NR4A1 Mediates Offspring Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mice

Background Intrauterine inflammation (IUI) is involved in the development of bronchopulmonary dysplasia (BPD). Previously, we observed BPD-like pathological changes in a mouse model of IUI. Here, we aimed to investigate key molecules involved in IUI-induced neonatal lung injury along with the long-term outcomes. Methods Pregnant C57BL/6 mice were randomly divided into control and IUI groups. Lung injury was evaluated using hematoxylin-eosin and Masson's trichrome staining. An NR4A1 overexpression plasmid was used to explore the downstream molecules acting in vitro. Accordingly, IUI-induced lung injury intervention was verified using Nr4a1 siRNA. Results The lungs of IUI-induced offspring showed a simple structure from postnatal day 1 to 6 months, with increased pulmonary fibrosis from 3 to 6 months postnatal. Postnatal NR4A1 intervention reversed IUI-induced neonatal lung injury. NR4A1 overexpression decreased cell proliferation and influenced the expression of epithelial-mesenchymal transition (EMT)-related key genes in MLE-12 cells. EREG is a downstream target of NR4A1, and blocking the receptor of EREG would recover the expression of EMT-related genes. Conclusion IUI induced BPD-like lung injury in neonates and pulmonary fibrosis in adults. The NR4A1-EREG-EGFR signaling pathway in pulmonary epithelial cells plays an important role in IUI-induced lung injury in offspring. The NR4A1 over-expression plasmid, created by cloning the open-reading frame into the pCMV-SPORT6 vector, was purchased from the DNA Library of Shanghai Jiaotong University (https://dnacore.shsmu.edu.cn). The JASPAR database was used to predict the potential transcription factor binding sites on the NR4A1 promoter, and NR4A1 overexpression plasmids were transfected into MLE-12 cells using Lipofectamine 2000 (Invitrogen), with the pCMV-SPORT6 vector set as a control.