Andrographolide protects against cigarette smoke-induced oxidative lung injury via augmentation of Nrf2 activity

Introduction

Chronic obstructive pulmonary disease (COPD) is a global health problem and is predicted to become the third leading cause of death by 2020 (Rabe et al., 2007). COPD is a chronic airway disease characterized by progressive airflow limitation associated with dysregulated inflammatory responses of the lungs and increased macrophage and neutrophil infiltration, leading to obstruction of small airways and destruction of lung parenchyma as a consequence of skewed activation of metalloproteases (Cosio et al., 2009; Brusselle et al., 2011; Mocchegiani et al., 2011). In sharp contrast to asthma, COPD is largely resistant to the anti-inflammatory effects of corticosteroids (Adcock and Barnes, 2008). At present, there are no specific treatments in halting the disease progression and suppressing the lung inflammation effectively in COPD (Barnes, 2010; Morjaria et al., 2010). Although the PDE4 inhibitor roflumilast has been newly approved for the treatment of COPD, it suffers from dose-limiting major side effects such as diarrhoea, headache, nausea and weight loss (Rabe, 2011; Page and Spina, 2012). There is an urgent need to discover novel compounds for COPD.

Cigarette smoking is a major risk factor for the development of COPD. Cigarette smoke is a rich source of potent oxidants and free radicals causing oxidative damage to airway epithelium and alveolar wall, infiltration of macrophages and neutrophils, and imbalance of oxidants and antioxidants (Cantin, 2010; Yao and Rahman, 2011). Pulmonary levels of oxidant biomarkers such as 3-nitrotyrosine (3-NT), 8-isoprostane and 8-hydroxydeoxyguanosine (8-OHdG) have been shown to correlate positively with COPD severity (Louhelainen et al., 2008; Yao and Rahman, 2011). Nuclear factor erythroid-2-related factor 2 (Nrf2) is a redox-sensitive transcription factor and is critical in protecting the lung against oxidative stress, as impaired Nrf2 has been implicated in several pulmonary diseases including acute respiratory distress syndrome, pulmonary fibrosis, asthma and COPD (Cho et al., 2006; Boutten et al., 2011). Nrf2 gene disruption resulted in enhanced susceptibility to emphysema after cigarette smoke exposure (Rangasamy et al., 2004). Nrf2 has been shown to regulate expression of antioxidants such as glutathione peroxidase (GPx) and glutathione reductase (GR) (Adair-Kirk et al., 2008; Hubner et al., 2009). Activation of Nrf2 by triterpenoids such as CDDO-imidazolide has demonstrated beneficial effects in a cigarette smoke-induced emphysema animal model (Sussan et al., 2009). Drug discovery of novel compounds with property of strengthening antioxidant defence is a vital strategy for COPD drug development.

Andrographolide is a labdane diterpene lactone isolated from the Andrographis paniculata plant (Lim et al., 2012). A. paniculata has long been used as herbal medicine for the prevention and treatment of upper respiratory tract infection in Asian countries and in Scandinavia (Coon and Ernst, 2004; Chang et al., 2008). Andrographolide has also been shown to possess hepatoprotective (Negi et al., 2008), antiviral (Lin et al., 2008), anti-cancer (Lim et al., 2012) and anti-inflammatory (Abu-Ghefreh et al., 2009; Bao et al., 2009) activities. Recently, it has been reported that andrographolide promoted Nrf2 nuclear translocation in a human endothelial cell line EA.hy926 by probing Nrf2 protein in the cell nuclear extracts and by measuring Nrf2-induced antioxidant response element (ARE) activation in cells transfected with ARE-luciferase construct (Yu et al., 2010).

We hypothesized that andrographolide may have protective effects against oxidative damage induced by cigarette smoke in COPD. In this study, we aimed to investigate potential antioxidative effects and mechanism of action of andrographolide in a cigarette smoke-induced lung injury mouse model. Our findings reveal for the first time that andrographolide can augment antioxidant gene expressions and activities probably via up regulation of Nrf2 activity in experimental models of cigarette smoke exposure. The reduction in cigarette smoke-induced lung oxidative damage conferred by andrographolide is probably associated with the significant decline in bronchoalveolar lavage (BAL) fluid inflammatory cell counts, chemokines, cytokines and proteases, and up-regulation of glutathione (GSH) redox defence system and heme oxygenase-1 (HO-1) expression. These findings support a novel therapeutic value for andrographolide in the treatment of COPD.

