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Anti-malarial drug artesunate protects against cigarette smoke-induced lung injury in mice.

ABSTRACT

Cigarette smoking is the primary cause of chronic obstructive pulmonary disease (COPD), which is mediated by lung infiltration with inflammatory cells, enhanced oxidative stress, and tissue destruction. Anti-malarial drug artesunate has been shown to possess anti-inflammatory and anti-oxidative actions in mouse asthma models. We hypothesized that artesunate can protect against cigarette smoke-induced acute lung injury via its anti-inflammatory and anti-oxidative properties. Artesunate was given by oral gavage to BALB/c mice daily 2 h before 4% cigarette smoke exposure for 1 h over five consecutive days. Bronchoalveolar lavage (BAL) fluid and lungs were collected for analyses of cytokines, oxidative damage and antioxidant activities. Bronchial epithelial cell BEAS-2B was exposed to cigarette smoke extract (CSE) and used to study the mechanisms of action of artesunate. Artesunate suppressed cigarette smoke-induced increases in BAL fluid total and differential cell counts; levels of IL-1 p, MCP-1, IP-10 and KC; and levels of oxidative biomarkers 8-isoprostane, 8-OHdG and 3-nitrotyrosine in a dose-dependent manner. Artesunate promoted anti-oxidant catalase activity and reduced NADPH oxidase 2 (NOX2) protein level in the lungs from cigarette smoke-exposed mice. In BEAS-2B cells, artesunate suppressed pro-inflammatory PI3 K/Akt and p44/42 MAPK signaling pathways, and increased nuclear Nrf2 accumulation in response to CSE. Artesunate possesses anti-inflammatory and anti-oxidative properties against cigarette smokeinduced lung injury, probably via inhibition of PI3K and p42/22 MAPK signaling pathways, augmentation of Nrf2 and catalase activities, and reduction of NOX2 level. Our data suggest that artesunate may have therapeutic potential for treating COPD.

Keywords:

Chronic obstructive pulmonary disease

Artemisinins

BEAS-2B cells

Nrf-2

NOX2

Introduction

Chronic obstructive pulmonary disease (COPD) is projected to become the third leading cause of death by 2020 (Rabe et al., 2007), and is characterized by increased macrophage and neutrophil infiltration, and progressive obstruction of small airways and destruction of lung parenchyma (Cosio et al., 2009; Brusselle et al., 2011). In contrast to asthma, COPD is more resistant to corticosteroids (Barnes, 2010; Morjaria et al., 2010). PDE4 inhibitor for the treatment of COPD shows significant side effects which limit its efficacy (Page and Spina, 2012). Thus, the discovery of novel compounds for COPD remains a major unmet need.

Cigarette smoke is the primary cause of COPD. Cigarette smoke is loaded with oxidants and free radicals which cause oxidative damage to bronchial epithelium and alveolar wall, infiltration of inflammatory cells, and tissue remodeling (Cantin, 2010; Yao and Rahman, 2011). Elevation of oxidative biomarkers 3-nitrotyrosine (3-NT), 8-isoprostane and 8-hydroxydeoxyguanosine (8-OHdG) within the lungs has been associated with COPD severity (Louhelainen et al., 2008; Yao and Rahman, 2011). Nuclear factor erythroid-2-related factor 2 (Nrf2), a redox-sensitive transcription factor important in preventing from oxidative stress, has been found to be impaired in COPD (Cho et al., 2006; Boutten et al., 2011). Genetic ablation of Nrf2 resulted in enhanced susceptibility to emphysema in a murine COPD model (Rangasamy et al., 2004). In mice exposed to cigarette smoke, activation of Nrf2 by CDDOimidazolide attenuated the development of emphysema (Sussan et al., 2009). Novel molecules with antioxidant properties represent a valuable class of compounds for COPD drug development.

