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Original Contribution
Glucosamine attenuates cigarette smoke-induced lung
inflammationby inhibiting ROS-sensitive inflammatory signaling
Yuh-Lin Wu a, An-Hsuan Lin a, Chao-Hung Chen b,c, Wen-Chien
Huang b,d, Hsin-Yi Wang a,Meng-Han Liu a, Tzong-Shyuan Lee a,n, Yu
Ru Kou a,n
a Department of Physiology, School of Medicine, National
Yang-Ming University, Taipei 112, Taiwanb Division of Thoracic
Surgery, Mackay Memorial Hospital, Taipei, Taiwanc Department of
Cosmetic Applications and Management, Mackay Medicine, Nursing and
Management College, Taipei, Taiwand Institute of Traditional
Medicine, School of Medicine, National Yang-Ming University, Taipei
112, Taiwan
a r t i c l e i n f o
Article history:Received 3 September 2013Received in revised
form18 December 2013Accepted 21 January 2014Available online 28
January 2014
Keywords:GlucosamineCigarette smokeLung inflammationNADPH
oxidaseReactive oxygen speciesAMP-activated protein
kinaseMitogen-activated protein kinasesNF-BSAT3Free radicals
a b s t r a c t
Cigarette smoking causes persistent lung inflammation that is
mainly regulated by redox-sensitivepathways. We have reported that
cigarette smoke (CS) activates a NADPH oxidase-dependent
reactiveoxygen species (ROS)-sensitive AMP-activated protein kinase
(AMPK) signaling pathway leading toinduction of lung inflammation.
Glucosamine, a dietary supplement used to treat osteoarthritis,
hasantioxidant and anti-inflammatory properties. However, whether
glucosamine has similar beneficialeffects against CS-induced lung
inflammation remains unclear. Using a murine model we show
thatchronic CS exposure for 4 weeks increased lung levels of
4-hydroxynonenal (an oxidative stressbiomarker), phospho-AMPK, and
macrophage inflammatory protein 2 and induced lung inflammation;all
of these CS-induced events were suppressed by chronic treatment
with glucosamine. Using humanbronchial epithelial cells, we
demonstrate that cigarette smoke extract (CSE) sequentially
activatedNADPH oxidase; increased intracellular levels of ROS;
activated AMPK, mitogen-activated protein kinases(MAPKs), nuclear
factor-B (NF-B), and signal transducer and activator of
transcription proteins 3(STAT3); and induced interleukin-8 (IL-8).
Additionally, using a ROS scavenger, a siRNA that targetsAMPK, and
various pharmacological inhibitors, we identified the signaling
cascade that leads toinduction of IL-8 by CSE. All these
CSE-induced events were inhibited by glucosamine pretreatment.Our
findings suggest a novel role for glucosamine in alleviating the
oxidative stress and lunginflammation induced by chronic CS
exposure in vivo and in suppressing the CSE-induced IL-8 in vitroby
inhibiting both the ROS-sensitive NADPH oxidase/AMPK/MAPK signaling
pathway and the down-stream transcriptional factors NF-B and
STAT3.
& 2014 Elsevier Inc. All rights reserved.
Cigarette smoking is the major etiological factor in the
devel-opment of chronic obstructive pulmonary disease (COPD), which
ischaracterized by persistent lung inflammation that results
inchronic bronchitis and emphysema [1,2]. The lung inflammation
induced by cigarette smoke (CS) is well recognized as
beingregulated by a complex mechanism involving various types of
cellsand inflammatory mediators [14]. For example, upon direct
stimu-lation by CS, lung epithelial cells produce inflammatory
chemokinesand cytokines, both of which play a crucial role in the
initiation andprogression of lung inflammation [2,59]. It is known
that theinduction of inflammatory mediators by CS in the lung is
mainlyregulated by redox-sensitive signaling pathways [35].
Initially, CSmay increase the intracellular levels of reactive
oxygen species (ROS)in lung cells via activation of NADPH oxidase
[6,1015]. NADPHoxidase, a membrane-bound enzyme complex, can
transport elec-trons across the plasma membrane, which reduces
oxygen to super-oxide; the product can then react, generating other
downstream ROS[15,16]. Subsequently, this increased intracellular
ROS may activatevarious ROS-sensitive signaling pathways, such as
the mitogen-activated protein kinases (MAPKs) and a number of
downstreamtranscriptional factors, such as nuclear factor-B (NF-B),
and
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/freeradbiomed
Free Radical Biology and Medicine
http://dx.doi.org/10.1016/j.freeradbiomed.2014.01.0260891-5849
& 2014 Elsevier Inc. All rights reserved.
Abbreviations:: COPD, chronic obstructive pulmonary disease; CS,
cigarettesmoke; CSE, cigarette smoke extract; MIP-2, macrophage
inflammatory protein 2;IL-8, interleukin-8; ROS, reactive oxygen
species; 4-HNE, 4-hydroxynonenal; AMPK,AMP-activated protein
kinase; MAPK, mitogen-activated protein kinase; ERK,extracellular
signal-regulated kinase; JNK, c-Jun N-terminal kinase; AP-1,
activatorprotein 1; NF-B, nuclear factor -light-chain enhancer of
activated B cells; STAT3,signal transducer and activator of
transcription proteins 3; HBEC, human bronchialepithelial cell;
PBS, phosphate-buffered saline; HE, hydroethidine; DHE,
dihy-droethidium; ETH, ethidium; DCFH-DA, dichlorofluorescein
diacetate; DCF,dichlorofluorescein; siRNA, small interfering RNA;
BALF, bronchoalveolar lavagefluid; LPS, lipopolysaccharide;
O-GlcNAc, O-linked-N-acetylglucosamine
n Corresponding authors. Fax: 886 2 2826 4049.E-mail addresses:
[email protected] (T.-S. Lee), [email protected] (Y. Ru Kou).
