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* Graduate Center for Nutritional Sciences, Department of
Surgery and
** Molecular and Cell Nutrition Laboratory, College of Agriculture, University of Kentucky, Lexington, KY 40546-0215
2To whom correspondence should be addressed. E-mail: bhennig{at}uky.edu.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B (NF-B) and activator protein-1 (AP-1). Because the transcription factor peroxisome proliferator activated receptor 
(PPAR) was reported to downregulate inflammatory pathways, we further investigated the effect of quercetin on PPAR. Porcine pulmonary-arterial endothelial cells were activated with linoleic acid in the presence or absence of quercetin. Oxidative stress was markedly induced by endothelial cell exposure to linoleic acid and diminished by treatment with quercetin as measured via the oxidation of 2',7'-dichlorofluorescin. Quercetin reduced linoleic acid–mediated binding activity of NF-B and AP-1 and mRNA levels of inflammatory genes such as interleukin-6 (IL-6) and vascular cell adhesion molecule-1 (VCAM-1). Cotreatment of linoleic acid plus quercetin or vitamin E also decreased linoleic acid–induced binding activity of PPAR. These data suggest that quercetin has potent antioxidative and anti-inflammatory properties and protects endothelial cells against linoleic acid–mediated cell dysfunction.
KEY WORDS: • atherosclerosis • fatty acids • quercetin • PPAR
Atherosclerotic lesions are thought to be initiated by vascular endothelial cell dysfunction. A damaged endothelium is less effective as a selectively permeable barrier to plasma components (1,2). The endothelium interacts with the blood and underlying tissues, serves as both a pro- and antithrombotic surface, and releases regulatory factors important in modulating vascular tone. Factors implicated in the pathogenesis of atherosclerosis include chronic and cumulative metabolic alterations of the endothelium induced by numerous activating molecules, such as certain lipids, prooxidants, and inflammatory cytokines. These risk factors may contribute to an overall cellular imbalance of the oxidative stress/antioxidant balance, thus leading to chronic activation of the endothelium and alterations of the endothelial barrier function, which can result in accelerated uptake of cholesterol-rich lipoproteins into the vessel wall.
After consumption of high-energy foods, triglyceride-rich lipoproteins are elevated, and hydrolysis of triglycerides by lipoprotein lipase occurs in proximity to the endothelial surface (3). An excessive local concentration of fatty acid anions may cause endothelial injury and therefore initiate the onset of atherosclerosis. PUFA are more susceptible to lipid peroxidation than SFA, in particular when insufficiently protected by antioxidants. Thus, if oxidative stress is a critical underlying parameter of atherosclerosis (4), then high serum PUFA concentrations may indicate a higher risk of atherosclerosis (5).
Antioxidants, such as vitamin E, can significantly reduce the linoleic acid–mediated endothelial cell activation and loss of endothelial integrity (6). Quercetin, like other polyphenolics, possesses high antioxidant abilities to inhibit free radical processes in cells (7), including the prevention of lipid peroxidation (8).
Numerous oxidative stress-sensitive transcription factors, such as nuclear factor-B (NF-B)3 and activator protein-1 (AP-1) (9) can mediate an inflammatory response due to oxidative stress by inducing gene transcription of adhesion molecules and cytokines such as vascular cell adhesion molecule-1 (VCAM-1) and interleukin-6 (IL-6). Antioxidants such as quercetin could protect plasma lipids from oxidation, thereby preventing the induction of inflammatory events. Thus, quercetin could help maintain the integrity of the endothelium.
There is increasing evidence that peroxisome proliferator activated receptors (PPARs) can modulate inflammatory events and are thus antiatherogenic (10). For example, PPAR can interfere negatively with NF-B, signal transducer and activator of transcription (STAT), and AP-1 signaling pathways (11,12). This could be by preventing transcription factors from binding to their target sequences (12,13), possibly through an interaction with a subunit of the transcription factors (e.g., p65) (12). Because most of the proinflammatory genes are under the control of the AP-1 and NF-B signaling pathways, and because PPARs can counterregulate a wide spectrum of proinflammatory genes, anti-inflammatory compounds may act through PPAR signaling. The binding pockets of PPARs are open to a variety of naturally occurring lipid-like substances acting as low-affinity ligands (14). Because quercetin is a lipophilic, polyphenolic substance with a chemical structure that could potentially fit the binding pocket of PPARs, we investigated whether the anti-inflammatory properties of quercetin could be through the activation of PPAR.
In this study we aimed to demonstrate that quercetin has endothelium-protective effects by preventing linoleic acid–induced oxidative stress formation and by decreasing the activation of oxidant-sensitive pathways.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The experimental media were composed of M-199 enriched with 5% (v:v) FBS and fatty acids (90 µmol/L). Fatty acids (>99% pure) were obtained from Nu-Chek-Prep. Preparations of experimental media with fatty acids were made as described previously (16). Thus, fatty acids were introduced into the media bound to serum albumin. Quercetin (10–50 µmol/L) and vitamin E (25 µmol/L) were added from stock solutions in dimethylsulfoxide (DMSO) and ethanol, respectively. Controls and fatty acid groups not treated with quercetin or vitamin E contained an equal amount of DMSO or ethanol. The final DMSO concentration in the media never exceeded 0.05% (v:v) in all treatment groups. For most experimental settings, cells were treated with quercetin and fatty acids for 6 h. Vitamin E was added 18 h before fatty acid treatment.
