Autophagy regulates hyperoxia-induced intracellular accumulation of surfactant protein C in alveolar type II cells.

Surfactant protein C (SP-C) deficiency is a risk factor for hyperoxia-induced bronchopulmonary dysplasia in newborn infants. However, the role of SP-C deficiency in the process is unclear. Here, using neonatal rat BPD model and MLE-12, mouse alveolar epithelial type II cell, we examined the changes of SP-C levels during hyperoxia. Immunohistochemistry, immunofluorescence, and ELISA analysis showed SP-C accumulation in alveolar epithelial type II cells. Electron microscopy further demonstrated the accumulation of lamellar bodies and the co-localization of lamellar bodies with autophagosomes in the cytoplasm of alveolar epithelial type II cells. The inhibition of autophagy with 3-Methyladenine and knockdown of Atg7 abolished hyperoxia-induced SP-C accumulation in the cytoplasm. Furthermore, inhibition of JNK signaling with SP600125 suppressed hyperoxia-induced Atg7 expression and SP-C accumulation. These findings suggest that hyperoxia triggers autophagy via JNK signaling-mediated Atg7 expression, which promotes the accumulation of SP-C within alveolar epithelial type II cells. Our data provide a potential approach for hyperoxic lung injury therapy by targeted pharmacological inhibition of autophagic pathway.

The role of placenta growth factor in the hyperoxia-induced acute lung injury in an animal model.

Prolonged exposure to hyperoxia leads to acute lung injury. Alveolar type II cells are main target of hyperoxia-induced lung injury. However, the cellular and molecular mechanisms remain unknown. Here, we aimed to investigate the role of placental growth factor (PLGF) in hyperoxia-induced lung injury. Using experimental hyperoxia-induced lung injury model of neonatal rat and mouse lung epithelial type II cells (MLE-12), we examined the levels of PLGF in bronchoalveolar lavage fluid and in the supernatants of MLE-12 cells. Our results revealed that exogenous PLGF induced hyperoxia-induced lung injury. Furthermore, PLGF triggered a shift of vinculin from insoluble to soluble cell fraction, similar to the observation under hyperoxia stimulation. Moreover, we observed significantly reduced phosphorylation of focal adhesion kinase and increased permeability in MLE-12 cells treated with PLGF. These results suggest that PLGF triggers focal adhesion disassembly in alveolar type II cells via inhibiting the activation of focal adhesion kinase. Our findings reveal a novel role of PLGF in hyperoxia-induced lung injury and provide a potential target for the management of hyperoxia-induced acute lung injury.

[Expression of ERK1/2 protein in lung tissues of newborn rats with hyperoxia-induced chronic lung disease].

OBJECTIVE: To study the expression of extracellular signal regulated protein kinase (ERK) 1/2 in lung tissues of newborn rats with chronic lung disease (CLD) caused by hyperoxia. METHODS: Forty-eight full-term newborn rats were randomly divided into two groups: hyperoxia and control. The two groups were exposed to a hyperoxic gas mixture (0.90 O(2)) for an induction of CLD and room air within 12 hrs after birth, respectively. The levels of ERK1/2 protein and mRNA in lung tissues were measured using immunohistochemistry, Western blot and real-time PCR methods on postnatal days 3, 7 and 14. The severity of pulmonary fibrosis was evaluated. RESULTS: The expression of p-ERK protein in lung tissues in the hyperoxia group was significantly higher than that in the control group on postnatal days 7 and 14 (P<0.01). There were no significant differences in the levels of total ERK1/2 protein and ERK1/2 mRNA. CONCLUSIONS: The activation of phosphorated ERK1/2 may lead to lung fibrosis caused by hyperoxia in newborn rats.

[Effects of TGF-ß1 on gene expression of connective tissue growth factor in lung fibroblasts].

