Hyperoxia-exposed Group Research Articles

Effects of the p16/cyclin D1/CDK4/Rb/E2F1 pathway on aberrant lung fibroblast proliferation in neonatal rats exposed to hyperoxia.

p16INK4a (p16) inhibits the vital G1 to S phase transition during cell cycle progression through the p16/cyclin D1/CDK4/retinoblastoma(Rb)/E2F1 pathway. Hyperoxia can suppress the G1/S checkpoint and induce more lung fibroblasts (LFs) to transition from the G1 phase to the S phase and undergo cell proliferation. The present study investigated the rate of p16 gene promoter methylation and the protein expression levels of p16, cyclin D1, CDK4, Rb and E2F1 in LFs from the lungs of rats exposed to hyperoxia and normoxia on postnatal days 3, 7 and 14. In the hyperoxia-exposed group, the methylation rate was 50 and 80% on days 7 and 14, respectively. Cyclin D1 and CDK4 overexpression was associated with p16 loss and Rb inactivation by phosphorylation. Rb phosphorylation induced E2F1 release in the G1 phase, which promoted cell proliferation. No methylation was observed in the normoxia-exposed group. These observations suggested that p16 loss may stimulate aberrant LF proliferation via the p16/cyclin D1/CDK4/Rb/E2F1 pathway.

Erythropoietin attenuates hyperoxia-induced lung injury by upregulating epidermal growth factor-like domain 7 in newborn rats.

The aim of the present study was to observe the effects of recombinant human erythropoietin (rhEPO) on the expression of epidermal growth factor-like domain 7 (EGFL7) and cell apoptosis in lung tissue following hyperoxic lung injury in newborn rats. The 96 Sprague-Dawley newborn rats were randomly divided into 4 groups (n=24) as follows: Room air-exposed control group, room air-exposed rhEPO-treated group, hyperoxia-exposed group and the hyperoxia-exposed rhEPO-treated group. Pups (n=8) from each group were sacrificed on postnatal days 3, 7 and 14. The pulmonary morphometric and microvessel density changes were observed. In addition, the mRNA and protein expression levels of EGFL7, B-cell lymphoma 2 (Bcl-2) and Bcl-2-like protein 4 (Bax) in lung tissue samples were measured. The rats in the hyperoxia-exposed group exhibited alveolar and pulmonary vascular dysplasia, as well as low mRNA and protein expression levels of EGFL7 and Bcl-2, in addition to high level of Bax in the lung tissue samples when compared with the room air-exposed control group (P<0.05). However, in the hyperoxia-exposed rhEPO-treated group the lung histopathology was improved, and the protein and mRNA expression levels of EGFL7 and Bcl-2 were increased compared with the hyperoxia-exposed group (P<0.05). Furthermore, the expression level of Bax was lower than that of the hyperoxia-exposed group (P<0.05). The present study demonstrated that rhEPO promotes alveolar development and increases pulmonary vascular density by upregulating the expression level of EGFL7 in hyperoxia-induced lung injury of newborn rats.

Effect of hyperoxia on cysteinyl aspartate specific proteinase-3 and proliferating cell nuclear antigen expressions in bone mesenchymal stem cells

Objective To investigate the effect of hyperoxia on the expressions of cysteinyl aspartate specific proteinase-3(Caspase-3) and proliferating cell nuclear antigen (PCNA) in bone marrow mesenchymal stem cells(BMSCs). Methods Primary BMSCs from SD rats were cultured in vitro, BMSCs grew to 70%-80% degrees of fusion and then were randomly divided into room air group and hyperoxia exposure group.Each group was divided into 5 sub-groups, and cultured for 0, 3, 6, 12, and 24 h respectively.Morphology investigation with inverted phase contrast microscope was adopted.The expressions of Caspase-3 and PCNA protein levels were detected by Western blot. Results (1)As time wenty by, compared with room air group, the gap between cells increased, some of the cells became circular, and cell detachment and floating appeared in the hyperoxia-exposure group (>12 h) compared with room air group.(2)As time went on, compared with the room air group, the expression levels of Caspase-3 protein in the hyperoxia-exposure group were increased, and the difference was significant after 6 hours of culture (0.27±0.04 vs 0.14±0.02, t=5.03, P=0.007). (3)Compared with room air group, the PCNA levels of the hyperoxiaexposure group(6 h) decreased, and the difference in PCNA protein expression levels was significant after 6 hours of culture (0.27±0.04 vs 0.38±0.04, t=3.37, P=0.028). Conclusions Hyperoxia exposure increases Caspase-3 expression levels and decreases PCNA expression, which may affect the proliferation and apoptosis of BMSCs. Key words: Hyperoxia; Bone mesenchymal stem cell; Proliferating cell nuclear antigen; Cysteinyl aspartate specific proteinase 3

