Development Of Hyperoxia-induced Lung Injury Research Articles

Expression changes of long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 and nuclear factor etythroid-2 related factor 2 and their significance in lung tissues of hyperoxia-induced premature newborn rats

Objective To observe the expressions of long noncoding RNA(lncRNA)metastasis-associated lung adenocarcinoma transcript 1(MALAT1)and nuclear factor etythroid-2 related factor 2(Nrf2) in lung tissues of hyperoxia-exposed premature neonatal rats, and explore the role of MALAT1 and Nrf2 in hyperoxia-induced lung injury. Methods Pregnant Sprague-Dawley (SD) rats were given cesarean section on the 21st day of gestation.After feeding for 24 h, a total of 80 premature rats were randomly divided into the air group and hyperoxia group.The rats in the air group were fed in the indoor environment[fraction of inspiration O2 (FiO2)=210 mL/L] and those in the hyperoxia group were fed in a high-oxygen box (FiO2>850 mL/L). Eight premature rats from each group were sacrificed and lung tissue samples were collected at 5 experimental time points (1st, 4th, 7th, 10th, 14th day ), respectively.Hematoxylin-eosin staining was used to observe pathological changes in lung tissues.Real-time quantitative polyme-rase chain reaction (qPCR)and Western blot were used to detect the expression level of MALAT1and Nrf2. Results Compared with the air group, the degree of alveolarization in lung tissues of the hyperoxia group rats was reduced, and radial alveolar count (RAC) decreased on the 1st day, but there was no significant difference between 2 groups (P>0.05), when they decreased on the 4th(3.14±0.23), 7th(5.25±0.38), 10th(4.41±0.44), 14th(3.41±0.13) day of exposure, the differences were statistically significant (all P 0.05). Compared with the air group, the mRNA expression of Nrf2 of hyperoxia group preterm rats decreased after the 1st day(0.791±0.031) of exposure, increased after the 4th(0.977±0.189), 7th(1.369±0.100), 10th(1.094±0.104) day, and the differences were statistically significant (all P 0.05). The hyperoxia group had significantly higher expression of free Nrf2 protein and lower expression of Nrf2-Keap1 protein than those of the air group at all time points.Within the hyperoxia group, the RNA expression of MALAT1 was positively correlated with RAC and Nrf2 (r=0.517, 0.533, all P<0.001). Conclusions Lung injury is gradually aggravated over the time of hyperoxia exposure.The level of lncRNA MALAT1 is associated with the severity of lung injury and the level of Nrf2 mRNA, suggesting that lncRNA MALAT1 and the Nrf2 target gene signaling pathway might be involved in the development of hyperoxia-induced lung injury in neonatal premature rats together. Key words: Hyperoxia-induced lung injury; Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1; Nuclear factor etythroid-2 related factor 2; Rat, preterm

Abstract P-601: FASUDIL, AN INHIBITOR OF RHO-ASSOCIATED COILED-COIL KINASE, ATTENUATES HYPEROXIA-INDUCED PULMONARY FIBROSIS IN NEONATAL RATS

Aims & Objectives: Oxygen therapy is important during the management of high-risk neonatal infants, such as those with preterm birth, low birth weight, and asphyxia. However, prolonged exposure to high oxygen concentrations can readily lead to diffuse nonspecific inflammation, which promotes airway remodeling and pulmonary fibrosis. The Rho/Rho-associated coiled-coil kinase (Rho/ROCK) signaling pathway plays an important role in numerous developmental and proliferative diseases. This study was performed to determine the efficacy of ROCK inhibitor fasudil in blocking the development of hyperoxia-induced lung injury and fibrosis in neonatal rats. Methods Neonatal rats were randomly divided into four groups: air + saline group, air + fasudil group, hyperoxia + saline group, and hyperoxia + fasudil group. The hyperoxia + saline and Hyp + fasudil groups were exposed to 95% oxygen for 21 days and administered intraperitoneal saline or fasudil once daily. The air + saline and air + fasudil group were exposed to 21% oxygen (room air) and administered the same volume of intraperitoneal saline or fasudil. Results Fasudil treated rats exhibited improved histopathological changes and decreased lung hydroxyproline content. Fasudil attenuated the protein level of alpha-smooth muscle actin, transforming growth factor-β1, and connective tissue growth factor. Additionally, fasudil reduced the activation of ROCK1 and myosin phosphatase targeting subunit 1 protein in the Rho/ROCK signaling pathway. Conclusions Fasudil may be a potentially effective therapeutic drug for hyperoxia-induced pulmonary fibrosis.

