Ferroptosis is Involved in Hyperoxic Lung Injury in Neonatal Rats.

PURPOSE: To evaluate whether ferroptosis is involved in hyperoxic acute lung injury (HALI) and its mechanisms through the HALI model. METHODS: HE staining was used to assess lung injury pathology after the establishment of neonatal rat HALI model. ELISA was used to detect ROS, GPX4, and GSH expression. Prussian blue staining and Western Blot were used to detect iron deposition and the expression of ferroptosis-related proteins, respectively. RESULTS: The HALI group showed pathological changes with larger and fewer alveoli and thicker alveolar septa after HE staining. Prussian blue staining detected significant iron deposition in the lung tissue of the HALI group. GPX4, GSH, GSS, and SLC7A11 expressions were significantly decreased in the HALI group than in the normal control group. In contrast, ROS, TFRC, FHC, and FLC expressions showed opposite results (p<0.05). CONCLUSION: Ferroptosis may be involved in the pathological process of hyperoxic lung injury in neonatal rats.

Single-cell transcriptomics reveals lasting changes in the lung cellular landscape into adulthood after neonatal hyperoxic exposure.

Ventilatory support, such as supplemental oxygen, used to save premature infants impairs the growth of the pulmonary microvasculature and distal alveoli, leading to bronchopulmonary dysplasia (BPD). Although lung cellular composition changes with exposure to hyperoxia in neonatal mice, most human BPD survivors are weaned off oxygen within the first weeks to months of life, yet they may have persistent lung injury and pulmonary dysfunction as adults. We hypothesized that early-life hyperoxia alters the cellular landscape in later life and predicts long-term lung injury. Using single-cell RNA sequencing, we mapped lung cell subpopulations at postnatal day (pnd)7 and pnd60 in mice exposed to hyperoxia (95% O2) for 3 days as neonates. We interrogated over 10,000 cells and identified a total of 45 clusters within 32 cell states. Neonatal hyperoxia caused persistent compositional changes in later life (pnd60) in all five type II cell states with unique signatures and function. Premature infants requiring mechanical ventilation with different durations also showed similar alterations in these unique signatures of type II cell states. Pathologically, neonatal hyperoxic exposure caused alveolar simplification in adult mice. We conclude that neonatal hyperoxia alters the lung cellular landscape in later life, uncovering neonatal programing of adult lung dysfunction.

The role of cytochrome P450 (CYP) enzymes in hyperoxic lung injury.

INTRODUCTION: Hyperoxic lung injury is a condition that can occur in patients in need of supplemental oxygen, such as premature infants with bronchopulmonary dysplasia or adults with acute respiratory distress syndrome. Cytochrome P450 (CYP) enzymes play critical roles in the metabolism of endogenous and exogenous compounds. AREAS COVERED: Through their complex pathways, some subfamilies of these enzymes may contribute to or protect against hyperoxic lung injury. Oxidative stress from reactive oxygen species (ROS) production is most likely a major contributor of hyperoxic lung injury. CYP1A enzymes have been shown to protect against hyperoxic lung injury while CYP1B enzymes seem to contribute to it. CYP2J2 enzymes help protect against hyperoxic lung injury by triggering EET production, thereby, increasing antioxidant enzymes. The metabolism of arachidonic acid to ω-terminal hydroxyeicosatetraenoic acid (20-HETEs) by CYP4A and CYP4F enzymes could impact hyperoxic lung injury via the vasodilating effects of 20-HETE. CYP2E1 and CYP2A enzymes may contribute to the oxidative stress in the lungs caused by ethanol- and nicotine-metabolism, respectively. EXPERT OPINION: Overall, the CYP enzymes, depending upon the isoform, play a contributory or protective role in hyperoxic lung injury, and are, therefore, ideal candidates for developing drugs that can treat oxygen-mediated lung injury.

Inhibition of CX3C receptor 1-mediated autophagy in macrophages alleviates pulmonary fibrosis in hyperoxic lung injury.

