Abstract: BACKGROUND: Ferroptosis-mediated ischemia-reperfusion injury plays a crucial role in the occurrence and progression in pressure ulcers, and there may be pressure ulcer-associated ferroptosis biomarkers, but the mechanism has not been elucidated.
OBJECTIVE: To investigate the molecular mechanisms underlying pressure ulcers using bioinformatic analysis, with a focus on identifying differentially expressed genes associated with ferroptosis during the process of pressure ulcer formation, thereby providing novel insights into the clinical treatment of pressure ulcers.
METHODS: The single-cell transcriptome sequencing dataset and ferroptosis-related genes were obtained and preprocessed from the Gene Expression Omnibus (GEO) and FerrDb databases. We performed clustering and proportion analyses, metabolic activity and pseudotime analysis, cell communication analysis, ferroptosis gene set cell population identification, and enrichment analysis to determine differentially expressed genes related to ferroptosis. Animal experiments were then conducted for further validation, with 20 Sprague-Dawley rats randomly assigned into a control group and a model group (n=10 per group). The control group received no treatment, while the model group underwent a cycle of ischemia-reperfusion to establish pressure ulcer models. Changes in differentially expressed genes and proteins in the wound tissues of pressure ulcer rats were detected using fluorescent quantitative PCR and western blot, respectively.
RESULTS AND CONCLUSION: The single-cell transcriptome sequencing data were clustered into six cell types, with a higher proportion of type 2 and type 3 keratinocytes observed in the pressure ulcer group. There was evident metabolic heterogeneity and evolutionary trajectory among cell populations. Type 2 and type 3 keratinocytes exhibited stronger cell communication, while type 2 keratinocytes demonstrating optimal ligand-receptor interactions. Type 2 keratinocytes demonstrated higher scores for ferroptosis, accompanied by significant upregulation or downregulation of specific genes. A total of 27 Gene Ontology enrichments, 20 Kyoto Encyclopedia of Genes and Genomes enrichments, and 24 ferroptosis-related differentially expressed genes, including glutathione peroxidase 4 (GPX4) and acyl-CoA synthetase long chain family member 4 (ACSL4), were identified. Animal experiments further confirmed the downregulation of GPX4, the ferroptosis-inhibiting protein, and the upregulation of ACSL4, the ferroptosis-promoting protein, in the model group. Overall, these findings indicate the presence of ferroptosis in pressure ulcer tissue. GPX4 and ACSL4 are important genes regulating ferroptosis in pressure ulcer tissues.

Key words: bioinformatics, single-cell transcriptome sequencing, pressure ulcer, ischemia-reperfusion, tissue injury, chronic wound, ferroptosis, oxidative stress, GPX4, ACSL4