OBJECTIVES: To investigate the effects of hyperoxia on the expression of N-methyl-D-aspartate receptor 1 (NMDAR1) and its synapse-associated molecules, including cannabinoid receptor 1 (CB1R), postsynaptic density 95 (PSD95), and synapsin (SYN), in the hippocampus of neonatal rats. METHODS: One-day-old Sprague-Dawley neonatal rats were randomly divided into a hyperoxia group and a control group (n=8 per group). The hyperoxia group was exposed to 80% ± 5% oxygen continuously, while the control group was exposed to room air, for 7 days. At 1, 3, and 7 days after hyperoxia exposure, hematoxylin and eosin (HE) staining was used to observe histopathological changes in the brain. The expression levels of NMDAR1, CB1R, PSD95, and SYN proteins and mRNAs in the hippocampus were detected by immunohistochemistry, Western blotting, and quantitative real-time PCR. RESULTS: After 7 days of hyperoxia exposure, the hyperoxia group showed decreased neuronal density and disordered arrangement in brain tissue. Compared with the control group, after 1 day of hyperoxia exposure, CB1R mRNA and both NMDAR1 and CB1R protein expression in the hyperoxia group were significantly downregulated, while SYN protein expression was significantly upregulated (P<0.05). After 3 days, mRNA expression of NMDAR1, CB1R, and SYN was significantly decreased (P<0.05); NMDAR1 and CB1R protein expression was significantly downregulated (P<0.05), while PSD95 and SYN protein expression was significantly upregulated (P<0.05). After 7 days of hyperoxia, the protein expression of NMDAR1 and CB1R was significantly upregulated (P<0.05). CONCLUSIONS Continuous hyperoxia exposure induces time-dependent changes in the expression levels of NMDAR1 and its synapse-associated molecules in the hippocampus of neonatal rats.
Animals ; Receptors, N-Methyl-D-Aspartate/genetics* ; Rats, Sprague-Dawley ; Hippocampus/pathology* ; Rats ; Animals, Newborn ; Receptor, Cannabinoid, CB1/genetics* ; Hyperoxia/metabolism* ; Disks Large Homolog 4 Protein/genetics* ; Synapsins/genetics* ; Synapses ; Male ; Female ; RNA, Messenger/analysis*

OBJECTIVE: To investigate the expression and possible regulatory mechanism of miR-34a in the lung tissue of neonatal rat model of bronchopulmonary dysplasia (BPD) induced by hyperoxia. METHODS: In the study, 80 newborn SD rats were randomly divided into hyperoxia group (FiO₂=60%) and air group (FiO₂=21%) within 2 hours after birth, 40 rats per group. Lung tissue samples of the SD rats in each group were extracted on the 1st, 7th, 14th and 21st days after birth, and the pathological changes of lung tissue were observed under light microscope after HE staining. The number of radial alveolar counts (RAC) and the mean alveolar diameter (MAD) and the thickness of alveolar septal thickness (AST) were measured to evaluate the development of alveoli. Real-time fluorescence quantitative PCR was used to detect the expression of miR-34a, angiopoietin-1 (Ang-1) and tyrosine kinase receptor-2 (Tie-2) in lung tissue of rats in hyperoxia group and air group at different time points. Enzyme-linked immunosorbent assay (ELISA) was used to detect the proteins expression of Ang-1 and Tie-2 in the lung tissues of the two groups at different time points. RESULTS: The weight of rats in the hyperoxia group on the 7th, 14th and 21st days after birth was significantly lower than that in the air group (P all < 0.05). With the prolongation of oxygen exposure, the number of alveoli decreased, the volume increased, the structure simplified, the alveolar cavity enlarged obviously and the alveolar septum thickened in the hyperoxia group. On the 7th, 14th and 21st days after birth, the RAC in the hyperoxia group was significantly lower than that in the air group (P all < 0.05). Compared with the air group, MAD and AST increased significantly on the 7th, 14th and 21st days after birth in the hyperoxia group, and the difference was statistically significant (P all < 0.05). The expression level of miR-34a in lung tissue of hyperoxia group was significantly higher than that of air group on the 7th, 14th and 21st days after birth, and the difference was statistically significant (P all < 0.05). Compared with the air group at the same time point, the expression levels of Ang-1 and Tie-2 mRNA and protein in the hyperoxia group were lower than those in the air group on the 14th and 21st days after birth (P all < 0.05). CONCLUSION The new BPD model of newborn SD rats can be successfully established by continuous exposure to 60% hyperoxia. The expression of miR-34a was up-regulated in the lung tissue of the new BPD model of neonatal rats. MiR-34a may play an important role in the occurrence and development of BPD by regulating Ang-1/Tie-2 signal pathway.
Animals ; MicroRNAs/metabolism* ; Bronchopulmonary Dysplasia/genetics* ; Hyperoxia/metabolism* ; Rats, Sprague-Dawley ; Animals, Newborn ; Rats ; Angiopoietin-1/genetics* ; Disease Models, Animal ; Receptor, TIE-2/genetics* ; Lung/pathology* ; Male

