Background: Studying effective interventions for hypoxia-induced injury is crucial, particularly in high-altitude areas. Symptoms stemming from intestinal injuries have a significant impact on the health of individuals transitioning from plains to plateau regions. This research explores the effects and mechanisms of nitrate supplementation in preventing hypoxia-induced intestinal injury. Methods: A hypoxia survival mouse model was established using 7% O-2 conditions. The intervention with 4 mM sodium nitrate (NaNO3) in drinking water commenced 7 days prior to hypoxia exposure. Weight monitoring, hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and intestinal permeability assays were employed for physiological, histological, and functional analyses. Quantitative PCR (qPCR), Western blot, and immunofluorescence were utilized to analyze the levels of tight junction (TJ) proteins and hypoxia-inducible factor 1 alpha (Hif 1 alpha). RNA sequencing (RNA-seq) identified nitrate's target, and chromatin immunoprecipitation (ChIP) verified the transcriptional impact of Hif 1 alpha on TJ proteins. Villin-cre mice infected with AAV9-FLEX-EGFP-Hif 1 alpha were used for mechanism validation. Results: The results demonstrated that nitrate supplementation significantly alleviated small intestinal epithelial cell necrosis, intestinal permeability, disruption of TJs, and weight loss under hypoxia. Moreover, the nitrate-triggered enhancement of TJs is mediated by Hif 1 alpha nuclear translocation and its subsequent transcriptional function. The effect of nitrate supplementation on TJs was largely attributed to the stimulation of the EGFR/PI3K/AKT/mTOR/Hif 1 alpha signaling pathways. Conclusion: Nitrate serves as a novel approach in preventing hypoxia-induced intestinal injury, acting through Hif 1 alpha activation to promote the transcription of TJ proteins. Furthermore, our study provides new and compelling evidence for the protective effects of nitrate in hypoxic conditions, especially at high altitudes.
