Novel mechanisms of intestinal flora regulation in high-altitude hypoxia.

Background: This study investigates the molecular mechanisms behind firmicutes-mediated macrophage (Mψ) polarization and glycolytic metabolic reprogramming through HIF-1α in response to intrinsic mucosal barrier injury induced by high-altitude hypoxia. Methods: Establishing a hypoxia mouse model of high altitude, we utilized single-cell transcriptome sequencing to identify key cell types involved in regulating intestinal mucosal barrier damage caused by high-altitude hypoxia. Through proteomic analysis of colonic tissue Mψ and metabolomic analysis of Mψ metabolites, we determined crucial proteins and metabolic pathways influencing intestinal mucosal barrier damage induced by high-altitude hypoxia. Mechanistic validation was conducted using RAW264.7 Mψ in vitro by assessing cell viability with CCK-8 assay following treatment with different metabolites. The hypoxia mouse model was further validated in vivo by transplanting gut microbiota of Firmicutes. Histological examinations through H&E staining assessed colonic cell morphology and structure, while the FITC-dextran assay evaluated intestinal tissue permeability. Hypoxia probe signal intensity in mouse colonic tissue was assessed via metronidazole staining. Various experimental techniques, including flow cytometry, immunofluorescence, ELISA, Western blot, and RT-qPCR, were employed to study the impact of HIF-1α/glycolysis pathway and different gut microbiota metabolites on Mψ polarization. Results: Bioinformatics analysis revealed that single-cell transcriptomics identified Mψ as a key cell type, with their polarization pattern playing a crucial role in the intestinal mucosal barrier damage induced by high-altitude hypoxia. Proteomics combined with metabolomics analysis indicated that HIF-1α and the glycolytic pathway are pivotal proteins and signaling pathways in the intestinal mucosal barrier damage caused by high-altitude hypoxia. In vitro cell experiments demonstrated that activation of the glycolytic pathway by HIF-1α led to a significant upregulation of mRNA levels of IL-1β, IL-6, and TNFα while downregulating mRNA levels of IL-10 and TGFβ, thereby promoting M1 Mψ activation and inhibiting M2 Mψ polarization. Further mechanistic validation experiments revealed that the metabolite butyric acid from Firmicutes bacteria significantly downregulated the protein expression of HIF-1α, GCK, PFK, PKM, and LDH, thus inhibiting the HIF-1α/glycolytic pathway that suppresses M1 Mψ and activates M2 Mψ, consequently alleviating the hypoxic symptoms in RAW264.7 cells. Subsequent animal experiments confirmed that Firmicutes bacteria inhibited the HIF-1α/glycolytic pathway to modulate Mψ polarization, thereby mitigating intestinal mucosal barrier damage in high-altitude hypoxic mice. Conclusion: The study reveals that firmicutes, through the inhibition of the HIF-1α/glycolysis pathway, mitigate Mψ polarization, thereby alleviating intrinsic mucosal barrier injury in high-altitude hypoxia.