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. 2018 Jan 26;50(1):e433.
doi: 10.1038/emm.2017.246.

Hydrogen-water Ameliorates Radiation-Induced Gastrointestinal Toxicity via MyD88's Effects on the Gut Microbiota

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Free PMC article

Hydrogen-water Ameliorates Radiation-Induced Gastrointestinal Toxicity via MyD88's Effects on the Gut Microbiota

Hui-Wen Xiao et al. Exp Mol Med. .
Free PMC article

Abstract

Although radiation therapy is a cornerstone of modern management of malignancies, various side effects are inevitably linked to abdominal and pelvic cancer after radiotherapy. Radiation-mediated gastrointestinal (GI) toxicity impairs the life quality of cancer survivors and even shortens their lifespan. Hydrogen has been shown to protect against tissue injuries caused by oxidative stress and excessive inflammation, but its effect on radiation-induced intestinal injury was previously unknown. In the present study, we found that oral gavage with hydrogen-water increased the survival rate and body weight of mice exposed to total abdominal irradiation (TAI); oral gavage with hydrogen-water was also associated with an improvement in GI tract function and the epithelial integrity of the small intestine. Mechanistically, microarray analysis revealed that hydrogen-water administration upregulated miR-1968-5p levels, thus resulting in parallel downregulation of MyD88 expression in the small intestine after TAI exposure. Additionally, high-throughput sequencing showed that hydrogen-water oral gavage resulted in retention of the TAI-shifted intestinal bacterial composition in mice. Collectively, our findings suggested that hydrogen-water might be used as a potential therapeutic to alleviate intestinal injury induced by radiotherapy for abdominal and pelvic cancer in preclinical settings.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Oral gavage with hydrogen-water protects mice against radiation-induced toxicity. Mice were treated with hydrogen-water 2 days before and 7 days after receiving 15 Gy TAI. (a) Direct examination of H2 concentrations over an 8-h period. (b) A mouse treated with 15 Gy TAI (left) and its littermate without irradiation (right). Note the change in fur color in the irradiated lower body. (c) Kaplan–Meier analysis of hydrogen-water- and normal water-treated mice after 15 Gy TAI. P<0.05 by log-rank test between TAI-exposed mice with or without hydrogen-water treatment, n=12. (d) Body weight was compared between hydrogen-water- and saline-treated mice after 15 Gy TAI. n=12; **P<0.01; Student’s t-test. (e) The level of MDA in the small intestine was compared among the healthy control, 12 Gy TAI and hydrogen-water groups. n=12; *P<0.05; **P<0.01;***P<0.005; Student’s t-test. (f) The expression levels of Nrf2 were assessed in small intestine tissue from the healthy control, 12 Gy TAI and hydrogen-water groups. n=12; *P<0.05; Student’s t-test.
Figure 2
Figure 2
Hydrogen-water administration improves GI function and epithelial integrity of irradiated mice. (a and b) Counts of droppings removed from the cage bedding each day from representative cages, n=6 mice per cage. Statistically significant differences are indicated: ***P<0.005; Student’s t-test. (c) Examples of intestinal tract from a total abdominal irradiated mouse and a TAI-exposed mouse with hydrogen-water treatment. (d) The morphology of small intestine in radiation-induced mice treated with normal water or hydrogen-water is shown by H&E staining. (eg) The expression levels of Glut1, Pgk1 and MDR1 were assessed by qRT-PCR in small intestine tissue from TAI mice without or with hydrogen-water treatment. Statistically significant differences are indicated: ***P<0.005; Student’s t-test.
Figure 3
Figure 3
Hydrogen-water upregulates the level of MyD88-targeting miR-1968-5p in the mouse small intestines. (a) The expression level of MyD88 was examined in the aforementioned small intestine tissues by qRT-PCR. Statistically significant differences are indicated: **P<0.01; ***P<0.005; Student’s t-test. (b) The expression of MyD88 was examined by western blotting in TAI-exposed mice with or without hydrogen-water treatment. (c) Alterations in miRNA expression in small intestine tissues from mice without or with TAI were assessed using microarray analysis. (d) The expression level of miR-1968-5p was examined by qRT-PCR in small intestine tissues from mice without TAI (Control, n=20), mice with TAI (TAI, n=20) and mice