Annals of Otology, Rhinology & Laryngology 122(9):595-600. © 2013 Annals Publishing Company. All rights reserved.
Caffeic Acid Phenethyl Ester Inhibits Diesel Exhaust Particle–Induced Inflammation of Human Middle Ear Epithelial Cells Via NOX4 Inhibition Sun-Young Jo; Naree Lee, MD; Sung-Moon Hong, MD; Hak Hyun Jung, MD; Sung-Won Chae, MD Objectives: Otitis media is one of the most common diseases in pediatric populations. Recent research on its pathogenesis has focused on air pollution. Chronic exposure to particulate air pollution is associated with the impairment of middle ear function. However, the mechanisms and the underlying inhibitory pathways, especially in the human middle ear, remain unknown. Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of propolis, a product of honeybee hives, which has anti-oxidative and anti-inflammatory activities. The aim of this study was to evaluate the inhibitory effect of CAPE on diesel exhaust particle (DEP)–induced inflammation of human middle ear epithelial cells and to determine the underlying pathway of the action of CAPE. Methods: The inflammatory damage caused by DEPs and the anti-inflammatory effects of CAPE were determined by measuring the levels of tumor necrosis factor α and nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4 with real-time reverse transcription polymerase chain reaction and Western blot analysis. The oxidative stress induced by DEPs and the anti-oxidative effects of CAPE were directly evaluated by measuring reactive oxygen species production by use of flow cytometric analysis of 2',7'-dichlorofluorescein diacetate. The effects of CAPE were compared with those of N-acetyl-L-cysteine, which has anti-oxidative and anti-inflammatory effects. Results: Use of CAPE significantly inhibited DEP-induced up-regulation of tumor necrosis factor α and NOX4 expression in a dose- and time-dependent manner. The accumulation of reactive oxygen species induced by DEPs was decreased by pretreatment with CAPE. The anti-inflammatory and anti-oxidative effects of CAPE were similar to those of N-acetyl-L-cysteine.
Conclusions: The inflammation induced by DEP is reduced by CAPE via the inhibition of NOX4 expression. These findings suggest that CAPE might be used as a therapeutic agent against DEP-induced inflammation of human middle ear epithelial cells. Key Words: diesel, nicotinamide adenine dinucleotide phosphate oxidase, otitis media.
DEP-induced MMP-1 is associated with increased extracellular signal–regulated kinase 1/2 phosphor ylation and the up-regulation of the expression and activity of the NADPH oxidase analog NOX4 in A549 cells.5 A previous study revealed a relationship between DEPs and a middle ear inflammatory response in human middle ear epithelial cells (HMEECs).2 It has been reported that the antioxidant caffeic acid is superior to p-coumaric and ferulic acids in inhibiting low-density lipoprotein oxidation, as well as in quenching radicals and singlet oxygen.6,7 In a previous study, caffeic acid phenethyl ester (CAPE) inhibited lipopolysaccharide-induced inflammation in HMEECs.8 However, the effects of caffeic acid on DEP-induced inflammation in HMEECs have not
Introduction
Otitis media (OM) is the most frequent disease of the middle ear mucosa caused by infection or inflammatory conditions, and is a common cause of hearing impairment in children.1 It can delay speech development and language acquisition, alter behavior, and influence the quality of life of patients.2 It can be caused by the infection of pathogens such as viruses, bacteria, and fungi. Cigarette smoking and air pollution can also induce inflammation in the middle ear that results in OM.3,4 Diesel exhaust particles (DEPs) are a component of air pollution that induce matrix metalloproteinase 1 (MMP-1) via the (reduced) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 4 redox–dependent mechanism in human lung epithelial cells.5 The
From the Division of Brain Korea 21 Program for Biomedical Science (Jo) and the Department of Otorhinolaryngology–Head and Neck Surgery, Korea University College of Medicine (Lee, Hong, Jung, Chae), Seoul, Korea. Correspondence: Sung-Won Chae, MD, Dept of Otorhinolaryngology–Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, 80 Guro-dong, Guro-gu, Seoul 152-703, South Korea.
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Jo et al, Caffeic Acid Phenethyl Ester sequences of primers
Primers Human NOX1 Human NOX4 Human NOX5 Human TNF-α Human GAPDH
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
Sequences AAG GAT CCT CCG GTT TTA CC TTT GGA TGG GTG CAT AAC AA CTC AGC GGA ATC AAT CAG CTG TG AGA GGA ACA CGA TCA GCC TTA CGT CTG TGC CGG CTT ATC CCA CTT CCA GAT ACA ACA TGA CTG CCA AGC CCT GGT ATG AGC CC AGG CGT TTG GGA AGG TTG GA GCA AAT TCC ATG GCA CCG T TCG CCC CAC TTG ATT TTG G
NOX — nicotinamide adenine dinucleotide phosphate oxidase; TNF — tumor necrosis factor; GAPDH — glyceraldehyde 3-phosphate dehydrogenase.
