H2 and Nitric Oxide Combo Therapy for Lung InjuryScientific Research

INTRODUCTION

Acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), represent a spectrum of common syndromes in critically ill patients with a mortality rate of 30% to 50% (^{1, 2}). Excessive cytokine-mediated inflammation plays a fundamental role in the pathogenesis of ALI (³). Moreover, nuclear factor κB (NF-κB) is a critical transcription factor required for maximal expression of many cytokines involved in the pathogenesis of ALI (⁴).

Recently, it is widely accepted that nitric oxide (NO) might exert an effective therapeutic role in ALI. However, it is well known that NO can produce both desirable and undesirable effects (^{5, 6}): NO has been reported to have both anti-inflammatory and cytocidal effects. The anti-inflammatory effects are presumably mediated by inhibition of platelet and neutrophil activation via enhanced guanylate cyclase (⁷) and/or poly(ADP-ribose) polymerase activity (⁸). The cytocidal effects are presumably mediated by reactive nitrogen species (RNS), such as peroxynitrite generation (⁹). In 2007, Ohsawa et al. first reported that hydrogen gas (H₂) has the potential to act as an antioxidant via selectively reducing the levels of hydroxyl radicals (•OH) and peroxynitrite (ONOO⁻) (¹⁰). Our studies have shown that H₂ inhalation significantly improves the survival rate and lung damage of septic mice (^{11, 12}). Moreover, we found that H₂ treatment also ameliorates the lipopolysaccharide (LPS)–induced ALI (¹²). Besides, some reports show that H₂ treatment can improve the hyperoxia- or ventilator-induced lung injury through reducing inflammation and oxidation (^14–16).

Thus, we hypothesized that the inhibitory effect of NO on inflammation may be enhanced by eliminating highly reactive by-products of NO inhalation, such as peroxynitrite, by adding H₂ to inhaled NO gas (¹⁷). The aim of the present study was to determine whether inhalation of NO combined with H₂ might be more effective in reducing lung injury and improving pulmonary inflammation in a murine model of LPS-induced ALI in comparison to inhalation of NO or H₂ alone. This study might establish a clinically applicable strategy for the treatment of ALI and provide a new avenue for the use of therapeutic gas in patients.

MATERIALS AND METHODS

Animals

Adult male C57BL/6 mice weighing 20 to 25 g were provided by the Laboratory Animal Center of the Academy of Military Medical Sciences in Beijing, China. Animals were housed under specific pathogen-free conditions at 20°C to 22°C with a 12:12-h light-dark cycle. Standard animal chow and water were freely available. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University and performed in accordance with the National Institutes of Health (Bethesda, Md) guidelines for the use of experimental animals.

Lipopolysaccharide-induced ALI

As described in previous study (^{13, 18, 19}), ALI was induced by intratracheal (i.t.) administration of LPS. Briefly, animals were anesthetized with sevoflurane. They were orally intubated with a sterile plastic catheter and intratracheally given a single dose of aerosolized LPS (25 μg/mouse; Escherichia coli 0111:B4; Sigma-Aldritch, St. Louis, Mo). Control mice were intratracheally given 50 μL of sterile phosphate-buffered saline (PBS).

Polymicrobial sepsis

Polymicrobial sepsis was induced by cecal ligation and puncture (CLP) as described in our previous study (¹¹). Briefly, we anesthetized mice deeply by intraperitoneal injection of 50 mg/kg pentobarbital sodium. We exposed the cecum by a 1-cm abdominal midline incision and subjected it to ligation below the ileocecal valve and a single through-and-through perforation of the ligated segment. We ligated the distal one half of the cecum and made a single puncture with a 21-gauge needle. A small amount of stool was extruded through the puncture site. We then replaced the cecum into the abdomen and closed the incision using a sterile 6-0 silk suture. One milliliter of prewarmed sterile saline was administered subcutaneously for fluid resuscitation. Animals with sham operation underwent the same procedure without CLP.