Methods

Cigarette smoke-induced lung injury and andrographolide treatment protocol

Mice were placed in a ventilated chamber filled with 4% cigarette smoke delivered by a peristaltic pump (Masterflex, Cole-Parmer Instrument Co., Niles, IL, USA) at a constant rate of 1 L·min⁻¹ as described (Chan et al., 2009). Total suspended particulate of 4% cigarette smoke was 493.5 ± 49.6 mg·m⁻³ (n = 4) recorded using the MicroDust Pro-aerosol monitor (Casella CEL, Bedford, UK). The level of total suspended particulates observed was comparable to that reported by other laboratories (Morris et al., 2008; Thatcher et al., 2008; Vlahos et al., 2010). To develop cigarette smoke-induced lung injury, 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 (Braber et al., 2011). Mice in the sham air group were simultaneously placed in another ventilated chamber but exposed to fresh air. Andrographolide (0.1, 0.5, and 1 mg·kg⁻¹; Sigma, St. Louis, MO, USA) (Figure 1A) or vehicle (1% DMSO) in 0.1 mL saline was given by i.p. injection 2 h before each cigarette smoke exposure as described by Bao et al. (2009). Mice were killed 24 h after the last cigarette smoke or sham air exposure, and lung samples were collected for various analyses.

Results

Andrographolide attenuates cigarette smoke-induced lung inflammation

Cotinine is a stable metabolite of nicotine, and it is often used as a surrogate maker for nicotine exposure (Benowitz, 2009). Plasma cotinine level in 4% cigarette smoke-exposed mice was sharply elevated above 100 ng·mL⁻¹. In contrast, plasma cotinine level in sham air control mice was undetectable (Figure 1B). BAL fluid was collected 24 h after the last cigarette smoke or sham air exposure. Cigarette smoke inhalation markedly increased total cell and neutrophil counts, with moderate but significant elevation in macrophage count, as compared with sham air control (Figure 1C). Andrographolide (0.1, 0.5 and 1 mg·kg⁻¹) significantly suppressed the total inflammatory cell and neutrophil counts in BAL fluid in a dose-dependent manner as compared with the DMSO control (Figure 1D). Andrographolide (1 mg·kg⁻¹) showed a moderate inhibitory effect on macrophage count but did not reach statistical significance.

BAL fluid levels of IL-1β, IP-10 (CXCL10), MCP-1 (CCL2) and KC (CXCL1) were significantly raised in cigarette smoke-exposed mice as compared with sham air control mice (Figure 2A). Andrographolide was able to abate the BAL fluid levels of IL-1β, IP-10, MCP-1 and KC in a dose-dependent manner, reaching significant inhibition at the dose of 1 mg·kg⁻¹.

In addition, andrographolide (1 mg·kg⁻¹) significantly suppressed the elevated gene expression of lung tissue GM-CSF, TNF-α and MIP-2α (as known as GRO-β or CXCL2) induced by cigarette smoking (Figure 2B). Cigarette smoke also up-regulated the gene expression of inducible NOS (iNOS), a pro-oxidant enzyme responsible for NO production and subsequent oxidative lung damage. Andrographolide markedly reversed the iNOS gene expression down to basal level (Figure 2B). Furthermore, the increase in gene expression of MMP-12 and tissue inhibitor of metalloproteinase-1 (TIMP-1), a metalloproteinase and an anti-metalloproteinase critical for lung remodelling and repair in COPD, by cigarette smoke was significantly inhibited by 1 mg·kg⁻¹ andrographolide (Figure 2B).

Andrographolide protects against cigarette smoke-induced oxidative lung damage

BAL fluid was used to assay for 3-NT, a product of protein nitration indicative of oxidative protein damage; 8-OHdG, a marker for oxidative DNA damage; and 8-isoprostane, an indicator for lipid peroxidation, using elisa. Cigarette smoking significantly elevated BAL fluid levels of 3-NT, 8-OHdG and 8-isoprostane as compared to the sham air control (Figure 3). Andrographolide markedly suppressed the levels of 3-NT, 8-OHdG and 8-isoprostane at all three doses used, ameliorating oxidative damage to proteins, DNA and lipids induced by cigarette smoking.