been studied. The aims of this study were to evaluate the inhibitory effect of CAPE on DEP-induced inflammation of HMEECs and to determine the underlying pathway of the action of CAPE. Materials and Methods
Cell Culture and Particles. The DEPs (National Institute of Standards and Technology, Gaithersburg, Maryland) used in this study were suspended in sterile saline solution (sodium chloride 0.9%) containing Tween 80 (0.01%) and sonicated for 15 minutes before use. We cultured HMEECs from the House Ear Institute at 37°C in a 1:1 mixed medium of 5% carbon dioxide in Dulbecco’s modified Eagle’s medium (Lonza, Basel, Switzerland) and bronchial epithelial cell basal medium (Lonza). The medium was supplemented with bovine pituitary extract (52 μg/ mL), hydrocortisone (0.5 μg/mL), human epidermal growth factor (0.5 ng/mL), epinephrine (0.5 mg/mL), transferrin (10 μg/mL), insulin (5 μg/mL), triiodothyronine (6.5 ng/mL), retinoic acid (0.1 ng/ mL), gentamicin (50 μg/mL), and amphotericin-B (50 ng/mL). The cells from passage 4 were used. Human middle ear epithelial cells were stimulated with DEPs (500 μg/mL) for 6 hours. To examine the effect of CAPE, we also pretreated HMEECs with CAPE (Sigma Aldrich, St Louis, Missouri; 100 μmol/L per milliliter) for 30 minutes. To examine the effect of N-acetyl-L-cysteine (NAC), we also pretreated HMEECs with NAC (Sigma Aldrich; 2.5 mmol/L per milliliter) for 30 minutes.
RNA Extraction and cDNA Synthesis. Total RNA was isolated from HMEECs with RNeasy Mini Kits (Qiagen GmbH, Hilden, Germany) and was reverse transcribed with Superscript III (Invitrogen, Life Technologies, Carlsbad, California) as described by the manufacturer. Total RNA 1 μg was reverse transcribed at 65°C for 10 minutes with 1 μg oligo(dT), 1 μg 10 mmol/L deoxyribonucleotide triphosphate mix (10 mmol/L each 2'-deoxyadenosine triphos-
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phate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and 2'-deoxythymidine triphosphate at neutral pH), and 13 μL distilled water and then incubated on ice for 1 minute. The contents of the tube were then collected and put in 4 μL 5X First-Strand Buffer, 1 μL 0.1 mol/L diothiothreitol, and 0.5 μL SuperScript III reverse transcriptase. The solution was mixed by gentle up-and-down pipetting and incubated at 50°C for 60 minutes. The reaction was inactivated by heating at 70°C for 15 minutes. Real-Time Polymerase Chain Reaction Analysis. NOX4 and tumor necrosis factor α messenger RNA (mRNA) expression was quantified by the polymerase chain reaction (PCR) ABI 7300 apparatus (Applied Biosystems, Life Technologies). Real-time PCR was carried out with Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Primer Express Software 3.0 (Applied Biosystems) was also used, and the primer sequences used in this study are shown in the Table. The reactions were performed in 96well plates with a final volume of 25 μL containing 12.5 μL of the SYBR Green PCR Master Mix, 1 μL of each gene-specific primer, and 2 μL of diluted complementary DNA. The reaction consisted of the initial denaturation at 95°C for 15 minutes, 40 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, and synthesis at 72°C for 1 minute. The SYBR Green fluorescence was measured at each extension step. The threshold cycle (Ct) value reflected the cycle number that saw significant increases in the reporter fluorescence signal above baseline. The NOX4 expression values were normalized to the amount of gly ceraldehyde 3-phosphate dehydrogenase (GAPDH): ΔCt = Ct of NOX4 − Ct of GAPDH. The fold change in NOX4 mRNA was 2(Ct of NOX4 – Ct of GAPDH). Protein Extraction and Western Blots. The protein extract was isolated from cells by means of lysis buffer containing 150 mmol/L sodium chloride, 1.0% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mmol/L Tris, pH 8.0 radioimmunoprecipitation assay buffer (Sigma Aldrich), and protease inhibitor cocktail (Sigma Aldrich). The total protein concentration was determined by the Bradford protein assay (BioRad, Hercules, California). Fifty micrograms of the total protein and sample buffer was denatured in a lithium dodecyl sulfate sample buffer (Invitrogen) at 95°C for 5 minutes, incubated on ice for 1 minute, and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis at 100 V for 1 hour. The protein sample was then transferred onto
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B
Fig 1. Expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and tumor necrosis factor α (TNF-α) messenger RNA (mRNA) levels in human middle ear epithelial cells (HMEECs) following diesel exhaust particle (DEP) exposure. A) Expression of NOX1, NOX4, and NOX5 in control and DEP-exposed groups. B) Expression of NOX4 and TNF-α in control and DEP-exposed groups with and without N-acetyl-L-cysteine (NAC) or caffeic acid phenylethyl ester (CAPE). These results were obtained from at least 10 independent experiments. Data shown are mean ± SD of 4 repeated experiments with duplicate samples. * — p < 0.05 compared with control group; † — p < 0.05 compared with DEPs group.