AIMS: To investigate the role of CX3CR1 in hyperoxic lung injury induced pulmonary fibrosis. MATERIALS AND METHODS: Hyperoxic lung injured mice were used as the disease model. Pulmonary fibrosis was determined by H&E and Masson's staining. Autophagy was investigated by western blot, immunofluorescence staining, and transmission electron microscopy. KEY FINDINGS: We observed that increased CX3CR1 expression corresponded with increased pulmonary fibrosis. Additionally, silencing of CX3CR1 significantly alleviated the fibrosis when compared to the control. We observed that exposure of mouse to hyperoxic environment increased macrophage levels along with an increased CD11b expression in the lung tissues. Subsequently, we also observed an increased expression of LC3-II and decreased p62 expression in hyperoxic mice models, suggesting the potential role of hyperoxia induced autophagy. CD11b and LC3/CX3CR1 were expressed and co-localized in a manner indicating CX3CR1 indeed does regulate macrophage autophagy in the hyperoxic lung injury model. We observed a decrease in hyperoxia-associated fibrosis, along with a decrease in autophagy when we used 3-MA (autophagy inhibitor) in our hyperoxic lung injury model. To elucidate the pathway through which CX3CR1 regulated autophagy, we further analyzed the Akt1 pathway. Our experimental results indicated that the Akt1 inhibitor (A-674563) did significantly decrease macrophage autophagy and fibrosis in hyperoxic mice models. SIGNIFICANCE: Thus, our data indicates a novel role of CX3CR1 in regulation of macrophage autophagy and promotion of pulmonary fibrosis in hyperoxic lung injured mice.

Attenuation of Hyperoxic Lung Injury in Newborn Thioredoxin-1-Overexpressing Mice through the Suppression of Proinflammatory Cytokine mRNA Expression.

The role of thioredoxin-1 (TRX), a small redox-active protein with antioxidant effects, during hyperoxic lung injury in newborns remains undetermined. We investigated TRX impact on hyperoxic lung injury in newborn TRX transgenic (TRX-Tg) and wildtype (WT) mice exposed to 21% or 95% O2 for four days, after which some mice were allowed to recover in room air for up to 14 days. Lung morphology was assessed by hematoxylin/eosin and elastin staining, as well as immunostaining for macrophages. The gene expression levels of proinflammatory cytokines were evaluated using quantitative real-time polymerase chain reaction. During recovery from hyperoxia, TRX-Tg mice exhibited an improved mean linear intercept length and increased number of secondary septa in lungs compared with the WT mice. Neonatal hyperoxia enhanced the mRNA expression levels of proinflammatory cytokines in the lungs of both TRX-Tg and WT mice. However, interleukin-6, monocyte chemoattractant protein-1, and chemokine (C-X-C motif) ligand 2 mRNA expression levels were reduced in the lungs of TRX-Tg mice compared with the WT mice during recovery from hyperoxia. Furthermore, TRX-Tg mice exhibited reduced macrophage infiltration in lungs during recovery. These results suggest that in newborn mice TRX ameliorates hyperoxic lung injury during recovery likely through the suppression of proinflammatory cytokines.

Nanoparticle Delivery of Proangiogenic Transcription Factors into the Neonatal Circulation Inhibits Alveolar Simplification Caused by Hyperoxia.

Rationale: Advances in neonatal critical care have greatly improved the survival of preterm infants, but the long-term complications of prematurity, including bronchopulmonary dysplasia (BPD), cause mortality and morbidity later in life. Although VEGF (vascular endothelial growth factor) improves lung structure and function in rodent BPD models, severe side effects of VEGF therapy prevent its use in patients with BPD.Objectives: To test whether nanoparticle delivery of proangiogenic transcription factor FOXM1 (forkhead box M1) or FOXF1 (forkhead box F1), both downstream targets of VEGF, can improve lung structure and function after neonatal hyperoxic injury.Methods: Newborn mice were exposed to 75% O2 for the first 7 days of life before being returned to a room air environment. On Postnatal Day 2, polyethylenimine-(5) myristic acid/polyethylene glycol-oleic acid/cholesterol nanoparticles containing nonintegrating expression plasmids with Foxm1 or Foxf1 cDNAs were injected intravenously. The effects of the nanoparticles on lung structure and function were evaluated using confocal microscopy, flow cytometry, and the flexiVent small-animal ventilator.Measurements and Main Results: The nanoparticles efficiently targeted endothelial cells and myofibroblasts in the alveolar region. Nanoparticle delivery of either FOXM1 or FOXF1 did not protect endothelial cells from apoptosis caused by hyperoxia but increased endothelial proliferation and lung angiogenesis after the injury. FOXM1 and FOXF1 improved elastin fiber organization, decreased alveolar simplification, and preserved lung function in mice reaching adulthood.Conclusions: Nanoparticle delivery of FOXM1 or FOXF1 stimulates lung angiogenesis and alveolarization during recovery from neonatal hyperoxic injury. Delivery of proangiogenic transcription factors has promise as a therapy for BPD in preterm infants.