Fractal analysis of alveolarization in hyperoxia-induced rat models of bronchopulmonary dysplasia.

No papers are available about potentiality of fractal analysis in quantitative assessment of alveolarization in bronchopulmonary dysplasia (BPD). Thus, we here performed a comparative analysis between fractal [fractal dimension (D) and lacunarity] and stereological [mean linear intercept (Lm), total volume of alveolar air spaces, total number of alveoli, mean alveolar volume, total volume and surface area of alveolar septa, and mean alveolar septal thickness] parameters in experimental hyperoxia-induced models of BPD. At birth, rats were distributed between the following groups: 1) rats raised in ambient air for 2 wk; 2) rats exposed to 60% oxygen for 2 wk; 3) rats raised in normoxia for 6 wk; and 4) rats exposed to 60% hyperoxia for 2 wk and to room air for further 4 wk. Normoxic 6-wk rats showed increased D and decreased lacunarity with respect to normoxic 2-wk rats, together with changes in all stereological parameters except for mean alveolar volume. Hyperoxia-exposed 2-wk rats showed significant changes only in total number of alveoli, mean alveolar volume, and lacunarity with respect to equal-in-age normoxic rats. In the comparison between 6-wk rats, the hyperoxia-exposed group showed decreased D and increased lacunarity, together with changes in all stereological parameters except for septal thickness. Analysis of receiver operating characteristic curves showed a comparable discriminatory power of D, lacunarity, and total number of alveoli; Lm and mean alveolar volume were less discriminative. D and lacunarity did not show significant changes when different segmentation thresholds were applied, suggesting that the fractal approach may be fit to automatic image analysis.

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.

Effects of Hyperoxia Exposure on Free Radicals Accumulation in Relation to Ultrastructural Pathological Changes of Diaphragm

COPD are associated with an increased load on the diaphragm leading to accumulation of reactive oxygen species (ROS) and the subsequent cellular damages and death. The pathological alterations inducted by ROS in the diaphragm during oxygen breathing are not known. The purpose of the present study was to examine the effects of hyperoxia exposure (HP) on free radicals (FR) accumulation in relation to the ultrastructural pathological alterations in the diaphragm. Twenty adult male rats were randomly assigned to two groups; control (C); and hyperoxia (HP). Animals of the HP were breathing 100% O2 for 72 hr continuously. Both serum and diaphragm tissue supernatant analysis showed significantly higher (p<0.05) FR in HP group, as compared with control group. Ultrastructure examinations showed that HP resulted in variety of pathological alterations in the mitochondria and endoplasmic reticulum that were associated with disarrangement of myofibrils, loss of I-banding for myosin, focal myolysis of the myofilaments, complete fragmentation of myosin, tearing of myofilamments from Z plates and tearing of the endothelial cell of the interstitial blood capillaries. Based on the results of the present study, it can be concluded that hyperoxia-induced acceleration ROS formation damaged the contractile apparatuses of the diaphragm and related endomembrane proteins that could involve intracellular calcium channels proteins.

