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The suppressive effects of metformin on inflammatory response of otitis media model in human middle ear epithelial cells
International Journal of Pediatric Otorhinolaryngology 89 (2016) 28e32
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: http://www.ijporlonline.com/
The suppressive effects of metformin on inflammatory response of otitis media model in human middle ear epithelial cells Jae Gu Cho, Jae Jun Song, June Choi, Gi Jung Im, Hak Hyun Jung, Sung Won Chae* Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 March 2016 Received in revised form 19 July 2016 Accepted 21 July 2016 Available online 25 July 2016
Objective: Metformin is a well-known anti-diabetic agent, but its mechanism is unclear. Recently, many reports have described the anti-inflammatory effects of metformin on various cell types, including human vascular smooth muscle cells and endothelial cells. This study was designed to investigate the antiinflammatory effect of metformin on lipopolysaccharide (LPS) induced inflammation in human middle ear epithelial cell lines (HMEECs). Methods: The effect of pretreatment by metformin (0, 1, 2, 4 mM) was evaluated by the inflammatory response in the HMEECs exposed to LPS (10 ng/ml). For verifying the suppression effect of metformin on the inflammatory cytokines, tumor necrosis factor-alpha (TNF-a) was evaluated by real-time polymerase chain reaction, and COX-2 protein was assessed by western blotting. Intracellular reactive oxygen species (ROS) was measured using 20 , 70 -dichlorofluorescein diacetate (DCFHDA) fluorocytometer. Results: Stimulation by LPS 10 ng/ml concentration showed 12.4 folds increase the expression of TNF-a mRNA compared to control on HMEECs. Pretreatment of metformin dose dependently suppressed the expression of TNF-a mRNA induced by LPS (2 mM, p ¼ 0.03). The amount of COX-2 protein production was significantly decreased by metformin pretreatment (4 mM, p ¼ 0.01). The production of ROS was decreased significantly by pretreatment of metformin (p ¼ 0.03). Conclusions: These findings suggest that the inflammatory response and oxidative stress induced by LPS could be suppressed by metformin in HMEECs. Therefore, metformin may have a therapeutic potential for the treatment of the otitis media. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Metformin Otitis media LPS Human middle ear epitheilial cells
1. Introduction Otitis media (OM) is the most common inflammatory disease in middle ear cavity. Most children experiences at least one episode of OM, even though the majority heals spontaneously. In some cases of them, the disease evolves into a more chronic phase with sustained inflammation and effusion material entrapped in the middle ear [1]. In recent report, children with OM have a poor quality of life and suffer from sleep, loss of appetite, otalgia, and behavioral problems [2]. A major cause of OME seems to be middle ear infections. The microbial products such as lipopolysaccharide(LPS) activate the inflammation in the first place, and pro-inflammatory mediators
* Corresponding author. Department of Otorhinolaryngology-Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, 80 Guro-dong, Gurogu, Seoul 152-703, South Korea. E-mail address: [email protected] (S.W. Chae). http://dx.doi.org/10.1016/j.ijporl.2016.07.025 0165-5876/© 2016 Elsevier Ireland Ltd. All rights reserved.
including bacterial components, cytokines, and chemokines have established the vicious cycle after the initiating factors are eliminated [3]. These causes of inflammation increase vascular permeability, chemotaxis, and production of reactive oxygen species (ROS), and are responsible for middle ear inflammation [4]. Therefore, modulation of these inflammatory factors may influence middle ear inflammation. The anti-inflammatory potential of metformin, a frontline drug for the treatment of type 2 diabetes mellitus, has been reported in many experimental models such as endothelial cells, human aortic smooth muscle cells, and activated macrophages [5e7]. In recent study, metformin inhibited the production of ROS from NADH in LPS-activated macrophage [8]. Aims of this study was to investigate the anti-inflammatory effect of metformin on LPS induced inflammation in HMEECs by measuring the expression of TNF-a mRNA, reactive oxygen species(ROS) suppression, and COX-2 protein production.
J.G. Cho et al. / International Journal of Pediatric Otorhinolaryngology 89 (2016) 28e32
2. Materials and methods 2.1. Cell culture HMEECs was maintained in a mixture of DMEM and BEBM (1:1) supplemented with bovine pituitary extract (52 mg/ml), hydrocortisone (0.5 mg/ml), hEGF (0.5 ng/ml), epinephrine 0.5 (mg/ml), transferrin (10 mg/ml), insulin (5 mg/ml), triiodothyronine (6.5 ng/ ml), retinoic acid (0.1 ng/ml), gentamycin (50 mg/ml), and amphotericin-B (50 ng/ml). All cells were grown in a humidified atmosphere at 37 C containing of 5% CO2 and 95% air. Growth media was changed every 3rd day. A bacterial endotoxin LPS (10 ng/ml, Sigma, St Louis, MO, USA) from Pseudomonas aeruginosa was used to induce inflammatory response and metformin (0, 1, 2, and 4 mM, Sigma) was used to inhibit inflammatory response. Horseradish peroxidase-labeled anti-rabbit antibody was used as secondary antibody (Amersham, Buckinghamshire, UK). 2.2. Real-time polymerase chain reaction (PCR) analysis
29
manufacturer's protocol (Bio-Rad, Hercules, CA, USA). Aliquots (25 mg protein) were mixed with sample buffer (BioRad) containing 2% mercaptoethanol, boiled for 5 min, and electrophoresed on 12% tris-HCl gels. After transfer to nitrocellulose membranes (Hybond ECL; Amersham Biosciences Corp.), membranes were blocked with PBS, 0.1% Tween-20 containing 5% (w/v) dry milk and 1% bovine serum albumin for 1 h at room temperature. Membranes were probed with COX-2 mouse anti-human antibody (clone 45 M1, 1:200; Neomarkers, Fermont, CA, USA), a-tubulin (Merck Biosciences, San Diego, CA, USA) or control goat IgG, followed by donkey antigoat IgG coupled to horseradish peroxidase (1:10,000 dilution; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), developed using an ECL detection kit (Pierce, Rockford, IL, USA) and exposed to X-ray film (XAR5, Kodak, Rochester, NY, USA). Densitometric analysis was performed using a Scion imager (Scion, Frederick, MD, USA). Relative protein expressions were calculated by determining the ratio of protein to atubulin. Results were obtained in 2 repeated experiments.
