The effects of gasotransmitters on bronchopulmonary dysplasia

Journal Pre-proof The effects of gasotransmitters on bronchopulmonary dysplasia Hai Lin, Xinbao Wang PII:

S0014-2999(20)30075-3

DOI:

https://doi.org/10.1016/j.ejphar.2020.172983

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EJP 172983

To appear in:

European Journal of Pharmacology

Received Date: 3 October 2019 Revised Date:

22 January 2020

Accepted Date: 31 January 2020

Please cite this article as: Lin, H., Wang, X., The effects of gasotransmitters on bronchopulmonary dysplasia, European Journal of Pharmacology (2020), doi: https://doi.org/10.1016/j.ejphar.2020.172983. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

The effects of gasotransmitters on bronchopulmonary dysplasia

Hai Lina, Xinbao Wangb*

a. Department of Traditional Chinese Medicine, Beijing Friendship Hospital, Capital Medical University, Beijing, P.R. China. b. Department of Pediatrics, Beijing Friendship Hospital, Capital Medical University, Beijing, P.R. China. Corresponding author: *Xinbao Wang, Department of Pediatrics, Beijing Friendship Hospital, Capital Medical University, Yongan Str. No. 95 West District, Beijing, P.R. China. Email: [email protected]

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Abstract Bronchopulmonary dysplasia (BPD), which remains a major clinical problem for preterm infants, is caused mainly by hyperoxia, mechanical ventilation and inflammation. Many approaches have been developed with the aim of decreasing the incidence of or alleviating BPD, but effective methods are still lacking. Gasotransmitters, a type of small gas molecule that can be generated endogenously, exert a protective effect against BPD-associated lung injury; nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) are three such gasotransmitters. The protective effects of NO have been extensively studied in animal models of BPD, but the results of these studies are inconsistent with those of clinical trials. NO inhalation seems to have no effect on BPD, although side effects have been reported. NO inhalation is not recommended for BPD treatment in preterm infants, except those with severe pulmonary hypertension. Both CO and H2S decreased lung injury in BPD rodent models in preclinical studies. Another small gas molecule, hydrogen, exerts a protective effect against BPD. The nuclear factor erythroid-derived 2 (Nrf2)/heme oxygenase-1 (HO-1) axis seems to play a central role in the protective effect of these gasotransmitters on BPD. Gasotransmitters play important roles in mammals, but further clinical trials are needed to explore their effects on BPD. Key words: bronchopulmonary dysplasia; nitric oxide; carbon monoxide; hydrogen sulfide; hydrogen 1. Introduction Bronchopulmonary dysplasia (BPD) was first reported by Northway in 1967 2

(Northway et al., 1967). Although more than 50 years have passed, BPD remains a major disease that causes mortality and morbidity among preterm infants. 1.1 Epidemiology and physiopathology of BPD and conventional BPD treatments The incidence of BPD among research centers differ. In the Israeli Neonatal Network, 13.7% of very low birth weight infants (birth weight<1500 g) and 31.0% of extremely low birth weight infants (birth weight<1000 g) among 12,139 surviving infants were diagnosed with BPD (Klinger et al., 2013). The incidence of BPD among very low birth weight infants in the Japanese Neonatal Network was 14.6% (Isayama et al., 2012), and the yearly incidence of BPD in the Vermont Oxford Network between 2000 and 2009 ranged from 26.2% to 30.4% (Horbar et al., 2012). Furthermore, up to 42% of extremely preterm infants (22–28 weeks gestation) were diagnosed with BPD in the National Institute of Child Health and Human Development-Neonatal Research Network (Stoll et al., 2010). The classic BPD observed in the 1960s is now also known as “old BPD” (Northway et al., 1967). Infants with old BPD exhibited increased infiltration of macrophages and foam cells in the alveolar lumen in addition to abnormally deposited collagen fibrils, elastin fibers, and reticulin. The pulmonary peripheral arteries underwent hypertrophy and remodeling. In 1998, Husain and colleagues found decreased and more diffuse alveolar septal fibrosis, decreased capillaries, and a significantly reduced radial alveolar count, but the alveolar size was significantly increased in surfactant-treated patients (Husain et al., 1998). Simplified pulmonary alveolar structures, decreased and dysmorphic vasculature, and a lesser degree of 3

fibrosis are the hallmarks of “new BPD” (Jobe, 1999). Hyperoxia, mechanical ventilation and inflammation are the main causes of BPD (Morty, 2018). In a hyperoxia-induced BPD rodent model, the radial alveolar count was significantly decreased, but the mean linear intercept was increased. The vascular density of the lung was significantly decreased, but the medial wall thickness and percentage of muscularized arterioles (>50% muscularization of the vessels) in peripheral lung tissues were significantly increased (Donda et al., 2018; Kurata et al., 2019). Macrophage infiltration into alveolar airspaces was also significantly increased. Although biomarkers of BPD are not fully understood, decreased vascular endothelial growth factor (VEGF) formation and increased platelet-derived growth factor levels contribute to BPD development (Liu et al., 1995; Jakkula et al., 2000). In addition, endothelin 1, interleukin (IL)-6 and IL-33 are biomarkers of BPD (El Shemi et al., 2017; Rivera et al., 2016). Many approaches aimed at decreasing the incidence of BPD and lung injury have been developed, but most of these approaches have been ineffective. Vitamin A and caffeine can decrease the incidence of BPD in some contexts (Pakvasa et al., 2018; Schwartz et al., 2017), but their effectiveness still needs to be verified. Dexamethasone can alleviate lung injury in BPD but has adverse effects, such as inhibited brain development and growth and increased risk of cerebral palsy (Doyle et al., 2017a, b). Thus, the need to explore effective therapies for BPD is urgent. 2. Gasotransmitters: nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H2S) 4