Hydrogen gas or NO treatment

According to our previous studies (^{11, 12, 20}), the animals were put in a sealed Plexiglas chamber with inflow and outflow outlets. Hydrogen gas or NO was supplied through a gas flowmeter respectively and delivered by air into the chamber through a tube at a rate of 4 L/min. The concentration of H₂ or NO in the chamber was continuously monitored with a commercially available detector and maintained during the treatment, respectively. Carbon dioxide was removed from the chamber gases with Baralyme. The animals without H₂ or NO treatment were exposed to room air in the chamber.

Experimental design

Experiment 1: effects of H₂ and/or NO treatment on LPS-induced ALI in mice

Eighty animals were randomly divided into five groups (n = 16 per group): PBS, LPS, LPS + NO, LPS + H₂, and LPS + NO + H₂ groups. The animals with NO and/or H₂ treatment were exposed to 20 ppm NO and/or 2% H₂ inhalation for 3 h starting at 5 min after i.t. administration of LPS, respectively. As a control, the animals from the PBS and LPS groups were given the treatment without NO and H₂ inhalation at the same time points. In this experiment, the oxygenation index (ratio of oxygen tension to inspired oxygen fraction [Pao₂/Fio₂]) was measured at 24 h after PBS or LPS administration. Moreover, the bronchoalveolar lavage fluid (BALF) was obtained for measuring the protein concentration as well as the number of total cells and polymorphonuclear neutrophils (PMNs) at 4 and 24 h after PBS or LPS administration (n = 6 per group at each time point). In addition, the lung samples were removed for evaluating the histopathology (n = 10 per group at 24 h), wet-to-dry (W/D) weight ratio (n = 6 per group at 24 h), and myeloperoxidase (MPO) activity (n = 6 per group at each time point).

Experiment 2: the underlying mechanisms of H₂ and/or NO treatment in LPS-induced ALI

Additional 60 animals were used in this experiment and were randomly assigned to five groups (n = 12 per group). The grouping method and experimental protocols were the same as in experiment 1. At 4 and 24 h after PBS or LPS administration, the inflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin 1β [IL-1β], IL-6, high-mobility group box 1 [HMGB1], IL-10) and chemokines (keratinocyte-derived chemokine [KC], macrophage inflammatory protein 1α [MIP-1α], macrophage inflammatory protein 2 [MIP-2], monocyte chemoattractant protein 1 [MCP-1]) in the BALF were measured (n = 6 per group at each time point). In addition, the lung samples were harvested for measuring the caspase 3 activity and NF-κB p65 DNA-binding activity.

Experiment 3: effects of H₂ and/or NO treatment beginning at 3 h after LPS administration on ALI in mice

Additional 50 animals were randomly assigned to five groups (n = 10 per group). The grouping method was the same as in experiment 1. The animals with NO and/or H₂ treatment were exposed to 20 ppm NO and/or 2% H₂ inhalation for 3 h starting at 3 h after LPS administration, respectively. As a control, the animals from the PBS and LPS groups were given the treatment without NO and H₂ inhalation at the same time points. In this experiment, the oxygenation index (Pao₂/Fio₂) and lung histopathology were measured at 24 h after PBS or LPS administration.

Experiment 4: effects of H₂ and/or NO treatment on polymicrobial sepsis–induced ALI in mice

Additional 50 animals were randomly assigned to five groups (n = 10 per group): sham, CLP, CLP + NO, CLP + H₂, and CLP + NO + H₂ groups. The animals with NO and/or H₂ treatment were exposed to 20 ppm NO and/or 2% H₂ inhalation for 3 h starting at 6 h after CLP operation, respectively. As a control, the animals from the sham and CLP groups were given the treatment without NO and H₂ inhalation at the same time points. In this experiment, the oxygenation index (Pao₂/Fio₂) and lung histopathology were measured at 24 h after sham or CLP operation.