Fractal analysis of alveolarization in hyperoxia-induced rat models of bronchopulmonary dysplasia

Many morphometric approaches have been proposed for quantitative evaluation of alveolarization in experimental models of bronchopulmonary dysplasia (BPD), such as alveolar number, alveolar size and variability, mean linear intercept (Lm), surface-to-volume ratio, or radial alveolar count. Conversely, no papers are available about potentiality of fractal analysis in this matter. Thus, in the present study, we performed a comparative analysis between Lm and fractal parameters (fractal dimension (D) and lacunarity (λ)), in experimental hyperoxia-induced models of BPD. At birth the newborn rats were randomly distributed between the following experimental groups: a) rats raised in ambient air for 2 weeks; b) exposed to 60% oxygen for 2 weeks; c) raised in normoxia for 6 weeks; d) exposed to 60% hyperoxia for 2 weeks and then to room air for further 4 weeks. Normoxic 6-weeks rats showed a significant decrease of Lm and a significant increase of D and λ with respect to normoxic 2-weeks rats, indicative of alveolar development. Hyperoxia-exposed rats of 2 weeks did not show significant changes in Lm and D with respect to normoxic rats equal in age, although λ was significantly lower in hyperoxic group. Conversely, in the comparison between 6-week rats, the hyperoxia-exposed group showed higher value of Lm and lower values of D and λ, when compared to the normoxic group, confirming a lower alveolar gas exchange surface-to-volume ratio and an airspace morphology of lower complexity. The analysis of the ROC curves showed a comparable discriminatory power of Lm and D as binary classifiers of the airspace morphology (areas under the curves of 0.815 and 0.753, respectively). Moreover, the values exhibited by the parameters when different segmentation thresholds were applied suggested that fractal parameters are less sensitive (D and λ showed a coefficient of variation of 0.005 and 0.009, respectively) than Lm (exhibiting a coefficient of variation of 0.05) to the bias eventually introduced in the analysis by approximations or errors in tissue segmentation. In conclusion, the present study demonstrated the ability of fractal analysis (D and λ) to identify developmental and hyperoxia-induced changes in alveolarization with discriminatory power similar to Lm. The fractal approach, however, appears more fit to automatic image analysis, being less influenced by approximations in thresholding and image editing.

Effect of Hyperoxia on the Viability and Proliferation of the Primary Type II Alveolar Epithelial Cells

To observe the effect of hyperoxia on the growth of type II alveolar epithelial cells (AEC II). The lungs of 19-day gestation fetal rats were primary cultured and the AEC II were purified by differential adhesion method. The cells were divided into control (normoxia) group and hyperoxia group. The cell growth, cell viability, cell apoptosis, and cell cycle were examined at 2, 4, 6, and 8 days of normoxia or hyperoxia exposure. The number of cells in hyperoxia-exposed group significantly decreased as compared to those of air control group. Number of cells in hyperoxia group was the highest at day 2 of exposure and gradually decreased with time. The viability of cells exposed to hyperoxia was substantially reduced compared with cells exposed to air. Percentage of cells in G1 phase and S phase in hyperoxia group increased gradually with increase in exposure duration and significant differences were seen at day 4 and day 6 compared with either the preceding time points and also with corresponding air-exposed cells. The percentage of both early apoptotic cells (Annexin-V(+)/PI(-)) and late apoptotic cells and necrotic cells (Annexin-V(+)/PI(+)) increased significantly in cells exposed to hyperoxia compared with cells exposed to air. Hyperoxia inhibits proliferation, viability and growth of AEC II and promotes apoptosis.

The expression of α-smooth muscle actin during the lung injury induced by hyperoxia

