American Journal of Respiratory Cell and Molecular Biology

Exposure to ozone has been associated with airway inflammation, oxidative stress, and bronchial hyperresponsiveness. The goal of this study was to examine whether these adverse effects of ozone could be prevented or reversed by hydrogen sulfide (H2S) as a reducing agent. The H2S donor sodium (NaHS) (2 mg/kg) or vehicle (PBS) was intraperitoneally injected into mice 1 hour before and after 3-hour ozone (2.5 ppm) or air exposure, and the mice were studied 24 hours later. Preventive and therapeutic treatment with NaHS reduced the ozone-induced increases in the total cells, including neutrophils and macrophages; this treatment also reduced levels of cytokines, including TNF-α, chemokine (C-X-C motif) ligand 1, IL-6, and IL-1β levels in bronchial alveolar lavage fluid; inhibited bronchial hyperresponsiveness; and attenuated ozone-induced increases in total malondialdehyde in bronchoalveolar lavage fluid and decreases in the ratio of reduced glutathione/oxidized glutathione in the lung. Ozone exposure led to decreases in the H2S production rate and in mRNA and protein levels of cystathionine-β-synthetase and cystathionine-γ-lyase in the lung. These effects were prevented and reversed by NaHS treatment. Furthermore, NaHS prevented and reversed the phosphorylation of p38 mitogen–activated protein kinase and heat shock protein 27. H2S may have preventive and therapeutic value in the treatment of airway diseases that have an oxidative stress basis.

The present study demonstrates that NaHS, an exogenous H2S donor, inhibits ozone-induced airway inflammation and bronchial hyperresponsiveness and protects the lungs against oxidative stress. H2S may have preventive and therapeutic value in the treatment of airway diseases that have an oxidative stress basis.

Ozone is a common air pollutant formed in the atmosphere through photochemical reactions of hydrocarbons and the oxides of nitrogen, both of which are released by motor vehicles and other combustion sources. High levels of ambient ozone have been linked to the worsening of symptoms and increased hospitalizations of patients with asthma (1) and chronic obstructive pulmonary disease (COPD) (2). Experimental ozone exposure at high concentrations can induce bronchial hyperresponsiveness (BHR) to bronchoconstrictor agents and neutrophilic airway inflammation in humans and animal species (3, 4). The mechanism underlying ozone-induced inflammation and BHR involves oxidative stress via the combined actions of ozone and its oxidation products (5, 6).

Oxidative stress, due to the excessive release of reactive oxygen species from inflammatory cells, immune cells, and structural cells in the airway, occurs in the airways and lungs of patients with asthma and COPD (7). Oxidative stress can lead to the amplification of inflammation and the induction of BHR. We previously showed that the adaptor protein MyD88 (which mediates the effects of Toll-like receptors [TLR2 and TLR4]) might be involved (8), and more recently, the p38 mitogen–activated protein kinase (MAPK) pathway was found to be important not only to the neutrophilic inflammatory response but also to the augmented contractile response induced by ozone (911).

Hydrogen sulfide (H2S), a novel endogenous signaling gasotransmitter (12), is generated from l-cysteine mainly by two enzymes, cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), both of which are present in most mammalian tissues, including the lungs. Accumulating data have indicated that H2S is implicated in various physiological and pathological processes of the respiratory system (1317). In clinical studies, serum H2S levels in patients with asthma or COPD have been associated with disease activity and severity (13, 14). It has been suggested that H2S could play antiinflammatory and antiremodeling roles in asthma (15) and COPD models (16, 17). For example, in an ovalbumin-induced rat model of asthma, sodium H2S (NaHS) administration attenuated airway inflammation and remodeling (15), whereas in a mouse model of tobacco smoke–induced emphysema, NaHS reduced tobacco smoke–induced airway inflammation, prevented the development of emphysema and pulmonary hypertension (16), and inhibited bronchial responsiveness (17).

To our knowledge, there have been no studies of the effects of H2S on ozone-induced airway inflammation and BHR. In the present study, H2S was administered exogenously before (preventive) and after (therapeutic) ozone exposure via an injection of NaHS, which has been widely studied as a donor of H2S. We evaluated the preventive and therapeutic effects of H2S on ozone-induced airway inflammation, BHR, and oxidative stress in a mouse model. We also determined the underlying intracellular signaling pathways that might be affected by H2S.

