Specific Dietary Polyphenols Attenuate Atherosclerosis in Apolipoprotein E–Knockout Mice by Alleviating Inflammation and Endothelial Dysfunction
2010; Lippincott Williams & Wilkins; Volume: 30; Issue: 4 Linguagem: Inglês
10.1161/atvbaha.109.199687
ISSN1524-4636
AutoresWai Mun Loke, Julie M. Proudfoot, Jonathan M. Hodgson, Allan J. McKinley, Neil J. Hime, Maria Magat, Roland Stocker, Kevin D. Croft,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoHomeArteriosclerosis, Thrombosis, and Vascular BiologyVol. 30, No. 4Specific Dietary Polyphenols Attenuate Atherosclerosis in Apolipoprotein E–Knockout Mice by Alleviating Inflammation and Endothelial Dysfunction Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessResearch ArticlePDF/EPUBSpecific Dietary Polyphenols Attenuate Atherosclerosis in Apolipoprotein E–Knockout Mice by Alleviating Inflammation and Endothelial Dysfunction Wai Mun Loke, Julie M. Proudfoot, Jonathan M. Hodgson, Allan J. McKinley, Neil Hime, Maria Magat, Roland Stocker and Kevin D. Croft Wai Mun LokeWai Mun Loke From the School of Medicine and Pharmacology (W.M.L., J.M.P., J.M.H., and K.D.C.), University of Western Australia, Perth; School of Biomedical, Biomolecular, and Chemical Sciences (W.M.L. and A.J.M.), University of Western Australia, Perth; and the Department of Pathology (N.H., M.M., and R.S.), Centre for Vascular Research, Sydney University, New South Wales, Australia. , Julie M. ProudfootJulie M. Proudfoot From the School of Medicine and Pharmacology (W.M.L., J.M.P., J.M.H., and K.D.C.), University of Western Australia, Perth; School of Biomedical, Biomolecular, and Chemical Sciences (W.M.L. and A.J.M.), University of Western Australia, Perth; and the Department of Pathology (N.H., M.M., and R.S.), Centre for Vascular Research, Sydney University, New South Wales, Australia. , Jonathan M. HodgsonJonathan M. Hodgson From the School of Medicine and Pharmacology (W.M.L., J.M.P., J.M.H., and K.D.C.), University of Western Australia, Perth; School of Biomedical, Biomolecular, and Chemical Sciences (W.M.L. and A.J.M.), University of Western Australia, Perth; and the Department of Pathology (N.H., M.M., and R.S.), Centre for Vascular Research, Sydney University, New South Wales, Australia. , Allan J. McKinleyAllan J. McKinley From the School of Medicine and Pharmacology (W.M.L., J.M.P., J.M.H., and K.D.C.), University of Western Australia, Perth; School of Biomedical, Biomolecular, and Chemical Sciences (W.M.L. and A.J.M.), University of Western Australia, Perth; and the Department of Pathology (N.H., M.M., and R.S.), Centre for Vascular Research, Sydney University, New South Wales, Australia. , Neil HimeNeil Hime From the School of Medicine and Pharmacology (W.M.L., J.M.P., J.M.H., and K.D.C.), University of Western Australia, Perth; School of Biomedical, Biomolecular, and Chemical Sciences (W.M.L. and A.J.M.), University of Western Australia, Perth; and the Department of Pathology (N.H., M.M., and R.S.), Centre for Vascular Research, Sydney University, New South Wales, Australia. , Maria MagatMaria Magat From the School of Medicine and Pharmacology (W.M.L., J.M.P., J.M.H., and K.D.C.), University of Western Australia, Perth; School of Biomedical, Biomolecular, and Chemical Sciences (W.M.L. and A.J.M.), University of Western Australia, Perth; and the Department of Pathology (N.H., M.M., and R.S.), Centre for Vascular Research, Sydney University, New South Wales, Australia. , Roland StockerRoland Stocker From the School of Medicine and Pharmacology (W.M.L., J.M.P., J.M.H., and K.D.C.), University of Western Australia, Perth; School of Biomedical, Biomolecular, and Chemical Sciences (W.M.L. and A.J.M.), University of Western Australia, Perth; and the Department of Pathology (N.H., M.M., and