Gasotransmitters, such as NO, CO and H2S, are endogenously and enzymatically generated small gas molecules. All gasotransmitters can freely permeate the membrane and do not depend on cognate membrane receptors (Wang, 2002). These gasotransmitters have specific targets and exert specialized functions at the physiological level (Table 1). In 1998, Robert F. Furchgott, Louis J. Ignarro, and Ferid Murad won a Nobel Prize in Medicine and Physiology for their discovery that endogenous NO, a well-known gas molecule, is a signaling molecule in the cardiovascular system (Hansson et al., 1998). Endogenous NO can be generated from L-arginine by nitric oxide synthase (NOS). There are three isoforms of NOS: neuronal nitric oxide synthase (nNOS or NOS I), inducible nitric oxide synthase (iNOS or NOS II), and endothelial nitric oxide synthase (eNOS or NOS III) (Bredt and Snyder, 1990; Farstermann and Kleinert, 1995). nNOS synthesizes NO in central and peripheral neural tissues (Bredt and Snyder, 1990; Mayer et al., 1990). iNOS, which was first isolated from murine macrophages activated by inflammatory mediators (Hevel et al., 1991; Stuehr et al., 1991), can be induced by inflammation in leukocytes, pulmonary epithelial cells and macrophages (Bandaletova et al., 1993). eNOS was first found in vascular endothelial cells (Forstermann et al., 1991). Endothelial cell-derived NO can regulate cardiovascular functions and exerts antithrombotic and anti-inflammatory effects on the surface of luminal tissue (Albrecht et al., 2003; Sase and Michel, 1995; Scarborough et al., 1998). Interestingly, fractional exhaled NO can also be detected in humans with airway inflammation, such as that in asthma, with a breath test. Asthma 5

is characterized as chronic airway inflammation and hyper-responsiveness with variable airflow limitation (Becker and Abrams, 2017; Podell, 1992). In asthma, mast cells and antigen-specific T-helper cell type 2 cells are activated, leading to the excessive production of cytokines such as IL-4 and IL-13 (Durrant and Metzger, 2010). These cytokines can upregulate the expression of iNOS, which increases the production of fractional exhaled NO in airway epithelial cells (Alving and Malinovschi, 2011; Alving et al., 1993). Fractional exhaled NO is an indicator used for asthma diagnosis (Chu et al., 2019; Lehtimaki et al., 2016; Miskoff et al., 2019; Pansieri et al., 2014). CO is a toxic gas (Lehr, 1970), and acute CO poisoning occurs when elevated CO binds normal hemoglobin (Hb), generating HbCO. The affinity of CO for Hb is nearly 250 times stronger than that of oxygen for Hb (Roughton, 1970). HbCO formation inhibits the binding of oxygen and Hb. The oxygen transportation function of Hb is reduced, resulting in tissue hypoxia (Lukin et al., 2000; Okada et al., 1976). However, CO can also be endogenously generated in vivo. CO can be produced from heme under the catalysis of heme oxygenase (HO), accompanied by bilirubin and iron byproducts (Tenhunen et al., 1968). CO, a second gasotransmitter, is a gaseous vascular modulator (Durante, 2002; Leffler et al., 2011) and exerts anti-inflammatory and antioxidative effects during stress (Kaczorowski and Zuckerbraun, 2007; Ryter et al., 2002). There are three HO isoforms: one inducible (HO-1) and two constitutive (HO-2, HO-3) isoforms (Maines et al., 1986; McCoubrey et al., 1997). HO-1 usually cannot be detected in normal tissues, except 6

the spleen, but HO-1 expression can be significantly enhanced by a series of stimuli that induce oxidative stress, such as heme, cytokines, lipopolysaccharide (LPS), and heat shock (Tenhunen et al., 1969). HO-1 is extensively localized in the cell membrane, nucleus, and mitochondria, where it plays important biological roles (Biswas et al., 2014; Lin et al., 2007; Ryter and Choi, 2009; Slebos et al., 2007). HO-1 exerts protective effects against oxidative stress and inflammation (Ghattas et al., 2002) (Almolki et al., 2004; Barrera et al., 2003). The expression of HO-2 in organs is usually constant, and HO-2 may be a physiological regulator (Wagener et al., 1999). HO-3 is a pseudogene derived from the HO-2 gene, and its function is not clear (Hayashi et al., 2004). H2S, a gas with a peculiar rotten egg smell, is toxic at high levels. H2S can be endogenously generated from cysteine under the catalysis of cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST) (Stipanuk, 1986; Stipanuk and Beck, 1982). Endogenous H2S is mainly produced by CBS and CSE. CBS is mainly located in the cytosol and highly expressed in neural and cardiac tissues (Quere et al., 1999). CSE is expressed in not only the cardiovascular system but also the liver, brain, colon and pancreas in mice (Linden et al., 2008) and the placenta, myometrium, and chorion in humans (Patel et al., 2009). MST can be found in the mitochondrial and cytosolic fractions and is mainly expressed in the renal proximal tubular epithelium, liver pericentral hepatocytes, cardiac cells, and brain neuroglial cells (Koj et al., 1975; Nagahara et al., 1998; Ogasawara et al., 1994). H2S regulates the vascular system (Wang, 2011); exerts 7

antihypertensive (Yang et al., 2008), antiatherosclerotic (Wang et al., 2009), antioxidative stress (Wang et al., 2011), and anti-inflammatory (Gong et al., 2010) effects; and alleviates myocardial and pulmonary vascular remodeling (Li et al., 2009). H2S is the third gasotransmitter (Wang, 2003). However, much regarding the effects of the clinical use of these gasotransmitters on BPD remains unknown. 3. Oxidative stress and BPD Oxidative injury can be caused by oxidative stress or nitrosative stress. Oxidative stress, a condition in which the concentration of reactive oxygen species is transiently or chronically enhanced, disturbs cellular metabolism and regulation and can even destroy cellular constituents (Lushchak, 2011). Reactive oxygen species are a series of reactive molecules and free radicals produced by the sequential reduction of molecular oxygen, including singlet oxygen, superoxide radical, hydroxyl radical and hydrogen peroxide, and can be produced by aerobic metabolism in cells (Lushchak, 2011, 2014). Oxidative stress results from the inability of antioxidant enzymes to neutralize the production of free radicals and the imbalance between oxidation and antioxidation (Perrone et al., 2012). Antioxidant enzymes, including superoxide dismutase (SOD), catalase, thioredoxin peroxidase, glutathione peroxidase (Gpx) and glutathione reductase, can convert oxygen radicals into less toxic substances or render them harmless to the body (Niki, 2016; Sies, 1993). Both malonic dialdehyde (MDA), the product of lipid peroxidation, and 8-hydroxydeoxyguanosine (8-OHdG), a biomarker of DNA damage (Wu et al., 2017), are often used to assess the degree of oxidative injury. 8