Activin A contributes to the development of hyperoxia-induced lung injury in neonatal mice.

Bronchopulmonary dysplasia (BPD) is one of the leading causes of morbidity and mortality in babies born prematurely, yet there is no curative treatment. In recent years, a number of inhibitors against TGFβ signaling have been tested for their potential to prevent neonatal injury associated with hyperoxia, which is a contributing factor of BPD. In this study, we assessed the contribution of activin A-a member of the TGFβ superfamily-to the development of hyperoxia-induced lung injury in neonatal mice. We placed newborn C57Bl6 mouse pups in continuous hyperoxia (85% O2) to mimic many aspects of BPD including alveolar simplification and pulmonary inflammation. The pups were administered activin A receptor type IIB-Fc antagonist (ActRIIB-Fc) at 5 mg/kg or follistatin at 0.1 mg/kg on postnatal days 4, 7, 10, and 13. Treatment with ActRIIB-Fc and follistatin protected against hyperoxia-induced growth retardation. ActRIIB-Fc also reduced pulmonary leukocyte infiltration, normalized tissue: airspace ratio and increased septal crest density. These findings were associated with reduced phosphorylation of Smad3 and decreased matrix metalloproteinase (MMP)-9 activity. This study suggests that activin A signaling may contribute to the pathology of bronchopulmonary dysplasia.

Effects of erythromycin on γ-glutamyl cysteine synthetase and interleukin-1β in hyperoxia-exposed lung tissue of premature newborn rats

ObjectiveTo explore the effect of erythromycin on hyperoxia-induced lung injury. MethodsOne-day-old preterm offspring Sprague-Dawley (SD) rats were randomly divided into four groups: group 1, air + sodium chloride; group 2, air + erythromycin;group 3, hyperoxia + sodium chloride; and group 4, hyperoxia + erythromycin. At one, seven, and 14 days of exposure, glutathione (GSH) and interleukin-1 beta (IL-1 beta) were detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), and bicinchoninic acid (BCA) was used to detect GSH protein. γ-glutamine-cysteine synthetase (γ-GCS) mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR). ResultsCompared with group 1, expressions of GSH and γ-GCS mRNA in group 3 were significantly increased at one and seven days of exposure (p < 0.05), but expression of γ-GCS mRNA was significantly reduced at 14 days; expression of IL-1 beta in group 3 was significantly increased at seven days of exposure (p < 0.05), and was significantly reduced at 14 days. Compared with group 3, expressions of GSH and γ-GCS mRNA in group 4 were significantly increased at one, seven, and 14 days of exposure (p < 0.05), but expressions of GSH showed a downward trend at 14 days; expression of IL-1 beta in group 4 was significantly reduced at one and seven days of exposure (p < 0.05). ConclusionsChanges in oxidant-mediated IL-1 beta and GSH are involved in the development of hyperoxia-induced lung injury. Erythromycin may up-regulate the activity of γ-GCS, increasing the expression of GSH, inhibiting the levels of oxidant-mediated IL-1 beta and alleviating hyperoxia-induced lung injury via an antioxidant effect.

