Experimental Study Of Tacrolimus On Pulmonary Dysplasia And Pulmonary Hypertension In Congenital Diaphragmatic Hernia

Background: Congenital diaphragmatic hernia(CDH)is a serious congenital malformation,the main pathological changes in CDH were defected in the development of the diaphragm and pulmonary hypoplasia,resulting in herniation of the abdominal viscera into the chest cavity,which lead to pulmonary arterial hypertension(PAH).Although many efforts and many studies have been done on pulmonary dysplasia and PAH in CDH,the mechanism of pulmonary development and PAH in CDH is not completely clear,and there are many difficulties and disputes in treatment on CDH.Therefore,its pathogenesis and treatment measures need to be further explored.Objective: This study aims to explores the effect and significance of prenatal administration of tacrolimus(FK506)on pulmonary development and PAH in Nitrofen-induced congenital diaphragmatic hernia rat model.To investigate the expression of BMPR2 pathway and its downstream related factors in the fetal lung tissue of CDH rats induced by Nitrofen,and to explore the mechanism of PAH and pulmonary hypoplasia in a nitrofen-induced rat model of CDH.Methods: 12 normal female SD rats in SPF grade were randomly divided into 4groups(3 rats in each group): Control group,CDH group,FK506 group and CDH+FK506 group,then CDH group and CDH+FK506 group were treated with Nitrofen,Control group and FK506 group were treated with olive oil at E9.5d.On E21.5 day,the fetal rats and lung tissue were removed by cesarean section after anesthesia,the lung weight、body weight and the occurrence of diaphragmatic hernia of fetal rats were recorded,The differences of pulmonary and vascular development in lung tissue among the groups were observed by HE staining.Double immunofluorescence staining with antibodies to CD31 and α-SMA.Quantitative real-time PCR(q RT-PCR)was used to detect the expression of BMPR2 m RNA,SMAD1 m RNA and SMAD5 m RNA in each group,and the protein expressions of BMPR2,SMAD1,p SMAD1,SMAD5 and p SMAD5 in each group were quantitatively analyzed by Western blot.Results:HE staining showed that the development of fetal lung in CDH group lags behind that of normal lung,The main pathological changes includethickening of the pulmonary interstitium,atrophy of the alveolar cavity,and pulmonary vascular remodeling,which could be alleviated by FK506 intervention.The results of q PCR showed that there was no statistical difference in the relative expression of SMD1 and SMAD5 m RNA among all groups(P > 0.05),but the relative expression of BMPR2 m RNA in CDH group was lower than that in Control group(P < 0.001),and the relative expression of BMPR2 m RNA in CDH+FK506 group was higher than that in CDH group(P < 0.001).The results of immunofluorescence staining showed that the expression of CD31 and α-SMA in CDH group was higher than that in Control group(P< 0.05),while the expression of CD31 and α-SMA in CDH+FK506 group was lower than that in CDH group(P < 0.05).Western blot results and semi-quantitative analyses showed that the expression levels of Bmpr2 and p-SMAD1 in CDH group were lower than those in CDH group,while the expression levels of Bmpr2 and p-SMAD1 in CDH+FK506 group were higher than those in CDH group.There was no significant difference in the expression of p-SMAD5 protein among the three groups.Conclusion: The results of this study show that although FK506 doesn’t reduce the incidence of diaphragmatic hernia,it can reduce the expression of α-SMA and CD31 and increase the expression of BMPR2 and its related factors in fetal lung tissue of Nitrofen-induced CDH rat model,so as to alleviate the fetal lung dysplasia of Nitrofen-induced CDH rat model,play a role of anti-vascular remodeling and reduce pulmonary vascular hypertension.It is suggested that FK506 intervention may play a role by regulating BMPR2 pathway in fetal lung tissue.