Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

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.     

Attenuation of lipid peroxidation and hyperlipidemia by quercetin glucoside in the aorta of high cholesterol-fed rabbit

Antioxidative activity of dietary flavonoids is suggested to be, at least partly, responsible for a wide variety of their biological effects relating to anti-atherosclerosis. However, it is not known whether dietary flavonoids reach to the target site and act as antioxidants. In this study, we tried to evaluate the antioxidative effect of quercetin 3-O-beta-D-glucoside (Q3G), a typical flavonoid present in vegetables, in rabbit aorta. New Zealand White rabbits were fed a control diet (control group), 2.0% cholesterol diet (HC group) and 2.0% cholesterol plus 0.1% Q3G (HC + Q3G group) for one month. The amounts of total cholesterol, triacylglycerol and total fatty acids in both the plasma and aorta were significantly lower in the HC + Q3G group as compared with the HC group. Quercetin was detected in the aorta of the HC + Q3G group after enzymatic deconjugation, indicating that quercetin accumulated as conjugated metabolites in the aorta. The contents of TBA-reacting substances (TBARS) and cholesteryl ester hydroperoxides (CEOOH) in the aorta of the HC + Q3G group were significantly lower than those in the HC group. The aorta of HC + Q3G group was more resistant than that of HC group in copper ion-induced lipid peroxidation ex vivo. HC + Q3G group accumulated a higher amount of vitamin E per total cholesterol than HC group in the aorta. These results strongly suggest that quercetin glucosides accumulate in the aorta as their metabolites and attenuate lipid peroxidation occurring in the aorta, along with the attenuation of hyperlipidemia.     

Protection by quercetin and quercetin 3-O-beta-D-glucuronide of peroxynitrite-induced antioxidant consumption in human plasma low-density lipoprotein.

Effect of quercetin and its conjugated metabolite quercetin 3-O-beta-D-glucuronide (Q3GA), on peroxynitrite-induced consumption of lipophilic antioxidants in human plasma low-density lipoprotein (LDL) was measured to estimate the role of dietary flavonoids in the defense system against oxidative modification of LDL based on the reaction of nitric oxide and superoxide anion. Synthesized peroxynitrite-induced consumption of endogenous lycopene beta-carotene and alpha-tocopherol was effectively suppressed by adding quercetin aglycone into LDL solution. Q3GA also inhibited the consumption of these antioxidants effectively. These results indicate that dietary quercetin is capable of inhibiting peroxynitrite-induced oxidative modification of LDL in association with lipophilic antioxidants present within this lipoprotein particle.     

Immunochemical detection of flavonoid glycosides: development, specificity, and application of novel monoclonal antibodies

Flavonoid-rich diets are expected to decrease the risk of cardiovascular diseases. The localization and target sites of flavonoids underlying the protective mechanism in vivo have not been fully investigated because the methods for detection of flavonoids have been limited to chemical analysis such as high-performance liquid chromatography. To further understand the actions of flavonoids in vivo, we developed a novel methodology that immunochemically evaluates flavonoids using specific antibodies. Quercetin-3-glucuronide (Q3GA), a major metabolite in human plasma, was coupled with keyhole limpet hemocyanin. Alternatively, the sugar moiety of quercetin-3-glucoside (Q3G) was succinylated and then coupled with a carrier protein. Using these two immunogens, we finally obtained two monoclonal antibodies, mAb14A2 and mAb11G6, from the immunogen using Q3GA and Q3G, respectively. Competitive enzyme-linked immunosorbent assay showed the unique difference in the specificity between the two similar antibodies: mAb14A2 recognized several quercetin-3-glycosides including Q3G and rutin but mAb11G6 was highly specific to the Q3G structure. The macrophage-derived foam cells in human atherosclerotic lesions were significantly stained with mAb14A2 but scarcely with mAb11G6. These results showed that the anti-flavonoid glycoside antibodies are useful tools for evaluating their localization in tissues and that the specificities strongly depend on the immunogen design for synthesizing the hapten-protein conjugates.     

Protection by quercetin and quercetin 3-O-β-D-glucuronide of peroxynitrite-induced antioxidant consumption in human plasma low-density lipoprotein

Effect of quercetin and its conjugated metabolite quercetin 3-O-β-D-glucuronide (Q3GA), on peroxynitrite-induced consumption of lipophilic antioxidants in human plasma low-density lipoprotein (LDL) was measured to estimate the role of dietary flavonoids in the defense system against oxidative modification of LDL based on the reaction of nitric oxide and superoxide anion. Synthesized peroxynitrite-induced consumption of endogenous lycopene β-carotene and α-tocopherol was effectively suppressed by adding quercetin aglycone into LDL solution. Q3GA also inhibited the consumption of these antioxidants effectively. These results indicate that dietary quercetin is capable of inhibiting peroxynitrite-induced oxidative modification of LDL in association with lipophilic antioxidants present within this lipoprotein particle.     