Free Radical Biology and Medicine 69 (2014) 208218
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ultimately promote inflammatory gene expression [3,4,1215].
Indeed,we have recently described a NADPH oxidase-dependent,
ROS-sensi-tive, AMP-activated protein kinase (AMPK) signaling
pathway invol-ving NF-B and signal transducer and activator of
transcriptionproteins 3 (STAT3) as its downstream transcriptional
factors; thissignaling pathway is crucial for CS-induced
interleukin-8 (IL-8)production in human lung epithelial cells [6].
Thus, the presenceof these ROS-related pathogenic mechanisms
suggests that therapeu-tic targeting of oxidative stress with
antioxidants to improve lunginflammation is recognized as being
beneficial when treatingCOPD [17].
Glucosamine is an amino-monosaccharide synthesized fromglucose
that is utilized for the biosynthesis of glycoproteins
andglycosaminoglycans [18]. It is a natural compound present in
mosthuman tissues with the highest concentrations being in
cartilage[19]. Owing to its basic role in cartilage and synovial
fluidsynthesis [20,21], and its anti-inflammatory effects on the
localcells present in joints [2224], glucosamine has long been used
asa dietary supplement when treating osteoarthritis. Recently,
sev-eral in vitro studies have suggested that glucosamine has
anantioxidant function that involves a number of mechanisms
thatimprove the redox balance in chondrocytes and cell types
otherthan lung cells [2528]. Additionally, growing evidence fromin
vitro studies has also indicated that glucosamine is able
tosuppress inflammatory responses in lung cells [29,30] and
othercell types [3136] when these responses are induced by
stimulisuch as lipopolysaccharide (LPS) or inflammatory
cytokines.Furthermore, in vivo investigations have demonstrated
that glu-cosamine is able to reduce inflammation in animal models
ofdiseases [23,32,3740]; however, such evidence for CS-inducedlung
inflammation is not yet available. The anti-inflammatoryeffects of
glucosamine found in the majority of these studies[29,3240] can be
mainly attributed to its ability to modulatethe signaling pathways
responsible for induction of inflammatorymediators. Thus, whether
glucosamine possesses antioxidantand anti-inflammatory effects on
CS-induced lung inflammationremains unclear.
The aims in this study were, first, to investigate the
antioxidantand anti-inflammatory effects of glucosamine on
CS-induced lunginflammation and, second, to determine the
therapeutic mechan-isms underlying these beneficial effects. We
employed an estab-lished murine model of chronic CS exposure [6,41]
to assess theinhibitory effects of glucosamine on oxidative stress,
the activationof AMPK, and various indices of lung inflammation.
Additionally,we used an established in vitro model of primary human
bronchialepithelial cells (HBECs) [6,42] to determine the
suppressive effectsof glucosamine on the activation of NADPH
oxidase, the increase inintracellular ROS, the activation of the
ROS-sensitive inflammatorysignaling pathway, and the induction of
IL-8 mediated by CSextract (CSE).
Materials and methods
Reagents
Antibodies and ELISA kits to measure IL-8, macrophage
inflam-matory protein 2 (MIP-2), and IL-1 were purchased from
R&DSystems (Minneapolis, MN, USA). Rabbit antibodies against
phospho-AMPK, AMPK, and STAT3 were obtained from Cell Signaling
(Beverly,MA, USA). Rabbit antibodies against 4-hydroxynonenal
(4-HNE) werepurchased from Abcam (Cambridge, MA, USA). Antibodies
againstp65, p47phox, and histone H1 as well as donkey anti-rabbit
IgGFITCantibodies were obtained from Santa Cruz Biotechnology
(Santa Cruz,CA, USA). Mouse antibody against -tubulin together with
curcumin,AG490, N-acetylcysteine, apocynin, and D-glucosamine
hydrochloride
were purchased from SigmaAldrich (St. Louis, MO, USA).
PD98059,SP600125, SB203580, BAY11-7085, and compound C were
obtainedfrom Calbiochem (San Diego, CA, USA). The EnzyChrom
NADP/NADPH assay kit was purchased from BioAssay Systems
(Hayward,CA, USA). Scramble, AMPK, and p47phox small interfering
RNAs(siRNAs) were purchased from Ambion (Austin, TX, USA).
INTERFERinsiRNA transfection reagent was obtained from Polyplus
(New York,NY, USA). The membrane-permeative probes hydroethidine
(HE) anddichlorofluorescein diacetate (DCFH-DA) were purchased from
Mole-cular Probes (Eugene, OR, USA).
Murine model of chronic CS exposure and glucosamine
treatment
All animal experiments were approved by the Animal Care andUse
Committee of the National Yang-Ming University. The murinemodel of
chronic CS exposure has been described in detailpreviously [6].
Briefly, male C57BL/6J mice at the age of 8 weeks(National
Laboratory Animal Center, Taipei, Taiwan) were ran-domly divided
into four groups for exposure to air or CS. Twogroups of mice were
treated with glucosamine (10 mg/mouse, ip)and the other two groups
were treated with vehicle (phosphate-buffered saline; PBS) every 2
days during the 4-week exposure.Thus the mice formed four groups,
namely airvehicle, CSvehicle, CSglucosamine, and airglucosamine.
Animals weregiven ad libitum access to food and water, and the
averaged bodyweights did not vary among the study groups after the
4-weekexposure. At each CS exposure, the mice were placed in
anexposure chamber and 750 ml of fresh CS generated from 1.5
cigar-ettes (Marlboro Red Label; 10.8 mg nicotine and 10.0 mg tar
percigarette) was delivered to the chamber. The CS passed out of
thechamber via four exhaust holes (1 cm) on the side panels.
Duringthe exposure, the mice were conscious and breathed
spontaneouslyin the chamber for 10 min. After exposure, the mice
were transferredto a new cage and allowed to inspire air normally.
The mice wereexposed at 1000 and 1600 hours each day for 4 weeks.
