臺灣博碩士論文加值系統

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-?羠 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-?羠 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 ?嵱 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 ?嵱 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 ?嵱) 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 ?嵱 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 ?嵱) 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.

中文摘要 I
Abstact V
總目錄 IX
圖目錄 XIV
附錄目錄 XVII
第一章 緒論 1
1.1發炎反應與粥樣動脈硬化之的進程 2
1.2 低密度脂蛋白 (low density lipoprotein) 的介紹 4
1.3低密度脂蛋白 (low density lipoprotein, LDL) 的氧化 5
1.4 發炎細胞激素簡介 6
1.5 單核球分化為巨噬細胞 8
1.6 清道夫受體與單核球分化成巨噬細胞的訊息傳遞 9
1.6.1清道夫受體CD36 (cluster of differentiation 36) 10
1.6.2清道夫受體SR-A (scavenger receptor class A type I/II) 12
1.7基質金屬蛋白分解酶和斑塊破裂 12
1.8 天然物的抗血管粥樣動脈硬化功效 13
1.9類黃酮之簡介 13
1.10 木犀草素 (luteolin)、芹黃素 (apigenin) 和白楊素 (chrysin) 15
1.11 研究動機 17
第二章 材料與方法 19
2.1木犀草素 (luteolin)、芹黃素 (apigenin) 和白楊素 (chrysin) 的來源 19
2.2 DPPH (α,α-diphenyl-β-pricrylhydrazy) 清除能力之測定 19
2.3以超高速離心機分離血漿中低密度脂蛋白 (low-density lipoprotein, LDL) 20
2.4 低密度脂蛋白之去鹽及濃縮 20
2.5 OxLDL的製備 21
2.6 脂質過氧化作用 (lipid peroxidation) 的測定 21
2.6.1 共軛雙烯的生成 (conjugated diene formation) 21
2.6.2 硫代巴比妥酸反應物 (thiobarbituric acid reactive substances; TBARS) 分析 22
2.6.3 脂質電泳 (electrophoresis of LDL) 22
2.7 細胞培養 (cell culture) 22
2.8 細胞存活率分析 (cell viability analysis) 23
2.9 OxLDL對THP-1細胞基因表現的影響 24
2.10 利用RT-Q-PCR分析基因表現 24
2.11 核蛋白之製備 (preparation of nuclear extract) 25
2.12 西方墨點法 (Western blot) 26
2.13 DiI-oxLDL的製備 27
2.14 THP-1 monocyte-to-macrophage differenciation和THP-1-derived macrophages對標定DiI的修飾型LDL的吞噬能力 27
2.15 細胞總蛋白製備 (preparation of total protein) 28
2.16 酵素電泳法 (zymography) 測定MMP-9酵素活性 28
2.17 統計分析 (statistical analysis) 29
第三章 結果 30
3.1抗氧化試管試驗 30
3.1.1 木犀草素 (luteolin)、芹黃素 (apigenin) 和白楊素 (chrysin) 清除自由基能力之影響 30
3.1.2 Luteolin、apigenin和chrysin抑制低密度脂蛋白 (LDL) 之過氧化 31
3.2 THP-1 monocytes 32
3.2.1 Luteolin、apigenin和chrysin對於THP-1 monocytes 經由oxLDL誘導細胞激素基因表現之影響 33
3.2.2 Luteolin、apigenin和chrysin對於THP-1 monocytes 經由oxLDL誘導轉錄因子NF-κB p65活性之影響 34
3.3 Monocyte-to-macrophage differentiation 34
3.3.1 Luteolin、apigenin和chrysin對於THP-1 monocyte-to-macrophage differentiation清道夫受體CD36和SR-A基因表現的影響 35
3.3.2 Luteolin、apigenin和chrysin對於THP-1 monocyte-to-macrophage differentiation分化指標CD14和CD68基因表現的影響 36
3.3.3 Luteolin、apigenin和chrysin對於受PMA刺激後的THP-1 monocyte-to-macrophage differentiation吞噬修飾型DiI-oxLDL能力的影響 37
3.4 THP-1-derived macrophages 38
3.4.1 Luteolin、apigenin和chrysin對於THP-1-derived macrophages由oxLDL誘導清道夫受體CD36和SR-A基因表現的影響 39
3.4.2 Luteolin、apigenin和chrysin對於THP-1-derived macrophages吞噬修飾型DiI-oxLDL能力的影響 40
3.4.3 Luteolin、apigenin和chrysin對於THP-1-derived macrophages經由oxLDL誘導p38、JNK與ERK蛋白質表現之影響 41
3.4.4 Luteolin、apigenin和chrysin對於THP-1-derived macrophages 經由oxLDL誘導轉錄因子NF-κB p65活性之影響 41
