Upregulated EFNB2 and EPHB4 promotes lung development in a nitrofen-induced congenital diaphragmatic hernia rat model

Congenital diaphragmatic hernia (CDH) is a common congenital malformation associated with high mortality rates, mainly due to pulmonary hypoplasia and persistent pulmonary hypertension following birth. The present study aimed to investigate abnormal lung development in a rat CDH model, and examine temporal and spatial changes in the expression of ephrin type-B receptor 4 (EPHB4) and ephrin-B2 (EFNB2) during fetal lung development, to elucidate the role of these factors during lung morphogenesis. Pregnant rats received nitrofen on embryonic day (E) 8.5 to induce CDH, and fetal lungs were collected on E13.5, E15.5, E17.5, E19.5, and E21.5. The mean linear intercept (MLI) and mean alveolar number (MAN) were observed in fetal lung tissue at E21.5 following hematoxylin and eosin staining. E13.5 fetal lungs were cultured for 96 h in serum-free medium and branch development was observed under a microscope. The gene and protein expression levels of EPHB4 and EFNB2 were assessed by reverse transcription-quantitative polymerase chain reaction analysis, and immunoblotting and immunohistochemistry, respectively. The fetal rat lungs were treated with EFNB2 and the activity of key signaling pathways was assessed. The lung index (lung weight/body weight) at E21.5 was significantly lower in the CDH rats, compared with that in the control fetal rats. The MLI and MAN were also lower in the CDH group. The number of lung terminal buds at E13.5 (embryonic stage), and the lung-explant perimeter and surface were all smaller in the CDH group rats than in the control group at the same age. Pulmonary hypoplasia was observed following 96 h of in vitro culture. No significant differences were found in the expression levels of EFNB2 and EPHB4 between the CDH and control groups at E13.5 (embryonic stage) or E15.5 (pseudoglandular stage), however, EFNB2 and EPHB4 were significantly upregulated at E17.5 (canalicular stage), and at E19.5 and E21.5 (saccular/alveolar stages). EFNB2 stimulated pulmonary branching and EFNB2 supplementation decreased the activity of p38, c-Jun NH2-terminal kinase, extracellular signal-regulated kinase, and signal transducer and activator of transcription. The CDH fetal rats developed pulmonary dysplasia at an early stage of fetal pulmonary development. Upregulated expression of EFNB2 and EPHB4 was observed in the rat lung of nitrofen-induced CDH, and the increased expression of EFNB2 promoted rat lung development in the nitrofen-induced CDH model.