Nitrosative stress is the result of reactive nitrogen species (RNS) overproduction. RNS can be produced when NO reacts with reactive oxygen species, and RNS at physiological concentrations are essential for normal biological functions. However, the overproduction of RNS can cause cell damage and even death. RNS are a series of compounds derived from NO, such as nitroxyl anion, S-nitrosothiols, and peroxynitrite. Increased eNOS expression significantly enhanced the production of NO and reduced oxidative stress in the aortas of db/db mice (Cheang et al., 2011). However, there is some controversy regarding the effect of eNOS on lung injury. Chronic eNOS overexpression was shown to decrease ventilation-induced lung injury by inhibiting inflammatory chemokine and cytokine generation, which is related to neutrophil infiltration in the lung air space (Takenaka et al., 2006). However, ventilator-induced lung injury was alleviated in eNOS-deficient mice (Vaporidi et al., 2010). The MDA and 8-isoprostane content was significantly increased in the bronchoalveolar lavage fluid (BALF) and lung tissue of wild-type mice subjected to ventilation but not in that of eNOS-deficient mice subjected to ventilation. The level of 8-isoprostane in the BALF was also lower in eNOS-deficient mice than in wild-type mice (Vaporidi et al., 2010). These data indicate that eNOS contributes to ventilation-induced lung injury by increasing superoxide production. iNOS was first found in macrophages and shown to be activated under proinflammatory conditions. Lesional iNOS can cause the abnormal production of NO radicals and superoxide radicals, which may react to peroxynitrite and cause cell damage (Ponnuswamy et al., 2009). Overexpression of iNOS in the rostral ventrolateral medulla caused 9

hypertension by increasing oxidative stress (Kimura et al., 2005). In contrast, inhibition of iNOS reduced oxidative stress in animals with lung inflammation (Marques et al., 2012). Therefore, iNOS plays an important role in the process of oxidative stress. Both reactive oxygen species and RNS can cause protein nitration and oxidation, lipid peroxidation and DNA damage, finally leading to cellular dysfunction or death (Capasso et al., 2019; O'Donnell et al., 1999; Tharmalingam et al., 2017). In lung injury induced in animals by ventilation, the levels of lipid oxidation products, MDA and 8-isoprostane in the BALF were significantly increased (Vaporidi et al., 2010), but SOD activity in lung tissues was decreased (Morton et al., 1999). Increased levels of plasma 8-isoprostane were also found in infants who developed BPD at 3 days of life (Ahola et al., 2003). The oxidative DNA damage marker 8-OHdG was significantly increased in infants who developed moderate/severe BPD at 7 days of life and shown to be an independent risk factor for BPD (Joung et al., 2011). The level of 3-nitrotyrosine was significantly increased in infants with BPD (Banks et al., 1998). 3-Nitrotyrosine, the product of the peroxynitrite reaction with protein tyrosine residues, is an index of peroxynitrite formation and oxidative stress (Ischiropoulos et al., 1992; Kooy et al., 1995). Therefore, oxidative stress and nitrosative stress are involved in the process of BPD. 4. NO and BPD 4.1 Preclinical effects of NO on BPD The preclinical effects of NO on BPD were mostly studied decades ago. iNOS 10

expression was found to be significantly increased in the whole lungs of preterm infants with BPD and to colocalize with cytokeratin, an epithelial cell marker (Davis et al., 2008). The expression of eNOS was also upregulated in large blood vessels and airway linings, but the expression of nNOS was not significantly different with the presence and absence of BPD according to the results of immunostaining. The complete absence of iNOS and partial eNOS deficiency exerted a protective effect on vascular and alveolar lung damage induced by VEGF overexpression in the developing mouse lung (Syed et al., 2016). Hyperoxia also reduced Ca2+-dependent NOS activity but increased Ca2+-independent NOS activity, accompanied by increased expression of eNOS and iNOS (Radomski et al., 1998). N(ω)-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of NOS) decreased epithelial proliferation and lung edema in some cases but increased metalloproteinase activity (Tourneux et al., 2009). Inhibition of NO synthesis by L-NAME increased the protein and mRNA expression of tumor necrosis factor (TNF)-α and IL-6, proinflammatory cytokines (Walley et al., 1999). Binding of the NF-kappaB (NF-κB) p50/p65 heterodimer was decreased by nitroprusside and increased by L-NAME. Interestingly, inhibition of iNOS by aminoguanidine or S-methylisothiourea sulfate significantly reduced neutrophil infiltration and lung injury and inhibited iNOS and nitrotyrosine production in lung injury induced by LPS in dogs (Numata et al., 1998). In addition, aminoguanidine or S-methylisothiourea sulfate significantly improved gas exchange, inhibited the decrease in oxygen partial pressures, and increased the lower mean arterial pressure. Therefore, iNOS may contribute to the process of lung injury 11

induced by LPS. iNOS inhibitors play a protective role against LPS-induced lung injury. L-Arginine (300 mg/kg i.p.) significantly reduced TNF-α and IL-1β production by alveolar macrophages in lung injury induced by endotoxin in rats (Meldrum et al., 1997). However, the NOS inhibitor N(G)-monomethyl-L-arginine reversed the protective effect of L-arginine. In conclusion, the anti-inflammatory effect of L-arginine is mediated by the NOS pathway. Exogenous substrates of NO can decrease inflammation in the process of lung injury induced by endotoxin. NO inhalation significantly decreased pulmonary arterial pressure and vascular resistance, improved ventilation-perfusion matching, and enhanced arterial oxygen tension in acute lung injury induced by ventilation (Hillman et al., 1995). Similarly, NO inhalation significantly alleviated hypercapnia, reduced leukocyte infiltration in BALF induced by hyperoxia (Cotton et al., 2006) and reduced the mean pulmonary arterial pressure and the increased ratio of pulmonary to systemic vascular resistance induced by hyperoxia (Mourani et al., 2004). In acute lung injury induced in rabbits by saline lavage, NO inhalation significantly reduced oxidative stress and decreased leukocyte infiltration in lung lavage fluid (Fioretto et al., 2012). NO inhalation (10 ppm) significantly reduced the expression of surfactant protein A but enhanced mannose binding in the lung tissues of neonatal rats (Du et al., 2006). However, treatment with Nw-nitro-L-arginine methyl ester (an inhibitor of eNOS) aggravated lung injury and even increased the inflammatory response induced by hyperoxia (Chang et al., 2001). These data indicate that endogenous NO mediates the process of hyperoxic lung injury. NO inhalation (10 ppm) partially improved alveolarization, 12

reduced right ventricular hypertrophy and pulmonary arterial wall thickness and increased the density of vessels in neonatal rats with BPD induced by bleomycin (Tourneux et al., 2009). In addition, NO inhalation significantly decreased fibrin deposition, inhibited leukocyte influx, and decreased alveolar septal thickness in a model of BPD in hyperoxic neonatal rats (ter Horst et al., 2007). Furthermore, NO inhalation significantly decreased the mRNA expression of the proinflammatory cytokines IL-6, cytokine-induced neutrophilic chemoattractant-1, and amphiregulin; reduced the increased mRNA expression of the fibrinolytic factors plasminogen activator inhibitor 1 and urokinase-type plasminogen activator receptor; decreased the mRNA expression of p21, a cell cycle inhibitor; and increased the expression of alveolar formation-related genes and fibroblast growth factor receptor-4 (ter Horst et al., 2007). In cultured alveolar epithelial cells (RLE-6TN cells), TGF-β1 significantly inhibited the expression and activity of eNOS but not those of iNOS (Vyas-Read et al., 2007). NOS inhibition by L-NAME led to obvious epithelial-mesenchymal transition and increased expression of α-smooth muscle actin. In contrast, exogenous NO treatment decreased the expression of α-smooth muscle actin and collagen I induced by TGF-β1, increased the expression of lamellar protein and E-cadherin, and enhanced synthesis of prosurfactant protein B (Vyas-Read et al., 2007). Therefore, endogenous NO mediates the process of epithelial-mesenchymal transition, and NO may play a protective role in interstitial lung diseases, such as BPD and idiopathic pulmonary fibrosis. An inhibitor of the VEGF receptor, SU-5416, could decrease the alveolar count and density of pulmonary vessels in neonatal rats 13

