Hydrogen gas ameliorates the LPS-induced BPD via inhibiting the activation of TNF-α/NF-κB inflammatory signaling pathway in placenta

Abstract

Abstract Objective To investigate the anti-inflammatory role of H2 in LPS-induced BPD via regulating TNF-α/NF-κB signaling pathway in placenta. Methods We induced a neonatal rat model of BPD by injecting lipopolysaccharide (LPS, 1ug) into the amniotic fluid at embryonic day 16.5(E16.5). Treatment of 30% hydrogen gas for 4 hours/day with continuously 5days. We primarily analyzed the neonatal outcomes and then compared inflammatory levels from Control group (CON), LPS group (LPS) and LPS with H2 inhalation group (LPS + H2). TUNEL and Hematoxylin-Eosin (HE) staining were performed to evaluate inflammatory and apoptotic levels. We further used RNA sequencing and ELISA assay to examine differentially expressed proteins and mRNA levels of tumor necrosis factor-α (TNF-α), nuclear factor kappa-B (NF-κB) (p65), interleukin (IL)-6, IL-18, IL-1β, C-C motif chemokine ligand 2(CCL2) and C-X-C motif chemokine ligand 1(CXCL1). Bioinformatics analysis (GO and KEEG) of RNA-seq and correlation analysis were applied to clarify the mechanisms of H2 anti-inflammatory effect on LPS-induced BPD. Results We found the H2 inhalation decreased production of inflammatory cytokines/chemokines (IL-6, IL-18, IL-1β, CCL2, CXCL1) in LPS-induced placenta to rescue from the BPD. Upon administration of H2, infiltration degree of LPS-induced placenta was reduced and infiltrating significantly narrowed down. Hydrogen normalized LPS-induced perturbed lung development, reduced lung apoptotic index, death ratio of fetus and neonate. Meanwhile, H2 also upregulated the survival ratio. RNA-seq and Elisa demonstrated that both mRNA and protein levels of TNF-α/NF-κB signaling pathway were activated by LPS, and H2 relieved the pro-inflammatory function of LPS on TNF-α/NF-κB-stimulated placenta. Correlation analysis showed a positive association of TNF-α vs both NF-κB and inflammatory cytokines/chemokines. Conclusion H2 inhalation alleviated LPS-induced BPD by inhibiting excessive pro-inflammatory cytokines and inflammatory chemokines via the TNF-α/NF-κB signaling pathway in placenta and may be a potential therapeutic strategy for BPD.