To investigate the differences in small intestinal toxicity and taxonomic composition, diversity, and functional pathways of gut microbiome and metabolome after different radiotherapies in mouse colorectal cancer (CRC) model.

Azoxymethane/dextran sodium sulfate (AOM/DSS)-induced mouse CRC model was treated with single pulse FLASH-RT (dose rate 100 Gy/s) or CONV-RT (dose rate 2 Gy/min) at whole abdomen. At 12 d after radiotherapy, sections of small intestinal tract tissue were dissected for hematoxylin and eosin (HE) staining and the fresh feces were collected for 16S ribosomal RNA (rRNA) microbiome sequencing and liquid chromatography and mass spectrometry (LC-MS) metabolomics sequencing to assess changes in the gut microbiota and metabolites. Microbial high-throughput 16S rRNA data was analyzed with QIIME2 and LEfSe softwares. ProteoWizard, XCMS and Ropls softwares were used for LC-MS analysis.

HE staining showed that FLASH-RT maintained small intestinal integrity and reduced the radiotherapy-induced injury. Sequencing analysis of gut fecal microbiome showed that phylum Bacteroidetes and genera Prevotella and Lactobacillus of microbial community were increased after FLASH-RT. Metabolomics sequencing analysis revealed that the metabolites after FLASH-RT were enriched in amino acid metabolism, while cholesterol metabolism was top enriched after CONV-RT.

FLASH-RT significantly mitigates the small intestine tissue damage compared with CONV-RT. FLASH-RT and CONV-RT have different impact on gut microbiota and its metabolites. Our results provide a theoretical basis for the early evaluation, prediction and individualized treatment of the irradiation effect after novel FLASH-RT on tumors through the evaluation of intestinal microbiota and metabolites.