polyvinylidene fluoride membrane with an electro blotting apparatus set at 80 V for 1 hour. Before use, the membrane was activated with methanol, blocked with 5% skim milk in TBS-T (Tris-buffered saline solution with Tween 20) for 1 hour, and then incubated with NOX4 (1:500 dilution; Santa Cruz Biotechnology, Dallas, Texas) and α-tubulin (1:1,000 dilution, Thermo Fisher Scientific, Waltham, Massachusetts) antibodies overnight at 4°C. After incubation with secondary antibody conjugated with horseradish peroxidase (1:2,000 dilution in 5% skim milk) for 1 hour, the signals on the membrane were detected with use of enhanced chemiluminescence luminol solution (KPL Inc, Gaithersburg, Maryland) and exposed to X-ray film (AGFA, Mortsel, Belgium). Measurement of ROS. The intracellular reactive oxygen species (ROS) produced in the DEP-exposed cells were measured with 2',7'-dichlorofluorescein diacetate (DCF(H)DA). Cells were seeded in 100-mm plates (1 × 106 cells per dish) and incubated in growth medium for 24 hours. In separate experiments, cells were pretreated with NAC (2.5 mmol/L) or CAPE (100 μmol/L) in each dish. After 30 minutes, the cells were stimulated with DEPs for 6 hours. The cells were then rinsed with phosphatebuffered saline solution, loaded with 50 μmol/L DCF(H)DA, and incubated for 30 minutes at 37°C. The DCF(H)DA fluorescence was measured with a Cytomics FC500 (Beckman Coulter, Brea, California). Data and Statistics. All data are expressed as mean ± SD. Statistical significance was assigned to p values of less than 0.05.
Results
DEP-Induced NOX4 mRNA Expression. The cells were stimulated with DEPs (500 μg/mL) for 6 hours. The expression of NOX4 was increased by DEP stimulation; however, NOX1 and NOX5 expressions were not affected by DEP stimulation (p < 0.05; Fig 1A). We also pretreated HMEECs with the known antioxidant NAC (2.5 mmol/L per milliliter; Sigma, A8199-10G) and CAPE (100 μg/mL) for 30 minutes before DEP stimulation (Fig 1B). The expressions of NOX4 and tumor necrosis factor α were down-regulated in cells pretreated with NAC or CAPE in comparison to those that received only DEP stimulation. These results were obtained from at least 10 independent experiments. DEP-Induced NOX4 Protein Expression. We performed Western blot analysis to confirm the effect of DEPs on NOX4 protein levels. The expression of NOX4 protein increased in cells stimulated by DEPs for 6 hours, but decreased in cells pretreated with NAC or CAPE (Fig 2). These results were obtained from at least 10 independent experiments. DEP-Induced Intracellular ROS Production. We measured ROS via fluorescence-activated cell sorting analysis. Reactive oxygen species were increased in cells stimulated with DEPs for 6 hours in comparison to the control cells (Fig 3A). However, ROS were decreased in cells that were pretreated by NAC or CAPE for 30 minutes (Fig 3B). The DEPstimulated cells had a median value of 21.7 ± 2.62, whereas those pretreated with NAC or CAPE before DEP stimulation had median values of 17.0 ± 2.41 and 13.8 ± 1.74, respectively (Fig 3C). These results were obtained from 20 independent experiments.
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Fig 2. Expression of NOX4 protein levels in HMEECs following DEP exposure with and without NAC and CAPE. These results were obtained from at least 10 independent experiments. * — p < 0.05 compared with control group; † — p < 0.05 compared with DEPs group.
Discussion
Otitis media is a common middle ear disease that might induce hearing impairment in children. It is one of the main reasons children in developed countries are prescribed antibiotics or undergo surgery.9 Exposure to traffic-related air pollution induces
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OM. Light-absorbing carbon and nitrogen dioxide are positively associated with traffic-related air pollution.10 Diesel, carbon monoxide, ozone, smoke, and sulfur dioxide also cause air pollution.11 The common pollutant diesel exhaust is composed of a core of elemental carbon and organic compounds including polycyclic aromatic hydrocarbons, nitro– polycyclic aromatic hydrocarbons, small amounts of sulfate, nitrate, metals, and other elements. Diesel exhaust is a major cause of inflammation and induces MMP-1 expression via extracellular signal– regulated kinase 1/2 activation in human lung epithelial cells.11 Our study focused on the relationship between DEPs and OM. Reactive oxygen species induce inflammatory responses in many diseases. Inflammation induced by ROS has been caused by the hydrogen peroxide– producing enzyme glucose oxidase in a skin model.12,13 Damage to DNA by ROS and nitrogen species can lead to inflammatory disease and even progression to cancer.14 Tumor necrosis factor α is a critical proinflammatory mediator leading to inflammation and is believed to be associated with tissue damage, fibrosis, and bone resorption in the middle ear in patients with OM.15 Tumor necrosis factor α increases ROS by inducing spermine oxidase in human lung epithelial cells.16 Reactive oxygen species are also known to induce OM.17 Several substances have been reported to induce ROS. For example, Helicobacter pylori has been associated with ROS activity and lipid peroxidation, which can lead to gastritis.18 The uncoupling of the
B
Fig 3. Role of reactive oxygen species in DEP-induced inflammation in HMEECs. A) Fluorescence intensity of control and DEP-exposed groups. B) Fluorescence intensity of DEPexposed groups with and without NAC or CAPE. C) Median value of fluorescence intensity relative to control. These results were obtained from 20 independent experiments. * — p < 0.05 compared with control group; † — p < 0.05 compared with DEPs group.