To explore the expression of α-smooth muscle actin (α-SMA) during the lung injury induced by hyperoxia in infantile rats. Sixty-four male Sprague-Dawley (SD) rats about 3 weeks were randomly assigned into normal control group which exposured to room air [fraction of inspired oxygen (FiO(2)) was 0.21] and hyperoxia exposure group (95%O(2)) according to random digits table. Eight rats in each group were randomly sacrificed at day 1, 7, 14 and 21.Pulmonary tissue remodeling was observed by hematoxylin-eosin (HE) staining. Immunohistochemistry method was performed to evaluate the expression of α-SMA in pulmonary tissue, further Western blotting was also made to determine the expression of α-SMA. The early histopathologic changes after HE were inflammation and edema in pulmonary tissue, while the later changes were interstitial hyperplasia and fibroblast proliferation. The expression of α-SMA was very slight in bronchial epithelium, alveolar epithelium and alveolar interstitium in normal control group, but increased with the time of hyperoxia exposure prolonged and peaked at 21st day. Western blotting detected that the expression of α-SMA after hyperoxia exposure for 1 day and 7 days in hyperoxia exposure group presented no difference compared with normal control group (1.02±0.12 vs. 1.00±0.13, 1.05±0.14 vs. 0.99±0.12, both P>0.05), but the expression of α-SMA after hyperoxia exposure for 14 days and 21 days was increased compared with normal control group (1.27±0.21 vs. 1.05±0.15, 2.26±0.28 vs. 1.05±0.14, P<0.05 and P<0.01). Pulmonary fibrosis remodeling was caused by hyperoxia exposure. The expression of α-SMA in pulmonary tissue in hyperoxia exposure groups obviously increased, and could play an important role in pulmonary fibrosis remodeling.

Protection of hyperoxia-induced lung injury by granulocyte-macrophage colony-stimulating factor via RAGE-NF-κB signaling pathway in newborn rats

To explore the effects of granulocyte-macrophage colony-stimulating factor (GMCSF) on hyperoxia exposure lung injury in newborn rats and elucidate its protective mechanism of operating via the signaling pathway of advanced glycation end products (RAGE)-NF-κB. Twenty-four 3-day-old SD rats from 3 litters were randomly divided into 3 groups. They were hyperoxia exposure plus GMCSF group (group A), hyperoxia exposure group (group B) and air exposure group (group C). The rats from groups A and B were placed in a sealed Plexiglas chamber with a minimal in-and-outflow, providing 6 - 7 exchanges per hour of chamber volume and maintaining O(2) levels above 95%. While the rats in group C only were exposed to air simultaneously. The rats in group A received subcutaneous injections of recombinant murine GMCSF (9 µg/kg) during hyperoxia exposure at 24 h, 72 h and 120 h respectively. And the rats in groups B and C received subcutaneous injections of saline vehicle alone at the same time point. Seven days later, all were sacrificed and immunohistochemistry was employed to assess the expression of RAGE in lung tissue. The levels of tumor necrosis factor-α in bronchoalveolar lavage fluid (BALF) and serum samples were detected by ELISA (enzyme-linked immunosorbent assay). The RAGE mRNA and NF-κB mRNA in tissue homogenates were detected by RT-PCR while RAGE and NF-κB by Western blot. Also the values of lung damage score were calculated with microscopic histology. The value of lung damage score in group C, B and A was 0.46 ± 0.20, 3.06 ± 0.33 and 2.31 ± 0.56 respectively, there was significantly difference among three groups (P = 0.000). The expression of RAGE mRNA and protein in three groups were 0.14 ± 0.02, 0.34 ± 0.06, 0.28 ± 0.04 and 0.30 ± 0.04, 0.76 ± 0.11, 0.55 ± 0.08 respectively. There were both significantly differences among three groups (P = 0.000, P = 0.000). The expression of NF-κB mRNA and protein in three groups were 0.41 ± 0.21, 0.90 ± 0.36, 0.69 ± 0.30 and 0.41 ± 0.26, 0.96 ± 0.43, 0.77 ± 0.33 respectively, there were both significantly difference among three groups (P = 0.000, P = 0.017). The level of TNF-α in BALF was 76 ± 10, 224 ± 42 and 143 ± 24 respectively, there was significantly difference among three groups (P = 0.000). All indicators above in group B and group A were significantly more than those in group C (all P < 0.05), while these indicators in group A were lower than those in group B. But there was no difference in the level of TNF-α of serum among three groups (P > 0.05). GMCSF may protect hyperoxia-induced lung injury via down-regulating the signaling pathway of RAGE-NF-κB.

