Study On The Role Of β-catenin In Hyperoxia-induced Bronchopulmonary Dysplasia On Newborn Rats

IntroductionBronchopulmonary dysplasia(BPD) is a multifactorial disease resulting from the impact of injury(including oxygen toxicity,barotrauma,volutrauma,and infection) on the immature lung.It is one of the most common and significant medical complications associated with preterm birth.Advances in perinatal medicine have resulted in increasing numbers of very low birth weight preterm infants who are at risk of BPD, and the incidence of BPD has increased.The histopathologic changes of severe airway injury and alternating sites of overinflation and fibrosis which used to be seen in older forms of BPD have been replaced by a milder form characterized by alveolar hypoplasia and variable interstitial cellularity and/or fibroproliferation.Mechanism of BPD included immature of the lung,injury and failured repairment after the injury.And we can comprehend it simply as the injury disturbed normal lung development and caused disorder of the repair.Injury caused by oxygen treatment and mechanical ventilation,oxygen toxicity and inflammation are thought to be the most contributing factors in the pathogenesis in BPD.But during the past years,treatments to reduce the injury such as antioxidant and anti-inflammatory have made no exact therapeutic effect.Further more,unlike injury to the adult lung that is essentially growth arrested,BPD indeed occurs in a growing lung with uncompleted morphogenesis.The spotlight focusing on impaired septation as a prominent feature of BPD prompts the neonatologist to question about disorders in the underlying molecular mechanisms,which are not only necessary for resolution and lung injury repair,but also for lung morphogenesis and development.Lung fibroblasts(LF) which can secrete elastin and participate in alveolarition,also can excrete collagen protein.and participate in lung fibrosis.It seems that LF plays an important role in the pathogenesis of BPD.The Wnt pathway has been identified as one of the numerous signaling pathways critical for precise temporal and spatial control of lung morphogenesis.The centrality ofβ-catenin as a key regulatory protein in the Wnt cascade is conferred by its capacity to tightly regulate nuclear transcription.Conditional targeted deletion ofβ-catenin from the alveolar epithelium of developing mouse embryos results in complete disruption of peripheral terminal alveolar saccule formation and disturbances of pulmonary vasculogenesis.β-catenin activation has been implicated in several chronic pulmonary disorders,like idiopathic pulmonary fibrosis(IPF) et al.We hypothesized thatβ-catenin signaling is involved in the reparative remodeling response to lung injury on an immature lung and actived in LE In this study,BPD was induced by hyperoxia exposure on neonatal rats which has been widely used for more than 20 years as a model to study associated cell and molecular alterations.We studied the expression of canonicalβ-catenin pathway molecules in BPD animal model and in fibroblasts from hyperoxia-exposed neonate rats.Material and Methods1.Animal modelsSeveral litters of Wistar pups were pooled together within 12 hours after birth and randomly divided into two groups:group A is the hyperoxia-exposed group and group B is the air-exposed group.Rats in group A were placed in an oxygen chamber into which oxygen was continuously delivered(FiO2=0.85+0.02)(OM-25ME oxygen monitor,USA).CO2 were kept below 0.5%(Dapex Gas monitor,USA).Temperature and humidity were maintained at 22～25℃and 60～70%,respectively.The chamber was opened for＜15min daily to switch dams between air and O2 environment to avoid the dams from oxygen toxicity.Except the FiO2,other details of the method and experimental control factors were similar in these two groups.2.Preparation of Lung SamplesPups from each group were killed on days 3,7,and 14,and a tracheal cannula was placed.An abdominal incision was made,the diaphragm was punctured carefully to collapse the lungs.Left lung was inflation-fixed via tracheal cannula using 4% paraformaldehyde for morphology observation and immunohistochemistry study.In some cases,after midline thoracotomy,blood was collected from right ventricle for blood cell count,then the pulmonary artery was cannulated and the left atrial appendage was clipped and the lungs were gently perfused with 10 ml of 0.9%saline to remove blood.Then put lungs in RNase-free Eppendorf tubes and stored at-80℃freezer for RT-PCR and Western blotting.3.Cell cultureCultures of rat LF were established by enzymatic dissociation of finely minced lung tissue removed from pups of each group on days 3,7,14.Experiments were performed with fibroblasts in the second or third passages.4.Experimental methods(1)The appearance and weight were monitored everyday.(2)Morphology observation:histological study,elastin staining,Masson staining.(3)Immunohistochemistry:measurement the expression levels ofα-SMA andβ-catenin.(4)Western blotting:measurement ofβ-catenin protein expression.(5)RT-PCR:measurement ofβ-catenin,TCF-1,LEF-1 mRNA expression.