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

OBJECTIVE: To investigate the effect of hyperoxia on intestinal metabolomics in mice. METHODS: Sixteen 8-week-old male C57BL/6 mice were randomly divided into hyperoxia group and control group, with 8 mice in each group. The hyperoxia group was exposed to 80% oxygen for 14 days. Mice were anesthetized and euthanized, and cecal contents were collected for untargeted metabolomics analysis by liquid chromatography-mass spectrometry (LC-MS) combined detection. Orthogonal partial least square discriminant analysis (OPLS-DA), volcano plot analysis, heat map analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used to analyze the effects of hyperoxia on metabolism. RESULTS: (1) OPLS-DA analysis showed that R²Y was 0.967 and Q² was 0.796, indicating that the model was reliable. (2) Volcano plot and heat map analysis showed significant statistical differences in the expression levels of metabolites between the two groups, with 541 up-regulated metabolites, 64 down-regulated metabolites, and 907 no differences, while the elevated 5-hydroxy-L-lysine was the most significant differential metabolite induced by high oxygen. (3) KEGG pathway enrichment analysis showed that porphyrin and chlorophyll metabolism (P = 0.005), lysine degradation (P = 0.047), and aromatic compound degradation (P = 0.024) were the targets affected by hyperoxia. (4) Differential analysis of metabolic products through KEGG enrichment pathway showed that hyperoxia had a significant impact on the metabolism of porphyrin and chlorophyll, lysine, and aromatic compounds such as benzene and o-cresol. CONCLUSIONS Hyperoxia significantly induces intestinal metabolic disorders. Hyperoxia enhances the metabolism of porphyrins and chlorophyll, inhibits the degradation of lysine, and delays the degradation of aromatic compounds such as benzene and o-cresol.
Mice ; Male ; Animals ; Lysine ; Hyperoxia ; Benzene ; Mice, Inbred C57BL ; Metabolic Diseases ; Oxygen ; Chlorophyll ; Porphyrins ; Biomarkers/metabolism*

Our previous studies have shown that calcitonin gene-related peptide (CGRP) exerts protective effects on the acute lung injury induced by oxidative stress. This study was aimed to investigate whether autophagy was involved in the protection of CGRP against oxidative stress-induced lung injury in neonatal rats. Newborn Sprague-Dawley (SD) rats were randomly divided into five groups: Control group, oxidative stress model group (Model group), Model + CGRP group, Model + CGRP + Rapamycin (an autophagy agonist) group, and Model + CGRP + LY294002 (an autophagy inhibitor) group. The model of hyperoxia-induced lung injury was established by continuous inhalation of oxygen (FiO₂ = 90%-95%) for 14 days in neonatal SD rats. Pathological changes of lung tissue were observed by hematoxylin and eosin (HE) staining, and mean linear intercept (MLI) was measured. The quantitative changes of autophagic vesicles (AV) in type II alveolar epithelial cells (AECII) were measured under the transmission electron microscope. The protein expressions of Caspase-3, Bcl-2, mTOR, and Beclin-1 in lung tissue lysates were detected by Western blot. The results showed that, compared to the Model group at the same time point, the number of AV in AECII and the expression level of Beclin-1 protein of the lung tissue were increased, while the expression level of mTOR protein was decreased, with alleviated pathological changes, reduced MLI value and Caspase-3 protein expression level, increased Bcl-2 protein expression level in the lung tissue of Model + CGRP group. In addition, we found that the protective effect of CGRP on hyperoxia-induced lung injury could be enhanced by autophagy activator Rapamycin and abolished by autophagy inhibitor LY294002. Together, these findings indicate that CGRP could attenuate hyperoxia-induced lung injury in neonatal rats by enhancing autophagy.
Acute Lung Injury/pathology* ; Animals ; Animals, Newborn ; Autophagy ; Calcitonin/metabolism* ; Calcitonin Gene-Related Peptide/metabolism* ; Caspase 3/metabolism* ; Hyperoxia/pathology* ; Lung/pathology* ; Lung Injury/prevention & control* ; Oxidative Stress ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Rats ; Rats, Sprague-Dawley ; Sirolimus/pharmacology*