3.4.5 抑制JNK、p38、ERK與NF-κB訊息傳遞對於THP-1-derived macrophages經由oxLDL誘導CD36 基因表現的影響 42
3.4.6 Luteolin、apigenin和chrysin對於THP-1-derived macrophages分泌的金屬蛋白酶MMP-9酵素活性之影響 42
第四章 討論 44
第五章 結論 52
參考文獻 53

1.Srivastava S, Sithu SD, Vladykovskaya E, et al. Oral exposure to acrolein exacerbates atherosclerosis in apoE-null mice. Atherosclerosis. 2011;215:301-308.
2.Shepherd J, Cobbe SM, Ford I, et al. Prevention of coronary heart disease with pravastatin in men with hypercholesterolemia. West of Scotland Coronary Prevention Study Group. N Engl J Med. 1995;333:1301-1307.
3.Breslow JL. Cardiovascular disease burden increases, NIH funding decreases. Nat Med. 1997;3:600-601.
4.Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993;362:801-809.
5.Ross R, Glomset JA. Atherosclerosis and the arterial smooth muscle cell: Proliferation of smooth muscle is a key event in the genesis of the lesions of atherosclerosis. Science. 1973;180:1332-1339.
6.Ross R. Atherosclerosis-an inflammatory disease. N Engl J Med. 1999;340:115-126.
7.Chiu JJ, Chien S. Effects of disturbed flow on vascular endothelium: pathophysiological basis and clinical perspectives. Physiol Rev. 2011;91:327-387.
8.Fuller C, Grundy S, Norkus E, Jialal I. Effect of ascorbate supplementation on low density lipoprotein oxidation in smokers. Atherosclerosis. . 1996;119:139-150.
9.Libby P. Inflammation in atherosclerosis. Nature. 2002;420:868-874.
10.Breslow JL. Mouse models of atherosclerosis. Science. 1996;272:685-688.
11.Stary HC, Chandler AB, Dinsmore RE, et al. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb Vasc Biol. 1995;15:1512-1531.
12.Beisiegel U, Weber W, Ihrke G, et al. The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein. Nature. 1989;341:162-164.
13.Gotto AM, Jr., Pownall HJ, Havel RJ. Introduction to the plasma lipoproteins. Methods Enzymol. 1986;128:3-41.
14.Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2005;3:28.
15.Puhl H, Waeg G, Esterbauer H. Methods to determine oxidation of low-density lipoproteins. Methods Enzymol. 1994;233:425-441.
16.Negre-Salvayre A, Coatrieux C, Ingueneau C, Salvayre R. Advanced lipid peroxidation end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for the inhibitors. Br J Pharmacol. 2008;153:6-20.
17.Brown DR. Neurodegeneration and oxidative stress: prion disease results from loss of antioxidant defence. Folia Neuropathol. 2005;43:229-243.
18.Chait A, Brazg RL, Tribble DL, Krauss RM. Susceptibility of small, dense, low-density lipoproteins to oxidative modification in subjects with the atherogenic lipoprotein phenotype, pattern B. Am J Med. 1993;94:350-356.
19.Hajjar DP, Haberland ME. Lipoprotein trafficking in vascular cells. Molecular Trojan horses and cellular saboteurs. J Biol Chem. 1997;272:22975-22978.
