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Cite this: Food Funct., 2014, 5, 2371
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Esculetin inhibits the inflammatory response by inducing heme oxygenase-1 in cocultured macrophages and adipocytes Younghwa Kim,a Yooheon Park,b Seulgi Namkoonga and Junsoo Lee*a Obesity is associated with chronic low-grade inflammation of adipose tissue. In this study, we investigated the anti-inflammatory effects of esculetin (ECT) through up-regulation of heme oxygenase-1 (HO-1) in cocultured macrophages and adipocytes. RAW264.7 macrophages and differentiated 3T3-L1 adipocytes were cocultured in serum-free Dulbecco's modified Eagle's medium with or without ECT for 24 h. Nitric oxide (NO), tumor necrosis factor-a (TNF-a), and monocyte chemoattractant protein-1 (MCP-1) production was measured in the coculture supernatant. ECT decreased the secretion of NO, TNF-a, and MCP-1. The expression of adipogenic proteins, including peroxisome proliferator-activated receptors g (PPARg) and CCAAT/enhancer binding protein a (C/EBPa) in cocultured adipocytes and inducible nitric oxide synthase (iNOS) in cocultured macrophages, was inhibited by ECT. Additionally, HO-1 expression was induced in cocultured macrophages and adipocytes. Silencing of HO-1 expression increased the
Received 24th April 2014 Accepted 3rd July 2014
production of NO, TNF-a, and MCP-1 in cocultured cells, in spite of the presence of ECT. This study
DOI: 10.1039/c4fo00351a
demonstrated that ECT exhibited anti-inflammatory properties by inhibiting the production of proinflammatory cytokines in the interaction between adipocytes and macrophages through HO-1
www.rsc.org/foodfunction
expression. ECT may have the potential to improve chronic inflammation in obesity.
Obesity is currently considered a major risk factor for cardiovascular diseases and type 2 diabetes.1 Adipogenesis is regulated by multiple transcription factors such as CCAAT/enhancer binding protein a (C/EBPa) and peroxisome proliferator-activated receptor g (PPARg). These transcriptional factors coordinate the expression of genes involved in creating and maintaining the adipocyte phenotype.2 Recent studies have reported that obesity is associated with chronic low-grade inammation in adipose tissue.3 Inammatory cytokines including tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) are secreted by adipose tissue.4 MCP-1 induces the inltration of macrophages into adipose tissue. Macrophages induce inammatory reactions by releasing various types of inammatory mediators, such as nitric oxide (NO), prostaglandins, and TNF-a. The inltrated macrophages promote inammatory conditions and eventually aggravate the obesity state.5 Therefore, anti-inammatory compounds could ameliorate obesity-related disorders
by inhibiting the expression of proinammatory factors in adipose tissue. Esculetin (6,7-dihydroxy-2H-1-benzopyran-2-one; ECT) is a coumarin derivative isolated from various natural plants such as Artemisia montana and Euphorbia lathyris.6,7 ECT has benecial biological and biochemical activities, such as antioxidant, antiproliferative, anticancer, anti-inammatory and neuroprotective activities.8–15 According to the previous study, ECT induces heme oxygenase-1 (HO-1) expression in vascular smooth muscle cells.16 HO-1 is a well-known rate-limiting enzyme that catalyzes the degradation of heme to carbon monoxide, free iron, and biliverdin. HO-1 has antioxidative, anti-inammatory, and antiapoptotic properties.17 In addition, HO-1 has been reported to decrease lipogenesis and adipocyte differentiation.18 Induction of HO-1 in cultured adipocytes is associated with increased adiponectin levels and decreased levels of the proinammatory cytokines such as TNF-a and IL6.19,20 Because of these characteristics of HO-1, we investigated the anti-inammatory activities of ECT through up-regulation of HO-1 in cocultured macrophages and adipocytes.