A complete description is available in the online supplement. All experimental procedures involving animals were approved by the Animal Ethics Committee of the institute.

Mice and Ozone Exposure

Ten-week-old male C57/BL6 mice (Harlan, Blackthorn, UK) were exposed to ozone mixed with air at a concentration 2.5 ppm for 3 hours. Control animals were exposed to air only. Mice were injected intraperitoneally with NaHS (2 mg/kg) or vehicle 1 hour before and after ozone or air exposure.

Bronchial Responsiveness

Twenty-four hours after exposure, mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and then transferred to a plethysmograph for the measurement of resistance and compliance (EMMS, Hants, UK) (8). The concentration of acetylcholine (ACh) required to increase lung resistance by 200% from baseline was calculated (PC200), and –log PC200 was taken as a measure of bronchial responsiveness.

Bronchoalveolar Lavage Fluid and Cell Counting

After anesthesia with an overdose of pentobarbitone, mice were lavaged with two 0.8-ml aliquots of PBS, and bronchoalveolar lavage (BAL) fluid was retrieved. The supernatant was stored at −80°C for further analysis. Total and differential cell counts were determined under a microscope.

Malondialdehyde and Cytokines in BAL Fluid

Concentrations of free and total malondialdehyde (MDA) in BAL fluid were measured using a HPLC system with fluorescent detection (18, 19). Total MDA included MDA molecules (free MDA) and MDA bound to proteins and other biomolecules.

Levels of chemokine (C-X-C motif) ligand 1 (CXCL1, KC), TNF-α, IL-6, and IL-1β in BAL fluid were measured using ELISA (R&D Systems, Abingdon, UK).

Analysis of Reduced and Oxidized Glutathione in Lung Tissue

The reduced/oxidized glutathione (GSH/GSSG) ratio in lung tissue was measured using the GSH/GSSG-412 kit (Oxis Research, Inc., Foster City, CA).

CBS and CSE Gene Expression

Total RNA in the lung was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA), and the cDNA was synthesized using the energicScriptcDNA synthesis kit (ShineGene Co., Ltd., Shanghai, China). Quantitative real-time PCR was conducted in triplicate using SYBR Green Supermix in the FTC-2000 sequence detection system (Funglyn Biotech, Inc., Toronto, ON, Canada). The β-actin gene was used as control.

H2S Production Assay

H2S production rate was measured using methylene blue assay (20).

Western Blot Analysis

Lung tissues were homogenized and qualified by bicinchoninic acid assay analysis. Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membrane and then incubated with primary antibodies against CBS and CSE (Novus Biologicals, Littleton, CO), glyceraldehyde 3-phosphate dehydrogenase, phospho-p38 MAPK, total-p38 MAPK, phospho-HSP27, and total-HSP27 (Cell Signaling Technology, Beverly, CA) for blot detection.

Statistical Analysis

Data are expressed as mean ± SEM. The statistical analysis was performed using SPSS 20.0 software (IBM Corp., Armonk, NY). Two-way ANOVA was performed for comparisons of %change in lung resistance between individual groups. One-way ANOVA with Bonferroni’s post hoc test (for equal variance) or Dunnett’s T3 post hoc test (for unequal variance) was performed for comparisons among multiple groups. P < 0.05 was considered significant.

Bronchial Responsiveness

There were no significant differences in baseline lung resistance values after PBS challenge in the experimental groups. After a single ozone exposure, PBS-pretreated ozone-exposed mice and PBS-treated ozone-exposed mice demonstrated a leftward shift of the concentration–response curve (Figures 1A and 1C), indicating an increase in bronchial responsiveness to ACh compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice (−logPC200: 1.91 ± 0.10 versus 2.46 ± 0.03, P < 0.001 [Figure 1B]; −logPC200: 1.78 ± 0.07 versus 2.35 ± 0.06, P < 0.001 [Figure 1D]). Pretreatment and treatment with NaHS did not affect bronchial responsiveness to ACh in air-exposed mice. Both preventive treatment and therapeutic treatment with NaHS inhibited the bronchial responsiveness of ozone-exposed mice compared with PBS-pretreated ozone-exposed mice and PBS-treated ozone-exposed mice (−logPC200: 2.35 ± 0.06 versus 1.91 ± 0.10, P < 0.01 [Figure 1B]; −logPC200: 2.33 ± 0.06 versus 1.78 ± 0.07, P < 0.001 [Figure 1D]).

