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.

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

Abstract 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.

Vitamin D/VDR signaling attenuates lipopolysaccharide­induced acute lung injury by maintaining the integrity of the pulmonary epithelial barrier.

Vitamin D and its receptor have a protective effect on epithelial barriers in various tissues. Low levels of vitamin D are associated with numerous pulmonary diseases, including acute lung injury (ALI) and acute respiratory distress syndrome. The present study investigated whether the vitamin D/vitamin D receptor (VDR) pathway may ameliorate lipopolysaccharide (LPS)­induced ALI through maintaining the integrity of the alveolar epithelial barrier. This was investigated by exposing wild­type (WT) and VDR knockout C57BL/6J mice to LPS, then comparing the healthy and LPS­treated mice lungs and bronchoalveolar lavage fluid (BALF). More specifically, lung histology, mRNA levels of proinflammatory cytokines and chemokines, and protein expression levels of tight junction proteins were determined. In addition, a vitamin D analog (paricalcitol) was administered to WT mice in order to investigate the effect of vitamin D on the alveolar epithelial barrier following exposure to LPS. VDR knockout mice exhibited severe lung injuries (P<0.001), increased alveolar permeability [demonstrated by a higher wet­dry ratio of lung weight (P<0.05), greater expression levels of BALF protein (P<0.001) and fluorescein isothiocyanate­conjugated 4 kDa dextran (P<0.001) leakage into the alveolar space], elevated proinflammatory cytokine and chemokine mRNA levels, as demonstrated by reverse transcription­quantitative polymerase chain reaction (P<0.05), and decreased protein and mRNA expression levels of occludin (P<0.01) and zonula occludens­1 (ZO­1; P<0.01) compared with WT mice. Paricalcitol treatment partially inhibited these pathological changes in WT mice by maintaining the mRNA and protein expression levels of occludin (P<0.01) and ZO­1 (P<0.05). A lack of VDRs in the pulmonary epithelial barrier appeared to compromise its defense, leading to more severe LPS­induced lung injury. Furthermore, vitamin D treatment alleviated LPS­induced lung injury and preserved alveolar barrier function. Therefore vitamin D treatment may present as a potential therapeutic strategy in ALI and acute respiratory distress syndrome.

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.

Association between ANKK1 (rs1800497) polymorphism of DRD2 gene and attention deficit hyperactivity disorder: a meta-analysis.

The role of dopamine neurotransmitter in attention deficit hyperactivity disorder (ADHD) remains controversial. Many molecular studies focusing on dopamine receptors have attempted to analyze the gene polymorphisms involved in dopaminergic transmission. Of these, rs1800497 (TaqIA) single nucleotide polymorphism (SNP) of the dopamine D2 receptor (DRD2) gene has been focused on by the most attention. However, this locus has recently been identified within the exon 8 of ankyrin repeat and kinase domain containing 1 (ANKK1), giving rise to a Glu713-to-Lys substitution in the putative ANKK1 protein. Thus, we performed a meta-analysis to determine whether ANKK1 polymorphism influences the risk of ADHD and examined the relationship between rs1800497 genetic variant and the etiology of ADHD. Relevant case-control studies were retrieved by database searches and selected according to established inclusion criteria. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the associations. Meta-regression, subgroup analysis, sensitivity analysis and cumulative meta-analysis were performed. A total of 11 studies with 1645 cases and 1641 controls were included. In the dominant model, the rs1800497 locus was associated with ADHD, with a pooled OR of 1.785 (95% CI=1.068-2.984, p=0.027). Subgroup analysis for ethnicity indicated that the polymorphism was associated with ADHD in Africans (OR=3.286, 95% CI=1.434-7.527, p=0.005), but not in East Asians (OR=1.513, 95% CI=0.817-2.805, p=0.188) and Caucasians (OR=1.740, 95% CI=0.928-3.263, p=0.084). However, the results of meta-regression indicated that publication date (p=0.601), source of controls (p=0.685), ethnicity (p=0.755) and diagnostic criteria (p=0.104) could not explain the potential sources of heterogeneity. This meta-analysis indicates that the rs1800497 locus may be associated with ADHD. These data provide possible references for future case-control studies in childhood disorders.

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 and function of aquaporin-1 in hyperoxia-exposed alveolar epithelial type II cells.

