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Keywords:

  • airway mucin;
  • MUC5AC;
  • wogonin;
  • NF-κB

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Conflict of Interest
  8. REFERENCES

In this study, we investigated whether wogonin significantly affects MUC5AC mucin gene expression and production in human airway epithelial cells. Confluent NCI-H292 cells were pretreated with wogonin for 30 min and then stimulated with tumor necrosis factor-α (TNF-α) for 24 h or the indicated periods. The MUC5AC mucin gene expression and mucin protein production were measured by RT-PCR and ELISA, respectively. We found that incubation of NCI-H292 cells with wogonin significantly inhibited mucin production and down-regulated MUC5AC gene expression induced by TNF-α in a dose-dependent fashion. To elucidate the action mechanism of wogonin, effect of wogonin on TNF-α-induced NF-κB signaling pathway was investigated by western blot analysis. Wogonin inhibited NF-κB activation induced by TNF-α. Inhibition of IKK by wogonin led to the suppression of IκB phosphorylation and degradation, p65 nuclear translocation and NF-κB-regulated gene expression. This, in turn, led to the down-regulation of MUC5AC protein production in NCI-H292 cells. Wogonin also inhibited the gene products involved in cell survival (Bcl-2) and proliferation (cyclooxygenase-2). These results suggest that wogonin inhibits the NF-κB signaling pathway, which may explain its role in the inhibition of MUC5AC mucin gene expression and production. Copyright © 2013 John Wiley & Sons, Ltd.


ABBREVIATIONS
ELISA

enzyme-linked immunosorbent assay

NF-κB

nuclear factor kappa B

PBS

phosphate-buffered saline

PVDF

polyvinylidene difluoride

RT-PCR

reverse transcription - polymerase chain reaction

SDS-PAGE

sodium dodecyl sulfate polyacrylamide gel electrophoresis

TNF-α

tumor necrosis factor-α

IκBα

inhibitory kappa B alpha

IKK

inhibitory kappa B kinase

INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Conflict of Interest
  8. REFERENCES

Airway mucus is a component of pulmonary innate immune function and plays a very important role in defense against invading pathogenic microbes, chemicals and particles. The protective function of airway mucus is attributed to the viscoelasticity of mucins. However, any abnormality in the quality or quantity of mucins not only causes altered airway physiology but may also impair host defenses often leading to serious airway pathology as exemplified in chronic bronchitis, cystic fibrosis, asthma and bronchiectasis (Adler and Li, 2001; Basbaum et al., 1999). Therefore, we suggest it is valuable to find the possible activity of controlling (inhibiting) the excessive mucin production (secretion) by the compounds derived from various medicinal plants. We have tried to investigate the possible activities of some natural products on mucin production (secretion) from airway epithelial cells. As a result of our trial, we previously reported that several natural compounds affected mucin production and/or secretion from airway epithelial cells (Heo et al., 2009; Lee et al., 2010; Lee et al., 2011a; Lee et al., 2011b). According to folk medicine and a number of reports, Scutellariae Radix has been used to control airway allergic or inflammatory diseases, and one of its components, wogonin, was reported to have diverse biological activities including antioxidative, anti-inflammatory and anticancer effects (Paoletti et al., 2009; Zhong et al., 2010; Clere et al., 2011; Zhou et al., 2011; McVean et al., 2002; Yin et al., 2001; Illek and Fischer, 1998). In our previous study, we demonstrated that wogonin inhibited epidermal growth factor (EGF)- or phorbol 12-myristate 13-acetate (PMA)-induced MUC5AC protein and gene expression (Kim et al., 2012). However, to the best of our knowledge, there are no reports about the effect of wogonin on mucin gene expression and production stimulated by a proinflammatory cytokine, tumor necrosis factor-α (TNF-α), from airway epithelial cells. Of the 21 mucin genes which identified until now, MUC5AC has been known as a major type of airway gel-forming mucin because it is highly expressed in the goblet cells (Song et al., 2003) and is regulated by proinflammatory cytokines (Takeyama et al., 2000). TNF-α is a well-known stimulant for secretion and gene expression of airway mucin (Shao et al., 2003; Song et al., 2003; Fischer et al., 1999). TNF-α level in sputum was reported to be increased, with further increases during exacerbation of diseases (Takeyama et al., 1999; Cohn et al., 2002). TNF-α converting enzyme mediated MUC5AC mucin expression in cultured human airway epithelial cells (Shao et al., 2003) and TNF-α induced MUC5AC gene expression in normal human airway epithelial cells (Song et al., 2003). It also induced mucin secretion from guinea pig tracheal epithelial cells (Fischer et al., 1999). Through binding its receptor, TNF-α activates several intracellular signal transduction cascades among which the NF-κB pathway is of pivotal importance. NF-κB is a heterodimer composed of p65, p50 and IκBα subunits present in the cytoplasm as an inactive state. In response to various stimuli, the IκBα subunit is phosphorylated and degraded, thereby facilitating the translocation of p50-p65 heterodimer to the nucleus. p50-p65 acts as a transcription factor regulating the expression of numerous genes including MUC5AC (Li and Verma, 2002). Therefore, in this study, we checked whether wogonin affects gene expression and production of airway mucin induced by TNF-α through affecting the activation of NF-κB pathway in NCI-H292 cells, a human pulmonary mucoepidermoid cell line, which are frequently used for the purpose of elucidating intracellular signaling pathways involved in airway mucin production and gene expression (Li et al., 1997; Takeyama et al., 1999; Shao et al., 2003)