20.Beutler B, Poltorak A. Positional cloning of Lps, and the general role of toll-like receptors in the innate immune response. Eur Cytokine Netw. 2000;11:143-152.
21.Kriegler M, Perez C, DeFay K, et al. A novel form of TNF/cachectin is a cell surface cytotoxic transmembrane protein: Ramifications for the complex physiology of TNF. Cell. 1988;53:45-53.
22.Chi D, Fitzgerald SM, Pitts S, et al. MAPK-dependent regulation of IL-1- and beta-adrenoreceptor-induced inflammatory cytokine production from mast cells: Implications for the stress response. BMC Immunology. 2004;5:22.
23.Ferguson-Smith AC, Chen Y-F, Newman MS, et al. Regional localization of the interferon-β2B-cell stimulatory factor 2/hepatocyte stimulating factor gene to human chromosome 7p15-p21. Genomics. 1988;2:203-208.
24.van der Poll T, Keogh CV, Guirao X, et al. Interleukin-6 gene-deficient mice show impaired defense against pneumococcal pneumonia. J Infect Dis. 1997;176:439-444.
25.Bessler H, Djaldetti R, Salman H, et al. IL-1 beta, IL-2, IL-6 and TNF-alpha production by peripheral blood mononuclear cells from patients with Parkinson''s disease. Biomed Pharmacother. 1999;53:141-145.
26.Barnes PJ, Karin M. Nuclear factor-kappaB: a pivotal transcription factor in chronic inflammatory diseases. N Engl J Med. 1997;336:1066-1071.
27.Kirkland TN, Viriyakosol S. Structure-function analysis of soluble and membrane-bound CD14. Prog Clin Biol Res. 1998;397:79-87.
28.Kitchens RL. Role of CD14 in cellular recognition of bacterial lipopolysaccharides. Chem Immunol. 2000;74:61-82.
29.Holness CL, Simmons DL. Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood. 1993;81:1607-1613.
30.Manduch M, Dexter DF, Jalink DW, et al. Undifferentiated pancreatic carcinoma with osteoclast-like giant cells: report of a case with osteochondroid differentiation. Pathol Res Pract. 2009;205:353-359.
31.Shiffman D, Mikita T, Tai JT, et al. Large scale gene expression analysis of cholesterol-loaded macrophages. J Biol Chem. 2000;275:37324-37332.
32.Kunjathoor VV, Febbraio M, Podrez EA, et al. Scavenger receptors class A-I/II and CD36 are the principal receptors responsible for the uptake of modified low density lipoprotein leading to lipid loading in macrophages. J Biol Chem. 2002;277:49982-49988.
33.Endemann G, Stanton LW, Madden KS, et al. CD36 is a receptor for oxidized low density lipoprotein. J Biol Chem. 1993;268:11811-11816.
34.Podrez EA, Hoppe G, O''Neil J, et al. Macrophage receptors responsible for distinct recognition of low density lipoprotein containing pyrrole or pyridinium adducts: models of oxidized low density lipoprotein. J Lipid Res. 2000;41:1455-1463.
35.Silverstein RL, Febbraio M. CD36, a scavenger receptor involved in immunity, metabolism, angiogenesis, and behavior. Sci Signal. 2009;2:re3.
36.Han J, Hajjar DP, Febbraio M, Nicholson AC. Native and modified low density lipoproteins increase the functional expression of the macrophage class B scavenger receptor, CD36. J Biol Chem. 1997;272:21654-21659.
37.Ley K, Miller YI, Hedrick CC. Monocyte and macrophage dynamics during atherogenesis. Arterioscler Thromb Vasc Biol. 2011;31:1506-1516.
38.Lipsky RH, Eckert DM, Tang Y, Ockenhouse CF. The carboxyl-terminal cytoplasmic domain of CD36 is required for oxidized low-density lipoprotein modulation of NF-kappaB activity by tumor necrosis factor-alpha. Recept Signal Transduct. 1997;7:1-11.
39.Han CY, Park SY, Pak YK. Role of endocytosis in the transactivation of nuclear factor-κB by oxidized low-density lipoprotein. Biochem. J. 2000;350:829-837.