Experiment 5: effects of subthreshold concentrations of H₂ and/or NO treatment on LPS-induced ALI in mice

Additional 50 animals were randomly assigned to five groups (n = 10 per group). The grouping method was the same as experiment 3. The animals with NO and/or H₂ treatment were exposed to 5 ppm NO and/or 1% H₂ inhalation for 3 h starting at 3 h after LPS administration, respectively. As a control, the animals from the PBS and LPS groups were given the treatment without NO and H₂ inhalation at the same time points. In this experiment, the oxygenation index (Pao₂/Fio₂) and lung histopathology were measured at 24 h after PBS or LPS administration.

Experiment 6: effects of subthreshold concentrations of H₂ and/or NO treatment on polymicrobial sepsis–induced ALI in mice

Additional 50 animals were randomly assigned to five groups (n = 10 per group). The grouping method was the same as in experiment 4. The animals with NO and/or H₂ treatment were exposed to 5 ppm NO and/or 1% H₂ inhalation for 3 h starting at 6 h after CLP operation, respectively. As a control, the animals from the sham and CLP groups were given the treatment without NO and H₂ inhalation at the same time points. In this experiment, the oxygenation index (Pao₂/Fio₂) and lung histopathology were measured at 24 h after sham or CLP operation.

Oxygenation index analysis

To evaluate the oxygenation capability of the lung, Pao₂/Fio₂ was calculated. At 24 h of PBS or LPS administration, animals were anesthetized and given endotracheal intubation with a 20-gauge catheter. They were subjected to mechanical ventilation with pure oxygen at 7 mL/kg. The respiratory rate was 120 breaths/min. The animals were ventilated for 15 min before blood gas sampling. The arterial blood was obtained from carotid artery and measured with a GEM Premier 3000 gas analyzer (Instrumentation Laboratory, Milan, Italy).

Cell counts and protein concentration in BALF

Animals were subjected to bronchoalveolar lavage for collecting BALF by the methods described previously (²¹). Bronchoalveolar lavage fluid was obtained by cannulating the trachea with a 20-gauge catheter. Two volumes of 0.5 mL of PBS (pH 7.4) were instilled, gently aspirated, pooled, and reaspirated. Lavage samples were centrifuged at 1,500g for 10 min at 4°C. The supernatant was stored at −20°C. Furthermore, the cell pellet was resuspended in PBS, and subsequently, the number of total cells was determined using a hemocytometer (Beckman Coulter, Inc, Fullerton, Calif). The slides were visualized using Wright-Giemsa staining (Fisher Scientific Co, Middletown, Va), and PMNs were identified by a certified laboratory technologist in a blinded fashion. Total protein concentration in the BALF was determined using a standard commercial kit (Bio-Rad Laboratories, Hercules, Calif).

Histologic examination

Lungs were harvested for observing morphologic alterations at 24 h after PBS or LPS administration. The samples were fixed with 10% formalin for 6 h at room temperature, embedded in paraffin, and sectioned at 5-μm thickness. After deparaffinization and rehydration, the sections were stained with hematoxylin-eosin. Histologic changes were evaluated by two pathologists who were blinded to the treatment regimen. A scoring system to grade the degree of lung injury was used, based on the following histologic features: edema, hyperemia and congestion, neutrophil margination and tissue infiltration, intra-alveolar hemorrhage and debris, and cellular hyperplasia. Each feature was graded as absent, mild, moderate, or severe, with a score of 0 to 3. A total score was calculated for each animal (¹⁰).

W/D weight ratio

To quantify the magnitude of pulmonary edema, we evaluated lung W/D weight ratio. The harvested wet lung was weighed and then placed in an oven for 24 h at 80°C and weighed when it was dried. The ratio of wet lung to dry lung was calculated (¹¹).

MPO activity

At 4 and 24 h after PBS or LPS administration, lungs were obtained and perfused with cold PBS to remove all blood, then weighed and stored at −80°C for no more than 1 week before the MPO assay. The supernatant from lung homogenate was prepared for detecting MPO activity (¹¹). Myeloperoxidase activity was defined as the quantity of enzyme degrading 1 μmol of peroxide per minute at 37°C and was expressed in unit per gram weight of wet tissue. The change in absorbance was measured spectrophotometrically at 590 nm by spectrophotometer (DU 640B; Beckman Coulter, Inc).