Antioxidant role of glutathione hormone and γ-glutamyl cysteine synthetase in hyperoxia-induced lung injury in premature newborn rats

Objective To explore the expression of glutathione hormone (GSH) and γ-glutamyl cysteine synthetase (γ-GCS) in premature newborn rats exposed to hyperoxia, and to study antioxidant role of GSH and γ-GCS in hyperoxia-induced lung injury. Methods One-day old preterm SD rats were divided randomly into 2 groups: hyperoxia group and air group. Newborn rats in hyperoxia group were continuously exposed to oxygen(oxygen>850 mL/L), and newborn rats in air group were exposed in room air. After 1, 7, 14 days of exposure, the preterm SD rats of 2 groups were killed, and whole lung of these rats were isolated. GSH and γ-GCS of pulmonary tissue homogenate were detected by double antibody sandwich enzyme linked immunosorbent assay (ELISA). Bicinchoninic acid (BCA) was used to detect GSH protein in lung tissue homogenate. Total lung RNA was extracted and γ-GCS mRNA was detected by reverse transcription polymerase chain reaction. Results 1. The results were detected by ELISA method and BCA method, compaired with air group, the expression of GSH protein in lung tissue induced by hyperoxia was significantly increased after 1, 7 days of exposure(all P 0.05). Conclusions The changes of GSH and γ-GCS in the lung of premature SD rats induced by oxidation outbreak participate in the development of hyperoxia-induced lung injury, the activity of γ-GCS may be increased by hyperoxia, and alleviate hyperoxia lung injury in premature rats through antioxidation of GSH. Key words: Hyperoxia; Lung injury; Glutathione hormone; γ-glutamyl cysteine synthetase

Sphingosine Kinase 1 Deficiency Confers Protection against Hyperoxia-Induced Bronchopulmonary Dysplasia in a Murine Model: Role of S1P Signaling and Nox Proteins

Bronchopulmonary dysplasia of the premature newborn is characterized by lung injury, resulting in alveolar simplification and reduced pulmonary function. Exposure of neonatal mice to hyperoxia enhanced sphingosine-1-phosphate (S1P) levels in lung tissues; however, the role of increased S1P in the pathobiological characteristics of bronchopulmonary dysplasia has not been investigated. We hypothesized that an altered S1P signaling axis, in part, is responsible for neonatal lung injury leading to bronchopulmonary dysplasia. To validate this hypothesis, newborn wild-type, sphingosine kinase1(-/-) (Sphk1(-/-)), sphingosine kinase 2(-/-) (Sphk2(-/-)), and S1P lyase(+/-) (Sgpl1(+/-)) mice were exposed to hyperoxia (75%) from postnatal day 1 to 7. Sphk1(-/-), but not Sphk2(-/-) or Sgpl1(+/-), mice offered protection against hyperoxia-induced lung injury, with improved alveolarization and alveolar integrity compared with wild type. Furthermore, SphK1 deficiency attenuated hyperoxia-induced accumulation of IL-6 in bronchoalveolar lavage fluids and NADPH oxidase (NOX) 2 and NOX4 protein expression in lung tissue. In vitro experiments using human lung microvascular endothelial cells showed that exogenous S1P stimulated intracellular reactive oxygen species (ROS) generation, whereas SphK1 siRNA, or inhibitor against SphK1, attenuated hyperoxia-induced S1P generation. Knockdown of NOX2 and NOX4, using specific siRNA, reduced both basal and S1P-induced ROS formation. These results suggest an important role for SphK1-mediated S1P signaling-regulated ROS in the development of hyperoxia-induced lung injury in a murine neonatal model of bronchopulmonary dysplasia.

Expression of thioredoxin and thioredoxin reductase in hyperoxia-induced lung injury of newborn rats