Protective effect of glutamine on hyperoxic lung injury of neonatal rats through endoplasmic reticulum stress pathway / 吉林大学学报(医学版)

Objective: To explore the protective effect of glutamine (GLN) on the hyperoxia-induced lung injury of the neonatal rats through endoplasmic reticulum stress (ERS) pathway, and to elucidate its mechanisms. Methods: A total of 90 Wistar rats were randomly divided into control group (FiO2 =21%), hyperoxia group (FiO2 85%), and hyperoxia+GLN group (Fi2 85%, the concentration of intraperitoneal injection of GLN was 0. 75 g · kg-1 · d-1); there were 30 rats in each group The body weights and water contents in the lung tissue of the neonatal rats were measured on the 3rd, 7th and 14th days of the experiment. HE staining was used to determine the morphology of lung tissue of the rats. The superoxide dismutase (SOD) activity in lung tissue of the rats was detected by nitro blue tetrazolium chloride (NBT), and the malondialdehyde (MDA) level was determined by thiobarbital acid (TBA). The expression levels of Caspase-12, GADD153, GRP78, Bel-2, and Bax in lung tissue of the rats were detected by Western blotting method. Results: Compared with control group at the same time, the body weights of the neonatal rats in hyperoxia group on the 3rd, 7th and 14th days were significantly decreased (P<0. 05), the water contents in lung tissue of the neonatal rats were increased (P<0. 05), the SOD activities were significantly decreased (P<0. 05), the levels of MDA in the lung tissue of the neonatal rats were increased (P<0. 05), the expressions levels of Caspase-12, GADD153, GRP78 and Bax proteins were significantly increased (P<0. 05), and the expression levels of Bcl-2 protein and the Bcl-2/Bax ratios were significantly decreased (P<0. 05). Compared with hyperoxia group at the same time, the body weights of the neonatal rats in hyperoxia + GLN group on the 3rd, 7th and 14th days were significantly increased (P<0. 05), the water contents in lung tissue of the neonatal rats were decreased (P<0. 05), the SOD activities were significantly increased (P< 0. 05), the levels of MDA in lung tissue of the neonatal rats were decreased (P<0. 05), the expression levels of Caspase-12, GADD153, GRP78 and Bax proteins were significantly decreased (P<0. 05), the expression levels of Bcl-2 protein and the Bcl-2/Bax ratios were increased (P<0. 05). The pathological sections of lung tissue of the rats in control group showed that lung tissue structure was regular, no alveolar edema was found, the alveolar size and alveolar septum were approximately the same, and no inflammatory cell infiltration was found; the histopathological sections of lung tissue of the rats in hyperoxia group showed swelling of brochial and alveolar epithelial cells, enlargement of alveolar lumen, edema of interstitial cells, inflammatory cell infiltration and fibrous exudation; the degrees of alveolar damage, the inflammatory exudation and the proliferation of fibrons tissue in hyperoxia+GLN group were alleviated which was between hyperoxia group and control group. Conclusion: GLN can alleviate the hyperoxia-induced lung tissue edema and inflammatory response of the neonatal rats, and one of mechanisms is that GLN can down-regulate the expression levels of Caspase-12, GADD153, GRP78 and Bax proteins and up-regulate the expression level of Bcl-2 protein through ERS pathway to protect hypoxic lung injury.

Effects of Lycopene in Hyperoxia-Induced Lung Injury in Newborn Rats.

The aim of this study was to evaluate the therapeutic effect of lycopene on a hyperoxia-induced lung injury model in rat pups. Full-term rat pups were included in the study 12-24 h after delivery. The pups were separated into 4 groups: normoxia control (NC), hyperoxia control (HC), hyperoxia + lycopene (HL), and normoxia lycopene (NL). The normoxia groups were housed in ambient air, and the hyperoxia groups in > 85% O2. HL and NL groups received 50 mg lycopene in oil/kg body weight/day delivered intraperitoneally (i.p.), the other groups received oil alone. On day 11, the rat pups were sacrificed and their lungs removed. Statistically significant injury was observed in all histological parameters measured (MLI, proliferating cell nuclear antigen (PCNA), and apoptosis) in the HC group (HC vs NC, p = 0.001). This injury could not be reversed with lycopene treatment (HC vs HL, 0.05; NC vs HL, p = 0.001). With hyperoxia, statistically significant decreases were observed in biochemical parameters in terms of SOD, MDA, and IL-6 values (HC vs NC: SOD, p = 0.02; MDA, p = 0.043; IL-6, p = 0.001). The use of lycopene did not provide any improvement in these values (HC vs HL, p > 0.05). Hyperoxia or lycopene had no effect on IL-1ß and GPx (p > 0.05). When comparing NC and NL groups, negative effects were observed in the group given lycopene in terms of MLI, PCNA, apoptosis, and IL-6 (all parameters, p = 0.001). We observed that 50 mg lycopene in oil/kg body weight/day given via i.p. had no curative effect on the hyperoxia-induced lung injury in newborn rats and may even induce adverse effects.