Caspase 3 activity

Lung homogenates were prepared, and caspase 3 activity was measured with caspase 3/CPP32 fluorometric assay kit (Biovision, Inc, Mountain View, Calif) in accordance with the manufacturer’s instructions (²²). The assay was run in duplicate.

Enzyme-linked immunosorbent assay

The cytokines and chemokines in the BALF were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits (mouse TNF-α, IL-1β, IL-6, IL-10, KC, MIP-1α, MIP-2, and MCP-1 ELISA kits are from R&D Systems [Minneapolis, Minn]; HMGB1 ELISA kit is from IBL [Hamburg, Germany]). All spectrophotometric readings were performed by a microplate reader (CA 94089; Molecular Devices, Sunnyvale, Calif). All experiments were performed according to the manufacturers’ instructions (^{11, 12}).

NF-κB activity

The DNA-binding activity of NF-κB in lung tissues was quantified by ELISA, using the TransAM NF-κB p65 transcription factor assay kit (Active Motif, Carlsbad, Calif). The nuclear extracts of lung tissues were prepared with a nuclear extract kit (Active Motif). According to the manufacturer’s instructions, all standards and samples were run in duplicate (²³).

Statistical analysis

All values, except for histologic scores, are presented as mean ± SEM. The histologic scores were analyzed with Kruskal-Wallis test followed by the Mann-Whitney U test with Bonferroni correction. The intergroup differences of the rest data were tested by one-way analysis of variance followed by least significant difference–t test for multiple comparisons. The statistical analysis was performed with SPSS 16.0 software (SPSS Inc, Chicago, Ill). In all tests, P < 0.05 was considered statistically significant.

RESULTS

Combination therapy with H₂ and NO attenuated LPS-induced lung injury in mice

In the present study, we first investigated the effects of 2% H₂ or 20 ppm NO inhalation on lung histopathology and function in mice with LPS challenge (Fig. 1). Lipopolysaccharide-challenged mice appeared to have significant lung injury characterized by alveolar wall thickening, infiltration of neutrophils into lung interstitium and alveolar space, and consolidation and alveolar hemorrhage. Hydrogen gas and/or NO treatment resulted in a reduction of infiltrated inflammatory cells and a marked improvement in lung architecture. Moreover, a scoring system to grade the degree of lung injury was used. Lipopolysaccharide-challenged mice showed significant increase in lung histologic scores (P < 0.05 vs. PBS group, n = 10 per group; Fig. 1A), which was reduced by H₂ or NO treatment (P < 0.05, n = 10 per group; Fig. 1A). Meanwhile, LPS-challenged mice showed significant increase in lung W/D ratio when compared with PBS group, which was also decreased by H₂ or NO treatment (P < 0.05, n = 6 per group; Fig. 1B). Interestingly, the Pao₂/Fio₂ was significantly decreased in LPS-challenged mice, which was improved by H₂ or NO treatment (P < 0.05, n = 6 per group; Fig. 1C). Furthermore, combination therapy with H₂ and NO could more effectively attenuate LPS-induced lung injury in mice (Fig. 1). In addition, to exclude that H₂ and/or NO inhalation might cause hypoxia in mice with LPS or PBS administration, the arterial blood gas was measured in all groups during H₂ and/or NO treatment. There were no differences in the levels of arterial pH, Pao₂, and Paco₂ among all groups (see Table, Supplemental Digital Content 1, at https://links.lww.com/SHK/A262). These results demonstrate that H₂ or NO treatment significantly improves lung histopathology and lung function in LPS-challenged mice, whereas combination therapy with H₂ and NO can more effectively attenuate LPS-induced lung injury.

