Identification of key genes and immune infiltration mechanisms in limb ischemia-reperfusion injury: a bioinformatics and experimental study.

AIM OF THE STUDY: To establish a cross-tissue bioinformatics model for identifying conserved key genes and immune infiltration mechanisms in ischemia-reperfusion injury (IRI) with experimental validation in limb IRI, including pharmacological targeting of the WNT5A/PLC pathway. MATERIALS AND METHODS: Transcriptomic data from CTGF-stimulated cardiac myocytes (GSE36073) were analyzed as a surrogate for limb IRI due to shared pathological mechanisms. Random forest, LASSO regression, algorithms identified feature genes, validated in a rat limb IRI model using RT-qPCR, and histology. Pharmacological inhibition (WNT5A inhibitor Box5, PLC inhibitor U-73122) was performed to assess pathway involvement. Immune cell infiltration patterns were analyzed via CIBERSORT. RESULTS: From 169 differentially expressed genes (116 upregulated, 53 downregulated), machine learning identified four key genes (WNT5A, PLCG, ITPR1, CAMK2A), significantly upregulated in experimental limb IRI (P<0.01). Pharmacological inhibition confirmed their functional roles: Box5 and U-73122 treatment reduced expression of WNT5A and PLC versus IRI controls (P<0.05), showing IRI-induced muscle fiber disruption, edema, and inflammation. Immune analysis revealed myeloid polarization shifts (increased M1, decreased M2 macrophages; P<0.05). WNT5A correlated negatively with memory immune cells, while PLCG, ITPR1, and CAMK2A correlated with lymphocyte subpopulations. CONCLUSION: We identified a conserved molecular signature across cardiac and skeletal muscle IRI, with WNT5A/PLC pathway components as mechanistically validated therapeutic targets. Our cross-tissue bioinformatic approach, reinforced by pharmacological and histological evidence, provides a novel framework for IRI analysis when direct patient data are unavailable. Combined targeting of macrophage polarization and cellular activation the WNT5A/PLC axis may offer synergistic therapeutic potential.