The aim of the present study was to investigate water transport dysfunction in alveolar epithelial type II cells (AECII), which were exposed to hyperoxia, and to investigate the mechanism of pulmonary edema resulting from hyperoxic lung injury. The lung cells of newborn rats were isolated for primary cell culture and divided into control and experimental groups. The control and experimental group cells were placed into a normoxic incubator (oxygen volume fraction, 0.21) or hyperoxic incubator (oxygen volume fraction, 0.9), respectively. Twenty-four, 48 and 72 h after cell attachment, the gene transcription and protein expression levels of aquaporin-1 (AQP1) were detected via quantitative polymerase chain reaction and western blot analysis. Flow cytometry was conducted to detect the volume of the cells in the experimental and control groups. In the present study, it was identified that AQP1 expression and cell volume were greater in the experimental group when compared with the control group. Thus, hyperoxia may disturb the gene expression regulation of AQP1 in AECII, resulting in water transport dysfunction. This may be one of the mechanisms underlying pulmonary edema caused by hyperoxic lung injury.

The mRNA expression of mitogen-activated protein kinase signal pathway related genes in the blood of arseniasis patients caused by burning coal / 中华预防医学杂志

<p><b>OBJECTIVE</b>To detect the mRNA expression of ERK1, ERK2, JNK1 and P38 gene in mitogen-activated protein kinase(MAPK) path way in the arseniasis patients caused by burning coal.</p><p><b>METHODS</b>70 arseniasis patients caused by burning coal at Jiaole village XingRen county in December 2006 were selected as case group, and another 30 villagers with similar living habits, matched gender and age, healthy physical condition without history of burning high arsenic coal were selected as control group from 12 km nearby the same village.Silver diethyl dithiocarbamate method (Ag-DDC) was taken to detect the arsenic contents in the environmental media, food, and arsenic level in the urine and hair of arseniasis patients.On the principle of informed consent, the peripheral blood was collected from the patients. The total RNA was extracted with Trizol method and cDNA was reversed from it. The mRNA expression of ERK1, ERK2, JNK1 and P38 gene in MAPK path way were tested by real-time fluorescent quantitative PCR (QT-PCR).</p><p><b>RESULTS</b>A total of 70 cases of arseniasis patients (31 cases of mild, 25 cases of moderate and 14 cases of severe) and 30 cases of control were chosen. The median (quartile) of arsenic contents in the indoor air, outdoor air, coal, chili and corn were 0.079 (0.053-0.117) mg/m(3) ,0.007 (0.002-0.015) mg/m(3) , 93.010 (39.460-211.740) mg/kg, 3.460(0.550-16.760) mg/kg and 1.500(0.300-4.140) mg/kg respectively. They were above the national health standards. The median (quartile) of arsenic contents in the soil, rice and drinking water were separately 12.130(4.230-24.820) mg/kg, 0.650(0.300-0.980) mg/kg and 0.043(0.012-0.089)mg/kg, which were within the national health standards. Compared with the control group ((26.97 ± 9.71)µg/g Cr), arsenic level in the patients' urine ((71.48 ± 22.74)µg/g Cr) increased significantly, the differences were significant (F = 90.38, P < 0.01). Compared with the control group ((1.58 ± 1.07)µg/g), arsenic level in the patients' hair ((4.45 ± 2.78) µg/g) increased significantly, the differences were significant (F = 48.22, P < 0.01). The relative expression amount of the median(quartile) for ERK2, JNK1 mRNA were 0.0667 (0.0378-0.1371) and 0.0013 (0.0009-0.0025), respectively. Compared with the control group 0.1744 (0.1009-0.1985) and 0.0022 (0.0017-0.0030) , only the decreases of ERK2, JNK1 mRNA expression was significant (χ(2) = 15.10, 14.25, P < 0.01), and no significance in the other index. ERK2 mRNA relative expression for mild, medium and severe groups were separately 0.0818 (0.0408-0.1509) ,0.0582 (0.0154-0.1699) and 0.0588 (0.0399-0.1034) . Compared with the control group (0.1744 (0.1099-0.1985) ), there was significant difference (Z = -2.89, -3.19, -2.67, P < 0.01). JNK1 mRNA relative expression were 0.0012 (0.0007-0.001 57), 0.0019 (0.0011-0.0035), 0.0013 (0.0010-0.0026), respectively. Compared with the control group (0.0022 (0.0017-0.0030) ), significances were found in the mild groups (Z = -3.72, P < 0.01).</p><p><b>CONCLUSIONS</b>Arsenic could induce the changes of ERK2 and JNK1mRNA expression in the MAPK path way in arseniasis patients.It suggests that the MAPK signaling pathway take part in the occurrence and development process of arseniasis caused by burning coal.</p>

