Proline-Mediated Inhibition of ATPIF1-mTOR Signaling Alleviates Radiation-Induced Macrophage Polarization and Colon Inflammation

Abstract Radiation therapy often triggers inflammatory complications such as radiation colitis, which is driven by persistent M1 macrophage polarization. While metabolic reprogramming is pivotal in macrophage activation, the role of amino acid metabolism—particularly proline—in regulating radiation-induced inflammation remains unexplored. In this study, bone marrow-derived macrophages (BMDMs) and RAW264.7 cells exposed to 4 Gy radiation exhibited robust M1 polarization (increasing from 13.5% in controls to 23.7% in F4/80 + CD86 + cells), elevated pro-inflammatory cytokines (TNF-α, IL-1β, IL-6; 3- to 6-fold increase), and mitochondrial dysfunction ( 30% decrease in ATP levels, 60% reduction in oxygen consumption rate [OCR], and 2-fold increase in mitochondrial ROS). Proline supplementation reversed these effects, suppressing M1 polarization by 50%, restoring ATP levels by 1.6-fold, and normalizing oxidative phosphorylation. Mechanistically, radiation upregulated ATPIF1 (1.8-fold increase) and phosphorylated mTOR (p-mTOR; 3.2-fold increase), while proline abolished these changes (ATPIF1 decreased by 30%, p-mTOR reduced by 50%). RNA-seq and metabolomics linked proline to ATP metabolism and amino acid homeostasis. ATPIF1 knockdown abolished radiation-induced pro-inflammatory effects and eliminated proline’s protective efficacy, confirming the ATPIF1-mTOR axis as the central regulatory mechanism. In a rat radiation colitis model, proline mitigated colon shortening, reduced histopathological damage, and decreased M1 macrophage infiltration. Collectively, our findings establish the ATPIF1-mTOR axis as a critical mediator of radiation-induced M1 macrophage polarization, which is effectively countered by proline supplementation.