OBJECTIVE: To study the effects of transforming growth factor-ß1 (TGF-ß1) on the gene expression of connective tissue growth factor (CTGF) in cultured lung fibroblasts of embryonic rats in vitro. METHODS: Wistar rats of embryonic 19 days were used for primary culture of lung fibroblasts (LFs). The cells in the experimental group were treated by different concentrations (1, 5 or 10 ng/mL) and different durations (12, 24 or 48 hrs) of TGF-ß1 to stimulate the LFs. The cells in the control group were cultured in serum-free medium. RT-PCR method was applied to detect CTGF mRNA expression in LFs. RESULTS: Compared with the control group, the levels of CTGF mRNA in LFs in the experimental group increased significantly (P<0.05). CTGF mRNA expression gradually increased with increasing concentration and duration of TGF-ß1 treatment (P<0.05). CONCLUSIONS: TGF-ß1 can stimulate CTGF gene expression in LFs and increase CTGF gene expression in a dose-and time-dependent manner.

[Expression of HoxB5, SPC and AQP5 in neonatal rats with hyperoxia-induced chronic lung disease].

OBJECTIVE: Alveolar epithelium impairment is one of pathological changes associated with chronic lung disease (CLD). Hoxb5 is one of the few homeobox genes strongly expressed in the developing lung. This study investigated the expression of HoxB5, SPC and AQP5 in rats with CLD in order to explore the role of Hoxb-5 in impairment and reparation of alveolar epithelium. METHODS: Eighty neonatal rats were randomly exposed to hyperoxia (model group) or to room air (control group) (n=40 each). The CLD model was induced by hyperoxia exposure. The expression of HoxB5, SPC and AQP5 protein and mRNA in the lung tissue was detected by immunohistochemistry and RT-PCR 1, 3, 7, 14 and 21 days after exposure. RESULTS: In the model group HoxB5 expression significantly decreased 7, 14 and 21 days after hyperoxia exposure. SPC expression decreased 3 days after hyperoxia exposure but increased significantly 7, 14 and 21 days after hyperoxia exposure as compared to the control group. AQP5 expression was progressively reduced with prolonged hyperoxia exposure. CONCLUSIONS: Hyperoxia exposure may lead to alveolar epithelial cell (AEC) damage in neonatal rats. The increased SPC expression and decreased AQP5 expression suggested that the ability of differentiation and transformation of AECII into AECI decreased in neonatal rats with CLD. The decreased HoxB5 expression following hyperoxia exposure might contribute to a decreased ability of differentiation of AECII.

[Effect of melatonin on hyperoxia-induced oxidant/antioxidant imbalance in the lung of neonatal rats with chronic lung disease].

OBJECTIVE: To 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). METHODS: Ninety 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. RESULTS: The 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). CONCLUSIONS: Melatonin may reverse oxidant/antioxidant imbalance in hyperoxia-induced lung disease, thus providing a protective effect against CLD in neonatal rats.

[Early clinical presentations and MRI characteristics in newborns with cerebral infarction].

OBJECTIVE: The present study aimed to characterize the clinical presentations and magnetic resonance imaging including conventional MRI and diffusion-weighted imaging (DWI) in newborns with cerebral infarction. METHODS: Clinical records of 16 newborn infants with cerebral infarction were reviewed. All cases underwent DWI examination in addition to conventional MRI examination [T1-weighted (T1W) and T2-weighted (T2W)]within 5 days after birth. Five patients received the second MRI examination at the age of 11 to 18 days. RESULTS: Eight patients had antenatal risk factors, 9 had intranatal risk factors, and no postnatal risk factors were found. Seizures as the first symptom were noted in 11 neonates, with a short duration and a low frequency. The first imaging examination (within 5 days) showed a slight hypointensity on T1W, a slight hyperintensity on T2W and significantly increased signal intensity with a clear boundary on DWI in the lesions. In the MRI re-examination, more obvious hypointensity on T1W and hyperintensity on T2W were noted, while hypointensity was shown on DWI in the lesions compared with the first imaging results. CONCLUSIONS: Seizures characterized by short duration and low frequency usually may be the first symptom in newborns with cerebral infarction. A hyperintensity on DWI was shown in the lesions at the early stage of neonatal cerebral infarction. A hypointensity on T1W and a hyperintensity on T2W were demonstrated in the lesions with increasing disease duration.