The controlanimals underwent identical procedures in another
chamber butwere exposed only to air. For each CS exposure, the
particleconcentration inside the exposure chamber was about 625
mg/m3
initially, but decreased overtime because the CS passed out of
thechamber via the exhaust holes. The HbCO levels immediately
afterthe 10-min exposure protocol for air- and CS-exposure mice
were0.4270.1 and 31.4873.92% (n6), respectively.
Preparation of bronchoalveolar lavage fluid (BALF) and lung
tissues
At the end of each experiment, the mice were euthanized withCO2
and a middle thoracotomy was performed. The left lung wasligated
and the right lung was lavaged four times with 0.6 ml ofwarm PBS
containing a complete protease inhibitor cocktail(Roche
Diagnostics, Mannheim, Germany). The BALF samples werethen
centrifuged at 350 g for 5 min at 4 1C, and the supernatant ofthe
first lavage fluid was stored at 80 1C for later analysis of
totalprotein using a Bio-Rad protein assay reagent (Bio-Rad
Laboratories,Hercules, CA, USA). The cell pellets of the BALF
samples wereresuspended in PBS for cell counting. Furthermore, the
right lungwas then stored at 80 1C for subsequent analysis. The
left lung wasfixed with 4% paraformaldehyde and embedded in
paraffin.
Immunohistochemical assessment
Formalin-fixed, paraffin-embedded tissue blocks of the leftlung
were cut into 8-m sections. Sections were
deparaffinized,rehydrated, and then covered with 3% H2O2 for 10
min. After beingblocked with bovine serum albumin, each slide was
first incubatedwith primary antibodies for 1 h at 37 1C, followed
by the corre-sponding secondary antibodies for an additional hour.
The color of
Y.-L. Wu et al. / Free Radical Biology and Medicine 69 (2014)
208218 209
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all of the sections was developed with 0.1% diaminobenzidine
andthen the sections were counterstained with hematoxylin; this
wasfollowed by examination under a microscope. The detected
signalwas digitally captured using an image analysis system
(Image-ProPlus 4.5, Media Cybernetics, Bethesda, MD, USA) as
describedpreviously [43]. The intensity of the immunoreaction
developedwithin epithelium was then assessed densitometrically.
Tenepithelial cells were analyzed for each section and three
differentsections were analyzed for each animal. The data were
averagedfor each animal and expressed in arbitrary units.
Measurement of an oxidative stress biomarker
Levels of 4-HNE, a product of lipid peroxidation [44], in
lungtissues were measured and this served as a biomarker of
oxidativestress as described previously [45].
Preparation of CSE
CSE was freshly prepared on the day of the experiment
aspreviously described [6,46], with some modifications. In
brief,1000 ml of the smoke generated from two burning
cigarettes(Marlboro Red Label; tar, 10.0 mg; nicotine, 0.8 mg;
size, 84 mm)without filters was sucked under a constant flow rate
(8 ml/s) into asyringe and then bubbled into a tube containing 20
ml serum-freemedium. The CSE solution was sterilized using a 0.22-m
filter(Millipore, Bedford, MA, USA) and the pH was adjusted to 7.4.
Theoptical density of the CSE solution was determined by measuring
theabsorbance at 302 [47] or 320 nm [48], which in reality showed
littledifference between preparations. This CSE solution was
considered100% CSE and was further diluted with serum-free medium
tovarious desired concentrations that were then used to treat
HBECsfor various durations. The CSE solution generally contains
water-
Fig. 1. Glucosamine (GS) suppression of the cigarette smoke
(CS)-induced increase in the 4-HNE and phospho-AMPK levels in lung
tissues, as well as lung inflammation, across thefour study groups.
Mice were chronically exposed to air or CS for 4 weeks. Two of the
four study groups received treatment with GS (10 mg/mouse, ip)
every 2 days during the4-week exposure duration. The expression of
(A) 4-HNE and (B) phospho-AMPK and AMPK in lung tissue was analyzed
by Western blotting. 4-HNE is a product of lipidperoxidation,
whereas AMPK is a key kinase involved in CS-induced lung
inflammation. The MIP-2 and IL-1 levels in (C and D) lung tissue
and in (E and F) bronchoalveolar lavagefluid (BALF) were determined
by ELISA. The (G) total cell counts, (H) differential cell counts,
and (I) total protein levels in the BALF were also determined. All
of the above were usedas indications of lung inflammation. Data in
each group are the mean7SEM from six mice. po0.05, significant
statistical difference between groups.
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208218210
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soluble components, such as ,-unsaturated aldehydes [48],
fromboth the particulate and the gas phases of whole CS.
Cell culture
HBECs (Cascade Biologics, Portland, OR, USA) were cultured
inepithelial cell growth medium (medium 200, Cascade
Biologics)containing 10% fetal bovine serum, 1 low-serum growth
supple-ment, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.25
g/mlamphotericin B (Biological Industries, Kibbutz Beit Haemek,
Israel)at 37 1C in an incubator with 5% CO2. Cells were pretreated
withN-acetylcysteine, glucosamine, apocynin, compound C, or
inhibi-tors before the CSE stimulation.
Measurement of intracellular ROS levels
The membrane-permeative probes HE and DCFH-DA were usedto assess
intracellular levels of superoxide (O2d) and hydrogenperoxide
(H2O2), respectively [49,50], using methods that havebeen described
previously [51]. HBECs were incubated in culturemedium containing
10 M DHE at 37 1C for 45 min. After stimula-tion with CSE for the
desired time, the cells were washed anddetached with trypsin/EDTA,
and the fluorescence intensity of thecells was then analyzed using
a multilabel counter (PerkinElmer,Waltham, MA, USA) at 530 nm
excitation and 620 nm emission forETH and at 488 nm excitation and
530 nm emission for DCF.Images of the cells were also obtained by
examining them usinga Nikon TE2000-U florescence microscope (Tokyo,
Japan).