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Conflict of Interest
  8. REFERENCES
Materials

All the chemicals and reagents used in this experiment including wogonin (purity: 95.0%) were purchased from Sigma (St. Louis, MO, U.S.A.) unless otherwise specified. Anti-NF-κB p65, anti-IkBα, anti-actin and anti-p84 antibodies were purchased from Santacruz Biotechnology (Santa Cruz, CA, U.S.A.). Phospho-specific anti-p65 (serine 536) and phospho-specific anti-IκBα (serine 32/36), anti-phospho-IKKα/β (Ser176/180) antibodies were purchased from Cell signaling Technology Inc. (Danvers, MA, U.S.A.).

Cell culture

NCI-H292 cells, a human pulmonary mucoepidermoid carcinoma cell line, were purchased from the American Type Culture Collection (Manassas, VA, U.S.A.) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) in the presence of penicillin (100 units/ml), streptomycin (100 µg/ml) and HEPES (25 mM) at 37 °C in a humidified 5% CO2, 95% air water-jacketed incubator. For serum deprivation, at 60% confluence, cultures were washed twice with phosphate-buffered saline (PBS) and recultured in RPMI 1640 with 0.2% FBS for 24 h.

Treatment of cells with wogonin

After 24 h of serum deprivation, cells were pretreated with varying concentrations of wogonin for 30 min and treated with TNF-α (10 ng/ml) for 24 h in serum-free RPMI 1640. Wogonin was dissolved in dimethylsulfoxide and treated in culture medium (final concentrations of dimethylsulfoxide were 0.5%). 0.5% dimethylsulfoxide did not affect mucin gene expression and production from NCI-H292 cells. After 24 h, cells were lysed with buffer solution containing 20 mM Tris, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA and protease inhibitor cocktail (Roche Diagnostics, IN, U.S.A.) and collected to measure the production of MUC5AC protein (in 24-well culture plate). The total RNA was extracted for measuring the expression of MUC5AC gene (in six-well culture plate) by using RT-PCR. For western blot analysis, cells were treated with wogonin for 24 h and then treated with TNF-α for the indicated periods.

