Article pubs.acs.org/JAFC
Quercetin, but Not Its Metabolite Quercetin-3-glucuronide, Exerts Prophylactic Immunostimulatory Activity and Therapeutic Antiinflammatory Effects on Lipopolysaccharide-Treated Mouse Peritoneal Macrophages Ex Vivo Yi-Ru Liao and Jin-Yuarn Lin* Department of Food Science and Biotechnology, National Chung Hsing University, 250 Kuokuang Road, Taichung 40227, Taiwan, Republic of China (ROC) ABSTRACT: This study investigated the prophylactic or therapeutic effects of quercetin (Q) and its metabolite quercetin-3glucuronide (Q3G) on lipopolysaccharide (LPS)-induced inflammation in mouse peritoneal macrophages ex vivo. Changes in pro- and antiinflammatory cytokine secretion profiles were determined. The results showed that Q or Q3G in vitro treatments lower than 50 μM did not exhibit cytotoxicity on macrophages. At noncytotoxic doses, Q and Q3G, particularly Q, administration in a prophylactic ex vivo model increased pro-/antiinflammatory cytokine secretion ratios by macrophages in the absence or presence of LPS. Quercetin, but not Q3G, administration in a therapeutic ex vivo model decreased pro-/ antiinflammatory cytokine secretion ratios in the absence or presence of LPS. Our results indicated that Q and Q3G administrations in a prophylactic manner might act as an immunostimulatory agent, but Q presented better ability than Q3G. Quercetin might have a therapeutic, but not prophylactic, effect on spontaneous or LPS-induced inflammation in vivo. KEYWORDS: cytokines, peritoneal macrophages, ex vivo models, quercetin, quercetin-3-glucuronide, lipopolysaccharide
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INTRODUCTION Quercetin, a flavonol, has been reported to have various physiological benefits. Quercetin has recently been found to possess antiinflammatory, antiproliferative, and antiatherosclerotic effects in humans and might be further applied to cancer prevention and therapy.1,2 Oral administration of quercetin has been found to inhibit bone loss in rat diabetic osteopenia models and improve sodium fluoride-induced oxidative stress in rat kidneys.3,4 Dietary quercetin supplementation for six weeks was found to lower mRNA steady levels in inflammatory genes interleukin (IL)-6, C-reactive protein (CRP), monocyte chemoattractant protein (MCP) 1, and acyloxyacyl hydrolase in the liver of mice fed a high fat diet, involving redox factor 1, a modulator of nuclear factor (NF)-κB signaling.5 Quercetin was found to inhibit nitric oxide production in endotoxin/cytokinestimulated microglia, protect hydrogen-induced apoptosis in human neuronal SH-SY5Y cells and reduce proinflammatory cytokines IL-6 and tumor necrosis factor (TNF)-α production in THP1 macrophages.6−8 Quercetin is undoubtedly considered a potent bioflavonoid, widely used in health foods and pharmacology. After digestion, dietary quercetin has been found metabolized into conjugated isorhamnetin and quercetin derivatives. It has been identified that quercetin 3-O-β-D-glucuronide serves as an antioxidative metabolite in rat plasma.9,10 It was also found that dietary quercetin could be metabolized in the eye lens, with quercetin and its metabolites active in inhibiting oxidative damage and preventing cataract formation.11 Among the active quercetin metabolites, quercetin 3-O-β-D-glucuronide, also called miquelianin or quercetin-3-glucuronide, is a flavonol glycoside and a type of phenolic compound in wine and some medicinal plants.12−14 Quercetin-3-glucuronide has been © 2014 American Chemical Society
recently isolated from various plants and found to have antioxidant, antiviral and antiinflammatory properties.15−17 As a quercetin metabolite in vivo and a natural product from plants, quercetin-3-glucuronide, a conjugated quercetin metabolite, seems to retain its ability to protect cellular and subcellular membranes from peroxidative attack from reactive oxygen species and peroxidative enzymes.18 The immunomodulatory and antiinflammatory potential of quercetin-3-glucuronide, however, remains unclear. Among the immune cell macrophages is the mature monocytes form, resident in almost all tissues and the body cavity, particularly the peritoneal cavity, to scavenge antigens in innate immunity. It is clear that macrophages are relatively long-lived and versatile cells that perform several different functions throughout the