with hydrogen-water treatment after TAI (TAI+HW, n=20) individually. Statistically significant differences are indicated: *P<0.05; **P<0.01; Student’s t-test. (e) The correlation between MyD88 mRNA expression and miR-1968-5p level was examined by qRT-PCR in 20 cases of small intestine tissues from mice with hydrogen-water treatment after TAI. **P<0.01; Pearson correlation coefficient, r=−0.7296. (f) The expression level of TLR4 was examined in the aforementioned small intestine tissues by qRT-PCR. Statistically significant differences are indicated: *P<0.05; ***P<0.005; Student’s t-test. (g) The expression level of TLR5 was examined in the aforementioned small intestine tissues by qRT-PCR. Statistically significant differences are indicated: *P<0.05; Student’s t-test.
Figure 4
Figure 4
MiR-1968-5p inhibits the expression of MyD88 by targeting its 3′UTR. (a and b) MiR-1968-5p inhibits the expression of MyD88 by targeting the predicted conserved miR-1968-5p-binding site at nucleotides 1595–1602 of the MyD88 3′UTR. The generated mutation sites at the MyD88 3′UTR seed region are indicated. The wild-type MyD88 3′UTR (or mutant) was inserted into the downstream of luciferase reporter gene in the pGL3-control vector. (c and d) The effect of miR-1968-5p (or anti-miR-1968-5p) on pGL3-MyD88 and pGL3- MyD88-mut reporters in 3T3 cells was measured by luciferase reporter assays. Statistically significant differences are indicated: *P<0.05; **P<0.01; Student’s t-test. (e and f) The effect of miR-1968-5p (or anti-miR-1968-5p) on the expression of MyD88 in 3T3 cells was measured by western blotting. Each experiment was repeated at least three times. NS, not significant.
Figure 5
Figure 5
Hydrogen-water treatment has no effect on the abundance of enteric bacteria. (a) The observed species number of intestinal bacteria in con and TAI-treated mice (with or without hydrogen-water oral gavage) was examined by 16S rRNA high-throughput sequencing after 5 days of TAI exposure. (b and c) The Shannon (b) and Simpson (c) diversity indices of intestinal bacteria in con and TAI-treated mice (with or without hydrogen-water oral gavage) were assessed by 16S rRNA high-throughput sequencing after 5 days of TAI exposure. For panels (ac), the top and bottom boundaries of each box indicate the 75th and 25th quartile values, respectively, and lines within each box represent the 50th quartile (median) value. Ends of whiskers mark the lowest and highest diversity values in each instance. (d) The relative abundance of enteric bacteria at the phylum level in con and TAI-treated mice (with or without oral gavage of hydrogen-water) was assessed using 16S high-throughput sequencing after irradiation at day 5. Statistically significant differences are indicated: Student’s t-test, n=4 per group.
Figure 6
Figure 6
Oral gavage with hydrogen-water retains the intestinal bacterial composition pattern impaired by TAI. (ac) Principal component and β diversity analyses were used to measure the shift in the intestinal bacterial composition profile in con and TAI-treated mice (with or without hydrogen-water oral gavage) after irradiation at day 5. Statistically significant differences are indicated: *P<0.05, ***P<0.001; Student’s t-test, n=4. For panel (b), the top and bottom boundaries of each box indicate the 75th and 25th quartile values, respectively, and lines within each box represent the 50th quartile (median) value. Ends of whiskers mark the lowest and highest diversity values in each instance. (d) Alterations in intestinal bacterial patterns at the genus level in con and TAI-treated mice (with or without hydrogen-water oral gavage) were assessed using 16S high-throughput sequencing after irradiation at day 5, n=4. The heatmap is color coded on the basis of row Z-scores. The mice with the highest and lowest bacterial levels are in red and blue, respectively. (e) The relative abundance of the top 10 bacteria at the genus level in con and TAI-treated mice (with or without hydrogen-water oral gavage) was assessed using 16S high-throughput sequencing after irradiation at day 5, n=4. (f) Linear discriminant analysis (LDA) effect size (LEfSe) results showed that the bacteria were significantly different in abundance between the TAI and hydrogen-water groups and indicated the effect size of each differentially abundant bacterial taxon in the small intestine (n=4). Statistically significant differences are indicated: Student’s t-test.

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