MUC5AC mucin analysis using ELISA

MUC5AC protein was measured by using ELISA. Cell lysates were prepared with PBS at 1:10 dilution, and 100 µl of each sample was incubated at 42 °C in a 96-well plate, until dry. Plates were washed three times with PBS and blocked with 2% BSA for 1 h at room temperature. Plates were again washed three times with PBS and then incubated with 100 µl of 45 M1, a mouse monoclonal MUC5AC antibody (1:200) (NeoMarkers, CA, U.S.A.) which was diluted with PBS containing 0.05% Tween 20 and dispensed into each well. After 1 h, the wells were washed three times with PBS, and 100 µl of horseradish peroxidase-goat anti-mouse IgG conjugate (1:3000) was dispensed into each well. After 1 h, plates were washed three times with PBS. Color reaction was developed with 3,3’,5,5’-tetramethylbenzidine peroxide solution and stopped with 1 N H2SO4. Absorbance was read at 450 nm.

Total RNA isolation and RT-PCR

Total RNA was isolated by using Easy-BLUE Extraction Kit (INTRON Biotechnology, Inc. Kyung-gi-do, Korea) and reverse transcribed by using AccuPower RT Premix (BIONEER Corporation, Daejeon, Korea) according to the manufacturer's instructions. 2 µg of total RNA was primed with 1 µg of oligo (dT) in a final volume of 30 µl (RT reaction). 2 µl of RT reaction product was PCR amplified in a 20 µl by using Thermoprime Plus DNA Polymerase (ABgene, Rochester, NY, U.S.A.). Primers for MUC5AC were (forward) 5’-TGA TCA TCC AGC AGG GCT-3’ and (reverse) 5’-CCG AGC TCA GAG GAC ATA TGG G-3’. As quantitative controls, primers for Rig/S15 rRNA, which encodes a small ribosomal subunit protein, a housekeeping gene that was constitutively expressed, were used. Primers for Rig/S15 were (forward) 5’-TTC CGC AAG TTC ACC TAC C-3’ and (reverse) 5’-CGG GCC GGC CAT GCT TTA CG-3’. The PCR mixture was denatured at 94 °C for 5 min followed by 35 cycles at 94 °C for 30s, 60 °C for 30s and 72 °C for 30s and for final extension,1 cycle at 72 °C for 10 min. After PCR, 15 µl of PCR products were subjected to 1% agarose gel electrophoresis and visualized with ethidium bromide under a transilluminator.

Preparation of nuclear and cytosolic extracts

NCI-H292 cells (confluent in 150 mm culture dish) were pretreated for 24 h at 37 °C with 20 μM of wogonin and then stimulated with TNF-α (50 ng/ml) for 30 min. After the treatment, the cells were harvested using 3 x trypsin-EDTA solution and then centrifuged in a microcentrifuge (1200 rpm, 3 min, 4 °C). The supernatant was discarded, and the cell pellet was washed by suspending in PBS. The cytoplamic and nuclear protein fraction were extracted using NE-PER® nuclear and cytoplasmic extraction reagent (Thermo-Pierce Scientific, Waltham, MA, U.S.A.) according to the manufacturer's instructions. Both extracts were stored at −20 °C. Protein content in extract was determined by Bradford method.

Preparation of whole cell extract

After the treatment of the cells with wogonin, media were aspirated, and the cells were washed with cold PBS. The cells were collected by scraping and centrifuged at 3000 rpm for 5 min. The supernatant was discarded. The cells were mixed with RIPA buffer (25 mM Tris-Hcl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) for 30 min with continuous agitation. The lysate was centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatant was used or immediately stored at −80 °C. Protein content in extract was determined by Bradford method.

Detection of proteins by western blot analysis

Cytosolic, nuclear and whole cell extracts containing proteins (each 20–60 µg as protein) were subjected to 7–15% SDS-PAGE and then transferred onto the PVDF membrane. The blots were blocked using 5% skim milk and probed with appropriate primary antibody in blocking buffer overnight at 4 °C. The membrane was washed with PBS and then probed with the secondary antibody conjugated with horseradish peroxidase (Calbiochem, CA, U.S.A.). Immunoreactive bands were detected by an enhanced chemiluminescence kit (Pierce ECL western blotting substrate, Thermo Scientific, Waltham, MA, U.S.A.).