(Tang et al., 2007). In addition, SU-5416 upregulated the apoptotic index of lung endothelial cells eight-fold and increased caspase-3 activity, while NO inhalation reversed the above effects of SU-5416. These data indicate that NO inhalation can reduce the apoptosis of lung endothelial cells induced by VEGF inhibition in neonatal rats, which may contribute to its protective effect in BPD. Therefore, endogenous NO is involved in the process of lung development. NO inhalation can alleviate lung injury, improve alveolarization and decrease pulmonary arterial remodeling. The mechanism of NO is related to its inhibitory effects on inflammation, reduction of lung endothelial cell apoptosis, and inhibition of epithelial-mesenchymal transition. 4.2 Clinical trials of the effect of NO on BPD The protective effect of NO has been confirmed in BPD animal models. However, these data are not consistent with those of clinical trials. The effect of clinical NO treatment for BPD is controversial. Inhaled NO can decrease the occurrence of BPD and may limit its severity (Truog, 2007). Yang and coworkers (2016) analyzed 22 randomized controlled trials, and the results showed that inhaled NO decreased the risk of BPD (RR: 0.88; P = 0.0007) and did not increase the risk of complications, such as necrotizing enterocolitis and retinopathy of prematurity. Inhaled NO could lead to earlier infant discharge and a shorter duration of oxygen therapy when therapy started from 7 to 21 days of age in infants at risk of BPD (Ballard et al., 2006). Askie and coworkers (2018) carried out a meta-analysis and showed that inhaled NO decreased the incidence of BPD or death among African American preterm infants compared with that in infants of other races (relative risk (RR), 0.77; 95% CI, 14

0.65-0.91). However, Donohue et al analyzed fourteen randomized controlled trials and found a 7% reduction in the occurrence of death and BPD together (RR, 0.93; 95% CI, 0.87-0.99) but no difference in the occurrence of death or BPD alone (Donohue et al., 2011). NO inhalation (20 ppm) improved oxygenation and increased PaO2 in preterm infants with severe BPD (Banks et al., 1999). However, this improvement in oxygenation conferred by NO inhalation did not alleviate acute lung injury or affect mortality but reduced the risk of severe respiratory failure in adult patients (Lundin et al., 1999). In contrast, most clinical trials have shown that inhaled NO does not decrease the incidence or severity of BPD or related deaths (Jensen and Kirpalani, 2014), (Jiang et al., 2016), affect pulmonary function or oxygenation during the development of BPD (Athavale et al., 2004), or decrease the incidence of mechanical ventilation (Kinsella et al., 2014). In addition, inhalation of NO did not affect short- or medium-term respiratory resistance or compliance in preterm infants using a ventilator (Di Fiore et al., 2007). Inhaled NO seemed to be safe in preterm infants without BPD but did not improve survival (Hasan et al., 2017). Some studies showed that NO inhalation beginning at day 7 led to more a severe course of BPD and increased the incidence of severe BPD or death (odds ratio: 2.24; 95% CI, 1.23-4.07) (Truog et al., 2014). These data indicated that NO inhalation may be harmful to preterm infants. Early treatment with inhaled NO seemed to increase the risk of severe intracranial hemorrhage, and later treatment with inhaled NO had no significant effect on the risk of BPD (Barrington and Finer, 2007). Therefore, inhaled NO is not recommended for BPD 15

treatment but benefits infants with severe pulmonary hypertension (Sweet et al., 2019). The mechanism by which the therapeutic effect of NO inhalation reduces BPD is not clear but may be related to the following factors. NO is a free radical because of the presence of one unpaired electron (Auger et al., 2011). NO can react with oxygen and produce nitrogen oxides, including peroxynitrite, which can cause tissue damage (Martinez and Andriantsitohaina, 2009). Hyperoxia or inflammation can increase the cGMP-dependent phosphodiesterase PDE5, decrease the intracellular cGMP response, and reduce responsiveness to exogenous to NO (Farrow et al., 2008). Discontinuation of NO inhalation can cause pulmonary hypertension rebound (Raffay et al., 2012). 5. The effect of CO on BPD When hyperoxic mice were exposed to 250 ppm CO, another endogenous gasotransmitter, for 1 h twice daily, alveolar simplification induced by hyperoxia was alleviated, and the mean linear intercept was decreased. In addition, CO treatment significantly reduced neutrophil and monocyte/macrophage infiltration in BALF and ameliorated the increased mRNA expression of proinflammatory cytokines (IL-1, IL-6, TNF-α, CCL-2, and CXCL1 and CXCL2) induced by hyperoxia in lung tissues but increased the mRNA expression of IL-10 and IL-13 (two “anti-inflammatory” cytokines) (Anyanwu et al., 2014). These data indicate that HO-1 deficiency aggravates lung injury in mice exposed to hyperoxia. Exogenous CO treatment could alleviate alveolar simplification induced by hypoxia, inhibit neutrophil and macrophage infiltration in BALF, and reduce pulmonary inflammation. 16

The volume of pleural effusion and total protein accumulation were significantly increased in the airways of rats with lung injury induced by hyperoxia but decreased in rats exposed to 250 ppm CO (Otterbein et al., 1999b). CO could dose-dependently (50-500 ppm) protect rats against lethal hyperoxia. In addition, CO exposure significantly inhibited lung hemorrhage, edema, and inflammatory cell infiltration induced by hyperoxia, accompanied by fibrin deposition, and decreased the cell apoptotic index. Interestingly, inhibition of endogenous CO by tin protoporphyrin (an inhibitor of HO enzymes) further aggravated lung tissue injury induced by hyperoxia, and even more pleural effusion was found in the airway. However, lung injury could be reversed by CO exposure. Therefore, endogenous CO is involved in the process by which lung injury is induced by hyperoxia. Furthermore, CO treatment alleviates lung injury by inhibiting inflammatory cell infiltration and reducing the lung cell apoptotic index. Studies have also found that all mice exposed to hyperoxia (>98% O2) died between 90 and 100 h, while 95% of mice exposed to hyperoxia and CO (>98% O2 containing 250 ppm CO) remained alive at 95 h, and 50% remained alive at 128 h (Otterbein et al., 2003). Therefore, CO exposure significantly protects mice against lethal hyperoxia. CO exposure also alleviated the increased wet/dry lung ratio and protein content in the BALF induced by hyperoxia and reduced formation of the lipid peroxidation product MDA. In addition, CO significantly reduced neutrophil infiltration and inhibited expression of the proinflammatory cytokines TNF-α, IL-1β, and IL-6. The mitogen-activated protein kinase (MAPK) pathway could be activated 17

