[Protective effect of N-acetylcysteine on hyperoxic lung injury and its relation with p38 mitogen-activated protein kinase signaling pathway].

OBJECTIVE: To investigate the expression of p38 mitogen-activated protein kinase (MAPK) in hyperoxic lung injury (HLI), and explore the protective effect of N-acetylcysteine (NAC) on HLI and its mechanism. METHODS: Thirty Wistar rats aged 3 weeks old were divided into five groups with 6 rats in each group according to random digits table: room-air group (A), hyperoxia injury group (B), hyperoxia+NAC group (C), hyperoxia+p38 MAPK inhibitor (SB203580) group (D), hyperoxia+NAC+SB203580 group (E). Rats in NAC groups were injected with NAC (200 mg/kg) intraperitoneally, and they received an intravenous injection of SB203580 (0.5 mg/kg) in SB203580 groups. The animals were sacrificed after 7 days of experiment. Lung pathology and grade of lung tissue injury were examined with light microscopy, lung wet/dry (W/D) ratio, total protein (TP) level in bronchoalveolar lavage fluid (BALF) and permeability coefficient were evaluated. The location and quantity of phosphorylation p38 MAPK (p-p38 MAPK) protein were detected by immunohistochemistry and Western blotting analysis respectively. RESULTS: The pathological changes in the lung in B group included severe alveolar oedema with inflammatory cells aggregation and red blood cell leakage, while the lung pathological pictures in C, D, E groups were improved significantly compared with B group. p-p38 MAPK positive cells increased in B group compared with those in A group, involving many types of pulmonary cells, especially in infiltrating inflammatory cells. In C, D, E groups, the positive cells remarkably decreased compared with B group. p-p38 MAPK content was higher in B group than that in A group (0.20+/-0.03 vs. 0.11+/-0.01, P<0.05), and p-p38 MAPK expressions in C, D, E groups decreased significantly compared with B group (0.16+/-0.02, 0.15+/-0.01, 0.14+/-0.02 vs. 0.20+/-0.03, all P<0.05), but were higher than those in A group (all P<0.05). There was no significant difference in p-p38 MAPK quantity among three groups. Changes in W/D ratio, TP and permeability coefficient among groups were comparable with those of p-p38 MAPK protein quantity. CONCLUSION: Reactive oxygen species (ROS) activated p38 MAPK signaling pathway. NAC may exert a protective effect on HLI through attenuation of hyperoxia-induced p38 MAPK activation.

N-acetylcysteine protects alveolar epithelial cells from hydrogen peroxide-induced apoptosis through scavenging reactive oxygen species and suppressing c-Jun N-terminal kinase.

The production of reactive oxygen species (ROS) during hyperoxia contribute to alveolar epithelial apoptosis. In the present study, the molecular mechanisms of oxidative stress-induced alveolar epithelial cell apoptosis were investigated. The cytoprotective effects of N-acetylcysteine (NAC) were evaluated. Treatments using 500 muM H(2)O(2) can induce primary alveolar type II epithelial cell apoptosis. During this procedure, c-Jun N-terminal kinase (JNK) was activated. SP600125, a specific inhibitor of JNK, can partially block H(2)O(2)-induced alveolar type II epithelial cells (ATII cells). SP600125 also attenuated Bax protein content and p53 nuclear accumulation induced by H(2)O(2). NAC (5 mM) pretreatment decreased H(2)O(2)-induced ATII cell apoptosis. The high level of intracellular reactive oxygen species (ROS) induced by H(2)O(2) was also attenuated by NAC pretreatment. Taken together, H(2)O(2) can induce primary ATII cells apoptosis and increase JNK phosphorylation. NAC, a precursor of glutathione (GSH) synthesis, can protect ATII cells from H(2)O(2)-induced apoptosis through scavenging ROS.

[The proliferation-promoting effects of calcitonin gene-related peptide on type II alveolar epithelial cell exposed to hyperoxia mediated by protein kinase C alpha pathway].