a
Materials and methods
Introduction
Department of Food Science and Technology, College of Agriculture, Life and Environment Sciences, Chungbuk National University, 52 Naesudong-ro, Heungdeok-gu, Cheongju, Chungbuk 361-763, Korea. E-mail: [email protected]. kr; Fax: +82-43-271-4412; Tel: +82-43-261-2566
b
Department of Food and Nutrition, Korea University, San 1, Jeongneung-Dong, Sungbuk-Gu, Seoul 136-703, Korea
This journal is © The Royal Society of Chemistry 2014
Chemicals Isobutylmethylxanthine (IBMX) and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modied Eagle's medium (DMEM), bovine serum (BS), fetal
Food Funct., 2014, 5, 2371–2377 | 2371
View Article Online
Food & Function
Published on 04 July 2014. Downloaded by University of Chicago on 31/10/2014 20:21:29.
bovine serum (FBS), trypsin–EDTA, and penicillin–streptomycin were purchased from Gibco BRL (Gaithersburg, MD, USA). Esculetin, antibodies against PPARg, C/EBPa, and b-actin, and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against inducible NO synthase (iNOS) was obtained from Cayman (Ann Arbor, MI, USA). ECL™ detection reagents were purchased from GE Healthcare (Buckinghamshire, UK). All other reagents and solvents used were of analytical and HPLC grade. Culture of RAW264.7 mouse macrophage The RAW264.7 mouse macrophage cell line was obtained from the Korean Cell Line Bank (Seoul, Korea) and maintained in DMEM supplemented with 10% heat-inactivated FBS, 100 U mL1 penicillin, and 100 mg mL1 streptomycin in a humidied atmosphere of 5% CO2 at 37 C. Culture and differentiation of 3T3-L1 cells 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (Manassas, VA, USA). The 3T3-L1 preadipocytes were cultured as previously described with minor modications.21 Briey, 3T3-L1 preadipocytes were maintained at 37 C in DMEM containing 10% BS. At 2 days post-conuence (designated day 0 of cell differentiation), adipocyte differentiation was induced with a mixture of IBMX (0.5 mM), dexamethasone (1 mM), and insulin (1 mg mL1) in DMEM containing 10% of FBS. On day 2, the medium was replaced with DMEM containing 10% of FBS and insulin (1 mg mL1) only. On day 4, the medium was replaced with DMEM containing 10% of FBS. Coculture of 3T3-L1 and Raw264.7 Adipocytes and macrophages were cocultured in a contact or transwell system as previously described, with modications.5 In the contact system, serum-starved differentiated 3T3-L1 cells were cultured in a 6-well plate, and macrophages (8 105 cells) were plated onto 3T3-L1 cells. The cells were cultured for 24 h with ECT. As a control, adipocytes and macrophages, the numbers of which were equal to those in the contact system, were cultured separately. In the transwell system, cells were cocultured by using transwell inserts with a 0.4 mm porous membrane (Costar Corning Inc., Corning, NY, USA) to separate adipocytes from macrophages. 3T3-L1 cells were differentiated on the lower well, and then differentiated 3T3-L1 cells were washed with serum-free DMEM and the medium was replaced with serum-free medium with ECT. At the same time, macrophages (8 105 cells) in the serum-free medium with ECT were plated onto insert wells, and then the inserts and wells containing different cell types were assembled. Aer incubation for 24 h, the supernatants of cocultured cells, 3T3-L1 cells in the lower wells, and macrophages in insert wells were harvested.
