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• W2014018523 abstract "Epidemiological studies suggest that the consumption of flavonoid-rich diets decreases the risk of cardiovascular diseases. However, the target sites of flavonoids underlying the protective mechanism in vivo are not known. Quercetin represents antioxidative/anti-inflammatory flavonoids widely distributed in the human diet. In this study, we raised a novel monoclonal antibody 14A2 targeting the quercetin-3-glucuronide (Q3GA), a major antioxidative quercetin metabolite in human plasma, and found that the activated macrophage might be a potential target of dietary flavonoids in the aorta. Immunohistochemical studies with monoclonal antibody 14A2 demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta, and that the intense staining was primarily associated with the macrophage-derived foam cells. In vitro experiments with murine macrophage cell lines showed that the Q3GA was significantly taken up and deconjugated into the much more active aglycone, a part of which was further converted to the methylated form, in the activated macrophages. In addition, the mRNA expression of the class A scavenger receptor and CD36, which play an important role for the formation of foam cells, was suppressed by the treatment of Q3GA. These results suggest that injured/inflamed arteries with activated macrophages are the potential targets of the metabolites of dietary quercetin. Our data provide a new insight into the bioavailability of dietary flavonoids and the mechanism for the prevention of cardiovascular diseases. Epidemiological studies suggest that the consumption of flavonoid-rich diets decreases the risk of cardiovascular diseases. However, the target sites of flavonoids underlying the protective mechanism in vivo are not known. Quercetin represents antioxidative/anti-inflammatory flavonoids widely distributed in the human diet. In this study, we raised a novel monoclonal antibody 14A2 targeting the quercetin-3-glucuronide (Q3GA), a major antioxidative quercetin metabolite in human plasma, and found that the activated macrophage might be a potential target of dietary flavonoids in the aorta. Immunohistochemical studies with monoclonal antibody 14A2 demonstrated that the positive staining specifically accumulates in human atherosclerotic lesions, but not in the normal aorta, and that the intense staining was primarily associated with the macrophage-derived foam cells. In vitro experiments with murine macrophage cell lines showed that the Q3GA was significantly taken up and deconjugated into the much more active aglycone, a part of which was further converted to the methylated form, in the activated macrophages. In addition, the mRNA expression of the class A scavenger receptor and CD36, which play an important role for the formation of foam cells, was suppressed by the treatment of Q3GA. These results suggest that injured/inflamed arteries with activated macrophages are the potential targets of the metabolites of dietary quercetin. Our data provide a new insight into the bioavailability of dietary flavonoids and the mechanism for the prevention of cardiovascular diseases. Flavonoids are widely distributed in plant foods and beverages and therefore are regularly ingested with the human diet. In 1936, Rusznyak and Szent-Gyoygi (1Rusznyak S. Szent-Gyorgyi A. Nature. 1936; 138: 798Crossref Scopus (140) Google Scholar) found citrus flavonoids reduced capillary fragility and permeability in blood vessels. Thereafter, a large number of biological activities of flavonoids have been described which overall are believed to be beneficial for good health. Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is a prime example of such a flavonoid and is bound to sugars in foods, mainly as β-glycosides. The quercetin glycosides occur in broccoli, apples, and especially in onions, with an abundance as high as 0.25-0.5 g/kg (2Hertog M.G. Hollman P.C. Katan M.B. J. Agric. Food Chem. 1992; 40: 2379-2383Crossref Scopus (1241) Google Scholar). The average daily intake of the flavonoids subclasses in The Netherlands is 23 mg (calculated as aglycones) of which quercetin supplies 16 mg (3Hertog M.G. Hollman P.C. Katan M.B. Kromhout D. Nutr. Cancer. 