Identification of quercetin 3-O-beta-D-glucuronide as an antioxidative metabolite in rat plasma after oral administration of quercetin

The potential beneficial effect of dietary quercetin (3,3',4',5,7-pentahydroxyflavone) has attracted much attention in relation to the prevention of cardiovascular disease. It is generally recognized that dietary quercetin is subject to metabolic conversion resulting in conjugated forms during absorption and circulation. However, no quercetin conjugates have yet been identified from biological fluids or tissues. In the present study, we isolated and characterized two quercetin conjugates from the plasma of quercetin-administered rats. The blood plasma was collected from 26 rats 30 min after oral administration of quercetin (250 mg/kg body weight), concentrated, dissolved in 2% acetic acid aqueous solution (pH 2.65), and extracted with ethyl acetate. Two compounds (P2, P3) were obtained from the extract by repeated reversed-phase HPLC. On the other hand, two quercetin glucuronides were synthesized chemically and identified as quercetin 3-O-beta-D-glucuronide (Q3GA) and quercetin 4'-O-beta-D-glucuronide (Q4'GA), as determined from FABMS, 1H- and 13C-NMR, and HMBC data. The retention times of P2 and P3 in the HPLC chromatogram corresponded to those of Q3GA and Q4'GA, respectively. FABMS data demonstrated that P2 and P3 are quercetin monoglucuronides. 1H-NMR data for P2 were completely in agreement with those for Q3GA. P2 was therefore identified as Q3GA. This is, to our knowledge, the first evidence that Q3GA accumulates in vivo after oral administration of quercetin. Q3GA is likely to act as an effective antioxidant in blood plasma low-density lipoprotein, because this conjugated metabolite was found to possess a substantial antioxidant effect on copper ion-induced oxidation of human plasma low-density lipoprotein as well as 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity.     

Identification of smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta with monoclonal antibody IIG10

We have developed a monoclonal antibody that specifically interacts with a surface antigen of human fibroblasts and smooth muscle cells. The antibody (antibody IIG10) recognizes a polypeptide of molecular mass 330,000, revealed by immunoblotting in fibroblast and smooth muscle cell extract, but not in vascular endothelial cells, peritoneal macrophages, peripheral blood lymphocytes nor hepatocytes. In tissue sections the antibody stained smooth muscle cells of myometrium, aorta and smaller blood vessels, and fibroblasts of connective tissue. Specificity of the antibody was further confirmed by double staining of aorta sections. Antibody IIG10 was used to identify smooth muscle-derived foam cells in the atherosclerotic plaque of human aorta.     

Effect of quercetin and its conjugated metabolite on the hydrogen peroxide-induced intracellular production of reactive oxygen species in mouse fibroblasts

To clarify the antioxidative role of quercetin metabolites in cellular oxidative stress, we measured the inhibitory effects of the quercetin aglycon and quercetin 3-O-beta-D-glucuronide (Q3GA), which is one of the quercetin metabolites in the blood after an intake of quercetin-rich food, on the production of hydrogen peroxide (H2O2)-induced intracellular reactive oxygen species in mouse fibroblast 3T3 cultured cells. When the cells were exposed to H2O2 in the presence of quercetin or Q3GA, Q3GA was found to be less effective than quercetin. In the case of a pretreatment with quercetin or Q3GA before the exposure, Q3GA, but not the quercetin aglycon, exerted an inhibitory effect, although its cellular uptake was unlikely. The quercetin aglycon appeared to fail in its antioxidative effect due to metabolic conversion into isorhamnetin conjugates, with substantial oxidative degradation resulting from the pretreatment. It is, therefore, suggested that quercetin metabolites take part in the protection of intracellular oxidative stress induced by the extraneous attack of H2O2.     