2.4. Measurement of ROS ®
Total RNA from HMEECs was extracted using RNeasy Mini Kits (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions (RNeasy handbook, June 2001). Total cellular RNA (15 mg) was reverse-transcribed for 60 min at 50 C in a 60 ml reaction mixture containing; 100U of Superscript-III reverse transcriptase (Invitrogen), 10 mM dithiothreitol, 1 mM of each of dATP, dTTP, dCTP, and dGTP, and 100 mg of Oligo-dT primer (Amersham Biosiences, Piscataway, NJ, USA) per milliliter. Reactions were stopped by heat inactivation for 10 min at 70 C. Real-time PCR was performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Reaction mixtures contained 12.5 ml of 2 SYBR Green Master Mix (containing; 200 mM of each of dATP, dGTP, and dCTP, 400 mM dUTP, 2 mM MgCl2, 0.125U uracil N-glycosylase, and 0.313U Ampliteq Gold DNA polymerase), 6.25 pmol of each sense and antisense primer, and 2 ml of cDNA in a final volume of 25 ml. Oligonucleotide primers were synthesized commercially at Bioneer Company (Daejon, Republic of Korea) as follows: sense primer; 50 ATC TTC TCG AAC CCC GAG TGA -30 and antisense primer; 50 - GGG TTT GCT ACA ACA TGG GC -30 for TNF- a; and sense primer; 50 -CGC CCT GTT CGC TCT GGG -30 and antisense primer; 50 - AGG AGG TCC GCA TGC TCA -30 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Reaction mixtures were incubated for 2 min at 55 C followed by 10 min at 95 C to activate the Ampliteq Gold DNA polymerase. The amplification profile used was; 40 cycles of 15sec of denaturation at 95 C followed by 1 min of annealing at 60 C for TNF- a and GAPDH. Data were analyzed using Sequence Detection System software (Applied Biosystems). Results were obtained in 3 repeated experiments with triplicate samples.
Cells plated in 100 mm dishes subjected to measurement of intracellular ROS using 20 ,70 -dichlorofluorescein diacetate (DCFHDA). HMEECs were seeded in 100 mm plates (1 106 cells/ dish) and incubated with growth media for 24hr. In separate experiments, cells were pretreated by metformin for 20 h. After 4 h LPS treated, cells were rinsed with PBS (Biosolution, BP007, Korea), loaded with 50 mM DCFHDA, and incubated for 30 min at 37 C. After which DCF fluorescence was measured by Cytomics FC500 (Beckman Coolter).
2.5. Statistics All data are expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to determine statistically significant dif’s F-test was used to correct for ferences between groups. Scheffe multiple comparisons when statistical significances were identified in the ANOVA. P < 0.05 for the null hypothesis was accepted as indicating a statistically significant difference.
2.3. Western blotting Cells were stimulated with LPS for 4 h and were pretreated for 20 h with four different concentration of 0, 1, 2, and 4 mM metformin. After the treatment of HMEECs with CSS, the medium was removed out and the cells were washed twice in PBS (10 mM, pH 7.4). The cells were incubated in 0.4 ml ice-cold lysis buffer (150 mmol/L NaCl, 20 mmol/L Tris [pH 7.5], 1 mmol/L EDTA, 0.1% Triton X-100) containing 0.5% protease inhibitor cocktail III (Calbiochem., San Diego, CA, USA). The cells were then centrifuged at 13,000 g for 25 min at 4 C, and the supernatant (total cell lysate) was collected, aliquoted and stored at 70 C. The protein concentration was determined by RC DC Protein Assay kit using the
Fig. 1. Inhibitory effect of metformin on LPS-induced TNF-a mRNA expression. Pretreatment with metformin dose-dependently suppressed TNF-a mRNA expression. In the 2 mM and 4 mM concentration of metformin pretreatment, there were statistically significant suppressive effect on expression of TNF-a mRNA. But there were no different effect between in the 2 and 4 mM concentration of metformin pretreatment. LPS: lipopolysaccharide (*: p < 0.05).
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J.G. Cho et al. / International Journal of Pediatric Otorhinolaryngology 89 (2016) 28e32
3. Results 3.1. The suppression of LPS-induced TNF-a mRNA by pretreatment of metformin To determine the effect of LPS to induce inflammation in HMEECs, the inflammatory response by LPS (4 h, 10 ng/ml) was evaluated on up-regulation of TNF-a mRNA, pro-inflammatory cytokine. TNF-a mRNA expression in HMEECs was significantly increased to 12.4 folds by the stimulation of LPS and metformin dosedependently suppressed LPS-induced TNF-a mRNA (Fig. 1). After pretreatment of 1 mM concentration of metformin, TNF-a mRNA was decreased to 60% compared to control, but it was not statistically significant decrease of expression of TNF-a mRNA. However in the 2 mM concentration of metformin pretreatment, it was
decreased to 40% compared to control, and it was statistically significant. In the 4 mM of metformin pretreatment, no more decrease was showed. In conclusion, the induction levels of TNF- a mRNA was significantly decreased in the two different treatments of concentration, 2 mM and 4 mM (P ¼ 0.03, 0.04) compared with control. 3.2. The effect of metformin on the LPS-induced COX-2 protein production The production of LPS-induced COX-2 protein was assessed after inhibition by metformin with 0, 1, 2 and 4 mM for 20 h. The expression of COX-2 protein was significantly decreased by pretreatment of 4 mM metformin (p ¼ 0.01) compared to control (Fig. 2). But the pretreatment of 1 and 2 mM metformin did not suppressed the expression of COX-2 protein. 3.3. The effect of metformin on production of LPS-induced ROS Production of ROS was measured by fluorescence-activated cell sorting (FACS) analysis. ROS was increased in stimulated cells by LPS for 4 h compared with that of untreated cells (Fig. 3A). Pretreatment of metformin suppressed ROS production induced by LPS (Fig. 3B). The production of ROS was significantly decreased by 36% (4 mM metformin, p ¼ 0.03) compared to stimulated cells by LPS. 4. Discussion
Fig. 2. Inhibitory effect of metforminon on LPS-induced COX-2 protein expression. Metformin pretreatment dose-dependently suppressed LPS-induced COX-2 protein expression. The difference of expression of COX-2 protein between in the pretreatment of metformin 2 and 4 mM was significant (p ¼ 0.043).