1993; 20: 21-29Crossref PubMed Scopus (1155) Google Scholar). Epidemiological evidence links diets rich in quercetin with decreased incidence of cardiovascular and neoplastic diseases (4Hertog M.G. Feskens E.J. Hollman P.C. Katan M.B. Kromhout D. Lancet. 1993; 342: 1007-1011Abstract PubMed Scopus (3976) Google Scholar, 5Hertog M.G. Feskens E.J. Hollman P.C. Katan M.B. Kromhout D. Nutr. Cancer. 1994; 22: 175-184Crossref PubMed Scopus (261) Google Scholar, 6Hertog M.G. Kromhout D. Aravanis C. Blackburn H. Buzina R. Fidanza F. Giampaoli S. Jansen A. Menotti A. Nedeljkovic S. Pekkarinen M. Simic B.S. Toshima H. Feskens E.J. Hollman P.C. Katan M.B. Arch. Intern. Med. 1995; 155: 381-386Crossref PubMed Scopus (1746) Google Scholar, 7Hertog M.G. Hollman P.C. Eur. J. Clin. Nutr. 1996; 50: 63-71PubMed Google Scholar, 8Keli S.O. Hertog M.G. Feskens E.J. Kromhout D. Arch. Intern. Med. 1996; 156: 637-642Crossref PubMed Google Scholar, 9Le Marchand L. Murphy S.P. Hankin J.H. Wilkens L.R. Kolonel L.N. J. Natl. Cancer Inst. 2000; 92: 154-160Crossref PubMed Scopus (482) Google Scholar). Because oxidative stress has been implicated in the pathogenesis of these diseases, the bioavailability of quercetin and other flavonoids has been investigated in relation to their antioxidant activities in vivo. The antioxidant potential of quercetin is related to the number and position of the free hydroxyl groups in the molecule (10Cao G. Sofic E. Prior R.L. Free Radic. Biol. Med. 1997; 22: 749-760Crossref PubMed Scopus (2174) Google Scholar); therefore, the regioselectivity of conjugation of the hydroxyl groups can be expected to modulate the biological activity of quercetin. Upon ingestion with the diet, quercetin glycosides are rapidly hydrolyzed during passage across the small intestine or by bacterial activity in the colon to generate quercetin aglycone, which is further metabolized in the so-called phase II reactions into the glucuronidated and/or sulfated derivatives. Alternatively, 3′- or 4′-hydroxyl group in the B-ring catechol moiety can also be methylated by catechol-O-methyltransferase (COMT) 2The abbreviations used are: COMTcatechol-O-methyltransferaseQ3GAquercetin-3-O-β-d-glucuronideHPLChigh performance liquid chromatographyHSAhuman serum albuminLPSlipopolysaccharideKLHkeyhole limpet hemocyaninBAECsbovine aortic endothelial cellsECDelectrochemical detectionELISAenzyme-linked immunosorbent assaymAbmonoclonal antibodyGAPDHglyceraldehydes-3-phosphate dehydrogenaseRTreverse transcriptionSR-Athe class A scavenger receptorPBSphosphate-buffered salineFITCfluorescein isothiocyanateLDLlow density lipoproteinDMEMDulbecco's modified Eagle's mediumFBSfetal bovine serumSR-Ascavenger receptor AHBSSHanks' balanced salt solutionIR3Gisorhamnetin-3-glucoside. activity. Previous reports have clearly shown that quercetin-3-glucuronide (Q3GA) and quercetin-3′-sulfate are the major quercetin conjugates in rat and human plasma, in which aglycone could not be detected (11Day A.J. Mellon F. Barron D. Sarrazin G. Morgan M.R. Williamson G. Free Radic. Res. 2001; 35: 941-952Crossref PubMed Scopus (417) Google Scholar, 12Moon J.H. Tsushida T. Nakahara K. Terao J. Free Radic. Biol. Med. 2001; 30: 1274-1285Crossref PubMed Scopus (249) Google Scholar). Although the biological activities of quercetin generally attenuate after conversion to the metabolites, the physiologically conceivable activities associated with oxidative stress for various quercetin metabolites have been reported (13Williamson G. Barron D. Shimoi K. Terao J. Free Radic. Res. 2005; 39: 457-469Crossref PubMed Scopus (194) Google Scholar). It is expected that Q3GA represents the radical scavenging actions of quercetin metabolites in vivo (12Moon J.H. Tsushida T. Nakahara K. Terao J. Free Radic. Biol. Med. 2001; 30: 1274-1285Crossref PubMed Scopus (249) Google Scholar), because it retains the catechol moiety (3′,4′-o-dihydroxyl group) responsible for radical scavenging of quercetin even after the enzymatic metabolism. catechol-O-methyltransferase quercetin-3-O-β-d-glucuronide high performance liquid chromatography human serum albumin lipopolysaccharide keyhole limpet hemocyanin bovine aortic endothelial cells electrochemical detection enzyme-linked immunosorbent assay monoclonal antibody glyceraldehydes-3-phosphate dehydrogenase