Paper
RAW264.7 cells (1 105 per well) were seeded in 96-well plates and incubated for 6 h. Cells were treated with or without lipopolysaccharide (LPS; 1 mg mL1) and different concentrations of ECT for 18 h. Then, 100 mL of culture medium from each sample was mixed with 50 mL of Griess reagent, and the mixture was incubated at 37 C for 10 min. The absorbance was read at 550 nm. The NO concentration was determined using sodium nitrite as the standard. HO-1 siRNA and transfection of cultured cells HO-1 Stealth RNAi™ siRNA and vehicle siRNA (Stealth RNAi Negative Control Duplexes) were purchased from Invitrogen (Invitrogen Corp., Carlsbad, CA, USA). For transfection, HO-1 siRNA or vehicle siRNA was combined with Lipofectamine 2000 (Invitrogen) and incubated for 20 min at room temperature to produce the transfection mixture. Then, the transfection mixture was added to differentiated adipocytes and macrophages. At 24 h aer the start of transfection, the medium was replaced with serum-free medium. HO-1 siRNA-transfected macrophages (1 105 per well) were added to HO-1 siRNAtransfected adipocytes with or without ECT in the contact system for 24 h. The efficiency of HO-1 knockdown was determined by western blot analysis and semiquantitative densitometry. MCP-1 and TNF-a immunoassay The supernatant in the contact or transwell system of cocultures was collected aer 24 h of incubation. MCP-1 and TNF-a concentrations in the coculture supernatants were measured with BD OptEIA kits (BD Biosciences Pharmingen, San Diego, CA, USA) for mouse MCP-1 and TNF-a. Western blotting Washed cell pellets were lysed in cell lysis buffer (iNtRON Biotech, Seongnam, Korea). The protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Scientic, Rockford, IL, USA). Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. Aer 1 h of incubation in blocking solution (5% skim milk), the membranes were incubated for 1 h at a 1 : 1000 dilution of primary antibodies at room temperature. The membranes were washed three times with Tween-20/Tris (hydroxymethyl) aminomethane-buffered saline (TTBS) and incubated in a 1 : 1000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were again washed three times with TTBS and then developed using ECL™ detection reagents. The autoradiograms were quantied by optical densitometry using the ImageJ program (NIH, Washington, DC, USA). Statistical analysis
NO assay NO production was determined by measuring the amount of nitrite, a relatively stable NO oxidation product. Briey,
2372 | Food Funct., 2014, 5, 2371–2377
All data presented are expressed as mean standard error and are representative of three or more independent experiments. Results were compared by one-way analysis of variance (ANOVA)
This journal is © The Royal Society of Chemistry 2014
View Article Online
Paper
using Duncan's multiple range tests of SAS 9.2 (SAS Institute, Cary, NC, USA). A P value of
Published on 04 July 2014. Downloaded by University of Chicago on 31/10/2014 20:21:29.
PAPER
Cite this: Food Funct., 2014, 5, 2371
View Journal | View Issue
Esculetin inhibits the inflammatory response by inducing heme oxygenase-1 in cocultured macrophages and adipocytes Younghwa Kim,a Yooheon Park,b Seulgi Namkoonga and Junsoo Lee*a Obesity is associated with chronic low-grade inflammation of adipose tissue. In this study, we investigated the anti-inflammatory effects of esculetin (ECT) through up-regulation of heme oxygenase-1 (HO-1) in cocultured macrophages and adipocytes. RAW264.7 macrophages and differentiated 3T3-L1 adipocytes were cocultured in serum-free Dulbecco's modified Eagle's medium with or without ECT for 24 h. Nitric oxide (NO), tumor necrosis factor-a (TNF-a), and monocyte chemoattractant protein-1 (MCP-1) production was measured in the coculture supernatant. ECT decreased the secretion of NO, TNF-a, and MCP-1. The expression of adipogenic proteins, including peroxisome proliferator-activated receptors g (PPARg) and CCAAT/enhancer binding protein a (C/EBPa) in cocultured adipocytes and inducible nitric oxide synthase (iNOS) in cocultured macrophages, was inhibited by ECT. Additionally, HO-1 expression was induced in cocultured macrophages and adipocytes. Silencing of HO-1 expression increased the
Received 24th April 2014 Accepted 3rd July 2014
production of NO, TNF-a, and MCP-1 in cocultured cells, in spite of the presence of ECT. This study
DOI: 10.1039/c4fo00351a
demonstrated that ECT exhibited anti-inflammatory properties by inhibiting the production of proinflammatory cytokines in the interaction between adipocytes and macrophages through HO-1
www.rsc.org/foodfunction
expression. ECT may have the potential to improve chronic inflammation in obesity.