BAL Cells

PBS-pretreated ozone-exposed mice and PBS-treated ozone-exposed mice demonstrated increases in total cell counts, macrophages, and neutrophils but not in lymphocytes or eosinophils in BAL fluid compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice (P < 0.01, P < 0.05, and P < 0.001, respectively [Figure 2A]; P < 0.001, P < 0.001, and P < 0.001, respectively [Figure 2B]). There were no effects of NaHS on BAL cells in air-exposed mice. NaHS pretreatment and NaHS treatment significantly reduced total cell counts, including macrophages and neutrophils in ozone-exposed mice, compared with PBS-pretreated ozone-exposed mice and PBS-treated ozone-exposed mice (Figures 2A and 2B).

BAL Cytokine Levels

Ozone exposure in PBS-pretreated mice and PBS-treated mice evoked significant increases in KC (P < 0.001 and P < 0.001 [Figures 3A and 3E]), TNF-α (P < 0.001 and P < 0.001 [Figures 3B and 3F]), IL-6 (P < 0.001 and P < 0.001 [Figures 3C and 3G]), and IL-1β (P < 0.001 and P < 0.001 [Figures 3D and 3H]) compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice. With NaHS treatment, the NaHS-pretreated air-exposed mice and the NaHS-treated air-exposed mice showed similar levels of cytokines compared with the PBS-pretreated air-exposed mice and the PBS-treated air-exposed mice (Figures 3A–3H), whereas NaHS-pretreated ozone-exposed mice and NaHS-treated ozone-exposed mice showed decreased levels of KC, TNF-α, IL-6, and IL-1β compared with PBS-pretreated ozone-exposed mice and PBS-treated ozone-exposed mice.

BAL MDA

Ozone exposure increased the total MDA concentrations in BAL fluid in the PBS-pretreated mice compared with PBS-pretreated air-exposed mice (P < 0.05 [Figure 4B]). Ozone exposure increased free MDA and total MDA in BAL fluid in the PBS-treated mice compared with the PBS-treated air-exposed mice (P < 0.05; P < 0.05 [Figures 4D and 4E]). NaHS-pretreated air-exposed mice and NaHS-treated air-exposed mice showed similar levels of free MDA and total MDA compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice (Figures 4A, 4B, 4D, and 4E). Pretreatment with NaHS significantly reduced total MDA concentrations in BAL (P < 0.01 [Figure 4B]) in ozone-exposed mice compared with PBS-pretreated ozone-exposed mice, whereas treatment with NaHS significantly reduced BAL free MDA concentrations (P < 0.05 [Figure 4D]) and total MDA concentrations (P < 0.05 [Figure 4E]) in ozone-exposed mice compared with PBS-treated ozone-exposed mice.

GSH/GSSG Ratio in the Lung

Ozone exposure significantly reduced the GSH/GSSG ratio in PBS-pretreated mice and PBS-treated mice compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice (P < 0.001 and P < 0.001 [Figures 4C and 4F]). Pretreatment and treatment with NaHS did not affect the GSH/GSSG ratio in air-exposed mice compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice. Preventive and therapeutic treatment with NaHS significantly prevented and reversed the GSH/GSSG ratio in ozone-exposed mice compared with PBS-pretreated ozone-exposed mice and PBS-treated mice ozone-exposed (P < 0.001 and P < 0.01 [Figures 4C and 4F]).

CSE and CBS mRNA and Protein Levels in Lung Tissue

Ozone exposure decreased the mRNA expression of CSE and CBS in PBS-pretreated and PBS-treated mice compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice (P < 0.001 and P < 0.001, respectively [Figures 5A and 5C]; P < 0.001 and P < 0.001, respectively [Figures 5B and 5D]). Pretreatment and treatment with NaHS did not affect the mRNA expression of CSE and CBS in air-exposed mice compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice. After preventive and therapeutic treatment with NaHS, the mRNA levels of CSE and CBS in ozone-exposed mice were significantly increased compared with PBS-pretreated ozone-exposed mice and PBS-treated ozone-exposed mice (P < 0.001 and P < 0.001, respectively [Figures 5A and 5C]; P < 0.05 and P < 0.05, respectively [Figures 5B and 5D]).