reverse transcription the class A scavenger receptor phosphate-buffered saline fluorescein isothiocyanate low density lipoprotein Dulbecco's modified Eagle's medium fetal bovine serum scavenger receptor A Hanks' balanced salt solution isorhamnetin-3-glucoside. Although the tissue distributions of quercetin and its metabolites have been assessed by a number of authors, only limited information on the localization of quercetin metabolites in the aorta is presently available. We have previously demonstrated that quercetin metabolites were present in the aorta of high cholesterol/quercetin glucoside-fed rabbits (14Kamada C. da Silva E.L. Ohnishi-Kameyama M. Moon J.H. Terao J. Free Radic. Res. 2005; 39: 185-194Crossref PubMed Scopus (101) Google Scholar), in which cholesterol accumulation in the aorta was significantly inhibited, suggesting the anti-atherosclerotic action of the quercetin metabolites in the aorta. To further understand the mechanism of the anti-atherosclerotic action of quercetin, it is necessary to know the target sites of the quercetin metabolites in the aorta. At present, a chromatographic technique such as high performance liquid chromatography (HPLC) or gas chromatography combined with mass spectrometry or electrochemical detection is the only analytical approach for evaluating quercetin and other flavonoids in biological samples. The immunochemical technique is a powerful tool for evaluating the localization of target molecules in tissue/cellular components; therefore, we developed a monoclonal antibody directed to a quercetin metabolite, Q3GA, and identified the activated macrophage cells as the potential target of dietary flavonoids in vivo. Materials—Quercetin dihydrate, acetobromo-α-d-glucuronic acid methyl ester, human serum albumin (HSA), lipopolysaccharides (LPS, from Escherichia coli), saccharic acid 1,4-lactone, and dinitrocatechol were purchased from Sigma. Quercetin-3-O-β-d-glucoside, quercetin-4′-O-β-d-glucoside, quercetin-3-O-sulfate, hyperoside, rutin, isorhamnetin (3′-methyl quercetin), and cyanidin-3-O-β-d-glucoside were obtained from Extrasynthese (Genay, France). Succinic anhydride was purchased from Wako Pure Chemicals (Osaka, Japan). Keyhole limpet hemocyanin (KLH), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide were obtained from Pierce. Quercetin-3-O-β-d-glucuronide (Q3GA) was chemically synthesized as reported previously (12Moon J.H. Tsushida T. Nakahara K. Terao J. Free Radic. Biol. Med. 2001; 30: 1274-1285Crossref PubMed Scopus (249) Google Scholar). Monoclonal murine antibody to scavenger receptor A (SR-A) was obtained from TransGenic Inc. (Hyogo, Japan). Rabbit polyclonal antibodies to CD36 and β-actin were obtained from Santa Cruz Biotechnology and BioLeg-end, respectively. Cell Culture—Bovine aortic endothelial cells (BAECs) (15Isshiki M. Ying Y.S. Fujita T. Anderson R.G. J. Biol. Chem. 2002; 277: 43389-43398Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) were cultured in Medium 199 (Sigma) containing 20% fetal bovine serum (FBS). RAW264 cell line was obtained from the Riken Cell Bank (Tsukuba, Ibaraki, Japan) and cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS. J774-1 was obtained from Cell Resource Center for Biomedical Research Institute of Development, Aging, and Cancer, Tohoku University (Sendai, Miyagi, Japan). Cells were cultured in an atmosphere containing 5% CO2 at 37 °C. All media contain 100 μg/ml penicillin and 100 units/ml streptomycin. Analysis of Quercetin Metabolites in Human Plasma—Healthy volunteers (provided informed consent) that fasted overnight were served 350-500 g of cooked onion paste (provided by Kagome Research Institute, Tochigi, Japan) roasted with salad oil. The onion amounts were designed so as to be almost the same per their body weights (∼7 g/kg). The quercetin content in the onion paste was analyzed by HPLC (16Moon J.H. Nakata R. Oshima S. Inakuma T. Terao J. Am. J. Physiol. 