Effect of quercetin and its glucuronide metabolite upon 6-hydroxydopamine-induced oxidative damage in Neuro-2a cells

Quercetin is ubiquitously distributed in plant foods. This antioxidative polyphenol is mostly converted to conjugated metabolites in the body. Parkinson disease (PD) has been suggested to be related to oxidative stress derived from abnormal dopaminergic activity. We evaluated if dietary quercetin contributes to the antioxidant network in the central nervous system from the viewpoint of PD prevention. A neurotoxin, 6-hydroxydopamine (6-OHDA), was used as a model of PD. 6-OHDA-induced H?O? production and cell death in mouse neuroblastoma, Neuro-2a. Quercetin aglycone suppressed 6-OHDA-induced H?O? production and cell death, although aglycone itself reduced cell viability at higher concentration. Quercetin 3-O-β-D-glucuronide (Q3GA), which is an antioxidative metabolite of dietary quercetin, was little incorporated into the cell resulting in neither suppression of 6-OHDA-induced cell death nor reduction of cell viability. Q3GA was found to be deconjugated to quercetin by microglial MG-6 cells. These results indicate that quercetin metabolites should be converted to their aglycone to exert preventive effect on damage to neuronal cells.     

Flavonoids protect LDL from oxidation and attenuate atherosclerosis

Consumption of some plant-derived flavonoids results in their absorption and appearance in plasma and tissues. The inverse relationship between dietary flavonoids consumption and cardiovascular diseases may be associated with the ability of flavonoids to attenuate LDL oxidation, macrophage foam cell formation and atherosclerosis. The effect of flavonoids on arterial cell-mediated oxidation of LDL is determined by their accumulation in the lipoprotein and in arterial cells, such as macrophages. Flavonoids can reduce LDL lipid peroxidation by scavenging reactive oxygen/nitrogen species, chelation of transition metal ions and sparing of LDL-associated antioxidants. They can also reduce macrophage oxidative stress by inhibition of cellular oxygenases [such as nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) oxidase] or by activating cellular antioxidants (such as the glutathione system). Thus, plant flavonoids, as potent natural antioxidants that protect against lipid peroxidation in arterial cells and lipoproteins, significantly attenuate the development of atherosclerosis.     

Effect of quercetin and its glucuronide metabolite upon 6-hydorxydopamine-induced oxidative damage in Neuro-2a cells

Abstract

Quercetin is ubiquitously distributed in plant foods. This antioxidative polyphenol is mostly converted to conjugated metabolites in the body. Parkinson disease (PD) has been suggested to be related to oxidative stress derived from abnormal dopaminergic activity. We evaluated if dietary quercetin contributes to the antioxidant network in the central nervous system from the viewpoint of PD prevention. A neurotoxin, 6-hydroxydopamine (6-OHDA), was used as a model of PD. 6-OHDA-induced H₂O₂ production and cell death in mouse neuroblastoma, Neuro-2a. Quercetin aglycone suppressed 6-OHDA-induced H₂O₂ production and cell death, although aglycone itself reduced cell viability at higher concentration. Quercetin 3-O-β-d-glucuronide (Q3GA), which is an antioxidative metabolite of dietary quercetin, was little incorporated into the cell resulting in neither suppression of 6-OHDA-induced cell death nor reduction of cell viability. Q3GA was found to be deconjugated to quercetin by microglial MG-6 cells. These results indicate that quercetin metabolites should be converted to their aglycone to exert preventive effect on damage to neuronal cells.     

AM-3K, an anti-macrophage antibody, recognizes CD163, a molecule associated with an anti-inflammatory macrophage phenotype.

CD163 is a member of the scavenger receptor cysteine-rich superfamily restricted to the monocyte/macrophage lineage and is thought to be a useful marker for anti-inflammatory or alternatively activated macrophages. In this study we used mass spectrometric analysis to determine that the antigen recognized by the antibody AM-3K, which we previously generated as a tissue macrophage-specific monoclonal antibody, was CD163. An anti-inflammatory subtype of macrophages stimulated by dexamethasone or interleukin-10 showed strong reactivity for AM-3K and increased expression of CD163 mRNA. Immunohistochemical staining of routinely processed pathological specimens revealed that AM-3K recognized a specialized subpopulation of macrophages. In granulomatous diseases such as tuberculosis, sarcoidosis, or foreign body reactions, tissue macrophages around granulomas, but not component cells of the granulomas such as epithelioid cells and multinucleated giant cells, showed positive staining for AM-3K. In atherosclerotic lesions, scattered macrophages in diffuse intimal lesions were strongly positive for AM-3K, whereas foamy macrophages in atheromatous plaques demonstrated only weak staining. We therefore suggest that, in routine pathological specimens, AM-3K is a useful marker for anti-inflammatory macrophages because these cells can be distinguished from inflammatory or classically activated macrophages. Because AM-3K cross-reacts with macrophage subpopulations in different animal species including rats, guinea pigs, rabbits, cats, dogs, goats, pigs, bovine species, horses, monkeys, and cetaceans, it will have wide application for detection of CD163 in various animals.     

