Abstract
Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.
Highlights
Polyphenols are a large family of natural compounds widely distributed in plant foods, and have been linked to improved human health through reduced chronic diseases, especially cardiovascular diseases
We have previously demonstrated that Q3GA accumulated in macrophage cells in the foamy macrophages in the human atherosclerotic lesions, and that the macrophage cells could deconjugate Q3GA into the aglycone in vitro [25], whereas the molecular mechanisms and biological consequences have been unclear
We demonstrated that LPSstimulation of the macrophages significantly enhanced the deconjugation of Q3GA and the accumulation of the aglycones, whereas zymography and an immunoblot analysis of the medium demonstrated that the LPS treatment did not significantly increase the secretion of the β-glucuronidase enzyme (Figure 3B), suggesting that β-glucuronidase activity was enhanced upon LPS treatment
Summary
Polyphenols are a large family of natural compounds widely distributed in plant foods, and have been linked to improved human health through reduced chronic diseases, especially cardiovascular diseases. Flavonoids are the most abundant polyphenols in our diets. Thereafter, a large number of biological activities of flavonoids ( called “vitamin P”) were described that are generally believed to be beneficial for good health. Quercetin (3,5,7,3’,4’pentahydroxyflavone) is the major flavonol-type flavonoid and is abundant in onion [3] and tea [4], which represent the major sources of flavonoids in the Dutch diet [5]. Epidemiological evidence links diets rich in quercetin and other flavonoids with the decreased incidence of cardiovascular, neoplastic, and neurodegenerative diseases [6,7,8,9,10,11,12,13]
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