[The changes and effects of metalloproteinase-2 and tissue inhibitors of metalloproteinase-1 protein and mRNA expression in the lung tissue of neonatal rats with chronic lung disease induced by hyperoxia].

OBJECTIVE: To investigate the dynamic changes and the effects of metalloproteinase-2 (MMP-2) and tissue inhibitors of metalloproteinase-2 (TIMP-2) mRNA in lungs of neonatal rats after inhaling high concentration of oxygen. METHODS: Full-term newborn rats were continuously exposed to oxygen (0.90-0.95) or room air (0.21O(2)) within 12 hours after birth. Lung histological study with hematoxylin-eosin staining (HE) and radical alveolar counts (RAC) were performed; the changes in MMP-2 and TIMP-2 protein and mRNA expression were measured by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) on 1, 3, 7, 14 and 21 days in hyperoxia groups and air inhalation controls. RESULTS: Compared with air inhalation controls, inflammation response was seen in early stage, the arrest of lung development was evident after 7 days of oxygen exposure, finally resulting in interstitial fibrosis; RAC began to decrease from 7 days in hyperoxia rats compared to air inhalation controls (P<0.05), more so on 14 days and 21 days (both P<0.01); MMP-2 protein and mRNA expression were higher on 3 days (P<0.05 and P<0.01), and protein decreased on 14 days (P<0.05), while mRNA didn't change; the expression of TIMP-2 protein and mRNA showed no change all the time. CONCLUSION: With prolonged hyperoxia, the balance of MMP-2/ TIMP-2 can not be maintained. Collagen breakdown is disturbed, which may lead to lung inflammatory injury in early stage and then collagen deposition in lung interstitium and lung development is arrested resulting in chronic lung disease.

[Effect of position on oxygenation in neonates after weaning from mechanical ventilation].

OBJECTIVE: What is the best suitable position for neonates who were weaned from mechanical ventilation has not been identified. This study aimed to evaluate the effect of the supine and prone positions on oxygenation in neonates within 6 hrs after weaning from mechanical ventilation. METHODS: Sixty neonates who were weaned from mechanical ventilation were randomly given prone or supine position (n=30 each). They all received oxygen inspiration and SPO2 was maintained in a normal range by adjusting the oxygen flow rate (FiO2). Blood PaO2 and PaCO2 levels were measured 1 and 6 hrs after weaning and then the alveolodouble ended arrowarterial oxygen partial pressure difference (A-aDO2), respiratory index and oxygenation index were calculated. RESULTS: Mean FiO2 used in the prone position group was significantly lower than that in the supine position group 1 and 6 hrs after weaning (P<0.01). The value of A-aDO2 in the prone position group 1 hr (171.06+/-86.55 vs 253.62+/-71.56; P<0.01) and 6 hrs after weaning (105.85+/-78.18 vs 208.48+/-86.80; P<0.01) were significantly lower than that in the supine position group. The respiratory index in the prone position group 1 and 6 hrs after weaning (2.16+/-1.24 and 1.35+/-1.11) was also reduced compared to 3.74+/-1.68 and 3.65+/-1.28 in the supine position group (P<0.01). In contrast, PaO2 in the prone position group 1 hr (88.70+/-32.65 vs 73.43+/-17.68; P<0.01) and 6 hrs (84.10+/-13.95 vs 70.20+/-20.27; P<0.01) after weaning was significantly higher than that in the supine position group. The oxygenation index in the prone position group 1 and 6 hrs after weaning (213.49+/-88.96 and 275.23+/-108.83) increased significantly compared to 141.54+/-43.25 and 160.62+/-63.03 in the supine position (P<0.01). CONCLUSIONS: The prone position is better than the supine position for the improvement of oxygenation within 6 hrs after weaning from mechanical ventilation in neonates.