40.van Berkel TJ, Out R, Hoekstra M, et al. Scavenger receptors: friend or foe in atherosclerosis? Current Opinion in Lipidology. 2005;16:525-535.
41.Janabi M, Yamashita S, Hirano K, et al. Oxidized LDL-induced NF-kappa B activation and subsequent expression of proinflammatory genes are defective in monocyte-derived macrophages from CD36-deficient patients. Arterioscler Thromb Vasc Biol. 2000;20:1953-1960.
42.Feng J, Han J, S FAP, et al. Induction of CD36 expression by oxidized LDL and IL-4 by a common signaling pathway dependent on protein kinase C and PPAR gama. ASBMB. 2000;41:688-696.
43.Libby P. Inflammation and Atherosclerosis. Circulation. 2002;105:1135-1143.
44.Janabi M, Yamashita S, Hirano K, et al. Oxidized LDL-induced NF-kappa B activation and subsequent expression of proinflammatory genes are defective in monocyte-derived macrophages from CD36-deficient patients. Arterioscler Thromb Vasc Biol. 2000;20:1953-1960.
45.Ceccopieri B, Marcomin AR, Vitagliano F, Fragapane P. Primary anorectal malignant melanoma: report of two cases. Tumori. 2000;86:356-358.
46.Suzuki H, Kurihara Y, Takeya M, et al. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature. 1997;386:292-296.
47.Babaev VR, Gleaves LA, Carter KJ, et al. Reduced atherosclerotic lesions in mice deficient for total or macrophage-specific expression of scavenger receptor-A. Arterioscler Thromb Vasc Biol. 2000;20:2593-2599.
48.Sakaguchi H, Takeya M, Suzuki H, et al. Role of macrophage scavenger receptors in diet-induced atherosclerosis in mice. Lab Invest. 1998;78:423-434.
49.Kuchibhotla S, Vanegas D, Kennedy DJ, et al. Absence of CD36 protects against atherosclerosis in ApoE knock-out mice with no additional protection provided by absence of scavenger receptor A I/II. Cardiovasc Res. 2008;78:185-196.
50.Osterud B, Bjorklid E. Role of Monocytes in Atherogenesis. Physiol Rev. 2003;83:1069-1112.
51.Galis ZS, Sukhova GK, Lark MW, Libby P. Increased Expression of Matrix Metalloproteinases and Matrix Degrading Activity in Vulnerable Regions of Human Atherosclerotic Plaques. J. Clin. Invest. 1994;94:2493-2503.
52.Poli G, Sottero B, Gargiulo S, Leonarduzzi G. Cholesterol oxidation products in the vascular remodeling due to atherosclerosis. Mol Aspects Med. 2009;30:180-189.
53.Libby P. Inflammation in atherosclerosis. Nature. 2002;420:868-874.
54.Reed J. Cranberry flavonoids, atherosclerosis and cardiovascular health. Crit Rev Food Sci Nutr. 2002;42:301-316.
55.Lo YH, Pan MH, Li S, et al. Nobiletin metabolite, 3'',4''-dihydroxy-5,6,7,8-tetramethoxyflavone, inhibits LDL oxidation and down-regulates scavenger receptor expression and activity in THP-1 cells. Biochim Biophys Acta. 2010;1801:114-126.
56.Wei HA, Lian TW, Tu YC, et al. Inhibition of low-density lipoprotein oxidation and oxidative burst in polymorphonuclear neutrophils by caffeic acid and hispidin derivatives isolated from sword brake fern (Pteris ensiformis Burm.). J Agric Food Chem. 2007;55:10579-10584.
57.Whitman SC, Kurowska EM, Manthey JA, Daugherty A. Nobiletin, a citrus flavonoid isolated from tangerines, selectively inhibits class A scavenger receptor-mediated metabolism of acetylated LDL by mouse macrophages. Atherosclerosis. 2005;178:25-32.