by hyperoxia, and the protein expression of extracellular regulated protein kinase (ERK)1/ERK2, c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase (MKK)3/6, and p38 MAPK in lung tissues was increased. Signs of lung injury (such as protein accumulation and leukocyte infiltration) were observed earlier in mkk3-/-mice exposed to hyperoxia than in wild-type mice exposed to hyperoxia (Otterbein et al., 2003). CO exposure did not affect the survival of mice deficient in jnk or mkk3 and did not inhibit the mRNA expression of proinflammatory cytokines. An inhibitor of p38 MAPK (SB203580) mimicked the effect of hyperoxia exposure on mkk3-/- mice. In cultured lung epithelial cells (A549 cells), CO could induce pan-p38 and MKK3 activation and increase cell viability, while SB203580 reversed these effects of CO. Therefore, the MKK3 MAPK pathway mediates the protective role of CO on lung injury induced by hyperoxia. In cultured mouse lung endothelial cells exposed to hyperoxia (95% O2, 5% CO2) for 3 h, cell death and cytochrome c release from mitochondria occurred in a time-dependent manner (Wang et al., 2007). HO-1 overexpression or CO (250 ppm) exposure significantly decreased cytochrome c release and reduced reactive oxygen species formation. In addition, CO inhibited apoptosis by reducing caspase-3 activation and suppressed extrinsic apoptosis signaling by inhibiting caspase-8 activation and transportation of the death-inducing signal complex from the Golgi to the plasma membrane. B-cell lymphoma-2 (Bcl-2) regulates intrinsic (mitochondria-dependent) apoptosis pathways, which include antiapoptotic proteins, such as Bcl-2 and Bcl-XL, and proapoptotic proteins, such as Bax, Bid, and Bad (Chipuk et al., 2010). Administration of CO inhibited Bid 18

activation and reduced Bax expression and translocation from the cytosol to mitochondria but induced the Bcl-XL/Bax interaction (Wang et al., 2007). Therefore, the protective effect of CO against hyperoxic injury underlies its inhibitory effect on apoptotic signaling. The protective effect of CO was also found in ventilator-induced lung injury. In mice with lung injury induced by a ventilator delivering a 12 ml/kg tidal volume of air for 6 h, CO exposure (250 parts per million) significantly reduced pulmonary edema and alveolar wall thickness and decreased neutrophil infiltration and IL-1β release in BALF (Faller et al., 2012). Pretreatment with CO inhalation for 1 h before ventilation had no protective effect on lung injury. However, after ventilation for 3 or 5 h, treatment with CO inhalation alleviated lung injury and decreased the total number of cells and neutrophil inflation. CO treatment exhibited the best protective effect when administered within the first hour of ventilation. In rats with ventilator-induced lung injury, CO inhalation dose-dependently decreased the level of the proinflammatory factor TNF-α and neutrophil leukocyte infiltration in BALF but did not affect hemodynamics or oxygenation (Dolinay et al., 2004). CO could stimulate p38 MAPK activation and increase the level of the anti-inflammatory molecule IL-10 in BALF but did not affect NF-κB activator protein-1 activation. An inhibitor of p38 MAPK, SB203580, reversed the CO-induced increase in IL-10. Therefore, the protective effect of CO on ventilator-induced lung injury is related to its anti-inflammatory role, which is related to the p38 MAPK pathway. CO inhalation significantly decreased IL-1β, monocyte chemotactic protein-1, and 19

macrophage inflammatory proteins in lung tissue homogenates and alleviated the increased expression of Egr-1 induced by ventilation (Hoetzel et al., 2008). Egr-1 is a proinflammatory regulator that modulates the generation of cytokines and chemokines. Egr-1–deficient mice could avoid lung injury with ventilation. Peroxisome proliferator-activated receptor (PPAR)-γ is thought to be an anti-inflammatory mediator that counteracts Egr-1. CO could induce the expression of PPAR-γ in mouse macrophages (RAW 264.7 cells). A specific inhibitor of PPAR-γ, GW9662, could abrogate the protective effect of CO in mice with lung injury induced by ventilation. Therefore, CO exerted a protective effect on lung injury through PPAR-γ and the downregulation of Egr-1. Studies have also indicated that the mRNA expression of caveolin-1 was increased in the lung tissues of mice on a ventilator and further increased with CO exposure (Hoetzel et al., 2009). However, lung injury was more aggravated in caveolin-1-deficient mice with mechanical ventilation than in wild-type mice with mechanical ventilation, and CO treatment failed to alleviate lung injury in caveolin-1-deficient mice with ventilation. Therefore, caveolin-1 is involved in the protective mechanism of CO against lung injury.

6. The effect of H2S on BPD H2S is involved in lung development. The expression of Cbs and Cth (key enzymes in H2S generation) was shown by immunofluorescence staining to be localized in lung vessels and the airway epithelium of mouse pups (Madurga et al., 2015). Dynamic changes in Cbs and Cth expression occur in the process of mouse lung alveolarization. 20

However, Cbs-/-or Cth-/-mouse pups exhibited impaired normal lung alveolarization and fewer but larger alveoli, accompanied by decreased peripheral lung vasculature but increased vascular wall thickness. In addition, inhibition of Cbs expression (by small interfering RNA, siRNA) or Cth expression (by DL-propargylglycine, PAG) repressed angiogenesis in lung endothelial cells. The number and length of endothelial tubes decreased, but administration of morpholin-4-ium 4-methoxyphenyl (morpholino) phosphinodithioate (GYY4137, a H2S donor) increased the formation of endothelial tubes (Madurga et al., 2015). These data indicate that the endogenous H2S pathway is involved in development of the pulmonary vascular and alveolarization. In a BPD mouse model developed via exposure to hyperoxia by the delivery of 85% O2 for 10 days, the number of total lung alveoli decreased by 56%, but the thickness of the alveolar septal wall increased by 29% (Madurga et al., 2014). GYY4137 treatment significantly improved pulmonary alveolarization and decreased leukocyte infiltration in the alveolar air space. The migration of cultured mouse alveolar type II cells could be stimulated by sodium hydrosulfide hydrate (NaHS) and inhibited by glibenclamide (Madurga et al., 2014). GYY4137 treatment significantly increased formation of a capillary-like network and decreased cellular reactive oxygen species production in human pulmonary artery endothelial cell injury induced by hyperoxia (Vadivel et al., 2014). Fewer pulmonary alveoli and a simpler alveolar structure were observed in neonatal rats exposed to hyperoxia (95% O2). GYY4137 treatment reversed the above effects of hyperoxia and preserved alveolar formation. In addition, GYY4137 reduced pulmonary hypertension, alleviated pulmonary artery remodeling, 21