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protein-2 gene also plays a role in ROS production.19 Zhang et al20 reported that NADPH oxidases rapidly increased the levels of ROS in lung epithelium. Electrons from NADPH oxidases are transferred through the enzyme to molecular oxygen and generate superoxide with the secondary production of ROS.21 Therefore, we focused on the connection between NOX and ROS generation.
Nicotinamide adenine dinucleotide phosphate oxi dase is a multi-subunit enzyme that catalyzes oxide ion production by the 1-electron reduction of oxygen by means of NADPH or (reduced) nicotinamide adenine dinucleotide (NADH). Evidence indicates that there is an entire family of NADPH oxidases, based on the discovery of gp91phox homologs.22 The family comprises 7 members, including NOX1, NOX2 (formerly termed gp91phox), NOX3, NOX4, NOX5, dual oxidase 1, and dual oxidase 2.23 NOX1 is found in colon and vascular cells and plays a role in host defense and cell growth; NOX2 is the catalytic subunit of the respiratory burst oxidase in phagocytes, but is also expressed in vascular, cardiac, renal, and neural cells; NOX3 is found in fetal tissue and the adult inner ear and is involved in vestibular function; NOX4, originally termed Renox (renal oxidase) because of its abundance in the kidney, is also found in vascular cells and osteoclasts; and NOX5 is a calcium ion–dependent homolog found in testis and lymphoid tissue, and also in vas-
cular cells.24 In this study, DEPs were cytotoxic and increased the levels of ROS and NOX4 expression in HMEECs. NOX4 is well known to generate high levels of ROS.
Propolis, a product derived from the hives of honeybees, possesses anti-inflammatory, antiviral, immunostimulatory, and carcinostatic activities.25 Caffeic acid phenylethyl ester is the active component of propolis. It has been demonstrated as a free radical scavenger and has antioxidant and anti-inflammatory effects.26 In this study, CAPE was used to reduce ROS expression and was found to decrease the levels of NOX4 mRNA and protein induced by DEPs. There were some limitations to this study. First, we used only immortalized HMEECs. Further studies, evaluating other cell lines and tissues, should be done. Second, studies focusing on the mechanism of DEP-induced inflammation are needed. It would also be interesting to study the effects of other antioxidants on OM. Conclusions
Inflammation induced by DEPs is reduced by CAPE via the inhibition of NOX4 expression. This finding suggests that CAPE could be used as a therapeutic agent against DEP-induced inflammation of HMEECs.
References
1. Rovers MM. The burden of otitis media. Vaccine 2008; 26(suppl 7):G2-G4.
2. Song JJ, Lee JD, Lee BD, Chae SW, Park MK. Effect of diesel exhaust particles on human middle ear epithelial cells. Int J Pediatr Otorhinolaryngol 2012;76:334-8.
3. Domagala-Kulawik J. Effects of cigarette smoke on the lung and systemic immunity. J Physiol Pharmacol 2008;59(suppl 6):19-34.
4. Shao MX, Nakanaga T, Nadel JA. Cigarette smoke induces MUC5AC mucin overproduction via tumor necrosis factor–alpha–converting enzyme in human airway epithelial (NCIH292) cells. Am J Physiol Lung Cell Mol Physiol 2004;287: L420-L427.
5. Amara N, Bachoual R, Desmard M, et al. Diesel exhaust particles induce matrix metalloprotease–1 in human lung epithelial cells via a NADP(H) oxidase/NOX4 redox–dependent mechanism. Am J Physiol Lung Cell Mol Physiol 2007;293: L170-L181.
6. Kikuzaki H, Hisamoto M, Hirose K, Akiyama K, Taniguchi H. Antioxidant properties of ferulic acid and its related compounds. J Agric Food Chem 2002;50:2161-8.
7. Meyer AS, Donovan JL, Pearson DA, Waterhouse AL, Frankel EN. Fruit hydroxycinnamic acids inhibit human lowdensity lipoprotein oxidation in vitro. J Agric Food Chem 1998; 46:1783-7. 8. Song JJ, Cho JG, Hwang SJ, Cho CG, Park SW, Chae
SW. Inhibitory effect of caffeic acid phenethyl ester (CAPE) on LPS-induced inflammation of human middle ear epithelial cells. Acta Otolaryngol 2008;128:1303-7. 9. Rovers MM, Schilder AG, Zielhuis GA, Rosenfeld RM. Otitis media. Lancet 2004;363:465-73. [Erratum in Lancet 2004;363:1080.] 10. Brauer M, Gehring U, Brunekreef B, et al. Traffic-related air pollution and otitis media. Environ Health Perspect 2006; 114:1414-8. 11. Wichmann HE. Diesel exhaust particles. Inhal Toxicol 2007;19(suppl 1):241-4. 12. Boldogh I, Bacsi A, Choudhury BK, et al. ROS generated by pollen NADPH oxidase provide a signal that augments antigen-induced allergic airway inflammation. J Clin Invest 2005; 115:2169-79. 13. Trenam CW, Dabbagh AJ, Morris CJ, Blake DR. Skin inflammation induced by reactive oxygen species (ROS): an invivo model. Br J Dermatol 1991;125:325-9. 14. Wiseman H, Halliwell B. Damage to DNA by reactive oxygen and nitrogen species: role in inflammatory disease and progression to cancer. Biochem J 1996;313:17-29. 15. Maes T, Provoost S, Lanckacker EA, et al. Mouse models to unravel the role of inhaled pollutants on allergic sensitization and airway inflammation. Respir Res 2010;11:7. 16. Babbar N, Casero RA Jr. Tumor necrosis factor–alpha increases reactive oxygen species by inducing spermine oxidase in
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human lung epithelial cells: a potential mechanism for inflammation-induced carcinogenesis. Cancer Res 2006;66:11125-30. 17. Döner F, Delibaş N, Dogru H, Yariktaş M, Demirci M. The role of free oxygen radicals in experimental otitis media. J Basic Clin Physiol Pharmacol 2002;13:33-40.