58.Wilcox LJ, Borradaile NM, de Dreu LE, Huff MW. Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP. Journal of Lipid Research. 2001;42:725-734.
59.Kawai Y, Nishikawa T, Shiba Y, et al. Macrophage as a target of quercetin glucuronides in human atherosclerotic arteries: implication in the anti-atherosclerotic mechanism of dietary flavonoids. J Biol Chem. 2008;283:9424-9434.
60.Wilcox LJ, Borradaile NM, de Dreu LE, Huff MW. Secretion of hepatocyte apoB is inhibited by the flavonoids, naringenin and hesperetin, via reduced activity and expression of ACAT2 and MTP. J Lipid Res. 2001;42:725-734.
61.Kuhnau J. The flavonoids. A class of semi-essential food components: their role in human nutrition. World Rev Nutr Diet. 1976;24:117-191.
62.Setchell KD, Cassidy A. Dietary isoflavones: biological effects and relevance to human health. J Nutr. 1999;129:758S-767S.
63.Sharma R, Tepas JJ, 3rd, Hudak ML, et al. Neonatal gut barrier and multiple organ failure: role of endotoxin and proinflammatory cytokines in sepsis and necrotizing enterocolitis. J Pediatr Surg. 2007;42:454-461.
64.Nijveldt RJ, Nood Ev, Hoorn DEv, et al. Flavonoids: a review of probable mechanisms of action and potential applications. Am J Clin Nutr. 2001;74:418-425.
65.Hu JP, Calomme M, Lasure A, et al. Structure-activity relationship of flavonoids with superoxide scavenging activity. Biol Trace Elem Res. 1995;47:327-331.
66.Jonathan E, Hicham K, Robert C, Catherine A. Structural dependence of flavonoid interactions with Cu2+ ions: implications for their antioxidant properties. Biochem. J. 1998;330:1173-1178.
67.Choi JS, Choi YJ, Park SH, et al. Flavones Mitigate Tumor Necrosis Factor-α-Induced Adhesion Molecule Upregulation in Cultured Human Endothelial Cells: Role of Nuclear Factor-κB. J. Nutr. 2004;134:1013-1019.
68.Lin Y, Shi R, Wang X, Shen H-M. Luteolin, a flavonoid with potentials for cancer prevention and therapy. Curr Cancer Drug Targets. 2008;8:634-646.
69.Choi JS, Choi YJ, Park SH, et al. Flavones mitigate tumor necrosis factor-alpha-induced adhesion molecule upregulation in cultured human endothelial cells: role of nuclear factor-kappa B. J Nutr. 2004;134:1013-1019.
70.Wang G, Xiao CQ, Li Z, et al. Effect of soy extract administration on losartan pharmacokinetics in healthy female volunteers. Ann Pharmacother. 2009;43:1045-1049.
71.Lin Y, Shi R, Wang X, Shen HM. Luteolin, a flavonoid with potential for cancer prevention and therapy. Curr Cancer Drug Targets. 2008;8:634-646.
72.Nimnual AS, Taylor LJ, Bar-Sagi D. Redox-dependent downregulation of Rho by Rac. Nat Cell Biol. 2003;5:236-241.
73.Robak J, Shridi F, Wolbis M, Krolikowska M. Screening of the influence of flavonoids on lipoxygenase and cyclooxygenase activity, as well as on nonenzymic lipid oxidation. Pol J Pharmacol Pharm. 1988;40:451-458.
74.Brown JE, Rice-Evans CA. Luteolin-rich artichoke extract protects low density lipoprotein from oxidation in vitro. Free Radic Res. 1998;29:247-255.
75.Xagorari A, Papapetropoulos A, Mauromatis A, et al. Luteolin inhibits an endotoxin-stimulated phosphorylation cascade and proinflammatory cytokine production in macrophages. J Pharmacol Exp Ther. 2001;296:181-187.
76.Chen CC, Chow MP, Huang WC, et al. Flavonoids inhibit tumor necrosis factor-alpha-induced up-regulation of intercellular adhesion molecule-1 (ICAM-1) in respiratory epithelial cells through activator protein-1 and nuclear factor-kappaB: structure-activity relationships. Mol Pharmacol. 2004;66:683-693.