OBJECTIVE: To explore the effects of calcitonin gene-related peptide (CGRP) on type II alveolar epithelial cell (AECII) exposed to hyperoxia, and to determine whether the mechanism is mediated by protein kinase C alpha/nuclear factor-KappaB (PKC alpha/NF-KappaB) signal pathway. METHODS: AECII were isolated from the lung of 21 days fetal rat and cultured for 15 hours to coalesce. Then AECII were randomly assigned into four groups: air, hyperoxia, O(2)/CGRP, and O(2)/CGRP8-37 (a receptor antagonist against CGRP). AECII were exposed to FiO(2) 21% (air) or 85% (hyperoxia) for 24 hours respectively. In O(2)/CGRP and O(2)/CGRP8-37 groups CGRP or both CGRP and CGRP8-37 were added into cultural fluid before placing the plate into 85% oxygen. Cell proliferation ability was determined by methyl thiazolyl tetrazolium (MTT) assay and cell cycles by flow cytometry. Western blotting was employed to detect the fraction of PKC alpha in membrane and cytosol, and translocation of NF-KappaB was observed under laser confocal microscopy. RESULTS: AECII in hyperoxia group showed a decreased viability of AECII [(68.752+/-5.766)% vs. (100.000+/-6.682)%] and had an enhanced percentage of G0/G1 phase [(80.652+/-6.253)% vs. (45.825+/-2.899)%] with a corresponding decline in percentage of S phase [(14.198+/-4.785)% vs. (27.470+/-2.775)%] and G2/M phases [(5.148+/-1.688)% vs. (26.708+/-1.863)%] compared with AECII in air (all P<0.01). Addition with CGRP before hyperoxia exposure promoted AECII proliferation [(94.813+/-6.102)%] and enhanced the cell proportions in S and G2/M phases [(30.547+/-9.861)% and (17.668+/-9.509)%, all P<0.01]. The ratio of membrane to cytoplasm fraction of PKC alpha declined (0.63+/-0.10 vs. 1.00+/-0.09) and the fluorescence of NF-KappaB in nucleus enhanced (22.98+/-2.20 vs. 14.54+/-2.35) in hyperoxia compared with that in air, while both the ratio of PKC alpha and intensity of NF-KappaB were increased in O(2)/CGRP group (1.41+/-0.23, 35.38+/-3.37) compared with those in hyperoxia (0.63+/-0.10, 22.98+/-2.20) and O(2)/CGRP8-37 groups (0.74+/-0.10, 24.88+/-1.81, all P<0.01). CONCLUSION: CGRP could promote proliferation of AECII when exposed to high oxygen tension. PKC alpha participates in the signal transduction process and NF-KappaB is a downstream molecular of PKC alpha, executing in part the function of PKC alpha signal.

[Expression and role of p38 mitogen-activated protein kinase in hyperoxia-induced lung injury juvenile rat model].

OBJECTIVE: Some research has shown that p38 mitogen-activated protein kinase (p38MAPK) plays important roles in lung injuries induced by various factors. Its expression and role in hyperoxia-induced lung injury remains unknown. This study investigated the expression and role of p38MAPK in hyperoxia-induced lung injury juvenile rat model. METHODS: Hyperoxia-induced lung injury rat model was prepared by 90% O(2) exposure. The location and expression of p38MAPK in lung tissues were detected by immunohistochemistry and Western blot respectively. Apoptosis index of lung was evaluated by TUNEL technique. The effect of SB203580, a p38MAPK inhibitor, on the apoptosis index of lung was observed. RESULTS: The expression of phosphor-p38MAPK increased obviously after hyperoxia. Positive phosphor-p38MAPK cells were mainly distributed in the alveolar, airway epithelial cells, pulmonary vascular endothelium cells and infiltrative inflammatory cells. The apoptosis index of lung also significantly elevated. SB203580 inhibited the activation of p38MAPK, and reduced the apoptosis index of lung. CONCLUSIONS: The phosphor-p38MAPK increased and was expressed in many kinds of lung cells in lung injury rat model. It may play a role in the induction of apoptosis in hyperoxia-induced lung injury.