OBJECTIVES: To study the role of adrenomedullin (ADM) in hyperoxia-induced lung injury by examining the effect of ADM on the expression of calcitonin receptor-like receptor (CRLR), receptor activity-modifying protein 2 (RAMP2), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB) in human pulmonary microvascular endothelial cells (HPMECs) under different experimental conditions. METHODS: HPMECs were randomly divided into an air group and a hyperoxia group ( RESULTS: Compared with the air group, the hyperoxia group had significant increases in the mRNA and protein expression levels of ADM, CRLR, RAMP2, ERK1/2, and PKB ( CONCLUSIONS ERK1/2 and PKB may be the downstream targets of the ADM signaling pathway. ADM mediates the ERK/PKB signaling pathway by regulating CRLR/RAMP2 and participates in the protection of hyperoxia-induced lung injury.
Adrenomedullin/genetics* ; Endothelial Cells ; Humans ; Hyperoxia/complications* ; Lung Injury ; Receptor Activity-Modifying Proteins

OBJECTIVES: To study the role of the low-density lipoprotein receptor-related protein 1 (LRP1)-proline-rich tyrosine kinase 2 phosphorylation (pPyk2)-matrix metalloproteinases 9 (MMP9) pathway in hyperoxia-induced lung injury in neonatal rats. METHODS: A total of 16 neonatal rats were randomly placed in chambers containing room air (air group) or 95% medical oxygen (hyperoxia group) immediately after birth, with 8 rats in each group. All of the rats were sacrificed on day 8 of life. Hematoxylin and eosin staining was used to observe the pathological changes of lung tissue. ELISA was used to measure the levels of soluble LRP1 (sLRP1) and MMP9 in serum and bronchoalveolar lavage fluid (BALF). Western blot was used to measure the protein expression levels of LRP1, MMP9, Pyk2, and pPyk2 in lung tissue. RT-PCR was used to measure the mRNA expression levels of LRP1 and MMP9 in lung tissue. RESULTS: The hyperoxia group had significantly higher levels of sLRP1 and MMP9 in serum and BALF than the air group ( CONCLUSIONS The activation of the LRP1-pPyk2-MMP9 pathway is enhanced in hyperoxia-induced lung injury in neonatal rats, which may be involved in the pathogenesis of bronchopulmonary dysplasia.
Animals ; Animals, Newborn ; Hyperoxia/complications* ; Lung ; Lung Injury/etiology* ; Matrix Metalloproteinase 9/genetics* ; Rats

OBJECTIVETo study the effect of rhubarb on neonatal rats with bronchopulmonary dysplasia (BPD) induced by hyperoxia.

METHODSA total of 64 rats (postnatal day 4) were randomly divided into four groups: air control, rhubarb control, hyperoxia model, and hyperoxia+rhubarb (n=16 each). The rats in the hyperoxia model and hyperoxia+rhubarb groups were exposed to hyperoxia (60% O2) to establish a BPD model. The rats in the rhubarb control and hyperoxia+rhubarb groups were given rhubarb extract suspension (600 mg/kg) by gavage daily. The pathological changes of lung tissue were evaluated by hematoxylin-eosin staining on postnatal days 14 and 21. The content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured by spectrophotometry. The mRNA and protein expression levels of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were determined by RT-PCR and Western blot respectively.

RESULTSThe hyperoxia model group showed reduced alveolar number, increased alveolar volume, and simplified alveolar structure, which worsened over the time of exposure to hyperoxia. These pathological changes were significantly reduced in the hyperoxia+rhubarb group. On postnatal days 14 and 21, compared with the air control and rhubarb control groups, the hyperoxia model group had significantly reduced radical alveolar count (RAC), significantly reduced activity of SOD in the lung tissue, and significantly increased content of MDA and mRNA and protein expression levels of TNF-α and IL-6 (P<0.05). Compared with the hyperoxia model group, the hyperoxia+rhubarb group had significantly increased RAC, significantly increased activity of SOD in the lung tissue, and significantly reduced content of MDA and mRNA and protein expression levels of TNF-α and IL-6 (P<0.05).

CONCLUSIONSRhubarb may play a protective role in rats with BPD induced by hyperoxia through inhibiting inflammatory response and oxidative stress.

Animals ; Animals, Newborn ; Bronchopulmonary Dysplasia ; metabolism ; pathology ; prevention & control ; Disease Models, Animal ; Hyperoxia ; complications ; Lung ; metabolism ; pathology ; Plant Extracts ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Rheum ; Superoxide Dismutase ; metabolism ; Tumor Necrosis Factor-alpha ; genetics

OBJECTIVETo explore the change of RAGE-NF-κB signaling pathway during the course of hyperoxia-induced lung injury in newborn rats, and the effect of glucocorticoid on this pathway.

