The Relationship Between RA And STRA6 In Retinoid Signaling Pathway And Lung Hypoplasia In The Nitrofen Model Of Congenital Diaphragmatic Hernia

Objectives1. To investigate the role of RA in the development of pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia.2. To investigate the STRA6 expression feature in different development stages of rat fetal lungs.3.To investigate the role of STRA6 plays in formation of pulmonary hypoplasia with CDH.1. establish animal model and divide into groupsTime-mated female Sprague-Dawley rats (N = 24) randomly divided into two groups: normal controls and nitrofen-induced group. Observation of sperm-positive vaginal smears was considered a proof of pregnancy and represented day 0.5 of gestation. Pregnant rats at day 9.5(D9.5) of gestation randomly received by gavage either a single-dose of 100 mg of 2,4-dichorophenyl-p-nitrophenylether (nitrofen) dissolved in 1 mL of olive oil or an equal volume of olive oil.Both controls and nitrofen-induced group were received 2 dams at embryonic D13.5 and D15.5,and 4 dams at D19.5 and D21.5, respectively. fetuses were both removed by caesarian section at D13.5,D15.5, D17.5and D21.5, respectively,whose Left lungs were utilized in next experiment . Lungs of D13.5 fetuses utilized in organ culture,which were randomly divided into four groups: normal controls group(n=6); normal controls +RA group(n=6) ; nitrofen-induced group(n=6);nitrofen-induced +RA group(n=6).all lungs of D15.5 fetuses and parts of lungs of D17.5,D17.5 fetuses utilized in QPCR, which were randomly divided into two groups: normal controls group(n=18) ; nitrofen-induced group(n=18). Parts of lungs of D17.5,D17.5 fetuses utilized in IMF, which were randomly divided into two groups: normal controls group(n=12) ; nitrofen-induced group(n=12).2.To investigate the role of RA in the development of pulmonary hypoplasia in nitrofen-induced hypoplastic foetal rat lung explantsFetuses were removed by caesarian section at D13.5 and washed three times by HBSS solution. Normal and nitrofen-exposed lungs were placed on translucent polycarbonate membrane dish inserts .The membranes were then placed on culture dishes and serum-free Dulbecco'smodified Eagle's culture medium was added until the lungs were lying at the air–medium interface. Culture medium was added 100 U/ml penicillin and 100 lg/ml streptomycin and was changedevery 24 h. Lungs were incubated at 37oC in 5%CO2 for 96 h. Exogenous RA 1 lM was diluted in ethanol and added daily to the culture medium at a final ethanol concentration of0.4%. Image analysis the development parameter(area and perimeter) in nitrofen-induced hypoplastic foetal rat lung explants.4. Detecting the expression of STRA6 mRNA in rat fetal lungs by real time Quantitative PCR.After extracted from each sample using Trizol reagent, the total mRNA was checked for integrity by 1% agarose gel electrophoresis, and its concentration was also detected by spectrophotometry. The total mRNA was used for quantification of STRA6 and reference geneβ-actin by Taqman probe-based two-step real time QPCR, and serial dilution of one sample RNA was prepared to create a standard curve for the relative quantification of mRNA in the samples. There is a duplicate for each sample in the experiments. The relative changes of STRA6 expression from QPCR were normalized toβ-actin expression in each sample. All relative expression levels in different gestational day points in two groups were compared with D21.5 in controls group (= 1.00).5. Detecting the expression of STRA6 protein in rat fetal lungs by immunofluorescenceCalculating immunofluorescence amounts of STRA6 protein in rat fetal lungs of normal controls and nitrofen-induced group at D17.5 and D21.5 by immunofluorescence.Results1. CDH rat model85 fetuses were collected from controls group, 30 from nitrofen-induced group at D13.5 and D15.5 , there were 45.9% fetuses fetuses had CDH in 61 fetuses (28/61)from nitrofen-induced group at D17.5 and D21.5. . All the defects in diaphragms occurred in left side. Generally, livers, stomachs and bowl could come into thoracic cavity through the defects in diaphragms.2.Studies of histologyThe development of lungs in normal controls group were mature, in nitrofen-induced group ,however, it happened pulmonary hypoplasia, reduced number of bronchus branch , most of bronchic cell collapsed, thickening of the alveolar septum , ductuli alveolares, bronchic cell and alveolar saccules alter like glandular organ, arteriole muscular layer became thickening and smooth muscle cell grew abnormality.3. the role of RA in the development of pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic herniaMorphology: In controls and controls+RA group,the development of the lung explants were mature and lung buds grew flourishing after 96 h of culture. Conversely, the Nitrofen group take place developmental necrotic and cellular necrosis gradually after 72 h , but the Nitrofen+RA group improve obviously. The development parameter: Retinoic acid significantly increased the number of lung area of nitrofen-exposed hypoplastic lungs after 96 h of culture(Nitrofen+RA: 1.867±0.032 mm2 vs Nitrofen :1.053±0.033 mm2,P<0.05), whilst the lung area of controls lungs did not change(controls+RA: 2.088±0.622 mm2 vs controls: 2.535±0.423,p>0.05) . Lung perimeter was also higher when RA was added to hypoplastic lungs compared to the non-treated ones, although it did not reach significance (Nitrofen+RA:6.237±0.581 mm vs Nitrofen:4.862±0.458mm mm; P=0.53); Lung area and perimeter were significant differences in controls and controls+RA groups compared with Nitrofen(respectively;P<0.05).4.Realtime QPCR detectionOn D15.5 , the relative mRNA expression levels of STRA6 were significantly decreased in the lungs of nitrofen(1.191±0.351)compared with controls(14.800±5.155)(P=0.000<0.01);STRA6 expression were fluctuated and significant difference in Nitrofen lungs (D17.5: 7.552±3.716 VS D15.5 and D21.5: 0.951±0.942; P=0.000<0.01) compared to the controls lungs were decreased gradually (D15.5 VS D17.5: 4.061±2.765 and D21.5: 1.000±0.449; P=0.000<0.01).5.IMF detectionOn D17.5,fluorescence amount of STRA6 protein were significantly increasing in the lungs of Nitrofen (1471.167±228.687) compared with controls (809.500±82.243) (P=0.000<0.01).ConclusionsRetinoic acid rescues lung hypoplasia in nitrofen-induced hypoplastic foetal rat lung explants(D13.5) .In earlier stage of lung morphogenesis (D15.5), Nitrofen could cause the lung cells retinol malassimilation and lead to lung hypoplasia with CDH by interfering with the STRA6 expression. However, STRA6 expression were increased during intermediate stage (D17.5), which could be a feedback reaction to the deficiency of lung retinol in Retinoid signaling pathway.