D-galactose protects the intestine from ionizing radiation-induced injury by altering the gut microbiome

Abstract

This article aims to investigate the protection of the intestine from ionizing radiation-induced injury by using D-galactose (D-gal) to alter the gut microbiome. In addition, this observation opens up further lines of research to further increase therapeutic potentials. Male C57BL/6 mice were exposed to 7.5 Gy of total body irradiation (TBI) or 13 Gy of total abdominal irradiation (TAI) in this study. After adjustment, D-gal was intraperitoneally injected into mice at a dose of 750 mg/kg/day. Survival rates, body weights, histological experiments and the level of the inflammatory factor IL-1β were observed after TBI to investigate radiation injury in mice. Feces were collected from mice for 16S high-throughput sequencing after TAI. Furthermore, fecal microorganism transplantation (FMT) was performed to confirm the effect of D-gal on radiation injury recovery. Intraperitoneally administered D-gal significantly increased the survival of irradiated mice by altering the gut microbiota structure. Furthermore, the fecal microbiota transplanted from D-gal-treated mice protected against radiation injury and improved the survival rate of recipient mice. Taken together, D-gal accelerates gut recovery following radiation injury by promoting the growth of specific microorganisms, especially those in the class Erysipelotrichia. The study discovered that D-gal-induced changes in the microbiota protect against radiation-induced intestinal injury. Erysipelotrichia and its metabolites are a promising therapeutic option for post-radiation intestinal regeneration.

Keywords: D-galactose (D-gal); Radiation injury; fecal microbiota transplantation; gut microbiota.

Fig. 1

D-gal injection increased the survival rate of irradiated mice and restored the structure of the mouse small intestine. (A) Experimental outline of irradiation and D-gal administration. D-gal was injected starting 7 days before irradiation and 7 days after TBI. (B-C) Kaplan–Meier survival curve (B) and body weights (C) of mice in the saline- and D-gal-treated groups after 7.5 Gy of TBI; There are 36 mice in each group. P = 0.0137 in (B). The significance analysis were obtained from Graphpad analysis by Log-rank (Mantel-Cox) test. (D) Representative images of =H&E-stained small intestine sections from the indicated groups. (E–I) The transcript levels of Glut1 (E), Mdr1 (F), Mus2 (G), Pgk1 (H) and Tff3 (I) in the small intestine of mice exposed to the indicated treatments. The error bars in graphs means standard deviation of indicated data. ^*  P < 0.05, ^**  P < 0.01; n = 12 animals per group. Data were analyzed for differences using independent samples t-test. Stars without a horizontal line were generated by comparing the IR-group in indicated treatment. Stars with a horizontal line mean that the two groups were compared. (J) Representative imagesof colons extracted from mice exposed to the indicated treatments on Day 15 after TBI. (K) The length of colons in the indicated groups. The error bars in graphs means standard deviation of indicated data. ^**  P < 0.01; n = 12 mice per group. Data were analyzed for differences using independent samples t-test. Stars without a horizontal line were generated by comparing the ‘Saline IR-’ group.

Fig. 2

D-gal injection alleviated radiation-induced small intestinal injury in mice. (A) Representative images of immunohistochemical staining showing the in situ expression of Lgr5 in the intestinal crypts of mice exposed to the indicated treatments. Small intestines were collected on the fifteenth day after the administration of 7.5 Gy of TBI and stained with H&E. (B) Statistical analysis of Lgr5-positive cells in immunohistochemistry assays. Data were collected from more than six images in the same field of view. The area fraction of Lgr5+ cells was analyzed using ImageJ software. The error bars in graphs means standard deviation of indicated data. (C) Representative images of immunofluorescence staining showing Ki67-positive cells in the small intestine of the indicated groups (red, Ki67; blue, DAPI). Yellow arrows indicate the location of Ki67. The small intestines were collected on the fifteenth day after irradiation in each group. The images were scanned and captured using a laser confocal microscope (Leica). (D) Statistical analysis of Ki67-positive cells in each group. Data were collected from more than six images in the same field of view. The data were analyzed using ImageJ software. The error bars in graphs means standard deviation of indicated data. (E) IL-1β levels in intestinal homogenates measured using ELISA. Small intestines were collected on the 15^th day after the administration of 7.5 Gy of TBI. The procedure was performed according to the instructions provided with the ELISA kit. Data were analyzed using a microplate reader. The error bars in graphs means standard deviation of indicated data. ^**  P < 0.01; n.s., not significant; n = 12 mice per group. Data in (B), (D) and (E) were analyzed for differences using independent samples t-test. Stars without a horizontal line were generated by comparing the ‘Saline IR-’ group. Stars with a horizontal line mean that the two groups were compared.