and provoked lung vascular growth in neonatal rats with lung injury induced by hyperoxia. Mice with lung injury induced by mechanical ventilation (a tidal volume of 12 ml/kg for 6 h) exhibited thickening of the alveolar wall and cellular infiltration. Inhalation of H2S (80 parts per million) significantly decreased the thickness of the alveolar wall and cellular infiltration (Faller et al., 2010). H2S inhalation prior to (1, 3, or 5 h) or after (5 or 10 h) mechanical ventilation could alleviate histological injury and reduce neutrophil influx in BALF and reactive oxygen species formation in the mouse lung (Faller et al., 2017). H2S also decreased the elevated levels of IL-1β and macrophage inflammatory protein-1 induced by mechanical ventilation in the lung tissue, accompanied by a reduction in neutrophil infiltration in the BALF. H2S treatment decreased the number of apoptotic cells and inhibited the expression of HO-1 in lung tissues (Faller et al., 2010). Further study showed that H2S enhanced formation of the antioxidant glutathione and inhibited formation of reactive oxygen species, but the above effects could be reversed by the phosphoinositide 3-kinase (PI3K)/Akt inhibitor LY294002 (Spassov et al., 2017). These data indicate that the Akt signaling pathway mediated the protective effect of H2S on ventilator-induced lung injury. H2S treatment could also upregulate the gene expression of Atf3, an anti-inflammatory and antiapoptotic regulator (Spassov et al., 2014). Additionally, intravenous administration of NaHS (36 µmol/kg/h) significantly alleviated ventilator-induced lung injury by reducing pulmonary inflammation, accompanied by decreases in the heart rate and body temperature (Aslami et al., 2010). A similar effect was also found in the 22

treatment of mice with ventilator-induced lung injury with mesoporous silica nanoparticles (DATS-MSN, a drug that slowly releases H2S). The results showed that DATS-MSN significantly alleviated ventilator-induced lung injury and improved respiratory function; reduced the TNF-α, IL-1α/β and IL-2 content; and inhibited the ventilator-induced activation of NF-κB in mouse lung tissues (Wang et al., 2017). Na2S pretreatment could also upregulate the antioxidant genes NQO1, GPX2, and GST-A4 and inhibit oxidative stress (Zysman et al., 2019). In conclusion, H2S not only alleviates ventilator-induced lung injury but also improves respiratory function. The mechanisms of H2S are related to its inhibition of pulmonary inflammation, cell apoptosis and oxidative stress.

7. The effect of hydrogen on BPD Hydrogen is another small gas molecule that exerts protective effects in BPD rodent models. In a BPD model induced by the intra-amniotic injection of LPS in neonatal rats, the alveolar size and mean linear intercept were first increased but then decreased with the oral intake of hydrogen water (Muramatsu et al., 2016). Oral intake of hydrogen water significantly decreased oxidative stress, reduced the levels of nitrotyrosine and 8-OHdG in lung tissues, and inhibited gene expression of fibroblast growth factor receptor 4, vascular endothelial growth factor receptor 2 and HO-1, but did not affect SOD1 gene expression in these rat models of LPS-induced BPD. In addition, in cultured A549 human lung adenocarcinoma epithelial cells, hydrogen reduced the production of reactive oxygen species induced by LPS 23

(Muramatsu et al., 2016). Therefore, hydrogen exerts a protective effect on BPD induced by LPS by decreasing reactive oxygen species production and upregulating the expression of FGFR4, VEGFR2, and HO-1. In rats with lung injury induced by hyperoxia (60 h), hydrogen-rich saline (i.p. injection) alleviated pulmonary edema, hemorrhage, and the infiltration of inflammatory cells (Sun et al., 2011). In addition, hydrogen significantly ameliorated the increased levels of TNF-α, IL-1β, and myeloperoxidase; reduced the percentage of apoptotic cells; and reduced the increases in SOD, MDA, and 8-OHdG levels induced by hyperoxia in lung tissues. In mice with lung injury induced by hyperoxia, hydrogen inhalation (2%) significantly improved blood oxygenation, improved lung dysfunction, and prolonged survival time (Kawamura et al., 2013). Hydrogen also inhibited alveolar capillary leakage and reduced lung edema and lung injury induced by hyperoxia. In addition, hydrogen decreased mRNA expression of the proinflammatory factors IL-1, IL-6, TNF-α, and intercellular cell adhesion molecule-1. Hydrogen also inhibited pulmonary epithelial cell apoptosis induced by hyperoxia, reduced the proportion of caspase 3-positive cells, upregulated the mRNA and protein expression of Bcl-2, and downregulated the mRNA and protein expression of Bax. Immunohistochemical analysis showed that hydrogen treatment significantly increased the proportion of HO-1-positive lung epithelial cells and the mRNA and protein expression of HO-1 in lung tissues. Therefore, hydrogen played a protective role in this BPD rodent model, inhibited oxidative stress, reduced pulmonary inflammation, and decreased cell apoptosis. In mice with ventilator-induced lung injury administered 2% hydrogen, the 24

DNA-binding activity of NF-κB was enhanced after 1 h but decreased after 2 h of ventilation (Huang et al., 2011). Hydrogen inhalation significantly increased the mRNA expression of Bcl-2, decreased the expression of Bax and reduced epithelial cell apoptosis. In addition, hydrogen inhalation significantly reduced pulmonary edema and inflammatory cell infiltration and decreased the mRNA expression of TNF-α. An inhibitor of NF-κB and SN50 reversed the above effects of hydrogen (Huang et al., 2011). Thus, the protective effect of hydrogen on ventilator-induced lung injury is related to NF-κB activation and Bcl-2/Bax regulation. Hydrogen inhalation significantly reduced ventilator-induced edema in lung tissues, alleviated alveolar capillary leakage and reduced the total number of cells and protein levels in BALF (Huang et al., 2010). Hydrogen inhalation also alleviated the increased mRNA expression of the inflammatory mediators Egr-1, TNF-α, IL-1β, and CCL2 induced by ventilation, reduced expression of the proapoptotic Bax gene, and upregulated expression of the antiapoptotic genes Bcl-2 and Bcl-XL. Additionally, hydrogen inhalation decreased apoptosis of epithelial cells and reduced formation of the lipid peroxidation product MDA. In rats with lung injury induced by one-lung ventilation, the oral intake of hydrogen water for 4 weeks before operation inhibited oxidative stress, decreased the activities of MDA and myeloperoxidase, and reduced the content of TNF-α, IL-1β, and IL-6 in lung tissues (Wu et al., 2015). Hydrogen also inhibited the activation of NF-κB. Therefore, the protective effect of hydrogen inhalation on ventilator-induced lung injury is related to its antioxidant, anti-inflammatory and antiapoptotic effects. 25