R.S.), Centre for Vascular Research, Sydney University, New South Wales, Australia. and Kevin D. CroftKevin D. Croft From the School of Medicine and Pharmacology (W.M.L., J.M.P., J.M.H., and K.D.C.), University of Western Australia, Perth; School of Biomedical, Biomolecular, and Chemical Sciences (W.M.L. and A.J.M.), University of Western Australia, Perth; and the Department of Pathology (N.H., M.M., and R.S.), Centre for Vascular Research, Sydney University, New South Wales, Australia. Originally published21 Jan 2010https://doi.org/10.1161/ATVBAHA.109.199687Arteriosclerosis, Thrombosis, and Vascular Biology. 2010;30:749–757Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 21, 2010: Previous Version 1 AbstractObjective— Animal and clinical studies have suggested that polyphenols in fruits, red wine, and tea may delay the development of atherosclerosis through their antioxidant and anti-inflammatory properties. We investigated whether individual dietary polyphenols representing different polyphenolic classes, namely quercetin (flavonol), (−)-epicatechin (flavan-3-ol), theaflavin (dimeric catechin), sesamin (lignan), or chlorogenic acid (phenolic acid), reduce atherosclerotic lesion formation in the apolipoprotein E (ApoE)−/− gene–knockout mouse.Methods and Results— Quercetin and theaflavin (64-mg/kg body mass daily) significantly attenuated atherosclerotic lesion size in the aortic sinus and thoracic aorta (P<0.05 versus ApoE−/− control mice). Quercetin significantly reduced aortic F2-isoprostane, vascular superoxide, vascular leukotriene B4, and plasma-sP-selectin concentrations; and augmented vascular endothelial NO synthase activity, heme oxygenase-1 protein, and urinary nitrate excretion (P<0.05 versus control ApoE−/− mice). Theaflavin showed similar, although less extensive, significant effects. Although (−)-epicatechin significantly reduced F2-isoprostane, superoxide, and endothelin-1 production (P<0.05 versus control ApoE−/− mice), it had no significant effect on lesion size. Sesamin and chlorogenic acid treatments exerted no significant effects. Quercetin, but not (−)-epicatechin, significantly increased the expression of heme oxygenase-1 protein in lesions versus ApoE−/− controls.Conclusion— Specific dietary polyphenols, in particular quercetin and theaflavin, may attenuate atherosclerosis in ApoE−/− gene–knockout mice by alleviating inflammation, improving NO bioavailability, and inducing heme oxygenase-1. These data suggest that the cardiovascular protection associated with diets rich in fruits, vegetables, and some beverages may in part be the result of flavonoids, such as quercetin.Atherosclerosis is a multifactorial disease developing over many years, with symptoms becoming apparent in the late stages of the disease. Inflammation,1 oxidative stress,2 and endothelial dysfunction3 are associated with the pathogenesis of atherosclerosis. Polyphenols are naturally occurring components of fruits and vegetables and are currently the focus of much nutritional and therapeutic interest. The results of population studies suggest that adopting polyphenol-rich diets may protect against cardiovascular disease,4–6 whereas animal and human intervention studies indicate cardiovascular protective effects of polyphenol-rich foods.7–11 Mechanisms by which these compounds exert their cardiovascular protective effects are inconclusive. It is widely hypothesized that dietary polyphenols protect against atherosclerosis by preventing 1 or more of the processes involved in disease progression, such as oxidative stress, inflammation, and endothelial dysfunction.12 However, there are many hundreds of polyphenols in the human diet, and it is not yet known if some compounds offer more cardiovascular protection