innate immune response and subsequent adaptive immune responses. It is recognized that macrophages are vital to immune response regulation and inflammation development. After activation, macrophages may secrete different cytokines, including pro- and antiinflammatory cytokines. Some cytokines, such as IL-1β, TNF-α, and IL-6, can be roughly classified as proinflammatory cytokines because they may highlight the way and trigger local inflammation within injured tissues.19 In contrast, IL-10 is recognized as an antiinflammatory cytokine that is produced by type 2 helper T (Th2) cells, T regulatory cells (Th3 cells), macrophages, and some B cells to inhibit the synthesis of Th1 and other cytokines and macrophage functions during the late inflammation Received: Revised: Accepted: Published: 2872
December 15, 2013 February 6, 2014 March 12, 2014 March 12, 2014 dx.doi.org/10.1021/jf405630h | J. Agric. Food Chem. 2014, 62, 2872−2880
Journal of Agricultural and Food Chemistry
Article
phase.20 Excessive macrophage activation is involved in many diseases, such as atherosclerosis. It has recently been suggested that activated macrophages within injured/inflamed arteries are the potential targets of dietary quercetin metabolites.21 The presence of 50 μM quercetin glucuronide regulates the coronary venular barrier function through improving bloodborne inflammatory mediators and pharmacological tools in a novel microvascular wall model.22 Taken together, inflamed macrophages may be selected as a good target for evaluating the antiinflammatory potential of quercetin and its metabolites. Changes in pro-/antiinflammatory cytokine secretion levels by target cells may be a good marker for inflammation status. Cumulative data imply that quercetin (Q) can be absorbed and metabolized into quercetin-3-glucuronide (Q3G) in vivo. However, little is known about the differences in antiinflammatory effects between Q and Q3G administration. To clarify this puzzle, this study investigated the prophylactic or therapeutic effects of Q and Q3G administration on lipopolysaccharide (LPS)-induced inflammation in peritoneal macrophages from female BALB/c mice using ex vivo models. Changes in pro-/antiinflammatory cytokine secretion profiles by macrophages were determined using enzyme-linked immuno-sorbent assay (ELISA) to unravel the possible immunomodulatory or antiinflammatory mechanisms of Q or Q3G.
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these experiments was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), National Chung Hsing University, Taiwan, ROC. Source of Mouse Primary Peritoneal Macrophages. The primary peritoneal macrophages from mice were collected according to the method described previously.23,24 Briefly, the adult female BALB/c mice were anesthetized with diethyl ether, bled using a retroorbital venous plexus puncture to collect blood, and immediately euthanized by CO2 inhalation. Peritoneal macrophages were prepared by lavaging the peritoneal cavity with 2 aliquots of 5 mL of sterile Hank’s balanced salts solution (HBSS) ((50 mL of 10× HBSS (Hyclone Laboratories Inc., South Logan, UT), 2.5 mL of antibiotic− antimycotic solution (100× PSA) containing 10 000 units of penicillin, 10 mg of streptomycin, and 25 μg of amphotericin B per mL in 0.85% saline (Atlanta Biologicals Inc., Norcross, GA); 20 mL of 3% bovine serum albumin (BSA, Sigma-Aldrich Co., St. Louis, MO) in PBS; 2.5 mL of 7.5% NaHCO3 (Wako, Osaka, Japan), 425 mL sterile water)) for a total of 10 mL through the peritoneum. The peritoneal lavage fluid was centrifuged at 400g for 10 min at 4 °C. The cell pellets were collected and resuspended in tissue culture medium (TCM, a serum replacement; Celox Laboratories Inc., Lake Zurich, IL); suspended in a medium consisting of 10 mL of TCM, 500 mL of Roswell Park Memorial Institute (RPMI) 1640 medium (Atlanta Biologicals Inc., Norcross, GA), and 2.5 mL of antibiotic−antimycotic solution (100× PSA). The peritoneal adherent cells (>90% of macrophages) from each animal were adjusted to 2 × 106 cells/mL in TCM medium with a hemocytometer using the trypan blue dye exclusion method. Mouse Peritoneal Macrophage Cultures with Q and Q3G and Cell Viability Determination. To evaluate the possible cytotoxic effect of the test samples, the cell viability of mouse primary peritoneal macrophages treated with Q or Q3G