METHODSTwenty-four Sprague-Dawley neonatal rats were randomly divided into three groups (n=8 each) : sham control (control group), hyperoxia-induced acute lung injury (model group) and glucocorticoid-treated acute lung injury (glucocorticoid group). Rats were sacrificed at 13 days after birth. RAGE and NF-κB expression levels in lung tissues were detected by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry analysis. The levels of tumor necrosis factor α (TNF-α) and sRAGE in bronchoalveolar lavage fluid (BALF) and serum were measured using ELISA. Lung damage was evaluated by histological examinations.

RESULTSRAGE and NF-κB mRNA and protein expression levels in lung tissues were significantly increased in the model and glucocorticoid groups compared with the control group (P<0.05). Serum RAGE concentrations were significantly increased but RAGE concentrations in BALF were significantly reduced in the model and glucocorticoid groups compared with the control group (P<0.05). RAGE and NF-κB expression at both mRNA and protein levels in lung tissues was significantly lower in the glucocorticoid group than in the model group (P<0.05). RAGE concentrations were significantly lower in serum (P<0.05), but were higher in BALF (P<0.05) in the glucocorticoid group than in the model group.

CONCLUSIONSRAGE-NF-κB pathway plays an important role in hyperoxia-induced lung injury in neonatal rats, and glucocorticoid administration may play a protective role against the lung injury by down-regulating RAGE-NF-κB signaling pathway.

Animals ; Animals, Newborn ; Glucocorticoids ; pharmacology ; Hyperoxia ; complications ; Lung Injury ; prevention & control ; NF-kappa B ; analysis ; genetics ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptor for Advanced Glycation End Products ; Receptors, Immunologic ; analysis ; genetics ; physiology ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; analysis

OBJECTIVETo explore the effect of silence of Pin1 expression on hyperoxia-induced apoptosis in alveolar epithelial cells A549.

METHODSA549 cells were divided into four groups: control, hyperoxia, negative lentivirus and Pin1-shRNA hyperoxia. The hyperoxia group was exposed to a mixture of 95%O2 and 5%CO2 for 10 minutes. Then cells were cultured in a closed environment. After 24 hours, the changes of morphology were observed under an inverted microscope. Cell apoptosis was detected by flow cytometry (FCM). The expression of X-linked inhibitor of apoptosis protein (XIAP) and Caspase-9 were detected by immunohistochemistry. The production of reactive oxygen species (ROS) and cellular mitochondria membrane potential (△Ψm) were determined by fluorescence microscopy.

RESULTSUnder the inverted microscope, the A549 cells grew slowly and the changes in morphology of the cells were most obvious in the hyperoxia and negative lentivirus groups. The changes in morphology of A549 cells were obviously improved in the Pin1-shRNA hyperoxia group. The FCM results showed that the apoptosis rate of A549 cells increased, Caspase-9 expression increased, XIAP expression decreased, mitochondrial ROS production increased and mitochondrial membrane potential decreased in the hyperoxia and negative lentivirus groups compared with the control group (P<0.05). Compared with the hyperoxia and negative lentivirus groups, the apoptosis rate of A549 cells decreased, Caspase-9 expression decreased, XIAP expression increased, mitochondrial ROS production decreased and mitochondrial membrane potential increased in the Pin1-shRNA hyperoxia group (P<0.05), although the levels of the indexes did not reach to those of the control group.

CONCLUSIONSSilencing of Pin1 could suppress hyperoxia-induced apoptosis of A549 cells.

Apoptosis ; Caspase 9 ; genetics ; Humans ; Hyperoxia ; pathology ; Membrane Potential, Mitochondrial ; NIMA-Interacting Peptidylprolyl Isomerase ; Peptidylprolyl Isomerase ; physiology ; Reactive Oxygen Species ; metabolism ; X-Linked Inhibitor of Apoptosis Protein ; genetics

OBJECTIVETo study the expression of fatty acid binding protein 4 (FABP4) in lungs and bronchoalveolar lavage fluid (BALF) of preterm rats exposed to 60% O2 and to elucidate the relationship between the changes of FABP4 expression and the pathogenesis of bronchopulmonary dysplasia (BPD).

METHODSHyperoxic lung injury was induced by exposing to 60% O2 in Spraque-Dawley rats within 6 hours after birth. Rats exposed to air were used as the control group. The lungs from groups aged postnatal days 3, 7 and 14 were removed and dissected from the main bronchi for analysis. Eight rats of each group were used to assess expression of FABP4 in lungs by immunohistochemistry and ELISA. Lung FABP4 mRNA levels were measured by semi-quantitative reverse transcription polymerase chain reaction. The levels of FABP4 in BALF were measured using ELISA.