18. Drake IM, Mapstone NP, Schorah CJ, et al. Reactive oxygen species activity and lipid peroxidation in Helicobacter pylori associated gastritis: relation to gastric mucosal ascorbic acid concentrations and effect of H pylori eradication. Gut 1998;42:768-71. 19. Arsenijevic D, Onuma H, Pecqueur C, et al. Disruption of the uncoupling protein–2 gene in mice reveals a role in immunity and reactive oxygen species production. Nat Genet 2000;26:435-9.
20. Zhang X, Shan P, Sasidhar M, et al. Reactive oxygen species and extracellular signal–regulated kinase 1/2 mitogen–activated protein kinase mediate hyperoxia-induced cell death in
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lung epithelium. Am J Respir Cell Mol Biol 2003;28:305-15.
21. Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 2004;4:181-9.
22. Geiszt M. NADPH oxidases: new kids on the block. Cardiovasc Res 2006;71:289-99. 23. Griendling KK. NADPH oxidases: new regulators of old functions. Antioxid Redox Signal 2006;8:1443-5. 24. Paravicini TM, Touyz RM. NADPH oxidases, reactive oxygen species, and hypertension: clinical implications and therapeutic possibilities. Diabetes Care 2008;31(suppl 2):S170S180. 25. Grunberger D, Banerjee R, Eisinger K, et al. Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 1988;44:230-2. 26. Parlakpinar H, Tasdemir S, Polat A, et al. Protective role of caffeic acid phenethyl ester (cape) on gentamicin-induced acute renal toxicity in rats. Toxicology 2005;207:169-77.
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Caffeic Acid Phenethyl Ester Inhibits Diesel Exhaust Particle–Induced Inflammation of Human Middle Ear Epithelial Cells Via NOX4 Inhibition Sun-Young Jo; Naree Lee, MD; Sung-Moon Hong, MD; Hak Hyun Jung, MD; Sung-Won Chae, MD Objectives: Otitis media is one of the most common diseases in pediatric populations. Recent research on its pathogenesis has focused on air pollution. Chronic exposure to particulate air pollution is associated with the impairment of middle ear function. However, the mechanisms and the underlying inhibitory pathways, especially in the human middle ear, remain unknown. Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of propolis, a product of honeybee hives, which has anti-oxidative and anti-inflammatory activities. The aim of this study was to evaluate the inhibitory effect of CAPE on diesel exhaust particle (DEP)–induced inflammation of human middle ear epithelial cells and to determine the underlying pathway of the action of CAPE. Methods: The inflammatory damage caused by DEPs and the anti-inflammatory effects of CAPE were determined by measuring the levels of tumor necrosis factor α and nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4 with real-time reverse transcription polymerase chain reaction and Western blot analysis. The oxidative stress induced by DEPs and the anti-oxidative effects of CAPE were directly evaluated by measuring reactive oxygen species production by use of flow cytometric analysis of 2',7'-dichlorofluorescein diacetate. The effects of CAPE were compared with those of N-acetyl-L-cysteine, which has anti-oxidative and anti-inflammatory effects. Results: Use of CAPE significantly inhibited DEP-induced up-regulation of tumor necrosis factor α and NOX4 expression in a dose- and time-dependent manner. The accumulation of reactive oxygen species induced by DEPs was decreased by pretreatment with CAPE. The anti-inflammatory and anti-oxidative effects of CAPE were similar to those of N-acetyl-L-cysteine.
Conclusions: The inflammation induced by DEP is reduced by CAPE via the inhibition of NOX4 expression. These findings suggest that CAPE might be used as a therapeutic agent against DEP-induced inflammation of human middle ear epithelial cells. Key Words: diesel, nicotinamide adenine dinucleotide phosphate oxidase, otitis media.