In the present study, the suppressive effect of metformin on LPS induced inflammation was demonstrated in the HMEECs for the first time. The elevation of inflammatory cytokines, such as TNF-a and COX-2, after exposure to LPS was suppressed by pretreatment of metformin, and the production of ROS induced by LPS also decreased. These results suggest that the inflammatory response of OM may be reduced by metformin. Several cytokines have been identified in middle ear effusions, e.g. IL-1b, IFN-g, IL-2, IL-6, IL-8, PAF, TNF-a, and FGF [9]. These cytokines are known to increase vascular permeability, chemotaxis,
Fig. 3. Production of reactive oxygen species (ROS) on LPS-induced in HMEECs. Assessed by measurement of 20 ,70 -20 ,70 -dichlorofluorescein diacetate (DCFDA) oxidation of live cells in FACS. (A) Fluorescence intensity of LPS treated cells were increased compared with that of control cells. Arrow is the control and blank arrow is LPS treated cells. (B) Fluorescence intensities of metformin pretreated cells were decreased dose-dependently. Arrow head is 2 mM of metformin, and black arrow is 4 mM of metformin pretreatment (p ¼ 0.03).
J.G. Cho et al. / International Journal of Pediatric Otorhinolaryngology 89 (2016) 28e32
and secretion activity, and thus, to cause OM and effusion. To demonstrate anti-inflammatory effect of metformin, TNF-a and COX-2 expression were detected by real-time PCR and western blot analysis. TNF-a is a critical pro-inflammatory mediator leading to inflammation, and is produced in response to stimuli triggered by bacterial LPS and viruses [10]. Moreover, TNF-a is believed to be associated with tissue damage, fibrosis, and bone resorption in the middle ear in OM [11]. COX-2 is the main contributor to prostaglandin production in acute and chronic inflammation [12]. As shown in the results, the expression of TNF-a mRNA and COX-2 protein induced by LPS were decreased significantly by pretreatment of metformin in HMEECs. Moreover, production of ROS induced by LPS was decreased by metformin. The oxidative stress has been implicated in the pathogenesis of otitis media [13]. ROS may cause tissue damage via lipid peroxidation, and can also enhance inflammatory response [14]. Therefore the modulation of ROS is one of the important factors that control the inflammations. Metformin is a widely prescribed oral anti-hyperglycemic agent as first line therapy for type 2 diabetes mellitus. Beside of antihypoglycemic effect, direct and indirect anti-inflammatory effects of metformin have been reported in in vitro and in vivo studies. Cao et al. have reported anti-inflammatory effect of metformin on vascular calcification in rat aortic smooth muscle cells [15]. In mouse lung tissue, metformin also suppressed ROS and allergic eosinophilic inflammation [16]. It has also been reported that metformin suppressed endotoxin induced uveitis and glial cell inflammation [17,18]. The activation of adenosine-monophosphate activated protein kinase(AMPK) constitutes the best known mechanism of metformin action [19]. AMPK is involved in the regulation of cellular metabolism and energy distribution. AMPK is an intracellular metabolism sensor that through the reduction of ATP-consuming process and the stimulation of ATP-generating pathways, maintains cellular energy homeostasis [20]. The activation of AMPK inhibit the production of proinflammatory cytokines IL-1b, IL-2, IL6 and TNF-a by negatively interfering with NF-kB, a signal transducer and activator of transcription (STAT), as well as AP-1 signaling pathways [15]. In this study, metformin suppressed the expression of TNF-a, a critical pro-inflammatory mediator, and COX-2, the main contributor to prostaglandin production, in LPS induced inflammation in HMEECs. ROS is produced from mitochondrial electron transport chain that synthesize ATP via ATP synthase. Metformin directly inhibits complex I(NADH:ubiquinone oxidorecuctase) of mitochondrial electron transport chain [21]. Complex I is the first of four complexes that comprise the electron transport chain, and generate ROS. In recent study, ROS from complex I was related to the induction of pro-inflammatory cytokines by LPS, and metformin could suppress the production of ROS and pro-inflammatory cytokines [8]. It was also reported that ROS was play role induced OM. Serum and mucosa malondialdehyde indicating free oxygen radicals production increased significantly in experimental otitis media [22]. In this experiments, it was verified that metformin could suppress the production of ROS. Some clinical studies or trial have evaluated the antiinflammatory effect of metformin. Metformin reduced C-reactive protein (CRP) compared with that in the placebo group at 12month in adults with impaired glucose tolerance [23]. But Pradhan et al. reported that no significant reduction in CRP or other inflammatory biomarkers was observed with treatment with metformin in patients with type 2 diabetes mellitus and elevated CRP level [24]. Although clinical studies have reported conflicting results as described above, direct and indirect anti-inflammatory effects of metformin have been reported in vitro and in vivo studies. This study has limitations that the mechanism of metformin is
31
