| 摘要: | 近年來許多研究報告指出，攝取富含類黃酮的食物可減低心血管疾病的罹患率。粥樣動脈硬化是發生在血管內壁的病變，研究證實主要由於低密度脂蛋白 (LDL) 氧化，並引發一連串的發炎反應所造成。粥狀動脈發生的進程包括以下幾個關鍵步驟：內皮細胞發生官能障礙並大量吸引單核球黏著、發炎單核球進入血管內膜分化成巨噬細胞、巨噬細胞吞噬oxLDL形成脂泡細胞並堆積在血管內壁形成斑塊 (plaque)、平滑肌細胞往內皮細胞外緣移動並纖維化生成纖維帽、脂泡細胞分泌發炎物質分解胞外基質弱化纖維帽、血管內壁產生缺口並引發一系列凝血反應生成血栓塞住血管。本論文的目的為探討三種黃酮 (Flavones) 木犀草素 (luteolin)、芹黃素 (apigenin) 和白楊素 (chrysin) 在預防粥樣動脈硬化 (atherosclerosis) 上的功效及其分子機轉。
本論文首先探討luteolin、apigenin和chrysin是否具有抗氧化活性，在試管試驗中luteolin具有極佳清除DPPH的能力，其IC50為9.11 μM，優於α-tocopherol的IC50 (37.46 μM)。此外，以銅離子誘導低密度脂蛋白氧化，探討受試物在試管中抑制LDL氧化之能力，結果顯示luteolin能有效抑制由銅離子誘導的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，至於apigenin和chrysin則無顯著效果。
本研究第二部份接著探討此三種類黃酮在人類單核球細胞株 (THP-1 monocytes) 被oxLDL刺激而引起的發炎反應是否有抑制效果。反轉錄定量聚合酶連鎖反應 (RT-Q-PCR) 的結果顯示，luteolin和apigenin都能抑制細胞激素TNF-α、IL-6及IL-1β基因表現。接著探討三種黃酮是否具有抑制轉錄因子NF-κB活化的能力，只有20 μM的luteolin有效的抑制核內NF-κB p65的蛋白質表現量，因此推測luteolin可能是經由抑制細胞核內的轉錄作用而導致相關發炎細胞激素無法生成。
第三部份探討三種黃酮在單核球分化成巨噬細胞 (THP-1 monocyte-to-macrophage differentiation) 階段是否具有抑制分化相關基因和oxLDL的吞噬。實驗結果證實，結果顯示luteolin能有效抑制30 nM PMA刺激下細胞中清道夫受體CD36 mRNA表現，並且三個黃酮皆可隨著濃度的提高而減少對DiI-oxLDL的吞噬，尤其以luteolin效果最為顯著 (p<0.01)。本研究接著利用50 μg/mL oxLDL和1.6 nM PMA共同作用誘導單核球分化成巨噬細胞，同樣測試在有oxLDL的情況之下清道夫受體CD36和SR-A以及分化指標CD14和CD68，結果發現luteolin能有效降低CD36及SR-A的表現，而apigenin和chrysin則以抑制分化指標CD14和CD68基因表現較為顯著。
本研究最後一部份探討此三種黃酮抑制PMA所誘導THP-1衍生的巨噬細胞 (THP-1-derived macrophages) 形成脂泡細胞的相關機轉。RT-Q-PCR的實驗結果顯示，luteolin和apigenin顯著的抑制CD36與SR-A基因表現 (p<0.05或p<0.01)，chrysin則無顯著效應；而另外加50 μg/mL oxLDL後，luteolin抑制CD36及SR-A效果最好 (p<0.01)，其次是apigenin，chrysin則無抑制SR-A表現。三種黃酮皆減少了THP-1-derived macrophages對DiI-oxLDL的吞噬 (p<0.01)。為探討三種黃酮在巨噬細胞抑制脂泡細胞形成的相關機轉，結果顯示三種黃酮皆可以抑制JNK和P38蛋白的磷酸化，而添加JNK與P38抑制劑後即抑制CD36表現，間接證明luteolin極為可能是經由阻斷JNK與P38的磷酸化而降低CD36表現，進而減少其吞噬oxLDL的能力和形成脂泡細胞。研究進一步發現luteolin和apigenin都會抑制NF-κB p65核蛋白的表現。最後分析三個黃酮對巨噬細胞所分泌之MMP-9的酵素活性影響，數據顯示luteolin和chrysin可以顯著抑制THP-1巨噬細胞分泌的MMP-9的活性。
整合以上結果，luteolin不僅可抑制脂質氧化，同時降低oxLDL的吞噬與脂泡細胞形成的關鍵基因CD36和MMP-9的表現，而apigenin和chrysin能抑制單核球分化指標和部分巨噬細胞上的清道夫受體的基因表現，因此木犀草素 (luteolin)、芹黃素 (apigenin) 和白楊素 (chrysin) 各有不同預防和治療粥樣動脈硬化之相關病症的功效。
Epidemiological and animal studies point to a possible protective effect of flavonoids against cardiovascular diseases. Oxidized LDL (oxLDL) is shown to be a major contributor to the development of atherosclerosis, as it enhances cellular cholesterol accumulation, as well as inflammatory and thrombotic processes. The early atherosclerotic plaque formation begins with the adherence of blood circulating monocytes to the vascular bed lining, the endothelium, followed by their migration to the sub-endothelial space. There, the monocytes differentiate into macrophages that accumulate massive amounts of lipoprotein-derived cholesterol and are converted into foam cells. The foam cells secrete pro-inflammatory cytokines that amplify the local inflammatory response in the lesion and may eventually cause plaque disruption. Flavonoids have been classified as anti-atherogenic agents that inhibit both foam cell formation and pro-inflammatory cytokine secretion. This anti-atherogenic feature of flavonoids appears to be related to their chemical structures. Luteolin, apigenin and chrysin are flavones, which differ only in the number of hydroxyl group on the B ring. In this report, we aim to study their atheroprotective effects and mechanisms.
