Abstract

Radiation proctitis (RP) is a frequent complication of pelvic radiotherapy that compromises treatment delivery and patient quality of life, yet the factors shaping injury severity remain incompletely defined. We prospectively profiled pretreatment fecal microbiomes and metabolomes from 55 patients and stratified them by outcome into mild versus severe RP. Baseline microbial composition showed Bacteroidales enriched in severe RP and Firmicutes enriched in mild cases. Multi-omics integration highlighted nicotinate/nicotinamide pathways; severe RP was characterized by concomitant reductions in both fecal and tissue NAD⁺ levels, along with an enrichment of microbial nicotinate/nicotinamide metabolism genes, primarily contributed by Bacteroides ovatus, B. xylanisolvens, and B. fragilis. In mice, fecal microbiota transplantation from severe-RP donors exacerbated radiation-induced colorectal injury and decreased colorectal NAD⁺, supporting a causal role for the microbiota. Gavage with Bacteroides similarly worsened pathology and lowered NAD⁺, whereas nicotinamide mononucleotide (NMN) supplementation attenuated the injury. Mechanistically, Bacteroides gavage reduced mitochondrial membrane potential, decreased the Lgr5⁺ stem-cell proportion and proliferative indices, associated with Wnt pathway modulation. NMN reversed these effects in parallel with NAD⁺ restoration. Together, these results identify a microbiota‒metabolite association wherein Bacteroidales enrichment is associated with NAD⁺ depletion, reduced mucosal proliferative capacity, and exacerbated radiation-induced colorectal injury. The work deepens insight into RP pathogenesis and suggests a potential basis for microbiome- and metabolite-targeted approaches to attenuate severe RP.