The Role Of BECN1 In Congenital Diaphragmatic Hernia Associated Pulmonary Hypoplasia

Objective:As a rare congenital birth defect,congenital diaphragmatic hernia(CDH)is characterized by the failure of diaphragm closure and the herniation of abdominal organs into the thoracic cavity,which interferes with the normal development of embryonic lung tissue.The prevalence of congenital diaphragmatic hernia is 1 per 4000-10000,and the mortality rate is as high as 1/3.At present,the pathogenesis of CDH is not clear.Pulmonary hypertension and pulmonary dysplasia often associated with CDH are important reasons for the high mortality of CDH.In recent years,the study of pulmonary development of CDH has become a new breakthrough,which is of great significance to reduce the mortality.Autophagy is a highly conservative intracellular regulatory process that plays a key role in lysosome conversion of cytoplasmic substances,including macromolecules and damaged organelles.Autophagy is induced by a variety of stimulants and has cytoprotective effects in many cases.Several autophagy-related genes(ATG)targeted homozygous deletions lead to embryonic or perinatal death in mice.More and more evidence show that loss of autophagy is associated with respiratory failure caused by abnormal lung development in the uterus.Beclin1 is considered to be a key regulator of autophagy formation.Conditioned loss of Beclin1 in lung epithelial cells of pregnant mice will lead to decrease of airway branches and damage of terminal balloon formation,leading to perinatal fatal respiratory distress,similar to pulmonary dysplasia in CDH.Therefore,this study observed the difference of Beclin1 expression during the development of fetal lung tissue of CDH and explored the role of Beclin1 and autophagy in the development of CDH lung tissue.Methods:1.To establish the fetal rat model of CDH.1)230g-260g female rat vaginal secretion smear on the day after copulation,if the sperm were observed,E0.5 day of pregnancy was determined.2)On the E8.5 day of pregnancy,nitrofen dissolved in edible oil(100mg/ml)1ml was given intragastrically to establish the fetal rat model of CDH,and the control group was given edible oil 1ml intragastric administration.3)the embryos were removed by cesarean section under anesthesia on the E13.5,E15.5,E17.5,E19.5 and E21.5of pregnancy respectively.By observing the integrity of the diaphragm,the fetal lung tissue samples with incomplete development of the diaphragm were collected and named as the CDH group.The diaphragm could not be observed on the E13.5 and E15.5 of pregnancy,therefore the fetal rat samples in the drug intragastric administration group were defaulted to the CDH group.After the samples were collected,immunohistochemistry and Western-blot test were used to verify the difference in the spatio-temporal expression of Beclin1between the CDH group and the control group.The difference in proliferation,apoptosis,development and autophagy between groups was verified on the E21.5.3.The E13.5 d fetal lung tissues of the CDH group and the control group were cultured in vitro.The expression of Beclin1 in the diaphragmatic hernia group was intervened,and the development of lung branches was observed After 96 hours(4 days)vitro culture.Western-blot experiment was used to verify the intervention effect and the changes of proliferation,apoptosis and autophagy.4)The experimental results were analyzed byχ~2 test and t test,and P<0.05 was regarded as statistically significant.Results:On the E13.5,E15.5,E17.5,E19.5 and E21.5 day of pregnancy,the expression of Beclin1 in the CDH group was significantly higher than that in the control group,and it was mainly expressed in fetal bronchial epithelial cell.On the 21st day of pregnancy,compared with the control group,the morphology of fetal lung tissue in CDH group was irregular,interstitial thickening,alveoli enlarged and fused,the expression of indicators representing the degree of development decreased,autophagy enhanced,at the same time,proliferation weakened,apoptosis increased,and the changes of epithelial cells were mainly.In the CDH group,the number of branches of lung tissue in vitro culture was significantly less than that in the control group.There was no significant change in pulmonary branches after increasing the expression of Beclin1 in the lung tissue of the CDH group with increased autophagy and proliferation,decreased apoptosis.After interfering the expression of Beclin1,the branches of embryonic lung tissue decreased significantly.Conclusion:The expression of Beclin1 increases during the development of fetal lung tissue of CDH,accompanied by autophagy,which may play a protective role in the branch and development of lung tissue.The protective effect may be limited when it reaches a certain level.