DEP-induced MMP-1 is associated with increased extracellular signal–regulated kinase 1/2 phosphor ylation and the up-regulation of the expression and activity of the NADPH oxidase analog NOX4 in A549 cells.5 A previous study revealed a relationship between DEPs and a middle ear inflammatory response in human middle ear epithelial cells (HMEECs).2 It has been reported that the antioxidant caffeic acid is superior to p-coumaric and ferulic acids in inhibiting low-density lipoprotein oxidation, as well as in quenching radicals and singlet oxygen.6,7 In a previous study, caffeic acid phenethyl ester (CAPE) inhibited lipopolysaccharide-induced inflammation in HMEECs.8 However, the effects of caffeic acid on DEP-induced inflammation in HMEECs have not
Introduction
Otitis media (OM) is the most frequent disease of the middle ear mucosa caused by infection or inflammatory conditions, and is a common cause of hearing impairment in children.1 It can delay speech development and language acquisition, alter behavior, and influence the quality of life of patients.2 It can be caused by the infection of pathogens such as viruses, bacteria, and fungi. Cigarette smoking and air pollution can also induce inflammation in the middle ear that results in OM.3,4 Diesel exhaust particles (DEPs) are a component of air pollution that induce matrix metalloproteinase 1 (MMP-1) via the (reduced) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) 4 redox–dependent mechanism in human lung epithelial cells.5 The
From the Division of Brain Korea 21 Program for Biomedical Science (Jo) and the Department of Otorhinolaryngology–Head and Neck Surgery, Korea University College of Medicine (Lee, Hong, Jung, Chae), Seoul, Korea. Correspondence: Sung-Won Chae, MD, Dept of Otorhinolaryngology–Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, 80 Guro-dong, Guro-gu, Seoul 152-703, South Korea.
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Jo et al, Caffeic Acid Phenethyl Ester sequences of primers
Primers Human NOX1 Human NOX4 Human NOX5 Human TNF-α Human GAPDH
Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
Sequences AAG GAT CCT CCG GTT TTA CC TTT GGA TGG GTG CAT AAC AA CTC AGC GGA ATC AAT CAG CTG TG AGA GGA ACA CGA TCA GCC TTA CGT CTG TGC CGG CTT ATC CCA CTT CCA GAT ACA ACA TGA CTG CCA AGC CCT GGT ATG AGC CC AGG CGT TTG GGA AGG TTG GA GCA AAT TCC ATG GCA CCG T TCG CCC CAC TTG ATT TTG G
NOX — nicotinamide adenine dinucleotide phosphate oxidase; TNF — tumor necrosis factor; GAPDH — glyceraldehyde 3-phosphate dehydrogenase.
been studied. The aims of this study were to evaluate the inhibitory effect of CAPE on DEP-induced inflammation of HMEECs and to determine the underlying pathway of the action of CAPE. Materials and Methods
Cell Culture and Particles. The DEPs (National Institute of Standards and Technology, Gaithersburg, Maryland) used in this study were suspended in sterile saline solution (sodium chloride 0.9%) containing Tween 80 (0.01%) and sonicated for 15 minutes before use. We cultured HMEECs from the House Ear Institute at 37°C in a 1:1 mixed medium of 5% carbon dioxide in Dulbecco’s modified Eagle’s medium (Lonza, Basel, Switzerland) and bronchial epithelial cell basal medium (Lonza). The medium was supplemented with bovine pituitary extract (52 μg/ mL), hydrocortisone (0.5 μg/mL), human epidermal growth factor (0.5 ng/mL), epinephrine (0.5 mg/mL), transferrin (10 μg/mL), insulin (5 μg/mL), triiodothyronine (6.5 ng/mL), retinoic acid (0.1 ng/ mL), gentamicin (50 μg/mL), and amphotericin-B (50 ng/mL). The cells from passage 4 were used. Human middle ear epithelial cells were stimulated with DEPs (500 μg/mL) for 6 hours. To examine the effect of CAPE, we also pretreated HMEECs with CAPE (Sigma Aldrich, St Louis, Missouri; 100 μmol/L per milliliter) for 30 minutes. To examine the effect of N-acetyl-L-cysteine (NAC), we also pretreated HMEECs with NAC (Sigma Aldrich; 2.5 mmol/L per milliliter) for 30 minutes.