8. Nrf2/HO-1 axis in BPD Nuclear factor erythroid-derived 2 (Nrf2) is a basic leucine zipper (bZIP) transcription factor that plays key roles in maintaining endogenous redox balance and regulating gene networks, including those involved in cell metabolism, cytoprotection, cancer and the immune process (Andrews et al., 1993; Loboda et al., 2016). Nrf2 is a pivotal regulator of antioxidant activity, as it induces antioxidants (SODs, Gpx and glutathione reductase), HO-1, and phase II enzymes (including quinone oxidoreductase and GSTs) (Loboda et al., 2016). Under pathological conditions, Nrf2 translocates from the cytosol to the nucleus and then binds small Maf proteins and specific antioxidant response elements to activate the transcription of cytoprotective genes, including HO-1, the most important cytoprotective gene (Hirotsu et al., 2012). Conditional deletion of Nrf2 in Clara cells augmented lung injury induced by hyperoxia and increased macrophage accumulation and epithelial sloughing in mouse lungs, accompanied by increased total cell infiltration in BALF (Reddy et al., 2011). Cell apoptosis and the inflammatory response were induced to a greater extent in mice with Nrf2 deficiency than in wild-type mice exposed to hyperoxia. Nrf2 deficiency even arrested alveolarization and aggravated lung edema and oxidation caused by hyperoxia (Cho et al., 2012). The survival rate of Nrf2-deficient mice was significantly lower than that of wild-type mice upon exposure to hyperoxia for 72 h (McGrath-Morrow et al., 2009). Nrf2 deficiency significantly increased the mean linear intercept and mean chord length of the lung alveoli, decreased the level of 26

surfactant protein C in the lungs, reduced Gpx2 and NQO1 production, and induced the expression of IL-6 (McGrath-Morrow et al., 2009). Therefore, Nrf2 deficiency enhances oxidative stress, increases cell death, augments lung injury, and arrests lung alveolarization. In contrast, inhibition of thioredoxin reductase-1 by aurothioglucose enhanced activation of Nrf2, accompanied by upregulation of NQO1 and HO-1 (Li et al., 2016). Nrf2 activation by aurothioglucose alleviated impaired alveolarization and reduced lung edema and inflammation induced by hyperoxia (Li et al., 2016; Tipple et al., 2007). In neonatal mice with lung injury induced by hyperoxia, the expression of HO-1 was found to be significantly increased (Anyanwu et al., 2014). In contrast, alveolar simplification was more aggravated, accompanied by an increase in macrophages in BALF, in HO-1 null (HO-1-/-) mice exposed to hyperoxia than in wild-type mice exposed to hyperoxia (Anyanwu et al., 2014). However, HO-1 overexpression increased the density of pulmonary vessels and alleviated pulmonary vascular remodeling and right ventricular hypertrophy in mouse pups exposed to hyperoxia (Fernandez-Gonzalez et al., 2012). HO-1 overexpression also decreased neutrophil accumulation, hemorrhage, edema, and alveolar septal thickness but did not alleviate alveolar simplification or affect ferritin and lactoferrin content. Therefore, HO-1 overexpression had a vascular protective effect in hyperoxia-induced BPD mice similar to the effect of exogenous CO in mice exposed to hyperoxia. In rats following intratracheal adenoviral ho-1 gene transfer, the mRNA and protein expression of HO-1 was significantly increased in the bronchiolar epithelium, accompanied by 27

alleviated lung injury, inhibited inflammation and decreased lung edema and hemorrhage induced by hyperoxia (Otterbein et al., 1999a). Furthermore, neutrophil influx was observed in the airway, and lung cell apoptosis was significantly reduced. However, ho-1 gene transfer did not affect antioxidant enzyme, manganese superoxide dismutase, or copper-zinc superoxide dismutase activities or the levels of L-ferritin and H-ferritin (Otterbein et al., 1999a). HO-1 overexpression inhibited human pulmonary epithelial cell growth, increased the number of cells in G0/G1 phase, and inhibited hyperoxic oxidant injury (Lee et al., 1996). Interestingly, hyperoxia significantly increased the expression of HO-1 and activation of signal transducer and activator of transcription 3 (STAT3) in mouse lung and endothelial cells. HO-1 or STAT3 overexpression had a protective effect against hyperoxic injury. HO-1 and CO did not protect STAT3 siRNA-treated murine lung endothelial cells or endothelial-specific STAT3-deficient mice against hyperoxic injury. However, STAT3 overexpression partially reduced cell apoptosis induced by hyperoxia in HO-1-deficient murine lung endothelial cells (Lee et al., 1996). Thus, endothelial STAT3 mediated the protective effects of HO-1 and CO, and the protection conferred by STAT3 was partially mediated by an HO-1-dependent pathway. Studies also found that pretreatment with an inducer of HO-1 or hemin could alleviate rat lung injury induced by ventilation, reduce the proportion of neutrophils in BALF and decrease myeloperoxidase activity in lung tissues (An et al., 2011). In addition, hemin reduced the levels of the proinflammatory cytokines TNF-α and IL-8 but increased the IL-10 content in BALF. The activity of MDA was also 28

inhibited by hemin. Activation of the Nrf2/HO-1 axis induced antioxidant and anti-inflammatory responses and therefore might be a novel therapy for BPD (Amata et al., 2017). Studies have shown that the HO-1/CO system is involved in the effects of NO, H2S and H2 on BPD (Figure 1). In neonatal rats, inhalation of NO (20 ppm) significantly increased the density of pulmonary vessels and radial alveolar count and enhanced lung alveolarization. In addition, expression of the VEGF, VEGFR-2, and HO-1 proteins was also significantly increased (Duong-Quy et al., 2014). Interestingly, HO-1 overexpression induced by (S)-6,7-dihydroxy-1-(4-hydroxynaphthylmethyl)-1,2,3,4-tetrahydroisoquinoline alkaloid negatively regulated the expression of iNOS in LPS-stimulated RAW 264.7 cells (Park et al., 2013). Sodium nitroprusside (an NO donor) significantly enhanced the expression of HO-1 but inhibited the expression of iNOS in the lung tissues of rats with LPS-induced acute lung injury (Xia et al., 2007). In cultured HeLa cells, HO-1 mRNA expression could be significantly increased by sodium nitroprusside, accompanied by upregulated expression of MAPKs, without influencing the SAPK/JNK pathway. PD98059 (an inhibitor of the ERK pathway) and SB203580 (an inhibitor of the p38 MAPK pathway) could reverse the above effect of sodium nitroprusside. Therefore, the NO donor sodium nitroprusside affected HO-1 expression via the ERK and p38 MAPK pathways (Chen and Maines, 2000). A further study also indicated that HO-1 expression was induced by sodium nitroprusside in RAW 264.7 cells via the cyclic adenosine 29