not verified in HMEECs. The protective effect of metformin in middle ear inflammation also remains to be determined in a suitable in vivo model. Despite these limitations, this in vitro study demonstrated that metformin has an anti-inflammatory and antioxidative effect on HMEECs. The results of this study demonstrate that metformin inhibited LPS-induced TNF-a mRNA expression, COX-2 protein and ROS production in HMEECs. Our findings provide an insight into the molecular mechanisms underlying the anti-inflammatory activities of metformin in relation to OM and other inflammatory disease states. Further studies are needed to determine how metformin inhibits the LPS-mediated inflammation in HMEECs, and to identify the genes regulated by metformin. References [1] J. Olsen, C.B. Pedersen, Secretory otitis media. IV. Medical consensus conference in Denmark, Ugeskr. Laeger 149 (1987) 2801e2802. [2] D.J. Grindler, S.J. Blank, K.A. Schulz, D.L. Witsell, J.E. Lieu, Impact of otitis media severity on Children's quality of life, Otolaryngol. Head. Neck Surg. 151 (2014) 333e340. [3] T. Ovesen, T. Ledet, Bacteria and endotoxin in middle ear fluid and the course of secretory otitis media, Clin. Otolaryngol. Allied Sci. 17 (1992) 531e534. [4] A. Harrison, L.O. Bakaletz, R.S. Munson Jr., Haemophilus influenzae and oxidative stress, Front. Cell Infect. Microbiol. 2 (2012) 40. [5] K. Isoda, J.L. Young, A. Zirlik, L.A. MacFarlane, N. Tsuboi, N. Gerdes, et al., Metformin inhibits proinflammatory responses and nuclear factor-kappaB in human vascular wall cells, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 611e617. [6] L. Li, J.C. Mamputu, N. Wiernsperger, G. Renier, Signaling pathways involved in human vascular smooth muscle cell proliferation and matrix metalloproteinase-2 expression induced by leptin: inhibitory effect of metformin, Diabetes 54 (2005) 2227e2234. [7] J.C. Mamputu, N.F. Wiernsperger, G. Renier, Antiatherogenic properties of metformin: the experimental evidence, Diabetes Metab. 29 (2003), 6S71eS76. [8] B. Kelly, G.M. Tannahill, M.P. Murphy, L.A. O'Neill, Metformin inhibits the production of reactive oxygen species from NADH: ubiquinone oxidoreductase to Limit induction of Interleukin-1beta (IL-1beta) and boosts Interleukin10 (IL-10) in lipopolysaccharide (LPS)-activated macrophages, J. Biol. Chem. 290 (2015) 20348e20359. [9] R.F. Yellon, G. Leonard, P.T. Marucha, R. Craven, R.J. Carpenter, W.B. Lehmann, et al., Characterization of cytokines present in middle ear effusions, Laryngoscope 101 (1991) 165e169. [10] D. Ophir, T. Hahn, A. Schattner, D. Wallach, A. Aviel, Tumor necrosis factor in middle ear effusions, Arch. Otolaryngol. Head. Neck Surg. 114 (1988) 1256e1258. [11] D.H. Kim, Y.S. Park, E.J. Jeon, S.W. Yeo, K.H. Chang, S.K. Lee, Effects of tumor necrosis factor alpha antagonist, platelet activating factor antagonist, and nitric oxide synthase inhibitor on experimental otitis media with effusion, Ann. Otol. Rhinol. Laryngol. 115 (2006) 617e623. [12] J.J. Song, H.W. Lim, K. Kim, K.M. Kim, S. Cho, S.W. Chae, Effect of caffeic acid phenethyl ester (CAPE) on H(2)O(2) induced oxidative and inflammatory responses in human middle ear epithelial cells, Int. J. Pediatr. Otorhinolaryngol. 76 (2012) 675e679. [13] F. Doner, N. Delibas, H. Dogru, M. Yariktas, M. Demirci, The role of free oxygen radicals in experimental otitis media, J. Basic Clin. Physiol. Pharmacol. 13 (2002) 33e40. [14] J.M. McCord, R.S. Roy, The pathophysiology of superoxide: roles in inflammation and ischemia, Can. J. Physiol. Pharmacol. 60 (1982) 1346e1352. [15] X. Cao, H. Li, H. Tao, N. Wu, L. Yu, D. Zhang, et al., Metformin inhibits vascular calcification in female rat aortic smooth muscle cells via the AMPK-eNOS-NO pathway, Endocrinology 154 (2013) 3680e3689. [16] M.C. Calixto, L. Lintomen, D.M. Andre, L.O. Leiria, D. Ferreira, C. Lellis-Santos, et al., Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice, PLoS One 8 (2013) e76786. [17] N.M. Kalariya, M. Shoeb, N.H. Ansari, S.K. Srivastava, K.V. Ramana, Antidiabetic drug metformin suppresses endotoxin-induced uveitis in rats, Invest Ophthalmol. Vis. Sci. 53 (2012) 3431e3440. [18] K. Labuzek, S. Liber, B. Gabryel, B. Okopien, Metformin has adenosinemonophosphate activated protein kinase (AMPK)-independent effects on LPS-stimulated rat primary microglial cultures, Pharmacol. Rep. 62 (2010) 827e848. [19] G. Zhou, R. Myers, Y. Li, Y. Chen, X. Shen, J. Fenyk-Melody, et al., Role of AMPactivated protein kinase in mechanism of metformin action, J. Clin. Invest 108 (2001) 1167e1174. [20] M.C. Towler, D.G. Hardie, AMP-activated protein kinase in metabolic control and insulin signaling, Circ. Res. 100 (2007) 328e341. [21] M.R. Owen, E. Doran, A.P. Halestrap, Evidence that metformin exerts its antidiabetic effects through inhibition of complex 1 of the mitochondrial
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respiratory chain, Biochem. J. 348 Pt 3 (2000) 607e614. €ner, N. Delibas¸, H. Dogru, M. Yariktas¸, M. Demirci, The role of free oxygen [22] F. Do radicals in experimental otitis media, J. basic Clin. physiology Pharmacol. 13 (2002) 33. [23] S. Haffner, M. Temprosa, J. Crandall, S. Fowler, R. Goldberg, E. Horton, et al., Intensive lifestyle intervention or metformin on inflammation and
coagulation in participants with impaired glucose tolerance, Diabetes 54 (2005) 1566e1572. [24] A.D. Pradhan, B.M. Everett, N.R. Cook, N. Rifai, P.M. Ridker, Effects of initiating insulin and metformin on glycemic control and inflammatory biomarkers among patients with type 2 diabetes: the LANCET randomized trial, JAMA 302 (2009) 1186e1194.