Objective To investigate the changes and potential roles of the expression of thioredoxin and thioredoxin reductase in the premature Newborn rat lung exposed to hyperoxia.Methods On the first day after ddivery,the preterm SD rats were randomay divided into air group and hyperoxia group(both n=64).The rats were kined on day 4.7,10 and 14 respectively.Total RNA of lung was isolated and Trx and TR mR-NA expression levels were detected by reverse transcription polymerase chain reaction(RT-PCR).The sections of lung were stained with HE method in order to assess lung histologic changes and examine lung radical alveolar counts(RAC).Results Rats in hyperoxia group showed typical lung injury,which Was characterized by alveolitis and delay of lung development.Compared with air group.RACs were significantly decreased in hyperoxia group(P＜0.05).RT-PCR results showed Trx and TR mRNA expression levels in hyperoxia group markedly increased,peaking at day 10(1.444±0.142 vs 0.609±0.078,P＜0.01)and day 7(2.035±0.160 vs 1.325±0.164,P＜0.01)respectively.Conclusion Trx and TR expression could be induced by hyperoxia,which might play an importandy protective role in the development of hyperoxia-induced lung injury. Key words: Hyperoxia; Premature; Thioredoxin; Lung iniury

The Contribution of Intermittent Hypoxemia to Late Neurological Handicap in Mice with Hyperoxia-Induced Lung Injury

Bronchopulmonary dysplasia (BPD) is considered by many to be an independent risk factor for poor neurodevelopment in premature infants. However, infants with BPD experience intermittent hypoxic episodes. This study was undertaken to determine whether intermittent hypoxic stress associated with BPD contributes to the development of neurological deficit. The model of BPD was produced in neonatal mice by exposure to hyperoxia (65% O₂) for 4 weeks. Arterial blood gases, pulmonary mechanics, and histopathology were used to define the degree of lung injury. The mice were subjected to brief (10 min/day) and intermittent (10 days) hypoxic stress (8% O₂) at different stages of the development of hyperoxia-induced lung injury. At 8 weeks of life, the neurofunction was assessed by water maze and rota-rod tests followed by cerebral morphological analysis using Nissl, bromodeoxyuridine, and caspase-3 immunostaining. Data were compared to naïve normoxic littermates and those mice that were exposed only to hyperoxia or intermittent hypoxia alone. Mice with BPD subjected to brief/intermittent hypoxia demonstrated a significantly poorer navigational memory performance as compared with normoxic mice and mice with BPD that were not subjected to intermittent hypoxia. The neurofunctional handicap in these mice was associated with significantly decreased brain weight and increased cerebral expression of caspase-3. Our results suggest that intermittent hypoxia associated with hyperoxia-induced lung injury, but not lung injury itself, results in significant neurological handicap in neonatal mice with BPD.

Existence of leukotoxin 9,10-epoxy-12-octadecenoate in lung lavages from rats breathing pure oxygen and from patients with the adult respiratory distress syndrome.

Pulmonary influxed neutrophils have been suggested to be involved in the development of hyperoxia-induced lung injury. We recently revealed that a highly toxic substance, 9,10-epoxy-12-octadecenoate, is biosynthesized by human neutrophils, thus it was named leukotoxin. Because hyperoxia-induced lung injury is a model of adult respiratory distress syndrome (ARDS), this study was designed to investigate whether or not leukotoxin is involved in the genesis of pulmonary oxygen toxicity and ARDS. After exposure to hyperoxia for 60 h, rats showed acute pulmonary edema, which was evidenced by increased lung weight, albumin concentrations, and angiotensin-converting enzyme (ACE) activities in lung lavages. These changes were correlated with an increased number of neutrophils. We detected leukotoxin in lung lavages of rats after exposure to hyperoxia for 60 h by high performance liquid chromatography and gas-chromatography/mass spectrometry. After intravenous injection of leukotoxin (100 mumol/kg) to rats, acute edematous lung injury occurred showing increases in lung weight, lung lavage albumin concentrations, and lung lavage ACE activities. In the lung lavages obtained from 5 patients with ARDS, significant increases in albumin concentrations and ACE activities were observed compared with those from subjects without pulmonary disease. Moreover, considerable amounts of leukotoxin, 38.5 +/- 21.9 nmol/lung lavage, were observed in the lavages from patients with ARDS. These findings suggest that leukotoxin plays an important role in the genesis of acute edematous lung damage in pulmonary oxygen toxicity, and that leukotoxin also links with the development of lung injury observed in patients with ARDS.