[Clinical evaluation of neonatal hypoglycemic brain injury demonstrated by serial MRIs].

OBJECTIVE: To study the relationship between clinical and imaging features in neonates with hypoglycemic brain injury. METHODS: Sixteen neonates with hypoglycemic brain injury received a MRI scan with the sequences of T1WI, T2WI and DWI within 48 hrs after admission. Of the 16 patients, 11 received second MRI scan at two weeks of their lives, and 3 received a third scan at ages of 1-5 months. RESULTS: Repeated seizures, lethargy and hypotonia were common clinical manifestations. Five severe hypoglycemia cases presented coma, respiratory failure and even cardiorespiratory arrest. The minimum mean value of whole blood glucose (WBG) in the 16 patients was 0.98+/-0.43 mmol/L, and that of the 5 severe cases was 0.72+/-0.42 mmol/L. EEG showed intermittent low voltage in the mild hypoglycemia cases. Flatten pattern and even electrocerebral silence was noted in the severe cases. Occipital and parietal cortexes (OPC) injuries were found in all of the 16 patients and 2 patients had concurrent periventricular white matter injury. A widespread involvement of cortex was found in the 5 severe hypoglycemia cases in which 1 showed widespread involvement of white matter, and 2 showed involvement of basal ganglia and thalamus. The 5 patients with widespread cortex injury and the 2 patients with OPC and periventricular white matter injury showed lower minimum WBG levels compared with those with OPC alone (0.71+/-0.35 mmol/L vs 1.19+/-0.42 mmol/L; t= 2.4124, P<0.05). The appearance of high-intensity signals on DWI was shown as early changes of signals in all of the 16 patients. The second MRI scan for 7 patients with OPC showed abnormal signals on T1WI and T2WI in 5 patients and abnormal signals on DWI in 3 cases. Cerebral atrophy and multicystic encephalomalacia were found in four patients with widespread involvement of cortex on DWI. In the follow-up one patient with OPC presented delayed myelination and one with concurrent white matter injury showed spastic diplegia. One patient with widespread involvement of cortex showed diffused encephalomalacia. CONCLUSIONS: The severity of hypoglycemic brain injury demonstrated by serial MRIs relates to the severity of hypoglycemia. The occipital and parietal areas are the most vulnerable following hypoglycemia in neonates. Severe hypoglycemic brain injury manifests as a widespread involvement of cortex, or combined with white matter, or basal ganglia and thalamus. DWI can show early hypoglycemic brain injury.

[Expression and role of connective tissue growth factor mRNA in premature rats with hyperoxia-induced chronic lung disease].

OBJECTIVE: To investigate the role of connective tissue growth factor (CTGF) in pulmonary fibrosis in rats with hyperoxia-induced chronic lung disease (CLD). METHODS: Eighty premature rats were randomly exposed to hyperxia (FiO2=0.90) or room air (control group) (n=40 each). CLD was induced by hyperoxia exposure. The expression of CTGF mRNA and transforming growth factor-beta 1 (TGF-beta 1), the levels of type I collagen and the proliferating cell nuclear antigen (PCNA) index were assayed with enzyme linked immunoadsorbent (ELISA), immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) on days 1, 3, 7, 14 and 21 after exposure. RESULTS: There were no differences in the levels of type I collagen, PCNA index, TGF-beta 1 protein and CTGF mRNA between the CLD and the control groups within 3 days after exposure. In the CLD group, the expression of TGF-beta 1 protein increased on the 7th day, remained at a higher level on the 14th and 21st day after exposure; the higher levels of type I collagen, PCNA index and CTGF mRNA were detected on the 14th day and peaked on the 21st day after exposure when comparing the control group. CTGF mRNA expression was positively correlated with type I collagen levels in the CLD group (gamma=0.89, P < 0.01). CONCLUSIONS: The expression of CTGF in lung tissues is associated with pulmonary fibrosis in CLD rats.

Gene expressions and roles of matrix metalloproteinases-8 and tissue inhibitor of metalloproteinases-1 in hyperoxia-induced pulmonary fibrosis in neonatal rats.