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: http://www.ijporlonline.com/
The suppressive effects of metformin on inflammatory response of otitis media model in human middle ear epithelial cells Jae Gu Cho, Jae Jun Song, June Choi, Gi Jung Im, Hak Hyun Jung, Sung Won Chae* Department of Otolaryngology-Head and Neck Surgery, Korea University College of Medicine, Seoul, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 March 2016 Received in revised form 19 July 2016 Accepted 21 July 2016 Available online 25 July 2016
Objective: Metformin is a well-known anti-diabetic agent, but its mechanism is unclear. Recently, many reports have described the anti-inflammatory effects of metformin on various cell types, including human vascular smooth muscle cells and endothelial cells. This study was designed to investigate the antiinflammatory effect of metformin on lipopolysaccharide (LPS) induced inflammation in human middle ear epithelial cell lines (HMEECs). Methods: The effect of pretreatment by metformin (0, 1, 2, 4 mM) was evaluated by the inflammatory response in the HMEECs exposed to LPS (10 ng/ml). For verifying the suppression effect of metformin on the inflammatory cytokines, tumor necrosis factor-alpha (TNF-a) was evaluated by real-time polymerase chain reaction, and COX-2 protein was assessed by western blotting. Intracellular reactive oxygen species (ROS) was measured using 20 , 70 -dichlorofluorescein diacetate (DCFHDA) fluorocytometer. Results: Stimulation by LPS 10 ng/ml concentration showed 12.4 folds increase the expression of TNF-a mRNA compared to control on HMEECs. Pretreatment of metformin dose dependently suppressed the expression of TNF-a mRNA induced by LPS (2 mM, p ¼ 0.03). The amount of COX-2 protein production was significantly decreased by metformin pretreatment (4 mM, p ¼ 0.01). The production of ROS was decreased significantly by pretreatment of metformin (p ¼ 0.03). Conclusions: These findings suggest that the inflammatory response and oxidative stress induced by LPS could be suppressed by metformin in HMEECs. Therefore, metformin may have a therapeutic potential for the treatment of the otitis media. © 2016 Elsevier Ireland Ltd. All rights reserved.
Keywords: Metformin Otitis media LPS Human middle ear epitheilial cells
1. Introduction Otitis media (OM) is the most common inflammatory disease in middle ear cavity. Most children experiences at least one episode of OM, even though the majority heals spontaneously. In some cases of them, the disease evolves into a more chronic phase with sustained inflammation and effusion material entrapped in the middle ear [1]. In recent report, children with OM have a poor quality of life and suffer from sleep, loss of appetite, otalgia, and behavioral problems [2]. A major cause of OME seems to be middle ear infections. The microbial products such as lipopolysaccharide(LPS) activate the inflammation in the first place, and pro-inflammatory mediators
* Corresponding author. Department of Otorhinolaryngology-Head and Neck Surgery, Guro Hospital, Korea University College of Medicine, 80 Guro-dong, Gurogu, Seoul 152-703, South Korea. E-mail address: [email protected] (S.W. Chae). http://dx.doi.org/10.1016/j.ijporl.2016.07.025 0165-5876/© 2016 Elsevier Ireland Ltd. All rights reserved.
including bacterial components, cytokines, and chemokines have established the vicious cycle after the initiating factors are eliminated [3]. These causes of inflammation increase vascular permeability, chemotaxis, and production of reactive oxygen species (ROS), and are responsible for middle ear inflammation [4]. Therefore, modulation of these inflammatory factors may influence middle ear inflammation. The anti-inflammatory potential of metformin, a frontline drug for the treatment of type 2 diabetes mellitus, has been reported in many experimental models such as endothelial cells, human aortic smooth muscle cells, and activated macrophages [5e7]. In recent study, metformin inhibited the production of ROS from NADH in LPS-activated macrophage [8]. Aims of this study was to investigate the anti-inflammatory effect of metformin on LPS induced inflammation in HMEECs by measuring the expression of TNF-a mRNA, reactive oxygen species(ROS) suppression, and COX-2 protein production.
J.G. Cho et al. / International Journal of Pediatric Otorhinolaryngology 89 (2016) 28e32
2. Materials and methods 2.1. Cell culture HMEECs was maintained in a mixture of DMEM and BEBM (1:1) supplemented with bovine pituitary extract (52 mg/ml), hydrocortisone (0.5 mg/ml), hEGF (0.5 ng/ml), epinephrine 0.5 (mg/ml), transferrin (10 mg/ml), insulin (5 mg/ml), triiodothyronine (6.5 ng/ ml), retinoic acid (0.1 ng/ml), gentamycin (50 mg/ml), and amphotericin-B (50 ng/ml). All cells were grown in a humidified atmosphere at 37 C containing of 5% CO2 and 95% air. Growth media was changed every 3rd day. A bacterial endotoxin LPS (10 ng/ml, Sigma, St Louis, MO, USA) from Pseudomonas aeruginosa was used to induce inflammatory response and metformin (0, 1, 2, and 4 mM, Sigma) was used to inhibit inflammatory response. Horseradish peroxidase-labeled anti-rabbit antibody was used as secondary antibody (Amersham, Buckinghamshire, UK). 2.2. Real-time polymerase chain reaction (PCR) analysis
29
manufacturer's protocol (Bio-Rad, Hercules, CA, USA). Aliquots (25 mg protein) were mixed with sample buffer (BioRad) containing 2% mercaptoethanol, boiled for 5 min, and electrophoresed on 12% tris-HCl gels. After transfer to nitrocellulose membranes (Hybond ECL; Amersham Biosciences Corp.), membranes were blocked with PBS, 0.1% Tween-20 containing 5% (w/v) dry milk and 1% bovine serum albumin for 1 h at room temperature. Membranes were probed with COX-2 mouse anti-human antibody (clone 45 M1, 1:200; Neomarkers, Fermont, CA, USA), a-tubulin (Merck Biosciences, San Diego, CA, USA) or control goat IgG, followed by donkey antigoat IgG coupled to horseradish peroxidase (1:10,000 dilution; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA), developed using an ECL detection kit (Pierce, Rockford, IL, USA) and exposed to X-ray film (XAR5, Kodak, Rochester, NY, USA). Densitometric analysis was performed using a Scion imager (Scion, Frederick, MD, USA). Relative protein expressions were calculated by determining the ratio of protein to atubulin. Results were obtained in 2 repeated experiments.