Relationship of DNA methylation, mRNA transcription and protein expression of glutathione-S-transferases-P1 gene and coal-pollution-borne endemic arsenism / 中国地方病学杂志

Objective To investigate DNA methylation in the promoter region,mRNA transcription and protein expression of glutathione-S-transferases-P1 (GSTP1) gene and their relation with arsenism.Methods In endemic coal-pollution-borne arsenism area,Jiaole village of Xinren county,Guizhou province,according to the diagnostic criteria of endemic arsenism(WS/T 211-2001),123 cases with endemic arsenism were selected and divided into three groups (mild arsenism group:42 cases,moderate arsenism group:41 cases and severe arsenism group:40 cases).Forty seven residents were selected as controls in a village about 12 km away from the endemic arsenism area.With the informed consent principle,peripheral blood of all respondents was collected in order to analyze DNA methylation and check mRNA.DNA methylation of GSTP1 gene promoter region in peripheral blood was assayed by PCR,and GSTP1 mRNA expression was assayed using real-time quantitative PCR.In addition,other cutaneous specimens originated from 53 cases with arsenism that accepted surgical treatment voluntarily were taken.Of these specimens,general pathological changes were 28 cases,precancerous 20 cases and cancerous 5 cases.Skin tissues of 15 cases of non-tumor surgery patients without abnormal pathological changes were as control group.GSTP1 protein expression in the skin tissue was detected using immunohistochemistry (IHC).Results Among different groups of arsenic poisoning,the positive rate of DNA methylation of GSTP1 gene was 28.57％(12/42) in the mild group,57.10％ (23/41) in the moderate group and 65.00％ (26/40) in the severe group.Compared with the control group (6.38％,3/47),the difference was statistically significant (x2 =7.792,26.000,33.412,all P ＜ 0.01).Among different groups of arsenic poisoning diagnosed by dermapathology,the positive rate of DNA methylation of GSTP1 gene was 21.43％(6/28) in the general pathological change group,50.00％(10/20) in the precancerous group and 80.00％(4/5) in the cancerous group.Compared with the control group(6.67％,1/15),the difference was statistically significant (x2 =3.562,7.468,10.756,all P ＜ 0.05).It showed that the positive rate of DNA methylation of GSTP1 gene increased with aggravation of the disease and dermatic lesion of arsenism (tendency x2 =38.239,x2 =13.659,all P ＜ 0.01).Compared with the control group(0.184 26),the expressions of GSTP1 mRNA in peripheral blood in moderate (0.087 77) and severe arsenic poisoning groups (0.056 93) were significantly reduced(all P ＜0.01),and that of severe group was significantly lower than that of the moderate group (P ＜ 0.01) ; compared with the control group(0.338 45) and the general lesion group(0.276 74),GSTP1 mRNA expression was significantly reduced in precancerous lesion group(0.104 81) and cancerous group(0.043 70),in which the cancerous group was significantly lower than that of the precancerous lesions.The difference of skin tissue GSTP1 protein expression rate between groups was statistically significant (x2 =20.948,P ＜ 0.05),in which the difference between the precancerous lesion group(65.00％,13/20),the cancer group (40.00％,2/5) and the control group(100.00％,15/15)was statistically significant (x2 =12.183,11.778,P ＜ 0.01).Spearman correlation analysis showed that the degree of skin lesion and the level of GSTP1 protein expression was negatively correlated (r =-0.520,P ＜ 0.05).Groups were divided according to DNA methylation of GSTP1 gene,and the mRNA and protein expression of GSTP1 in methylation group(0.038 40,57.14％) was significantly lower compared with that of unmethylated group(0.187 07,95.74％; Z =9.032,x2 =23.134,all P ＜ 0.01).Conclusions Arsenism may lead to DNA methylation of human GSTP1 gene promoter region,thereby inhibiting expression of mRNA and protein.GSTP1 gene plays an important role in arsenism or carcinogenic process.