2000; 279: R461-R467Crossref PubMed Google Scholar) after the acid hydrolysis of the quercetin mono- and di-glucosides in the methanolic extract of the paste and determined to be ∼32.9 mg/100 g fo onion paste (as the equivalent for the quercetin aglycone). Before and 1.5 h after intake, heparinized blood was collected from each subject, and the plasma was obtained by centrifugation. The quercetin metabolites in the plasma were extracted with 5 volumes of methanol, and the methanolic fractions were evaporated under an N2 stream and dissolved in 20% aqueous acetonitrile containing 0.5% phosphoric acid. For analysis of aglycone, plasma samples were mixed with an equal volume of ethyl acetate and centrifuged. After two extractions, the ethyl acetate layers were collected, evaporated under an N2 stream, and dissolved in HPLC solvent. Ten μl of the sample was injected into an HPLC-electrochemical detection (ECD) system (ESA, Cambridge, MA) equipped with a TSK-gel ODS-80Ts column (4.6 × 150 mm). The separation of the compounds was carried out by a gradient elution. Solvent A was 20% aqueous acetonitrile containing 0.5% phosphoric acid, and solvent B was 100% acetonitrile containing 0.5% phosphoric acid. The gradient program was as follows: 0-10 min, 1% B; 10-20 min, linear gradient to 25% B; 20-25 min, linear gradient to 1% B; 25-30 min, hold; flow rate, 0.8 ml/min. Electrochemical detection was performed with a coulometric electrode at 150 mV. Cell-mediated LDL Oxidation—LDL (d = 1.063-1.093 g/ml) was isolated from healthy human volunteers by sequential ultracentrifugation and dialyzed in phosphate-buffered saline. The LDL concentrations were determined by measuring the protein contents using a BCA protein assay kit (Pierce). BAECs in a 60-mm dish were treated with LDL (200 μg/ml in FBS-free Medium 199) in the presence of 5 μm Cu2+ and the different concentrations of Q3GA and the related compounds at 37 °C. After incubation, the oxidation reaction was terminated by adding 0.1 volume of 1 mm EDTA, 10 μm 2,6-di-tert-butyl-p-cresol solution. The LDL oxidation was measured as the thiobarbituric acid-reactive substances with fluorescent detection (excitation 515 nm, emission 553 nm). Tetraethoxypropane was used as the standard compound that readily decomposes into the thiobarbituric acid-reactive malondialdehyde, a representative aldehyde formed in oxidized LDL, during the assay processes. Preparation of Monoclonal Antibody to Q3GA—To prepare the immunogen, the synthesized Q3GA was conjugated with KLH by a carbodiimide procedure. Briefly, the carboxylic derivatives of Q3GA (11.0 μmol) was activated by incubating with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (11.0 μmol) in the presence of N-hydroxysuccinimide (11.0 μmol) in dimethylformamide (200 μl) at room temperature overnight. A 100-μl aliquot of the mixture was added to 360 μl of KLH (10 mg/ml) or HSA (10 mg/ml) in phosphate-buffered saline (PBS) and incubated at room temperature for 4 h. After incubation, the proteins were dialyzed to PBS at 4 °C for 2 days. The obtained Q3GA-KLH conjugate (0.6 mg/ml in PBS) was emulsified with an equal volume of adjuvant. Six-week-old female BALB/c mice were intraperitoneally immunized with this emulsion (100 μl). The mice were repeatedly boosted with the immunogens (0.2 mg/ml) emulsified with an equal volume of adjuvant every 2 weeks. In the final boost, 100 μl of the immunogens (0.5 mg/ml in PBS) without adjuvant was intravenously injected. Three days after the final boost, one of the mice was sacrificed, and the spleen was removed. The spleen cells were fused with P3U1 myeloma cells in the presence of polyethylene glycol 1500 (Roche Applied Science) and cultured in the hypoxanthine/aminopterin/thymidine medium for the selection of the hybridomas. After a week, the immunoreactivities of the culture supernatants were screened by enzymelinked immunosorbent assay (ELISA) as follows. Fifty μl of the antigens (5 μg/ml) in PBS were coated in wells and incubated at 37 °C for 1 h. After washing three times with PBS containing 0.05% Tween 20 (TPBS), the wells were blocked with a 4% aqueous solution of Block Ace (Dainihon Seiyaku, Osaka, Japan) at 37 °C for 1 h. After washing, 100 μl of the primary antibody in TPBS was added, and the wells were incubated at 37 °C for 2 h. After washing, 100 μl of the peroxidase-labeled anti-mouse IgG goat antibody (Chemicon International, Temecula, CA) with a 1:5000 dilution was added and incubated at 37 °C for 1 h. The color-developing reaction was performed by the addition of 100 μl of the TMB substrate solution (within TMB substrate reagent