Artesunate is a semi-synthetic derivative of artemisinin, the principal active component of the plant Artemisia annua. Artesunate is used clinically for the treatment of malaria and has established a good safety record (Gordi and Lepist, 2004). Aside from being an anti-malarial, artesunate has exhibited antiinflammatory (Cheng et al., 2011), anti-oxidative (Efferth et al., 2007; Sieber et al., 2009), anti-cancer (Krishna et al., 2008) and anti-viral effects (Efferth et al., 2008). We have recently reported anti-oxidative actions of artesunate in a mouse asthma model, through promoting nuclear translocation of Nrf2 (Ho et al., 2012).

In this study, we hypothesized that artesunate can attenuate cigarette smoke-induced acute lung injury in mice. Our findings reveal for the first time that artesunate significantly decreased bronchoalveolar lavage (BAL) fluid inflammatory cell counts, and cytokine and chemokine levels, and lung protease expression, in an experimental model of cigarette smoke-induced lung injury. The protective effects exhibited by artesunate may be associated with inhibition of phosphoinositol 3-kinase (PI3K) and p44/42 mitogen-activated protein kinase (MAPK) pathways, augmentation of Nrf2 expression and catalase activity, and down-regulation of NADPH oxidase 2 (NOX2) expression. These data support a novel therapeutic value of artesunate in the treatment of COPD.

Materials and methods

Animals

Female BALB/c mice of 6 to 8 weeks old (Animal Resources Centre, Canning Vale, Western Australia, Australia) were maintained in a 12 h light-dark cycle with food and water available ad libitum. Animal experiments were performed according to the institutional guidelines for Animal Care and Use Committee of the National University of Singapore.

Cigarette smoke-induced lung injury and artesunate treatment protocol

Mice were placed in a ventilated chamber filled with 4% cigarette smoke delivered using peristaltic pumps (Masterflex, Cole-Parmer Instrument Co., Niles, IL, USA) at a constant rate of 1 l [min.sup.-1] as described (Guan et al., 2013). Mice were exposed to 10 sticks of 3R4F research cigarettes (Tobacco and Health Research Institute, University of Kentucky, Lexington, KY, USA) over a period of 60 min a day for five consecutive days. Mice in the sham air group were simultaneously placed in another ventilated chamber but exposed to fresh air. 3R4F is the most commonly used cigarette in COPD research and a comprehensive analysis of its mainstream smoke chemistry has been reported using mass spectrometry (Roemer et al., 2012). Artesunate (10, 30, and 100 mg [kg.sup.-1]; Sigma, St. Louis, MO, USA) or vehicle (60% PEG) in 0.1 ml saline was given by oral gavage 2 h before each cigarette smoke exposure. Mice were sacrificed 24 h after the last cigarette smoke or sham air exposure, and lung samples were collected for various biochemical analyses.

BAL fluid analyses

Tracheotomy was performed and a cannula was inserted into the trachea. Ice-cold PBS was instilled into the lungs and BAL fluid was collected. BAL fluid total and differential cell counts were determined. BAL fluid levels of keratinocyte chemoattractant (KC), IFN[gamma]-inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1) and IL-1[beta] were measured using ELISA (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. BAL fluid levels of oxidative damage markers were measured using enzyme immunoassays kits for 3-NT (Upstate/Millipore, Billerica, MA, USA), 8-isoprostane and 8-OHdG (Cayman Chemicals, Ann Arbor, MI, USA) according to the manufacturer's instructions.

Lung tissue analyses

Frozen lung tissues were homogenized in PBS, and the supernatants were used to determine the activities of catalase, superoxide dismutase (SOD), glutathione peroxidase (GPx) using enzymatic assay kits (Cayman Chemicals) according to the manufacturer's instructions. Total mRNA was also extracted from mouse lung tissues using TRlzol reagent (Invitrogen, Carlsbad, CA, USA) and then used for first-strand cDNA synthesis. Primers for inflammatory and antioxidant genes are listed in Table 1. Template cDNA in PCR mixture containing SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA) was amplified and quantitated using a sequence detector (ABI 7500 Cycler; Applied Biosystems). The mRNA expression levels for all samples were normalized to the level of the housekeeping gene 18S.