than others.We selected several common dietary polyphenols (structures in Figure 1 ), such as quercetin (a flavonol found in the diet from fruits, vegetables, and tea), (−)-epicatechin (a flavan-3-ol from cocoa and tea), theaflavin (a dimeric catechin from black tea), sesamin (a lignan from sesame seeds), and chlorogenic acid (a phenolic acid from coffee and some fruits) for this study. Grape polyphenols (including quercetin) have been shown to improve the lipoprotein profile and reduce plasma inflammatory biomarkers and oxidized low-density lipoprotein (LDL) in healthy human subjects, which may decrease cardiovascular disease risk.7,8 Quercetin reduces blood pressure and improves endothelial function in the rat.13 Previous human intervention studies have indicated that (−)-epicatechin from cocoa improves endothelial function9 and reduces inflammation.10 In a short-term human intervention study, we demonstrated that quercetin and, to a lesser extent, (−)-epicatechin are able to augment NO production and reduce endothelin-1 (ET-1), whereas epigallocatechin gallate had no effect.14 In vitro studies with leukocytes indicate that the anti-inflammatory activity of flavonoids may be dissociated from their antioxidant activity.15 Theaflavin was included in our study because it is a major constituent of black tea, which is widely consumed in Western countries and may offer similar antioxidant potency as green tea catechins.16 Black tea consumption has been shown to improve endothelial function in patients with coronary artery disease.11 Sesamin is a bioactive lignan in sesame seeds; it was shown to reduce LDL cholesterol and interfere with the metabolism of the antioxidant γ-tocopherol.17 Chlorogenic acid, a major component of coffee and some fruits, can act as an antioxidant in vitro.18 Given concerns about the bioavailability of polyphenols in vivo,19 we examined their effects when incorporated individually into the diet of a well-established mouse model of atherosclerosis. We determined if these pure polyphenols prevent or reduce the formation of atherosclerotic lesions in apolipoprotein E (ApoE)−/− mice and investigated the mechanisms by which these compounds may exert their antiatherosclerotic effects. This study provides insight into the potential beneficial effects of consuming polyphenol-rich diets. Download figureDownload PowerPointFigure 1. Structures of polyphenols used in the study.MethodsMaterialsChemicals and reagents were purchased from Sigma Aldrich, St Louis, Mo; and Cayman Chemical, Ann Arbor, Mich. High-purity solvents were from Univar (Perth, Western Australia). Sesamin and theaflavin were provided by Suntory (Japan) and Unilever (Netherlands), respectively. All compounds had greater than 95% purity based on high-performance liquid chromatographic analysis.C57BL/6J and ApoE−/− MiceThe present study was approved by and performed under the guidelines of the Animal Ethics Committees of the University of Western Australia and Royal Perth Hospital. A total of 150 four-week-old male ApoE−/− mice and 25 C57BL/6J mice were obtained from the Animal Resource Centre, Canningvale, Australia. The genetic background for the ApoE−/− is C57BL/6J. They have been backcrossed to the C57BL/6J 10 times. The mice were housed in groups of 5 and placed on a nonpurified stock diet of AIN, 93 mol/L (Glenforrest Stockfeeds, Perth, Western Australia) (calculated nutritional parameters in Supplemental Table 1; available online at http://atvb.ahajournals.org). A total of 125 ApoE−/− mice were randomized to receive quercetin, (−)-epicatechin, theaflavin, sesamin, or chlorogenic acid (1.3 mg/d; 64-mg/ kg body mass; n=25 for each treatment group). These levels are approximately equivalent to 350 mg/d in humans.20 The treatment