OBJECTIVE: Extracellular matrix (ECM) deposition is a major reason of pulmonary fibrosis in hyperoxia-induced lung injury. However, the relevant mechanism has not been identified. This study examined the gene expressions of matrix metalloproteinases-8 (MMP-8, a catabolic enzyme of type I collagen) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in neonatal rats with hyperoxia-induced pulmonary injury in order to explore the role of MMP-8 and TIMP-1 in pulmonary fibrosis. METHODS: Eighty term newborn rats were randomly exposed to hyperoxia (FiO2=0.90, hyperoxia group)and to room air (FiO2=0.21, control group)(n=40 each). Lung injury was induced by hyperoxia exposure. The content of type I collagen and the expressions of type I collagen protein and MMP-1 mRNA and TIMP-1 mRNA were assayed with enzyme linked immunoadsorbent (ELISA), immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) respectively on days 1, 3, 7, 14 and 21 after exposure. RESULTS: The content of type I collagen and the expression of type I collagen protein in the hyperoxia group were statistically higher than those in the control group at 14 and 21 days post-exposure. The MMP-8 mRNA expression decreased while the TIMP-1 mRNA expression increased significantly in the hyperoxia group as compared to the control group at 14 and 21 days post-exposure. CONCLUSIONS: Hyperoxia exposure down-regulates MMP-8 mRNA expression and up-regulates TIMP-1 mRNA expression. This results in a reduction of ECM degradation, thereby ECM deposition occurs in lung tissue, which may be an important mechanism of pulmonary fibrosis following hyperoxia-induced lung injury.

[Protection of captopril against chronic lung disease induced by hyperoxia in neonatal rats].

OBJECTIVE: This study examined the protein and mRNA contents of angiotesin converting enzyme (ACE), angiotensin II (Ang II) and type I collagen and the changes of lung histomorphology in neonatal rats with hyperoxia-induced chronic lung disease (CLD) and investigated the protection of captopril against CLD and the possible mechanism. METHODS: A total of 240 term neonatal Wistar rats were randomly assigned into air, model, normal saline and captopril-treated groups (n=60 each). The air group was exposed to room air (FiO2=0.21) immediately after birth. The other three groups were exposed to hyperoxia (FiO2=0.9) for 21 days to induce lung injury. The captopril-treated group received captopril daily (30 mg/kg) by intragastric administration between the 7th and 21st days of hyperoxia exposure. The normal saline group was administrated with normal saline instead. At each time interval of 1, 3, 7, 14 and 21 days after experiment, six rats of each group were randomly chosen and sacrificed. The protein and mRNA levels of ACE, Ang II and type I collagen were measured by enzyme-linked immunosorbentassay, radio-immunity technique and RT-PCR. The changes of lung histomorphology were observed under a light microscope. RESULTS: The protein and mRNA expressions of ACE, Ang II and type I collagen increased significantly in the model and normal saline groups on the 14th and peaked on the 21st days of exposure compared with those of the air group (P < 0.05 or 0.01). Captopril treatment reduced significantly the protein and mRNA expressions of ACE, Ang II and type I collagen compared the model and normal saline groups on the 14th and 21st days, although the values were significantly higher than the air group (P < 0.05 ). The histopathologic examination demonstrated broadened lung interstitium and reduced alveolar quantity and lung fibrosis was developed in the model and normal saline groups on the 14th day of exposure. Captopril treatment obviously alleviated the changes of lung histomorphology. CONCLUSIONS: Captopril can inhibit the protein and mRNA expressions of ACE, Ang II and type I collagen and alleviate lung fibrosis in neonatal rats with hyperoxia-induced lung injury/CLD. This may contribute to one of the possible mechanisms underlying the protective effects of captopril against lung injury/CLD.

[Effect of losartan on lung fibrosis in neonatal rats with hyperoxia-induced chronic lung disease].

