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*

High concentrations of oxygen, indispensable for the treatment of severe hypoxemia from neonatal as well as adult respiratory distress syndrome, increase the risk of oxygen toxicity. Biochemical mechanisms are lipid peroxidation, protein sulfhydryl oxidation, enzyme inactivation, and DNA damage. Recent reports suggest that cytokines might be involved in free radical injury as well as in adaptive response to hyperoxic injury. However, actual signal transduction pathways involving cytokines have not yet been clarified. In this study we exposed cultured human umbilical vein endothelial cells (HUVECs) to either ambient air or 100% oxygen, and compared for the rate of DNA synthesis ([3H]thymidine uptake) at different time points up to 72 h. After exposing the cells to each treatment condition, we extracted RNA, constructed complementary DNA using reverse transcriptase, amplified the specific DNA segments of cytokines by polymerase chain reaction (PCR), and used the PCR products for gel electrophoresis to examine the bands which signified mRNA levels of corresponding cytokines. There was a significant decrease in the rate of DNA synthesis as early as 24 h. The mRNA expression of IL-1 beta and TNFa seemed less influenced by hyperoxia, while IL-8 and TGF beta showed marked increase in mRNA levels at 6 h of 100% oxygen exposure.
Cells, Cultured ; Cytokines/genetics* ; DNA/biosynthesis ; Endothelium, Vascular/metabolism* ; Human ; Hyperoxia/metabolism* ; RNA, Messenger/analysis* ; Umbilical Veins

OBJECTIVETo explore the effect of low-concentration lipopolysaccharide (LPS) pretreatment on hyperoxia-induced immature brain injury in neonatal mice and explore and the related mechanisms.

METHODSForty-eight neonatal mice on postnatal day 3 (PND3) were randomized into normal control group, LPS (0.3 mg/kg) group, hyperoxia group (hyperoxia exposure for 24 h), and hyperoxia+LPS group (hyperoxia exposure for 24 h 30 min after 0.3 mg/kg LPS treatment). At PND5, all the neonatal mice were sacrificed to examine the morphological changes of microglia in the periventricular white matter using Tomato lectin staining, measure malondialdehyde (MDA) content in the immature brain, detect mRNA expression of tumor necrosis factor-α (TNF-α) using real-time PCR, and determine caspase-3 protein expression with Western blotting.

RESULTSCompared with the control group, exposures to LPS, hyperoxia, and both all resulted in microglia activation in the periventricular white matter. The number of activated microglia, MDA content, TNF-α mRNA expression and caspase-3 protein expression in the immature brain were significantly higher in hyperoxia group than in the control group and LPS group (P<0.05). LPS pretreatment significantly enhanced hyperoxia-induced microglia activation in the immature brain (P<0.05).

CONCLUSIONHyperoxia causes immature brain injury mediated by microglia activation, and LPS pretreatment can enhance such brain injury in neonatal mice.

Animals ; Animals, Newborn ; Brain ; metabolism ; pathology ; Caspase 3 ; metabolism ; Hyperoxia ; Lipopolysaccharides ; adverse effects ; Malondialdehyde ; metabolism ; Mice ; Mice, Inbred C57BL ; Microglia ; metabolism ; pathology ; Tumor Necrosis Factor-alpha ; metabolism

Antioxidants ; metabolism ; therapeutic use ; Bronchopulmonary Dysplasia ; drug therapy ; etiology ; metabolism ; Humans ; Hyperoxia ; complications ; Infant, Newborn ; Lung ; metabolism ; Reactive Oxygen Species ; metabolism

OBJECTIVEResolution of alveolar damage after lung injury requires finely orchestrated processes that include coordinated and effective tissue reconstruction to reestablish a functional barrier. Reconstitution of denuded type I alveolar epithelial cell that undergo apoptotic and necrotic death after lung injury is required in many pulmonary diseases. Disruption of distal airway development and type II-type I alveolar epithelial cell differentiation after lung injury and disordered repair of the alveolus after injury is one of the predominant pathological characteristics of chronic lung disease (CLD) of premature infants. HoxB5 belongs to the Hox gene family encoding transcription factors known for their role in skeletal patterning and the elaboration of organs. HoxB5 is required for embryonic respiratory tract morphogenesis. The present study aimed to test the hypothesis that HoxB5 may participate in the etiology of CLD and to understand possible mechanism.

