Objective <p>Radiation-induced myocardial fibrosis (RIMF) significantly impacts the long-term quality of life and non-tumor mortality in patients undergoing thoracic tumor radiotherapy. Despite its clinical significance, the mechanisms underlying RIMF formation remain inadequately understood, highlighting the importance of investigating its pathological mechanisms and identifying potential intervention targets.</p> Methods <p>An RIMF model was established in C57BL/6 mice, with interventions administered via subcutaneous intraperitoneal injections of HMGB1 neutralizing antibody (HMNA). The validity of the RIMF model and the effects of HMNA intervention were evaluated using hematoxylin and eosin (HE), Masson’s trichrome staining, and echocardiography. Additionally, western blot, qPCR, ELISA, CCK8 assay, and tissue immunofluorescence were employed to investigate the relationship between radiation and cardiac injury, as well as the role of HMGB1 in RIMF formation, both in vitro and in the C57BL/6 mouse model.</p> Results <p>HE and Masson’s trichrome staining, coupled with echocardiographic assessments, demonstrated that the early phase HMNA intervention group (RT + HMNA) exhibited significantly reduced cardiac tissue damage and fibrosis, and improved cardiac function compared to the radiation treatment group (RT). Experiments conducted on cardiomyocytes (H9C2) and in the mouse model, utilizing western blot, qPCR, ELISA, and tissue immunofluorescence demonstrated that radiation induced a substantial release of HMGB1 from cardiomyocytes into the extracellular space and triggered overexpression of the membrane receptor TLR4. Western blot analysis demonstrated that the hearts of the RT group exhibited significantly elevated levels of TLR4, NF-κB, PAI-1, and collagen I compared to the control group. In contrast, these levels were significantly lower in the RT + HMNA group. CCK8 assay results indicated that the proliferation activity of myofibroblasts was significantly higher in the active HMGB1 culture group (DMEM + HMGB1) than in the group without HMGB1 (DMEM).</p> Conclusion <p>Early phase HMNA intervention during radiation exposure can effectively reduce fibrosis in RIMF, thereby improving cardiac function. Radiation induces cardiomyocytes to release substantial amounts of HMGB1 into the extracellular space. HMGB1 likely mediates the formation of RIMF through the HMGB1/TLR4/NF-kB/PAI-1 pathway in target cells and promotes myofibroblast proliferation.</p>