Ozone exposure significantly decreased the protein expression of CSE and CBS in PBS-pretreated mice and PBS-treated mice compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice (P < 0.01 and P < 0.001, respectively [Figures 5E and 5G]; P < 0.001 and P < 0.001, respectively [Figures 5F and 5H]). The protein levels of CSE and CBS in air-exposed mice were not significantly affected by pretreatment and treatment with NaHS. NaHS treatment prevented and reversed the decreases in the protein levels of CSE and CBS in ozone-exposed mice compared with PBS-pretreated ozone-exposed mice and PBS-treated ozone-exposed mice (P < 0.01 and P < 0.05, respectively [Figures 5E and 5G]; P < 0.05 and P < 0.05, respectively [Figures 5F and 5H]).

H2S Production Rate in Lung Tissue

Ozone exposure inhibited the production rate of H2S in the lung tissue of PBS-pretreated mice and PBS-treated mice compared with that of PBS-pretreated air-exposed mice and PBS-treated air-exposed mice (P < 0.01 [Figure 6A]; P < 0.01 [Figure 6B]). Pretreatment and treatment with NaHS did not affect the production rate of H2S in air-exposed mice, but it increased the production of H2S in ozone-exposed mice compared with PBS-pretreated ozone-exposed mice and PBS-treated ozone-exposed mice (P < 0.001 [Figure 6A]; P < 0.01 [Figure 6B]).

p38 MAPK/HSP27 Phosphorylation

Ozone exposure resulted in a significant induction of p38 MAPK and HSP27 phosphorylation in PBS-pretreated mice and PBS-treated mice compared with PBS-pretreated air-exposed mice and PBS-treated air-exposed mice (P < 0.001 and P < 0.001, respectively [Figures 7A and 7C]; P < 0.001 and P < 0.01, respectively [Figures 7B and 7D]). The phosphorylation levels of p38 MAPK or HSP27 in air-exposed mice were not affected by pretreatment and treatment with NaHS. However, the increased phosphorylation of p38MAPK/HSP27 in ozone-exposed mice was attenuated by pretreatment and treatment with NaHS compared with PBS-pretreated ozone-exposed mice and PBS-treated ozone-exposed mice (P < 0.001 and P < 0.001, respectively [Figures 7A and 7C]; P < 0.001 and P < 0.05, respectively [Figures 7B and 7D]).

In the present study, we found that the H2S donor NaHS, administered before and after ozone exposure, inhibited ozone-induced airway inflammation and BHR and reduced oxidative stress. Furthermore, NaHS prevented and reversed ozone-induced decreases in the expression of CBS and CSE and in the H2S production rate in lung tissue. More importantly, preventive and therapeutic treatment with NaHS inhibited ozone-induced phosphorylation of p38 MAPK and HSP27. Hence, we conclude that H2S protects the lungs against oxidative stress via the antioxidant activity of H2S and by its inhibitory effects on ozone-activated p38 MAPK/HSP27 pathways.

H2S is known as a highly toxic gas that can do serious harm to human health. However, H2S has also been considered a third physiological gaseous mediator after CO and NO. The role of H2S in inflammation is controversial. H2S has been shown to be proinflammatory in various animal models of inflammation, such as acute pancreatitis and associated lung injury (21), endotoxic shock (22), and carrageenan-induced hindpaw edema (23). However, experimental studies have also indicated that H2S has antiinflammatory effects in acute lung injury (24, 25), COPD (16, 17), asthma (15, 26), and pulmonary hypertension (27). These conflicting observations might be due to the multifaceted role of H2S in inflammation and to differences in experimental conditions and the animal models used. In our acute ozone exposure model, we observed that preventive and therapeutic treatment with NaHS reduced the inflammatory cells as well as the release of cytokines, including KC, TNF-α, IL-6, and IL-1β, in the BAL fluid, indicating that H2S had protective antiinflammatory effects.

