Vitamin D Improves Lung Pathology Of Neonatal Rats With BPD And Its Mechanisms

Objects:Bronchopulmonary dysplasia（BPD）is a major chronic pulmonary complication in preterm infants.It is characterized by premature alveolar developmental arrest with enlarged alveolar spaces,decreased terminal space numbers and reduced secondary septa counts.Antenatal exposure to bacterial infections and inflammation has been an important risk factor for BPD development.Our previous studies observed the overexpression of inflammatory cytokines（IL-1β、IFN-γ、TNF-α）in lung tissue of neonatal rats with lipopolysaccharide（LPS）-induced lung injury,a model for BPD.1,25（OH）₂D₃,the biologically active form of Vitamin D（VitD）,is not only involved in the metabolism between calcium and phosphorus in mineral and skeletal homeostasis,but also regulates the differentiation,growth,and function of a broad range of cells of the immune system,including secretion of inflammtory cytokines such as Interferon-gamma（IFN-γ）and Interleukin-1beta（IL-1β）.We try to investigate the role of 1,25（OH）₂D₃ on amelioration of LPS-induced alveolar arrest of neonatal rats with BPD,and explore the potential mechanisms.Methods:Part 1:LPS was injected into the amniotic sac of pregnant SD rats at 16.5 days post coitum with 1,25（OH）₂D₃ intervention and lung sections of neonatal rats stained with hematoxylin and eosin（H&E）were analyzed to determine whether VitD have a certain improving effect on LPS-induced alveolar arrest of neonatal rats.Part 2:Through Real-time PCR and ELISA,we detected the levels of mRNA and protein expression of IFN-γ、IL-4 in the neonatal rat lung tissue and spleen to observe whether VitD could depress the overexpression of IFN-γin lung tissue induced by LPS and degree of inflammatory response in lung tissue and body.Moreover,we analyzed the relationshp between methylation percentage of the vitamin D response element（VDRE）of IFN-γpromoter region combinated with VDR and VitD by BSP.We also verified VitD could change the methylation status of VDRE in IFN-γpromoter region to regulate the expression of IFN-γin rat bone marrow macrophages by nonspecific methylation inhibitor 5-aZac.Part 3:After LPS or LPS+VitD stimulation,the activation pro-Caspase1 and secretion IL-1βof RAW264.7 cells were detected by Western blot and ELISA.We investigated the inhibition effection of VitD on LPS-induced pyroptosis.Part 4:After LPS or LPS+VitD stimulation,rat bone marrow macrophages were detected on the technology of RNA-sequence to analyze the regulation of VitD treatment based on cell inflammation model.Results:Part 1:Co-treatment with theVitD and LPS increased terminal airspace counts（P<0.05）,discrease mean linear intercept（P<0.05）,compared to LPS treatment alone,suggesting VitD effectively alleviated the lung alveolar structure simplification induced by LPS.Part 2:VitD increased the methylation percentage of VDRE in IFN-γpromoter region,compared to LPS treatment（82.92±0.08 vs 94.08±1.7,P<0.05）,depressed the expression level of IFN-γin lung tissue of neonatal rats with BPD,and resulted in inhibition of inflammatory response in lung tissue and body.Part 3:The activated pro-Caspase1 levels and IL-1βexpression levels of RAW264.7 were significantly decreased by VitD treatment,resulting in depressing LPS-induced pyroptosis.Part 4:Based on the technology of RNA-Sequence,we found VitD could regulated the expression of many genes to decrease the expression level of IFN-γand IL-1β。Conclusion:Our study demonstrated VitD effectively alleviated the LPS-induced lung alveolar structure simplification by down-regulating the expression of IFN-γand IL-1β.The relative mechanisms are as follows:1)VitD could reduce the IFN-γexpression in lung tissue by increasing the methylation of the VDRE of VDR combinated with IFN-γpromoter region.Then VitD could depress the inflammation response in lung tissue and body.2)VitD could decrease the activation of pro-Caspase1 and secretion of IL-1βresulting in depressing the pyroptosis in cells.3)The results of RNA-Seq shows VitD could decrease the expression level of IFN-γand IL-1βby regulating the expression of many relative genes.