Integrated Analysis of Single‐Cell and Bulk RNA Sequencing Data Reveals Cellular Senescence‐Related Signatures Predicting New Potential Therapeutic Drugs in Hepatic Ischemia‐Reperfusion Injury

ABSTRACT Three human bulk RNA sequencing (RNA‐seq) datasets from the GEO database, comprising liver tissues from 76 patients with hepatic ischemia–reperfusion injury (IRI) and 80 controls, were analyzed to identify differentially expressed genes related to cellular senescence (DEG‐CSRGs). A total of 19 DEG‐CSRGs were identified out of 866 cellular senescence‐related genes, and key hub genes (e.g., JUN, FOS, ATF3) were subsequently screened. In parallel, a single‐cell RNA sequencing (scRNA‐seq) analysis of a mouse hepatic IRI model was conducted, profiling 4998 immune cells from control and IRI liver tissues. The analysis revealed the central roles of macrophages, monocytes, and neutrophils in the IRI process, with significant upregulation of hub gene expression in these immune cell populations. Pseudotime trajectory and intercellular communication analyses further elucidated the dynamic transitions and interactions among immune cells during IRI progression. Drug‐gene interaction prediction indicated fluoxetine (FLX) as a potential therapeutic candidate, and its binding affinity to hub genes was supported by molecular docking and molecular dynamics simulations. Bioinformatics predictions were experimentally validated using in vivo mouse models ( n = 5 per group) and in vitro RAW264.7 macrophage cell assays (qPCR: n = 6 per group; Western blot: n = 3 per group), confirming that FLX mitigated liver injury and suppressed the expression of cellular senescence‐related factors.