Effects of hyperoxia on erythropoietin receptor expression in lung development of neonatal rats

Oxygen toxicity is thought to be a major contributing factor in the pathogenesis of bronchopulmonary dysplasia (BPD). Animal experiments reveal that erythropoietin (EPO) may have protective effects against hyperoxic lung injury, but the mechanisms remain unknown. The aim of this study was to evaluate effects of hyperoxia on erythropoietin receptor expression in lung development of neonatal rats. Several litters of Wistar pups were pooled together within 12 hours after birth and randomly divided into two groups (n = 24 in each): air-exposed control group and hyperoxia-exposed group. In hyperoxia-exposed group, the rats were exposed to 85% oxygen. Pups (n = 8) from each group were sacrificed on postnatal days 3, 7, and 14. The pulmonary histological and morphometric changes were observed after hematoxylin-eosin (HE) staining under light microscope. Radical alveolar counts (RAC) were compared between the two groups to evaluate the differences of alveolarization. Expressions of platelet endothelial cell adhesion molecule-1 (PECAM-1) and erythropoietin receptor (EPOR) in lung tissue were measured by immunohistochemistry. Expressions of EPOR mRNA and EPOR protein were measured by RT-PCR and Western blotting. In hyperoxia-exposed group, there were a few inflammatory cells infiltration in interstitium on day 3 and inflammatory response worsened on day 7. Alveolar and capillary hypoplasia and interstitial fibrosis were evident on day 14. RAC increased in air-exposed control group along with the age in days. RAC decreased from day 7 in hyperoxia-exposed group compared with air-exposed control group [(6.85 ± 1.04) vs. (7.33 ± 1.0), P < 0.01], which was more evident on day 14 [(6.20 ± 1.58) vs. (9.07 ± 0.69), P < 0.001]. Expression of PECAM-1 protein increased in air-exposed control group along with the age in days. But in hyperoxia-exposed group, it decreased on day 7 and 14 [(15.14 ± 1.51) vs. (31.47 ± 2.43), (11.04 ± 1.76) vs. (41.41 ± 3.83), P < 0.001] compared with air-exposed control group. Expression of EPOR on day 3 in air-exposed control group was the strongest and weakened gradually with the increase of postnatal days. Expression of EPOR in hyperoxia-exposed group decreased on day 3 and became more evident on day 7 and day 14 compared with air-exposed control group [(1.62 ± 0.04) vs. (1.82 ± 0.06), P < 0.05; (0.48 ± 0.01) vs. (1.10 ± 0.07), (0.39 ± 0.04) vs. (0.87 ± 0.03), P < 0.001]. Expression of EPOR mRNA on day 3 in air-exposed control group was the strongest and was decreased significantly in hyperoxia-exposed group compared with air-exposed control group at all time points [(0.87 ± 0.07) vs. (1.1 ± 0.17), (0.18 ± 0.07) vs. (0.36 ± 0.08), P < 0.01;(0.14 ± 0.05) vs. (0.36 ± 0.09), P < 0.001]. EPOR may participate in the modulation of normal lung development. Depressed expression of EPOR and PECAM-1 may be involved in the pathogenesis of alveolar and capillary hypoplasia induced by hyperoxia.

Expression of hypoxia-induced mitogenic factor in mouse bronchopulmonary dysplasia model and its significance

Objective To study the expression of hypoxia-induced mitogenic factor (HIMF) in mouse bronchopulmonary dysplasia ( BPD) model and its significance. Methods Thirty-two Kunming neonatal mice were randomly divided into normoxia exposure group (21% 02,n=16) and hyperoxia exposure group (85% O2,n=16). After exposure for 7, 14, 21 and 28 days, HE staining was applied to observe the morphological changes of lung tissues. The mRNA and protein levels of HIMF were detected by real-time reverse transcription-polymerase chain reaction ( RT-PCR) and Western blotting, respectively. Results In normoxia exposure group, the alveoli was gradually mature and well-distributed, with thin interstitial tissue. However, in hyperoxia exposure group, the increased septa] wall thickness and alveolar fusion were observed, and the radical alveolar count was decreased by 31.1%-65.3%. As compared with normoxia exposure group, the mRNA and protein levels of HIMF were enhanced by 2.435-2.528 times (P<0.05) and 2.45-5.42 times (P<0.05) at the 7th, 14th and 21st day after exposure, but decreased by 52. 81 % (P<0. 05) and 74. 60% at the 28th day after exposure. Conclusion The mouse BPD model was successfully established, and HIMF may participate in the pathogenesis of BPD. Key words: Hypoxia-induced mitogenic factor; Bronchopulmonary dysplasia; Gene expression