OBJECTIVE: In addition to regulating blood pressure, angiotensin II is involved in lung fibrogenesis. This study aimed to explore the effect of losartan, an angiotensin II type 1 receptor antagonist, on lung fibrosis in neonatal rats with hyperoxia-induced chronic lung disease (CLD) and its possible mechanisms. METHODS: Neonatal Wistar rats were randomly divided into four groups within 24 hrs after birth: room air exposure, hyperoxia exposure (85%-90% O2), hyperoxia exposure + losartan, and hyperoxia exposure + placebo. Losartan (5 mg/kg/d) or placebo was administered beginning on the 6th day after birth. After 7, 14 and 21 days of exposure, 8 rats in each group were sacrificed. Lung histological changes were evaluated by hematoxylin-eosin staining. Levels of hydroxyproline (HYP), superoxide dismutase (SOD) and malondialdehyde (MDA) in lung tissues were determined by spectroscopy. RESULTS: Hyperoxia exposure resulted in decreased alveolar septation, enlarged terminal air space, increased collagen deposition, pulmonary hemorrhage, and pulmonary consolidation. In the hyperoxia exposure + losartan group, the alveolar septum became thinner and lung fibrosis was alleviated, but the alveolar space was not obviously deflated and the number of secondary septum was not increased. Hyperoxia exposure increased significantly the HYP contents in lung tissues 14 and 21 days after exposure. Addition of losartan to the hyperoxia exposure resulted in decreased HYP contents (471.46 +/- 30.63 mu g/kg vs 545.15 +/- 34.90 mu g/kg for hypoxia alone; P < 0.01) after 21 days of exposure. SOD activity increased 7 days after hyperoxia exposure and then decreased to levels similar to the air exposure group. MDA levels increased to a peak at 7 days and remained at higher levels through 21 days of exposure when compared with the air exposure group (P < 0.01). Losartan treatment significantly increased SOD activities (82.94 +/- 4.62 U/mg protein vs 67.78 +/-8.02 U/mg protein; P < 0.01) and decreased MDA levels (30.54 +/- 5.89 nmol/mg protein vs 48.75 +/- 8.09 nmol/mg protein, P < 0.01) compared with the hyperoxia exposure group 21 days after exposure. CONCLUSIONS: Losartan attenuated lung fibrosis in neonatal rats with hyperoxia-induced CLD, possibly through an increase of antioxidase enzyme activity and reduction of lipid peroxidation.

[Expression and roles of CDK4 and p21 in lung tissues of premature rats with hyperoxia-induced chronic lung disease].

OBJECTIVE: The pathogenic mechanisms of chronic lung disease (CLD) of premature infants are not fully understood. Its final pathological changes have been shown to relate with lung cell proliferation. The aim of the study was to explore the relationship of the expression of cyclin dependent kinase 4 (CDK4) mRNA and cyclin dependent kinase inhibitor (CDKI) p21 mRNA with lung cell proliferation. METHODS: Eighty premature rats were randomly assigned to a hyperoxia group and a control group (n=40 each). The hyperoxia group was exposed to high concentration of oxygen (FiO2 >0.90 and developed CLD. The control group was exposed to room air (FiO2 =0.21). The rats were randomly subdivided into groups sacrificed at 1, 3, 7, 14 and 21 days of exposure. Lung tissues were collected and made into 5 microm sections. Expression of proliferating cell nuclear antigen (PCNA) in lung tissues was detected by immunohistochemistry. Dynamic expression of CDK4 mRNA and p21 mRNA were detected by in situ hybridization. Lung histomorphology and fibrosis were observed by microscopy. RESULTS: Expression of PCNA and CDK4 mRNA of lung cells in the hyperoxia group increased significantly after 7 days of exposure, and further increased after 14 and 21 days compared with controls. p21 mRNA expression in the hyperoxia group significantly increased after 1 and 3 days of exposure. After 7 days, p21 mRNA expression progressively decreased, but remained at a higher level than control through 21 days of exposure. PCNA expression was positively correlated to CDK4 mRNA expression (r=0.83, P < 0.05) and negatively correlated to p21 mRNA expression (r=-0.81, P < 0.05) in the hyperoxia group between 7 and 21 days of exposure. CONCLUSIONS: Hyperoxia can induce lung cell proliferation in premature rats. The over-expression of CDK4 gene and the decreased expression of p21 gene in lung tissues may be associated with lung cell proliferation induced by hyperoxia.

