Glucuronides of phytoestrogen flavonoid enhance macrophage function via conversion to aglycones by β-glucuronidase in macrophages - PubMed (original) (raw)

Glucuronides of phytoestrogen flavonoid enhance macrophage function via conversion to aglycones by β-glucuronidase in macrophages

Atsushi Kaneko et al. Immun Inflamm Dis. 2017 Sep.

Abstract

Introduction: Flavonoids are converted to inactive metabolites like glucuronides in the gut, and circulate mainly as glucuronides in blood stream, resulting in low concentrations of active aglycones in plasma. It is therefore unclear how oral flavonoids exert their effects in tissues. We recently reported the plasma pharmacokinetics of some flavonoids and suggested the possibility that the absorbed flavonoids modified macrophage functions leading to enhance bacterial clearance. We aimed to confirm their pharmacological profiles focusing on tissue macrophages.

Methods: Pseudoinfection was induced by intradermal injection of FITC-conjugated and killed Staphylococcus aureus into the ears of mice treated with or without genistein 7-O-glucuronide (GEN7G, 1 mg/kg, i.v.). FACS analysis was performed on single cell suspensions dispersed enzymatically from the skin lesions at 6 h post pseudoinfection to evaluate phagocytic activities of monocytes/macrophages (CD11b+ Ly6G- ) and neutrophils (CD11b+ Ly6G+ ). Phagocytosis of the FITC-conjugated bacteria by four glucuronides including GEN7G was evaluated in cultures of mouse macrophages.

Results: After GEN7G injection, genistein was identified in the inflamed ears as well as GEN7G, and the phagocytic activity of CD11b+ Ly6G- cells was increased. GEN7G was converted to genistein by incubation with macrophage-related β-glucuronidase. Macrophage culture assays revealed that GEN7G increased phagocytosis, and the action was dampened by a β-glucuronidase inhibitor. Binding of aglycones to estrogen receptors (ERs), putative receptors of flavonoid aglycones, correlated to biological activities, and glucuronidation reduced the binding to ERs. An ER antagonist suppressed the increase of macrophage function by GEN7G, whereas estradiol enhanced phagocytosis as well.

Conclusions: This study suggests a molecular mechanism by which oral flavonoids are carried as glucuronides and activated to aglycones by β-glucuronidase in tissue macrophages, and contributes to the pharmacological study of glucuronides.

Keywords: estrogen receptor; macrophages; β-glucuronidase.

© 2017 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

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Figures

Figure 1

Figure 1

Conversion of intravenously administered genistein 7‐_O_‐glucuronide to aglycone in the skin. Particles of FITC‐conjugated and killed S. aureus (67 µg/10 µl/site) were injected intradermally into the one side of the ears of mice. Genistein 7‐_O_‐glucuronide (GEN7G) dissolved in saline was administered intravenously to the mice at a dose of 1 mg/10 mL/kg immediately and 3 h after the pseudoinfection. The inflamed and non‐inflamed ears were cut off at 6 h after the pseudoinfection, and stored at −80°C until use. The ears were homogenized in PBS containing 20 mmol/L saccharic acid 1,4‐lactone, followed by measurement of GEN7G and genistein by LC‐MS/MS. Data are shown as mean ± SEM of triplicate tests.

Figure 2

Figure 2

Dose‐ and time‐dependency of genistein 7‐_O_‐glucuronide on macrophage phagocytosis. RAW264.7 cells were cultured for 1, 2, and 3 days in the presence of a suboptimal dose of IFN‐γ (0.5 ng/mL), followed by examination of phagocytic activity. Dose‐dependency (A) and time‐dependency (B) of genistein 7‐_O_‐glucuronide (GEN7G) and/or genistein were examined. Data are shown as mean ± SEM of triplicate tests. **P < 0.01 significance versus vehicle control by Dunnett's test. #, ##P < 0.05, 0.01 significance versus vehicle control by Student's _t_‐test, respectively.

Figure 3

Figure 3

Morphological changes in RAW264.7 cells due to genistein and genistein 7‐_O_‐glucuronide. RAW264.7 cells were cultured for 3 days in the presence of a suboptimal dose of IFN‐γ (0.5 ng/mL) plus the indicated test sample. Bars: 50 µm.