The epidemiology of acute respiratory distress syndrome in pediatric intensive care units in China.

OBJECTIVE: To assess the incidence of, predisposing factors for, and the rates and relative risks of mortality from acute respiratory distress syndrome (ARDS) in pediatric patients. DESIGN: A prospective study in 12 consecutive months from 2004 to 2005 in 25 pediatric intensive care units (PICUs). PATIENTS AND SETTING: ARDS was diagnosed according to the 1994 American-European Consensus Conference definitions, applied to all severely ill admissions between 1 month and 14 years of age. The PICUs were in major municipalities and provincial cities, and half were university affiliated. MEASUREMENTS AND RESULTS: From a total of 12,018 admissions, 7,269 were severely ill. One hundred and five (1.44%) patients developed ARDS and 64 (61.0%) died, which accounts for 13.2%, of the total ICU death (n = 485, 6.7%) or a nine times relative risk of dying. The median age at onset of ARDS was 24 months and 40% were less than 12 month old. Median time from PICU admission to the onset of ARDS was 16 h, and in 63% <24 h. Pneumonia (55.2%) and sepsis (22.9%) were the major predisposing factors for ARDS. These were respectively 14 and 5 times as high a death rate as those of the severely ill patients without ARDS. CONCLUSIONS: ARDS has a high mortality in these Chinese PICUs, especially in those with pneumonia and sepsis, and adequate management including lung protective ventilation strategy is required.

[Influence of 60% oxygen inhalation on type II alveolar epithelial cells and protective role of calcitonin gene-related peptide from their damage induced in premature rat].

OBJECTIVE: To study in vitro the influence of 60% oxygen and the protective effect of calcitonin gene-related peptide (CGRP) on type II alveolar epithelial cells (AEC II) isolated from the lung of premature rat. METHODS: AEC II were isolated from the lung of 19-day rat fetus, and they were then cultured in six-well plates. The cells were randomly divided into four groups: air group, hyperoxia group, hyperoxia plus CGRP group, hyperoxia plus CGRP and CGRP8-37 (CGRP receptor antagonist) group. Cells of air group and hyperoxia group were exposed to 21% air or 60% oxygen, respectively, while in hyperoxia plus CGRP group CGRP was added, and in hyperoxia plus CGRP and CGRP8-37 group CGRP and CGRP8-37 were added before exposure to 60% oxygen. Cells in four groups were cultured for 24 hours, and then ground into homogenates for detection of malondialdehyde (MDA), total antioxidant capacity (TAOC) and superoxide dismutase (SOD) with ultraviolet spectrophotometer. Reactive oxygen species (ROS) and apoptosis rate of AEC II were analyzed by flow cytometry and the mRNA level of surfactant associated protein C (SPC) was measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: The levels of ROS, MDA and apoptosis rate were increased whereas TAOC, SOD and SPC mRNA expression declined in hyperoxia group compared with those in air group (all P<0.01). In contrast, MDA, ROS and apoptosis rate were significantly lower and levels of TAOC, SOD and SPC mRNA expression were significantly higher in hyperoxia plus CGRP group than those in hyperoxia group (all P<0.01). The differences in 6 parameters above between hyperoxia group and hyperoxia plus CGRP and CGRP8-37 group were not statistically significant. CONCLUSION: Exposure of AEC II from immature rat to 60% oxygen for 24 hours may produce oxidative injury, inducing apoptosis and decrease in SPC mRNA level of AEC II of premature rat in vitro, while CGRP may play a protective role against hyperoxic lung injury by its antioxidant property, and also inhibition of AEC II apoptosis and promotion of the SPC mRNA expression.

[Changes of aquaporin expression during lung development in rats].