Effect of exogenous hydrogen sulfide on iNOS/NO in neonatal rats with hyperoxia lung injury / 中国新生儿科杂志

Objective To study the effect of hydrogen sulfide (H2S) on iNOS/NO in neonatal rats with hyperoxia-induced lung injury.Method Eighty full-term neonatal SD rats were randomly assigned into 4 groups (each group 20 rats including control group,hyperoxia group,NaHS + hyperoxia group,PPG + hyperoxia group.Rats in NaHS + hyperoxia group had 90 μmol/kg NaHS injected intraperitoneally,those in PPG + hyperoxia group had PPG 50 mg/kg injected,and those in the other 2 groups had the same amount of 0.9％ normal saline injected.Except for the control group that exposed to air,the other three groups were exposed to 95％ O2 for 7 days.Then pulmonary histopathology was studied by HE staining,the ratios of lung wet/dry weight (W/D) were determined as measurement of the severity of pulmonary edema,maleic dialdehyde (MDA),iNOS activity and NO levels in lung tissue were measured using commercial kits,iNOS mRNA expression was detected by Real-time PCR.Analysis of variance and LSD-t test were used for statistical analysis.Result (1) Compared with control group,the hyperoxia group showed erythrocyte extravasation and leukocyte infiltration in the alveoli with inflammatary cell infiltrations,alveolar septum edema,whereas pathological injury changes induced by hyperoxia were alleviated by NaHS and the damage was exacerbated by PPG.(2) In hyperoxia group,H2S was decreased compared with control group (117.6±20.4 μmol/L vs.184.3 ± 13.7 μmol/L).In NaHS + hyperoxia group,H2S was apparently increased compared with the hyperoxia group (247.3 ±32.4 μmol/L vs.117.6 ±20.4 μmol/L),while in PPG + hyperoxia group H2S was decreased compared with the hyperoxia group (89.2 ± 8.3 μmol/L vs.117.6 ±20.4 μmol/L) (P ＜0.01).(3) In the hyperoxia group,the ratios of lung W/D (5.81 ±0.22),the contents of MDA (1.69 ± 0.14) nmol/ml,iNOS activity (2.24 ± 0.19) U/mg prot,NO levels (22.37 ±3.04) × 10-3 μmol/g prot,iNOS mRNA expression (1.43 ±0.09) showed significant increase respectively (P ＜ 0.01) compared with the control group (5.06 ± 0.15),(0.78 ± 0.08)nmol/ml,(1.18 ± 0.18) U/mg prot,(7.49 ± 1.91) × 10-3 μmol/g prot,(0.90 ± 0.08).NaHS administration showed a significant decrease in lung W/D,lung tissue MDA content,iNOS activity,NO level,iNO SmRNA expression (5.59 ±0.19),(1.44±0.11) nmol/ml,(1.84 ±0.27) U/mg prot,(14.23 ±2.00)× 10-3 μmol/g prot,(1.28 ±0.10) compared with the hyperoxia group (P ＜0.01).The above markers were significantly increased after PPG administration (6.18 ± 0.26),(1.99 ± 0.19) nmol/ml,(2.66 ± 0.23) U/mgprot,(30.94 ±3.31) × 10-3 μmol/g prot,(1.73 ±0.06) (P ＜0.01).Conclusion Exogenous H2S can relieve hyperoxia-induced lung injury by down-regulating the expression of iNOS mRNA,decreasing iNOS activity and decreasing NO production.

Oridonin protects the lung against hyperoxia-induced injury in a mouse model.

Hyperoxic acute lung injury (HALI) is caused by prolonged exposure to high oxygen partial pressure. This study was undertaken to investigate the protective effects of oridonin on HALI in a mouse model. Mice were randomly divided into three groups: the control group, HALI group and oridonin (ORI) group. HALI was induced by exposing mice to pure oxygen at 2.5 atmospheres absolute (ATA) for six hours in the HALI and ORI groups. In the ORI group, mice were intraperitoneally injected with ORI at 10 mg/kg twice daily after hyperoxic exposure. Animals were sacrificed 24 hours after the hyperoxia exposure, followed by bronchoalveolar lavage fluid (BALF). Lungs were then collected. Each lung was processed for HE staining and detection of wet-to-dry weight ratio. The lactate dehydrogenase (LDH) activity and protein content of BALF were determined, and the contents of malonaldehyde (MDA), glutathione (GSH), tumor necrosis factor alpha (TNF-?) and interleukin-10 (IL-10) in the lung were measured. Our results showed prolonged exposure to hyperoxia significantly damaged the lung, caused lung edema, increased MDA and TNF-?, and reduced GSH and IL-10 in the lung. However, post-exposure treatment with oridonin was able to improve lung pathology, attenuate lung edema, reduce MDA and TNF-?, and increase GSH and IL-10 in the lung. These findings suggest that oridonin can protect the lung against hyperoxia-induced injury in mice.