Determination of NADPH oxidase activity
The activity of NADPH oxidase was examined using an Enzy-Chrom
NADP/NADPH assay kit according to the manufacturer'sinstructions.
This assay kit simply measures the NADP/NADPHconcentration in the
samples based on a glucose dehydrogenasecyclic reaction, in which
the formed NADPH reduces a formazanreagent. The intensity of the
reduced product color, measured at565 nm, is proportionate to the
NADP/NADPH concentration inthe samples. Thus, the intracellular
NADP or NADPH wasextracted from cellular lysates (1 mg). The
extraction was thensubjected to this assay kit to measure the
change in NADP/NADPH ratio to reflect the relative NADPH oxidase
activity.
Extraction of membrane proteins
The membrane extracts were prepared as described
previously[6,42]. Briefly, cells were rinsed with ice-cold PBS and
then lysedin hypotonic lysis buffer (1 mM TrisHCl in PBS containing
1 g/mlleupeptin, 10 g/ml aprotinin, 1 mM phenylmethylsulfonyl
fluor-ide) on ice for 5 min, followed by centrifugation at 5000 rpm
for5 min. Next, the harvested supernatant was centrifuged at45,000
rpm for 75 min. The pellet was collected and lysed withSDS lysis
buffer (1% Triton X-100, 0.1% SDS, 0.1% sodium deox-ycholate, 1
g/ml leupeptin, 10 g/ml aprotinin, 1 mM phenyl-methylsulfonyl
fluoride) and the mixture was used as themembrane protein extract.
Protein concentrations were deter-mined by the Bio-Rad protein
assay (Richmond, CA, USA).
Western blot analysis
Aliquots of cell lysates, tissue lysates, or membrane
proteinextracts were separated by 812% SDSPAGE and then
trans-blotted onto an Immobilon-P membrane (Millipore). After
beingblocked with 5% skim milk, the blots were incubated with
variousprimary antibodies and then the appropriate secondary
antibo-dies. The specific protein bands were detected using an
enhanced
chemiluminescence kit (PerkinElmer), which was followed by
thequantification with the ImageQuant 5.2 software (Healthcare
Bio-Sciences, Philadelphia, PA, USA).
Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNAs were isolated from cells using Tri reagent
andconverted into cDNA with reverse transcriptase (New
EnglandBiolabs, Ipswich, MA, USA) using oligo(dT) as the primer.
Theresultant cDNAs were then used as templates for the
semiquantita-tive PCR. PCR was performed in a DNA thermal cycler
(BiometraTpersonal, Laprepco, Horsham, PA, USA) using the following
pro-gram: 94 1C for 2 min, followed by 35 cycles of 94 1C for 30 s,
58 1Cfor 30 s, 72 1C for 1 min, and then a final single cycle of 72
1C for10 min. The nucleotide sequences of the primers were as
follows:IL-8, sense, 50-ACTTCCAAGCTGGCCGTGGCT-30, antisense,
50-TCACTGG-CATCTTCACTGATT-30, and glyceraldehyde-3-phosphate
dehydrogen-ase (GAPDH), sense, 50-TGTTCCAGTATGACTCCACTC-30,
antisense,50-TCCACCACCCTGTTGCTGTA-30.
Determining the concentration of MIP-2 and IL-1
The concentrations of MIP-2 and IL-1 in BALF and in lungtissue
were measured using an ELISA kit according to the manu-facturer's
instructions.
Small interfering RNA transfection
HBECs were transfected with scramble or AMPK siRNA
usingINTERFERin siRNA transfection reagent for 24 h. The
nucleotidetarget sequences for human AMPK were sense,
GAUUUCGGAUUAU-CUAAUATT, and antisense, UAUUAGAUAAUCCGAAAUCGG.
Statistical analysis
The results are presented as the mean7SEM. Statistical
eva-luations involved one-way ANOVA followed by Dunnett's test
orFisher's least significant difference procedure for multiple
compar-isons as appropriate. Differences were considered
statisticallysignificant at po0.05.
Results
Suppressive effects of glucosamine on CS-induced oxidative
stress,AMPK phosphorylation, and lung inflammation in mice
Compared to the air control animals with or without
theglucosamine treatment, mice chronically exposed to CS for 4
weekswere found to have increased levels of 4-HNE in their
lungs(Figs. 1A; a biomarker of oxidative stress). Additionally,
glucosa-mine treatment had a tendency to decrease the
CS-inducedincreases in H2O2 levels of BALF and lung tissues,
although thedifferences did not attain significance (Supplementary
Fig. S1).This was accompanied by higher levels of phospho-AMPK
(Fig. 1B),MIP-2 (Figs. 1C; a human IL-8 homolog), and IL-1 (Fig.
1D) in theirlungs. Furthermore, the levels of MIP-2 (Fig. 1E) and
IL-1 (Fig. 1F)in the BALF from the CS-exposure animals were also
increased inparallel with higher BALF total cell counts (Fig. 1G),
higher BALFdifferential cell counts (Fig. 1H), and an increased
total protein inthe BALF (Fig. 1I; an index of lung vascular
permeability). Impor-tantly all of the above were significantly
attenuated by the chronicglucosamine treatment. Further
immunohistochemical analysisshowed stronger staining for
phospho-AMPK and MIP-2 in theepithelial cells and infiltrating
inflammatory cells in mouse lungsections that had been exposed to
CS compared to the air control
Y.-L. Wu et al. / Free Radical Biology and Medicine 69 (2014)
208218 211
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animals with or without the glucosamine treatment (Fig. 2).The
CS-induced expression of phospho-AMPK and MIP-2 wasgreatly reduced
by the chronic treatment with glucosamine(Fig. 2). For the air, CS,
CSglucosamine, and airglucosaminegroups, the relative expression
levels of phospho-AMPK that werepositive for immunostaining were
1.070.2-, 2.370.6-, 1.470.2-,and 1.270.2-fold (n6), respectively.