2.4. Measurement of ROS ®
Total RNA from HMEECs was extracted using RNeasy Mini Kits (Qiagen GmbH, Hilden, Germany), according to the manufacturer's instructions (RNeasy handbook, June 2001). Total cellular RNA (15 mg) was reverse-transcribed for 60 min at 50 C in a 60 ml reaction mixture containing; 100U of Superscript-III reverse transcriptase (Invitrogen), 10 mM dithiothreitol, 1 mM of each of dATP, dTTP, dCTP, and dGTP, and 100 mg of Oligo-dT primer (Amersham Biosiences, Piscataway, NJ, USA) per milliliter. Reactions were stopped by heat inactivation for 10 min at 70 C. Real-time PCR was performed using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Reaction mixtures contained 12.5 ml of 2 SYBR Green Master Mix (containing; 200 mM of each of dATP, dGTP, and dCTP, 400 mM dUTP, 2 mM MgCl2, 0.125U uracil N-glycosylase, and 0.313U Ampliteq Gold DNA polymerase), 6.25 pmol of each sense and antisense primer, and 2 ml of cDNA in a final volume of 25 ml. Oligonucleotide primers were synthesized commercially at Bioneer Company (Daejon, Republic of Korea) as follows: sense primer; 50 ATC TTC TCG AAC CCC GAG TGA -30 and antisense primer; 50 - GGG TTT GCT ACA ACA TGG GC -30 for TNF- a; and sense primer; 50 -CGC CCT GTT CGC TCT GGG -30 and antisense primer; 50 - AGG AGG TCC GCA TGC TCA -30 for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Reaction mixtures were incubated for 2 min at 55 C followed by 10 min at 95 C to activate the Ampliteq Gold DNA polymerase. The amplification profile used was; 40 cycles of 15sec of denaturation at 95 C followed by 1 min of annealing at 60 C for TNF- a and GAPDH. Data were analyzed using Sequence Detection System software (Applied Biosystems). Results were obtained in 3 repeated experiments with triplicate samples.
Cells plated in 100 mm dishes subjected to measurement of intracellular ROS using 20 ,70 -dichlorofluorescein diacetate (DCFHDA). HMEECs were seeded in 100 mm plates (1 106 cells/ dish) and incubated with growth media for 24hr. In separate experiments, cells were pretreated by metformin for 20 h. After 4 h LPS treated, cells were rinsed with PBS (Biosolution, BP007, Korea), loaded with 50 mM DCFHDA, and incubated for 30 min at 37 C. After which DCF fluorescence was measured by Cytomics FC500 (Beckman Coolter).
2.5. Statistics All data are expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to determine statistically significant dif’s F-test was used to correct for ferences between groups. Scheffe multiple comparisons when statistical significances were identified in the ANOVA. P < 0.05 for the null hypothesis was accepted as indicating a statistically significant difference.
2.3. Western blotting Cells were stimulated with LPS for 4 h and were pretreated for 20 h with four different concentration of 0, 1, 2, and 4 mM metformin. After the treatment of HMEECs with CSS, the medium was removed out and the cells were washed twice in PBS (10 mM, pH 7.4). The cells were incubated in 0.4 ml ice-cold lysis buffer (150 mmol/L NaCl, 20 mmol/L Tris [pH 7.5], 1 mmol/L EDTA, 0.1% Triton X-100) containing 0.5% protease inhibitor cocktail III (Calbiochem., San Diego, CA, USA). The cells were then centrifuged at 13,000 g for 25 min at 4 C, and the supernatant (total cell lysate) was collected, aliquoted and stored at 70 C. The protein concentration was determined by RC DC Protein Assay kit using the
Fig. 1. Inhibitory effect of metformin on LPS-induced TNF-a mRNA expression. Pretreatment with metformin dose-dependently suppressed TNF-a mRNA expression. In the 2 mM and 4 mM concentration of metformin pretreatment, there were statistically significant suppressive effect on expression of TNF-a mRNA. But there were no different effect between in the 2 and 4 mM concentration of metformin pretreatment. LPS: lipopolysaccharide (*: p < 0.05).