Suplatast tosilate protects the lung against hyperoxic lung injury by scavenging hydroxyl radicals.

Prolonged exposure to hyperoxia produces extraordinary amounts of reactive oxygen species (ROS) in the lung and causes hyperoxic lung injury. Although supraphysiological oxygen is routinely administered for the management of respiratory failure, there is no effective strategy to prevent hyperoxic lung injury. In our previous study, we showed that suplatast tosilate, an asthma drug that inhibits T helper 2 (Th2) cytokines, ameliorated bleomycin-induced lung injury and fibrosis through Th2-independent mechanisms. Because bleomycin also generates ROS, we hypothesized that suplatast tosilate might have antioxidant activity and protect the lung against hyperoxic lung injury. To test this hypothesis, mice exposed to hyperoxia were given suplatast tosilate through drinking water. Treatment with suplatast tosilate significantly prolonged mouse survival, reduced the increases in the numbers of inflammatory cells, levels of the pro-inflammatory cytokines/chemokines IL-6 and MCP-1, and protein in bronchoalveolar lavage fluid, and ameliorated lung injury in histological assessment. Suplatast tosilate treatment also significantly inhibited hyperoxia-induced elevations in the levels of 8-hydroxydeoxyguanosine, a marker of oxidative DNA damage, in bronchoalveolar lavage fluid and 8-isoprostane, a marker of lipid peroxidation, in lung tissue. This finding suggests that suplatast tosilate exerts an antioxidant activity in vivo. In addition, we investigated whether suplatast tosilate has a scavenging effect on hydroxyl radical, the most reactive and harmful ROS, using electron paramagnetic resonance spin-trapping. Suplatast tosilate was shown to scavenge hydroxyl radicals in a dose-dependent manner, and its reaction rate constant with hydroxyl radical was calculated as 2.6×1011M-1S-1, which is faster than that of several well-established antioxidants, such as ascorbate, glutathione, and cysteine. These results suggest that suplatast tosilate protects the lung against hyperoxic lung injury by decreasing the degree of oxidative stress induced by ROS, particularly by scavenging hydroxyl radicals. Suplatast tosilate might become a potential therapeutic for hyperoxic lung injury.

MiR-196a regulates heme oxygenase-1 by silencing Bach1 in the neonatal mouse lung.

In the lung, heme oxygenase-1 (HO-1) is developmentally regulated, with its highest expression in the first days of life. In addition, neonatal mice have limited HO-1 induction in hyperoxia compared with adults. However, few reports have addressed the functional effect of microRNAs (miRNAs) in the regulation of HO-1 in vivo. The aims of the present study were to characterize changes in lung miRNA expression during postnatal development and in response to hyperoxic exposure, and to identify miRNAs that target lung HO-1 gene expression. Neonatal (<12 h old) and adult (2 mo old) mice were exposed to room air or hyperoxia (95% oxygen) for 72 h. TaqMan low-density array rodent miRNA assays were used to calculate miRNA expression changes between control and hyperoxia groups in neonatal and adult lungs. In neonates, we identified miR-196a, which binds to the 3'-untranslated region of the transcriptional repressor BTB and CNC homology 1 (Bach1) and regulates its expression, and subsequently leads to higher levels of lung HO-1 mRNA compared with levels in adults. Despite the increase at baseline, miR-196a was degraded in hyperoxia resulting in limited HO-1 induction in neonatal mice lungs. Furthermore, the developmental differences in lung HO-1 gene expression can be explained in part by the variation in miRNA-196a and its effect on Bach1. This report is the first to show developmental differences in lung miR-196a and its effect on Bach1 and HO-1 expression at baseline and in hyperoxia.

Beneficial Effect of Etanercept on Hyperoxic Lung Injury Model in Neonatal Rats.