was determined using 3-(4,5-dimethylthiazol-2-diphenyl)-2,5-tetrazolium bromide (MTT) assay. Briefly, peritoneal macrophages (2 × 106 cells/mL in TCM medium, 50 μL/well) in the absence or presence of Q (0.8, 8, 40, 100, and 200 μM in TCM medium; 50 μL/well), or Q3G (0.8, 1.6, 8, 40, 100, and 200 μM in TCM medium; 50 μL/well), respectively, were cocultured in 96-well plates at 37 °C in a humidified incubator with 5% CO2 and 95% air for 48 h. An endotoxin and B-cell mitogen, lipopolysaccharide (LPS, Sigma-Aldrich Co., L-2654, St. Louis, MO) at a final concentration of 2.5 μg/mL in culture was selected as a positive control in every individual experiment. Aliquots of 10 μL of 5 mg/mL MTT (Sigma, St. Louis, MO) in PBS were added to each well in the 96-well plate. The plates were incubated for another 4 h. The culture medium was then discarded. The plates were carefully washed with PBS buffer twice. Aliquots of 100 μL of DMSO were added to each well and oscillated for 30 min to extract formed insoluble formazan. The absorbance was measured at 550 nm on a plate reader (ELISA reader, ASYS Hitech, GmbH, Austria). The cell viability was calculated using the absorbance (A) at 550 nm; cell viability (% of control) = [(Asample − Asample blank)/(Acontrol − Ablank)] × 100. The remaining cell viability showed that Q or Q3G treatments lower than 50 μM did not have cytotoxicity on peritoneal macrophages. Antiinflammation Assessments of Q and Q3G Administrations Ex Vivo. To verify the antiinflammatory potential of Q and Q3G, Q and Q3G were prepared and administered to mouse primary macrophages in the absence or presence of LPS under two different ex vivo experimental models, including prophylactic and therapeutic models. Dexamethasone (Dex), a potent glucocorticoid drug having antiinflammatory and immunosuppressant functions, was chosen as a positive control in this study. A Prophylactic Ex Vivo Model. To avoid excessive high concentrations administered in vivo, noncytotoxic concentrations of Q or Q3G at 25 and 10 μM based on the in vitro study were selected to conduct antiinflammation assessments ex vivo. Accordingly, concentrations of 25 and 10 μM Q or Q3G responded to 1 × 106 cells/mL in vitro. On the basis of our preliminary results, we presumed that there are a total of 6 × 106 peritoneal macrophages in a 20 g normal mouse, reflecting its corresponding doses of Q or Q3G were 0.15 and 0.06 μmol/mouse for treating a total of 6 × 106 peritoneal macrophages/mouse in vivo. In our preliminary results, intraperitoneal
MATERIALS AND METHODS
Chemicals. Quercetin (Q) is a typical flavonoid existing in many plants. Quercetin-3-glucuronide (Q3G) is a major quercetin metabolite found in the serum in humans. In this study, both quercetin dihydrate (C15H10O7·2H2O; 338.27 g/mol) (Sigma-Aldrich Co., Steinheim, Switzerland) and quercetin-3-glucuronide (C21H18O13; 478.37 g/mol) (Carbosynth Limited, Berkshire, UK) were purchased at the highest available purity (>98%, HPLC). The chemical structures of Q and Q3G are shown in Figure 1. Quercetin was dissolved in
Figure 1. The chemical structures of quercetin and quercetin-3glucuronide. dimethyl sulfoxide (DMSO, Wako, Osaka, Japan) to prepare a stock solution at a concentration of 20 mM and sterilized using a filter (Millipore, Carrigtwohill, Cork, Ireland) with 0.2 μm pore size. Quercetin-3-glucuronide (Q3G) was dissolved in phosphate-buffered saline (PBS, 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4, 0.2 μm filtered) to prepare a stock solution at a concentration of 2 mM and sterilized using a filter (Millipore, Carrigtwohill, Cork, Ireland) with 0.2 μm pore size. The stock solution was stored at −80 °C for future use. Experimental Mice. Female BALB/cByJNarl mice (7 weeks old) were obtained from the National Laboratory Animal Center, National Applied Research Laboratories, National Science Council in Taipei, Taiwan, ROC, and maintained in the Department of Food Science and Biotechnology at National Chung Hsing University, Taiwan, ROC. The mice were housed and kept on a chow diet (laboratory standard diet, Diet MF 18, Oriental Yeast Co., Ltd., Osaka, Japan). The animal room was kept on a 12-h-light and 12-h-dark cycle. Constant temperature (25 ± 2 °C) and relative humidity (50−75%) were maintained. After acclimation for 1 week, the mice were sacrificed to obtain peritoneal macrophages. BALB/c strain mice weighting 20−25 g were used throughout the experiment. The animal use protocol for 2873