In the first part of this work, we examined the effects of three flavones on the inhibition of LDL oxidation. In vitro experiments demonstrated that luteolin was scavenger of DPPH (1,1-Diphenyl-2-picrylhydrazyl), with IC50 of 9.11 μM. Copper-induced LDL oxidation was also inhibited by luteolin as measured by decreased formation of malondialdehyde, reduced electrophoretic mobility and delayed formation of conjugated dienes.
In the second part of this work, we investigated the effects of three flavones on the oxLDL induced TNF-, IL-6 and IL-1, mRNA expression and NF-B activation in the THP-1 monocytes. RT-Q-PCR showed that luteolin significantly inhibited oxLDL-stimulated TNF-, IL-6 and IL-1 mRNA expression. Furthermore, oxLDL-mediated NF-B activation was markedly inhibited by luteolin.
The inhibition of monocyte differentiation and ingestion of oxLDL is an effective way to prevent or delay the inflammatory process that leads to the development of atherosclerosis. Two scavenger receptors, CD36 and SR-A, have been centrally implicated in oxLDL uptake process. In the next section, THP-1 monocytes were pretreated with three flavones for 30 min before cotreated with PMA (30 nM) or PMA (1.6 nM) supplemented with oxLDL (50 μg/ml) for differentiation. It was found that mRNA expression of CD36 and SR-A was significantly inhibited by 10 M luteolin (p<0.05 or p<0.01) during THP-1 monocyte differentiation (1.6 nM PMA plus 50 μg/ml oxLDL). RT-Q-PCR also showed 10 M apigenin and chrysin significantly inhibited CD14 and CD68 mRNA expression induced by PMA (1.6 nM) plus oxLDL (p<0.05). All three flavones (10 M) markedly decreased the uptake of DiI-oxLDL related to the effect on the scavenger receptors or differentiation markers (p<0.05 or p<0.01).
RT-Q-PCR analyses revealed that 20 M luteolin reduced the CD36 and SR-A mRNA level in THP-1-derived macrophages in the presence and absence of oxLDL. The CD36 mRNA expression was also significantly affected by 20 apigenin and chrysin when oxLDL was present in the cells. To further investigate the effects of three flavones on DiI-oxLDL uptake, it was found that the accumulation of DiI-oxLDL was significantly attenuated by three flavones (10 or 20 ). Addition of JNK inhibitor (SP600125)、P38 inhibitor (SB203580) or NF-κB inhibitor (BAY 11-7082), luteolin suppressed oxLDL-mediated expression of CD36, indicating upregulation of CD36 may be mediated through JNK and P38 signaling pathway. Western blot also revealed that three flavones (20 M) reduced oxLDL-induced JNK, P38 phosphorylation and nuclear NF-κB p65 protein.
Apoptosis of macrophages and/or foam cells creates a necrotic core. Thinning and erosion of the fibrous cap in unstable plaques, through matrix degradation by proteases, ultimately results in plaque rupture and thrombosis of the artery. The activity of MMP-9 (matrix metalloproteinase-9) was suppressed in THP-1-derived macrophages by luteolin and chrysin as measured by zymography (p<0.01).
This result indicates flavones, despite structural similarities, exert different atheroprotective effects and mechanisms. |