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3. Results 3.1. The suppression of LPS-induced TNF-a mRNA by pretreatment of metformin To determine the effect of LPS to induce inflammation in HMEECs, the inflammatory response by LPS (4 h, 10 ng/ml) was evaluated on up-regulation of TNF-a mRNA, pro-inflammatory cytokine. TNF-a mRNA expression in HMEECs was significantly increased to 12.4 folds by the stimulation of LPS and metformin dosedependently suppressed LPS-induced TNF-a mRNA (Fig. 1). After pretreatment of 1 mM concentration of metformin, TNF-a mRNA was decreased to 60% compared to control, but it was not statistically significant decrease of expression of TNF-a mRNA. However in the 2 mM concentration of metformin pretreatment, it was
decreased to 40% compared to control, and it was statistically significant. In the 4 mM of metformin pretreatment, no more decrease was showed. In conclusion, the induction levels of TNF- a mRNA was significantly decreased in the two different treatments of concentration, 2 mM and 4 mM (P ¼ 0.03, 0.04) compared with control. 3.2. The effect of metformin on the LPS-induced COX-2 protein production The production of LPS-induced COX-2 protein was assessed after inhibition by metformin with 0, 1, 2 and 4 mM for 20 h. The expression of COX-2 protein was significantly decreased by pretreatment of 4 mM metformin (p ¼ 0.01) compared to control (Fig. 2). But the pretreatment of 1 and 2 mM metformin did not suppressed the expression of COX-2 protein. 3.3. The effect of metformin on production of LPS-induced ROS Production of ROS was measured by fluorescence-activated cell sorting (FACS) analysis. ROS was increased in stimulated cells by LPS for 4 h compared with that of untreated cells (Fig. 3A). Pretreatment of metformin suppressed ROS production induced by LPS (Fig. 3B). The production of ROS was significantly decreased by 36% (4 mM metformin, p ¼ 0.03) compared to stimulated cells by LPS. 4. Discussion
Fig. 2. Inhibitory effect of metforminon on LPS-induced COX-2 protein expression. Metformin pretreatment dose-dependently suppressed LPS-induced COX-2 protein expression. The difference of expression of COX-2 protein between in the pretreatment of metformin 2 and 4 mM was significant (p ¼ 0.043).
In the present study, the suppressive effect of metformin on LPS induced inflammation was demonstrated in the HMEECs for the first time. The elevation of inflammatory cytokines, such as TNF-a and COX-2, after exposure to LPS was suppressed by pretreatment of metformin, and the production of ROS induced by LPS also decreased. These results suggest that the inflammatory response of OM may be reduced by metformin. Several cytokines have been identified in middle ear effusions, e.g. IL-1b, IFN-g, IL-2, IL-6, IL-8, PAF, TNF-a, and FGF [9]. These cytokines are known to increase vascular permeability, chemotaxis,
Fig. 3. Production of reactive oxygen species (ROS) on LPS-induced in HMEECs. Assessed by measurement of 20 ,70 -20 ,70 -dichlorofluorescein diacetate (DCFDA) oxidation of live cells in FACS. (A) Fluorescence intensity of LPS treated cells were increased compared with that of control cells. Arrow is the control and blank arrow is LPS treated cells. (B) Fluorescence intensities of metformin pretreated cells were decreased dose-dependently. Arrow head is 2 mM of metformin, and black arrow is 4 mM of metformin pretreatment (p ¼ 0.03).
J.G. Cho et al. / International Journal of Pediatric Otorhinolaryngology 89 (2016) 28e32
and secretion activity, and thus, to cause OM and effusion. To demonstrate anti-inflammatory effect of metformin, TNF-a and COX-2 expression were detected by real-time PCR and western blot analysis. TNF-a is a critical pro-inflammatory mediator leading to inflammation, and is produced in response to stimuli triggered by bacterial LPS and viruses [10]. Moreover, TNF-a is believed to be associated with tissue damage, fibrosis, and bone resorption in the middle ear in OM [11]. COX-2 is the main contributor to prostaglandin production in acute and chronic inflammation [12]. As shown in the results, the expression of TNF-a mRNA and COX-2 protein induced by LPS were decreased significantly by pretreatment of metformin in HMEECs. Moreover, production of ROS induced by LPS was decreased by metformin. The oxidative stress has been implicated in the pathogenesis of otitis media [13]. ROS may cause tissue damage via lipid peroxidation, and can also enhance inflammatory response [14]. Therefore the modulation of ROS is one of the important factors that control the inflammations. Metformin is a widely prescribed oral anti-hyperglycemic agent as first line therapy for type 2 diabetes mellitus. Beside of antihypoglycemic effect, direct and indirect anti-inflammatory effects of metformin have been reported in in vitro and in vivo studies. Cao et al. have reported anti-inflammatory effect of metformin on vascular calcification in rat aortic smooth muscle cells [15]. In mouse lung tissue, metformin also suppressed ROS and allergic eosinophilic inflammation [16]. It has also been reported that metformin suppressed endotoxin induced uveitis and glial cell inflammation [17,18]. The activation of adenosine-monophosphate activated protein kinase(AMPK) constitutes the best known mechanism of metformin action [19]. AMPK is involved in the regulation of cellular metabolism and energy distribution. AMPK is an intracellular metabolism sensor that through the reduction of ATP-consuming process and the stimulation of ATP-generating pathways, maintains cellular energy homeostasis [20]. The activation of AMPK inhibit the production of proinflammatory cytokines IL-1b, IL-2, IL6 and TNF-a by negatively interfering with NF-kB, a signal transducer and activator of transcription (STAT), as well as AP-1 signaling pathways [15]. In this study, metformin suppressed the expression of TNF-a, a critical pro-inflammatory mediator, and COX-2, the main contributor to prostaglandin production, in LPS induced inflammation in HMEECs. ROS is produced from mitochondrial electron transport chain that synthesize ATP via ATP synthase. Metformin directly inhibits complex I(NADH:ubiquinone oxidorecuctase) of mitochondrial electron transport chain [21]. Complex I is the