dx.doi.org/10.1021/jf405630h | J. Agric. Food Chem. 2014, 62, 2872−2880
Journal of Agricultural and Food Chemistry
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administrations with Q or Q3G, even at 0.30 μmol/mouse, over 24 h did not significantly affect the body weight and cause toxic signs in the experimental mice. Each animal received an appropriate concentration of Q or Q3G dissolved in a volume of 100 μL of sterilized PBS using aliquots from a single lot of Q or Q3G (both at 0.06 and 0.15 μmol/ mouse), respectively, to evaluate the prophylactic (preventive) Q or Q3G effects on LPS-induced inflammation ex vivo. The negative control animals received the same volume of PBS, and the positive control animals received the same volume of Dex (0.03 μmol/mouse). At 12 h after Q, Q3G, PBS, or Dex injection, all of the experimental animals were sacrificed. The peritoneal macrophages were collected as described previously. The isolated macrophages were cultured in the absence or presence of LPS (Sigma-Aldrich Co., L-2654, St. Louis, MO) at a final concentration of 25 μg/mL in 6-well plates. The plate was incubated at 37 °C in a humidified incubator with 5% CO2 and 95% air for 48 h. The plates were centrifuged at 400g for 10 min. The cell culture supernatants were collected and stored at −80 °C for cytokine assays. A Therapeutic Ex Vivo Model. We found that administration with an intraperitoneal (i.p.) injection of LPS at a concentration of 10 mg/ kg body weight (BW) might induce acute systemic inflammation.25,26 To obtain inflamed macrophages in vivo, a series of preliminary experiments were conducted to determine the effective LPS dosage for inducing moderate systemic inflammation and inflamed macrophages. We found that mice administered with an intraperitoneal injection of LPS at a concentration of 8 mg/kg BW over 12 h could induce moderate systemic inflammation and inflamed macrophages in mice. Therefore, LPS i.p. was administered at a concentration of 8 mg/kg BW for 12 h to induce inflammation in vivo. We further presumed a total of 6 × 106 peritoneal macrophages in a 20 g normal mouse, reflecting a corresponding concentration about 25 μg/mL LPS to stimulate 1 × 106 peritoneal macrophages/mL in vitro. Dex was found to have antiinflammatory effects to inhibit LPS-induced inflammation in J774 macrophages at 0.1−10 μM in vitro.27 We adopted 1 μM Dex as a positive control to treat inflamed peritoneal macrophages in vitro. To evaluate the therapeutic (curative) effects of Q or Q3G on LPSinduced inflammation ex vivo, each animal was first challenged with Escherichia coli LPS (O127:B8, Sigma-Aldrich Co., L-3129, St. Louis, MO) to induce moderate systemic inflammation to obtain inflamed macrophages in vivo. Mice were given an intraperitoneal injection of LPS from E. coli O127:B8 at a dose of 8 mg/kg body weight (BW). Each animal received this dose of LPS in a volume of 100 μL of LPS dissolved in sterilized PBS using aliquots from a single lot of LPS. The negative control animals received the same volume of PBS. At 12 h after LPS or PBS injection, all experimental animals were sacrificed. The inflamed or normal peritoneal macrophages were collected. The isolated inflamed or normal macrophages (1 × 106 cells/mL) were cultured in the absence or presence of Q or Q3G at the indicated final concentrations of 0.4, 0.8, 4, 20, and 50 μM in 6-well plates. Dex (1 μM) was chosen as a positive control. The plate was incubated at 37 °C in a humidified incubator with 5% CO2 and 95% air for 48 h. The plates were centrifuged at 400g for 10 min. The supernatants in cell cultures were collected and stored at −80 °C for cytokine assays. Proinflammatory and Antiinflammatory Cytokine Level Measurement in Macrophage Cultures Using ELISA. The macrophage culture supernatants in each individual treatment were collected to measure proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and antiinflammatory cytokine (IL-10) levels using sandwich ELISA kits. The TNF-α, IL-1β, IL-6, and IL-10 concentrations were assayed according to the cytokine ELISA protocol from the manufacturer’s instructions (mouse DuoSet ELISA Development system, R&D Systems, Minneapolis, MN). The sensitivity of these cytokine assays was
