Mechanism of blueberry anthocyanins in ameliorating radiation-induced intestinal injury through gut microbiota modulation.

Abstract

Radiation-induced intestinal injury denotes structural and functional impairment of the intestines resulting directly or indirectly from ionizing radiation, such as that employed in radiation therapy for malignant tumors in the abdominal or pelvic regions, within the treatment area. It is a prevalent and severe consequence of radiation therapy for abdominal and pelvic tumors, greatly affecting treatment outcomes and patients' quality of life. Western medicine primarily employs symptomatic management, including medical drug therapy, hyperbaric oxygen therapy, nutritional support therapy, endoscopic and surgical interventions, as well as stem cell transplantation. However, current therapeutic agents for RIII fall far short of achieving ideal outcomes. Previous studies indicate that blueberry anthocyanins (BA) possess not only anticancer properties but also potent anti-inflammatory, antioxidant, and anti-radiation activities. Seventy male C57BL/6J mice (6-8 weeks old) were categorized into five groups: a normal control group (Con), an irradiation-only group (IR), groups administered low-dose BA prior to and following irradiation (IR+BA-L, 100 mg/kg bw), a high-dose BA group (IR+BA-H, 200 mg/kg bw), and a group receiving the clinical radiation protection agent amphotericin (IR+WR-2721, 30 mg/kg bw). Furthermore, a group for antibiotic-induced gut microbiota depletion and a validation group for fecal microbiota transplantation (FMT) were established. A model of radiation-induced intestinal damage was created utilizing a single 14 Gy dose of abdominal X-ray irradiation. The irradiation field encompassed a 2.5 cm by 20 cm region extending from the pubic symphysis to the xiphoid process. BA was supplied through oral gavage from 14 days prior to irradiation until 3.5 days post-irradiation at a dose of 0.2 mL/day per mouse, whereas the normal control group received saline via gavage for 14 consecutive days. Tissue samples were obtained 3.5 days post-irradiation. Through the assessment of survival rates, body mass, intestinal histopathology, inflammatory cytokine concentrations (IL-1β, IL-6, TNF-α), oxidative stress indicators (SOD, MDA), and intestinal barrier proteins (occludin, claudin-1), alongside immunohistochemical analysis of the proliferative cell marker Ki67 and the Paneth cell marker lysozyme, the proliferation and differentiation of intestinal stem cells will be evaluated. Furthermore, 16S rDNA sequencing was executed on colonic contents, and non-targeted metabolomics analysis was undertaken on colonic tissue to assess alterations in gut microbiota and host metabolism. Mice treated with BA intervention exhibited significant alleviation of intestinal injury, manifested as reduced weight loss, increased survival rate, markedly elongated colon length, substantially mitigated intestinal damage, and elevated histological scores. Concurrently, BA therapy downregulated levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6, while decreasing elevated SOD content and MDA levels. Immunohistochemical analysis revealed that BA intervention significantly increased Ki67 + proliferating cells and Lysozyme + Paneth cells in the crypt region of the colon. Western blot analysis demonstrated that BA upregulated the levels of intestinal tight junction proteins Occludin and Claudin-1. 16S rDNA sequencing revealed that BA improved gut microbiota composition by increasing the abundance of beneficial bacteria and reducing pathogenic bacteria. Metabolomic analysis revealed altered metabolic patterns in mouse colon tissue following both radiation exposure and BA intervention. BA restored levels of 53 potential biomarkers. Finally, fecal microbiota transplantation (FMT) validated BA's protective effect against RIII via the gut microbiota. The results of this study show that blueberry anthocyanin (BA) exerts significant protective effects against radiation-induced intestinal injury (RIII). It ameliorates RIII by modulating gut microbiota composition and associated metabolites. FMT experiments further validate that BA's protective action depends on gut microbiota regulation. The findings suggest that BA, as a gut microbiota-modulating agent, holds promise for preventing and treating RIII.