ABSTRACT
INTRODUCTION: Hydrogen (H2) is regarded as a novel therapeutic agent against several diseases owing to its inherent biosafety. Bronchopulmonary dysplasia (BPD) has been widely considered among adverse pregnancy outcomes, without effective treatment. Placenta plays a role in defense, synthesis, and immunity, which provides a new perspective for the treatment of BPD. This study aimed to investigate if H2 reduced the placental inflammation to protect the neonatal rat against BPD damage and potential mechanisms. METHODS: We induced neonatal BPD model by injecting lipopolysaccharide (LPS, 1 µg) into the amniotic fluid at embryonic day 16.5 as LPS group. LPS + H2 group inhaled 42% H2 gas (4 h/day) until the samples were collected. We primarily analyzed the neonatal outcomes and then compared inflammatory levels from the control group (CON), LPS group and LPS + H2 group. HE staining was performed to evaluate inflammatory levels. RNA sequencing revealed dominant differentially expressed genes. Bioinformatics analysis (GO and KEGG) of RNA-seq was applied to mine the signaling pathways involved in protective effect of H2 on the development of LPS-induced BPD. We further used qRT-PCR, Western blot and ELISA methods to verify differential expression of mRNA and proteins. Moreover, we verified the correlation between the upstream signaling pathways and the downstream targets in LPS-induced BPD model. RESULTS: Upon administration of H2, the inflammatory infiltration degree of the LPS-induced placenta was reduced, and infiltration significantly narrowed. Hydrogen normalized LPS-induced perturbed lung development and reduced the death ratio of the fetus and neonate. RNA-seq results revealed the importance of inflammatory response biological processes and Toll-like receptor signaling pathway in protective effect of hydrogen on BPD. The over-activated upstream signals [Toll-like receptor 4 (TLR4), nuclear factor kappa-B p65 (NF-κB p65), Caspase1 (Casp1) and NLR family pyrin domain containing 3 (NLRP3) inflammasome] in LPS placenta were attenuated by H2 inhalation. The downstream targets, inflammatory cytokines/chemokines [interleukin (IL)-6, IL-18, IL-1ß, C-C motif chemokine ligand 2 (CCL2) and C-X-C motif chemokine ligand 1 (CXCL1)], were decreased both in mRNA and protein levels by H2 inhalation in LPS-induced placentas to rescue them from BPD. Correlation analysis displayed a positive association of TLR4-mediated signaling pathway both proinflammatory cytokines and chemokines in placenta. CONCLUSION: H2 inhalation ameliorates LPS-induced BPD by inhibiting excessive inflammatory cytokines and chemokines via the TLR4-NFκB-IL6/NLRP3 signaling pathway in placenta and may be a potential therapeutic strategy for BPD.
Subject(s)
Bronchopulmonary Dysplasia , Hydrogen , Inflammation , Lipopolysaccharides , NF-kappa B , NLR Family, Pyrin Domain-Containing 3 Protein , Placenta , Signal Transduction , Toll-Like Receptor 4 , Female , Pregnancy , Lipopolysaccharides/toxicity , Hydrogen/pharmacology , Hydrogen/therapeutic use , Animals , Placenta/metabolism , Placenta/drug effects , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 4/genetics , Signal Transduction/drug effects , Rats , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , NF-kappa B/metabolism , Inflammation/metabolism , Inflammation/drug therapy , Administration, Inhalation , Bronchopulmonary Dysplasia/metabolism , Bronchopulmonary Dysplasia/chemically induced , Bronchopulmonary Dysplasia/drug therapy , Bronchopulmonary Dysplasia/prevention & control , Interleukin-6/metabolism , Interleukin-6/genetics , Rats, Sprague-Dawley , Disease Models, AnimalABSTRACT
OBJECTIVE: To analyze the influence of optimal codon usage on the expression levels and immunogenicity of DNA vaccines, encoding the human papillomavirus type 6b (HPV 6b) E7 gene. METHODS: The full length E7 gene of HPV 6b was modified to substitute human preferred codon for rarely used codon, and three mutations were introduced into the pRB binding site of HPV 6b E7 to eliminate its transformation potential. The codon optimized and mutated E7 gene (hu-mE7) were cloned into the Kpn I and EcoR I site of the pcDNA3 mammalian expression vector, the in vitro expression of the hu-mE7 gene and the immunogenicity of hu-mE7 DNA vaccine were compared with the wt-E7gene. RESULTS: The in vitro expression of pcDNA3-hu-mE7 was much higher than the classical wt-E7 plasmid in monkey COS-1 cell line. Mice immunized intramuscularly with the pcDNA3-hu-mE7 showed that the codon modified E7 gene induced a stronger IFN-gamma ratios than the wt-E7 gene. CONCLUSIONS: These results suggest that the optimized codon usage contributes to the enhancement of gene expression and immunogenicity of HPV 6b E7 gene.
