Ferroptosis is a newly recognized form of regulated cell death that is characterized by severe lipid peroxidation initiated by iron overload and the generation of reactive oxygen species (ROS). However, the role of iron in ionizing radiation (IR)-induced intestinal injury has not been fully illustrated yet. In this study, we found that IR induced ferroptosis in intestinal epithelial cells, as indicated by the increase in intracellular iron levels and lipid peroxidation, upregulation of prostaglandin-endoperoxide synthase 2 (PTGS2) mRNA, reduced glutathione peroxidase 4 (GPX4) mRNA and glutathione (GSH) levels, and significant mitochondrial damage. In addition, the iron chelator deferoxamine (DFO) attenuated IR-induced ferroptosis and intestinal injury in vitro and in vivo. Intriguingly, pharmacological inhibition of autophagy with 3-methyladenine (3-MA) mitigated IR-induced ferritin downregulation, iron overload and ferroptosis. IR increased the levels of nuclear receptor coactivator 4 (NCOA4) mRNA and protein. NCOA4 knockdown significantly inhibited the reduction of ferritin, decreased the level of intracellular free iron, and mitigated ferroptosis induced by IR in HIEC cells, indicating that NCOA4-mediated autophagic degradation of ferritin (ferritinophagy) was required for IR-induced ferroptosis. Furthermore, cytoplasmic iron further activated mitoferrin2 (Mfrn2) on the mitochondrial membrane, which in turn increased iron transport into the mitochondria, resulting in increased ROS production and ferroptosis. In addition, mice fed with an iron-deficient diet for 3 weeks showed a significant reversal in the intestinal injury induced by abdominal IR exposure. Taken together, ferroptosis is a novel mechanism of IR-induced intestinal epithelial cytotoxicity, and is dependent on NCOA4-mediated ferritinophagy.

Radiation-induced intestinal injury (RIII) occurs during instances of intentional or accidental radiation exposure. However, there are few effective treatments available for the prevention or mitigation of RIII currently. (-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, possesses potent antioxidant activity and has been shown to be effective in ameliorating many oxidative stress-related diseases. The therapeutic effects and mechanism of EGCG on RIII have not yet been determined. In the present study, we investigated whether EGCG confers radioprotection against RIII. Our data demonstrated that administration of EGCG not only prolonged the survival time of lethally irradiated mice, but also reduced radiation-induced intestinal mucosal injury. Treatment with EGCG significantly increased the number of Lgr5⁺ intestinal stem cells (ISCs) and their progeny Ki67⁺ cells, and reduced radiation-induced DNA damage and apoptosis. Besides, EGCG displayed the same radioprotective effects in human intestinal epithelial HIEC cells as in mice, characterized by a decrease in the number of γH2AX foci and ferroptosis. Moreover, EGCG decreased the level of reactive oxygen species (ROS) and activated the transcription factor Nrf2 and its downstream targets comprising antioxidant proteins Slc7A11, HO-1 and GPX4. Treatment with the Nrf2 inhibitor ML385 abolished the protective effects of EGCG, indicating that Nrf2 activation is essential for EGCG activity. Taken together, our findings demonstrated that EGCG protects against RIII by scavenging ROS and inhibiting apoptosis and ferroptosis through the Nrf2 signal pathway, which could be a promising medical countermeasure for the alleviation of RIII.

The intestine is prone to radiation damage in patients undergoing radiotherapy for pelvic tumors. However, there are currently no effective drugs available for the prevention or treatment of radiation-induced enteropathy (RIE). In this study, we aimed at investigating the impact of indole-3-carboxaldehyde (I3A) derived from the intestinal microbiota on RIE. Intestinal organoids were isolated and cultivated for screening radioprotective tryptophan metabolites. A RIE model was established using 13 Gy whole-abdominal irradiation in male C57BL/6J mice. After oral administration of I3A, its radioprotective ability was assessed through the observation of survival rates, clinical scores, and pathological analysis. Intestinal stem cell survival and changes in the intestinal barrier were observed through immunofluorescence and immunohistochemistry. Subsequently, the radioprotective mechanisms of I3A was investigated through 16S rRNA and transcriptome sequencing, respectively. Finally, human colon cancer cells and organoids were cultured to assess the influence of I3A on tumor radiotherapy. I3A exhibited the most potent radioprotective effect on intestinal organoids. Oral administration of I3A treatment significantly increased the survival rate in irradiated mice, improved clinical and histological scores, mitigated mucosal damage, enhanced the proliferation and differentiation of Lgr5⁺ intestinal stem cells, and maintained intestinal barrier integrity. Furthermore, I3A enhanced the abundance of probiotics, and activated the AhR/IL-10/Wnt signaling pathway to promote intestinal epithelial proliferation. As a crucial tryptophan metabolite, I3A promotes intestinal epithelial cell proliferation through the AhR/IL-10/Wnt signaling pathway and upregulates the abundance of probiotics to treat RIE. Microbiota-derived I3A demonstrates potential clinical application value for the treatment of RIE.