compounds were blended with the mouse feed, which was ground into pellets and stored at 0°C until used. The control groups of 25 ApoE−/− mice and 25 C57BL/6J wild-type mice received the blended and pelletized mouse feed with no compounds added. The mice began to receive the prescribed treatment at the age of 6 weeks, until the end of the study. Food, fluid intake, and body weight were monitored on a regular basis throughout the study. Urine was collected from each group in metabolic cages at the ages of 16 and 26 weeks. After 10 weeks of treatment, when the mice were aged 16 weeks, 5 from each group were killed for analysis of early lesion development and plasma and aortic biochemistry studies. The remaining mice were killed for the same analyses at the age of 26 weeks (ie, after 20 weeks of treatment). Animal numbers were based on the power analysis performed on the desired end points (P<0.008 for multiple comparisons between the treatment and control groups) and on a previous study that showed significant differences in lesion area by the age of 26 weeks.21Plasma and Aortic Tissue SamplingNonfasting mice from each group were studied at the ages of 16 weeks (n=5) and 26 weeks (n=20). Mice were anesthetized with pentobarbital sodium (Nembutal), and the abdominal and thoracic cavities were opened by ventral incision. A blood sample was obtained via vena cava puncture and collected into 50-μL EDTA, 1 g/10 mL in 0.9% saline. The blood plasma was stored after the addition of butylated hydroxytoluene, 8 μg/mL, at −80°C. The aortic sinus and the thoracic and abdominal aorta were removed and stripped of any external fatty deposits. Aortas for histopathologic and biochemical analysis were prepared as previously described.21,22Histological Analysis of Mouse Aorta SpecimensThe size of atherosclerotic lesions in the mouse aorta was determined by measuring the cross-sectional lesion area using procedures described previously.22 The aorta was rinsed in phosphate-buffered saline (PBS) after removal from the phosphate-buffered formaldehyde (4% by volume; pH, 7.0 to 7.3) and processed as described in the Supplemental Material. For immunohistochemistry of heme oxygenase-1 (HO-1), paraffin-embedded thoracic aorta specimens were sectioned every 400 μm, proximally from the third pair of intercostal arteries, for a total distance of 2800 μm. HO-1 protein was detected with anti–rat HO-1 polyclonal rabbit antibody (SPA-895; Stressgen, Ann Arbor, Mich) (final concentration, 10 μg/mL), applied for 24 hours at 4°C. Bound HO-1 antibody was detected with biotinylated anti–rabbit goat IgG (Dako, Glostrup, Denmark) (final concentration, 3.8 μg/mL), applied for 1 hour at room temperature, and the ABC detection method (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, Calif). Counterstaining was achieved with hematoxylin and Scott blue solution.Plasma Cholesterol and Aortic Fatty Acid CompositionThe total cholesterol content of the mouse plasma samples was measured using a commercially available cholesterol assay kit (Boehringer Mannheim, Germany). Aortic tissue was thawed, weighed, and homogenized in 2 mL of PBS and extracted using ice-cold Folch solution (chloroform-methanol, 2:1 by volume containing 0.1-mmol/L butylated hydroxytoluene). The chloroform layer (containing the F2-isoprostanes and fatty acids) was collected, dried, and analyzed, as described in detail in the Supplemental Material. For the distribution of lipoprotein classes, mouse plasma was fractionated by fast protein liquid chromatography using 2 columns (Superose HR6 10/30; Amersham Biosciences, Uppsala, Sweden) in series at a flow rate of 30 mL/h. Plasma samples from 5 mice in the same group (80 μL each) were pooled to give 400 μL, were diluted with 400 μL of PBS, and