OBJECTIVE: To study the expression and significance of ubiquitin-specific protease 7 (USP7) and the key factors of the Wnt signaling pathway in the lung tissue of preterm rats after hyperoxia exposure. METHODS: A total of 180 preterm neonatal Wistar rats were randomly divided into an air control group, an air intervention group, a hyperoxia control group, and a hyperoxia intervention group, with 45 rats in each group. Lung injury was induced by hyperoxia exposure in the hyperoxia groups. The preterm rats in the intervention groups were given intraperitoneal injection of the USP7 specific inhibitor P5091 (5 mg/kg) every day. The animals were sacrificed on days 3, 5, and 9 of the experiment to collect lung tissue specimens. Hematoxylin-eosin staining was used to observe the pathological changes of lung tissue. RT-PCR and Western blot were used to measure the mRNA and protein expression levels of USP7 and the key factors of the Wnt signaling pathway β-catenin and α-smooth muscle actin (α-SMA) in lung tissue. RESULTS: The air groups had normal morphology and structure of lung tissue; on days 3 and 5, the hyperoxia control group showed obvious alveolar compression and disordered structure, with obvious inflammatory cells, erythrocyte diapedesis, and interstitial edema. On day 9, the hyperoxia control group showed alveolar structural disorder and obvious thickening of the alveolar septa. Compared with the hyperoxia control group at the corresponding time points, the hyperoxia intervention group had significantly alleviated disordered structure, inflammatory cell infiltration, and bleeding in lung tissue. At each time point, the hyperoxia groups had a significantly lower radial alveolar count (RAC) than the corresponding air groups ( CONCLUSIONS Hyperoxia exposure can activate the Wnt/β-catenin signaling pathway, and USP7 may participate in hyperoxic lung injury through the Wnt/β-catenin signaling pathway. The USP7 specific inhibitor P5091 may accelerate the degradation of β-catenin by enhancing its ubiquitination, reduce lung epithelial-mesenchymal transition, and thus exert a certain protective effect against hyperoxic lung injury.
Animals ; Animals, Newborn ; Hyperoxia/physiopathology* ; Lung/physiopathology* ; Random Allocation ; Rats ; Rats, Wistar ; Thiophenes/pharmacology* ; Ubiquitin-Specific Peptidase 7/metabolism* ; Ubiquitin-Specific Proteases ; Wnt Signaling Pathway

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 study the expression of SUMO-modified CCAAT enhancer binding protein α (C/EBPα) in preterm rat model of bronchopulmonary dysplasisa (BPD) induced by hyperoxia exposure and its role.

METHODSEighteen preterm rats were randomly divided into an air group and a hyperoxia group (n=9 each). The model of BPD was prepared in preterm rats exposed to hyperoxia. The rats from the two groups were sacrificed on postnatal days 4, 7 and 14 respectively (3 rats at each time) and lung tissues were harvested. Periodic acid-Schiff (PAS) staining was used to observe the differentiation of rat lung tissues. Ki67 expression was detected by immunohistochemistry. Western blot was used to measure the protein expression of small ubiquitin-related modifier-1(SUMO1) and C/EBPα. A co-immunoprecipitation assay was performed to measure the protein expression of SUMO-modified C/EBPα.

RESULTSCompared with the air group, the hyperoxia group showed a decreased glycogen content in the lung tissue on postnatal day 4, and an increased content on postnatal days 7 and 14. Over the time of hyperoxia exposure, the hyperoxia group showed an increased expression of Ki67 in the lung tissue compared with the air group at all time points. Compared with the air group, the protein expression of C/EBPα increased on postnatal day 4 and decreased on postnatal days 7 and 14 in the hyperoxia group (P<0.05). The hyperoxia group had significantly upregulated expression of SUMO1 and SUMO-modified C/EBPα compared with the air group at all time points (P<0.05). In the hyperoxia group, the protein expression of SUMO-modified C/EBPα was positively correlated with the glycogen content (r=0.529, P<0.05) and the expression of Ki67 (r=0.671, P<0.05).