The acute radiation-induced intestinal injury (RIII) has raised much concerns and is influenced by non-cytocidal radiation effects including the perturbations in gut microbiota. Although a number of studies have reported alteration in gut microbiota following radiation, little is known about its dynamic variation in the progression of acute RIII. In this study, mouse model were treated with total body irradiation (TBI) of 0, 4, 8 and 12 Gy, and the intestinal tissues and fecal samples were collected at 6 h, 3.5 d and 7 d post radiation. We found that the intestinal injuries were manifested in a radiation dose-dependent manner. Results from 16S rRNA gene sequencing demonstrated that the diversity of gut microbiota was not significantly affected at the prodromal stage of acute RIII, after 6 h of radiation. At the critical stage of acute RIII, after 3.5 d of radiation, the composition of gut microbiota was correlated with the radiation dose. The Pearson’s correlation analysis showed that the relative abundances of phylum Proteobacteria, genera Escherichia-Shigella and Eubacterium xylanophilum_group, and species Lactobacillus murinus exhibited linear correlations with radiation dose. At the recovery stage of acute RIII, after 7 d of radiation, the diversity of gut microbiota decreased as a whole, among which the relative abundance of phyla Proteobacteria and Bacteroides increased, while that of phylum Tenericutes and genus Roseburia decreased. The intra-gastric administration of compound probiotics for 14 days improved the survival duration of mice exposed to 9 Gy TBI, alleviated the intestinal epithelial injury and partially restored the diversity of gut microbiota. Our findings suggest that acute RIII is accompanied by the dysbiosis of gut microbiota, including its decreased diversity, reduced abundance of beneficial bacteria and increased abundance of pathogens. The gut microbiota cannot be used as sensitive biomarkers at the prodromal stage in acute RIII, but are potential biomarkers at the critical stage of acute RIII. The dysbiosis is persistent until the recovery stage of acute RIII, and interventions are needed to restore it. The administration of probiotics is an effective strategy to protect against acute RIII and subsequent dysbiosis.

Colorectal cancer (CRC) is still one of the most important neoplasias causing human death. Multidisciplinary therapy has won consensus in the management of CRC, of which, radiotherapy occupies an important position. However, radioresistance is still a major obstacle in local control of CRC. Overexpression of long non-coding RNA HOTAIR has been found to correlate with tumorigenesis and poor prognosis in several types of cancer. In the present study, we analyzed HOTAIR expression levels of 53 CRC patients in tumor and adjacent normal tissue by real-time quantitative PCR. Knockdown of HOTAIR by RNA interference was performed to explore its roles in cell proliferation, migration, invasion, apoptosis and radiosensitivity. Results showed that CRC patients had higher HOTAIR expression in tumor tissues compared with adjacent normal tissues. In vitro, downregulation of HOTAIR reduced proliferation, migration and invasiveness while enhanced apoptosis and radio-sensitivity of CRC cells. Taken together, our findings suggest that long non-coding RNA HOTAIR expression is closely associated with tumor invasion and radiosensitivity, indicating the potential role in diagnostics and therapeutics of CRC.

Radiation-induced intestinal injury (RIII) commonly occur during abdominal-pelvic cancer radiation therapy; however, no effective prophylactic or therapeutic agents are available to manage RIII currently. This study aimed to clarify the potential of probiotic consortium supplementation in alleviating RIII. Male C57BL/6J mice were orally administered a probiotic mixture comprising Bifidobacterium longum BL21, Lactobacillus paracasei LC86, and Lactobacillus plantarum Lp90 for 30 days before exposure to 13 Gy of whole abdominal irradiation. The survival rates, clinical scores, and histologic changes in the intestines of mice were assessed. The impacts of probiotic consortium treatment on intestinal stem cell proliferation, differentiation, and epithelial barrier function; oxidative stress; and inflammatory cytokines were evaluated. A comprehensive examination of the gut microbiota composition was conducted through 16S rRNA sequencing, while changes in metabolites were identified using liquid chromatography-mass spectrometry. The probiotic consortium alleviated RIII, as reflected by increased survival rates, improved clinical scores, and mitigated mucosal injury. The probiotic consortium treatment exhibited enhanced therapeutic effects at the histologic level compared with individual probiotic strains, although there was no corresponding improvement in survival rates and colon length. Moreover, the probiotic consortium stimulated intestinal stem cell proliferation and differentiation, enhanced the integrity of the intestinal epithelial barrier, and regulated redox imbalance and inflammatory responses in irradiated mice. Notably, the treatment induced a restructuring of the gut microbiota composition, particularly enriching short-chain fatty acid-producing bacteria. Metabolomic analysis revealed distinctive metabolic changes associated with the probiotic consortium, including elevated levels of anti-inflammatory and antiradiation metabolites. The probiotic consortium attenuated RIII by modulating the gut microbiota and metabolites, improving inflammatory symptoms, and regulating oxidative stress. These findings provide new insights into the maintenance of intestinal health with probiotic consortium supplementation and will facilitate the development of probiotic-based therapeutic strategies for RIII in clinical practice.