Effect of lipopolysaccharide via intranasal instillation on lung development in neonatal mice and its mechanism

Abstract:

Objective：To investigate the effect of lipopolysaccharide（LPS） via intranasal instillation on the lung development in neonatal mice，and to explore the role of postnatal pulmonary inflammation in the development of bronchopulmonary dysplasia and its mecha－nism. Methods：A total of 20 C57BL/6 mice on postnatal day 1（P1） were randomly divided into LPS group and saline group. The LPS group was given LPS（25 mg/kg/day） via intranasal instillation from P1 to postnatal day 13（P13），while the saline group was given an equal volume of saline via intranasal instillation from P1 to P13. The mice were randomly sacrificed on postnatal day 14（P14） for col－lection of the lung tissue. The morphological changes in the lung tissue were observed by HE staining. The expression of cluster of differentiation 31（CD31） was measured by immunohistochemistry to determine pulmonary microvascular density. The mRNA expres－sion of vascular endothelial growth factor receptor 2（VEGFR-2） and macrophage inflammatory protein-1 alpha（MIP-1α） was deter－mined by quantitative real-time PCR（qRT-PCR）. The protein expression of nuclear factor kappa-B p65 subunit（P65），nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor，alpha（IκBα），phosphorylated P65（Ser536），phosphorylated Iκ－Bα（Ser32），and VEGFR-2 was measured by Western blot. Results：HE staining showed that compared with the saline group，the LPS group showed alveolar enlargement and inflam－matory cell infiltration around blood vessels；the LPS group had a significantly lower radial alveolar count（4.533±0.166 vs. 8.377±0.290，P=0.000） and a significantly higher mean linear intercept（54.890±2.074 vs. 32.750±0.787，P=0.000）. The immunohistochemistry results of CD31 showed that the LPS group had a signifi－cantly lower microvascular density than the saline group（3.387±0.007 vs. 5.631±0.014，P=0.000）. qRT-PCR showed that compared with the saline group，the LPS group had significantly lower mRNA expression of VEGFR-2 and significantly higher mRNA expres－sion of MIP-1α（P=0.000；P=0.000）. Western blot showed that the LPS group had significantly increased protein expression of P65，Ser536，and Ser32 and significantly reduced protein expression of IκBα and VEGFR-2 compared with the saline group（all P=0.000）. Conclusion：LPS via intranasal instillation can inhibit the lung development in neonatal mice and postnatal pulmonary inflammation may be involved in the development of bronchopulmonary dysplasia. The mechanism may be related to downregulation of VEGFR-2，activation of NF-κB，and upregulation of MIP-1α.