Figure 4

Figure 4

Involvement of signaling via nuclear estrogen receptor on macrophage activation of genistein 7‐_O_‐glucuronide. RAW264.7 cells were cultured for 3 days in the presence of a suboptimal dose of IFN‐γ (0.5 ng/mL) with genistein 7‐_O_‐glucuronide (GEN7G, 30 µmol/L), estradiol (1, 3, 10, 30 µmol/L), or vehicle. The estrogen receptor antagonist ICI‐182780 was added at 5 µmol/L, three times on days 0, 1, and 2. Phagocytosis (A and C) and CD88 expression (B) were analyzed, showing data as mean ± SEM of triplicate tests. The reproducibility was confirmed in several separate examinations. **P < 0.01 significance versus vehicle control by Dunnett's test, #, ##P < 0.05, 0.01 significance by Student's _t_‐test, respectively.

Figure 5

Figure 5

Molecular‐based interaction of flavonoids to estrogen receptors. (A and B) Aliquots of human estrogen receptor‐α (ERα) and estrogen receptor‐β (ERβ) were incubated for 2 h with 0.5 nmol/L [3H]‐estradiol in the presence of the test flavonoid at the indicated concentrations. Receptor proteins were filtered and washed, the filters are then counted to determine specifically bound [3H]‐estradiol. The concentration‐dependent curves are shown for ERα (A) and ERβ (B). (C) A β‐arrestin assay was performed using CHO‐K1 cell engineered to co‐express ProLink‐tagged GPR30 (human G‐coupled estrogen receptor 1), and β‐galactosidase acceptor‐tagged β‐arrestin. The cells were seeded in 384‐well microplates and incubated with the test flavonoid at the indicated concentrations, followed by incubation with a detection reagent cocktail for 1 h. Chemiluminescent signals were measured and are shown as the percentage activity. The concentration‐dependent curves are shown in GPR30. (D) A list of the half maximal inhibitory concentration (IC50) is shown. N = 2 in all assays.

Figure 6

Figure 6

Measurement of β‐glucuronidase in macrophage sources. (A) Total RNAs were prepared from proliferating RAW264.7 cells, followed by preparation of cDNAs. TaqMan gene expression assays were performed using TaqMan primers for Gusb (β‐glucuronidase) and Gapdh (glyceraldehyde‐3‐phosphate dehydrogenase). N = 2. (B) RAW264.7 cells cultured overnight was cultured for an additional 24 h after replacing the culture fluid with fresh medium. The culture supernatants and 0.1% Triton X‐treated cellular lysates were harvested. The amounts of β‐glucuronidase per well were measured by ELISA method. N = 3, (C) normal ears were cut off, homogenized, and β‐glucuronidase per mg protein was measured. N = 1.

Figure 7

Figure 7

Deconjugation of genistein 7‐_O_‐glucuronide to aglycone in RAW264.7 cells. Multiple reaction monitoring (MRM) chromatograms of genistein standard (A), genistein 7‐_O_‐glucuronide (GEN7G) standard (B), the supernatants incubated for 24 h in 24‐well plates under the following conditions: live RAW264.7 cells alone seeded at 1 × 105 cells/0.5 mL/well (C), 30 µmol/L GEN7G alone (D), 30 µmol/L GEN7G, and the live cells (E), 30 µmol/L GEN7G incubated for 24 h with the cell lysate prepared by sonicating RAW264.7 cells seeded at 1 × 105 cells/well (F).

Figure 8

Figure 8

Involvement of deconjugation process in macrophage activation of genistein 7‐_O_‐glucuronide. RAW264.7 cells were cultured for 3 days in the presence of a suboptimal dose of IFN‐γ (0.5 ng/mL) with or without 30 µmol/L genistein 7‐_O_‐glucuronide (GEN7G). A β‐glucuronidase inhibitor 1‐((6,8‐dimethyl‐2‐oxo‐1,2‐dihydroquinolin‐3‐yl)methyl)‐3‐(4‐ethoxyphenyl)‐1‐(2‐hydroxyethyl) thiourea was added at 10 µmol/L every day. The deconjugated genistein (A) in the culture fluids was measured by LS/MS/MS, and phagocytosis (B) and CD88 expression (C) of cells were evaluated. Data are shown as mean ± SEM of Ta tests. The reproducibility was confirmed in several separate examinations. ND: Not detected, ##P < 0.01 significance by Student's _t_‐test.

Figure 9

Figure 9

Possible mechanisms of macrophage activation by genistein 7‐_O_‐glucuronide. (1) GEN7G infiltrates to the inflamed sites through increased microvascular permeability; (2) GEN7G is exposed to resident macrophages and converted to genistein by β‐glucuronidase of resident macrophages intra‐ and extracellularly; (3) thereafter, resident macrophages are more easily activated by genistein and GEN7G than neutrophils that accumulate later in the inflamed sites.

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