Measurement of oxidative stress. Cellular oxidation was determined by 2',7'-dichlorofluorescein (DCF) fluorescence as described by Mattson et al. (17). This measurement of cell oxidation utilizes an imaging technique based on the conversion of 2',7'-dichlorofluorescin into fluorescent 2',7'-dichlorofluorescein as a result of activation with reactive oxygen species (ROS), primarily peroxyl radicals and peroxides. After treatment of endothelial cells with linoleic acid for 6 h, cells were loaded with 100 µmol/L 2,7-dichlorofluorescin diacetate (Molecular Probes) by incubation for 30 min. Before analysis for oxidative stress, cells were washed 3 times in HEPES buffer. In experiments utilizing H2O2 as an inducer of oxidative stress, 0.1 mmol/L H2O2 in HEPES buffer was applied to cells after DCF-staining. Imaging studies employed a multiwell fluorescent plate reader (Molecular Devices). The dye was excited at 490 nm, and emission was filtered using a 510-nm barrier filter.
Transcription factor (NF-B, AP-1, and PPAR) activation studies: electrophoretic mobility shift assay (EMSA).
Nuclear extracts containing active proteins were prepared from cells according to the method of Dignam et al. (18). Nuclear extracts were incubated for 25 min with 32P-end-labeled oligonucleotide probes containing enhancer DNA element NF-B (5' AGTTGAGGGGACTTTCCCAGGC 3'), AP-1 (5' CGCTTGATGAGTCAGCCGGAA 3') (Promega) or PPAR (5' AGGTCAAAGGTCA 3') (Santa Cruz). Incubation at room temperature was performed in the presence of nonspecific competitor DNA. After binding, the complexed and uncomplexed DNA in the mixture were resolved by electrophoresis in a 6.5% (wt:v) nondenaturing polyacrylamide gel and visualized by autoradiography. Control reactions using a 200-fold molar excess of unlabeled oligonucleotide probes or a supershift assay were performed to demonstrate the specificity of the shifted DNA-protein complexes for NF-B, PPAR and AP-1, respectively.
Il-6 and VCAM-1 expression studies. Total RNA was extracted from endothelial cells by the use of TRI reagent (Sigma) according to the manufacturer’s protocol. Gene expression was determined through RT-PCR as described earlier (19). The following primers were employed in the PCRs; IL-6 forward: 5' AAT TCG GTA CAT CCT CGA CG 3', reverse: 5' GCG CAG AAT GAG ATG AGT TG 3', VCAM-1 forward: 5' ATGACA TGC TTG AGC CAG G 3', reverse: 5' GTG TCT CCT TCT TTG ACA CT 3', ß-actin forward: 5' GGG ACC TGA CCG ACT ACC TC3', reverse: 5' GGG CGA TGA TCT TGA TCT TC3'. The amplified PCR products were electrophoresed on a 2% (wt:v) tris-borate EDTA agarose gel, stained with SYBR Gold (Molecular Probes) and visualized by using phosphorimaging technology (FLA-5000; Fuji).
Statistical analysis. The data were quantified and analyzed using the Scion Image and Sigma Stat software, respectively. Comparisons between treatments were made by 1- or 2-way ANOVA with post-hoc comparisons of the means made by Tukey’s tests. A statistical probability of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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B and AP-1.
Linoleic acid increased the DNA binding activity of both transcription factors NF-B and AP-1 as determined by EMSA. Cotreatment of quercetin and linoleic acid for 6 h downregulated the activation of NF-B and AP-1 (Figs. 2A and B, respectively). Consistent with the observations made in measuring oxidative stress, 25 µmol/L appeared to be the most effective concentration of quercetin with no additional benefit of higher concentrations to downregulate NF-B (Fig. 2A). Maximal downregulation of linoleic acid–induced AP-1 binding activity already occurred at 10 µmol/L quercetin (Fig. 2B).
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B and AP-1 binding activity or VCAM-1 mRNA. Indeed, quercetin was effective only when applied at higher concentrations (50 µmol/L).
Quercetin and vitamin E decrease linoleic acid–induced PPAR binding activity.