Cigarette smoke extract and bronchial epithelial cell culture

Cigarette smoke extract (CSE) was freshly prepared by bubbling smoke from one cigarette to 10 ml of RPMI1640 medium supplemented with 10% FBS at a rate of one cigarette every 10 min as described (Kode et al., 2008). The resulting medium was adjusted to pH 7.4 and sterile-filtered through a 0.45-mm Acrodisc filter (Pall, Ann Arbor, MI, USA). Control medium was prepared in the same way as CSE by bubbling fresh air into 10 ml of RPMI medium. Human BEAS-2B bronchial epithelial cells (American Type Tissue Collection, Rockville, MD, USA) were cultured in RPMI 1640 supplemented with 10% FBS. Cells were pre-incubated with 30 [micro]M artesunate or vehicle control (0.05% DMSO) for 2 h before exposure to 2% CSE for indicated times.

Immunoblotting

Frozen lung tissues were homogenized in 1% Triton X-100 and Halt Protease Inhibitor Cocktail Kit in phosphate-buffered saline lysis buffer (Thermo Scientific, Rockford, IL, USA). BEAS-2B cells were lysed in M-PER[R] Mammalian Protein Extraction Reagent and Halt Protease Inhibitor Cocktail Kit (Thermo Scientific) for total protein extraction. Cytoplasmic and nuclear protein extractions were performed using a nuclear extraction kit (Active Motif, Carlsbad, CA, USA). Total protein extracts (20 |xg per lane) were separated by 10% SDS-PAGE and immunoblots were probed with antiNOX2 antibody (1:500, Abeam, Cambridge, MA, USA), or anti-Akt (1:1000), anti-phospho-Akt (1:1000), anti-44/42 MAPK(1:1000), anti-phospho-44/42 MAPK (1:1000), or anti-(3-actin (1:10,000) antibodies (Cell Signaling Technology, Beverly, MA, USA). For Nrf2 protein analysis, both cytoplasmic and nuclear proteins (30p.g per lane) were separated by 10% SDS-PAGE and immunoblots were probed with anti-Nrf2 antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA, USA), or anti-TATA box binding protein (TBP, 1:2000, Abeam) antibody. Band intensity was quantitated using ImageJ software (NIH) as described previously (Guan et al., 2013).

Statistical analysis

Data are presented as means [+ or -] SEM. One-way ANOVA followed by Dunnett's test was used to determine significant differences between treatment groups. Significant level was set at p < 0.05.

Results

Artesunate attenuates cigarette smoke-induced lung inflammation

BALB/c mice were exposed to 4% cigarette smoke or air for 1 h daily over five consecutive days. Cigarette smoke inhalation markedly increased total BAL fluid cell counts, especially neutrophil count, and moderately but significantly enhanced macrophage and lymphocyte counts (Fig. 1A). Artesunate (10, 30 and 100 mg kg'1) significantly suppressed the total and neutrophil counts, in BAL fluid in a dose-dependent manner (Fig. IB). Significant inhibition of BAL fluid macrophage count was observed with the highest dose of artesunate.

BAL fluid levels of IL-ip, IP-10, MCP-1 and KC were significantly raised in cigarette smoke-exposed mice, and artesunate was able to abate the cytokine levels in a dose-dependent manner (Fig. 2A). Artesunate significantly suppressed cigarette smoke-induced pulmonary gene expression of TNF-[alpha], MIP-2[alpha], TGF-[beta], metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1 (Fig. 2B). Lung expression of GM-CSF was also repressed but did not reach statistical significance. Upon cigarette smoke exposure, inducible NOS (iNOS), an enzyme responsible for NO production and subsequent oxidative damage was up-regulated, and artesunate markedly reduced the iNOS gene expression in the lungs (Fig. 2B). In contrast, there appeared to be no induction of GPx gene expression upon cigarette smoke exposure, and artesunate caused a slight rise in GPx expression.