OBJECTIVE: Many studies have shown that tissue development is closely correlated with fluid transport. Aquaporins (AQPs) are a group of cell membrane proteins that actively and selectively transport water. This study aimed to investigate the changes of AQPs expression during lung development in rats in order to elucidate the role of AQPs in the rat lung development. METHODS: AQP1, AQP3, AQP4 and AQP5 proteins and mRNA in the lung cell membrane were measured by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) respectively in the 20-day-old embryo (E20), 7-day-old newborn rat, and one-month-old young and adult rats. The correlation between AQPs expression and lung development was studied. RESULTS: With increasing age, the lung development showed a dynamic and successive course, with the most rapid from the fetus to the newborn rat, and then a slowed down afterwards. AQPs mRNA was weakly expressed in the lung of the E20 group. Lung AQPs mRNA and protein increased rapidly after birth until adulthood. The AQPs distribution patterns in the lung were unique with no duplication. There was a positive correlation between AQPs expression and lung development (P<0.05). CONCLUSIONS: In addition to being involved in the transepithelial transport of water in the lung, AQPs is also related to its development.

[Oxidative stress induced the expression of Bax and p53 during the apoptosis of alveolar type II epithelial cell].

OBJECTIVE: To investigate the effects of oxidative stress on the survival and apoptosis of alveolar epithelial type II (ATII) cells, as well as the mechanisms of apoptosis. METHODS: 500 mumol/L H(2)O(2) was added into primary ATII cells at different times and cell viability, apoptotic ratio and the expression of Bax and p53 were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry (FCM) and Western blotting analysis, respectively. The change in mitochondrial membrane potential (MMP) was detected by fluorescence microscopy and FCM. RESULTS: The cell viability and MMP were decreased by H(2)O(2) compared with the controls (F(1)=85.211, F(2)=72.453, respectively, both P<0.05). The cell apoptotic ratios were increased with the time of the stimulation prolonged compared with the controls (F=54.002, P<0.05). H(2)O(2) increased Bax and p53 protein levels (F(1)=28.118, F(2)=43.456, both P<0.05). CONCLUSION: High level of oxidative stress can inhibit ATII cells proliferation, and induce cells apoptosis and decrease the MMP. Up-regulation of the expression of Bax and p53 may contribute to its apoptosis effects.

[Apoptosis of alveolar type II epithelial cells regulated by extracellular signal-regulated kinase under oxidative stress].

OBJECTIVE: To investigate survival and apoptotic responses of alveolar type II epithelial cells (AT II cells) under oxidative stress and the regulation mechanism mediated by extracellular signal-regulated kinase (ERK). METHODS: Primary passage of cultured rat AT II cells were challenged with hydrogen peroxide (H(2)O(2)), and the cells were pretreated with specific inhibitor of ERK (PD 98059) in another group. Cell viability, apoptotic rate and the expression of phosphorylated ERK1/2 (p-ERK1/2)were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and Western blotting analysis, respectively. RESULTS: Compared with control group, decreased cell viability and increased apoptotic rate in AT II cells occurred in dose-dependent manner when treated with H(2)O(2) 500 and 1,000 micromol/L (all P<0.05), but no differences were found when H(2)O(2) were 10 and 100 micromol/L in concentrations. When the cells were treated with 500 micromol/L H(2)O(2) for 30 minutes, no differences in cell viability and apoptotic rate were found compared with control group,but reduced cell viability and increased apoptotic rate were found when the duration was 180 minutes, and it was in time-dependent manner (both P<0.05). The expression of p-ERK peaked at 30 minutes after stimulation by 500 micromol/L H(2)O(2). When PD 98059 was added, it enhanced apoptotic rate after H(2)O(2)-exposure. CONCLUSION: Apoptosis can be induced by H(2)O(2) in AT II cells in dose-and time-dependent manners. ERK signaling pathway plays a role in the regulation of apoptosis and may be protective for AT II cells under oxidative stress.

[Expression and modulation of aquaporin 5 in hyperoxia induced lung injury].