PURPOSE: To determine whether prophylaxis with etanercept, an anti-inflammatory drug, would decrease the severity of lung injury in a neonatal rat model of bronchopulmonary dysplasia (BPD); MATERIALS AND METHODS: Rat pups were divided into three groups: pups exposed to room air (group 1; n = 10), to hyperoxia + placebo (group 2; n = 9), and to hyperoxia + etanercept (group 3; n = 8). Lung morphology was assessed by alveolar surface area percentage, which is a measure of alveolar size. The severities of lung inflammation and antioxidant capacity were assessed by quantifying tumor necrosis factor-α (TNF-α), transforming growth factor-ß (TGF-ß), malondialdehyde (MDA), and superoxide dismutase (SOD) from lung homogenate; RESULTS: The percentage of alveolar surface areas were significantly higher in group 3 compared to group 2 (p = .004) and similar in both group 1 and group 3 (p = .21). The mean level of lung MDA was significantly higher in group 2 compared to group 1 and group 3 (p < .05 for both). Lung homogenate SOD activities in group 3 was significantly higher than group 2 (p < .001). Furthermore, group 3 pups had lower levels of TNF-α and TGF-ß in lung homogenate than that in group 2 (p < .05 for both) but similar in both group 1 and group 3; CONCLUSION: Etanercept has favorable effects on alveolarization as well as inflammation and oxidative stress markers in a neonatal rat model of BPD.

Dexpanthenol therapy reduces lung damage in a hyperoxic lung injury in neonatal rats.

OBJECTIVE: Dexpanthenol (Dxp) plays a major role in cellular defense and in repair systems against oxidative stress and inflammatory response and it has not yet been evaluated in treatment of bronchopulmonary dysplasia (BPD). We tested the hypothesis that proposes whether Dxp decreases the severity of lung injury in an animal model of BPD. METHODS: Forty rat pups were divided into four groups: control, control + Dxp, hyperoxia and hyperoxia + Dxp. All animals were processed for lung histology and tissue analysis. The degree of lung inflammation, oxidative and antioxidant capacity was assessed from lung homogenates. RESULTS: Lung injury score and alveol diameter increased in the hyperoxia group (p < 0.001). Median level of malondialdehyde, total oxidant status and oxidative stress indexes was significantly higher in the hyperoxia group compared to the other groups. The median superoxide dismutase activity in the hyperoxia group was notably less than those of control + Dxp and hyperoxia + Dxp groups (p < 0.01). Similarly, lung catalase, glutathione (GSH) peroxidase and reduced GSH activities in the hyperoxia group were significantly lower than other groups. Furthermore, the hyperoxia + Dxp group had lower tumor necrosis factor-α and interleukin-1ß median levels compared to the hyperoxia group (p = 0.007). CONCLUSION: Dxp treatment results in less emphysematous change as well as decrease in inflammation and oxidative stress markers in an animal model of BPD.

Treatment of hyperoxic acute lung injury with stem cell transplantation in murine models: a brief review.

Hyperoxia has been a prominent clinical concern with the emergence of the intensive care unit and prolonged mechanical ventilation, along with the increasing use of hyperbaric oxygen therapy. Indeed, prolonged breathing of a high oxygen partial pressure may cause hyperoxic acute lung injury (HALI). To date, HALI has been a focus in the fields of pediatric and pulmonary medicine. However, no effective strategies have been developed for therapy for HALI due to the complicated mechanisms underlying the pathogenesis of HALI. In recent years, increasing studies have employed cell-based therapy for HALI. In this review, we summarize findings from available studies on therapy for HALI using stem cells in murine models and, based on concerns in this field, present our findings on cell-based therapy for HALI.

Influence of cyclooxygenase-2 inhibitor on the expressions of surfactant protein B and transforming growth factor β1 of newborn rats in hyperoxia environment / 中华实用儿科临床杂志