3',5'-monophosphate/protein kinase A/MAPK pathway but not the cyclic guanosine monophosphate pathway (Kim et al., 2006). Therefore, HO-1 is involved in the protective role of NO. H2S treatment could upregulate the CO content and HO-1 protein expression in pulmonary hypertension induced by high pulmonary blood flow, and these effects could be reversed by propargylglycine (an inhibitor of the key H2S enzyme cystathionine-gamma-lyase) (Li et al., 2007). In mice with lung injury induced by hyperoxia, H2S (i.p. injection of 0.56 mol/L NaHS at a dosage of 0.1 ml/kg/day) treatment significantly increased Nrf2 translocation to the nucleus and enhanced HO-1 activity in lung tissues, accompanied by decreased MDA and peroxynitrite production and NADPH oxidase activity (Li et al., 2013). H2S treatment inhibited the mRNA expression of IL-1β, monocyte chemotactic protein-1, and macrophage inflammatory protein-2 and enhanced IL-10 expression in lung tissues. Interestingly, H2S reduced the protein expression of iNOS in lung tissues and decreased the nitrate and nitrite content in the BALF of mice (Li et al., 2013). In mice with lung injury induced by LPS, GYY4137 (a slow-releasing H2S donor) significantly alleviated lung injury; reduced lung hemorrhage, edema, and alveolar wall thickness; and decreased neutrophil infiltration and inflammatory mediators (TNF-α, IL-6, prostaglandin E2 and nitrite) in BALF (Jiang et al., 2019). The protein expression of HO-1 in lung tissues was significantly increased by GYY4137. However, the protective effect of GYY4137 could be abrogated by tin protoporphyrin (an inhibitor of HO-1). Therefore, HO-1 mediates the protective role of GYY4137 against LPS-induced lung injury. 30

HO-1 gene expression is regulated by Nrf2 in the process of oxidative stress. Hydrogen treatment increased the expression of Nrf2 but showed no protective effects in Nrf2-/- mice with lung injury induced by hypoxia. In rats with BPD induced by the intra-amniotic injection of LPS, HO-1 gene expression in the lungs could be induced by the oral intake of hydrogen-rich water (Muramatsu et al., 2016). Activation of the Nrf2/HO-1 pathway was also observed to be part of the protective role of hydrogen inhalation in lung injury induced by seawater instillation in rabbits (Diao et al., 2016). Hydrogen treatment significantly upregulated the mRNA and protein expression of the Nrf2-dependent genes Nqo1, GSTA2, UGT1A6, and Prdx-1 and increased the activity of HO-1 in the lung tissues of rats exposed to hyperoxia (Kawamura et al., 2013). However, lung injury was worse in Nrf2-/- mice exposed to hyperoxia than in wild-type mice exposed to hyperoxia, and the protective role of hydrogen on lung injury was abrogated in Nrf2-/- mice. Interestingly, hydrogen decreased the MDA content and number of 8-OHdG-stained cells in the lung tissues of Nrf2-/- mice exposed to hyperoxia but did not affect Nrf2-dependent genes (HO-1, Nqo1, and GSTA2) (Kawamura et al., 2013). Thus, hydrogen confers a protective effect against lung injury induced by hyperoxia via both an Nrf2-dependent pathway and its free radical-scavenging effect. Inhalation of 2% H2 significantly alleviated lung injury, reduced the content of the proinflammatory cytokines TNF-α and IL-6, enhanced IL-10, decreased oxidative stress, reduced MDA production, and increased the antioxidant enzymes superoxide dismutase and catalase in the lungs of mice with sepsis induced by cecal ligation and puncture (Yu et al., 2019). Hydrogen treatment 31

also increased HO-1 levels in wild-type mice but not Nrf2 knockout mice, and the protective effect of hydrogen on lung injury was not observed in Nrf2 knockout mice. Therefore, Nrf2 plays a key role in the protective effects of hydrogen against lung injury induced by sepsis.

9. Perspective and challenges The protective role of NO has been clarified in preclinical studies, but the results of these studies are not consistent with those of clinical trials. A previous meta-analysis showed that inhaled NO does not reduce the incidence of BPD or related deaths but may increase the risk of severe intracranial hemorrhage. Therefore, inhaled NO is not recommended for BPD treatment in preterm infants. Studies have also found that CO, H2S, and hydrogen exert protective effects in rodent BPD models. However, their effectiveness and safety in clinical use still need to be clarified. When and how to use these gases in the clinic are questions that remain to be answered. Nrf2/HO-1 seem to play a central role in the mechanisms of all these gases, but the crosstalk among them is unknown. The differences among the effects of these gases on BPD also need to be explored. Although the effects of gasotransmitters on BPD have attracted great interest worldwide, further work is needed.

Declarations of interest: The authors declare that there are no conflicts of interest.

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58

Figure 1 The effect of NO, H2S and H2 on the HO-1/CO system (Amata et al., 2017).

59

Table 1 Metabolism of Gasotransmitters

NO

CO

H2S

NO

Heme

cystathionine γ-lyase

synthase

oxygenases

cystathionine β-synthase

Substrate

L-arginine

Heme

L-cysteine

Scavenger

Hemoglobin Hemoglobin

Protein targets

cGMP

cGMP

KATP channel

KCa channel

KCa channel

cAMP

Seconds

Minutes

Minutes

Generate Enzyme

Half time in

Hemoglobin

solution Abbreviation: cAMP, cyclic adenosine monophosphate; cGMP, cyclic guanosine monophosphate; CO, carbon monoxide; H2S, hydrogen sulfide;KATP, adenosine triphosphate-sensitive potassium channel; KCa, calcium activated potassium channels; NO, nitric oxide (Wang, 2002)

Author statement

Manuscript title: The effects of gasotransmitters on bronchopulmonary dysplasia

Hai Lin has drafted the manuscript and carefully revised the content. Dr. Xinbao Wang have made substantial contributions to design and review of the work. All of the authors approved the final version to be published in European Journal of Pharmacology. The authors declare there is no conflict of interest. All persons who have made substantial contributions to the work reported in the manuscript, including those who provided editing and writing.

Xinbao Wang Ph. D.