BHR is a state characterized by a heightened bronchoconstrictor response to a variety of stimuli. BHR is a hallmark of asthma, and it is also a well-recognized effect of ozone exposure, likely resulting from an increase in airway smooth muscle contractility (10). The neutrophil accumulation induced by ozone exposure also contributes to BHR (28, 29). KC and IL-6 are two important chemoattractants for neutrophil recruitment (8, 30), whereas TNF-α and IL-1β play roles in the development of BHR (3133). Our results showed that NaHS inhibited ozone-induced BHR, which was correlated with reduced proinflammatory cytokine production and decreased neutrophilic inflammation.

Ozone exposure induced oxidative stress in mice, indicated by the increased total MDA in the BAL fluid and the decreased GSH/GSSG ratio in the lung tissue. MDA is a reliable oxidative damage marker of lipid peroxidation and could play an important role in the early inflammatory response to ozone (34). GSH is an abundant soluble thiol in cells that protects against oxidative stress by acting as a substrate for glutathione peroxidase and by scavenging free radicals (35). The ratio of GSH/GSSG has been regarded as a marker of oxidative stress (36). In our study, after acute ozone exposure, the antioxidant defense system in the mice was impaired and oxidative stress occurred, as reflected by the decrease in the GSH/GSSG ratio in lung tissues. Our results showed that pretreatment and treatment with NaHS prevented and reversed the ozone-induced increase in MDA and the decrease in the GSH/GSSG ratio. These findings are most likely attributable to the antioxidative capability of NaHS. As a small gaseous molecule, H2S could freely cross the plasma membrane and the mitochondrial membrane to scavenge reactive oxygen species and reactive nitrogen species (37). H2S could promote the production of reduced GSH by enhancing cystine/cysteine transporters and redistributing GSH to mitochondria (38). In addition, H2S could increase the activity of antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and glutathione reductase (39).

In the present study, we investigated the signaling mechanisms by which H2S could inhibit ozone-induced inflammation, oxidative stress, and BHR. We previously showed that the p38 MAPK pathway was rapidly activated after ozone exposure and that this activation subsequently played an important role in the development of cytokine production, airway inflammation, and BHR (11). Moreover, activated p38 MAPK might be involved in the increased airway smooth muscle contractile responses after ozone challenge via p38 MAPK–dependent phosphorylation of the actin-binding protein HSP27 (10). Based on these previous studies, we explored the influence of H2S on the phosphorylation of p38 MAPK and HSP27 in an ozone model. The results of the present study showed that preventive and therapeutic treatment with NaHS inhibited the phosphorylation of p38 MAPK and HSP27 induced by ozone, suggesting that H2S suppressed ozone-activated p38 MAPK and HSP27 pathways, which might be one of important mechanisms underlying the protective effects of H2S against ozone-induced airway inflammation and BHR. Our results are supported by previous studies that show that H2S can inhibit p38 MAPK activity, particularly in airway smooth muscle cells (40).

We also investigated the effects of ozone exposure on the mRNA and protein levels of the enzymes CBS and CSE, which are important to the synthesis of H2S in the body. The study showed that ozone exposure reduced the mRNA and protein levels of CBS and CSE in lung tissue, consistent with previous studies by other investigators (16, 24). The decreased expression of CBS and CSE was related to the decreased H2S production rate in the lung tissue. This finding could indicate that the level of H2S might be low in lung tissue, although we did not measure the concentrations of H2S in the serum or lung tissue. We also found that the exogenous administration of NaHS could prevent and reverse the decreased expression of CBS and CSE in the lungs, which is important for maintaining the production of H2S.

Our findings demonstrate that NaHS, an exogenous H2S donor, inhibits ozone-induced airway inflammation and BHR and protects the lungs against oxidative stress. These effects could be attributed, at least in part, to the antioxidant effects of H2S and its capacity to suppress the p38 MAPK/HSP27 pathway. These findings suggest that H2S may be a promising preventive and therapeutic agent for inflammatory airway diseases that have an oxidative stress basis.

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*These authors contributed equally to this work.

Correspondence and requests for reprints should be addressed to Xin Zhou, M.D., Department of Respiratory Medicine, Shanghai First People’s Hospital, Shanghai Jiao Tong University, No. 100, Haining Road, Shanghai, 200080, China. E-mail:

This work was supported by grant no. 81100024 from the National Natural Science Foundation of China and by grant no. 2011274 from the Shanghai Health Bureau.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2013-0415OC on July 10, 2014

Author disclosures are available with the text of this article at www.atsjournals.org.

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