(6)Fibroblast was cultured and detected by morphology observation through inverted microscope and immunofluorescence staining to vimentin(a marker of LF). MTT method was used to evaluate the proliferative activity of LF.(7)Immuocytochemistry:measurement the expression levels ofα-SMAβ-catenin and vimentin.(8)Western blotting:measurement ofβ-catenin protein expression.(9)RT-PCR:measurement ofβ-catenin,TCF-1,LEF-1 mRNA expression.5.Statistical analysisNormally distributed data are expressed as the mean±SEM and were assessed for significance by Student's t test or ANOVA with post-hoc continuity correction for multiple comparisons as indicated in the text.Non-normally distributed data were assessed for significance using the Wilcoxon rank sum test.Statistical calculations were performed using SPSS13.0 so,ware.Statistical difference was accepted at p＜0.05.Results1.General status and weight changes Rats from group A presented dyspnea since 3 days exposure to hyperoxia.On the 7^th day of oxygen exposure they began to appear fatigue,pale,and present tachypnea and cyanosis for different degree.After 14 days exposure some even had to rely on high oxygen and become worse with the time gone.The control groups didn't have the appearances above.At the beginning there was no difference in average birth weight between the two groups on 3^rd day.From 7 days of oxygen exposure,weights of the experimental groups showed a significant decrease compared with control groups at the sams time period(p＜0.05).2.Lung morphology(1)Changes of lung pathology.On the 3rd day of the experiment,the irregular structure of pulmonary alveoli with small alveolar lumen and thick alveolar septum was observed in each group.On the 7th day,alveoli structure of the control group became regular.While in the hyperxia group,the septum became thinner and decreased in their numbers,large numbers of inflammatory cells infiltrated into the alveoli and septum.On the 14th days,alveoli were regular in size in the control group.However in the hyperxia group,the alveolar cavity became larger,along with thinned alveolar septum,decreased numbers of alveoli,and increased numbers of local pulmonary interstitial fibroblasts.(2)Changes of elastin expression.Elastin was expressed on the tips of septation between two alveolars,like mushrooms in group B on the 7th and 14th day.But expressed in the thick alveolar septa on the same experiment times.(3)Masson staining.Lungs in group B had thin alveolar septa and minimal collagen staining.Lungs in group A had fine strands or thick bundles of collagen in the thick alveolar septa on day 14.3.LF identification,proliferative activityLF was determined by its morphology:fusiform shape,has long apophysis and orbicular-ovate nucleus on the centre;distributed like corona radiate or palisade. Vimentin staining was positive.MTT method showd that LF from group A grown quicker than which from group B at eath experiment point. 4.Changes ofα-SMA protein expressionOn lung tissue,α-SMA expressed on myofibroblast and vascular smooth muscle cell.In group B,it was expressed on the tips of septation between two alveolars on the 7th and 14th day,as elastin staining.But in group A,it was expressed in the thick alveolar septa on the same experiment times,as collagen staining.On LF,it was expressed in the cytoplasm in almost ce11(＞95%),and increased in group A on day 7 and 14.5.Changes ofβ-catenin expressionThe expression was increased in total lung expression ofβ-catenin after 3 days' hyperoxia exposure.This peak occurred on the 14th day.Increased interstitial cell-predominant immunostaining ofβ-catenin was detected by light microscopic evaluation of immunostained lung sections on the 7th and 14th days in hyperoxia group compared with control.And it was observed that significantly increased nuclear colocalization ofβ-catenin in lung interstitial cells on the 7th and 14th days in hyperxia group compared with 21%02 control(P＜0.01).In the cultured LF,the expression ofβ-catenin was significantly increased on each time points after hyperxia exposure,and nuclear colocalization was obviously.6.Changes of TCF-1 and LEF-1 mRNA expressionβ-actin normalized mRNA expression by RT-PCR of LEF-1 was increased on the 3^rd,7^th and 14t^th days in hyperxia group and LF from those groups,which were consistent with the period of maximalβ-catenin expression.While 13-actin normalized mRNA expression by RT-PCR of TCF-1 had no change during each experiment time point.Conclusions1.The histopathological changes of lung injury induced by hyperoxia(FIO₂=0.85) are similar to the characteristics of BPD in preterm infants.2.Disturbed distribution of MF may connected with the alveolarition.3.The proliferation of MF is connected with the repairment after lung injury,and thus made a very important role of MF in the development of BPD.4.Wnt/β-catenin pathway is involved in alveolarition and the development of BPD.5.Wnt/β-catenin pathway worked through 13-catenin/LEF-1.6.LF worked as MF in the alveolarition and the development of BPD.7.Wnt/β-catenin pathway may be involved in the proliferation and activation of MF.8.Wnt/β-catenin worked in BPD maybe through the mechanism which involved in the proliferation,activation and migration of the MF.