Similarly, the relativeexpression levels of MIP-2 that were
positive for immunostainingwere 1.070.3-, 4.070.9-, 1.470.2-, and
1.070.2-fold (n6),respectively.
Inhibitory effects of glucosamine on CSE-induced IL-8expression
in HBECs
Analysis of the cell lysates showed that exposure of HBECs
tovarious concentrations (0.75, 1.5, and 3%) of CSE for 24 h
increasedthe level of IL-8 protein (Fig. 3A). In addition, exposure
of HBECs to3% CSE for up to 24 h time-dependently elevated the
level of IL-8protein (Fig. 3B) and, similarly, 3% CSE for up to 18
h time-dependently increased the level of IL-8 mRNA level (Fig.
3C). As3% CSE resulted in the most profound IL-8 induction, 3% CSE
wastherefore chosen as the standard treatment for all
subsequentexperiments throughout this study. In the presence of the
ROSscavenger N-acetylcysteine (1, 2, 4 mM) or in the presence
ofglucosamine (0.0125, 0.05, 0.2 mM), CSE-induced IL-8
proteinexpression was significantly inhibited, with the most
dramaticinhibition occurring at 4 and 0.2 mM, respectively (Figs.
3D and E).In addition, the CSE induction of IL-8 mRNA expression
was alsoattenuated by pretreatment with glucosamine at 0.2 mM (Fig.
3F).As a result, N-acetylcysteine at 4 mM and glucosamine at 0.2
mMwere used in all subsequent experiments of this study.
Suppressive effect of glucosamine on the CSE-induced
NADPHoxidase-dependent increase in intracellular levels of ROS in
HBECs
Within 30 min after exposure, CSE was found to cause anincrease
in intracellular levels of O2d (ETH; Fig. 4A) and of H2O2
(DCF; Fig. 4B) in HBECs and these responses were
significantlyreduced not only by pretreatment with N-acetylcysteine
andapocynin (an inhibitor of NADPH oxidase), but also by
pretreat-ment with glucosamine (Fig. 4). Because translocation of
thep47phox subunit from the cytosol to the membrane is requiredfor
activation of NADPH oxidase [15,16], we further studied
thesuppressive effect of glucosamine on the CSE-induced activation
ofNADPH oxidase. Exposure of HBECs to CSE for 15 min
significantlyincreased the presence of p47phox in the membrane
compartment,but decreased the presence of p47phox in the cytosol
(Fig. 5A). Thistranslocation of p47phox was prevented by
pretreatment withglucosamine (Fig. 5A). Additionally, exposure of
HBECs to CSE for15 min also caused an increase in NADPH oxidase
activity; this wasinhibited not only by pretreatment with apocynin,
but also bypretreatment with glucosamine (Fig. 5B). These findings
suggestthat glucosamine may prevent increases in intracellular ROS
via asuppression of the CSE-induced activation of NADPH
oxidase.
Inhibitory effect of glucosamine on the CSE-induced
ROS-sensitiveAMPK/MAPK signaling pathway and the induction of IL-8
in HBECs
We then went on to investigate the inhibitory effect
ofglucosamine on the CSE-evoked ROS-sensitive signaling pathwaythat
leads to IL-8 expression in HBECs. We found that CSE-inducedIL-8
expression was inhibited by pretreatment with the AMPKinhibitor
compound C (Fig. 6A), at a concentration that couldeffectively
suppress CSE-induced AMPK activation (phosphoryla-tion) (Fig. 6B).
Similarly, CSE-induced IL-8 expression was sup-pressed by
pretreatment with AMPK siRNA (Fig. 6D), at aconcentration that
resulted in a significant reduction in theamount of AMPK protein
available for phosphorylation (Fig. 6C).Having established such
inhibition, importantly, CSE-inducedAMPK phosphorylation was found
to be prevented, not only bypretreatment with N-acetylcysteine
(Fig. 6E), but also by pretreat-ment with glucosamine (Fig. 6F).
These findings suggest thatglucosamine is able to inhibit
CSE-induced IL-8 expression viathe suppression of the activation of
ROS-sensitive AMPK signaling.
Fig. 2. Glucosamine (GS) suppression of the cigarette smoke
(CS)-induced increase in levels of phospho-AMPK and MIP-2 in lung
epithelial cells in representative lungsections obtained from four
mice. The expression was detected by immunostaining with antibodies
against phospho-AMPK or MIP-2. Specificity of immunostaining
wasconfirmed using an IgG-negative control. Mice were chronically
exposed to air or CS for 4 weeks and treated with GS (10 mg/mouse,
ip) every 2 days during the exposure.The original magnification of
each image is 200 .
Y.-L. Wu et al. / Free Radical Biology and Medicine 69 (2014)
208218212
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It has been reported that the MAPKs (ERK, JNK, and p38) arealso
important for the induction of inflammatory mediators by
CS[5,1214]. We found that pretreatment of HBECs with an
ERKinhibitor (PD98059) or a JNK inhibitor (SP600125) was able
tosignificantly reduce CSE-mediated IL-8 expression; however,
pre-treatment with a p38 inhibitor (SB203580) failed to produce
suchan effect (Fig. 7A). Indeed, CSE was able to induce JNK and
ERKactivation (phosphorylation), which peaked at 4 h (Figs. 7B and
E).Importantly, the CSE-induced JNK and ERK phosphorylation
wassignificantly reduced not only by pretreatment with compound
C(Figs. 7C and F), but also by pretreatment with glucosamine(Figs.
7D and G). These findings suggest that glucosamine is ableto
inhibit CSE-induced IL-8 expression via suppression of
theactivation of MAPK signaling, which is downstream of AMPK.
Inhibitory effects of glucosamine on the CSE-induced
activationof NF-B and STAT3
NF-B and STAT3 have been reported to be the
transcriptionalfactors downstream of the CSE-induced activation of
AMPKsignaling [6]. Indeed, pretreatment of HBECs with the
NF-Binhibitor BAY11-7085 or the JAK/STAT inhibitor AG490 is able
tosignificantly reduced CSE-induced IL-8 expression (Fig. 8A).