Artesunate protects against cigarette smoke-induced oxidative lung damage

Lung level of NADPFI oxidase 2 (NOX2), an important prooxidant (Lee and Yang, 2012), was noticeably up-regulated in response to cigarette smoke exposure. Artesunate significantly suppressed NOX2 expression back to basal level (Fig. 3A). These data suggest that artesunate is able to attenuate oxidative damage to proteins, DNA and lipids induced by cigarette smoke. 3-NT is a marker for protein nitrosative damage catalyzed by NO; 8-OHdG is an oxidative DNA damage marker, and 8-isoprostane is a marker for lipid peroxidation (Yao and Rahman, 2011). Cigarette smoke drastically elevated the levels of 3-NT, 8-OHdG and 8-isoprostane in BAL fluid, and artesunate dose-dependently down-regulated these markers (Fig. 3B). Cigarette smoke induced a marked increase in lung catalase activity. Artesunate treatment further increased the lung catalase activity, which may partially explain its anti-oxidative effects observed. In contrast, lung SOD and GPx activities were not altered by acute cigarette smoke exposure or by artesunate treatment (Fig. 3C).

Artesunate modulates pro-inflammatory and oxidative signaling pathways

It has been demonstrated that PI3K/Akt and p44/42 MAPK pathways are activated in COPD (Mercer et al., 2004; Ito et al.,

2007). In BEAS-2B cells exposed to 2% CSE, increases in phosphoAkt and phospho-p44/42 MAPK were observed within 1-5 min (Fig. 4A). Pretreatment with 30p,M artesunate markedly reduced CSE-induced phosphorylation of Akt and p44/42 MAPK to basal levels, and thereby suppressed PI3K/Akt and p44/42 MAPK signaling pathways. On the other hand, exposing BEAS-2B cells to 2% CSE resulted in a marked Nrf2 nuclear accumulation at 8h, but artesunate had no effect on nuclear Nrf2 level at this time point. However, at 24 h after CSE exposure, artesunate drastically elevated Nrf2 nuclear accumulation (Fig. 4B). Notably, cytoplasmic levels of Nrf2 were similar across all treatment groups. Our data suggest that the anti-oxidative effects of artesunate in cigarette smoke-induced lung injury may be mediated by the actions of nuclear Nrf2.

Discussion

Our data reveal for the first time that anti-malarial drug artesunate can suppress cigarette smoke-induced airway inflammation and oxidative damage in mice, probably via inhibition of pro-inflammatory signaling pathways and augmentation of antioxidative defense. Cigarette smoke contains substantive amounts of toxic oxidants and free radicals including reactive aldehyde, quinone, hydroquinone, semiquinone and superoxide (Cantin, 2010; Yao and Rahman, 2011), which trigger off multiple series of pro-inflammatory responses. Activation of neutrophils and macrophages induces further production of endogenous reactive oxygen species (ROS) and reactive nitrogen species (RNS) causing damage the lungs. Both neutrophils and macrophages are also rich sources of tissue proteases such as elastase and MMP, which promotes destruction of alveolar walls. KC and MIP-2[alpha] are essential for neutrophil infiltration into the lungs, while MCP-1 and IP-10 are vital for recruitment of macrophages (Cosio et al., 2009; Brusselle et al., 2011). Clinical findings show that sputum samples collected from COPD patients contained high levels of KC and MCP-1 (Traves et al., 2002). Our data revealed for the first time that artesunate prevented cigarette smoke-induced lung infiltration of neutrophils and, to a lesser extent, macrophages in a dose-dependent manner and was able to abate KC, IP-10, MCP-1, IL-1(3, TNF-[alpha] and MIP-2[alpha] levels in lungs from cigarette smoke-exposed mice.

We observed a strong increase in lung MMP-9 expression along with an adaptive elevation in TIMP-1 level in cigarette smoke-exposed mice. MMP-9 belongs to a large family of zinc-dependent proteinases that can degrade collagen and elastin, attributable to cigarette smoke-induced emphysema and airflow limitation in smokers (Yao et al., 2013; Ishii et al., 2014). Alveolar macrophages from COPD patients contain higher levels of MMP-9 compared with those from normal subjects (Russell et al., 2002; Ishii et al., 2014). Sputum MMP-9 levels in COPD are directly associated with the severity of emphysema (Chaudhuri et al., 2013). Artesunate markedly suppressed the lung level of MMP-9. In addition, cigarette smoke-induced TGF-[beta]1 gene expression was down-regulated by artesunate. It has been shown that TGF-[beta]1 can be proteolytically activated by MMP-9 (Yu and Stamenkovic, 2000). Our findings implicate a protective role of artesunate against airway remodeling and emphysema in COPD.