set, BD Biosciences). The binding of the antibody to the antigen was evaluated by measuring the absorbance at 450 nm. The immunoreactive hybridomas were then cloned by the limited dilution method. After repeated screening and cloning, a monoclonal antibody mAb14A2 was finally obtained. The antibody was purified by ammonium sulfate precipitation from the culture supernatant and used in the following experiment. Competitive ELISA—For competitive ELISA, the reaction of the primary antibody was carried out in the absence or presence of competitors. The competitive reactions were performed in PBS containing 1% HSA at 37 °C for 90 min. The cross-reactivity of the antibody to the competitors was expressed as B/B0, in which B is the amount of the antibody bound to the coating antigen in the presence of the competitor, and B0 is in the absence of a competitor. Immunohistochemistry—This investigation was carried out on aortic wall samples obtained during autopsy from patients with generalized arteriosclerosis. Each autopsy was performed at Tokyo Women's Medical University after the patients' family members granted informed consent according to the established guideline. Each sample was prepared for 10% formalin-fixed, paraffin-embedded materials and for frozen materials embedded in the optimum cutting temperature compound (Sakura Finetechnical, Tokyo, Japan) at -80 °C. Multiple 6-μm-thick sections were cut from these paraffin-embedded and frozen materials and used for the histopathological and immunohistochemical examinations. The paraffin-embedded sections were deparaffinized in xylene and ethanol, rehydrated in distilled water. Frozen sections were dried, postfixed or not in 10% formalin, and rehydrated. These prepared sections were quenched for 10 min with 3% hydrogen peroxide for inhibiting the endogenous peroxidase activity, rinsed in PBS, pretreated for 30 min at room temperature with 5% skim milk in PBS, and treated with the avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA). Sections were then incubated overnight at 4 °C with the primary antibodies such as the mAb14A2 at a dilution of 1:200 and mouse monoclonal IgG1 against CD68 (Clone KP-1; DakoCytomation, Kyoto, Japan) at a dilution of 1:10,000. Sections processed with omission of the primary antibodies or incubated with 5% skim milk in PBS served as negative reaction controls. Antibody binding was visualized by the avidin-biotin-immunoperoxidase complex method using the appropriate Vectastain ABC kit (Vector Laboratories). Immunohistochemical localization of Q3GA was verified by comparison of consecutive sections stained with hematoxylin-eosin and immunostained for CD68. 3,3′-Diaminobenzidine tetrahydrochloride was used as the chromogen, and hematoxylin was used as the counterstain. Immunostained sections were observed with a light microscope (Olympus, Tokyo, Japan). In addition, the location of Q3GA immunoreactivity in macrophages was strictly identified by the double immunofluorescence method on frozen sections. In brief, sections were postfixed for 10 min at 4 °C in 100% acetone, rehydrated, rinsed in PBS, pretreated for 10 min at room temperature with 5% skim milk in PBS, and incubated overnight at 4 °C with the mAb14A2 and rabbit polyclonal IgG against CD68, simultaneously (catalog number sc-9139; Santa Cruz Biotechnology, Santa Cruz, CA). Sections were then rinsed in PBS and incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG and Cy3-conjugated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) at the same time. Double-immunostained sections were observed with a fluorescence microscope (Nikon, Tokyo, Japan). The appearance of yellowish signals at merging FITC (green) and Cy3 (red) was considered as the co-localization of Q3GA and CD68. Competitive experiments to confirm the specificity of the immunostaining with mAb14A2 were also performed with the antibody in the presence of 100 μm Q3GA. Normal mouse IgG was used as the negative control. Immunohistochemical localization of the immunoreaction product deposits was verified by light microscopy on consecutive sections with hematoxylineosin or immunostained for CD68. To confirm the immunostaining of the conjugate metabolites with mAb14A2, the sections were treated for 60 min with β-glucuronidase (>600 