Research On The Mechanism Of YAP-TEAD Regulating CTGF In Pulmonary Dysplasia Of Congenital Diaphragmatic Hernia Model

Background : Congenital diaphragmatic hernia(CDH)is a congenital malformation disease due to abnormal embryonic development,is one of the neonatal critical diseases.It is an important task for clinical and scientific researchers to explore the pathogenesis of CDH and find effective treatment methods.Hippo signaling pathway is a highly conserved inhibitory pathway,which has been found to play an important role in lung development in recent years.Objective: In this study,the key effect complex of Hippo signaling pathway YAP-TEAD and target factor CTGF were studied in fetal rat models of congenital diaphragmatic hernia at different gestational ages,and the possible pathogenesis of pulmonary dysplasia of diaphragmatic hernia was analyzed and discussed,which may be helpful to explain the pathogenesis and find accurate treatment methods in the future.Methods: 15 normal SPF grade SD pregnant rats were treated with Nitrofen and olive oil at E9.5d,and divided into CDH group and Control group.Fetal rats were removed by cesarean section under anesthesia at E17.5d,E19.5d,and E21.5d,to obtain fetal lung tissue,and fetal weight,lung weight,and teratogenic conditions recorded at the same time.HE staining was used to observe the difference in lung development in different gestational ages and different groups.Real-time fluorescence quantitative PCR was used to detect the expression of YAP,TEAD(1-4),and CTGF mRNA,and to analyze the correlation of each indicator.YAP,pYAP,TEAD4,and CTGF were qualitatively analyzed by immunohistochemical staining to observe their distribution.Western blot was used to quantitatively analyze the expression of each protein in the histochemistry for further verification.Results: The teratogenic conditions of each gestational age in the CDH group were consistent with the literature reports,and the modeling was successful.The lung index of the CDH group was lower than that of the Control group,indicating the presence of pulmonary dysplasia in the CDH group.HE staining showed that the lung development of the CDH group at each gestational age was slow and lagged,such as pulmonary dysplasia and pulmonary vascular heterogeneity.Transcription and protein levels showed that there was no statistical difference in the expression of all indicator E17.5d(pseudo-glandular stage),while there was a difference in most indicators at E19.5d(canalicular stage)and E21.5d(saccular stage).At the canalicular stage,the relative mRNA expression levels of TEAD(1,3,4)and CTGF in CDH group were all increased,while the immunohistochemical results showed that the expression of YAP was decreased,and that of CTGF was decreased,and Western blot semi-quantitative results showed that YAP was decreased.At the saccular stage,YAP mRNA in the CDH group increased,and CTGF mRNA showed decreased expression.The immunohistochemical results showed that YAP expression decreased,pYAP expression increased,TEAD4 expression decreased,and CTGF expression also decreased.The semi-quantitative results of Western blot were similar,showing that YAP decreased,pYAP increased,and CTGF expression decreased.Besides,in the TEAD family,TEAD(1,3,4)mRNA almost all showed an upward trend with the increase of gestational age,while TEAD2 showed a downward trend,suggesting that TEAD2 was different from other members.Conclusion: Nitrofen-induced CDH fetal rat model can better simulate human congenital diaphragmatic hernia.The key factors in Hippo signaling pathway,such as YAP,TEAD,and CTGF,participated in and affected the lung tissue development process of the CDH fetal rat model.Overactivation of Hippo signaling pathway in the saccular stage was speculated to be one of the mechanisms of lung dysplasia in CDH fetal rats.