Exploring the mechanisms of protective effect of high-energy X-ray FLASH radiotherapy on intestine through multi omics analysis.

BACKGROUND: The aim of this study is to investigate the potential mechanisms underlying the protective effects of high-energy X-ray FLASH radiotherapy (FLASH-RT) on intestine through multi-omics analysis. METHODS: This study utilized syngeneic colon carcinoma mouse models of CT26 and MC38 to evaluate the therapeutic efficacy of FLASH-RT versus conventional dose rate radiotherapy (CONV-RT) by monitoring survival, tumor size, and body weight. Furthermore, healthy C57BL/6 female mice received whole-abdominal irradiation with either FLASH-RT, CONV-RT, or sham irradiation to compare differences in normal tissue protection. 72 h post-irradiation, intestinal contents from mice were collected for metagenomic analysis, and intestinal tissue was harvested for non-targeted metabolic and single-cell sequencing analyses. RESULTS: In CT26 and MC38 models, both CONV-RT and FLASH-RT have demonstrated similar anti-tumor efficacy. Compared with CONV-RT, whole-abdominal FLASH-RT significantly alleviated acute intestinal injury in mice, as evidenced by better preservation of crypt numbers and villus architecture in the FLASH group. Metagenomic analysis revealed that the relative abundance of the gut-protective bacterium Ligilactobacillus ruminis was significantly higher in the FLASH group than in the CONVgroup. Non-targeted metabolomic profiling identified 34 differential metabolites, of which 29 were upregulated and 5 were downregulated in the FLASH group. Notably, the abundance of 2-hydroxyglutarate, a metabolite associated with the butyrate metabolism pathway, was significantly elevated in the FLASH group compared with the CONV group (p < 0.05). Single-cell sequencing data revealed notable differences in cell distribution and proportions between the groups, with a higher proportion of fibroblasts, proliferative cells, macrophages, and CD4 + T cells in the FLASH group compared to the CONV and control groups. Immunofluorescence analysis revealed a significantly greater number of Lgr5⁺ intestinal stem cells in the FLASH group compared to the CONV group. Conversely, immunohistochemical analysis demonstrated stronger p50/p65 staining intensity in the CONV group relative to the FLASH group. CONCLUSIONS: This study confirms that FLASH-RT, compared to CONV-RT, maintains equivalent antitumor efficacy while mitigating damage to normal intestinal tissues. Moreover, it preliminarily reveals that the protective mechanism of FLASH-RT is multifaceted, involving remodeling of the microbiota-metabolite axis, attenuation of inflammatory responses, and enhanced preservation of stem cells.