first of four complexes that comprise the electron transport chain, and generate ROS. In recent study, ROS from complex I was related to the induction of pro-inflammatory cytokines by LPS, and metformin could suppress the production of ROS and pro-inflammatory cytokines [8]. It was also reported that ROS was play role induced OM. Serum and mucosa malondialdehyde indicating free oxygen radicals production increased significantly in experimental otitis media [22]. In this experiments, it was verified that metformin could suppress the production of ROS. Some clinical studies or trial have evaluated the antiinflammatory effect of metformin. Metformin reduced C-reactive protein (CRP) compared with that in the placebo group at 12month in adults with impaired glucose tolerance [23]. But Pradhan et al. reported that no significant reduction in CRP or other inflammatory biomarkers was observed with treatment with metformin in patients with type 2 diabetes mellitus and elevated CRP level [24]. Although clinical studies have reported conflicting results as described above, direct and indirect anti-inflammatory effects of metformin have been reported in vitro and in vivo studies. This study has limitations that the mechanism of metformin is
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not verified in HMEECs. The protective effect of metformin in middle ear inflammation also remains to be determined in a suitable in vivo model. Despite these limitations, this in vitro study demonstrated that metformin has an anti-inflammatory and antioxidative effect on HMEECs. The results of this study demonstrate that metformin inhibited LPS-induced TNF-a mRNA expression, COX-2 protein and ROS production in HMEECs. Our findings provide an insight into the molecular mechanisms underlying the anti-inflammatory activities of metformin in relation to OM and other inflammatory disease states. Further studies are needed to determine how metformin inhibits the LPS-mediated inflammation in HMEECs, and to identify the genes regulated by metformin. References [1] J. Olsen, C.B. Pedersen, Secretory otitis media. IV. Medical consensus conference in Denmark, Ugeskr. Laeger 149 (1987) 2801e2802. [2] D.J. Grindler, S.J. Blank, K.A. Schulz, D.L. Witsell, J.E. Lieu, Impact of otitis media severity on Children's quality of life, Otolaryngol. Head. Neck Surg. 151 (2014) 333e340. [3] T. Ovesen, T. Ledet, Bacteria and endotoxin in middle ear fluid and the course of secretory otitis media, Clin. Otolaryngol. Allied Sci. 17 (1992) 531e534. [4] A. Harrison, L.O. Bakaletz, R.S. Munson Jr., Haemophilus influenzae and oxidative stress, Front. Cell Infect. Microbiol. 2 (2012) 40. [5] K. Isoda, J.L. Young, A. Zirlik, L.A. MacFarlane, N. Tsuboi, N. Gerdes, et al., Metformin inhibits proinflammatory responses and nuclear factor-kappaB in human vascular wall cells, Arterioscler. Thromb. Vasc. Biol. 26 (2006) 611e617. [6] L. Li, J.C. Mamputu, N. Wiernsperger, G. Renier, Signaling pathways involved in human vascular smooth muscle cell proliferation and matrix metalloproteinase-2 expression induced by leptin: inhibitory effect of metformin, Diabetes 54 (2005) 2227e2234. [7] J.C. Mamputu, N.F. Wiernsperger, G. Renier, Antiatherogenic properties of metformin: the experimental evidence, Diabetes Metab. 29 (2003), 6S71eS76. [8] B. Kelly, G.M. Tannahill, M.P. Murphy, L.A. O'Neill, Metformin inhibits the production of reactive oxygen species from NADH: ubiquinone oxidoreductase to Limit induction of Interleukin-1beta (IL-1beta) and boosts Interleukin10 (IL-10) in lipopolysaccharide (LPS)-activated macrophages, J. Biol. Chem. 290 (2015) 20348e20359. [9] R.F. Yellon, G. Leonard, P.T. Marucha, R. Craven, R.J. Carpenter, W.B. Lehmann, et al., Characterization of cytokines present in middle ear effusions, Laryngoscope 101 (1991) 165e169. [10] D. Ophir, T. Hahn, A. Schattner, D. Wallach, A. Aviel, Tumor necrosis factor in middle ear effusions, Arch. Otolaryngol. Head. Neck Surg. 114 (1988) 1256e1258. [11] D.H. Kim, Y.S. Park, E.J. Jeon, S.W. Yeo, K.H. Chang, S.K. Lee, Effects of tumor necrosis factor alpha antagonist, platelet activating factor antagonist, and nitric oxide synthase inhibitor on experimental otitis media with effusion, Ann. Otol. Rhinol. Laryngol. 115 (2006) 617e623. [12] J.J. Song, H.W. Lim, K. Kim, K.M. Kim, S. Cho, S.W. Chae, Effect of caffeic acid phenethyl ester (CAPE) on H(2)O(2) induced oxidative and inflammatory responses in human middle ear epithelial cells, Int. J. Pediatr. Otorhinolaryngol. 76 (2012) 675e679. [13] F. Doner, N. Delibas, H. Dogru, M. Yariktas, M. Demirci, The role of free oxygen radicals in experimental otitis media, J. Basic Clin. Physiol. Pharmacol. 13 (2002) 33e40. [14] J.M. McCord, R.S. Roy, The pathophysiology of superoxide: roles in inflammation and ischemia, Can. J. Physiol. Pharmacol. 60 (1982) 1346e1352. [15] X. Cao, H. Li, H. Tao, N. Wu, L. Yu, D. Zhang, et al., Metformin inhibits vascular calcification in female rat aortic smooth muscle cells via the AMPK-eNOS-NO pathway, Endocrinology 154 (2013) 3680e3689. [16] M.C. Calixto, L. Lintomen, D.M. Andre, L.O. Leiria, D. Ferreira, C. Lellis-Santos, et al., Metformin attenuates the exacerbation of the allergic eosinophilic inflammation in high fat-diet-induced obesity in mice, PLoS One 8 (2013) e76786. [17] N.M. Kalariya, M. Shoeb, N.H. Ansari, S.K. Srivastava, K.V. Ramana, Antidiabetic drug metformin suppresses endotoxin-induced uveitis in rats, Invest Ophthalmol. Vis. Sci. 53 (2012) 3431e3440. [18] K. Labuzek, S. Liber, B. Gabryel, B. Okopien, Metformin has adenosinemonophosphate activated protein kinase (AMPK)-independent effects on LPS-stimulated rat primary microglial cultures, Pharmacol. Rep. 62 (2010) 827e848. [19] G. Zhou, R. Myers, Y. Li, Y. Chen, X. Shen, J. Fenyk-Melody, et al., Role of AMPactivated protein kinase in mechanism of metformin action, J. Clin. Invest 108 (2001) 1167e1174. [20] M.C. Towler, D.G. Hardie, AMP-activated protein kinase in metabolic control and insulin signaling, Circ. Res. 100 (2007) 328e341. [21] M.R. Owen, E. Doran, A.P. Halestrap, Evidence that metformin exerts its antidiabetic effects through inhibition of complex 1 of the mitochondrial
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