Increased oxygen tension attenuates acute ultraviolet-B-induced skin angiogenesis and wrinkle formation

Acute ultraviolet (UV)-B irradiation causes skin wrinkle formation associated with hyperplasia of cutaneous blood vessels. This study reports that increased dermal oxygen tension attenuates acute UVB-induced angiogenesis and wrinkle formation. Twenty-four hairless mice (HOS:HR-1) were assigned to 3 groups: 1) control group, 2) UVB-irradiated (UVB) group, and 3) UVB-irradiated and hyperoxia-exposed (UVB+HO) group. The backs of the mice were exposed to UVB irradiation 3 times per week for a 5-wk period. To increase dermal oxygen tension, the mice were exposed to hyperoxia (90% oxygen) for 2 h immediately after each UVB irradiation. Hyperoxic exposure increased dermal oxygen tension by about 10 times compared with the control level. Degree of wrinkle formation and epidermal thickness increased significantly after a 5-wk UVB-irradiation period, whereas hyperoxic exposure attenuated these increases. Tissue adenosine triphosphate concentration and angiogenesis increased significantly only in the UVB group compared with the control group. Although the expression of hypoxia inducible factor-1alpha mRNA, a key molecule for angiogenesis, increased significantly in the UVB and UVB+HO groups compared with the control group, the protein level increased significantly only in the UVB group. The activity of matrix metalloproteinase-2 and -9, critical molecules for angiogenesis, did not increase in the UVB and UVB+HO groups compared with the control group. Active type 1 collagenase activity and soluble collagen content in all of the groups were roughly similar. These results suggest that increased dermal oxygen tension attenuates angiogenesis and wrinkle formation following acute UVB irradiation.

Impact of docosahexaenoic acid on oxidation in newborn rats with high oxygen resuscitation

Objective To evaluate the impact of docosahexaenoic acid (DHA) on the level of oxidative and ant+oxidative factors in newborn rats with high oxygen resuscitation. Methods Pregnant rats were divided randomly into regular diet group and DHA supplement group (DHA 100 mg/d, Qd, administrated through gavage). Within 24 h after birth, the newborn rats were given low concentration oxygen (8%) for 2 h. Then, the newborn rats were divided into groups exposed to 100% O2 or room air. There were 4 groups: regular diet + hyperoxia-exposed group, regular diet + room air group, DHA + hyperoxia-exposed group and DHA+room air group. The activities of superoxide dismutase (SOD), maleic dialdehyde (MDA) and glutathione peroxidase (GSH-Px) in newborn rats exposed to high oxygen or room air for 1 d, 3 d, 7 d and at the time of immediately after asphyxia were measured by spectrophotometry. Results At day 1,3 and 7, SOD activities in newborn rats of DHA+ hyperoxia-exposed group were (93. 7± 16.8) U/ml, (128.2±11.5) U/ml and (170.9±9.0) U/ml, higher than those in DHA+room air group [(73.7± 21.5) U/mt, (112.2±19. 9) U/ml and (124.2±24. 6) U/ml, respectively] (P 0. 05). Conclusions Dietary DHA during pregnancy and breast feeding period might protect newborn rats against hyperoxic damage by increasing antioxidase level and decreasing peroxide level when exposed to high oxygen. Key words: Docosahexaenoic acids; Hyperoxia; Asphyxia; Animals, newborn

Melatonin protects against oxidative damage in a neonatal rat model of bronchopulmonary dysplasia