OBJECTIVE: To explore the expression and the modulation of aquaporin 5(AQP5) in hyperoxia induced lung injury. METHODS: Sixty four Wistar rats of 2 weeks old, were randomly assigned to following groups (n=8): air group, hyperoxia 3, 7, 14 days groups, air + dexamethasone (Dex), hyperoxia 3, 7, 14 days + Dex groups. The rats were kept in oxygen chamber at normal pressure (O(2)> or =95%) in hyperoxia groups, and in normal pressure air (O(2)=21%) in room-air group, and the rats in Dex groups were injected with Dex (5 mg.kg(-1).d(-1)) intraperitoneally for 3 consecutive days in room-air or hyperoxia exposure. The expression of AQP5 mRNA level and the location were detected by reverse transcription-polymerase chain reaction and immunohistochemistry respectively. RESULTS: AQP5 was strongly labeled in alveolar epithelial type I cells, and was also expressed in the secretory epithelium plasma membrane in the airway. The location of AQP5 in hyperoxia groups was not changed, but the expression of AQP5 mRNA had a notable gradual decline when the time of hyperoxia exposure was prolonged, compared to control group (all P<0.05). There was no difference in AQP5 mRNA level between hyperoxia groups and hyperoxia + Dex groups at different time points (all P<0.05). CONCLUSION: The significant decrease in AQP5 may be an important factor of pulmonary edema formation in hyperoxia-induced lung injury. Dex does not have effect on modulating the AQP5 expression in acute lung injury.

[Study on the relationship between cytosolic phospholipase A2-gamma activation and myocardial cell injury during cardiopulmonary bypass].

OBJECTIVE: To investigate the relationship between cytosolic phospholipase A(2)-gamma(cPLA(2)-gamma) activation and alteration of myocardial ultrastructure during cardiopulmonary bypass (CPB) in the operation of ventricular septal defect. METHODS: Myocardial tissues from the right atria of 12 patients undergone ventricular septal defect were collected before and after CPB, cPLA(2)-gamma gene expression was detected by reverse transcription-polymerase chain reaction (RT-PCR) and analysis was carried out using gel image analysis software. Meanwhile, alteration of myocardial ultrastructure was observed under electron microscopy. RESULTS: cPLA(2)-gamma gene expressions were statistically significant between two groups (P<0.05), and they were higher in patients after CPB than those prior to CPB. The alteration of myocardial ultrastructure after CPB included hypertrophy of nucleus, swelling under plasma membrane, chromatin margination and dilatation of smooth endoplasmic reticulum (SER) in myocardial cell, structures degeneration, hyperplasia of mitochondria as well as swelling, and myocardial fibre dissolve, etc. CONCLUSION: Increased cPLA(2)-gamma gene expression in myocardial tissue after CPB might play an important role in damage of membrane integrity, energy decompensation and myocardial contraction dysfunction.

[Clinical study on respiratory mechanics of synchronized intermittent mandatory ventilation in premature infants with respiratory failure].

OBJECTIVE: To evaluate the clinical significance of synchronized intermittent mandatory ventilation (SIMV) in prematures infants necessitating assistant ventilation. METHODS: Forty-two premature infants were studied in whom SIMV was used in 24 and intermittent mandatory ventilation (IMV) was used in 18. The parameters of fractional concentration of inspired oxygen (FiO(2)), peak inspiratory pressure (PIP), expiratory tidal volume (V(Texp)), oxygen index (OI), compliance of the respiratory system (Crs) and airway resistance (Raw) were compared. The frequency of administration of sedatives and incidence rate of complications were also compared between two groups. RESULTS: In SIMV group, PIP, V(Texp), Raw and Crs were significant better after 2 hours ventilation (all P<0.05). There were significant difference in FiO(2) after 12 hours and OI at 24 hours in two groups. They were better in SIMV group. Sedatives were less used in SIMV group than IMV group (P<0.05). CONCLUSION: In premature infants, SIMV could reduce FiO(2), PIP, OI, Raw and improve V(Texp), Crs more rapidly when assistant ventilation is necessary. The sedatives are used less in patients on SIMV.