RNA Extraction and cDNA Synthesis. Total RNA was isolated from HMEECs with RNeasy Mini Kits (Qiagen GmbH, Hilden, Germany) and was reverse transcribed with Superscript III (Invitrogen, Life Technologies, Carlsbad, California) as described by the manufacturer. Total RNA 1 μg was reverse transcribed at 65°C for 10 minutes with 1 μg oligo(dT), 1 μg 10 mmol/L deoxyribonucleotide triphosphate mix (10 mmol/L each 2'-deoxyadenosine triphos-
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phate, deoxyguanosine triphosphate, deoxycytidine triphosphate, and 2'-deoxythymidine triphosphate at neutral pH), and 13 μL distilled water and then incubated on ice for 1 minute. The contents of the tube were then collected and put in 4 μL 5X First-Strand Buffer, 1 μL 0.1 mol/L diothiothreitol, and 0.5 μL SuperScript III reverse transcriptase. The solution was mixed by gentle up-and-down pipetting and incubated at 50°C for 60 minutes. The reaction was inactivated by heating at 70°C for 15 minutes. Real-Time Polymerase Chain Reaction Analysis. NOX4 and tumor necrosis factor α messenger RNA (mRNA) expression was quantified by the polymerase chain reaction (PCR) ABI 7300 apparatus (Applied Biosystems, Life Technologies). Real-time PCR was carried out with Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Primer Express Software 3.0 (Applied Biosystems) was also used, and the primer sequences used in this study are shown in the Table. The reactions were performed in 96well plates with a final volume of 25 μL containing 12.5 μL of the SYBR Green PCR Master Mix, 1 μL of each gene-specific primer, and 2 μL of diluted complementary DNA. The reaction consisted of the initial denaturation at 95°C for 15 minutes, 40 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 1 minute, and synthesis at 72°C for 1 minute. The SYBR Green fluorescence was measured at each extension step. The threshold cycle (Ct) value reflected the cycle number that saw significant increases in the reporter fluorescence signal above baseline. The NOX4 expression values were normalized to the amount of gly ceraldehyde 3-phosphate dehydrogenase (GAPDH): ΔCt = Ct of NOX4 − Ct of GAPDH. The fold change in NOX4 mRNA was 2(Ct of NOX4 – Ct of GAPDH). Protein Extraction and Western Blots. The protein extract was isolated from cells by means of lysis buffer containing 150 mmol/L sodium chloride, 1.0% Igepal CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mmol/L Tris, pH 8.0 radioimmunoprecipitation assay buffer (Sigma Aldrich), and protease inhibitor cocktail (Sigma Aldrich). The total protein concentration was determined by the Bradford protein assay (BioRad, Hercules, California). Fifty micrograms of the total protein and sample buffer was denatured in a lithium dodecyl sulfate sample buffer (Invitrogen) at 95°C for 5 minutes, incubated on ice for 1 minute, and separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis at 100 V for 1 hour. The protein sample was then transferred onto
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Fig 1. Expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and tumor necrosis factor α (TNF-α) messenger RNA (mRNA) levels in human middle ear epithelial cells (HMEECs) following diesel exhaust particle (DEP) exposure. A) Expression of NOX1, NOX4, and NOX5 in control and DEP-exposed groups. B) Expression of NOX4 and TNF-α in control and DEP-exposed groups with and without N-acetyl-L-cysteine (NAC) or caffeic acid phenylethyl ester (CAPE). These results were obtained from at least 10 independent experiments. Data shown are mean ± SD of 4 repeated experiments with duplicate samples. * — p < 0.05 compared with control group; † — p < 0.05 compared with DEPs group.
polyvinylidene fluoride membrane with an electro blotting apparatus set at 80 V for 1 hour. Before use, the membrane was activated with methanol, blocked with 5% skim milk in TBS-T (Tris-buffered saline solution with Tween 20) for 1 hour, and then incubated with NOX4 (1:500 dilution; Santa Cruz Biotechnology, Dallas, Texas) and α-tubulin (1:1,000 dilution, Thermo Fisher Scientific, Waltham, Massachusetts) antibodies overnight at 4°C. After incubation with secondary antibody conjugated with horseradish peroxidase (1:2,000 dilution in 5% skim milk) for 1 hour, the signals on the membrane were detected with use of enhanced chemiluminescence luminol solution (KPL Inc, Gaithersburg, Maryland) and exposed to X-ray film (AGFA, Mortsel, Belgium). Measurement of ROS. The intracellular reactive oxygen species (ROS) produced in the DEP-exposed cells were measured with 2',7'-dichlorofluorescein diacetate (DCF(H)DA). Cells were seeded in 100-mm plates (1 × 106 cells per dish) and incubated in growth medium for 24 hours. In separate experiments, cells were pretreated with NAC (2.5 mmol/L) or CAPE (100 μmol/L) in each dish. After 30 minutes, the cells were stimulated with DEPs for 6 hours. The cells were then rinsed with phosphatebuffered saline solution, loaded with 50 μmol/L DCF(H)DA, and incubated for 30 minutes at 37°C. The DCF(H)DA fluorescence was measured with a Cytomics FC500 (Beckman Coulter, Brea, California). Data and Statistics. All data are expressed as mean ± SD. Statistical significance was assigned to p values of less than 0.05.
Results
DEP-Induced NOX4 mRNA Expression. The cells were stimulated with DEPs (500 μg/mL) for 6 hours. The expression of NOX4 was increased by DEP stimulation; however, NOX1 and NOX5 expressions were not affected by DEP stimulation (p < 0.05; Fig 1A). We also pretreated HMEECs with the known antioxidant NAC (2.5 mmol/L per milliliter; Sigma, A8199-10G) and CAPE (100 μg/mL) for 30 minutes before DEP stimulation (Fig 1B). The expressions of NOX4 and tumor necrosis factor α were down-regulated in cells pretreated with NAC or CAPE in comparison to those that received only DEP stimulation. These results were obtained from at least 10 independent experiments. DEP-Induced NOX4 Protein Expression. We performed Western blot analysis to confirm the effect of DEPs on NOX4 protein levels. The expression of NOX4 protein increased in cells stimulated by DEPs for 6 hours, but decreased in cells pretreated with NAC or CAPE (Fig 2). These results were obtained from at least 10 independent experiments. DEP-Induced Intracellular ROS Production. We measured ROS via fluorescence-activated cell sorting analysis. Reactive oxygen species were increased in cells stimulated with DEPs for 6 hours in comparison to the control cells (Fig 3A). However, ROS were decreased in cells that were pretreated by NAC or CAPE for 30 minutes (Fig 3B). The DEPstimulated cells had a median value of 21.7 ± 2.62, whereas those pretreated with NAC or CAPE before DEP stimulation had median values of 17.0 ± 2.41 and 13.8 ± 1.74, respectively (Fig 3C). These results were obtained from 20 independent experiments.