RESULTSFABP4 immunoreactivity was detected in the majority of alveolar macrophages, bronchial epithelial cells and endothelial cells. FABP4 protein levels in lung tissues in the hyperoxic exposure group increased significantly compared with the control group on days 3, 7 and 14 after birth (P<0.05), and FABP4 mRNA levels in lung tissues also increased significantly in the hyperoxic exposure group compared with the control group on days 7 and 14 after birth (P<0.05). The hyperoxic exposure group demonstrated increased FABP4 levels in BALF compared with the control group on days 7 and 14 after birth (P<0.05).

CONCLUSIONSFABP4 levels increase in preterm rat lungs after hyperoxic lung injury, which may contribute to the pathogenesis of BPD.

Animals ; Bronchopulmonary Dysplasia ; etiology ; Fatty Acid-Binding Proteins ; analysis ; genetics ; Female ; Hyperoxia ; metabolism ; Lung ; chemistry ; Lung Injury ; metabolism ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; physiology

BACKGROUNDExposure of adult mice to more than 95% O2 produces a lethal injury by 72 hours. Nuclear factor kappa B (NF-κB) is a transcriptional factor that plays a key role in the modulation of cytokine networks during hyperoxia-induced acute lung injury (ALI). Osteopontin (OPN) is a phosphorylated glycoprotein produced principally by macrophages. Studies have reported that exogenous OPN can maintain the integrity of the cerebral microvascular basement membrane and reduce brain damage through inhibiting NF-κB activities in the brain after subarachnoid hemorrhage. However, it is not clear whether OPN can reduce lung injury during ALI by inhibiting transcriptional signal pathways of NF-κB and consequent inhibition of inflammatory cytokines. Thus we examined the effects and mechanisms of recombinant OPN (r-OPN) on ALI.

METHODSNinety-six mice were randomly divided into phosphate buffered saline (PBS) and r-OPN groups. Mice were put in an oxygen chamber (>95% O2) and assessed for lung injury at 24, 48, and 72 hours. Expressions of NF-κB, matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9), and tissue inhibitors of MMP-2 and MMP-9 (TIMP-1, TIMP-2) mRNA in lungs were examined with RT-PCR. Expression and distribution of NF-κB protein in lungs were measured with immunohistochemistry.

RESULTSExposure to hyperoxia for 72 hours induced more severe lung injury in the PBS group compared with the r-OPN group. Expression of NF-κB mRNA in the PBS group exposed to hyperoxia for 48 and 72 hours was significantly higher than the r-OPN group (P < 0.05). With 72-hour exposure, expression of TIMP-1 mRNA in the r-OPN group was significantly higher than that of the PBS group (P < 0.05). Expression of TIMP-2 mRNA in the r-OPN group at 48 and 72 hours was significantly higher than those in the PBS group (P < 0.05). After 72-hour exposure, expression of NF-κB protein in airway epithelium in the PBS group was significantly higher than that in the r-OPN group (P < 0.05).

CONCLUSIONr-OPN can inhibit the release and activation of MMPs through inhibition of the expression of NF-κB and promotion of the expression of TIMPs, and alleviate hyperoxia-induced ALI.

Acute Lung Injury ; genetics ; metabolism ; Animals ; Hyperoxia ; metabolism ; physiopathology ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Mice ; NF-kappa B ; genetics ; metabolism ; Osteopontin ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; metabolism

OBJECTIVETo study the effect of hyperoxia exposure on the expression of p53 and proliferating cell nuclear antigen (PCNA) in fetal rat lung fibroblasts (LFs).

METHODSPrimary rat embryonic LFs were cultured in vitro. LFs grew to subconfluence and then were randomly divided into air and hyperoxia exposure (95% O₂, 5% CO₂) groups. After LFs were cultured for 12 and 24 hours, the proliferation was analyzed by MTT. p53 mRNA level was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). p53 and PCNA protein levels were determined by Western blot.

RESULTSAfter 12 and 24 hours of culture the growth inhibition rate of LFs was 8% and 23% respectively in the hyperoxia exposure group. p53 mRNA and protein levels increased significantly (P<0.01) in the hyperoxia exposure group after 12 and 24 hours of culture compared with the air exposure group. Hyperoxia exposure decreased PCNA expression after 24 hours of culture (P<0.01).

CONCLUSIONSHyperoxia exposure increases p53 level and decreases PCNA expression, resulting in inhibitions of LFs proliferation and DNA repair.

Animals ; Cell Proliferation ; Female ; Fibroblasts ; metabolism ; Hyperoxia ; metabolism ; pathology ; Lung ; cytology ; metabolism ; Proliferating Cell Nuclear Antigen ; analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Tumor Suppressor Protein p53 ; analysis ; genetics