Obesity is currently considered a major risk factor for cardiovascular diseases and type 2 diabetes.1 Adipogenesis is regulated by multiple transcription factors such as CCAAT/enhancer binding protein a (C/EBPa) and peroxisome proliferator-activated receptor g (PPARg). These transcriptional factors coordinate the expression of genes involved in creating and maintaining the adipocyte phenotype.2 Recent studies have reported that obesity is associated with chronic low-grade inammation in adipose tissue.3 Inammatory cytokines including tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) are secreted by adipose tissue.4 MCP-1 induces the inltration of macrophages into adipose tissue. Macrophages induce inammatory reactions by releasing various types of inammatory mediators, such as nitric oxide (NO), prostaglandins, and TNF-a. The inltrated macrophages promote inammatory conditions and eventually aggravate the obesity state.5 Therefore, anti-inammatory compounds could ameliorate obesity-related disorders
by inhibiting the expression of proinammatory factors in adipose tissue. Esculetin (6,7-dihydroxy-2H-1-benzopyran-2-one; ECT) is a coumarin derivative isolated from various natural plants such as Artemisia montana and Euphorbia lathyris.6,7 ECT has benecial biological and biochemical activities, such as antioxidant, antiproliferative, anticancer, anti-inammatory and neuroprotective activities.8–15 According to the previous study, ECT induces heme oxygenase-1 (HO-1) expression in vascular smooth muscle cells.16 HO-1 is a well-known rate-limiting enzyme that catalyzes the degradation of heme to carbon monoxide, free iron, and biliverdin. HO-1 has antioxidative, anti-inammatory, and antiapoptotic properties.17 In addition, HO-1 has been reported to decrease lipogenesis and adipocyte differentiation.18 Induction of HO-1 in cultured adipocytes is associated with increased adiponectin levels and decreased levels of the proinammatory cytokines such as TNF-a and IL6.19,20 Because of these characteristics of HO-1, we investigated the anti-inammatory activities of ECT through up-regulation of HO-1 in cocultured macrophages and adipocytes.
a
Materials and methods
Introduction
Department of Food Science and Technology, College of Agriculture, Life and Environment Sciences, Chungbuk National University, 52 Naesudong-ro, Heungdeok-gu, Cheongju, Chungbuk 361-763, Korea. E-mail: [email protected]. kr; Fax: +82-43-271-4412; Tel: +82-43-261-2566
b
Department of Food and Nutrition, Korea University, San 1, Jeongneung-Dong, Sungbuk-Gu, Seoul 136-703, Korea
This journal is © The Royal Society of Chemistry 2014
Chemicals Isobutylmethylxanthine (IBMX) and dimethyl sulfoxide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco's modied Eagle's medium (DMEM), bovine serum (BS), fetal
Food Funct., 2014, 5, 2371–2377 | 2371
View Article Online
Food & Function
Published on 04 July 2014. Downloaded by University of Chicago on 31/10/2014 20:21:29.
bovine serum (FBS), trypsin–EDTA, and penicillin–streptomycin were purchased from Gibco BRL (Gaithersburg, MD, USA). Esculetin, antibodies against PPARg, C/EBPa, and b-actin, and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody against inducible NO synthase (iNOS) was obtained from Cayman (Ann Arbor, MI, USA). ECL™ detection reagents were purchased from GE Healthcare (Buckinghamshire, UK). All other reagents and solvents used were of analytical and HPLC grade. Culture of RAW264.7 mouse macrophage The RAW264.7 mouse macrophage cell line was obtained from the Korean Cell Line Bank (Seoul, Korea) and maintained in DMEM supplemented with 10% heat-inactivated FBS, 100 U mL1 penicillin, and 100 mg mL1 streptomycin in a humidied atmosphere of 5% CO2 at 37 C. Culture and differentiation of 3T3-L1 cells 3T3-L1 preadipocytes were purchased from the American Type Culture Collection (Manassas, VA, USA). The 3T3-L1 preadipocytes were cultured as previously described with minor modications.21 Briey, 3T3-L1 preadipocytes were maintained at 37 C in DMEM containing 10% BS. At 2 days post-conuence (designated day 0 of cell differentiation), adipocyte differentiation was induced with a mixture of IBMX (0.5 mM), dexamethasone (1 mM), and insulin (1 mg mL1) in DMEM containing 10% of FBS. On day 2, the medium was