METHODSPremature rat pups were taken out surgically at gestational age 21 d. CLD was induced by hyperoxia exposure in neonatal premature rats. Eighty premature rats were randomly exposed to hyperoxia (FiO2 = 0.90, CLD group) and to room air (FiO2 = 0.21, control group) (n = 40 each). Lung specimens were obtained respectively on days 1, 3, 7, 14 and 21 after exposure. Histopathologic changes was assayed after hematoxylin and eosin (HE) staining and pulmonary development was evaluated by lung coefficient and radical alveolar counts (RAC), dynamic changes of RAC were observed; and the expression of HoxB5, AQP-5, and SP-B mRNA were assayed by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSThere were no significant differences in the RAC and the expression level of HoxB5, AQP-5, and SP-B mRNA between the CLD and the control groups within 3 days after birth (P > 0.05). However, compared to the control group, the RAC of the CLD group was reduced (6.35 +/- 0.83 vs. 7.67 +/- 0.52), and the expression of HoxB5 (0.98 +/- 0.14 vs. 1.20 +/- 0.16), AQP-5 (0.78 +/- 0.11 vs. 1.04 +/- 0.19) mRNA were significantly lower (P < 0.05), while the expression of SP-B mRNA was increased on the 7th day (P < 0.05). On the 14th day, the RAC and the expression of HoxB5, AQP-5 mRNA of CLD group were significantly lower than those of the control group (P < 0.05), and the expression of SP-B mRNA continued to increase (P < 0.05). On the 21st day, the expression of HoxB5, AQP-5 mRNA decreased to the nadir (0.64 +/- 0.11 vs. 1.18 +/- 0.13 and 0.67 +/- 0.12 vs. 0.97 +/- 0.01, respectively) (P < 0.01), on the same day the expression of SP-B mRNA reached to the pinnacle (1.43 +/- 0.07 vs. 1.12 +/- 0.09) (P < 0.01). The expression of HoxB5 mRNA was positively correlated with RAC in the CLD group (r = 0.685, P < 0.01).

CONCLUSIONSWith hyperoxia exposure, the mRNA expression of specific marker of type I alveolar epithelial cell, AQP-5, was decreased while specific marker of type II alveolar epithelial cell, SP-B, was increased; and the expression of HoxB5 mRNA in lung tissues kept on decreasing. Decreased expression of HoxB5 may associate with the disruption of type II-I alveolar epithelial cell differentiation and thus may play an important role in inhibition of lung development with CLD. The altered Hox gene expression may predispose lung pathology.

Animals ; Animals, Newborn ; Female ; Homeodomain Proteins ; genetics ; metabolism ; Hyperoxia ; complications ; Lung ; growth & development ; metabolism ; pathology ; Lung Diseases ; etiology ; metabolism ; pathology ; Pregnancy ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the protective effect of hyperoxia solution on acute lung injury caused by phosgene poisoning by observing the changes of PaO2 and malondialdehyde (MDA) contents, superoxide dismutase (SOD) activity in serum and Glutathione (GSH/GSSG) contents in lung tissues.

METHODSThe rabbits were divided into normal control group, hyperoxia solution (H0) and balance salt (BS) groups. Group HO and Group BS inhaled phosgene and the former was given intravenously hyperoxia solution (which was replaced by balance salt solution in Group BS). The content of MDA and the activity of SOD in serum were observed at different time points, the amount of GSH and GSSG in lung tissue were also measured.