units/ml) from Helix pomatia in 0.1 m sodium acetate buffer, pH 5.0, prior to the reaction with mAb14A2. Analysis for Cellular Uptake of Quercetin-3-glucuronide—RAW264 macrophages were grown to confluence in DMEM containing 10% FBS on 60-mm dish in an atmosphere containing 5% CO2 at 37 °C. The cells were treated with or without LPS (1 μg/ml) in 2 ml of DMEM with 10% FBS. After a 24-h incubation, the cells were washed twice with 1 ml of FBS-free media, after which the media were exchanged with FBS-free DMEM containing 20 μm Q3GA. Following a 4-h incubation, the cell were washed three times with 1 ml of Hanks' balanced salt solution (HBSS), scraped from the dish, and resuspended in 200 μl of methanol/acetic acid (100:1). Q3GA and its cellular metabolites were then extracted by sonication for 1 min using an Astrason XL2020 ultrasonic processor (Heat Systems-Ultrasonics, Farmingdale, NY) at level 6. After centrifugation, the supernatants were collected, evaporated under an N2 stream, and dissolved in 20% aqueous acetonitrile containing 0.5% phosphoric acid. The samples were injected into the HPLC-ECD system as already described. Quantitation of quercetin compounds was performed using standard curves developed by the peak areas of authentic compounds (Q3GA, quercetin, and isorhamnetin). Immunocytochemical detection of Q3GA with mAb14A2 was also performed. Cells were cultured on coverslips in a 24-well plate. After treatment of cells with or without LPS/Q3GA as described above, cells were washed with HBSS three times and then fixed for 10 min in 4% paraformaldehyde in PBS on ice. To prevent nonspecific antibody binding, the cells were washed twice in PBS and blocked for 1 h at room temperature with 1% skim milk in PBS. Membranes were permeabilized by exposing the fixed cells to PBS containing 0.2% Triton X-100 for 2 min on ice. The cells were then incubated in the primary antibody (mAb14A2) in PBS containing 3% bovine serum albumin overnight at 4 °C. The cells were then incubated for 1 h in the presence of FITC-labeled anti-mouse IgG (Dako Japan Co., Ltd., Kyoto, Japan), rinsed with PBS, and mounted on glass slides using Dako Cytomation fluorescent mounting medium. Images of cellular immunofluorescence were acquired using a Leica TCS-NT confocal laser scanning microscope. The DNA was also stained with propidium iodide. β-Glucuronidase Activity—Intracellular β-glucuronidase activity was measured by a colorimetric analysis using phenolphthalein mono-β-glucuronide (Sigma) as the substrate. Briefly, 30 μg of cell-free extracts, prepared by repeated freezing and thawing of cells, were mixed with 0.6 mm phenolphthalein mono-β-glucuronide in 100 μl of 0.1 m sodium phosphate buffer, pH 5.0. After incubation at 37 °C for 30 min followed by adding 200 μl of 0.1 m sodium phosphate buffer, pH 11.0, the absorbance at 550 nm indicating the formation of phenolphthalein aglycone was measured. Extracellular activity was evaluated using Q3GA as the substrate. The culture medium was removed from the dishes and then incubated with 50 μm Q3GA at 37 °C for 1 h. After incubation, the formed aglycone was extracted twice with ethyl acetate, evaporated, and dissolved in mobile phase for HPLC-ECD analysis. Ten ml of the samples was injected into HPLC-ECD system as described above. The activity was expressed as the conversion rate (%) of Q3GA into the aglycone. Expression of Scavenge Receptors in Macrophage Cells—The RAW264 cells cultured in a 35-mm dish were washed twice with FBS-free DMEM, after which the media were exchanged with FBS-free DMEM containing the indicated concentrations of Q3GA dissolved in 5 μl of dimethyl sulfoxide (Me2SO). After a 30-min preincubation, the cells were treated with or without oxidized LDL (100 μg/ml). The oxidized LDL was prepared upon incubation of LDL (1 mg/ml) with 5 μm Cu2+ in PBS at 37 °C for 24 h followed by dialysis in PBS at 4 °C for 4 days. Following incubation, the cells were lysed, and the total RNA was isolated and spectrophotometrically quantified. The expression levels of the scavenger receptors and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected by a reverse transcription (RT)-PCR. The RT reaction was performed with 10 μg of total RNA and an oligo(dT) primer using the first strand cDNA