[Study on effect of dexamethasone on the expression of matrix metalloproteinase and its tissue inhibitors in hyperoxia-induced lung injury].

OBJECTIVE: To observe the effect of dexamethasone on the mRNA expression of matrix metalloproteinase (MMPs) and tissue inhibitor of metalloproteinase (TIMPs) in the lung tissue and to explore the protective mechanism of dexamethasone in hyperoxia-induced lung injury. METHODS: Thirty-two two-week old Wistar rats were randomly divided into atmospheric-air group (n=16) and hyperoxia group (n=16). After 7 days of continuous exposure to high concentration O (2)(>95%), the lung wet/dry(W/D) ratio, the protein content in bronchoalveolar lavage fluid(BALF) and histopathological changes of the lung were measured in 16 rats(8 in each group). The lung tissue specimens of the other 16 rats were cultured, 8 among which served as atmospheric-air control group, the remainder in the hyperoxia group were divided into hyperoxia control group,hyperoxia+dexamethasone (1 x 10(-8) mol/L) group, hyperoxia+dexamethasone (1 x 10(-6) mol/L) group, and hyperoxia+dexamethasone (1 x 10(-4) mol/L) group. Eight samples were examined in each group. After cultured for 24 hours, the lung tissue were collected and its mRNA expression of MMP-2, MMP-9, TIMP-1 and TIMP-2 were determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: 1. Pulmonary edema, hemorrhage and extensive inflammatory cells infiltration were observed in hyperoxia group, but no such changes were found in the atmospheric-air group. The lung W/D and the protein content in BALF in hyperoxia group were significantly higher than those in atmospheric air groups. 2. The mRNA expressions of MMP-2, MMP-9, TIMP-1, TIMP-2 and the ratio of MMP-2/TIMP-2, MMP-9/TIMP-1 were significantly higher in the hyperoxic group than those in the atmospheric-air group. 3. Dexamethasone could down-regulate the mRNA expressions of MMP-2 and MMP-9 in a concentration dependent manner. The mRNA expressions of TIMP-1, TIMP-2 also could be reduced by dexamethasone. Decreasing ratios of MMP-2/TIMP-2 and MMP-9/TIMP-1 were found in correlation with increasing concentration of dexamethasone. CONCLUSION: Dexamethasone can reduce the mRNA expressions of MMPs as well as regulate the balance of MMPs/TIMPs, which may be one of the mechanism of its protective effect on hyperoxia-induced lung injury.

[Evaluation of antibacterial activity of amoxycillin sodium and clavulanate potassium and the pharmacoeconomics in the therapy of acute respiratory infection].

OBJECTIVE: To explore the antibacterial activity of amoxycillin sodium and clavulanate potassium (trade name: Anqi) in vitro and the pharmacoeconomics in the therapy of acute respiratory infection. METHODS: Minimal inhibition concentration (MIC), minimal bactericidal concentration (MBC) and bactericidal curve of amoxycillin sodium and clavulanate potassium against common pathogens were determined and compared with some other same kind of antibiotics without beta-Lactamase inhibitor. Eighty cases diagnosed as respiratory infection were randomly divided into 4 groups: group 1 was treated with i.v. Anqi; group 2 was treated with i.v. Anqi and oral consecutive strategy; group 3 was treated with iv ampicillin and sulbactam; group 4 was treated with i.v. cefuroxime. The clinical therapeutic effects were observed and cost-effectiveness analyzed. RESULTS: In terms of MIC, MBC and bactericidal curve of 135 bacterial strains, Anqi was superior to the other same-kind antibiotics without beta-lactamase inhibitor, this effect was especially obvious on Klebsiella pneumoniae and Escherichia coli which can produce extended spectrum beta-lactamases (ESBLs). The cost-effectiveness of the consecutive therapy group was the best. CONCLUSION: Anqi has a wide antimicrobial spectrum and strong effect on the bacteria producing ESBLs, the consecutive therapy strategy should be clinically recommended.