77.Kumazawa Y, Kawaguchi K, Takimoto H. Immunomodulating effects of flavonoids on acute and chronic inflammatory responses caused by tumor necrosis factor alpha. Curr Pharm Des. 2006;12:4271-4279.
78.Kotanidou A, Xagorari A, Bagli E, et al. Luteolin reduces lipopolysaccharide-induced lethal toxicity and expression of proinflammatory molecules in mice. Am J Respir Crit Care Med. 2002;165:818-823.
79.Tormakangas L, Vuorela P, Saario E, et al. In vivo treatment of acute Chlamydia pneumoniae infection with the flavonoids quercetin and luteolin and an alkyl gallate, octyl gallate, in a mouse model. Biochem Pharmacol. 2005;70:1222-1230.
80.Chen CY, Peng WH, Tsai KD, Hsu SL. Luteolin suppresses inflammation-associated gene expression by blocking NF-kappaB and AP-1 activation pathway in mouse alveolar macrophages. Life Sci. 2007;81:1602-1614.
81.Xagorari A, Roussos C, Papapetropoulos A. Inhibition of LPS-stimulated pathways in macrophages by the flavonoid luteolin. Br J Pharmacol. 2002;136:1058-1064.
82.Joussen AM, Rohrschneider K, Reichling J, et al. Treatment of corneal neovascularization with dietary isoflavonoids and flavonoids. Exp Eye Res. 2000;71:483-487.
83.Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med. 1995;1:27-31.
84.Naderi GA, Asgary S, Sarraf-Zadegan N, Shirvany H. Anti-oxidant effect of flavonoids on the susceptibility of LDL oxidation. Mol Cell Biochem. 2003;246:193-196.
85.Jeong YJ, Choi YJ, Choi JS, et al. Attenuation of monocyte adhesion and oxidised LDL uptake in luteolin-treated human endothelial cells exposed to oxidised LDL. Br J Nutr. 2007;97:447-457.
86.Shin EK, Kwon HS, Kim YH, et al. Chrysin, a natural flavone, improves murine inflammatory bowel diseases. Biochem Biophys Res Commun. 2009;381:502-507.
87.Bae Y, Lee S, Kim SH. Chrysin suppresses mast cell-mediated allergic inflammation: involvement of calcium, caspase-1 and nuclear factor-kappaB. Toxicol Appl Pharmacol. 2011;254:56-64.
88.Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 1993;362:801 - 809.
89.Burits M, Bucar F. Antioxidant activity of Nigella sativa essential oil. Phytother Res. 2000;14:323-328.
90.Hermann M, Gmeiner B. Altered susceptibility to in vitro oxidation of LDL in LDL complexes and LDL aggregates. Ann N Y Acad Sci. 1993;683:363-364.
91.Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254.
92.Devaraj S, Hugou I, Jialal I. Alpha-tocopherol decreases CD36 expression in human monocyte-derived macrophages. J Lipid Res. 2001;42:521-527.
93.Esterbauer H, Striegl G, Puhl H, Rotheneder M. Continuous monitoring of in vitro oxidation of human low density lipoprotein. Free Radic Res Commun. 1989;6:67-75.
94.Carmichael J, DeGraff WG, Gazdar AF, et al. Evaluation of a tetrazolium-based semiautomated colorimetric assay: assessment of chemosensitivity testing. Cancer Res. 1987;47:936-942.
95.Wang X, Seed B. A PCR primer bank for quantitative gene expression analysis. Nucleic Acids Res. 2003;31:e154.
96.Teupser D, Thiery J, Walli AK, Seidel D. Determination of LDL- and scavenger-receptor activity in adherent and non-adherent cultured cells with a new single-step fluorometric assay. Biochim Biophys Acta. 1996;1303:193-198.
97.Verouti SN, Fragopoulou E, Karantonis HC, et al. PAF effects on MCP-1 and IL-6 secretion in U-937 monocytes in comparison with OxLDL and IL-1beta effects. Atherosclerosis. 2011;219:519-525.