Combination therapy with H₂ and NO reduced the cells and protein in the BALF of LPS-challenged mice

As shown in Figure 2, LPS-challenged mice showed the significant increase in total cells, PMNs, and total protein in the BALF at 4 and 24 h (P < 0.05 vs. PBS group, n = 6 per group), which were markedly reduced by H₂ or NO treatment alone (P < 0.05, n = 6 per group). Moreover, both H₂ and NO treatment together could more significantly reduce the total cells, PMNs, and total protein in the BALF of LPS-challenged mice. These data further indicate that combination therapy with H₂ and NO can more effectively attenuate LPS-induced lung inflammation and injury in mice.

Combination therapy with H₂ and NO decreased LPS-induced neutrophil recruitment into the lungs

We also detected the lung MPO activity, an indicator of neutrophil infiltration, at 4 and 24 h after PBS or LPS administration (Fig. 2). The lung MPO activity of LPS-challenged mice dramatically increased (P < 0.05 vs. PBS group, n = 6 per group), which was also inhibited by H₂ and/or NO treatment (P < 0.05, n = 6 per group). These results suggest that combination therapy with H₂ and NO can more effectively attenuate lung inflammation in LPS-challenged mice.

Combination therapy with H₂ and NO ameliorated the nitrotyrosine in lung tissues of LPS-challenged mice

Reactive nitrogen species generation mediated by NO inhalation, such as peroxynitrite, has cytocidal effects. In this study, we found that the nitrotyrosine level was significantly increased in the lungs of LPS-challenged animals, which was further increased in the animals after NO inhalation treatment (P < 0.05, n = 6 per group; Fig. 3). However, H₂ treatment significantly decreased the nitrotyrosine level in the lungs of LPS-challenged animals (P < 0.05, n = 6 per group; Fig. 3). Combination therapy with H₂ and NO could also significantly ameliorate the nitrotyrosine in lung tissues of LPS-challenged mice.

Combination therapy with H₂ and NO downregulated the cytokines and chemokines in the BALF of LPS-challenged mice

As depicted in Figure 4, we found that the levels of both proinflammatory cytokines (TNF-α, IL-1β, IL-6, and HMGB1) and anti-inflammatory cytokine (IL-10) in the BALF were significantly increased at 4 and 24 h in LPS-challenged mice (P < 0.05 vs. PBS group, n = 6 per group). Hydrogen gas or NO treatment alone markedly downregulated the levels of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and HMGB1) in the BALF of LPS-challenged mice (P < 0.05, n = 6 per group), whereas H₂ or NO treatment alone had no significant effects on anti-inflammatory cytokine IL-10 (P > 0.05, n = 6 per group). Furthermore, LPS-challenged mice exhibited the significantly increased levels of chemokines (KC, MIP-1α, MIP-2, and MCP-1) in the BALF at 4 and 24 h (P < 0.05 vs. PBS group, n = 6 per group), which were also reduced by H₂ or NO treatment alone (P < 0.05, n = 6 per group). Moreover, both H₂ and NO treatment together could more effectively reduce proinflammatory cytokines and chemokines and increase anti-inflammatory cytokine in the BALF of LPS-challenged mice. These results demonstrate that combination therapy with H₂ and NO can more effectively downregulate the cytokines and chemokines in the BALF of LPS-challenged mice.

Combination therapy with H₂ and NO inhibited LPS-induced pulmonary NF-κB DNA-binding activity

In LPS-challenged mice, the lung NF-κB p65 DNA-binding activity was significantly elevated at 4 and 24 h (P < 0.05 vs. PBS group, n = 6 per group). However, H₂ and/or NO treatment significantly inhibited the lung NF-κB DNA-binding activity of LPS-challenged mice (P < 0.05, n = 6 per group; Fig. 5). Combination therapy with H₂ and NO could more significantly inhibit the lung NF-κB activation in LPS-challenged mice.

Combination therapy with H₂ and NO prevented lung cell apoptosis of LPS-challenged mice

Moreover, we investigated the effects of H₂ treatment on pulmonary cell apoptosis in LPS-challenged mice by caspase 3 activity (Fig. 6). We found that caspase 3 activity was significantly increased in the lungs of LPS-challenged animals, which was prevented by H₂ or NO treatment alone (P < 0.05, n = 6 per group; Fig. 6). However, both H₂ and NO treatment simultaneously could more effectively ameliorate LPS-induced pulmonary cell apoptosis (Fig. 6). These results indicate that the i.t. administration of LPS increases the pulmonary cell apoptosis, which can be significantly alleviated by H₂ and/or NO treatment.