RESULTS(1) The serum MDA contents increased and PaO2, SOD activity decreased significantly in Group HO and Group BS along with time increasing as compared with control group. The contents of GSH in lung tissue decreased in two groups compared with that in control group, however the contents of GSSG ascended instead. (2) At 3 and 8 h of the experiment, PaO2 of Group HO [(9.91 +/- 0.49), (9.15 +/- 0.46) mm Hg respectively] were significantly higher than those of Group BS [(9.03 +/- 0.76), (8.11 +/- 0.57) mm Hg respectively] (P < 0.01). The contents of MDA of Group HO (3.66 +/- 0.35), (5.31 +/- 0.15) micromol/L respectively] were lower than those of Group BS [(4.32 +/- 0.26), (7.4 +/- 0.33) micromol/L respectively] (P < 0.01). SOD activity in Group HO [(237.37 +/- 29.96), (208.10 +/- 18.80) NU/ml respectively] were higher than those of Group BS [(195.02 +/- 21.44), (144.87 +/- 21.26) NU/ml respectively] (P < 0.05 or P < 0.01). The content of GSSG lung tissue in Group HO (423.67 +/- 38.21) micromol/L were lower than those of Group BS (523.85 +/- 43.14) mol/L (P < 0.01). There were no significant differences in the content of GSH in lung tissues between Group HO and group BS.

CONCLUSIONHyperoxia solution can reduce acute lung injury of rabbits following phosgene poisoning.

Acute Lung Injury ; etiology ; metabolism ; pathology ; Animals ; Glutathione Peroxidase ; metabolism ; Hyperoxia ; Lung ; drug effects ; metabolism ; pathology ; Malondialdehyde ; analysis ; Oxygen ; administration & dosage ; pharmacology ; Phosgene ; poisoning ; Rabbits ; Superoxide Dismutase ; metabolism

OBJECTIVETo study the effect of melatonin, a potent antioxidant both in vitro and in vivo, on hyperoxia-induced oxidant/antioxidant imbalance in the lung of neonatal rats with chronic lung disease (CLD).

METHODSNinety neonatal rats were randomly divided into three groups (n=30 each): air-exposed, hyperoxia-exposed, melatonin-treated (4 mg/kg melatonin was administered 30 minutes before hyperoxia exposure and once daily after exposure). CLD was induced by hyperoxia exposure (FiO2=0.85). Lung specimens were obtained 3, 7, and 14 days after exposure (n=10 each) for histopathologic examination. The levels of total antioxydase capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), myeloperoxidase (MPO), catalase (CAT), nitrite/nitrate, and malondialdehyde (MDA) in the lung were assayed by the spectrophotometer.

RESULTSThe histopathologic examination showed that lung damage was obviously alleviated in the melatonin-treated group. The levels of T-AOC, GSH-Px, SOD and CAT in the lung were significantly higher in the melatonin-treated group than those in the other two groups at all time points (p<0.05). The levels of MPO, nitrite/nitrate and MDA in the lung increased significantly in the untreated hypoxia-exposed group compared with those in the air-exposed group at all time points (p<0.05 or 0.01), while the levels of MPO, nitrite/nitrate and MDA in the melatonin-treated group were significantly reduced as compared with the untreated hypoxia-exposed group (p<0.05).

CONCLUSIONSMelatonin may reverse oxidant/antioxidant imbalance in hyperoxia-induced lung disease, thus providing a protective effect against CLD in neonatal rats.

Animals ; Animals, Newborn ; Antioxidants ; pharmacology ; Chronic Disease ; Female ; Hyperoxia ; metabolism ; Lung ; metabolism ; Lung Diseases ; metabolism ; Male ; Malondialdehyde ; analysis ; Melatonin ; pharmacology ; Nitric Oxide ; analysis ; Peroxidase ; metabolism ; Rats ; Rats, Wistar