98.Fuhrman B, Partoush A, Volkova N, Aviram M. Ox-LDL induces monocyte-to-macrophage differentiation in vivo: Possible role for the macrophage colony stimulating factor receptor (M-CSF-R). Atherosclerosis. 2008;196:598-607.
99.Huang RS, Hu GQ, Lin B, et al. MicroRNA-155 silencing enhances inflammatory response and lipid uptake in oxidized low-density lipoprotein-stimulated human THP-1 macrophages. J Investig Med. 2010;58:961-967.
100.Barolet AW, Babaei S, Robinson R, et al. Administration of exogenous endothelin-1 following vascular balloon injury: early and late effects on intimal hyperplasia. Cardiovasc Res. 2001;52:468-476.
101.Kishimoto Y, Tani M, Uto-Kondo H, et al. Astaxanthin suppresses scavenger receptor expression and matrix metalloproteinase activity in macrophages. Eur J Nutr. 2010;49:119-126.
102.Orrenius S, Gogvadze V, Zhivotovsky B. Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol. 2007;47:143-183.
103.de Whalley CV, Rankin SM, Hoult JR, et al. Flavonoids inhibit the oxidative modification of low density lipoproteins by macrophages. Biochemical Pharmacology. 1990;39:1743-1750.
104.Safari MR, Sheikh N. Effects of flavonoids on the susceptibility of low-density lipoprotein to oxidative modification. Prostaglandins Leukot Essent Fatty Acids. 2003;69:73-77.
105.Huang X. Iron overload and its association with cancer risk in humans: evidence for iron as a carcinogenic metal. Mutat Res. 2003;533:153-171.
106.Rafat Husain S, Cillard J, Cillard P. Hydroxyl radical scavenging activity of flavonoids. Phytochemistry. 1987;26:2489-2491.
107.Terashima M, Kakuno Y, Kitano N, et al. Antioxidant activity of flavonoids evaluated with myoglobin method. Plant Cell Rep. 2012;31:291-298.
108.Zarev S, Bonnefont-Rousselot D, Jedidi I, et al. Extent of copper LDL oxidation depends on oxidation time and copper/LDL ratio: chemical characterization. Arch Biochem Biophys. 2003;420:68-78.
109.Ziouzenkova O, Sevanian A, Abuja PM, et al. Copper can promote oxidation of LDL by markedly different mechanisms. Free Radic Biol Med. 1998;24:607-623.
110.Zhou F, Chen S, Xiong J, et al. Luteolin Reduces Zinc-Induced Tau Phosphorylation at Ser262/356 in an ROS-Dependent Manner in SH-SY5Y Cells. Biol Trace Elem Res. 2012.
111.Qi L, Pan H, Li D, et al. Luteolin improves contractile function and attenuates apoptosis following ischemia-reperfusion in adult rat cardiomyocytes. Eur J Pharmacol. 2011;668:201-207.
112.Liao PH, Hung LM, Chen YH, et al. Cardioprotective effects of luteolin during ischemia-reperfusion injury in rats. Circ J. 2011;75:443-450.
113.Lien EJ, Ren S, Bui HH, Wang R. Quantitative structure-activity relationship analysis of phenolic antioxidants. Free Radic Biol Med. 1999;26:285-294.
114.Lv L, Zhang Y, Kong Q. Luteolin prevents LPS-induced TNF-alpha expression in cardiac myocytes through inhibiting NF-kappaB signaling pathway. Inflammation. 2011;34:620-629.
115.Zhu LH, Bi W, Qi RB, et al. Luteolin inhibits microglial inflammation and improves neuron survival against inflammation. Int J Neurosci. 2011;121:329-336.
116.Seelinger G, Merfort I, Schempp CM. Anti-oxidant, anti-inflammatory and anti-allergic activities of luteolin. Planta Med. 2008;74:1667-1677.
117.Brody JS, Spira A. State of the art. Chronic obstructive pulmonary disease, inflammation, and lung cancer. Proc Am Thorac Soc. 2006;3:535-537.