were filtered through a 0.45-μm filter; 450 μL of diluted plasma was applied to the column and eluted with PBS containing 1-mmol/L EDTA (pH, 7.2). Fractions, 0.5 mL, were collected into tubes containing butylated hydroxytoluene, 20 μL, at 4 mg/mL. Protein was monitored continuously by absorbance at 280 nm, and total cholesterol was measured in each of the fractions using an enzymatic method (Roche Diagnostics GmbH, Mannheim, Germany).Oxidative StressVascular oxidative stress was assessed by measuring F2-isoprostanes in aortic tissue by gas chromatography–mass spectrometry using a previously described method.23 Aortic F2-isoprostanes were measured in lipid extracts from the mouse aortas and corrected for arachidonic acid. Vascular superoxide was assessed by lucigenin-derived chemiluminescence,24 as described in detail in the Supplemental Material. The chemiluminescence signal was inhibited by 90% in control incubations, with superoxide dismutase added.Ex Vivo Vascular Leukotriene B4 ProductionThe effect of the polyphenol treatments on 5-lipoxygenase enzyme activity was determined by measuring vascular leukotriene B4 (LTB4) production ex vivo, as described in detail in the Supplemental Material.Plasma-Soluble P-SelectinThe long-term effects of treatment on platelet reactivity were investigated by measuring the plasma concentrations of soluble P-selectin (sP-selectin) using a commercially available mouse sP-selectin enzyme immunoassay kit (R&D Systems, Minneapolis, Minn).Vascular Endothelial NO Synthase Activity, Urinary Nitrite, Nitrate, and ET-1NOS activity in aortic homogenates was determined by monitoring the conversion of L-[3H]arginine to L-[3H]citrulline using an NOS activity assay kit (Cayman Chemical). Results were expressed as picomoles of L-citrulline per milligram of protein per 60 minutes. Nitrite and nitrate concentrations in urine were determined using a previously published gas chromatographic–mass spectrometric method.25 Results were corrected for creatinine levels. ET-1 was measured in urine with a commercially available ET-1 (mouse) enzyme immunoassay kit (Assay Design, Ann Arbor, Mich). ET-1 concentrations were corrected for creatinine levels.Statistical AnalysisStatistical analyses were performed using SPSS version 15 (SPSS Inc, Chicago, Ill). Data are presented as mean±SEM. A 1-way ANOVA26 with post hoc analyses using the Tukey honestly significant difference test was used to compare treatments. Initially, the ApoE−/− control mice were compared with the C57BL/6J mice. The effect of polyphenol treatments in the ApoE−/− mice were then compared with effects in the ApoE−/− control mice. The results analyzed were considered significantly different when P<0.05.ResultsAnimals and Polyphenol DietsThere was no difference in body mass between C57BL/6J and ApoE−/− mice over 26 weeks (from a mean body mass of 9.5± 0.5 g at week 4 to 28.0±0.3 g at week 26). The daily intake of the polyphenols was calculated for each group based on the daily consumption of the mouse feed (mean daily intake in mg/kg of body mass: quercetin, 63.8±0.5; (−)-epicatechin, 64.0±0.3; theaflavin, 63.5±0.8; sesamin, 63.7±0.4; and chlorogenic acid, 63.8±0.6). No significant difference between the groups was observed.Aortic Lesion AnalysesThe lesion area in the transverse section of the aorta was expressed as a percentage of the lesion to the total area of the aortic tissue. No significant atherosclerotic lesion was observed in C57BL/6J and ApoE−/− mice at the age of 16 weeks (data not shown). At the age of 26 weeks, ApoE−/− mice exhibited significantly greater lesions at the aortic sinus and the thoracic aortic region just below the aortic arch compared with the C57BL/6J control mice (Figure 2A and B; P<0.05 versus C57BL/6J