Effect of a conjugated quercetin metabolite, quercetin 3-glucuronide, on lipid hydroperoxide-dependent formation of reactive oxygen species in differentiated PC-12 cells

To assess the efficacy of conjugated quercetin metabolites as attenuators for oxidative stress in the central nervous system, we measured the 13-hydroperoxyoctadecadienoic acid (13-HPODE)-dependent formation of reactive oxygen species (ROS) in pheochromocytoma PC-12 cells in the presence of quercetin 3-O-β-glucuronide (Q3GA) and related compounds. A 2',7'-dichlorofluorescin (DCFH) assay showed that Q3GA significantly suppressed the formation of ROS, when it was coincubated with 13-HPODE (coincubation system). However, it was less effective than quercetin aglycon in the concentration range from 0.5 to 10 μM. In an experiment in which the cells were incubated with the test compounds for 24 h before being exposed to 13-HPODE, Q3GA was also effective in suppressing the formation of ROS in spite that little Q3GA was taken up into the cells. These results suggest that antioxidative metabolites of quercetin are capable of protecting nerve cells from attack of lipid hydroperoxides.     

Effect of a conjugated quercetin metabolite,quercetin 3-glucuronide,on lipid hydroperoxide-dependent formation of reactive oxygen species in differentiated PC-12 cells

To assess the efficacy of conjugated quercetin metabolites as attenuators for oxidative stress in the central nervous system, we measured the 13-hydroperoxyoctadecadienoic acid (13-HPODE)-dependent formation of reactive oxygen species (ROS) in pheochromocytoma PC-12 cells in the presence of quercetin 3-O-β-glucuronide (Q3GA) and related compounds. A 2′,7′-dichlorofluorescin (DCFH) assay showed that Q3GA significantly suppressed the formation of ROS, when it was coincubated with 13-HPODE (coincubation system). However, it was less effective than quercetin aglycon in the concentration range from 0.5 to 10 μM. In an experiment in which the cells were incubated with the test compounds for 24 h before being exposed to 13-HPODE, Q3GA was also effective in suppressing the formation of ROS in spite that little Q3GA was taken up into the cells. These results suggest that antioxidative metabolites of quercetin are capable of protecting nerve cells from attack of lipid hydroperoxides.     

Immunochemical detection of food-derived polyphenols in the aorta: macrophages as a major target underlying the anti-atherosclerotic activity of polyphenols

It has been suggested that polyphenol-rich diets decrease the risk of cardiovascular diseases. Although studies of the bioavailability of polyphenols, particularly their absorption and metabolism, have been reported recently, the tissue and cellular distributions underlying their biological mechanisms remain unknown. It is difficult to evaluate the specific localization of tissue and/or cellular polyphenols, because the method is limited to chromatography. To overcome these difficulties, we have developed anti-polyphenol antibodies to characterize immunohistochemically the localization of polyphenols and their metabolites in vivo. Two novel monoclonal antibodies were raised against quercetin and tea catechins, which represent flavonoid-type polyphenols distributed in foods and beverages, and are expected to exhibit anti-oxidative and anti-inflammatory activities in vivo. Using these antibodies, we identified activated macrophages as a specific target of these flavonoids during the development of atherosclerotic lesions. This review describes recent findings on the molecular actions of flavonoids that underly their anti-atherosclerotic activity in vivo.     

Total Flavonoids of Engelhardia roxburghiana Wall. Leaves Alleviated Foam Cells Formation through AKT/mTOR-Mediated Autophagy in the Progression of Atherosclerosis

Engelhardia roxburghiana Wall. is a traditional Chinese medicine used for treating cardiovascular diseases. Our previous study has implicated potential effects of total flavonoids of Engelhardia roxburghiana Wall. (TFER) against hyperlipidemia. The aim of the study is to uncover the effects and underlying mechanisms of TFER on foam cells formation after atherosclerosis. We used high fat diet (HFD) induced Apoe-/- mice and oxidized density lipoprotein (ox-LDL) induced THP-1 cells to mimic process of atherosclerosis in vivo and in vitro, respectively. Lipid accumulation, inflammation response, autophagosomes formation and expressions of autophagy related target genes were assessed. Our present study demonstrated TFER (500 mg/kg) alleviated macrophage infiltration and lipid accumulation in thoracic aortas of HFD-treated mice. In ox-LDL-treated THP-1 cells, MDC staining and Western blot analysis all indicated that the TFER (200 μg/ml) reduced foam cells formation and IL-1β releasing, activated autophagy through suppressing AKT/mTOR signaling, significantly regulating expressions of AKT, p-AKT, mTOR, p-mTOR, Beclin 1, LC3-II, p62. It is suggested that TFER alleviated atherosclerosis progression in vivo and in vitro through reducing foam cells formation and inflammatory responses, and the possible mechanism may be due to the activation of macrophage autophagy by inhibiting AKT and mTOR phosphorylation.     