PURPOSE: Hyperoxia has the chief biological effect of cell death. We have previously reported that cathepsin B (CB) is related to fetal alveolar type II cell (FATIIC) death and pretreatment of recombinant IL-10 (rIL-10) attenuates type II cell death during 65%-hyperoixa. In this study, we investigated what kinds of changes of CB expression are induced in FATIICs at different concentrations of hyperoxia (65%- and 85%-hyperoxia) and whether pretreatment with rIL-10 reduces the expression of CB in FATIICs during hyperoxia. MATERIALS AND METHODS: Isolated embryonic day 19 fetal rat alveolar type II cells were cultured and exposed to 65%- and 85%-hyperoxia for 12 h and 24 h. Cells in room air were used as controls. Cytotoxicity was assessed by lactate dehydrogenase (LDH) released into the supernatant. Expression of CB was analyzed by fluorescence-based assay upon cell lysis and western blotting, and LDH-release was re-analyzed after preincubation of cathepsin B-inhibitor (CBI). IL-10 production was analyzed by ELISA, and LDH-release was re-assessed after preincubation with rIL-10 and CB expression was re-analyzed by western blotting and real-time PCR. RESULTS: LDH-release and CB expression in FATIICs were enhanced significantly in an oxygen-concentration-dependent manner during hyperoxia, whereas caspase-3 was not activated. Preincubation of FATIICs with CBI significantly reduced LDH-release during hyperoxia. IL-10-release decreased in an oxygen-concentration-dependent fashion, and preincubation of the cells with rIL-10 significantly reduced cellular necrosis and expression of CB in FATIICs which were exposed to 65%- and 85%-hyperoxia. CONCLUSION: Our study suggests that CB is enhanced in an oxygen-concentration-dependent manner, and IL-10 has an inhibitory effect on CB expression in FATIICs during hyperoxia.
Animals ; Cathepsin B/*genetics/metabolism ; *Down-Regulation ; Gene Expression Regulation ; Hyperoxia/*genetics ; Interleukin-10/*pharmacology/physiology ; L-Lactate Dehydrogenase/metabolism ; Necrosis/chemically induced ; Oxygen/metabolism ; Rats

OBJECTIVETo study the effects of prolonged 85% oxygen exposure on lung vascular development and the expression of angiopoietin-1 (Ang-1) in the neonatal rat lungs.

METHODSNinety-six Sprague-Dawley rat pups were randomly exposed to air (control group) and 85% oxygen (experimental group) 6 hrs after birth. The rats were sacrificed 3, 7 and 14 days after exposure and their lungs were sampled. The lung sections were stained with hematoxylin and eosin for histological evaluation and analysis of vessel volume density. Expressions of angiopoietin-1 (Ang-1) in lung tissue were measured by immunohistochemistry. Expression of Ang-1 protein and mRNA was detected by Western Blot and Real time-PCR.

RESULTSAfter being exposed to 85% oxygen for 14 days, lung tissues had pathological changes as "new" bronchopulmonary dysplasia (BPD). The RAC on day 7 and day 14 in experimental group decreased significantly as compared with the control group [(10.55 ± 0.13) vs. (11.74 ± 0.19), (12.47 ± 0.05) vs. (15.03 ± 0.16), P < 0.05]. The X-ray showed that the diameter of lung vessel was much smaller and the vessels had less branches in experimental group compared with the control group on day 14. The vessel volume density on day 14 in experimental group decreased significantly as compared with the control group [(3.55 ± 0.09) vs. (6.03 ± 0.16), P < 0.05]. Immunohistochemistry and Western blotting showed that the expressions of Ang-1 protein on day 7 and day 14 in the experimental group decreased significantly as compared with the control group [(4.27 ± 0.34) vs. (3.10 ± 0.29), P < 0.05, (5.65 ± 0.49) vs. (3.21 ± 0.28), P < 0.01], [(0.88 ± 0.31) vs. (0.41 ± 0.12), P < 0.05, (0.90 ± 0.29) vs. (0.21 ± 0.06), P < 0.01]. The expressions of Ang-1 mRNA on day 7 and day 14 in the experimental group also decreased significantly as compared with the control group [(0.85 ± 0.14) vs. (0.44 ± 0.21), P < 0.05, (0.87 ± 0.24) vs. (0.24 ± 0.05), P < 0.01].

