| 摘要: | 動脈粥狀硬化(atherosclerosis)為現代人健康及生命的隱形殺手。動脈粥狀硬化通常會引起血栓(thrombosis)、中風(stroke)與心肌梗塞(myocardial infraction)等多項併發症。根據研究顯示，低密度脂蛋白(Low Density Lipoprotein；LDL)的氧化修飾及巨噬泡沫細胞(macrophage foam cell)的產生，是早期動脈粥狀硬化的主要特徵。藉由天然抗氧化物的作用，可清除自由基、抑制LDL的氧化、預防泡沫細胞形成，因此可預防動脈粥狀硬化產生。
本研究第一部份是以體外抗氧化實驗評估山奈酚(Kaempferol)及鼠李檸檬素(Rhamnocitrin)的自由基清除、抑制LDL氧化及調控泡沫細胞形成相關基因表達之能力。實驗顯示，山奈酚及鼠李檸檬素有清除DPPH (1,1-Diphenyl-2-picrylhydrazyl)的能力， IC50分別為26.10±1.33 mM及28.38±3.07 mM。此外，山奈酚及鼠李檸檬素皆可有效抑制銅離子誘導的LDL的氧化：可抑制脂質過氧化產物 - 丙二醛 (Malondialdehyde, MDA) 的產生、減低 apolipoprotein B (apoB)表面電荷的改變、也會延緩共軛雙烯(conjugated diene) 的形成。
本研究第二部份乃以THP-1衍生之巨噬細胞爲研究對象，利用反轉錄定量聚合酶鏈鎖反應 (reverse transcription quantitative PCR, RT-Q-PCR) 探討山奈酚及鼠李檸檬素對泡沫細胞形成相關基因的mRNA表達的影響。研究顯示，在氧化型LDL(ox-LDL)存在時，山奈酚及鼠李檸檬素(20 mM) 可顯著誘導ABC轉運蛋白A1 (ATP binding cassette transporter A1, ABCA1)的 mRNA表現；但對ox-LDL所誘導產生的清道夫受體 (scavenger receptor, 即CD36) 的 mRNA 表現卻無明顯影響。
進一步以流式細胞儀分析THP-1 細胞衍生之巨噬細胞的CD36蛋白的表現發現，雖然單獨加入山奈酚(20 mM)會誘導CD36的蛋白表現，但在ox-LDL存在時，山奈酚或鼠李檸檬素(20 mM)對於ox-LDL所誘導產生的CD36的蛋白表現並無顯著影響。
為了解山奈酚或鼠李檸檬素對巨噬細胞攝入ox-LDL的影響，本研究進一步利用DiI螢光標定的ox-LDL輔以流式細胞儀分析發現，無論山奈酚或鼠李檸檬皆能顯著降低巨噬細胞內ox-LDL的攝入量。綜合以上結果推論，山奈酚、鼠李檸檬可能藉由促進ABCA1的表達，將細胞內的膽固醇排出，因此降低ox-LDL的攝入量，進而抑制泡沫細胞的形成。
綜合以上結果，山奈酚、鼠李檸檬素不僅可有效清除自由基、抑制LDL的氧化，還可抑制泡沫細胞的形成，因此補充富含山奈酚、鼠李檸檬素的食物可能可以預防動脈粥狀硬化及相關併發症的產生。
Atherosclerosis, the most common form of cardiovascular disease, is the leading cause of death worldwide. Atherosclerosis is also one of the primary causes of thrombosis, stroke, and myocardial infraction. There are a number of studies revealing that the oxidative modification of low density lipoprotein and foam cell formation plays a key role in the pathogenesis of atherosclerosis. Antioxidative reagents, which can effectively inhibit LDL oxidation and foam cell formation, may prevent atherosclerosis via reducing early atherogenesis, and slowing down the progression to advance stages.
The aim of the study is to investigate the effects of kaempferol and rhamnocitrin on atherogenesis. The first part of in vitro experiments demonstrated that Kaempferol and rhamnocitrin were scavengers of DPPH (1,1-Diphenyl-2-picrylhydrazyl), with IC50 26.10±1.33 mM and 28.38±3.07 mM, respectively. Copper-induced low density lipoprotein (LDL) oxidation was also inhibited by kaempferol and rhamnocitrin as measured by decreased formation of malondialdehyde, reduced electrophoretic mobility and delayed formation of conjugated dienes.
Many studies suggested that macrophages played critical pathogenic roles in the foam cell formation. Reverse tranascription quantitative PCR (RT-Q-PCR) was used to elucidate the expression of CD36, ABCA1 and adipophilin mRNA in THP-1 derived macropahges. Current results revealed that the expression of ATP binding cassette transporter A1 (ABCA1) was significantly induced by the addition of kaempferol and rhamnocitrin (20 mM) in the presence of ox-LDL. On the other hand, kaempferol and rhamnocitrin did not exert significant effect on ox-LDL induced CD36, scavenger receptor, mRNA expression.
Flow cytometry analyses revealed that the CD36 protein level was induced by kaempferol. However, it was not significantly affected by kaempferol or rhamnocitrin (20 mM) when ox-LDL was present in THP-1 derived macrophages. To further investigate the effects of kaempferol and rhamnocitrin on ox-LDL uptake, it was found that the accumulation of DiI-ox-LDL was significantly attenuated by kaempferol and rhamnocitrin (20 mM). All of these results indicated that kaempferol and rhamnocitrin inhibited form cell formation in THP-1 derived macrophages possibly through up-regulation of ABCA-1 mRNA expression.
Current study indicates that the consumption of kaempferol and rhamnocitrin-rich food may prevent atherosclerosis through their antioxidant activity and inhibitory activity on foam cell formation. |
