黃同醇抑制動脈粥樣硬化機轉之探討

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標題:	黃同醇抑制動脈粥樣硬化機轉之探討 Anti-atherogenic mechanism of flavonols
作者:	連子偉 Tzi-wei Lian
貢獻者:	吳明娟嘉南藥理科技大學：生物科技研究所
關鍵字:	清道夫受體動脈粥樣硬化 flavonols CD36
日期:	2007
上傳時間:	2008-12-03 11:16:39 (UTC+8)
摘要:	動脈粥樣硬化是個複雜的慢性發炎的疾病，也是造成血管栓塞、中風與心肌梗塞等心血管疾病之元兇。低密度脂蛋白之氧化修飾是引發動脈粥樣硬化的早期關鍵步驟。血管粥狀硬化的病灶(atherosclerotic lesion)-脂肪條紋是因為血管內膜的巨噬細胞透過清道夫受體吞噬氧化的低密度脂蛋白，進而轉變成脂泡細胞，堆積於血管內膜所造成的。CD36是清道夫受體B群的一員，主要分佈於巨噬細胞上，專司辨識與吞噬氧化型低密度脂蛋白。CD36的表現會因為過氧化體增生活化受體(PPAR之配位體(ligands)，例如15-deoxy-12,14-prostaglandin J2 (15dPGJ2)的存在而活化。本研究之目的有二: 前半段以體外實驗探討非瑟酮(fisetin)、山奈酚(kaempferol)、桑色素(morin) 、楊梅素(myricetin) 以及槲皮素(quercetin)抑制低密度脂蛋白氧化的活性。結果發現五種黃酮醇皆可有效抑制銅離子誘導的LDL氧化所產生的丙二醛(MDA)與apolipoprotein B表面電荷的改變，並且延緩共軛雙烯(conjugated diene)的生合成。其中以非瑟酮(fisetin) 以及槲皮素(quercetin)之抑制效果最好，顯示B環的3’, 4’ di-OH (catechol)可能參與銅離子的螯合。第二部份主要探討黃酮醇是否能抑制U937單核球細胞分化為巨噬細胞及吞噬ox-LDL的能力與CD36表現的影響。結果發現，黃酮醇中山奈酚(kaempferol)具有減少U937單核球細胞分化為巨噬細胞的能力(p＜0.01)。槲皮素(quercetin)則是會增加U937單核球細胞衍生為巨噬細胞之比例及單核球上清道夫受體CD36之表現( p＜0.01)。非瑟酮(fisetin)會增加單核球及巨噬細胞上清道夫受體CD36之表現( p＜0.01)。而在ox-LDL吞噬分析試驗中，得知黃酮醇對於U937衍生之巨噬細胞吞噬DiI-ox-LDL的能力有明顯下降之趨勢 (p<0.01)。RT-Q-PCR顯示添加20M的槲皮素(quercetin)會增加U937所衍生之巨噬細胞的CD36 mRNA之表現 (p<0.01); 但是添加20M的楊梅素(myricetin)卻能有效抑制CD36 mRNA之表現(p<0.01)。流式細胞儀分析發現20M的山奈酚(kaempferol)與槲皮素(quercetin)可以誘導U937所衍生之巨噬細胞的CD36蛋白質表現(p<0.01)；但20M的非瑟酮(fisetin)與楊梅素(myricetin)均顯著抑制CD36蛋白質表現(分別為 p<0.05 p<0.01)。因此，非瑟酮(fisetin)與楊梅素(myricetin)不僅能抑制巨噬細胞吞噬ox-LDL，並能降低CD36之蛋白質表現。此外，添加20M的非瑟酮(fisetin)、桑色素(morin)及楊梅素(myricetin)，也能有效抑制15dPGJ2 活化PPAR所誘導的CD36蛋白質的表現 (p<0.05, p<0.05 , p<0.01)；但是山奈酚(kaempferol)與槲皮素(quercetin)反而增加15dPGJ2 活化所誘導的CD36蛋白質的表現 (p<0.01)。至於CD36 mRNA上，卻發現這五種黃酮醇均可抑制由15dPGJ2 活化所誘導的CD36 mRNA的生合成。由以上實驗結果得知，非瑟酮(fisetin)、桑色素(morin)及楊梅素(myricetin)不僅可抑制LDL的氧化，同時對於ox-LDL的吞噬與脂泡細胞形成的關鍵基因-CD36的表達，均可有效地抑制。因此，多食用富含非瑟酮(fisetin)、桑色素(morin)及楊梅素(myricetin)成分的飲食，可能具有預防罹患動脈粥樣硬化之相關病症的幼纂C Atherosclerosis is a complex and chronic inflammatory disease. It is one of the primary causes of thrombosis, stroke and myocardial infraction. Oxidation of low density lipoprotein is a critical early stage in the pathogenesis of atherosclerosis. The earliest atherosclerotic lesion is the “fatty streak”, the intimal collections of lipid-laden macrophages and smooth muscle cells. CD36, a class B scavenger receptor, is a macrophage recptor which binds native and oxidized low density lipoprotein (ox-LDL), and plays a critical role in atherosclerotic foam cell formation. On the other hand, the effect of ox-LDL on CD36 is due, in part, to its ability to activate the transcription factor, PPAR(peroxisome proliferator activated receptor-). PPARligands, such as 15dPGJ2 (15-deoxy12,14-prostaglandin J2) also increase CD36 expression. Thus, inhibition of LDL oxidation and CD36 expression is one of the major issues in the prevention of initiation and progression of atherosclerosis Flavonols are phenolic substances isolated from a wide range of plants. In this study, the anti-atherogenic mechanism of five flavonols, namely fisetin, kaempferol, morin, myricetin and quercetin, was investigated. In vitro experiments demonstrated that the five flavonols effectively inhibited MDA formation and LDL surface net charge change as well as notably delayed conjugated dienes synthesis during copper-induced LDL oxidation. Among them, quercetin and fisetin exhibited stronger antioxidant activity than the other flavonols. Cell culture experiment was first employed in U937 cells to investigate the effects of flavonol treatment on PMA-stimulated cell differentiation. Flow cytometry analyses revealed that kaempherol (20 M) decreased the ratio of differentiated cells as compared with control (p<0.01); on the other hand, quercetin not only increased the ratio but also induce cell surface CD36 protein expression in non-differentiated cells. Fisetin (20 M) induced cell surface CD36 protein expression in both non-differentiated and differentiated cells (p<0.01). In the next section, U937 monocytes were differentiated into macrophages before treated with flavols (20 M). It was found that cell surface CD36 protein expression was significantly inhibited by fisetin and myricetin (20 M) (p<0.05 and p<0.01, respectively) in U937-derived macrophages. RT-Q-PCR showed myricetin (20 M) significantly inhibited CD36 mRNA expression (p<0.01). Furthermore, when PPAR was further activated by 15dPGJ2, fisetin, morin and myricetin markedly inhibited CD36 protein expression (p<0.05, p<0.05 and p<0.01, respectively). RT-Q-PCR showed that all five flavonols significantly inhibited 15dPGJ2-induced CD36 mRNA expression. To further investigate whether flavonols directly inhibited PPAR activation in U937-derived macrophages, the PPRE binding activity of nuclear extract was analyzed. It was found that only kaempherol and quercetin (20 M) significantly inhibited PPAR activation (p<0.05 and 0.01). Finally, the ultimate effect of flavonols on form cell formation was investigated in U937-derived macrophages. All five flavonols (20 M) markedly decreased the uptake of DiI-oxLDL regardless of effect on CD36 expression. In conlusion, consumption of food rich in flavonols may prevent atherosclerosis through their antioxidant and inhibitory activity on foam cell formation.
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