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Fig 2. Expression of NOX4 protein levels in HMEECs following DEP exposure with and without NAC and CAPE. These results were obtained from at least 10 independent experiments. * — p < 0.05 compared with control group; † — p < 0.05 compared with DEPs group.
Discussion
Otitis media is a common middle ear disease that might induce hearing impairment in children. It is one of the main reasons children in developed countries are prescribed antibiotics or undergo surgery.9 Exposure to traffic-related air pollution induces
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OM. Light-absorbing carbon and nitrogen dioxide are positively associated with traffic-related air pollution.10 Diesel, carbon monoxide, ozone, smoke, and sulfur dioxide also cause air pollution.11 The common pollutant diesel exhaust is composed of a core of elemental carbon and organic compounds including polycyclic aromatic hydrocarbons, nitro– polycyclic aromatic hydrocarbons, small amounts of sulfate, nitrate, metals, and other elements. Diesel exhaust is a major cause of inflammation and induces MMP-1 expression via extracellular signal– regulated kinase 1/2 activation in human lung epithelial cells.11 Our study focused on the relationship between DEPs and OM. Reactive oxygen species induce inflammatory responses in many diseases. Inflammation induced by ROS has been caused by the hydrogen peroxide– producing enzyme glucose oxidase in a skin model.12,13 Damage to DNA by ROS and nitrogen species can lead to inflammatory disease and even progression to cancer.14 Tumor necrosis factor α is a critical proinflammatory mediator leading to inflammation and is believed to be associated with tissue damage, fibrosis, and bone resorption in the middle ear in patients with OM.15 Tumor necrosis factor α increases ROS by inducing spermine oxidase in human lung epithelial cells.16 Reactive oxygen species are also known to induce OM.17 Several substances have been reported to induce ROS. For example, Helicobacter pylori has been associated with ROS activity and lipid peroxidation, which can lead to gastritis.18 The uncoupling of the
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Fig 3. Role of reactive oxygen species in DEP-induced inflammation in HMEECs. A) Fluorescence intensity of control and DEP-exposed groups. B) Fluorescence intensity of DEPexposed groups with and without NAC or CAPE. C) Median value of fluorescence intensity relative to control. These results were obtained from 20 independent experiments. * — p < 0.05 compared with control group; † — p < 0.05 compared with DEPs group.
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protein-2 gene also plays a role in ROS production.19 Zhang et al20 reported that NADPH oxidases rapidly increased the levels of ROS in lung epithelium. Electrons from NADPH oxidases are transferred through the enzyme to molecular oxygen and generate superoxide with the secondary production of ROS.21 Therefore, we focused on the connection between NOX and ROS generation.
Nicotinamide adenine dinucleotide phosphate oxi dase is a multi-subunit enzyme that catalyzes oxide ion production by the 1-electron reduction of oxygen by means of NADPH or (reduced) nicotinamide adenine dinucleotide (NADH). Evidence indicates that there is an entire family of NADPH oxidases, based on the discovery of gp91phox homologs.22 The family comprises 7 members, including NOX1, NOX2 (formerly termed gp91phox), NOX3, NOX4, NOX5, dual oxidase 1, and dual oxidase 2.23 NOX1 is found in colon and vascular cells and plays a role in host defense and cell growth; NOX2 is the catalytic subunit of the respiratory burst oxidase in phagocytes, but is also expressed in vascular, cardiac, renal, and neural cells; NOX3 is found in fetal tissue and the adult inner ear and is involved in vestibular function; NOX4, originally termed Renox (renal oxidase) because of its abundance in the kidney, is also found in vascular cells and osteoclasts; and NOX5 is a calcium ion–dependent homolog found in testis and lymphoid tissue, and also in vas-
cular cells.24 In this study, DEPs were cytotoxic and increased the levels of ROS and NOX4 expression in HMEECs. NOX4 is well known to generate high levels of ROS.
Propolis, a product derived from the hives of honeybees, possesses anti-inflammatory, antiviral, immunostimulatory, and carcinostatic activities.25 Caffeic acid phenylethyl ester is the active component of propolis. It has been demonstrated as a free radical scavenger and has antioxidant and anti-inflammatory effects.26 In this study, CAPE was used to reduce ROS expression and was found to decrease the levels of NOX4 mRNA and protein induced by DEPs. There were some limitations to this study. First, we used only immortalized HMEECs. Further studies, evaluating other cell lines and tissues, should be done. Second, studies focusing on the mechanism of DEP-induced inflammation are needed. It would also be interesting to study the effects of other antioxidants on OM. Conclusions
Inflammation induced by DEPs is reduced by CAPE via the inhibition of NOX4 expression. This finding suggests that CAPE could be used as a therapeutic agent against DEP-induced inflammation of HMEECs.
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