CONCLUSIONSHyperoxia may induce over-proliferation and differentiation disorders of alveolar epithelial cells in preterm rat model of BPD, possibly through an increased expression of SUMO-modified C/EBP&alpha.

Animals ; Animals, Newborn ; Bronchopulmonary Dysplasia ; etiology ; metabolism ; pathology ; CCAAT-Enhancer-Binding Protein-alpha ; metabolism ; Cell Proliferation ; Disease Models, Animal ; Hyperoxia ; complications ; pathology ; Ki-67 Antigen ; analysis ; Pulmonary Alveoli ; pathology ; Rats ; Rats, Sprague-Dawley ; Sumoylation

OBJECTIVETo investigate the protective effect of prostaglandin E1 (PGE-1) against brain injury induced by hyperoxia in neonatal rats and observe the changes in the expression of glucose-regulated protein 78 (GRP78) and cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), and to provide a theoretical basis for the clinical application of PGE-1 in the treatment of neonatal brain injury induced by hyperoxia.

METHODSSixty neonatal Wistar rats were randomly divided into air control group, hyperoxic brain injury model group, and hyperoxic brain injury+PGE-1 group. All rats except those in the air control group were treated to establish a hyperoxic brain injury model. From the first day of modeling, the rats in the hyperoxia brain injury+PGE-1 group were intraperitoneally injected with PGE-1 2 μg/kg daily for 7 consecutive days, while the other two groups were treated with normal saline instead. The water content of brain tissue was measured; the pathological changes of brain tissue were evaluated by hematoxylin-eosin staining; the apoptosis of brain cells was assessed by nuclear staining combined with TUNEL staining; the protein expression of GRP78 and CHOP in brain tissue was measured by Western blot.

RESULTSThe water content of brain tissue in the hyperoxic brain injury model group was significantly higher than that in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the water content of brain tissue in the hyperoxic brain injury+PGE-1 group was significantly higher than that in the air control group (P<0.05). The pathological section of brain tissue showed inflammatory cell infiltration and mild cerebrovascular edema in the brain parenchyma in the hyperoxic brain injury model group; the periparenchymal inflammation and edema in the hyperoxic brain injury+PGE-1 group were milder than those in the hyperoxic brain injury model group. The apoptosis index of brain tissue in the hyperoxic brain injury model group was significantly higher than that in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the apoptosis index of brain tissue in the hyperoxic brain injury+PGE-1 group was significantly higher than that in the air control group (P<0.05). The protein expression of GRP78 and CHOP in brain tissue was significantly higher in the hyperoxic brain injury model group than in the hyperoxic brain injury+PGE-1 group and air control group (P<0.05); the protein expression of GRP78 and CHOP was significantly higher in the hyperoxic brain injury+PGE-1 group than in the air control group (P<0.05).

CONCLUSIONSPGE-1 has a protective effect against hyperoxia-induced brain injury in neonatal rats, which may be related to the inhibition of cell apoptosis by down-regulating the expression of GRP78 and CHOP.

Alprostadil ; therapeutic use ; Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Brain ; pathology ; Brain Injuries ; metabolism ; pathology ; prevention & control ; Heat-Shock Proteins ; analysis ; Hyperoxia ; complications ; Neuroprotective Agents ; therapeutic use ; Rats ; Rats, Wistar ; Transcription Factor CHOP ; analysis

OBJECTIVETo investigate the expression of long non-coding RNA NANCI in lung tissues of neonatal mice with hyperoxia-induced lung injury and its regulatory effect on NKX2.1.

METHODSA total of 48 neonatal C57BL/6J mice were randomly divided into an air group and a hyperoxia group, with 24 mice in each group. Each group was further divided into 7-day, 14-day, and 21-day subgroups, with 8 mice in each subgroup. The mice in the air group were fed in the indoor environment (FiO=21%) and those in the hyperoxia group were fed in a high-oxygen box (oxygen concentration: >95%). The mice were sacrificed at each time point and lung tissue samples were collected. Hematoxylin and eosin staining was used to observe pathological changes in lung tissues. RT-qPCR and Western blot were used to measure the mRNA and protein expression of NANCI and NKX2.1.