Fig. 3

D-gal injection influenced the microorganism structure of the gut microbiota in irradiated mice. (A–C) PCA analysis of the gut microbiota in mice from the ‘saline IR+’ and ‘D-gal IR+’ groups on Day 0 (A), Day 5 (B) and Day 10 (C) post-irradiation; n ≥ 10 mice per group. (D–F) PCoA analysis of the gut microbiota in mice from the ‘saline IR+’ and ‘D-gal IR+’ groups on Day 0 (D), Day 5 (E) and Day 10 (F) post-irradiation; n ≥ 10 mice per group. The difference in beta diversity was indicated by PERMANVO and is shown as the P value in the PCoA plot.

Fig. 4

D-gal altered the dominant bacteria in the intestinal microbiome. (A) Intestinal flora composition at genus level 10 days post-irradiation in ‘Saline IR-’ and ‘Saline IR+’ groups. (B) Linear discriminant analysis effect size (LEfSe) of gut microbiota in the saline group before and after 13Gy TAI. (C) Intestinal flora composition at the genus level measured at 10 days post-irradiation in the ‘Saline IR-’ and ‘D-gal IR-’ groups. (D) LEfSe of gut microbiota in the ‘Saline IR-’ and ‘D-gal IR-’ groups before and after the administration of 13 Gy of TAI. (E) Intestinal flora composition at the genus level measured at 10 days post-irradiation in the ‘Saline IR+’ and ‘D-gal IR+’ groups. (F) LEfSe of the gut microbiota in the aforementioned groups.

Fig. 5

FMT from D-gal-treated mice alleviated radiation-induced small intestine injury. (A) Schematic of the FMT assay. Mice were provided antibiotics in the drinking water for 4 weeks. Beginning on the day before irradiation, recipient mice were administered the microbiota mix from donor mice by oral gavage daily for 10 days. (B-C) Kaplan–Meier survival curve (B) and body weight (C) of mice in the FMT-saline and FMT-D-gal groups after the administration of 7.5 Gy of TBI; The number of mice in each group are as followed: Control: n = 37; FMT-Saline IR+: n = 36, P = 0.0015; FMT-D-gal IR+: n = 54, P < 0.0001. (D) Representative images of HE-stained small intestine sections from the indicated groups. Small intestines were collected on the seventh day after the administration of 13 Gy of TAI. Tissues were collected from at least 6 mice in each group. (E–I) The transcript levels of Glut1 (E), Mdr1 (F), Mus2 (G), Pgk1 (H) and Tff3 (I) in the small intestine of mice in different groups. The error bars in graphs means standard deviation of indicated data. ^*  P < 0.05, ^**  P < 0.01; n ≥ 7 mice per group. (J–K) IL-1β and IL-6 levels in intestinal homogenates measured using ELISA. The error bars in graphs means standard deviation of indicated data. ^*  P < 0.05, ^**  P < 0.01; n ≥ 7 mice per group. Data in (E)–(K) were analyzed for differences using independent samples t-test. Stars without a horizontal line were generated by comparing the IR-group in indicated treatment (E–I) or ‘Control IR-’ group (J and K). Stars with a horizontal line mean that the two groups were compared.

Fig. 6

D-gal altered the transcriptome of HIEC-6 cells. (A) Differentially expressed genes in subcluster 3 in the indicated groups. (B) Variations in the expression of 1073 genes identified in (A). (C) The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the genes shown in (A). (D) Differentially expressed genes in subcluster seven in the indicated groups. (E) Variations in the expression of the 663 genes identified in (D). (F) KEGG pathway enrichment analysis of the genes shown in (D).