Angptl4 protects against severe proinflammatory effects of saturated fat by inhibiting fatty acid uptake into mesenteric lymph node macrophages

Dietary saturated fat is linked to numerous chronic diseases, including cardiovascular disease. Here we study the role of the lipoprotein lipase inhibitor Angptl4 in the response to dietary saturated fat. Strikingly, in mice lacking Angptl4, saturated fat induces a severe and lethal phenotype characterized by fibrinopurulent peritonitis, ascites, intestinal fibrosis, and cachexia. These abnormalities are preceded by a massive acute phase response induced by saturated but not unsaturated fat or medium-chain fat, originating in mesenteric lymph nodes (MLNs). MLNs undergo dramatic expansion and contain numerous lipid-laden macrophages. In peritoneal macrophages incubated with chyle, Angptl4 dramatically reduced foam cell formation, inflammatory gene expression, and chyle-induced activation of ER stress. Induction of macrophage Angptl4 by fatty acids is part of a mechanism that serves to reduce postprandial lipid uptake from chyle into MLN-resident macrophages by inhibiting triglyceride hydrolysis, thereby preventing macrophage activation and foam cell formation and protecting against progressive, uncontrolled saturated fat-induced inflammation.     

Heterogeneity of macrophage populations in human lymphoid tissue and peripheral blood

Human lymphoid tissue and peripheral blood leukocytes were stained with six monoclonal antibodies directed against monocyte/macrophage populations. The staining pattern described by each of these monoclonal reagents was compared with the distribution of morphologically distinguishable tissue macrophages. The results show that there exists considerable heterogeneity of tissue macrophages based on the expression of surface and/or cytoplasmic antigens; furthermore, the distribution of cells bearing particular antigenic determinants is associated with distinct regions in normal lymphoid tissue. Double staining methods demonstrated that these antibodies bind to different, as well as to identical, macrophage populations. OKM-1 antibody binds predominantly to sinus histiocytes and tingible body macrophages. The Leu M-1 reagent stains interdigitating reticulum cells, while the KiM-4 antibody labels follicular dendritic cells. Leu M-3 antibody identifies cells predominantly in the germinal center, and histiocytes lining the sinuses. Both CM-1 and BRL-M.1 appear to stain tissue macrophages distributed throughout the lymphoid tissue.     

Paraoxonases and cardiovascular diseases: pharmacological and nutritional influences

PURPOSE OF REVIEW: To summarize the new articles published in the last year on paraoxonases, including their expression in cardiovascular diseases, and regulation by pharmacological and nutritional means. RECENT FINDINGS: The elucidation of the crystal structure of the paraoxonase 1 (PON1) gene, obtained by directed evolution, shows that it consists of a six-bladed beta-propeller with a unique active site. PON1 is present in HDL but also in lipoprotein-deficient serum, in VLDL and in chylomicrons. PON1 protects lipids in lipoproteins, in macrophages and in erythrocytes from oxidation. Cellular PON2 and PON3 were also shown to reduce oxidative stress. Beyond its antioxidative properties, PON1 possesses additional antiatherogenic properties against macrophage foam cell formation: attenuation of cholesterol and oxidized lipids influx, inhibition of macrophage cholesterol biosynthesis and stimulation of macrophage cholesterol efflux. The PON1 gene is regulated by Sp1 and protein kinase C, whereas the PON2 gene in macrophages is regulated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. PON1 activity and mass are both reduced in cardiovascular diseases and the hypocholesterolemic drugs, statins, increase serum PON1 activity (by reducing oxidative stress, or by upregulating hepatic PON1 expression). Expression of cellular PON2, like PON1, was upregulated by statins. Nutritional antioxidants, such as polyphenols, increase PON1 mRNA expression and activity, by an aryl hydrocarbon receptor-dependent mechanism. SUMMARY: The elucidation of PON1 structure and its active center has enabled a better understanding of its mechanism of action, including its physio-pathological substrate(s). Some drugs and nutrients including dietary antioxidants and polyphenols considerably increase the activities of paraoxonases which, in turn, can reduce oxidative stress and atherosclerosis development.