Andrographolide augments the GPx and GR activities

Cigarette smoke exposure induced a marked adaptive increase in the lung catalase activity as compared with sham air. Andrographolide (1 mg·kg⁻¹) significantly reduced the catalase activity. In contrast, lung SOD activity was not altered by cigarette smoke challenge or by andrographolide treatment (Figure 4). The activity of GPx, an antioxidant enzyme capable of reducing H₂O₂ to H₂O by oxidizing GSH, was not altered by cigarette smoke but was substantially augmented in cigarette smoke-exposed mouse lungs by andrographolide. On the other hand, lung GR activity was markedly abated by cigarette smoke exposure, but andrographolide treatment totally restored GR activity (Figure 4). In brief, andrographolide promoted the GSH-related enzyme activities, which may be attributable to its antioxidant effects.

Andrographolide promotes nuclear Nrf2 accumulation and GSH level

To investigate if the antioxidative effects of andrographolide were mediated through Nrf2 activation, we studied nuclear Nrf2 translocation and transactivation in BEAS-2B human bronchial epithelial cells. Addition of 2% CSE to BEAS-2B cells resulted in marked nuclear translocation of Nrf2 at 4 h, and andrographolide (30 μM) had no effect on nuclear Nrf2 level. In contrast, at 24 h after CSE exposure, nuclear Nrf2 dropped to the basal level, but andrographolide treatment maintained the nuclear Nrf2 level elevated in BEAS-2B cells (Figure 5A). In contrast, cytoplasmic levels of Nrf2 were very low in control and all treatment groups (Figure 5A). Using Nrf2 transactivation assay, Nrf2 binding to ARE remained significantly higher in andrographolide-treated BEAS-2B cells 24 h after 2% CSE exposure (Figure 5B). Furthermore, cellular GSH level was also strongly elevated by andrographolide in BEAS-2B cells exposed to 2% CSE for 24 h (Figure 5C). Our data suggest that andrographolide is able to sustain nuclear Nrf2 accumulation and activation in cells exposed to cigarette smoke, leading to augmented production of GSH.

Andrographolide augments Nrf2-regulated gene targets

To investigate biological responses to nuclear Nrf2 accumulation and activation, we examined Nrf2-specific antioxidant gene expressions including glutamate-cysteine ligase catalytic (GCLC) subunit, glutamate-cysteine ligase modifier (GCLM) subunit, GR, GPx-2 and HO-1 in BEAS-2B cells exposed to 2% CSE for 12 h and 24 h. Andrographolide significantly enhanced the expression of antioxidants GCLM, GCLC, GR, GPx-2 and HO-1 in CSE-exposed BEAS-2B cells (Figure 6). Notably, andrographolide augmented gene expression of both GPx-2 and HO-1 by about 8- to 12-fold as compared with DMSO controls. Our results clearly indicate that andrographolide is a potent enhancer of antioxidant expression in response to cigarette smoke.

Discussion

Cigarette smoke contains high concentrations of potent oxidants and free radicals including reactive aldehyde, quinone, hydroquinone, semiquinone and superoxide (Cantin, 2010; Yao and Rahman, 2011). Exposure to cigarette smoke causes lung epithelial cell damage and triggers production of pro-inflammatory cytokines and chemokines, leading to inflammatory cell infiltration and activation, especially the neutrophils and macrophages. Both neutrophils and macrophages are rich sources of endogenous reactive oxygen species (ROS), reactive nitrogen species (RNS) and tissue proteases such as elastase and metalloproteinases, entailing additional oxidant burden to the lungs and facilitating destruction of alveolar walls. KC, GM-CSF and MIP-2α are pivotal for neutrophil lung infiltration and MCP-1 and IP-10 are essential for recruitment of macrophages and monocytes (Louhelainen et al., 2008; Cosio et al., 2009; Brusselle et al., 2011). Our findings reveal for the first time that andrographolide prevented cigarette smoke-induced lung infiltration of neutrophils and, to a lesser extent, macrophages in a dose-dependent manner and was able to suppress the increase of KC, IP-10, MCP-1, IL-1β, GM-CSF, TNF-α and MIP-2α in lungs from cigarette smoke-exposed mice.

We also observed a sharp rise in lung MMP-12 expression together with an adaptive increase in lung TIMP-1 expression in cigarette smoke-exposed mice. MMP-12 is able to degrade elastin, disrupting lung architecture leading to airspace enlargement (Mocchegiani et al., 2011). Mice deficient in MMP-12 were protected against cigarette smoke-induced lung inflammation and emphysema (Hautamaki et al., 1997). An association study has strongly suggested that MMP12 plays a role in determining lung function and susceptibility to COPD in adult smokers (Hunninghake et al., 2009). Andrographolide reversed the elevation of lung MMP-12 expression and the adaptive increase in TIMP-1 in cigarette smoke-exposed mice. These findings implicate a protective role of andrographolide against airway inflammation, remodelling and emphysema in COPD.