Relationship between glutathione S-transferase GSTO 1 Glu155 △Glu genetic polymorphism and arsenic poisoning caused by coal-burning / 中国地方病学杂志

ObjectiveTo investigate glutathione S-transferase GSTO 1 Glu155△Glu genetic polymorphism and risks of arsenic poisoning caused by coal-burning in Guizhou.Methods GSTO1 Glu155 △Glu gene polymorphism was analyzed by polymerase chain reaction-with confronting two-pair primers among one hundred and thirty arsenic poisoning patients and one hundred and thirty healthy controls.The results were verified by DNA sequencing.The association between different genotypes and arsenic poisoning was analyzed by unconditional Logistic regression model.ResultsThe results of Glu/Glu and Glu/△Glu genotype detected by this method were consistent with those of DNA sequencing.The frequencies of GSTO1 Glu/Glu genotype and Glu/△Glu genotype were 94.85％(92/97) and 5.15％(5/97) in the patients,99.15％(117/118) and 0.85％(1/118) in the controls,respectively.The difference was statistically significant(x2 =3.896,P ＜ 0.05).△Glu/△Glu genotype was not found in both patients and controls.After age and sex adjusting,GSTO1 Glu155 △Glu polymorphism was found to be a risk factor of arsenic poisoning [odds ratio (OR) =1.85,95％ confidence interval (CI):1.39 - 17.48].ConclusionsThe study finds that GSTO1 Glu 155 △ Glu polymorphism is associated with risk of arsenic poisoning.The relationship between them should be further studied through increasing sample size.

[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.

[Early prediction of the injured regions in neonatal brain with hypoxic-ischemic encephalopathy by diffusion weighted imaging and measuring their apparent diffusion coefficient].

OBJECTIVE: To elucidate that diffusion weighted imaging (DWI) can be used to predict the injured regions of neonatal brain with hypoxic-ischemic encephalopathy (HIE) in the early phase of injury, and to measure the apparent diffusion coefficient (ADC) values in the multiple regions of the brain. METHOD: The participants in this study were twenty-six infants with HIE from neonatology ward hospitalized between July 2006 and July 2009. Nineteen patients had severe HIE, and seven had moderate HIE. DWI and conventional magnetic resonance imaging (MRI) were performed for each case within the first 72 hrs. The ADC values of eight regions of interest (ROIs) were measured in ten cases with severe HIE (ADC values group). ROIs included posterior limb of internal capsule (PLIC), ventrolateral thalami, basal ganglia, perirolandic cortex, occipital cortex, centrum semiovale, brainstem, and frontal white matter. Twelve neonates were enrolled as the control subjects. RESULTS: During the first 72 hrs, the conventional MRI of 26 patients showed subarachnoid hemorrhage in 5, subdural hemorrhage in 2, and mild high signal intensity in the cortex of only one patient. In the 19 cases with severe HIE, abnormal signal intensities were seen in ventrolateral thalami and perirolandic cortex of 17 patients (89%), and the remaining 2 infants showed abnormal cortex and subcortical white matter. In 7 cases with moderate HIE, 4 had abnormal signal intensity in the cortex and subcortical white matter, 2 had abnormal periventricular white matter, and only one showed abnormal signal intensity in the ventrolateral thalami and perirolandic cortex. In the ADC values group, the average ADC values of posterior limb of internal capsule (PLIC), ventrolateral thalami, basal ganglia, perirolandic cortex, occipital cortex, centrum semiovale, brainstem, and frontal white matter respectively were 0.68 (0.56 - 0.88), 0.73 ± 0.13, 0.67 ± 0.11, 0.78 ± 0.22, 0.90 ± 0.16, 0.87 ± 0.21, 0.73 ± 0.19, 1.32 ± 0.22 × 10(-3) mm(2)/S. In the control group, the average ADC values of posterior limb of internal capsule (PLIC), ventrolateral thalami, basal ganglia, perirolandic cortex, occipital cortex, centrum semiovale, brainstem, and frontal white matter respectively were 0.96 (0.95 - 1.02), 1.02 ± 0.90, 1.15 ± 0.99, 1.08 ± 0.07, 1.09 ± 0.08, 1.39 ± 0.20, 0.96 ± 0.05, 1.58 ± 0.18× 10(-3) mm(2)/S. There was statistically significant difference in the average ADC values between each of 8 ROIs of infants with HIE and healthy neonates (P < 0.01). CONCLUSION: In the first days after birth, the major injured regions of severe HIE were ventrolateral thalami and perirolandic cortex, the minor injured regions were cortex and subcortical white matter. Multiple regions of moderate HIE were injured, including cortex with subcortical white matter, periventricular white matter, and ventrolateral thalami with perirolandic cortex. The ADC values of the regions with abnormal signal intensity decreased, also some regions with the normal signal intensity.

