| BackgroundsBronchopulmonary dysplasia (BPD) is a common complication of preterm birth that contributes significantly to morbidity and mortality in neonatal intensive care units. In spite of many advances in neonatal medicine in the past few decades, like the introduction of better MV strategies and the use of surfactant and antenatal steroids, the incidence of BPD has not declined. The incidence of BPD in the United States is about 10,000-15,000 new cases each year out of which the majority of those affected have a birth weight<1250g. Pulmonary and neurodevelopmental sequelae of this devastating disease extend even into adulthood. Bronchopulmonary dysplasia (BPD), which affects prematurely born infants with acute respiratory failure that receive oxygen therapy, first described by William (Bill) Northway and colleagues in 1967. BPD results from life-saving interventions, such as mechanical ventilation and oxygen supplementation used to manage preterm infants with acute respiratory failure. This damage to the developing lung is exacerbated by barotrauma and volutrauma caused by positive-pressure mechanical ventilation, the associated oxygen toxicity stunts the post-natal development of the lung and also by inflammation. The BPD is characterized by both pulmonary microvascular and alveolarization dysplasia, show the alveolar structure simplification and capillaries decreased production.Bronchopulmonary dysplasia (BPD) is the most common chronic respiratory disease affecting infants wherein the developmental program of the lung is altered secondary to preterm birth of the baby (1). Lung development progresses in five distinct stages:embryonic, pseudoglandular, canalicular, saccular, and alveolar (2,3). Human preterm babies who develop BPD are born in the late canalicular or early saccular stage of lung development. The late canalicular stage is characterized by development of the primitive alveoli and the alveolar capillary barrier, and the differentiation of type Ⅰ and type Ⅱ pneumocytes. The early saccular stage is marked by initiation of surfactant production, pulmonary vascularization, and enlargement of terminal airways. With preterm birth, this programed development is disrupted, and in the setting of inflammation [whether it is due to infection, mechanical ventilation (MV), or hyperoxia] causes impaired alveolarization leading to BPD.The exact etiology and pathogenesis of BPD is still not very clear, which is mainly on the basis of genetic susceptibility, barotrauma, volutrauma, intrauterine infection, inflammation, oxidative stress factors as the common role in the development of immature lung tissue due to the alveoli and lung microvascular developmental disorders and abnormal lung tissue injury after repair results. The study found that pulmonary microvascular developmental disorders are closely associated with the occurrence/development of BPD, regulating gene expression in lung microvascular development imbalances may interfere with the normal development of lung microvascular and slow down the alveolar process, it played a key role in the pathogenesis of BPD. Several researches have showed that in the process of lung development, the formation of pulmonary vascular can promote alveolar growth, and maintain the normal structure of the alveolar. Furthermore, pulmonary vascular net expand at the same time alveolar differentiated, thus form the effective blood barrier. Premature babies because lung has not yet developed, assisted ventilation treatment could reduce pulmonary vascular bed and alveolar stunted, lead to insufficient blood exchange appear a series of clinical symptoms, pulmonary microvascular developmental genes and proteins also become a focus of research domestic and overseas in recent years. Premature inhaled high concentration oxygen or long-term mechanical ventilation, a large number of oxygen free radicals and inflammation of the lung tissue of medium, cause inflammation cells in the lungs, eventually lead to the occurrence of BPD. Visible, oxidative stress and the inflammatory response played a crucial role in the development of BPD.Transcription factor CASZ1 also called ZNF693, is a newly discovered a transcription factor, belongs to the zinc finger gene family, a member of its protein products as long as by combined with DNA or polymerization and the important role of transcription factors, participation of value-added, apoptosis and differentiation of normal cells. Charpentier, etc found that CASZ1 mainly expressed in vascular endothelial cells, CASZ1 regulate the behavior of the endothelial cells to promote the formation of a vascular network and branch through the EGFL7/RhoA pathways, CASZ1 also can adjusted endothelial cell adhesion to promote vascular bud by RhoA, these studies have shown that CASZ1 in microvascular budding and reconstruction plays an important role, it is necessary for the formation of blood vessels and morphogenesis.Pre-B cell colony enhancing factor (PBEF) is also known as visfation, or nicotinamide phosphoribosyl transferase, it was discovered in recent years that mainly composed of mature fat cells and activation of inflammatory cells secrete, systemic inflammation factors of multiple organ all can express and related with inflammation, cell growth, angiogenesis and apoptosis, and so on the many kinds of function of proteins. There are cells in vitro experimental studies have found that inflammatory stimulation can make the alveolar epithelial cells and pulmonary arteriole PBEF endothelial cells such as a large number of tables, and reduce the expression of PBEF can reduce the cells related to damage caused by inflammation stimulation, suggesting that PBEF may involve including pulmonary microvascular damage, alveolar epithelial cell damage, increased permeability on the respiratory system disease, such as multiple pathophysiologic process.This research we use the high volume fraction of 80% oxygen to build the newborn rat model of BPD and observed the newborn rat lung tissue pathology change, microvascular density measurement, gene and protein expression of transcription factor CASZ1 and inflammatory factor PBEF genes. To explore the expression of CASZ1 and the impact on pulmonary microvascular development in lung tissue of newborn rats with BPD and explore the expression and significance of PBEF in the lungs of newborn rats BPD.Obiectives1、We use the high volume fraction of 80% oxygen to build the newborn rat model of BPD and