Molecular Mechanism of the <scp>IRF1</scp> / <scp>NFE2L1</scp> ‐ <scp>DT</scp> / <scp>ALKBH5</scp> /Cx43 Axis in Radiation‐Induced Injury in Vascular Endothelial Cells Through Pyroptosis

Radiotherapy effectively eradicates tumor cells but can also trigger pyroptotic damage in vascular endothelial cells. This study investigates the role of interferon regulatory factor 1 (IRF1) in radiation-induced endothelial injury, aiming to provide mechanistic insights for optimizing radiotherapy. Human umbilical vein endothelial cells (HUVECs) were exposed to x-ray irradiation, after which cell viability, lactate dehydrogenase (LDH) release, γ-H2AX foci formation, and the expression of pyroptosis-associated proteins (NLRP3, Cleaved Caspase-1, GSDMD-N, IL-1β, IL-18) were assessed. Expression levels of IRF1, NFE2L1-DT, PELP1, ALKBH5, and Cx43 were quantified. Chromatin enrichment of IRF1 at the NFE2L1-DT promoter and IRF1-NFE2L1-DT interactions were examined, along with NFE2L1-DT or ALKBH5 binding to PELP1. The enrichment of m⁶A modifications on Cx43 transcripts was also evaluated. X-ray exposure reduced HUVEC viability, elevated LDH release, increased γ-H2AX foci, and upregulated IRF1, along with pyroptosis markers. Silencing IRF1 reversed these changes. Mechanistically, IRF1 directly bound to and increased NFE2L1-DT expression. NFE2L1-DT interacted with PELP1 to enhance the binding of PELP1 to and ALKBH5 mRNA, thus upregulating ALKBH5 expression. ALKBH5-mediated m⁶A demethylation subsequently downregulated Cx43 expression. Overexpression of NFE2L1-DT or ALKBH5, or silencing Cx43, attenuated the protective effects of IRF1 silencing against radiation-induced damage. These findings indicate that radiation-induced IRF1 upregulation leads to endothelial injury by promoting pyroptosis through the NFE2L1-DT/ALKBH5/Cx43 axis.