118.Karin M, Lawrence T, Nizet V. Innate immunity gone awry: linking microbial infections to chronic inflammation and cancer. Cell. 2006;124:823-835.
119.Park CM, Jin KS, Lee YW, Song YS. Luteolin and chicoric acid synergistically inhibited inflammatory responses via inactivation of PI3K-Akt pathway and impairment of NF-kappaB translocation in LPS stimulated RAW 264.7 cells. Eur J Pharmacol. 2011;660:454-459.
120.Funakoshi-Tago M, Nakamura K, Tago K, et al. Anti-inflammatory activity of structurally related flavonoids, Apigenin, Luteolin and Fisetin. Int Immunopharmacol. 2011;11:1150-1159.
121.Liu S, Wang S, Wu Y, et al. Production of proinflammatory cytokines in the human THP-1 monocyte cell line following induction by Tp0751, a recombinant protein of Treponema pallidum. Sci China Life Sci. 2010;53:229-233.
122.Yue TL, Wang X, Sung CP, et al. Interleukin-8. A mitogen and chemoattractant for vascular smooth muscle cells. Circ Res. 1994;75:1-7.
123.Eguchi A, Murakami A, Li S, et al. Suppressive effects of demethylated metabolites of nobiletin on phorbol ester-induced expression of scavenger receptor genes in THP-1 human monocytic cells. Biofactors. 2007;31:107-116.
124.Lo YH, Pan MH, Li S, et al. Nobiletin metabolite, 3'',4''-dihydroxy-5,6,7,8-tetramethoxyflavone, inhibits LDL oxidation and down-regulates scavenger receptor expression and activity in THP-1 cells. Biochim Biophys Acta. 2010;1801:114-126.
125.Yen JH, Weng CY, Li S, et al. Citrus flavonoid 5-demethylnobiletin suppresses scavenger receptor expression in THP-1 cells and alters lipid homeostasis in HepG2 liver cells. Mol Nutr Food Res. 2011;55:733-748.
126.Morihara N, Ide N, Weiss N. Aged garlic extract inhibits CD36 expression in human macrophages via modulation of the PPARgamma pathway. Phytother Res. 2010;24:602-608.
127.Granados-Principal S, Quiles JL, Ramirez-Tortosa CL, et al. Squalene ameliorates atherosclerotic lesions through the reduction of CD36 scavenger receptor expression in macrophages. Mol Nutr Food Res. 2012;56:733-740.
128.Collot-Teixeira S, Martin J, McDermott-Roe C, et al. CD36 and macrophages in atherosclerosis. Cardiovasc Res. 2007;75:468-477.
129.Ando C, Takahashi N, Hirai S, et al. Luteolin, a food-derived flavonoid, suppresses adipocyte-dependent activation of macrophages by inhibiting JNK activation. FEBS Lett. 2009;583:3649-3654.
130.Nickel T, Schmauss D, Hanssen H, et al. oxLDL uptake by dendritic cells induces upregulation of scavenger-receptors, maturation and differentiation. Atherosclerosis. 2009;205:442-450.
131.Kim SH, Shin KJ, Kim D, et al. Luteolin inhibits the nuclear factor-kappa B transcriptional activity in Rat-1 fibroblasts. Biochem Pharmacol. 2003;66:955-963.
132.Lu L, Liu H, Peng J, et al. Regulations of the key mediators in inflammation and atherosclerosis by aspirin in human macrophages. Lipids Health Dis. 2010;9:16.
133.Singh U, Dasu MR, Yancey PG, et al. Human C-reactive protein promotes oxidized low density lipoprotein uptake and matrix metalloproteinase-9 release in Wistar rats. J Lipid Res. 2008;49:1015-1023.
134.Negre Salvayre A, Coatrieux C, Ingueneau C, Salvayre R. Advanced lipid peroxidation end products in oxidative damage to proteins. Potential role in diseases and therapeutic prospects for the inhibitors. Br J Pharmacol. 2008;153:6-20.