Anti-inflammatory, anti-proliferative and anti-atherosclerotic effects of quercetin in human in vitro and in vivo models
Introduction
Cardiovascular disease (CVD) is an important health problem and one of the main causes of morbidity and mortality in industrialized countries. Specific dietary patterns are positively associated with cardiovascular risk factors [1] and the development of atherosclerosis [2]. For example, diets with a high saturated fat content coincide with elevated levels of circulating C-reactive protein (CRP), a sensitive inflammatory marker predictive of future CVD [3], and with an increased incidence of cardiovascular events [1], [2]. On the other hand, diets rich in fruit and vegetables are inversely related to inflammatory CVD markers and atherosclerosis [1], [2]. A clear understanding of the mechanisms underlying the protective effects of food constituents has not been reached thus far, and the role of individual dietary factors remains enigmatic.
Among the dietary constituents that may influence development of CVD are polyphenols, and epidemiological studies of diet-disease relationships support a possible protective role [4], [5]. However, controlled human studies testing the effect of specific polyphenol-rich foods on circulating risk factors for CVD (e.g. CRP, fibrinogen) are not consistent, with some studies showing reductions in risk factor concentrations [6], [7], while others report a lack of effect [8].
Quercetin is a potent dietary polyphenol that can exert anti-inflammatory, anti-proliferative and anti-oxidative effects [9], [10], [11]. The combination of these putatively beneficial properties makes quercetin a most promising ‘nutriceutical’ for CVD prevention. However, a substantial part of the experimental evidence that suggests health effects comes from in vitro studies and short-term in vivo studies, that often require relatively high doses of compound and lack clear-cut biological endpoints.
In this study, we evaluated quercetin in cultured human cells and two humanized models of CVD, with particular emphasis on the inflammatory aspects of the disease. In cell culture, we have analyzed the anti-oxidative and anti-inflammatory properties of quercetin. In these assays, vitamin E (α-tocopherol) and genistein served as an anti-oxidative and anti-inflammatory reference control, respectively. Direct anti-inflammatory effects of quercetin in vivo were investigated in mice expressing the human CRP gene, both before and after cytokine administration. Of note, the murine form of CRP is not an acute phase reactant or inflammatory marker. Long-term effects of quercetin on the development of atherosclerosis were investigated in human ApoE*3Leiden transgenic mice (ApoE*3L), a model with humanized lipoprotein metabolism [12]. Sodium salicylate served as an anti-inflammatory reference control in the in vivo experiments.
We confirm the putative direct anti-inflammatory effects of quercetin in short-term in vitro and in vivo experiments and show that long-term quercetin treatment attenuates atherosclerotic lesion formation. Bioinformatical pathway analysis of aortic microarray gene expression data revealed that quercetin affects vascular cell proliferation and inflammatory state.
Section snippets
Endothelial cell culture assay for quantifying oxidative stress
Oxidative stress was determined essentially as described [13]. Briefly, endothelial cells from human umbilical veins (HUVEC) were isolated and confluent HUVEC monolayers of passage 1 or 2 were used. The hydrogen peroxide (H2O2)-induced oxidative stress was assessed with a fluorescence assay using fluorophore C11-BODIPY581/591. HUVEC were incubated for 30 min at 37 °C with C11-BODIPY581/591 (1 μmol/L). Subsequently, cells were washed with PBS and oxidative stress was induced with PBS containing 5
Quercetin exerts anti-oxidative and anti-inflammatory effects in cultured human cells
The anti-oxidative and anti-inflammatory capacity of quercetin was first evaluated in human cell culture models and was analyzed together with two reference control compounds with established anti-oxidative (vitamin E) or anti-inflammatory (genistein) effects.
To evaluate its anti-oxidative capacity, we measured the effect of quercetin on H2O2-induced lipid peroxidation using fluorophore C-11 BODIPY581/591. Pre-incubation of HUVEC with quercetin for 18 h dose-dependently (1-100 μmol/L) protected
Discussion
Polyphenols such as quercetin have become the subject of considerable attention, mainly because of their broad spectrum of putatively beneficial effects for vascular health (‘nutriceutical’). Here, we focused on the anti-inflammatory and anti-atherogenic effects of the polyphenol quercetin. Using human CRP transgenic mice, we show that short-term (14 days) treatment with dietary quercetin fully quenches the cytokine-induced expression of human CRP, a sensitive inflammation marker and risk
Funding
This work was supported by the Nederlandse Organisatie voor Toegepast Wetenschappelijk Onderzoek (TNO) research project ‘New Initiative Systems Biology/Personalized Health’.
Author contribution
LV and SZ carried out the in vivo experiments. SZ, PW, and LV were responsible for data collection and analysis. MvE analyzed the micro-array gene expression data. RK, MM and TK designed the study, analyzed data and wrote the manuscript.
Conflict of interest
The authors declare to have no conflict of interest.
Acknowledgements
We thank Karin Toet, Erik Offerman and Annie Jie for excellent technical assistance.
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