Effect of dexamethasone on the content of pulmonary surfactant protein D in young rats with acute lung injury induced by lipopolysaccharide.

OBJECTIVE: Pulmonary surfactant protein-D (SP-D) is regarded as a valuable biomarker in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). This study was to explore the changes of SP-D content in lung tissue following ALI and the effect of dexamethasone (Dex) on the SP-D content in young rats. METHODS: One hundred and forty-four 21-day-old Sprague-Dawley rats were randomly assigned into control, ALI and Dex-treated groups. ALI was induced by intraperitoneal injection of lipopolysaccharide (LPS) (4 mg/kg) in the rats from the ALI and Dex-treated groups. Normal saline was given for the control group. Dex (5 mg/kg) was administered 1 hr after LPS injection in the Dex-treated group. At each time interval of 6, 12, 24, 36, 48 and 72 hrs after LPS injection, eight rats of each group were randomly chosen and sacrificed. Western blot was employed to detect the content of SP-D in lung tissues. RESULTS: The pulmonary SP-D content decreased significantly at 36, 48 and 72 hrs after LPS administration in the ALI group, and reduced to a nadir (0.92 +/-0.11 vs 3.27 +/- 0.52) at 48 hrs compared with that of the control group (P < 0.01). The SP-D content in the Dex-treated group increased significantly at 36,48 and 72 hrs after LPS administration when compared with the ALI group (P < 0.01). A significant difference in the SP-D content between the Dex-treated and the control group was noted only at 72 hrs after LPS administration (P < 0.05). CONCLUSIONS: The SP-D content in lung tissue was reduced following ALI in young rats at the early stage. Early administration of Dex can significantly increase the pulmonary SP-D content.

Effects of dexamethasone on the ultrastructure of alveolar type II cells in young rats with lipopolysaccharide-induced acute lung injury.

OBJECTIVE: Alveolar type II (AT II) cells play a crucial role in the maintenance of pulmonary surfactant homeostasis and pulmonary immunity. The effects of dexamethasone (Dex) on the ultrastructure of AT II cells after acute lung injury remain unknown. This study focused on the ultrastructural changes caused by acute lung injury and on the effects of Dex administration on these ultrastructural changes in young rats. METHODS: Seventy-two 21-day-old Sprague-Dawley rats were randomly divided into control, acute lung injury and Dex-treated groups. Rats in the lung injury group were intraperitoneally injected with 4 mg/kg lipopolysaccharide (LPS) in order to induce acute lung injury, while the control rats were injected with the same amount of normal saline (NS). The Dex-treated group was injected first with LPS followed 1 hr later by Dex (5 mg/kg) injection. Eight rats in each group were sacrificed 24, 48 and 72 hrs after LPS or NS injection. Lung samples were obtained from the lower parts of left lungs and fixed with 2.5% glutaraldehyde for transmission electron microscope examination. RESULTS: Microvilli of AT II cells disappeared and the number of lamellar bodies (LBs) increased in the lung injury group 24 hrs after LPS injection. The ring-like arrangement of LBs around nuclei was present until 48 hrs after LPS injection. By 48 hrs after LPS injection, giant LBs with vacuole-like abnormalities appeared. The shape of nuclei became irregular and the border of the nuclei became blurred. By 72 hrs after LPS injection, the number of LBs was obviously reduced; nucleoli disappeared; and karyolysis occurred in some of the nuclei. In contrast, in the Dex-treated group, LBs crowded on one side of AT II cells and exocytosis appeared on the same side by 24 hrs after LPS injection. By 48 hrs, the number of LBs was reduced. The number of mitochondria increased, and some of them became swollen and enlarged. However, by 72 hrs, the number of LBs increased and the ring-like arrangement of LBs around the nucleus again appeared. CONCLUSIONS: Ultrastructural changes of AT II cells following lung injury induced by LPS were time-dependent in young rats. Dex may ameliorate AT II cell injury and promote functional restoration of AT II cells in LPS-induced acute lung injury.