Statistical analysis

Means of individual group were converted to percent control and expressed as mean ± S.E.M. The difference between groups was assessed using one-way ANOVA and Duncan's Multiple Range test as a post-hoc test. p < 0.05 was considered as significantly different.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Conflict of Interest
  8. REFERENCES

Effect of wogonin on TNF-α- induced MUC5AC mucin production

Wogonin inhibited TNF-α-induced MUC5AC mucin production. The amounts of MUC5AC mucin in the cells of wogonin-treated cultures were 100 ± 10%, 350 ± 37%, 216 ± 14%, 145 ± 22% and 89 ± 34% for control, TNF-α 0.2 nM only, TNF-α plus wogonin 5 × 10−6 M, TNF-α plus wogonin 10−5 M and TNF-α plus wogonin 2 ×10−5 M, respectively (Fig. 1). Cell viability was checked by sulforhodamine B (SRB) assay, and there was no cytotoxic effect of wogonin, at 5, 10 and 20 μM (data were not shown).

image

Figure 1. Effect of wogonin on TNF-α-induced MUC5AC mucin production. NCI-H292 cells were pretreated with various concentrations of wogonin (5, 10 and 20 μM) for 30 min and then stimulated with TNF-α (10 ng/ml) for 24 h. Cell lysates were collected for measurement of MUC5AC mucin production by ELISA. Three independent experiments were performed, and the representative data were shown. Each bar represents a mean ± S.E.M. of three to four culture wells in comparison with that of control set at 100%. * significantly different from control (p < 0.05). + significantly different from TNF-α alone (p < 0.05). (cont: control, concentration unit is μM.)

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Effect of wogonin on TNF-α-induced MUC5AC mucin gene expression

MUC5AC gene expression induced by TNF-α was also inhibited by pretreatment with 10 μM and 20 μM of wogonin (Fig. 2).

image

Figure 2. Effect of wogonin on TNF-α-induced MUC5AC mucin gene expression. NCI-H292 cells were pretreated with wogonin at indicated concentrations for 30 min and then stimulated with TNF-α (10 ng/ml) for 24 h. MUC5AC mucin gene expression was measured by RT-PCR. As quantitative control, Rig/S15 rRNA, which encodes a small ribosomal subunit protein, a housekeeping gene that was constitutively expressed, was used. Three independent experiments were performed, and the representative data were shown. (cont: control, Wog: wogonin, concentration unit is μM.)

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Effects of wogonin on TNF-α-induced nuclear translocation and phosphorylation of NF-κB p65

Nuclear translocation of NF-kB p65 by TNF-α was inhibited by pretreatment with 20 μM of wogonin (Fig. 3 (A)). In the nuclear fraction of the TNF-α only-treated cells, there was an increase in nuclear translocation of p65 gradually and optimal level was reached at 30 min. However, in the cells treated with wogonin plus TNF-α, the level of p65 was gradually decreased as compared to the TNF-α only-treated cells (Fig. 3(A)). TNF-α-induced phosphorylation of p65 was gradually increased and reached optimal level at 30 min. However, wogonin completely blocked phosphorylation of p65 (Fig. 3(B)).

image

Figure 3. Effects of wogonin on TNF-α-induced nuclear translocation and phosphorylation of NF-κB p65. (A) NCI-H292 cells were either untreated or pretreated with 20 μM wogonin for 24 h at 37 °C and then stimulated with TNF-α (50 ng/ml) for the indicated times. Nuclear protein extracts were prepared and resolved on 10% SDS-PAGE, transferred onto a PVDF membrane, probed with antibody against p65. The results shown are the representative of three independent experiments. To ensure equal protein loading, the membrane was reprobed with anti-p84 antibody. (B) NCI-H292 cells were incubated with 20 μM wogonin for 24 h at 37 °C and then stimulated with TNF-α (50 ng/ml) for the indicated times. Nuclear protein extracts were prepared and subjected to western blot analysis using phospho-specific p65 (Ser 536) antibody. The results shown are the representative of three independent experiments. As a loading control, p84 levels were analyzed.