observed the newborn rat lung tissue pathology change, microvascular density measurement, as well as CASZ1 and PBEF genes and protein expression.2、Analysis the relationship between CASZ1 protein expression levels and pulmonary microvascular density in the newborn rat lung tissue of hyperoxic group, and explore the possible mechanism of CASZ1 in the development of BPD.3、Observe the dynamic change of PBEF expression in the development of BPD, analyze its possible role in the process of BPD.Methods1、48 newborn rats were randomly divided into two groups:hyperoxic group and control group within 24 hours of postnatal age,24 rats each group. The rats in hyperoxic group were exposed to high oxygen volume fraction of 800 mL/L. The rats in control group were exposed to normal oxygen, other experiments controlling factor is the same as the experimental group.2、These newborn rats were then sacrificed at the 3、7、14 days after experiments. Following deep anesthesia with pentobarbital (60 mg/kg, intraperitoneal), open the chests and the left lungs were excised and fixed overnight in 4% paraformaldehyde at 4℃. The tissues were dehydrated, transparentized and embedded with ethanol, xylene and paraffin, respectively. Sections (4 μm) were cut from the paraffin blocks and stained with hematoxylin and eosin (H&E) for histological changes. A quantitative analysis of the radial alveolar count (RAC) and the mean septal wall thickness (ST) was carried out as previously described,they were used to evaluate the effect of hyperoxia on lung histological damage.3、Immunohistochemical method is applied to detect the distribution and expression of CD31,CASZ1 and PBEF of two groups newborn rats in lung tissue at each time point. We detect the pulmonary microvascular density by measuring the CD31 endothelial cells of the immune staining positive area relative to the total area of the lung parenchyma cell.4、Bronchoalveolar lavage fluid was collected at the 3、7、14 days after experiments, and use the ELISA method to detect the PBEF protein concentration in bronchoalveolar lavage fluid of newborn rats at each time point.5、The mRNA expression of CASZ1 and PBEF were determined by quantitation Real-Time PCR method.6、The protein expression of CASZ1 and PBEF were determined by Western blot method.7、The results are shown as the means ± SD. Statistical analysis was performed on SPSS 17.0 statistical software. One-way ANOVA test and the student’s t-test were used for data analysis. A value of P<0.05 was considered statistically significance.Results1、As time went on, the lung tissue gradually mature, the size of alveolar was uniformity, the alveolar structures of newborns was regular and the radial alveolar count increases gradually in control group. However, in the hyperoxic group, the alveoli integrated gradually, lung interval fracture, alveolar uneven size, radial alveolar count decreased significantly, the septal wall became thickness, there was a tremendous amount of inflammatory cell infiltration. On days 14,the hyperoxia group have a similar BPD pathological changes in the lung tissue structure.2、The radial alveolar count (RAC) and the mean septal wall thickness (ST) between the two groups was no difference on the 3 days; 7 days and 14 days the hyperoxic group RAC decreased significantly and ST markedly thickened compared with control group (P< 0.05)3、Immunohistochemical staining results showed that:1) CD31 is widely expressed in the cytoplasm of pulmonary microvascular endothelial cells, With the days going by, the CD31 expression level increased and the microvascular density dropped significantly at each time point compared with the control group (P<0.05);2) CASZ1 is mainly expressed in bronchial epithelial cells, alveolar epithelial cells and pulmonary vascular endothelial cells in the lung tissue. High oxygen group 3 days,7 days,14 days CASZ1 integral optical density value of positive cells were significantly lower than the corresponding control group (P< 0.01);3) PBEF expression in lung tissue in bronchial epithelial cells, alveolar epithelial cells and inflammatory cells, The PBEF expression in both hyperoxic group and the control group has a gradually rising trend. The PBEF positive cells integral optical density value in the hyperoxic group were higher than the corresponding of the control group at 3 days,7 days and 14 days. The difference was statistically significant (P< 0.05).4、At the 3 days,7 days the newborn rat lung tissue concentration PBEF protein in bronchoalveolar lavage fluid has no statistical difference between the two groups, but the PBEF protein concentration in BALF of hyperoxia group was significantly higher than that of control group at the 14 days (P< 0.01).5、Compared with the control group, CASZ1 gene and protein expression levels of the hyperoxic group were significant low (P< 0.01); The PBEF gene and protein expression level of the hyperoxic group were significant higher than the control group (P< 0.05).6、High oxygen group CASZ1 protein expression level in newborn rat lung tissue was positively correlated with pulmonary microvascular density (r= 0.519, P = 0.009< 0.01).Conclusions1、The hyperoxic group, after 14 days exposed to 80% oxygen continuously,the alveoli integrated gradually, lung interval fracture, alveolar uneven size, radial alveolar count decreased significantly, the septal wall became thickness, there was a tremendous amount of inflammatory cell infiltration. On the days 14, the rats had a similar pathological change in the lung tissue structure to BPD.2、The CASZ1 mRNA and protein expression gradually decreases with the extension of high oxygen exposure time, all time points were significantly lower than the control group. This research suggests that hyperoxia can inhibit the expression of transcription factors CASZ1, CASZ1 participated in the development of BPD.3、The CASZ1 protein expression level in lung tissue of the hyperoxic group were correlated with pulmonary microvascular density positively, this speculated that high oxygen hinder the development of pulmonary vascular through the expression of CASZ1. consequently, lead to the occurrence of BPD.4、The results showed that the expression of PBEF in experimental is significantly higher than normal control group (P< 0.05), indicating that PBEF is involved in the development of BPD, but the specific mechanisms of how PBEF take part in the BPD is unclear, we needs further research. |
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