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

OBJECTIVE: 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. METHODS: 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. RESULTS: 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]. CONCLUSIONS: 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.

[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.

[Anti-inflammatory effects of erythropoietin on hyperoxia-induced bronchopulmonary dysplasia in newborn rats].

OBJECTIVE: Bronchopulmonary dysplasia (BPD) is a multifactorial disease resulting from the impact of injury (including oxygen toxicity, barotrauma, volutrauma, and infection) on the immature lung. Oxygen toxicity is thought to be a major contributing factor in the pathogenesis in BPD. Previous animal studies have shown that exposure to hyperoxia in the neonatal period causes lung structural changes that are similar to the histology seen in human infants with BPD. Erythropoietin (EPO) has pleiotropic actions including antioxidant, anti-apoptotic, anti-inflammatory and angiogenic effects. Animal experiments reveal that EPO may have protective effects on hyperoxic lung injury, but the mechanisms remain unknown. The aim of the study was to evaluate the anti-inflammatory effects and understand mechanism of action of EPO on the hyperoxia-induced BPD in newborn rats. METHOD: Several litters of Wistar pups were pooled together within 12 hours after birth and randomly divided into four groups: I. air-exposed control group, II. air-exposed human recombinant erythropoietin (rhEPO)-treated group, III. hyperoxia-exposed placebo group and IV. hyperoxia-exposed rhEPO-treated group . Group III and IV rats were exposed to 85% oxygen. Group II and IV rats received rhEPO (1200 IU/kg) subcutaneously on postnatal days 0 and 2. Group I and III received 0.9% saline in the same way. Pups from each group were sacrificed on days 3, 7, and 14. Blood hemoglobin concentration, hematocrit and platelet count were determined by blood cell analyzer. Total protein content in bronchoalveolar lavage fluid (BALF) and myeloperoxidase (MPO) were measured by biochemical assay. Changes of monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) mRNA expressions were measured by RT-PCR. RESULT: In group III, there were a few inflammatory cells infiltrations in interstitium on day 3 and inflammatory response worsened on day 7. Alveolar and capillary hypoplasia and interstitial fibrosis were evident on day 14. The pathological changes were ameliorated greatly in group IV and the survival was prolonged. There were no abnormal raises of hemoglobin concentration, hematocrit and platelet count in group IV compared with group I. Total protein concentration in BALF was measured as a marker for capillary leakage. MPO is a major constituent of neutrophil cytoplasmic granules and its activity therefore is a direct measure of neutrophil presence and an indirect indicator of lung injury. Total protein concentration and MPO in BALF were greatly depressed in group IV compared with group III, P<0.05, P<0.001. The upregulation of genes of CINC-1 and MCP-1 was closely related with lung inflammation caused by oxidative stress. MCP-1 and CINC-1 mRNA expression were detected and it was found that their changes were in line with the degree of lung inflammation. MCP-1 and CINC-1 mRNA expression increased in group III compared with group I especially on day 7, P<0.01 or <0.001. The changes of MCP-1 and CINC-1 mRNA were positively correlated with changes of MPO in BALF covering all groups on days 3, 7 and 14, respectively (r = 0.391, P<0.05; r = 0.701, P<0.01; r = 0.600, P<0.01; r = 0.471, P<0.01; r = 0.789, P<0.01; r = 0.588, P<0.01). CONCLUSION: EPO could significantly reduce the lung inflammatory cell infiltration, and capillary endothelial cell injury in hyperoxic lung injury in newborn rats. The mechanism may be related with the inhibition of MCP-1 and CINC-1 gene expression by EPO.