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Effects of wogonin on TNF-α-induced IκBα phosphorylation, degradation and phosphorylation of IKKα/β

TNF-α increased phosphorylation of IκBα to its maximum level within 5 min, which was then decreased. However, preincubation of NCI-H292 cells with wogonin prior to TNF-α exposure dramatically abrogated the phosphorylation of IκBα (Fig. 4 (A)). At 5 min after treatment, TNF-α showed the maximal induction of IκBα degradation. However, preincubation of NCI-H292 cells with wogonin prior to TNF-α exposure suppressed IκBα degradation (Fig. 4 (B)). Activation of IKK depends on phosphorylation and TNF-α activated IKKα/β. However, preincubation of NCI-H292 cells with wogonin prior to TNF-α exposure suppressed IKKα/β activation (Fig. 4 (C)).

image

Figure 4. Effects of wogonin on TNF-α-induced IκBα phosphorylation, IκBα degradation and phosphorylation of IKKα/β. (A) NCI-H292 cells were incubated with 20 μM wogonin for 24 h and treated with 50 ng/ml TNF-α for the indicated times. Cytoplasmic extracts were fractionated and then subjected to western blot analysis using phospho-specific IκBα (Ser 32/36) antibody. The results shown are the representative of three independent experiments. Equal protein loading was evaluated by β-actin levels. (B) NCI-H292 cells were incubated with 20 μM wogonin for 24 h and treated with 50 ng/ml TNF-α for the indicated times. Cytoplasmic extracts were prepared and analyzed by western blot using antibody against anti-IκBα. The results shown are the representative of three independent experiments. Equal protein loading was evaluated by β-actin levels. (C) NCI-H292 cells were incubated with 20 μM wogonin for 24 h and treated with 25 ng/ml TNF-α for the indicated times. Whole cell lysates (100 µg) were prepared and then subjected to western blot analysis using phospho-specific IKKα/β (Ser 176/180) antibody. The results shown are the representative of three independent experiments. Equal protein loading was evaluated by β-actin.

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Effects of wogonin on the expression of TNF-α-induced NF-κB-dependent gene products involved in the antiapoptosis and proliferation of NCI-H292 cells

TNF-α induced the expression of Bcl-2 protein. However, wogonin suppressed TNF-α-induced expression of Bcl-2 protein in a time-dependent fashion (Fig. 5 (A)). TNF-α induced the expression of COX-2 protein. However, wogonin suppressed TNF-α-induced expression of COX-2 protein (Fig. 5 (B)).

image

Figure 5. Effects of wogonin on the expression of TNF-α-induced NF-κB-dependent gene product involved in the antiapoptosis and proliferation of NCI-H292 cells. (A) NCI-H292 cells were incubated with 20 µ M wogonin for 6 h and then treated with 20 ng/ml TNF-α for the indicated times. Whole cell extracts were prepared and analyzed by Western blot using the indicated antibodies. The results shown are the representative of three independent experiments. (B) NCI-H292 cells were incubated with 20 µ M wogonin for 6 h and then treated with 20 ng/ml TNF-α for the indicated times. Whole cell extracts were prepared and analyzed by Western blot using the indicated antibodies. The results shown are the representative of three independent experiments.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Conflict of Interest
  8. REFERENCES