mice). Lesion formation at both locations was significantly reduced in ApoE−/− mice fed a diet containing quercetin or theaflavin (Figure 2A and B, 60% to 80% and 45% to 55%, respectively; P<0.05 versus ApoE−/− control mice). Examples of micrographs for aortic cross sections stained for lipid lesions are shown in Supplemental Figure I (available online at http://atvb.ahajournals.org). Treatment with (−)-epicatechin, sesamin, and chlorogenic acid appeared to diminish lesion formation (14%, 40%, and 29%, respectively, versus ApoE−/− mice); however, these differences were not statistically significant. Download figureDownload PowerPointFigure 2. A, Cross-sectional lesion area (percentage of total cross-sectional area) in the aortic sinus from C57BL and ApoE−/− mice after 20 weeks of different dietary treatments: C57BL/ control (n=20), ApoE/control (n=20), ApoE/quercetin (n=18), ApoE/(−)-epicatechin (n=19), ApoE/theaflavin (n=20), ApoE/sesamin (n=18), and ApoE/chlorogenic acid (n=19). B, Cross-sectional lesion area (percentage of total cross-sectional area) in the thoracic region just below the aortic arch from C57BL and ApoE−/− mice after 20 weeks of different dietary treatments: C57BL/control (n=20), ApoE/control (n=20), ApoE/quercetin (n=18), ApoE/(−)-epicatechin (n=19), ApoE/theaflavin (n=20), ApoE/sesamin (n=18), and ApoE/chlorogenic acid (n=19). In A and B, bars with the same subscript are not significantly different from each other using 1-way ANOVA analysis with the Tukey honestly significant difference post hoc analysis.In a subgroup of mice, the expression of HO-1 protein in aortic sections was examined. Aortic sections from apoE−/− mice showed greater staining for HO-1 than those of C57BL/6J mice, with HO-1 staining limited largely to atherosclerotic lesions. In apoE−/− mice fed the quercetin-supplemented diet, HO-1 protein was significantly increased compared with either the apoE−/− control (P<0.05) or the epicatechin-fed apoE−/− mice (P<0.01) (Figure 3). Download figureDownload PowerPointFigure 3. Examples of aortic lesion HO-1 staining (brown) in C57BL wild-type sections (n=72), ApoE−/− control (n=56), quercetin-fed mice (n=64), and epicatechin-fed mice (n=88). Data presented (×40 magnification) are the sum of HO-1 stain (pixels) per lesion tissue area. Few sections from C57BL wild-type mice had lesions or HO-1 staining. Sections without lesions or HO-1 staining were assigned a value of 0. *Quercetin vs ApoE−/− (P<0.05), quercetin vs epicatechin (P<0.01), and quercetin vs C57BL control (P<0.001).Plasma Cholesterol and Aortic Fatty Acid CompositionApoE−/− mice had significantly elevated plasma concentrations of cholesterol compared with the C57BL/6J mice (Supplemental Table II). The polyphenols did not significantly affect plasma cholesterol concentrations or lipoprotein distribution (Supplemental Figure II) in the ApoE−/− mice after 20 weeks of treatment. None of the polyphenol treatment exerted a significant effect on the fatty acid composition in the aorta compared with the control apoE−/− mice (data not shown).Vascular Oxidative StressAt the age of 26 weeks, ApoE−/− mice had significantly higher concentrations of aortic F2-isoprostanes than C57BL/6J mice (Figure 4A, P<0.05). Diets incorporating quercetin or (−)-epicatechin significantly reduced aortic F2-isoprostane concentrations in ApoE−/− mice (Figure 4A, P<0.05 versus ApoE−/− control mice). Theaflavin, sesamin, and chlorogenic acid treatments did not show any significant effect on aortic F2-isoprostane concentrations. Aortic tissues from the ApoE−/− mice had a slightly elevated superoxide level compared with C57BL/6J mice at the age of 26 weeks (Figure 4B), although this was not significantly different. Quercetin and (−)-epicatechin treatments