Objective To investigate the effects of selective cyclooxygease-2 inhibitor on pulmonary surfactant protein(SP-B) and transforming growth factor(TGF-β1) of hyperoxic lung injury in newborn rats.Methods One hundred and five SD rats were randomly divided into 3 groups (35 cases in each group):air group (group Ⅰ),in which the rats were exposed to room air;hyperoxia group(group Ⅱ),in which the rats were exposed to hyperoxia(850 mL/L oxy gen);Celecoxib group(group Ⅲ),in which the rats were exposed to heyperoxia(850 mL/L oxygen) and intraperitoneally injected with 5 mg/kg Celecoxib.The lungs of rats were removed on 3 d,7 d,14 d after birth and the following indices were measured:lung section from the lower right lung were stained with HE,and the histological changes was examined;the contents of SP-B and TGF-β1 in the bronchoalveolar lavage fluid of left lung was determinated by using enzyme-linked immunosorbent assay (ELISA);right upper lung was immunohistochemically stained to measure the contents of SP-B and TGF-β1,quantitative real-time PCR(RT-PCR) was used to detect the mRNA expression of SP-B and TGF-β1.Results There were no inflammatory cells and exudation in the lung in group Ⅰ;in group Ⅱ,the structure disorder,pulmonary edema,and inflammatory infiltrates were found;but the damage was obviously alleviated in group Ⅲ.Protein expression could be better detected by ELISA,at the time of 14 day,SP-B was expressed at different levels in3 groups:(29.93±6.40) ng/L in group Ⅰ,(18.20 ±3.70) ng/L in group Ⅱ and (19.63 ±10.20) ng/L in group Ⅲ,SP-B level in group Ⅱ was significantly lower than that in group Ⅰ (t =13.152,P ＜ 0.01),and the expres sion in group Ⅲ was significantly higher than that in group Ⅱ (t =5.190,P ＜ 0.01).TGF-β1 was expressed at different levels in 3 groups:(34.73 ±2.30) μg/L in group Ⅰ,(41.66 ± 1.80) μg/L in group Ⅱ and (38.03 ±0.20) μg/L in group Ⅲ,and the level of TGF-β1 was significantly higher in group Ⅱ than that in group Ⅰ (t =6.584,P ＜ 0.01),but the expression of group Ⅲ was significantly lower than that in group Ⅱ (t =5.609,P ＜ 0.01).The expression of mRNA was detected by RT-PCR,and at the time of 14 day,SP-B mRNA was expressed at different levels in 3 groups:3.14 ±0.10 in group Ⅰ,0.81 ±0.06 in group Ⅱ and 1.12 ±0.06 in group Ⅲ,and SP-B level in group Ⅱ was significantly lower than that in the group Ⅰ (t =55.050,P ＜0.01),and the expression in group Ⅲ was significantly higher than that in group Ⅱ (t =10.305,P ＜ 0.01).TGF-β1 mRNA was expressed at different levels in the 3 groups:1.94 ±0.03 in group Ⅰ,13.26 ±0.43 in group Ⅱ and 6.49 ±0.26 in group Ⅲ,the level of TGF-β1 was significantly higher in group Ⅱ than that in group Ⅰ (t =75.471,P ＜ 0.01),while the expression of group Ⅲ was significantly lower than that in group Ⅱ (t =38.470,P ＜ 0.01).Conclusions Cyclooxygenase-2 inhibitor can attenuate hyperoxic lung injury in rats,and the mechanism might be related to the reduction of prostaglandin.

Bacillus Calmette-Guerín vaccination: a novel therapeutic approach to preventing hyperoxic lung injury.

OBJECTIVE: A growing body of evidence suggests that vaccinations play a role in the normal maturation of the immune system and in both the development and balance of immune regulatory pathways that can impact health later in life. This study aimed to evaluate the effects of Bacillus Calmette-Guerín (BCG) vaccine on the hyperoxia-induced neonatal rat lung injury. METHODS: Four groups were defined as hyperoxia-exposed BCG-vaccinated, hyperoxia-exposed placebo, room air-exposed control and room air-exposed BCG-vaccinated group. The validity of the hyperoxia-induced lung injury model used in this study was confirmed by histological and immunohistochemical test. Gene expression related with cytokine and growth factor was evaluated by real-time reverse transcription polymerase chain reaction. RESULT: The mean alveolar surface area and quantification of secondary crest formation in the oxygen-exposed placebo group was significantly lower than that of the oxygen-exposed BCG-vaccinated group. Compared to the oxygen-exposed placebo group, the oxygen-exposed BCG-vaccinated group showed a significantly decreased alveolar septal fibrosis and smooth muscle actin expression. The expression of genes VEGF, FGF-BP1, IL-13, and NFκB1 (p50) in the lungs of the hyperoxia-exposed BCG-vaccinated group was significantly higher than that of the hyperoxia-exposed placebo group. CONCLUSION: Results suggest that BCG vaccination can protect against neonatal hyperoxic lung injury. These benefits may be interpreted to coincide with its immunomodulatory effects on pro-inflammatory and anti-inflammatory cytokine balance and expression of growth factors.

Substance P protects against hyperoxic-induced lung injury in neonatal rats.