Wogonin was reported to have various biological effects especially in conjunction with anti-inflammatory, anticancer and antioxidative effects (Paoletti et al., 2009; Zhong et al., 2010; Clere et al., 2011; Zhou et al., 2011; McVean et al., 2002; Yin et al., 2001; Illek and Fischer, 1998) and wogonin inhibited EGF- or PMA-induced MUC5AC protein and gene expression in our previous study (Kim et al., 2012). However, as aforementioned in introduction, there is no report about the potential effect of wogonin on mucin gene expression and production stimulated by a proinflammatory cytokine, TNF-α, from cultured airway epithelial cells. As can be seen in results, wogonin suppressed the expression of MUC5AC mucin gene and the production of MUC5AC mucin protein, induced by TNF-α (Fig. 1, 2). There was no cytotoxic effect of wogonin at the concentrations of 5, 10 and 20 μM, based on the data from SRB assay (data were not shown). This result suggests that wogonin can regulate mucin gene expression and production of mucin protein induced by TNF-α, by directly acting on airway epithelial cells. Next, wogonin was reported to inhibit TNF-α- induced NF-kB activation in microglial cells and aortic smooth muscle cells (Piao et al., 2008; Lee et al., 2006). Therefore, to determine the effect of wogonin on NF-kB activation followed by TNF-α treatment in mucin-producing NCI-H292 cells, we examined whether wogonin inhibits TNF-α-induced nuclear translocation of NF-κB p65 by western blot. As shown in Fig. 3 (A), nuclear translocation of NF-kB p65 by TNF-α was inhibited by pretreatment with 20 μM of wogonin. In the nuclear fraction of the TNF-α only-treated cells, there was an increase in nuclear translocation of p65 gradually and reached optimal level at 30 min. However, in the cells treated with wogonin plus TNF-α, the level of p65 was gradually decreased as compared to the TNF-α only-treated cell. Transcriptional activity of p65 largely depends on its phosphorylation. For this reason, we investigated the effect of wogonin on TNF-α-induced phosphorylation of p65. As shown in Fig. 3 (B), TNF-α-induced phosphorylation of p65 was gradually increased and reached optimal level at 30 min. However, wogonin completely blocked the phosphorylation of p65. NF-κB activation involves the phosphorylation of inhibitory kappa B alpha (IκBα) by inhibitory kappa B kinases (IKKs), resulting in IκBα degradation. As a result, NF-κB subunits are released and translocated to the nucleus. Therefore, we investigated the effect of wogonin on the phosphorylation of IκBα. As shown in Fig. 4 (A), TNF-α increased phosphorylation of IκBα to its maximal level within 5 min, which was then decreased. However, preincubation of NCI-H292 cells with wogonin prior to TNF-α exposure dramatically abrogated the phosphorylation of IκBα. Next, IκBα degradation is required for the activation of NF-κB. Consequently, we determined whether wogonin inhibits TNF-α-induced NF-κB activation by inhibition of IκBα degradation. As shown in Fig. 4 (B), at 5 min after treatment, TNF-α showed the maximal induction of IκBα degradation. However, preincubation of NCI-H292 cells with wogonin prior to TNF-α exposure suppressed the degradation of IκBα. Most agents that activate NF-κB through a common pathway based on phosphorylation, proteaseome mediated degradation of IκB. The key regulatory step in this pathway involves the activation of IKK complex. Activation of IKK depends on phosphorylation. Therefore, we investigated whether wogonin inhibits the TNF-α-induced activity of IKKα/β. As can be seen in results, TNF-α activated the IKKα/β, although wogonin suppressed its activation (Fig. 4 (C)). We also found that the suppression of NF-κB activation correlated with the suppression of expression of the gene product, Bcl-2, known to inhibit apoptosis. Suppression of this gene product was accompanied by enhancement of the apoptotic effects of TNF-α. Wogonin has antiproliferative effect because this flavonoid down-regulates the expression of COX-2 (Fig. 5 (A), (B)). Overall, these results indicate that mucin production might be inhibited by wogonin through NF-κB, because it suppressed the NF-κB-regulated gene products. Taken together, our findings suggest that wogonin can inhibit MUC5AC mucin production and down-regulates MUC5AC gene expression by inhibiting TNF-α-induced NF-κB activity in NCI-H292 cells. At the same time, the result from this study suggests a possibility of using wogonin as a new efficacious mucoregulator for pulmonary diseases especially in conjunction with inflammation, although further studies are essential.

Conflict of Interest

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Conflict of Interest
  8. REFERENCES

The authors have declared that there are no conflicts of interest.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Conflict of Interest
  8. REFERENCES
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