Oxidative stress plays an important role in the pathogenesis of bronchopulmonary dysplasia (BPD). Melatonin (MT) has direct and indirect free radical detoxifying activity. The present study was to investigate whether treatment with MT would attenuate hyperoxia-induced lung injury and the effect of MT on imbalance of oxidants/antioxidants in the lung of neonatal rats. BPD was induced by exposure to hyperoxia in neonatal rats (n=90). The rats were divided randomly into three groups (n=30 each): air-exposed control group, hyperoxia-exposed group, and hyperoxia-exposed MT-treated group. Lung specimens were obtained respectively on day 3, day 7, and day 14 after exposure (n=10 each). Activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), and levels of myeloperoxidase (MPO), nitrite/nitrate, and malondialdehyde (MDA) were assayed. Histopathologic changes were observed in the tissues stained with hematoxylin and eosin and Masson's trichrome stain. Increased levels of MPO, nitrite/nitrate, and MDA in the hyperoxia-exposed rats were significantly reduced by MT (P<0.05). Activities of GSH-Px, SOD, and CAT which did not change after exposure to hyperoxia were increased by MT (P<0.05). Furthermore, BPD associated histopathological alterations such as reduced total number of alveoli and interstitial fibrosis were obviously abated in the MT-treated group. MT can reverse oxidants/antioxidants imbalance in damaged lung tissue and thus exert a beneficial effect on hyperoxia-induced lung disease in neonatal rats. With regard to humans, there may be a protective effect of MT on BPD.

Protective effect of Radix Salviae Milliorrhizae Extract (764-3) on hyperoxia-induced acute lung injury of neonatal rats

Objective To study the protective effect of Radix Salviae Milliorrhizae Extract (764-3) on hyperoxia-induced acute lung injury of neonatal rats and to compare the effect of it with that of superoxide dismutase (SOD).Methods Hyperoxia lung injury model was established by putting Sprague Dawley neonatal rats into self-made oxygen box within high-concentration oxygen(≥90 % O2)for 7 days. Interfered with SOD and 764-3 respectively, the lung index, contents of malondialdehyde (MDA), activity of SOD, radical alveolar counts (RAC) and pathological changes have been compared. Meanwhile, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) was utilized to detect the number of apoptosis of lung cells. Results On the seventh day, the contents of MDA and the number of apoptosis of lung cells of SOD prevention group and 764-3 prevention group were lower than those of the hyperoxia-exposed group( P 0.05).However, compared with those of hyperoxia-exposed group, the lung index of 764-3 prevention group showed insignificant difference(P >0.05). Conclusion Radix Salviae Milliorrhizae Extract (764-3) may have certain protective effect on hyperoxia-in-duced acute lung injury. Key words: Hyperoxia; Acute lung injury; Radix Salviae Milliorrhizae Extract; Rat

Effect of dexamethasone on aquporin-1 expression in the long of hyperoxia-exposed newborn rats

Objective To investigate the effect of dexamethasone on aquporin-1(AQP1) expression in the lung of hyperoxia-exposed newborn rats and to explore the protective mechanism of dexamethasone in lung injury.Methods Thirty-two newhorn Wistar rats were randomly divided into 4 groups:room-air group, hyperoxia-exposure group, room-air group treated with dexamethasone and hyperoxia-exposure group treated with dexamethasone. Lungs were harvested on the third day. The lung AQP1 mRNA expression and the protein location were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. In addition, the wet-to-dry weight ratio(W/D), the protein content of broncho alveolar lavage fluid (BALF), lung permeation index and histological changes in lung were measured respectively. Results On the third day after hyperoxia-exposure, lung tissues appeared congestion, inflammatory cells exudation and edema. The lung W/D, the protein content in BALF and the lung permeation index in hyperoxia group were significantly higher than those in room-air group. In contrast, lung injury degree relieved in hyperoxia group treated with dexamethasone, The expressions of AQP1 mRNA in room-air group, hyperoxia group and hyperoxia group tteated with dexamethasone were 0.70 ±0.04, 0.42 ± 0.03 and 1.04 ± 0.04 respectively, and there was aignificant difference ( all p < 0.05). Compared with room-air group, the expressions of AQP1 mRNA was lower than that in hyperoxia group, while the expression was upregulated in hyperoxia group treated with dexamethasone. The changes of AQP1 protein level was persistent with the changes of AQP1 mRNA expression. Conclusion The expressions of lung AQP1 downregulates in hyperoxia-induced lung injury. Dexamethasone can upregulates the expressions of lung AQP1, which might be one of the protective mechanisms against hyperoxia-induced lung injury. Key words: Aquaporin-1; Hyperoxia; Lung injury; Dexamethasone; Newborn rat

