川陳皮素的代謝產物3,4-dihydroxy-5,6,7,8-tetrametho...

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Title:	川陳皮素的代謝產物3,4-dihydroxy-5,6,7,8-tetrametho... Nobiletin metabolite, 3,4-dihydroxy-5,6,7,8-tetramethoxyflavone, inhibits LDL oxidation and suppresses scavenger receptor expression in THP-1 macrophages
Authors:	駱雅萱
Contributors:	嘉南藥理科技大學：生物科技系暨研究所吳明娟
Keywords:	DiI-acLDL CD36 SR-A DiI-oxLDL 清道夫受體 (scavenger receptor) 多甲氧基類黃酮 (polymethoxylated flavone) 低密度脂蛋白 (low-density lipoproteinLDL) 粥樣動脈硬化生成 (atherogenesis CD36 PPARg scavenger receptor A (SR-A) DiI-acLDL atherogenesis DiI-oxLDL low-density lipoprotein (LDL)
Date:	2009
Issue Date:	2011-10-27 14:43:15 (UTC+8)
Abstract:	流行病學研究報告指出，攝取富含類黃酮的食物可減低心血管疾病的罹患率。本論文的目的為探討柑橘的多甲氧基類黃酮 (polymethoxylated flavone, PMF) 川陳皮素 (nobiletin, NOB) 及其代謝產物3',4'-dihydroxy- 5,6,7,8-tetramethoxyflavone (DTF) 在預防粥樣動脈硬化 (atherosclerosis) 上的功效及其分子機轉。本論文主要探討此兩種多甲氧基類黃酮對於低密度脂蛋白 (low density lipoprotein, LDL) 的氧化修飾、單核球分化成巨噬細胞 (monocyte-to-macrophage) 以及脂泡細胞形成 (foam cell formation) 等三個階段的抑制功效。　　本研究首先在試管中利用銅離子 (Cu2+) 誘導LDL氧化時會伴隨生成共軛雙烯 (conjugated diene) 和硫代巴比妥酸反應物 (TBARS) 及apolipoprotein B電荷改變的特性，以分光光度法 (spectophotometry) 及脂質電泳，評估此兩種多甲氧基類黃酮的抗氧化能力。結果顯示DTF因為B環為鄰苯二酚 (catechol) 結構，因此比相同濃度的母體化合物 (parent compound) NOB及對照組DL--tocopherol抑制LDL氧化的效果顯著為佳。本研究第二部份接著探討此兩種多甲氧基類黃酮在THP-1單核球分化成巨噬細胞階段是否具有抑制功效。反轉錄定量聚合酶連鎖反應 (RT-Q-PCR) 的結果顯示，DTF可有效並隨劑量顯著地降低PMA (30 nM) 所誘導的分化指標基因 (differentiation marker)-CD11b，及清道夫受體 (scavenger receptors) 包括 SR-A、CD36與lectin-like oxidized LDL receptor-1 (LOX-1) 等基因的mRNA表現。然而，NOB卻僅對LOX-1的mRNA表現有抑制功效。進一步以流式細胞儀分析細胞表面CD36及SR-A蛋白的表現也有相似的結果。　　OxLDL也可誘導THP-1單核球在低濃度PMA (1 ng/ml) 條件下，分化成巨噬細胞。RT-Q-PCR的結果顯示，DTF雖然可顯著抑制oxLDL誘導的清道夫受體的mRNA表現，但是卻無法抑制CD11b的表現。因此可知，PMA與oxLDL所誘導單核球分化成巨噬細胞的機轉並不完全相同。雖然已知oxLDL是PPAR的促效劑，可啟動CD36的轉錄。但添加PPAR的拮抗劑antagonist)-GW9662並無法抑制CD36的表現，此結果顯示，除了PPAR的活化之外，還有其他分子機轉參與oxLDL所誘導的單核球的分化。　　螢光顯微鏡的觀察及流式細胞儀的分析結果也發現，DTF可有效減少PMA所誘導THP-1單核球對DiI標示 (labeled) 的oxLDL及乙醯化低密度脂蛋白 (acLDL) 的吞噬，且抑制功效隨濃度遞增而變強。而NOB雖然對CD36及SR-A的表現無抑制效果，卻也能抑制修飾型低密度脂蛋白的吞噬。　　本研究最後一部份探討此兩種多甲氧基類黃酮對於THP-1所衍生的巨噬細胞的清道夫受體的表現，及吞噬修飾型LDL的能力的影響。RT-Q-PCR的實驗結果顯示DTF比NOB更顯著的減少了清道夫受體CD36與SR-A的mRNA表現，流式細胞儀的結果也支持DTF能顯著降低巨噬細胞吞噬修飾型LDL的能力。轉錄分析的結果則顯示，DTF可以抑制CD36基因表現的降低的原因，可能有部分是由於PPAR活性被抑制而造成。綜合以上結果，NOB的代謝產物DTF不僅具強抗氧化力，也能抑制單核球分化過程衍生的、及巨噬細胞上的清道夫受體的基因表現，並進而減低修飾型LDL的吞噬，因此具有防止脂泡細胞生成的幼纂C There is accumulating evidence that LDL oxidation is essential for atherogenesis, and antioxidants that prevent oxidation may either decelerate or reduce atherogenesis. Current results demonstrated that 3',4´dihydroxy-5,6,7,8- tetramethoxyflavone (DTF), the major metabolite of nobiletin (a citrus polymethoxylated flavone), effectively inhibited Cu2+-mediated LDL oxidation. This inhibition was assessed by the decrease in the related parameters: conjugated dienes, thiobarbituric acid reactive substances and relative electrophoretic mobility (REM). Monocyte-to-macrophage differentiation plays a vital role in early atherogenesis. The differentiation of monocytic THP-1 cell line was induced by phorbol 12-myristate 13-acetate (PMA; 30 nM), a potent activator of PKC, or PMA (1.6 nM) in combination with oxLDL (50 g/ml) for 24 h. Treatment of THP-1 monocytes with DTF (10-20 M) dose-dependently attenuated differentiation along with the reduced gene expression of scavenger receptors, CD36, SR-A and LOX-1. Incubation of monocyte-derived macrophages with DiI-modified LDL (acetylated LDL and oxidized LDL, 20 g/ml) led to lipoprotein uptake and the addition of DTF (10-20 M) blunted LDL uptake as shown by flow cytometry and fluorescence. Transactivation analyses revealed that the inhibition of PMA-stimulated monocyte differentiation by DTF was mediated at least in part through interference with PPARγ. In conclusion, the combination of antioxidant and inhibition of monocyte differentiation as well as foam cell formation properties of DTF, makes it especially beneficial against atherogenesis.
Relation:	校內校外均不公開，學年度：97，94 頁
Appears in Collections:	[Dept. of Biotechnology (including master's program)] Dissertations and Theses

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