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

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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: tslee@ym.edu.tw (T.-S. Lee), yrkou@ym.edu.tw (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

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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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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

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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 .

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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).

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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.

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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