發炎相關基因表現,進而抑制IL-1β所誘發之發炎反應。黃芩及wogonin除了能對NF-κB活性有抑制作用外,對p38路徑也有抑制作用,因此wogonin及黃芩除了在肝臟發炎疾病外,在其它發炎相關疾病當中具有開發的潛力。 wogonin及黃芩同時具有抗B肝病毒及抗發炎的功效,比起其它只有單一療效的化合物,更具有發展的前景。

IL-1β and CC-chemokines are proinflammatory cytokines, play an essential role in many chronic inflammatory disease. CC-chemokines are regulated by IL-1β, which triggers the invasion of immune cells into a site of infection. In previous study from our laboratory, it has been found that Chinese herbal medicine Scutellariae radix and one of its constituent wogonin possess anti-HBV activity. In this study, IL-1β-induced CC-chemokines MIP-1β expression in Huh7 cells was selected as a cell model to study the anti-inflammatory activity of Scutellaria radix and one of its constituent wogonin. Firstly, Scutellaria radix and wogonin suppression of IL-1β-induced MIP-1β gene expression were examined. Secondly, to demonstrate the inhibition of IL-1β-induced MIP-1β gene expression was using MIP-1β promoter transcription factors, c-jun and NF-κB.
Wogonin and Scutellariae radix suppressed IL-1β-induced MIP-1β expression in Huh7 cells. In order to investigate the molecular mechanism of wogonin and Scutellariae radix, quantitative-PCR analysis of the MAPK inhibitors and NF-κB inhibitor revealed that IL-1β-induced MIP-1β expression through activating JNK and NF-κB signaling pathway. Utilizing Western blotting, it was found that wogonin and Scutellariae radix had no effect on IL-1β-induced c-jun protein expression and phosphorylation level, whereas wogonin and Scutellariae radix suppressed IL-1β-induced p38 phosphorylation level and p65-induced NF-κB activation.
NF-κB and p38 pathway played central role in regulating the expression of a variety of genes that control immune responses, even if p38 pathway had nothing to do with MIP-1β expression. To examine how wogonin and Scutellariae radix regulate NF-κB activation, focused on (1) NF-κB translocation; (2) NF-κB transactivation activity; (3) NF-κB DNA binding activity. Using immunofluorescence and Western blotting to analyze the distribution of p65 protein, wogonin and Scutellariae radix did not inhibit IL-1β-induced NF-κB nuclear translocation; using one hybrid assay to analysis p65 transactivation activity, Huh7 cells were transiently transfected with a 5×Gal4 response elements-luciferase reporter gene, ectopic expression of a fusion protein containing the DNA binding domain of Gal4 and transaactivation domains of p65 could up regulate reporter gene, wogonin and Scutellariae radix exhibited inhibition on transactivation activity of p65. Lastly, Electrophoretic mobility shift assay (EMSA) revealed that wogonin and Scutellariae radix reduced NF-κB binding to theκB site on MIP-1β promoter when p65 was overexpressed. Therefore, ectopic expression of p65 couldn’t abolish the suppressive effect of wogonin and Scutellariae radix on p65-induced MIP-1β promoter activation and mRNA expression.
In this study, the molecular mechanism on Scutellariae radix and wogonin regulated anti-inflammatory activity were focused, and it was found that wogonin and Scutellariae radix blocked IL-1β-induced inflammation through NF-κB transactivation and suppression of DNA-binding activity. Besides NF-κB activity, wogonin and Scutellariae radix also inhibited IL-1β-induced p38MAPK phosphorylation level. For this reason, except chronic hepatic inflammation disease, wogonin and Scutellariae radix are potential candidates for drug development to suppress other inflammatory disease.
The dual function of wogonin and Scutellariae radix in hepatic cells, including anti-HBV activity and anti-inflammatory activity, have more potential to be developed into therapeutic agents for HBV-related hepatic inflammatory diseases than drugs that only possess single function.

中文摘要1-2
英文摘要3-4
壹、緒論5-10
貳、材料與方法11-22
參、結果23-32
肆、討論33-37
伍、參考文獻38-42
陸、圖表43-78

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