We revealed that cigarette smoke exposure increased oxidative damage to protein, DNA, and lipid in the lung as indicated by the increased levels of biomarkers 3-NT, 8-OHdG and 8-isoprostane, and artesunate potently suppressed their expression levels. The protective effects of artesunate against nitrosative damage can be partly explained by its inhibitory effect on the iNOS expression, leading to a drop in NO production, peroxynitrite formation and protein nitration at tyrosine residues. Besides, NOX family of enzymes promotes ROS production and are important components for oxidative stress. NOX2 is the first member described and has been studied extensively (Lee and Yang, 2012). It has been reported that inhibition of NOX2 reduces ROS production in mouse lung (Lee et al., 2013). Cigarette smoke induced an increase in NOX2 level in the lungs but was effectively reduced by artesunate to the basal level. The lowered oxidative damage in artesunate-treated mice may also be partly due to inhibition of NOX2.

Catalase is a metalloprotein oxidoreductase which is important for neutralization of [H.sub.2][O.sub.2] into [H.sub.2]O and [O.sub.2] (Rahman et al., 2006). Mice exposed to acute cigarette smoke showed enhanced catalase activity, probably a defensive physiological response. There were no significant changes to the SOD and GPx activities upon cigarette smoke exposure, which is consistent with our recent report (Guan et al., 2013). Artesunate markedly boosted catalase activity further, thus bolstering the antioxidant defense system and curbing oxidative damage to the lungs. Taken together, our findings strongly showed protective effects of artesunate against cigarette smoke-induced airway inflammation and oxidative damage.

To investigate the molecular mechanisms of anti-inflammatory and antioxidative actions of artesunate, we studied the effects of artesunate on PI3K/Akt and p44/42 MAPK pro-inflammatory signaling pathways and on nuclear translocation of redox-sensitive transcription factor Nrf2 in CSE-treated BEAS-2B human bronchial epithelial cells. In peripheral lung tissues from COPD patients, PI3K pathway was found drastically activated (To et al., 2010), and PI3K inhibitors may have therapeutic value for COPD (Ito et al., 2007; To et al., 2010). On the other hand, highly elevated p44/42 MAP kinase activity was observed in lung tissues from emphysema patients and in CSE-exposed human airway cells (Mercer et al., 2004; Shen et al., 2011). We observed strong inhibition of CSE-induced phosphorylation of Akt and p44/42 MAP kinase in BEAS-2B cells by artesunate, indicating that artesunate may attain its anti-inflammatory effects in COPD by suppressing activation of P13K/Akt and p44/42 MAP kinase signaling pathways.

Nrf2 plays a predominant role in antioxidant protection in cigarette smoke-exposed lungs (Yao and Rahman, 2011). In patients with advanced COPD, Nrf2 activity is reduced in peripheral lung tissues, leading to reduced antioxidant activity and persistent oxidative stress and damage (Malhotra et al., 2008). Nrf2 gene disruption resulted in enhanced susceptibility to emphysema after cigarette smoke exposure (Louhelainen et al., 2008). Sulforaphane, an Nrf2 activator, was able to enhance Nrf2 antioxidant defence in response to cigarette smoke in human epithelial cells (Malhotra et al., 2008). Artesunate strongly promoted Nrf2 nuclear stabilization and accumulation in BEAS-2B cells at 24 h after CSE exposure. This finding is line with our recent report showing artesunate's ability to elevate Nrf2 nuclear level in mouse asthma model (Ho et al., 2012). Nrf2 is responsible for regulating the expression of antioxidants such as catalase (Ma, 2013). Our findings reveal that the anti-oxidative action of artesunate in COPD may be mediated by its activation of Nrf2.

In conclusion, our data illustrate the potential therapeutic value of oral artesunate in treating COPD through its anti-oxidative and anti-inflammatory properties. Artesunate was able to attenuate the initial stages of oxidative stress in an acute cigarette smoke exposure model. To further validate its therapeutic value in COPD, a chronic cigarette smoke-induced lung injury mouse model needs to be developed (Lugade et al., 2011) in order to evaluate the antiCOPD effects of artesunate.