replaced with DMEM containing 10% of FBS and insulin (1 mg mL1) only. On day 4, the medium was replaced with DMEM containing 10% of FBS. Coculture of 3T3-L1 and Raw264.7 Adipocytes and macrophages were cocultured in a contact or transwell system as previously described, with modications.5 In the contact system, serum-starved differentiated 3T3-L1 cells were cultured in a 6-well plate, and macrophages (8 105 cells) were plated onto 3T3-L1 cells. The cells were cultured for 24 h with ECT. As a control, adipocytes and macrophages, the numbers of which were equal to those in the contact system, were cultured separately. In the transwell system, cells were cocultured by using transwell inserts with a 0.4 mm porous membrane (Costar Corning Inc., Corning, NY, USA) to separate adipocytes from macrophages. 3T3-L1 cells were differentiated on the lower well, and then differentiated 3T3-L1 cells were washed with serum-free DMEM and the medium was replaced with serum-free medium with ECT. At the same time, macrophages (8 105 cells) in the serum-free medium with ECT were plated onto insert wells, and then the inserts and wells containing different cell types were assembled. Aer incubation for 24 h, the supernatants of cocultured cells, 3T3-L1 cells in the lower wells, and macrophages in insert wells were harvested.
Paper
RAW264.7 cells (1 105 per well) were seeded in 96-well plates and incubated for 6 h. Cells were treated with or without lipopolysaccharide (LPS; 1 mg mL1) and different concentrations of ECT for 18 h. Then, 100 mL of culture medium from each sample was mixed with 50 mL of Griess reagent, and the mixture was incubated at 37 C for 10 min. The absorbance was read at 550 nm. The NO concentration was determined using sodium nitrite as the standard. HO-1 siRNA and transfection of cultured cells HO-1 Stealth RNAi™ siRNA and vehicle siRNA (Stealth RNAi Negative Control Duplexes) were purchased from Invitrogen (Invitrogen Corp., Carlsbad, CA, USA). For transfection, HO-1 siRNA or vehicle siRNA was combined with Lipofectamine 2000 (Invitrogen) and incubated for 20 min at room temperature to produce the transfection mixture. Then, the transfection mixture was added to differentiated adipocytes and macrophages. At 24 h aer the start of transfection, the medium was replaced with serum-free medium. HO-1 siRNA-transfected macrophages (1 105 per well) were added to HO-1 siRNAtransfected adipocytes with or without ECT in the contact system for 24 h. The efficiency of HO-1 knockdown was determined by western blot analysis and semiquantitative densitometry. MCP-1 and TNF-a immunoassay The supernatant in the contact or transwell system of cocultures was collected aer 24 h of incubation. MCP-1 and TNF-a concentrations in the coculture supernatants were measured with BD OptEIA kits (BD Biosciences Pharmingen, San Diego, CA, USA) for mouse MCP-1 and TNF-a. Western blotting Washed cell pellets were lysed in cell lysis buffer (iNtRON Biotech, Seongnam, Korea). The protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Scientic, Rockford, IL, USA). Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred onto nitrocellulose membranes. Aer 1 h of incubation in blocking solution (5% skim milk), the membranes were incubated for 1 h at a 1 : 1000 dilution of primary antibodies at room temperature. The membranes were washed three times with Tween-20/Tris (hydroxymethyl) aminomethane-buffered saline (TTBS) and incubated in a 1 : 1000 dilution of horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The membranes were again washed three times with TTBS and then developed using ECL™ detection reagents. The autoradiograms were quantied by optical densitometry using the ImageJ program (NIH, Washington, DC, USA). Statistical analysis
NO assay NO production was determined by measuring the amount of nitrite, a relatively stable NO oxidation product. Briey,
2372 | Food Funct., 2014, 5, 2371–2377
All data presented are expressed as mean standard error and are representative of three or more independent experiments. Results were compared by one-way analysis of variance (ANOVA)
This journal is © The Royal Society of Chemistry 2014
View Article Online
Paper
using Duncan's multiple range tests of SAS 9.2 (SAS Institute, Cary, NC, USA). A P value of