synthesis kit. The PCRs were carried out using 0.75 μl of cDNA in 24 μl of 10 mm Tris-HCl, pH 9.0, containing 50 mm KCl, 0.1% Triton X-100, 1.5 mm MgCl2, 200 μm dNTPs, 1 μm of each forward and reverse primer, and 2 units of rTaqDNA polymerase (Toyobo Co., Osaka, Japan). The reactions were heated at 94 °C for 5 min and then immediately cycled 24 times (SR-A), 23 times (CD36), or 21 times (GAPDH) through a 50-s denaturing step at 94 °C, a 50-s annealing step at 51 °C (SR-A), 60 °C (CD36), or 64 °C (GAPDH), and a 50-s extension step at 72 °C. After the cycling procedure, a final 10-min elongation step at 72 °C was performed. The following primers were used as follows: SR-A, 5′-ATGACAGAGAATCAGAGG-3′ (forward) and 5′-CCCTCTGTCTCCCTTTTC-3′ (reverse) (PCR product 855 bp); CD36, 5′-CCCAGTCACTTGTGTTTTGAAC-3′ (forward) and 5′-GAACCTTTGAAGGCTTACATCC-3′ (reverse) (PCR product 246 bp); GAPDH, 5′-AACCCATCACCATCTTCCAGGAGC-3′ (forward) and 5′-CACAGTCTTCTGAGTGGCAGTGAT-3′ (reverse) (PCR product 350 bp). Quantitative real time RT-PCR was performed using TaqMan® gene expression assay and TaqMan® universal PCR master mix reagents (Applied Biosystems). The RT reaction was performed with 1 μg of total RNA and random primer using the high capacity cDNA reverse transcription kit (Applied Biosystems). The amplification of PCR products was monitored by Applied Biosystems 7500 real time PCR system. The reaction conditions for RT and PCR were based on the protocols provided by Applied Biosystems. Relative levels of gene expression for each sample were calculated using comparative Ct method. The target gene expression in each sample was normalized to GAPDH Ct values. Data are expressed as the means ± S.D. of three separate experiments. Immunoblot Analysis—The cells were washed twice with HBSS and lysed with RIPA lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm EDTA) containing 1 mm phenylmethylsulfonyl fluoride. The protein samples were boiled with reducing sample buffer for 5 min. The samples (10 μg) were run on 10% SDS-polyacrylamide gels, transferred to a poly(vinylidene) fluoride membrane (Hybond-P, GE Healthcare), incubated at room temperature for 1 h with a blocking reagent (EzBlock, ATTO Corp., Tokyo, Japan) in TTBS (Tris-buffered saline containing 0.05% Tween 20) for blocking, washed in TTBS, and treated with primary antibody at 4 °C overnight. After washing, blots were further incubated for 1 h at room temperature with secondary antibody coupled to horseradish peroxidase in TTBS. After washing, the membrane was visualized by using ECL-Plus detection reagent. Cholesterol Accumulation in RAW264 Cells—RAW264 cells in 35-mm dish were treated with or without Q3GA in FBS-free DMEM for 24 h. After washing twice with HBSS, cells were treated with oxidized LDL (0.2 mg/ml) for 4 h. After washing, total cholesterol was extracted three times with 1 ml of n-hexane/isopropyl alcohol (3:2, v/v). The extracts were dried up and then saponified in 10 m KOH/ethanol (1:9, v/v) at 90 °C for 1 h. Free cholesterol was extracted with 1 ml of ether, evaporated, and then dissolved in ice-cold acetone. Then 20 μl of the supernatant was injected onto TSK-gel Octyl-80Ts column (4.6 × 150 mm) equilibrated with acetonitrile/methanol/water (46:45:9) at a flow rate of 1 ml/min with UV detection at 210 nm. Statistical Analysis—Data from real time PCR and cholesterol accumulation were expressed as the mean ± S.D. Comparisons were analyzed with the Student's t test. A p value < 0.05 was considered statistically significant. Q3GA as a Major Quercetin Metabolite in Human Plasma—It is known that most of the quercetin is metabolized to the glucuronides, sulfates, and/or methylated form during absorption and circulation (17da Silva E.L. Piskula M.K. Yamamoto N. Moon J.H. Terao J. FEBS Lett. 1998; 430: 405-408Crossref PubMed Scopus (161) Google Scholar), and therefore the quercetin aglycone could not be detected in human and rat plasma (11Day A.J. Mellon F. Barron D. Sarrazin G. Morgan M.R. Williamson G. Free Radic. Res. 2001; 35: 941-952Crossref Pu" @default.
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• W2014018523 title "Macrophage as a Target of Quercetin Glucuronides in Human Atherosclerotic Arteries" @default.
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