significantly attenuated vascular superoxide (Figure 4B, P<0.05 versus ApoE−/− control mice); no significant effect was observed for the other treatments. Download figureDownload PowerPointFigure 4. A, Aortic F2-isoprostane concentrations of C57BL and ApoE−/− mice, expressed per unit mass of aortic arachidonic acid, after 20 weeks of different dietary treatments: C57BL/control (n=20), ApoE/control (n=19), ApoE/quercetin (n=18), ApoE/(−)-epicatechin (n=18), ApoE/theaflavin (n=19), ApoE/sesamin (n=18), and ApoE/chlorogenic acid (n=18). B, Superoxide anion radical production in abdominal aortic tissues from C57BL and ApoE−/− mice after 20 weeks of different dietary treatments: C57BL/control (n=20), ApoE/control (n=19), ApoE/quercetin (n=19), ApoE/(−)-epicatechin (n=18), ApoE/theaflavin (n=20), ApoE/sesamin (n=18), and ApoE/chlorogenic acid (n=19). C, Ex vivo LTB4 production in abdominal aortic tissues from C57BL and ApoE−/− mice after 20 weeks of different dietary treatments: C57BL/control (n=20), ApoE/control (n=20), ApoE/quercetin (n=19), ApoE/(−)-epicatechin (n=19), ApoE/theaflavin (n=20), ApoE/sesamin (n=19), and ApoE/chlorogenic acid (n=18). D, Plasma sP-selectin concentrations of C57BL and ApoE−/− mice (μM creatinine) after 20 weeks (mice aged 26 weeks) of different dietary treatments: C57BL/control (n=20), ApoE/control (n=20), ApoE/quercetin (n=19), ApoE/(−)-epicatechin (n=19), ApoE/theaflavin (n=20), ApoE/sesamin (n=18), and ApoE/chlorogenic acid (n=18). In A through D, bars with the same subscript are not significantly different from each other using 1-way ANOVA analysis with the Tukey honestly significant difference post hoc analysis.Ex Vivo Vascular LTB4 Production and Plasma-sP-SelectinAortic tissues from ApoE−/− mice produced significantly higher amounts of LTB4 compared with the C57BL/6J mice (Figure 4C, P<0.05). Quercetin and theaflavin treatment significantly reduced LTB4 in the aortic tissues (Figure 4C, P<0.05).ApoE−/− mice expressed significantly higher plasma concentrations of sP-selectin than C57BL/6J mice at the age of 26 weeks (P<0.005) (Figure 4D). Treatment with quercetin, (−)-epicatechin, and theaflavin significantly lowered the plasma sP-selectin concentrations (P<0.005 versus ApoE−/− control mice). Sesamin and chlorogenic acid did not significantly affect plasma sP-selectin concentrations compared with the ApoE−/− control mice (Figure 4D).Vascular Endothelial NO Synthase Activity and Urinary Nitrite, Nitrate, and ET-1At the age of 26 weeks, the vascular endothelial NO synthase (eNOS) activity of ApoE−/− mice in the control group was significantly lower compared with the C57BL/6J mice (P<0.05, Figure 5A). Quercetin and theaflavin significantly increased eNOS activity in the aortic tissues (P<0.05 versus ApoE−/− control mice, Figure 5A), whereas the other polyphenols showed insignificant elevations in eNOS activity. The increase in eNOS activity significantly correlated with the elevation in excretion of nitrate in the polyphenol-treated ApoE−/− mice at 26 weeks (R=0.65, P<0.001). All 5 treatments elevated the urinary nitrate concentration, with quercetin- or theaflavin-treated ApoE−/− mice having significantly higher urinary nitrate concentrations compared with the ApoE−/− control mice (P<0.05, Supplemental Table III). However, there was no significant difference in urinary nitrite between any of the treatment groups (Supplemental Table III). Download figureDownload PowerPointFigure 5. A, Vascular eNOS activity of C57BL and ApoE−/− mice after 20 weeks (mice aged 26 weeks) of different dietary treatments: C57BL/control (n=5), ApoE/control (n=5), ApoE/quercetin (n=5), ApoE/(−)-epicatechin (n=5), ApoE/theaflavin (n=5), ApoE/sesamin (n=5), and ApoE/chlorogenic acid (n=5). B, Urinary