CONCLUSIONSProlonged exposure of high concentration of oxygen may cause impairment of lung vascular development by inhibiting expression of Ang-1 in neonatal rats, which is likely to contribute to pathogenesis of BPD.

Angiopoietin-1 ; metabolism ; Animals ; Animals, Newborn ; Hyperoxia ; Lung ; blood supply ; metabolism ; Pulmonary Artery ; growth & development ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo date, studies on the roles of angiopoietin-2 (ang-2) during the retinal vascular development focused on either double transgenic mice with inducible expression of ang-2 or ang-2 gene knockout mice or ang-2 gene deficient mice, and others focused on cell culture. Few studies have been done in animals with normal genetic expression or oxygen-induced animal model. In this study, the hyperoxia-induced model of retinopathy was established to investigate the expression of ang-2 in the retinae of model rats with retinopathy of prematurity (ROP) and explore the expression relationship between ang-2 in the retinae and retinal vascular development.

METHODSEighty newborn Sprague-Dawley rats were randomly divided into hyperoxia and normoxia control groups, then each group was further divided into 4, 7, 10, 14 and 20 days subgroups. Beginning on day 1 of life, 40 rats in hyperoxia group were exposed to hyperoxia [(75+/-2)% O2] for 7 days, and subsequently were moved into room air, thus ROP model was set up. An additional 40 rats were raised in room air served as age matched controls. Rats of postnatal day4 (d 4), d 7, d 10, d 14 and d 20 were sacrificed respectively. Rats left eyes were obtained, the anterior half of the eyeball and the vitreous were removed, then the retinae were gently scraped off the choroid, and used to delineate retinal vascular development by adenosine diphosphatase (ADPase) staining. Right eyes were embedded in paraffin and frozen sections were stained with immunohistochemistry to measure the expression of ang-2 in the retinae. Meanwhile, the number of new vascular cell nuclei extending into the internal limiting membrane in cross-sections was counted after HE staining to assess retinal neovascularization.

RESULTSThe retinal vasculature of the rats began to develop after birth, grew to most of the retinae on the 7th day, and was developed around 20th day. There was a large area of persistent central retinal vaso-obliteration in the retinal vasculature in rats after exposure to hyperoxia for 7 days, and the developed vessels were distorted. Hyperoxia induced neovascularization occurred on the 10th day and reached maximum neovascular response on d 14. The result of HE staining showed that the highest number of neovascular nuclei per cross section extending into the vitreous in hyperoxia model was seen on d 14 as compared with that of the same age of control groups and subretinal hematocele was observed in hyperoxia model of d 10 group. The expression of ang-2 in control group was the strongest in the retina on the 7th day, and was the least on the 20th day. In hyperoxia group, the expression of ang-2 on the 7th day was the weakest, the optical density value (OD value) was 82.106 62+/-6.302 40, significantly less than that of the age-matched normoxia control group, 115.448 33+/-9.373 10, and lower than that of hyperoxia model d 10, 124.425 95+/-10.572 09, and d 14, 117.685 20+/-10.578 48 (P<0.001). After the rats had been returned to room air for 3 days, the expression of ang-2 increased significantly and reached the peak on d10, it's OD value was 110.07 039+/-12.59 962, the result had significant difference as compared with the age-matched control group, 124.42 595+/-10.572 09 (P<0.05) and this strong expression remained till d 14 in model group.

CONCLUSIONHyperoxia induced neovascularization and inhibited the expression of ang-2 in the retinae; and the neovascularization is in accordance with the change of angiopoietin-2 expressed in retinae, suggesting that there is a close relationship between the expression of ang-2 and retinal vascular development.

Angiopoietin-2 ; metabolism ; Animals ; Animals, Newborn ; Disease Models, Animal ; Female ; Hyperoxia ; metabolism ; Oxygen ; administration & dosage ; adverse effects ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Retina ; growth & development ; metabolism ; Retinal Neovascularization