Combination therapy with H₂ and NO attenuated LPS- and polymicrobial sepsis–induced lung injury in mice

In the present study, we also investigated the effects of 2% H₂ or 20 ppm NO inhalation beginning at 3 h after LPS administration on lung histopathology and function in mice with LPS-induced lung injury. Hydrogen gas and/or NO treatment at 3 h after LPS administration still resulted in a reduction of infiltrated inflammatory cells and a marked improvement in lung architecture. Hydrogen gas or NO treatment significantly reduced the LPS-induced increase in lung histologic scores (P < 0.05, n = 10 per group; Fig. 7A) and also markedly improved the Pao₂/Fio₂ in LPS-challenged mice (P < 0.05, n = 6 per group; Fig. 7B). Combination therapy with H₂ and NO could more effectively attenuate LPS-induced lung injury in mice (Fig. 7). Furthermore, we investigated the effects of 2% H₂ or 20 ppm NO inhalation on lung histopathology and function in mice with polymicrobial sepsis–induced lung injury. Hydrogen gas or NO treatment also significantly reduced the polymicrobial sepsis–induced increase in lung histologic scores (P < 0.05, n = 10 per group; Fig. 7C) and also markedly improved the Pao₂/Fio₂ in polymicrobial sepsis–challenged mice (P < 0.05, n = 6 per group; Fig. 7D). Combination therapy with H₂ and NO could more effectively attenuate polymicrobial sepsis–induced lung injury in mice (Fig. 7). These results demonstrate that H₂ or NO posttreatment significantly improves lung histopathology and lung function in LPS- and polymicrobial sepsis–challenged mice, whereas combination therapy with H₂ and NO can more effectively attenuate LPS- and polymicrobial sepsis–induced lung injury.

Combination therapy with subthreshold concentrations of H₂ and NO synergistically attenuated LPS- and polymicrobial sepsis–induced lung injury in mice

We further investigated the effects of subthreshold concentrations of 1% H₂ or 5 ppm NO inhalation on lung histopathology and function in mice with LPS- and polymicrobial sepsis–induced lung injury. Here, we found that 1% H₂ or 5 ppm NO treatment alone did not improve lung histopathology and function in mice with LPS- and polymicrobial sepsis–induced lung injury. Combination therapy with subthreshold concentrations of H₂ and NO could synergistically attenuate LPS- and polymicrobial sepsis–induced lung injury in mice (Fig. 8).

Taken together, combination therapy with H₂ and NO can significantly inhibit the NF-κB activation as well as reduce the production of proinflammatory cytokines and chemokines in the lungs of LPS-challenged mice. Moreover, combination therapy with H₂ and NO can significantly prevent neutrophil accumulation in the lungs as well as decrease lung edema, vascular permeability, and cell apoptosis in mice subjected to LPS. Importantly, both H₂ and NO treatment together can synergistically significantly improve lung histopathology and function in mice with LPS exposure and polymicrobial sepsis. These results suggest that combination therapy with H₂ and NO may be useful as a novel anti-inflammatory therapy to treat ALI.

DISCUSSION

In the current study, we found that (1) the mice with i.t. administration of LPS exhibited significant lung injury, which was significantly improved by 2% H₂ and/or 20 ppm NO treatment for 3 h starting at 5 min or 3 h after LPS administration; (2) H₂ and/or NO treatment inhibited LPS-induced pulmonary early and late NF-κB activation; (3) H₂ and/or NO treatment downregulated pulmonary inflammation and cell apoptosis; (4) H₂ and/or NO treatment also significantly attenuated lung injury in polymicrobial sepsis; and (5) combination therapy with subthreshold concentrations of H₂ and NO could synergistically attenuate LPS- and polymicrobial sepsis–induced lung injury. In conclusion, these results demonstrate that combination therapy with H₂ and NO could more significantly ameliorate LPS- and polymicrobial sepsis–induced ALI, perhaps by reducing lung inflammation and apoptosis, which may be associated with the decreased NF-κB activity.