PPAR protects cells against proinflammatory and prooxidative insults, and linoleic acid is a natural ligand for this transcription factor. Therefore, the effects of linoleic acid and/or quercetin on PPAR DNA binding activity were assessed in the present study. Exposure to linoleic acid for 6 h markedly induced PPAR activity (Figs. 4A and B). Although quercetin alone did not affect binding of this transcription factor, it diminished PPAR activation in cells cotreated with linoleic acid. To assess whether the effects of quercetin on activation of PPAR were specific, cells were pretreated with another antioxidant, vitamin E (25 µmol/L for 18 h) and treated with linoleic acid for 6 h. Similar to the effects exerted by quercetin, vitamin E also protected against linoleic acid–induced binding activity of PPAR (Fig. 4B).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The antioxidant properties of quercetin also could be responsible in part for the anti-inflammatory effects observed in the present study. We showed that quercetin can downregulate the linoleic acid–induced activation of both NF-B and AP-1. These are oxidative stress–sensitive transcription factors that can therefore be modified by oxidants and antioxidants. The precise way in which linoleic acid induces an inflammatory response in endothelial cells is not clear. However, oxidation products of linoleic acid might be directly involved in the activation of NF-B. Lipid peroxides rather than H2O2 were suggested to mediate the activation of NF-B in response to oxidative stress (24). It is possible that lipid metabolites and derivatives, including oxidized fatty acids, can induce the inflammation, in addition to the native linoleic acid itself. Furthermore, quercetin was suggested to suppress inhibitory IB kinase (25) and c-Jun kinase (26), which subsequently could lead to suppression of NF-B and AP-1 activation. Activation of NF-B leads to expression of inflammatory cytokines and adhesion molecules, resulting in recruitment of monocytes and accelerated development of atherosclerosis (27). Because most of the proinflammatory genes are under the control of the AP-1 and NF-B signaling pathways (28) and quercetin can counterregulate these pathways, the potent anti-inflammatory properties of quercetin are clearly demonstrated. In support of its anti-inflammatory properties, we showed that quercetin can block the linoleic acid–induced expression of both VCAM-1 and IL-6, a downstream event of NF-B activation (29).
Because PPAR can interfere negatively with NF-B, STAT, and AP-1 signaling pathways (11,12), we further investigated whether quercetin can affect PPAR binding activity. Even though both PPAR
and PPAR were reported to be anti-inflammatory and antiatherogenic, PPAR is involved in lipid metabolism and could therefore be activated independently of fatty acid modification by oxidation. Thus, the present study focuses on PPAR. Although quercetin has a chemical structure that could potentially be a ligand for PPAR, exposure to quercetin alone did not activate this transcription factor. Consistent with the known overall effects of fatty acids on PPAR activation, treatment with linoleic acid induced PPAR binding activity in endothelial cells. However, cotreatment with quercetin markedly downregulated linoleic acid–induced PPAR activation. These results are consistent with earlier reports showing that quercetin can mediate downregulation of PPAR in transiently transfected macrophages (30) and in murine epidermal keratinocytes (31). Because linoleic acid can induce oxidative stress and inflammation and at the same time activate PPAR, we suspected that lipid oxidation is involved in activating the PPAR pathway. To further address the question whether the decreased PPAR activation observed in the present study is specific for quercetin, endothelial cells were treated with another antioxidant (vitamin E) followed by exposure to linoleic acid. Vitamin E also prevented the linoleic acid–mediated activation of PPAR. These data suggest that quercetin can reduce the linoleic acid–mediated activation of PPAR due to its antioxidant effect and the prevention of lipid oxidation. Previous studies indicated that oxidized products of linoleic acid are good ligands for PPARs (32). Quercetin may act as an antioxidant; thus the decreased formation of oxidized products of linoleic acid might be a reason for the lower binding of linoleic acid to PPAR in the presence of quercetin.
These data suggest that the quercetin-mediated protection observed against linoleic acid–induced endothelial cell activation is independent of PPAR signaling. In addition, it appears that the diminished PPAR DNA binding activity observed in cells exposed to linoleic acid plus quercetin may be related to a general antioxidant effect of this polyphenolic. Indeed, PPARs may act as critical rescue molecules by downregulating oxidative stress–sensitive and inflammatory signaling pathways. In cells that are protected by antioxidants such as quercetin and vitamin E, oxidative stress–sensitive pathways, including PPAR, are less likely to become activated.
Overall, our data suggest that quercetin is a potent antioxidant and anti-inflammatory substance, which can protect the endothelium against oxidative stress and inflammatory events, despite downregulation of PPAR activation. Specifically, quercetin inhibited linoleic acid–induced activation of oxidative stress–sensitive pathways, such as NF-B and AP-1, and inflammatory genes, such as VCAM-1 and IL-6. These data support the hypothesis that plant phenolics such as quercetin can help prevent the development of atherosclerosis by downregulating the expression of inflammatory cytokines and adhesion molecules.
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FOOTNOTES |
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3 Abbreviations used: AP-1, activator protein-1; DCF, 2',7'-dichlorofluorescein; DMSO, dimethylsulfoxide; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; IL-6, interleukin-6; NF-B, nuclear factor-B; PPAR, peroxisome proliferator activated receptor; ROS, reactive oxygen species; STAT, signal transducer and activator of transcription; VCAM-1, vascular cell adhesion molecule-1.
Manuscript received 9 October 2003. Initial review completed 11 November 2003. Revision accepted 11 January 2004.
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