Differential expression of mitochondrial proteome and NADH-dehydrogenase subunit 4 in type II alveolar epithelial cell of primary fetal rat exposed to hyperoxia

Objective To explore the effect of hyperoxia exposure on the mitochondrial proteome and the expression of NADH-dehydrogenase subunit 4 (ND4) of type Ⅱ alveolar epithelial cells (AEC Ⅱ) of primary fetal rat. Methods The primary fetal rats (gestation 19～20 d) AEC Ⅱ were cultured in vitro and then were randomly divided into 2 groups. Air-exposed control group and hyperoxia-exposed group. The differential expression of mitoehondrial proteome was analyzed by two-dimensional electrophoresis. The expression of ND4 mRNA and protein was determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. Results Compared with air control group, we found 55 protein spots with differential expression after 24 h of hyperoxia. After exposure to hyperoxia for 12 h and 24 h, the expression of AEC Ⅱ ND4 mRNA (1.20±0. 12 vs 4.61±0. 43,0. 69±0. 07 vs 2.15± 0. 14, PF 0.05 for all). Conclusions The changes of AEC Ⅱ mitoehondrial (including respiratory chain) function might play a critical role in the development of hyperoxia lung injury. Key words: Hyperoxia; Pulmonary alveoli; Epithelial cells; Mitochondria/proteins; Proteome; NADH-dehydrogenase

Mechanism of retinoic acid and mitogen-activated protein kinases regulating hyperoxia lung injury

To investigate the protective effect of retinoic acid (RA) on hyperoxic lung injury and the role of RA as a modulator on mitogen-activated protein kinases (MAPKs), gastation 21 d Sprague-Dawley (SD) fetuses (term = 22 d) were delivered by hysterotomy. Within 12-24 h of birth, premature rat pups were randomly divided into 4 groups (n=12 each): air-exposed control group (group I); hyperoxia-exposed group (group II), air-exposed plus RA group (group III), hyperoxia-exposed plus RA group (group IV). Group I, III were kept in room air, and group II, IV were placed in 85 % oxygen. The pups in groups III and IV were intraperitoneally injected with RA (500 microg/kg every day). All lung tissues of premature rat pups were collected at the 4th day after birth. Terminal transferase d-UTP nick end labeling (TUNEL) staining was used for the detection of cell apoptosis. The expression of PCNA was immunohistochemically detected. Western blot analysis was employed for the determination of phosphorylated and total nonphosphorylated ERKs, JNKs or p38. Our results showed that lungs from the pups exposed to hyperoxia for 4 d exhibited TUNEL-positive nuclei increased markedly throughout the parenchyma (P<0.01), and decreased significantly after RA treatment (P<0.01). The index of PCNA-positive cells was significantly decreased (P<0.01), and was significantly increased by RA treatment (P<0.01). The air-space size was significantly enlarged, secondary crests were markedly decreased in hyperoxia-exposed animals. RA treatment improved lung air spaces and secondary crests in air-exposed pups, but had no effect on hyperoxia-exposure pups. Western blotting showed that the amounts of JNK, p38 and ERK proteins in hyperoxia-exposure or RA-treated lung tissues were same as those in untreated lung tissues (P>0.05), whereas activation of these MAPKs was markedly altered by hyperoxia and RA. After hyperoxia exposure, p-ERK1/2, p-JNK1/2 and p-p38 were dramatically increased (P<0.01), whereas p-JNK1/2 and p-p38 were markedly declined and p-ERK1/2 was further elevated by RA treatment (P<0.01). It is concluded that RA could decrease cell apoptosis and stimulate cell proliferation under hyperoxic condition. The protection of RA on hyperoxia-induced lung injury was related to the regulation of MAP kinase activation.