Oxidative stress emitted by the cigarette smoke and generated from the infiltrated macrophages and neutrophils play a pivotal role in the pathogenesis in COPD. Excessive accumulation of ROS and RNS in the lungs results in protein denaturation, lipid peroxidation and DNA damage as determined by the levels of 3-NT (Sugiura and Ichinose, 2011), 8-isoprostane (Louhelainen et al., 2008) and 8-OHdG (Tzortzaki et al., 2011; Aoshiba et al., 2012), respectively, in BAL fluid or in induced sputum. We observe that andrographolide completely blocked the increase of all three oxidant biomarkers in BAL fluid from cigarette smoke-exposed mice in a dose-dependent manner. The suppression of 3-NT level can be partly explained by the inhibitory effect of andrographolide on the iNOS expression, leading to a drop in NO production, peroxynitrite formation and protein nitration of tyrosine residues (Sugiura and Ichinose, 2011). Alternatively, andrographolide has also demonstrated potent antioxidant activity by enhancing both GPx and GR activities in lung tissues from cigarette smoke-exposed mice. GPx and GR play a critical role in generating GSH, an endogenous non-protein thiol with potent free radical scavenging capacity (Biljak et al., 2010; Cantin, 2010; Gould et al., 2011).

To investigate the molecular mechanism of antioxidant action for andrographolide, we studied the effects of andrographolide on nuclear translocation and activation of the redox-sensitive transcription factor Nrf2 and the gene expression of Nrf2-regulated antioxidant gene targets in CSE-treated BEAS-2B human bronchial epithelial cells. Nrf2 plays a predominant role in antioxidant protection against oxidative damage in the lungs incurred by cigarette smoking (Boutten et al., 2011). In line with other studies (Lee and Lee, 2011; Taguchi et al., 2011), cytoplasmic levels of Nrf2 in BEAS-2B cells in control and all treatment groups were almost undetectable. It is mainly due to rapid ubiquitination and proteasomal degradation of Nrf2 targeted by Kelch-like ECH-associated protein 1 (Keap1), a E3 ubiquitin ligase. Andrographolide strongly promoted Nrf2 nuclear stabilization and accumulation, and binding to ARE in BEAS-2B cells exposed to CSE for 24 h. Indeed, andrographolide has been shown to elevate Nrf2 nuclear level in human endothelial cells (Yu et al., 2010). Furthermore, in the process of developing a reporter gene assay for monitoring Nrf2 activation, andrographolide was found to be the most potent Nrf2 activator among 2000 biologically active compounds tested (Smirnova et al., 2011). In addition, andrographolide noticeably enhanced cellular level of GSH in BEAS-2B cells in response to CSE, providing a powerful first-line antioxidant defence against cigarette smoke-induced epithelial damage. Andrographolide has also been shown to promote GSH level in nicotine-stimulated peripheral blood lymphocytes (Das et al., 2009). Our findings strongly support the view that activation of Nrf2 pathway is an attractive therapeutic approach to the treatment of COPD (Boutten et al., 2011).

In patients with advanced COPD, Nrf2 activities are reduced in peripheral lungs, resulting in reduced antioxidant responses and persistent oxidative stress. The reduction of Nrf2 activity may be due to a drop in the Nrf2-positive regulator DJ-1 level, which permits destabilization of Nrf2 protein via rapid proteasomal degradation by Keap1 (Malhotra et al., 2008). In animals, Nrf2 gene disruption resulted in enhanced susceptibility to emphysema after cigarette smoke exposure (Rangasamy et al., 2004). Nrf2 activator such as sulforaphane was able to enhance Nrf2 antioxidant defence in response to cigarette smoke in DJ-1-disrupted human epithelial cells, indicating that Nrf2 is an effective target for COPD drug development (Malhotra et al., 2008). In the present study, we used acute cigarette smoke exposure and, instead of a drop in Nrf2 activity, we observed a slight promotion of nuclear Nrf2 level. Andrographolide further enhanced Nrf2 protein nuclear accumulation and activity, and Nrf2 antioxidant defence. To ensure therapeutic value of andrographolide in COPD, a chronic cigarette smoke-induced lung injury mouse model needs to be developed with clinical features similar to humans such as reductions in Nrf2 protein and activities, and the activator effect of andrographolide on Nrf2 antioxidant defence can then be evaluated in vivo.

