Preliminary mechanistic exploration of mitochondrial function in intestinal protection mediated by high-energy X-ray FLASH radiotherapy

Purpose Ultra-high dose rate (UHDR) radiation retains tumor-killing efficacy while reducing toxicity to normal tissues, holding a promising transformative radiotherapy paradigm. This study aimed to explore the potential role of mitochondria in intestinal protection conferred by high-energy X-ray FLASH radiotherapy (FLASH-RT) and the associated signaling pathways. Method The entire abdomen of healthy female C57BL/6 mice was irradiated using three modes: ultra-high dose rate irradiation (FLASH-RT), conventional dose rate irradiation (CONV-RT), and sham irradiation (Control). Mouse survival status and body weight changes were monitored within 15 days post-irradiation. At 72 hours post-irradiation, whole blood was collected for hematological analysis, and intestinal tissues were harvested for pathological detection, transmission electron microscopy (TEM) observation of mitochondrial changes, and two types of mitochondria- targeted metabolomic assays. Results A Compact single High-energy X-ray Source FLASH-RT device(CHEx-FLASH) was used, with a dose rate of 200 Gy/s. At 15 days post-irradiation, the survival rates of the Control group (100%, 10/10) and FLASH-RT group (80%, 8/10) were significantly higher than that of the CONV-RT group (30%, 2/10). Body weight decreased in the early post-irradiation period across groups, but the decline was milder in FLASH-RT with greater late-stage recovery. Hematological results at 72 hours showed that CONV-RT induced more severe bone marrow suppression compared to FLASH-RT. Intestinal histopathological analysis revealed that FLASH-RT alleviated intestinal inflammation and promoted enterocyte proliferation, while DNA double-strand breaks and apoptosis levels did not differ significantly between the two irradiated groups. FLASH-RT mitigated mitochondrial damage, reduced reactive oxygen species (ROS) levels and mildly activated mitophagy. Mitochondria-related energy metabolomics sequencing of intestinal tissues showed that the mitochondrial damage marker malonic acid was significantly lower in FLASH-RT than in CONV-RT, and differentially expressed metabolites were primarily enriched in mitochondrial antioxidant pathways. Additionally, the increased expression of the antioxidant protein NRF2 and superoxide dismutase ༈SOD) were verified. Conclusion CHEx-FLASH achieves UHDR irradiation and alleviates radiation-induced intestinal injury. The protective effect of FLASH-RT on intestinal tissues may be mediated by mitigating mitochondrial damage and enhancing antioxidant pathways through improved mitochondrial energy metabolism.