RESULTSThe air group had the highest mRNA expression of NANCI and NKX2.1 at 7 days and the same level of mRNA expression at 14 and 21 days. Compared with the air group, the hyperoxia group had significant reductions in the degree of alveolarization and radial alveolar count (RAC) in lung tissues (P<0.05), and in the hyperoxia group, RAC gradually decreased over the time of hyperoxia exposure (P<0.05). The hyperoxia group had significantly lower mRNA and protein expression of NANCI and NKX2.1 than the air group at all time points (P<0.05). In both groups, the relative mRNA and protein expression of NANCI and NKX2.1 gradually decreased over the time of hyperoxia exposure (P<0.05). The expression of NKX2 was positively correlated with that of NANCI (r=0.585, P=0.003), and the expression of NKX2 and NANCI was positively correlated with RAC in the hyperoxia group (r=0.655 and 0.541 respectively, P<0.05).

CONCLUSIONSNANCI may be involved in the development of immature lung tissues. Lung injury is gradually aggravated over the time of hyperoxia exposure. The levels of NANCI and NKX2.1 are associated with the severity of lung injury, suggesting that the NANCI/NKX2.1 target gene signaling pathway might be involved in the development of hyperoxia-induced lung injury in neonatal mice.

Animals ; Animals, Newborn ; Female ; Hyperoxia ; complications ; Lung ; metabolism ; Lung Injury ; etiology ; Male ; Mice ; Mice, Inbred C57BL ; Nuclear Proteins ; physiology ; RNA, Long Noncoding ; physiology ; Signal Transduction ; physiology ; Thyroid Nuclear Factor 1 ; Transcription Factors ; physiology

OBJECTIVETo study the association between endoplasmic reticulum stress (ERS) pathway mediated by inositol-requiring kinase 1 (IRE1) and the apoptosis of type II alveolar epithelial cells (AECIIs) exposed to hyperoxia.

METHODSThe primarily cultured AECIIs from preterm rats were devided into an air group and a hyperoxia group. The model of hyperoxia-induced cell injury was established. The cells were harvested at 24, 48, and 72 hours after hyperoxia exposure. An inverted phase-contrast microscope was used to observe morphological changes of the cells. Annexin V/PI double staining flow cytometry was performed to measure cell apoptosis. RT-PCR and Western blot were used to measure the mRNA and protein expression of glucose-regulated protein 78 (GRP78), IRE1, X-box binding protein-1 (XBP-1), and C/EBP homologous protein (CHOP). An immunofluorescence assay was performed to measure the expression of CHOP.

RESULTSOver the time of hyperoxia exposure, the hyperoxia group showed irregular spreading and vacuolization of AECIIs. Compared with the air group, the hyperoxia group showed a significantly increased apoptosis rate of AECIIs and significantly increased mRNA and protein expression of GRP78, IRE1, XBP1, and CHOP compared at all time points (P<0.05). The hyperoxia group had significantly greater fluorescence intensity of CHOP than the air group at all time points. In the hyperoxia group, the protein expression of CHOP was positively correlated with the apoptosis rate of AECIIs and the protein expression of IRE1 and XBP1 (r=0.97, 0.85, and 0.88 respectively; P<0.05).

CONCLUSIONSHyperoxia induces apoptosis of AECIIs possibly through activating the IRE1-XBP1-CHOP pathway.

Animals ; Apoptosis ; Cells, Cultured ; Endoplasmic Reticulum Stress ; physiology ; Endoribonucleases ; physiology ; Epithelial Cells ; physiology ; Female ; Hyperoxia ; metabolism ; pathology ; Multienzyme Complexes ; physiology ; Protein-Serine-Threonine Kinases ; physiology ; Pulmonary Alveoli ; pathology ; Rats ; Rats, Sprague-Dawley ; Transcription Factor CHOP ; physiology ; X-Box Binding Protein 1 ; physiology