In line with the Nrf2 activation by andrographolide in BEAS-2B cells, we have also observed strong inductions of Nrf2-regulated gene targets including GCLC, GCLM, GR, GPx-2 and HO-1 (Adair-Kirk et al., 2008; Hubner et al., 2009) by andrographolide in response to CSE. GCL is the rate-limiting enzyme in the GSH biosynthesis pathway. This enzyme is a heterodimer comprising of the catalytic subunit GCLC and the regulatory subunit GCLM (Biljak et al., 2010; Gould et al., 2011). Overexpression of GCL in human granulosa tumour cells has been shown to increase total GSH levels and protect against H₂O₂-induced cell death (Cortes-Wanstreet et al., 2009). In addition to being the most abundant cellular free radical scavenger, GSH also functions as a reducing substrate in the redox cycle to facilitate the reduction of H₂O₂ by GPx to H₂O and formation of glutathione disulphide (GSSG), to prevent peroxide-induced DNA damage, lipid peroxidation and protein degradation. GSSG is a substrate for GR to regenerate GSH to sustain the redox cycle (Biljak et al., 2010; Cantin, 2010). GPx and GR are important endogenous antioxidant enzymes responsible for the oxidative balance in the lungs in response to cigarette smoke (Biljak et al., 2010; Gould et al., 2011). It has been reported that GPx activity was markedly decreased in patients with COPD (Biljak et al., 2010). It is noteworthy that andrographolide was able to augment the GPx-2 expression level by at least 12-fold in BEAS-2B cells in response to CSE stimulation. The observed slow rate of GPx-2 gene induction starting at 24 h is consistent with that reported by Singh et al. (2006) that Nrf2 activation-induced GPx-2 expression occurred after 24 h and peaked at 72 h. The slow induction of GPx-2 might be due to the slow degradation rate of this protein (Chu et al., 1999). Our findings clearly indicate that activation of Nrf2 activity leading to up-regulation of GSH level is an antioxidant mechanism of action for andrographolide against cigarette smoking.

Aside from the GSH cycle, andrographolide also induced a rapid eightfold increase in HO-1 gene expression in BEAS-2B cells exposed to CSE within 12 h. Our findings are in line with those reported that HO-1 mRNA induction peaked at 6–8 h and returned to basal level at 24 h in human lung fibroblasts stimulated with CSE (Baglole et al., 2008) and in human bronchial epithelial cells treated with Nrf2 activator (Kumar et al., 2011). Under oxidative stress condition, free heme, a pro-oxidant catalysing production of free radicals, is released from hemoproteins to induce oxidative damages. HO-1 can be rapidly induced under the control of the Nrf2 transcription factor to catabolize heme into labile iron (Fe), carbon monoxide and biliverdin. The latter end product can be further converted by biliverdin reductase into bilirubin. All three reactive products have been shown to possess antioxidant and anti-inflammatory actions (Fredenburgh et al., 2007; Gozzelino et al., 2010). Patients with COPD showed reduced levels of HO-1 in BAL fluid alveolar macrophages (Fredenburgh et al., 2007). Adenovirus-mediated gene transfer of HO-1 in mice ameliorated elastase-induced inflammation and airspace enlargement (Shinohara et al., 2005). Polymorphisms of the HO-1 promoter associated with reduced HO-1 expression have been linked to increased susceptibility to emphysema development (Exner et al., 2004). The antioxidant actions of andrographolide against cigarette smoke-induced lung injury may also be linked to augmented HO-1 expression.

In conclusion, this study reveals for the first time that andrographolide can augment antioxidant gene expression and activities probably via up regulation of Nrf2 activity in experimental models of cigarette smoke exposure. The antioxidant mechanisms of action for andrographolide are summarized in Figure 7. In addition, inhibition of NF-κB is another well-established mechanism of action for andrographolide to bring about anti-inflammatory effects (Bao et al., 2009; Lim et al., 2012), which may contribute to some of the protective effects observed in the present study. The reduction in cigarette smoke-induced lung oxidative damage afforded by andrographolide is likely associated with the significant decline in BAL fluid inflammatory cell counts, in chemokine, cytokine and protease productions, and the up-regulation of GSH redox defence system and HO-1 expression. These findings support a novel therapeutic value for andrographolide in the treatment of COPD.

Conflicts of interest

None.