It is well-known that gram-negative organisms account for approximately half of the infections predisposing to ALI, such as pneumonia or sepsis (^1–3). Endotoxin (LPS) is a critical mediator of organ dysfunction and death associated with infections of gram-negative organisms. Some features of ALI/ARDS can be reproduced by administration of LPS, which induces the expression of inflammatory cytokines and chemokines and upregulate leukocyte adhesion molecules, resulting in lung injury and dysfunction (¹⁸). It is well established that i.t. administration of LPS can induce a model of ALI (¹⁸). In the present study, a mouse model of ALI was successfully produced by i.t. administration of aerosolized LPS according to our previous report (^{13, 18, 19}). We found that lung injury, characterized by increased lung water content, disruption of lung architecture, extravasation of red blood cells, and accumulation of inflammatory cells, was present at 24 h after LPS administration, which is consistent with other studies (^{18, 19}). In addition, well-accepted and widely used CLP is considered to be a clinically relevant model for studying the pathogenesis and treatment of sepsis. Therefore, we further used a murine model of CLP-induced lung injury in this study.

Nitric oxide is a potent endogenous vasodilator that can be exogenously administered via inhalation. Inhaled NO has been used for treatment of ALI/ARDS (^24–26). Inhaled NO has the ability to provide selective pulmonary vasodilatation, improve ventilation-perfusion mismatch, and subsequently ameliorate the elevated pulmonary vascular resistance and pulmonary hypertension seen in lung injury (²⁵). Importantly, NO is involved in both the production of and protection from oxidative injury, regulates both immune and inflammatory responses, decreases neutrophil sequestration in lungs, and decreases edema formation and lung injury. Nitric oxide alters the immune function by modifying the release of cytokines and other components of the inflammatory cascade from alveolar macrophages and inhibits the active adhesion molecules and the neutrophil oxidative burst involved in neurophil migration. However, inhaled NO can be rapidly converted to active intermediates, including nitrogen dioxide, peroxynitrite, and nitrotyrosine in the presence of superoxide, which can result in deleterious adverse effects including further lung tissue damage, impaired surfactant function, or aggravated circulatory failure. Moreover, inhaled NO rapidly binds to hemoglobin, with high affinity, to form methemoglobin at doses of 40 ppm or more. Clinically significant methemoglobinemia from inhaled NO administered at doses of 20 ppm or less to patients with ARDS is uncommon (incidence <1%) and more likely to occur at doses greater than 80 ppm. Therefore, we used 20 ppm NO inhalation in this present study.

Recent studies show that H₂ has antioxidant, anti-inflammatory, and antiapoptotic properties (²⁷). We and other researchers have found that H₂ inhalation can attenuate many kinds of lung injuries caused by ventilator, transplantation, hyperoxia, irradiation, and sepsis (^{11, 14, 15, 28, 29}). Interestingly, H₂ specifically quenches exclusively detrimental ROS and RNS, such as •OH and ONOO⁻. Therefore, combination therapy with NO and H₂ inhalation might be more effective in improving lung injury and inflammation. According to our previous studies (^{11, 12, 22}), 2% H₂ was used in this study. In the present study, i.t. administration of LPS can induce lung injury characterized by the deterioration of lung histopathology and histologic scores and increase in lung W/D weight ratio, as well as total protein in the BALF, and lung function, which was improved by H₂ or NO inhalation alone.