Abbreviations: 3-NT, 3-nitrotyrosine; 8-OHdG, 8-hydroxydeoxyguanosine; BAL, bronchoalveolar lavage; COPD, chronic obstructive pulmonary disease; CSE, cigarette smoke extract; GPx, glutathione peroxidase; iNOS, inducible nitric oxide synthase; IP-10, IFN--y-inducible protein 10; KC, keratinocyte chemoattractant; MCP-1, monocyte chemoattractant protein-1; NOX, NADPH oxidase; Nrf2, nuclear factor erythroid-2-related factor 2; PI3K, phosphoinositide 3-kinase; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; TBP, TATA box binding protein; TIMP-1, tissue inhibitor of metalloproteinase-1.

http://dx.doi.org/10.1016/j.phymed.2014.07.018

Conflict of interest statement

None.

Acknowledgement

This research work was supported in part by a research grant NMRC/CBRG/0027/2012 from the National Medical Research Council of Singapore and by a COT grant HQ/S10-095COT0.21.

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ARTICLE INFO

Article history:

Received 30 May 2014

Received in revised form 11 July 2014

Accepted 22 July 2014

David S.W. Ng (a,1), Wupeng Liao (a,1), W.S. Daniel Tan (a), Tze Khee Chan (a), Xin Yi Loh (a), W.S. Fred Wong (a,b), *

(a) Department of Pharmacology, Yong Loo Lin School of Medicine, National University Health System, Singapore, Singapore

(b) Immunology Program, Life Science Institute; National University of Singapore, Singapore, Singapore

* Corresponding author at: Department of Pharmacology, Yong Loo Lin School of Medicine, Immunology Program. Center for Life Sciences, National University of Singapore, 28 Medical Drive, #03-05, Singapore 117456, Singapore.

Tel.: +65 6516 3263; fax: +65 6873 7690.

E-mail address: phcwongf@nus.edu.sg (W.S.F. Wong).

(1) Co-first authors: David S.W. Ng and Wupeng Liao are co-first authors for the manuscript.

Table 1
Primer sets for reverse transcriptase-polymerase chain reaction
analysis.

Targets         Sequences

                Forward

mMMP-9          5'-CGCTCATGTACCCGCTGTAT-3'
mTIMP-l         5'-GTGGGAAATGCCCCAGAT-3'
mGM-CSF         5'-GGGCGCCTTGAACATGAC-3'
mTNF-[alpha]    5'-TCGAGTGACAAGCCCGTAGC-3'
mMIP-2[alpha]   5'-AGTGAACTGCCCTGTCAATGC-3'
miNOS           5'-cgggCAAACATCACATTCAGATCCCG-3'
hGPx            5'-CTTCGGACACCTCAGACGG-3'
TGF-[beta]      5'-CCTCGAGACAGGCCATTTGT-3'
18S             5'-GCCGCTAGAGGTGAAATTCTTG-3'

Targets

                Reverse

mMMP-9          5'-TGTCTGCCGCACTCAAAGAC-3'
mTIMP-l         5'-GGGCATATCCACAGAGGCTTT-3'
mGM-CSF         5'-TTGTGTTTCACAGTCCGnTCC-3'
mTNF-[alpha]    5'-CTCAGCCACTCCAGCTGCTC-3'
mMIP-2[alpha]   5'-AGG CAA ACT TTT TGA CCG CC-3'
miNOS           5'-TATATTGCTGTGGCTCCCATGTT-3'
hGPx            5'-GCTCTTTCTCCCCGTTCACA-3'
TGF-[beta]      5'-AAACCGACCTTTGCCAATGC-3'
18S             5'-CATTCTTGGCAAATGCTTTCG-3'
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Author:Ng, David S.W.; Liao, Wupeng; Tan, W.S. Daniel; Chan, Tze Khee; Loh, Xin Yi; Wong, W.S. Fred
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Oct 15, 2014
Words:4539
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