Addi-tionally, CSE exposure caused an increase in nuclear
expression of
NF-B p65 (Fig. 8B) and STAT3 (Fig. 8C), which peaked at 12
h.Importantly, CSE-mediated nuclear translocation of both p65
andSTAT3 was prevented by pretreatment with glucosamine (Figs.
8Dand E). These findings suggest that glucosamine is able to
inhibitCSE-induced IL-8 expression via suppression of the
activation ofNF-B and STAT3.
Discussion
Our in vivo study demonstrates that chronic CS exposure for4
weeks increases the lung levels of 4-HNE and phospho-AMPK inmice
(Figs. 1 and 2). Additionally, chronic CS exposure inducedlung
inflammation, as is evidenced by the increase in lung levelsof
MIP-2 and IL-1, the infiltration of inflammatory cells, and
theincreased vascular permeability (Figs. 1 and 2). All of
theseCS-induced events were suppressed by chronic treatment
withglucosamine. We then used the in vitro model to investigate
thetherapeutic mechanisms underlying the beneficial effects
ofglucosamine. We employed HBECs to study the induction of IL-8by
CSE. We did this because the alleviation of CS lung inflamma-tion
by glucosamine in mice is associated with a reduced expres-sion of
phospho-AMPK and MIP-2 in lung epithelium (Fig. 2) andbecause IL-8
produced by lung epithelial cells is known to be
Fig. 3. Glucosamine (GS) inhibition in relation to cigarette
smoke extract (CSE)-induced IL-8 expression in HBECs. (A) Cells
were exposed to 04.5% CSE for 24 h. (B and C)Cells were incubated
with medium alone or 3% CSE for indicated times. (D and E) Cells
were incubated with or without 3% CSE for 24 h with pretreatment
with vehicle,N-acetylcysteine (NAC; a ROS scavenger; 14 mM) or GS
(0.01250.2 mM). (F) Cells were incubated with or without 3% CSE for
18 h with pretreatment with vehicle or GS(0.2 mM). Protein (A, B,
D, and E) and mRNA (C and F) levels in the cell lysates were
analyzed byWestern blotting and RT-PCR, respectively. Cells were
pretreated with NAC orGS before CSE stimulation. Data in each group
are the mean7SEM from four independent experiments. *po0.05 vs
control (A, D, E, and F) or time 0 (B and C). #po0.05 vsCSE without
drug pretreatment (D, E, and F).
Y.-L. Wu et al. / Free Radical Biology and Medicine 69 (2014)
208218 213
-
important to the induction of lung inflammation by CS [2,59].
Wedemonstrate that exposure of HBECs to CSE sequentially
activatedNADPH oxidase; increased intracellular ROS level;
activated AMPK,MAPKs, NF-B, and STAT3; and induced IL-8 (Figs. 38).
Using aROS scavenger, a siRNA targeting AMPK, and various
pharmacolo-gical inhibitors, we determined the signaling cascade of
thevarious participants in CSE-induced IL-8 expression.
Specifically,the signaling pathway involves ROS-mediated NADPH
oxidase/AMP/MAPK signaling with NF-B and STAT3 as the
downstreamtranscriptional factors. Thus, whereas these findings
confirm theimportance of AMPK [6] and MAPKs [3,4,1215] in the
CS-inducedlung inflammation, we have further identified that AMPK
is akinase that acts upstream of the MAPKs. Importantly, all of
theseCSE-induced intracellular events were suppressed by
pretreatmentwith glucosamine. Accordingly, our findings suggest
that glucosa-mine is able to suppress the oxidative stress and lung
inflammation
induced by chronic CS exposure in vivo and is also able to
suppressCSE-induced IL-8 expression in vitro via its antioxidant
activity andthe inhibition of ROS-sensitive inflammatory
signaling.
Our study seems to be the first to report that glucosaminehas
both antioxidant and anti-inflammatory activities againstCS-induced
lung inflammation and that the latter is likely to belinked to the
former because the CS-evoked inflammatory signal-ing we have
observed is ROS sensitive. These two beneficialactivities of
glucosamine have been suggested separately byprevious studies that
have focused on stimuli other than CS insult.Using culture medium
alone without cells, we demonstrated thatchallenge with CSE or H2O2
(30 M) could increase the level ofH2O2 in culture medium, which was
prevented by incubation withglucosamine (Supplementary Fig. S2),
suggesting the ex vivoantioxidant activity. Glucosamine has been
shown in vitro to haveantioxidant activity via the chelation of
ferrous ions [25,26], the
Fig. 4. Glucosamine (GS) inhibition in relation to cigarette
smoke extract (CSE)-induced NADPH oxidase-dependent increase in
intracellular levels of ROS in HBECs. Cellswere incubated with
medium alone or exposed to 3% CSE for 30 min. Except for the
control group, cells were pretreated with N-acetylcysteine (NAC; a
ROS scavenger;2 mM), apocynin (APO; an inhibitor of NADPH oxidase;
150 M), or GS (0.2 mM). Levels of O2 and H2O2, respectively, were
measured by HE/ETH and DCFH-DA/DCF assaysand expressed as mean
fluorescence intensity. Cells were pretreated with NAC, GS, or APO
before CSE stimulation. Data in each group are the mean7SEM from
fourindependent experiments. *po0.05 vs medium alone; #po0.05 vs
CSE without drug pretreatment.
Fig. 5. Glucosamine (GS) inhibition in relation to cigarette
smoke extract (CSE)-induced activation of NADPH oxidase in HBECs.
Cells were incubated with medium alone orexposed to 3% CSE for 15
min with pretreatment with apocynin (APO; an inhibitor of NADPH
oxidase; 150 M) or GS (0.2 mM). (A) Protein levels were analyzed by
Westernblotting. (B) NADPH oxidase activity was analyzed by a
NADP/NADPH assay kit. Data in each group are the mean7SEM from four
independent experiments. Cells werepretreated with APO or GS before
CSE stimulation. *po0.05 vs medium alone.