Expression dynamics of caveolin-1 in fibroblasts of newborn rats with chronic lung disease and its impact on lung fibroblast proliferation.

PURPOSE:: To evaluate the changes of caveolin-1 in lung fibroblasts in newborn Wistar rats when exposed to hyperoxic conditions, as well as lung fibroblasts cell cycle. METHODS:: One hundred newborn Wistar rats were randomly divided (50 rats/group) into experimental and control groups, exposed to hyperoxic conditions or normal air, respectively. The fraction of inspired oxygen (FiO2) in the experimental group was 90%, whereas this value was 21% in the control group. Lung fibroblasts were collected on days 3, 7, and 14 of the experiment. Caveolin-1 expression dynamics in lung fibroblasts was assayed in each group by immunofluorescence and Western blot analyses. Flow cytometry (FCM) was used to assess the proportions of lung fibroblasts at different stages of the cell cycle. RESULTS:: On day 3, no significant difference in caveolin-1 expression was observed between the hyperoxic and control groups; however, on days 7 and 14, caveolin-1 expression was significantly lower in the hyperoxic group than in the control (P<0.05). No apparent differences were observed in caveolin-1 expression in the control group at the different time points. Using FCM analysis, we showed that the proportion of lung fibroblasts in G0/G1 phase in the hyperoxic group decreased compared to that of the control group on day 7, while the proportion of S-phase cells increased (P<0.05). These differences were more significant when the groups were compared on day 14 (P<0.01). CONCLUSION:: After seven days the exposure to hyperoxic conditions, lung fibroblasts proliferated and caveolin-1 expression decreased.

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats.

AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue. METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (LPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.

[Relationship between expression of aquaporin-1, -5 and pulmonary edema in hyperoxia-induced lung injury in newborn rats].

OBJECTIVE: Aquaporin (AQP) is a group of cell membrane transporting proteins. The study was designed to investigate the changes of AQP1 and AQP5 in the lung tissue under hyperoxia and their roles in pulmonary edema. METHODS: Two hundred newborn rats were randomized into different oxygen concentrations exposure: FiO2=0.80 (Experimental group 1), FiO2=0.60 (Experimental group 2), FiO2=0.40 (Experimental group 3) and FiO2=0.21 (Air control group). Rats were sacrificed at 1, 3, 5, 7 and 14 days after the beginning of experiment (10 rats each time point). The expressions of AQP1 and AQP5 were examined by Western Blot. The ratio of lung wet weight to lung dry weight (wet-to-dry weight ratio, W/D), and the protein content in bronchoalveolar lavage fluid (BALF) were measured. RESULTS: Compared with the Air control group, the W/D ratio and the protein content in BALF in the three experiment groups increased significantly and the increased extent was positively related to the duration and the oxygen concentration of hyperoxia-exposure. The expression of AQP1 in the experimental groups began to decrease at the 3rd day and significant differences were found at the 5th and the 7th days after hyperoxia-exposure compared with that in the Air control group (P < 0.05). The AQP1 expression was restored somewhat at the 14th day after hyperoxia-exposure, but it was still lower in the Experimental groups 1 and 2 than that in the Air control group (P < 0.05). The expression of AQP5 in the experimental groups were reduced compared with that in the Air control group 3 days after hyperoxia-exposure and the decrease of AQP5 expression was associated with duration of hyperoxia-exposure. The comparison among three experimental groups showed that the decrease of AQP1 and AQP5 expressions was associated with the concentration of hyperoxia-exposure. CONCLUSIONS: The expressions of AQP1 and AQP5 decreased in hyperoxia-induced lung injury and correlated with the severity of pulmonary edema.