HSPH1 and DNAJB1 as potential key regulators in hepatic ischemia-reperfusion injury

Hepatic ischemia-reperfusion injury (HIRI) is a common and inevitable pathological event during liver transplantation and hepatectomy, which significantly impairs postoperative liver function recovery and patient prognosis. However, the molecular and cellular mechanisms of HIRI have not been fully elucidated and further research is urgently needed. In recent years, the rapid development of bioinformatics analysis technology and the research method combining multi-dimensional data mining with experimental verification have become important strategies for exploring the mechanisms of complex diseases. Building on this, this study aims to screen and analyze the potential roles and mechanisms of key regulatory factors in the process of HIRI through systematic bioinformatics analysis and experimental verification, providing a basis for clarifying its pathogenesis and finding potential therapeutic targets. In this study, two transcriptome microarray datasets (GSE14951 and GSE7706) of human liver tissue were systematically analyzed, and candidate genes related to IRI were initially screened through differential expression analysis. Combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, protein-protein interaction (PPI) network construction and modular analysis were performed to identify potential key regulatory factors, ultimately highlighting HSPH1 and DNAJB1. Subsequently, based on a mouse liver IRI model, paired reperfusion tissue samples from clinical liver transplant patients, and an AML12 cell hypoxia/reoxygenation (H/R) model, the two genes were experimentally validated from multiple perspectives, including transcriptional expression, protein levels, and subcellular localization. A combination of quantitative PCR (qPCR), Western blotting, immunohistochemistry (IHC), immunofluorescence (IF), and co-immunoprecipitation (Co-IP) was employed to comprehensively evaluate their expression dynamics, subcellular distribution, and protein-protein interaction characteristics. Differential expression analysis identified 154 genes with consistent expression trends across the two datasets, which were significantly enriched in metabolic, stress response, inflammatory, and protein folding pathways. PPI network construction and module analysis further identified HSPH1 and DNAJB1 as core components of a heat shock protein interaction cluster. Validation using a mouse IRI model, paired reperfusion tissue samples from clinical liver transplant patients, and an AML12 cell hypoxia/reoxygenation (H/R) model demonstrated that both genes were significantly upregulated under IRI conditions, localized in the cytosol, and exhibited co-localization and physical interaction. Transcription factor prediction analysis suggested that STAT3 and NR1I2 might be involved in their transcriptional regulation. In conclusion, HSPH1 and DNAJB1 are co-expressed and physically interact in hepatic IRI, suggesting that they may be involved in the regulation of protein homeostasis and cellular stress responses related to liver IRI, providing important experimental evidence for a deeper understanding of the molecular characteristics of liver IRI.