ET-1 concentrations of C57BL and ApoE−/− mice after 20 weeks (mice aged 26 weeks) of different dietary treatments: C57BL/control (n=5), ApoE/control (n=5), ApoE/quercetin (n=5), ApoE/(−)-epicatechin (n=5), ApoE/theaflavin (n=5), ApoE/sesamin (n=5), and ApoE/chlorogenic acid (n=5). In A and B, bars with the same subscript are not significantly different from each other using 1-way ANOVA analysis with the Tukey honestly significant difference post hoc analysis.Urinary concentrations of ET-1 were significantly increased in the ApoE−/− mice at the age of 26 weeks compared with C57BL/6J mice (P<0.05); this was attenuated with quercetin and (−)-epicatechin dietary treatments (P<0.05, Figure 5B).Correlation Between Lesion Size and BiomarkersLesion size at the aortic sinus was significantly correlated with aortic F2-isoprostanes (R=0.29, P<0.001), aortic eNOS activity (R=−0.67, P<0.001), and urinary nitrate (R=−0.52, P<0.005); the lesion amounts at the thoracic aorta were significantly correlated to aortic F2-isoprostanes (R=0.22, P<0.01), aortic superoxide (R=0.20, P<0.05), aortic LTB4 (R=0.21, P<0.01), aortic eNOS activity (R=−0.38, P<0.05), and urinary nitrate (R=−0.38, P<0.05) (Supplemental Table IV).DiscussionOur study has shown that particular dietary polyphenols are bioactive molecules that can inhibit the development of atherosclerosis. In particular, quercetin and theaflavin significantly attenuated lesion formation in ApoE−/− mice. Previous studies have shown that polyphenol-rich beverages, such as red wine,21 dealcoholized red wine,22 and tea,27 can inhibit atherosclerosis in ApoE−/− mice. These beverages contain a complex mixture of polyphenolic compounds. Our study suggests that certain individual polyphenols can attenuate atherosclerosis, and these compounds may represent some of the active components of polyphenol-rich beverages. Compounds such as quercetin and theaflavin, which inhibit atherosclerosis, may do so through many pathways, including inhibition of inflammation (LTB4), improvement of NO bioavailability, and propagation of HO-1 expression (Table). Table. Effects of Specific Polyphenols on Tested Pathways at Week 26*VariableQuercetinEpicatechinTheaflavinSesaminChlorogenic AcideNOS indicates endothelial NO synthase; ET, endothelin; HO, heme oxygenase; LTB4, leukotriene B4; ND, not determined.*Data are expressed as percentage change compared with the apolipoprotein E−/− mice fed a control diet.†P<0.05 vs apolipoprotein E−/−control mice.Aortic sinus lesion formation−79†−14−56†−41−24Thoracic aorta lesion formation−57†−15−56†−24−43Plasma cholesterol0.1−24−2−199Aortic F2-isoprostanes−60†−77†−39−279HO-1 protein190†4NDNDNDAortic superoxide−42†−41†−10−24−15Aortic LTB4−53†−34−47†−188Plasma-soluble P-selectin−29†−33†−26†−7−7Urinary nitrate90†4465†4121Vascular eNOS activity1446†631923†305466Urinary ET-1−52†−51†−1−4−40Lipid peroxidative damage may be a critical step in the pathogenesis of atherosclerosis.2 The well-recognized antioxidant activity of many polyphenols has led to the proposal that polyphenol protection against atherosclerosis may involve their antioxidant properties.28 Quercetin and catechins in red wine and tea have been shown to inhibit atherosclerosis in ApoE−/− mice while also reducing LDL susceptibility to oxidation.27,29 Quercetin-3-O-glucuronide (a major in vivo quercetin metabolite) was shown to localize within activated macrophages in human atherosclerotic lesions and to prevent the uptake of oxidized LDL through the downregulation of scavenger receptors.30 Black tea consumption decreased lipoprotein oxidation in New Zealand white rabbits31; and theaflavin, a major polyphenol present in black tea, was shown to be as effective as c
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