The aim of the study was to investigate the effects of substance P (SP) in hyperoxia-induced lung injury in newborn rats. Thirty-two rat pups were randomly divided into four groups: normoxia/saline, normoxia/SP, hyperoxia/saline and hyperoxia/SP. In a separate set of experiments, the neonatal rat pups were exposed to 21% or >95% O2 for 14 days with or without intraperitoneal administration of SP. On day 14, the animals were sacrificed and the lungs were processed for histology and biochemical analysis. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was used for the detection of apoptosis. Antioxidant capacity was assessed by glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD), oxidative stress was assessed by determining the extent of formation of malondialdehyde (MDA), activities of NADPH oxidase activity, and formation of reactive oxygen species (ROS). The activity of phospho-p38 (p-p38) and -ERK1/2 (p-ERK1/2) proteins and expression of NF-E2-related factor 2 (NRF2) were detected by Western blot, and the expression of p-p38 was detected by immunofluorescence analysis. Compared with the hyperoxia treatment, the lung damage was significantly ameliorated following the SP treatment. Furthermore, the lungs from the pups exposed to hyperoxia TUNEL-positive nuclei increased markedly and decreased significantly after SP treatment. The levels of MDA decreased and that of GSH-Px and SOD increased following the SP treatment. The SP treatment significantly suppressed the activity of NADPH oxidase and reduced ROS production. SP stimulation may result in blocking p38 MAPK and ERK signaling pathways, and the activities of p-p38 and p-ERK, and expression of NRF2 decreased following the SP treatment. These findings indicate that SP can ameliorate hyperoxic lung injury through decreasing cell apoptosis, elevating antioxidant activities, and attenuating oxidative stress.

Adenosine promotes vascular barrier function in hyperoxic lung injury.

Hyperoxic lung injury is characterized by cellular damage from high oxygen concentrations that lead to an inflammatory response in the lung with cellular infiltration and pulmonary edema. Adenosine is a signaling molecule that is generated extracellularly by CD73 in response to injury. Extracellular adenosine signals through cell surface receptors and has been found to be elevated and plays a protective role in acute injury situations. In particular, ADORA2B activation is protective in acute lung injury. However, little is known about the role of adenosine signaling in hyperoxic lung injury. We hypothesized that hyperoxia-induced lung injury leads to CD73-mediated increases in extracellular adenosine, which is protective through ADORA2B signaling pathways. To test this hypothesis, we exposed C57BL6, CD73(-/-), and Adora2B(-/-) mice to 95% oxygen or room air and examined markers of pulmonary inflammation, edema, and monitored lung histology. Hyperoxic exposure caused pulmonary inflammation and edema in association with elevations in lung adenosine levels. Loss of CD73-mediated extracellular adenosine production exacerbated pulmonary edema without affecting inflammatory cell counts. Furthermore, loss of the ADORA2B had similar results with worsening of pulmonary edema following hyperoxia exposure without affecting inflammatory cell infiltration. This loss of barrier function correlated with a decrease in occludin in pulmonary vasculature in CD73(-/-) and Adora2B(-/-) mice following hyperoxia exposure. These results demonstrate that exposure to a hyperoxic environment causes lung injury associated with an increase in adenosine concentration, and elevated adenosine levels protect vascular barrier function in hyperoxic lung injury through the ADORA2B-dependent regulation of occludin.

Sustained hyperoxia-induced NF-κB activation improves survival and preserves lung development in neonatal mice.

Oxygen toxicity contributes to the pathogenesis of bronchopulmonary dysplasia (BPD). Neonatal mice exposed to hyperoxia develop a simplified lung structure that resembles BPD. Sustained activation of the transcription factor NF-κB and increased expression of protective target genes attenuate hyperoxia-induced mortality in adults. However, the effect of enhancing hyperoxia-induced NF-κB activity on lung injury and development in neonatal animals is unknown. We performed this study to determine whether sustained NF-κB activation, mediated through IκBß overexpression, preserves lung development in neonatal animals exposed to hyperoxia. Newborn wild-type (WT) and IκBß-overexpressing (AKBI) mice were exposed to hyperoxia (>95%) or room air from day of life (DOL) 0-14, after which all animals were kept in room air. Survival curves were generated through DOL 14. Lung development was assessed using radial alveolar count (RAC) and mean linear intercept (MLI) at DOL 3 and 28 and pulmonary vessel density at DOL 28. Lung tissue was collected, and NF-κB activity was assessed using Western blot for IκB degradation and NF-κB nuclear translocation. WT mice demonstrated 80% mortality through 14 days of exposure. In contrast, AKBI mice demonstrated 60% survival. Decreased RAC, increased MLI, and pulmonary vessel density caused by hyperoxia in WT mice were significantly attenuated in AKBI mice. These findings were associated with early and sustained NF-κB activation and expression of cytoprotective target genes, including vascular endothelial growth factor receptor 2. We conclude that sustained hyperoxia-induced NF-κB activation improves neonatal survival and preserves lung development. Potentiating early NF-κB activity after hyperoxic exposure may represent a therapeutic intervention to prevent BPD.