Y.-L. Wu et al. / Free Radical Biology and Medicine 69 (2014)
208218214
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scavenging of intracellular ROS [26,27], the enhancement
ofreduced glutathione levels [27], the upregulation of
hemeoxygenase-1 (an antioxidant enzyme) activity [28], and the
down-regulation of the NADPH oxidase system [28], all using cell
typesother than lung cells. On the other hand, glucosamine has
alsobeen shown to have anti-inflammatory properties via the
suppres-sion of inflammatory mediator production in lung cells
[29,30], inmacrophages [31,32], in microglial cells [33,34], in
prostate cancercells [35], and in aortic smooth muscle cells [36],
after inductionby LPS or inflammatory cytokines. Furthermore, in
vivo investiga-tions have demonstrated that glucosamine is able to
reduceinflammation in animal models of disease, including arthritis
inrats [23], trauma-hemorrhage in rats [32], colitis in rats [37],
brainischemia/reperfusion injury in rats [38], atherosclerosis in
rabbits[39], and LPS-induced lung inflammation in rats [40]. The
anti-inflammatory effects of glucosamine in most of these
studies[29,3240] are mainly attributable to its ability to
modulatevarious participants, such as MAPKs and NF-B, in the
signalingpathways that are responsible for induction of
inflammatorymediators. Thus, our findings are in good agreement
with theabove-reported observations.
In this study, two CSE-mediated early events were demon-strated
to be modulated by glucosamine. Within 15 min of CSE
exposure, activation of NADPH oxidase was observed, which
wasevidenced by both the translocation of the p47phox subunit
fromthe cytosol to the membrane compartment, which is an
essentialevent forming the active NADPH oxidase [16], and an
increase inNADPH oxidase activity (Fig. 5). Additionally, within 30
min ofexposure, CSE was able to elevate the intracellular level of
ROS as aresult of the activation of NADPH oxidase; this was
confirmed bythe finding that the increase in intracellular ROS was
significantlyreduced by an inhibitor of NADPH oxidase (Fig. 4).
Importantly,these two early events were both attenuated by
glucosamine, whichsuggests that glucosamine may initially inhibit
the CS-inducedactivation of NADPH oxidase and that a consequence of
this is thealleviation of downstream events, including the
generation ofintracellular ROS, the activation of ROS-sensitive
inflammatorysignaling, and, ultimately, the induction of lung
inflammation. Never-theless, we cannot exclude the possibility that
glucosamine maydirectly scavenge intracellular ROS [26,27], which,
in turn, would leadto a suppression of the CS-induced activation of
ROS-sensitiveinflammatory signaling. The exact mechanism by which
glucosamineinhibits the CS-induced activation of NADPH oxidase
remains elusive.However, exogenous glucosamine is known to be taken
into cells bymembrane glucose transporters [38,52] and these
proteins areexpressed by a number of different types of mammalian
cells,
Fig. 6. Glucosamine (GS) inhibition in relation to cigarette
smoke extract (CSE)-induced NADPH oxidase-dependent ROS-sensitive
AMPK activation in HBECs. (A) Cells wereincubated with medium alone
or exposed to 3% CSE for 24 h with pretreatment with compound C (an
AMPK inhibitor; 5 M). (B, E, and F) Cells were incubated with
mediumalone or exposed to 3% CSE for 30 min with pretreatment with
N-acetylcysteine (NAC; a ROS scavenger; 2 mM), compound C (5 M), GS
(0.2 mM), or their vehicle. (C and D)Cells were pretreated with
2550 nM AMPK (C), with 50 nM AMPK (D), or with scramble siRNAs.
Cells were then incubated with medium alone or exposed to 3% CSE
for 24 h.Protein levels were analyzed by Western blotting. Cells
were pretreated with NAC, compound C, or GS before CSE stimulation.
Data in each group are the mean7SEM from fourindependent
experiments. *po0.05 vs medium alone (A, B, D, E, F) or without
siRNA (C); #po0.05 vs CSE without pretreatment with drug (A, B, E,
F) or siRNA (C, D).
Y.-L. Wu et al. / Free Radical Biology and Medicine 69 (2014)
208218 215
-
including lung epithelial cells [53,54]. Additionally, the
influx ofglucosamine is likely to increase the intracellular level
of proteinO-linked-N-acetylglucosamine (O-GlcNAc) [32,36,55,56],
which isknown to suppress inflammation of the cardiovascular
system[32,36,55] and other organs [38,56]. O-GlcNAc is known to act
as astress response factor that induces the posttranslational
modificationof a diverse array of cytoplasmic and nuclear proteins,
includingMAPKs and NF-B [32,36,55]. In fact, glucosamine has been
reportedto suppress the expression of p22phox (another subunit of
NADPHoxidase) in chondrocytes [28], LPS- or cytokine-mediated
phos-phorylation of MAPKs in HBECs [29] and prostate cancer cells
[35],LPS-induced IB phosphorylation in cardiomyocytes [32],
TNF--induced p65 phosphorylation in aortic smooth muscle cells
[36],and LPS-induced nuclear translocation and DNA binding of p65
toNF-B consensus sequence in microglial cells or
macrophages[34,38]. Thus, it is also possible that glucosamine, via
the action ofO-GlcNAc, may directly modulate NADPH oxidase as well
as thekinases and transcriptional factors that are involved in
CS-evokedinflammatory signaling. If this is the case, the cytosolic
componentsof NADPH oxidase (p40phox, p47phox, and p67phox) [15],
AMPK,MAPKs, and NF-B would be potential targets for protein
interac-tion with O-GlcNAc.