A comparison of the effects of inhaled NO with those of inhaled H₂ in this study revealed that they exerted equivalent lung-protective effect. The inflammatory markers were similarly eliminated. A major difference between the effects of NO and H₂ inhalation is the production of nitrotyrosine in the lung tissue affected by LPS administration in NO breathing. The increased amount of nitrotyrosine produced by inhaled NO shows that some adverse effects on signal trafficking or cellular function may occur sooner or later through the RNS reactions with the tyrosine at the active site of vital enzymes or cellular components. Moreover, other potential methods to reduce peroxynitrite production while maintaining the beneficial effects of inhaled NO might be to use a peroxynitrite decomposition catalyst or increase the cGMP concentration in the cells by a soluble guanylyl cyclase activator or phosphodiesterase 5 inhibitor, because these enhancers of NO effects via cGMP induction might enable reduction of the inhaled NO concentration, thereby decreasing peroxynitrite formation and minimizing the adverse effects of NO breathing. In this study, we also found that both NO and H₂ treatment could synergistically attenuate LPS- and polymicrobial sepsis–induced lung injury. This result indicates that NO and H₂ can also improve lung injury through a synergistic protective mechanism.

Pulmonary cell apoptosis is considered to be important in the pathogenesis of ALI (³⁰). At the molecular level, apoptosis is activated by the aspartate-specific cysteine protease cascade, including caspases 3 and 12 (³¹). Caspase 3 is considered to be the most important of the executioner caspases (³¹). We found that caspase 3 activity was dramatically increased in the lungs of LPS-challenged mice, which was prevented by H₂ or NO treatment alone. Both NO and H₂ treatment together could more effectively ameliorate lung cell apoptosis in LPS-challenged mice.

Neutrophilic inflammation is associated with ALI/ARDS (^{3, 18}). Inhaled LPS can cause neutrophilic inflammation and decrements in pulmonary function, which is caused by the recruitment of neutrophils from the vascular space to the airspace (^{3, 18}). In this study, mice exposed to LPS exhibited a massive recruitment of inflammatory cells including neutrophils and macrophages in the airways. We found that the total cells and PMNs in the BALF of LPS-challenged mice were significantly increased, which were attenuated by H₂ or NO treatment alone. Furthermore, we investigated lung neutrophil infiltration by measuring the activity of lung MPO, a neutrophil-specific enzyme. Hydrogen gas or NO treatment alone prevented the increase in lung MPO activity in LPS-challenged mice. Moreover, we found that H₂ or NO treatment dramatically prevented the increase in proinflammatory cytokines (TNF-α, IL-1β, IL-6, and HMGB1) in the BALF of LPS-challenged mice. Our results demonstrated that both H₂ and NO treatment at the same time could more significantly ameliorate the LPS-induced lung neutrophil infiltration and inflammation.

The increase in alveolar neutrophils is due to the enhanced chemokines. Some studies have shown that this cell infiltration was associated with the increase in BALF levels of chemoattractant cytokines such as KC, MIP-1α, MCP-1, and MIP-2 (^{3, 4, 18}). Keratinocyte-derived chemokine and MIP-2 are major chemokines for neutrophils. Macrophage inflammatory protein 1α is involved in the acute inflammatory state in the recruitment and activation of polymorphonuclear leukocytes. Moreover, MCP-1 is a chemokine important in the recruitment and adherence of monocytes and neutrophils to endothelium. In the present study, we found that the chemokines (KC, MIP-1α, MIP-2, and MCP-1) in the BALF are significantly elevated in mice subjected to LPS, which were prevented by H₂ or NO treatment alone. Nuclear factor κB is a critical transcription factor that is required for the expression of many cytokines and chemokines in the pathogenesis of ALI (⁴). Nuclear factor κB regulates gene expression of cytokines, chemokines, and adhesion molecules. In this study, H₂ or NO treatment alone inhibited the lung NF-κB DNA-binding activity in LPS-challenged mice. Interestingly, our results further indicated that both H₂ and NO treatment more significantly reduced the LPS-induced lung inflammation through downregulation of neutrophil recruitment as well as proinflammatory cytokines and chemokines.

In conclusion, in low concentration, H₂ is neither explosive nor dangerous. Thus, combination therapy with inhaled H₂ and NO may represent a promising future therapeutic option for ALI, and H₂ eliminates the adverse by-products of NO exposure. This study supports the view that NO and H₂ are suggestive partners that can be used as a mixture for breathing.