A rat study has indicated that intraperitoneal doses of
gluco-samine are completely absorbed, whereas oral doses show
lowbioavailability, which indicates that the gut is a site of
presystemicloss [57]. For this reason, we used intraperitoneal
administrationas the route for glucosamine treatment in vivo.
Additionally, thedosage of glucosamine (glucosamine HCl) used in
this study(10 mg/mouse; every 2 days over a 4-week CS exposure) was
welltolerated by our animals and is similar to those suggested by
otherstudies that have focused on the immunomodulatory functions
of
glucosamine in mice (10 mg/mouse/day for 34 days, ip) [58]
oranti-inflammatory function of glucosamine in rats (1000 mg/kg/day
for 7 days, ip) [40]. Similarly, the concentrations of glucosa-mine
(0.01250.2 mM) that were used for the in vitro studies werenot
higher than those employed in our previous
investigations'experimental models using lung epithelial cells
(0.150 mM)[29,40,59] or other cell types (110 mM) [35].
In our in vivo study, we found that the glucosamine treatmentin
mice had a trend to decrease the CS-induced increases in H2O2levels
of BALF and lung tissues, although the differences did notattain
significance. It is known that ROS have a short life span inaqueous
solutions [60] and they react quickly with various sub-strates
including lipids to form lipid peroxidation products, such as4-HNE
[44]. The inability of these data on ROS levels to fit wellwith our
other findings may be due to the limitation of ourmeasurements. We
speculate that, when we measured their levels,the ROS in BALF and
tissue samples had already decayed duringthe process of preparing
these samples. We, however, demon-strated that CS exposure
increased the lung tissue level of 4-HNE,which was attenuated by
the glucosamine treatment. This findingindicates that the lungs of
animals treated with glucosamine had areduced oxidative stress
after CS exposure. This reduction inoxidative stress seems to
correlate well with the attenuation ofCS-induced lung inflammation
achieved by the glucosaminetreatment. It has been reported that
4-HNE can increase theproduction of IL-8 in a human macrophagic
cell line [48]. However,our in vitro study show that CSE exposure
did not alter theexpression of 4-HNE in HBECs, compared to the
control(Supplementary Fig. S3).
In summary, our findings suggest a novel role for
glucosamineregarding the alleviation of oxidative stress and lung
inflammation
Fig. 7. Glucosamine (GS) inhibition in relation to cigarette
smoke extract (CSE)-induced AMPK-dependent activation of MAPKs in
HBECs. (A) Cells were incubated withmedium alone or exposed to 3%
CSE for 24 h, with or without pretreatment with MAPK inhibitor (SB,
SB203580, a p38 inhibitor, 10 M; PD, PD98059, an ERK inhibitor,10
M; SP, SP600125, a JNK inhibitor, 10 M) for 30 min. (B and E) Cells
were incubated with medium alone or exposed to 3% CSE for the
indicated times. (C, D, F, and G) Cellswere incubated with medium
alone or exposed to 3% CSE for 4 h, with or without pretreatment
with compound C (an AMPK inhibitor; 5 M) or GS (0.2 mM) for 30 min.
Cellswere pretreated with MAPK inhibitors, compound C, or GS before
CSE stimulation. Protein levels were analyzed by Western blotting.
Data in each group are the mean7SEMfrom four independent
experiments. *po0.05 vs medium alone (A, C, D, F, and G) or time 0
(B and E). #po0.05 vs CSE without treatment with drugs (A, C, D, F,
and G).
Y.-L. Wu et al. / Free Radical Biology and Medicine 69 (2014)
208218216
-
induced by chronic CS exposure in vivo and the suppression of
theCSE-induced IL-8 in vitro by inhibiting both ROS-sensitive
NADPHoxidase/AMPK/MAPK signaling and their downstream
transcrip-tional factors NF-B and STAT3. Our findings clearly
support thepossibility of using glucosamine to ameliorate lung
inflammationin smokers and that glucosamine treatment may be a
potentialtherapy option when treating COPD.
Acknowledgments
The authors are grateful to Dr. Ralph Kirby, Department of
LifeSciences, National Yang-Ming University, for his help in
languageediting. This study was supported by Grants NSC
101-2320-B-010-042-MY3, NSC 100-2628-B-001-MY2, NSC
102-2628-B-010-001-MY3, and NSC 100-2320-B-010-018-MY3 from the
NationalScience Council, Taiwan; Grant MMH10193 from the
MackayMemorial Hospital, Taipei, Taiwan; and a grant from the
Ministryof Education, Aim for the Top University Plan, Taiwan.
Appendix A. Supplementary data
Supplementary data associated with this article can be found
inthe online version at
http://dx.doi.org/10.1016/j.freeradbiomed.2014.01.026.
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Glucosamine attenuates cigarette smoke-induced lung inflammation
by inhibiting ROS-sensitive inflammatory signalingMaterials and
methodsReagentsMurine model of chronic CS exposure and glucosamine
treatmentPreparation of bronchoalveolar lavage fluid (BALF) and
lung tissuesImmunohistochemical assessmentMeasurement of an
oxidative stress biomarkerPreparation of CSECell cultureMeasurement
of intracellular ROS levelsDetermination of NADPH oxidase
activityExtraction of membrane proteinsWestern blot analysisReverse
transcription-polymerase chain reaction (RT-PCR)Determining the
concentration of MIP-2 and IL-1Small interfering RNA
transfectionStatistical analysis
ResultsSuppressive effects of glucosamine on CS-induced
oxidative stress, AMPK phosphorylation, and lung inflammation in
miceInhibitory effects of glucosamine on CSE-induced IL-8
expression in HBECsSuppressive effect of glucosamine on the
CSE-induced NADPH oxidase-dependent increase in intracellular
levels of ROS in...Inhibitory effect of glucosamine on the
CSE-induced ROS-sensitive AMPK/MAPK signaling pathway and the
induction of IL-8...Inhibitory effects of glucosamine on the
CSE-induced activation of NF-B and STAT3
DiscussionAcknowledgmentsSupplementary dataReferences