Antibiotic Alleviates Radiation-Induced Intestinal Injury by Remodeling Microbiota, Reducing Inflammation, and Inhibiting Fibrosis

Abstract

Radiation-induced intestinal injury is a common complication of abdominal radiation therapy. However, the pathological features of radiation-induced intestinal injury and its therapeutic regimen are not very clear. The aim of this study was to investigate the effects of antibiotic pretreatment on radiation-induced intestinal injury. Abdominal radiation disrupted the intestinal microbiota balance and significantly reduced bacterial diversity in mice. Antibiotic cocktail (Abx) pretreatment effectively removed the intestinal microbiota of mice, and metronidazole also reduced the diversity of intestinal bacteria to some extent. Two antibiotic pretreatment regimens improved the reconstitution ability of the gut microbiota in mice after radiation. Further experiments showed that Abx pretreatment effectively reduced the content of lipopolysaccharide (LPS) and inhibited the TLR4/MyD88/NF-κB signaling pathway in the ileum. In addition, Abx pretreatment regulated macrophage cell polarization in the ileum, downregulated TGF-β1, phosphorylated Smad-3 and α-SMA protein levels, and upregulated E-cadherin protein expression. Eventually, Abx pretreatment significantly improved the survival rate and attenuated intestinal injury of mice after radiation by reducing inflammation and preventing intestinal fibrosis. These results revealed that antibiotic pretreatment can effectively alleviate gut microbiota turbulence and intestinal damage caused by abdominal radiation in mice. Collectively, these findings add to our understanding of the pathogenesis of radiation enteritis.

Figure 1

Abdominal radiation changes the gut microbiota of mice. (A) Venn diagram illustrating overlap of gut microbiota OTUs for pre- and postradiation groups. (B) Alpha-diversity of the gut microbiota community for Pre- and Postradiation groups. (C) PCA of gut microbiota for Pre- and Postradiation group. (D) Heat map analysis of gut microbiota for pre- and postradiation groups. The results were expressed as mean ± SEM. n = 4/3. *P < 0.05.

Figure 2

Antibiotic pretreatment 14d alters gut microbiota of mice (A) Venn diagram illustrating overlap of gut microbiota OTUs for Pre.Con14, Pre.MDE14, and Pre.Abx14 groups. (B) Alpha-diversity of the gut microbiota community for Pre.Con14, Pre.MDE14, and Pre.Abx14 groups. (C) PCA of gut microbiota for Pre.Con14, Pre.MDE14, and Pre.Abx14 groups. (D) Heat map analysis of gut microbiota for Pre.Con14, Pre.MDE14, and Pre.Abx14 groups. The results were expressed as mean ± SEM. n = 4. *P < 0.05.

Figure 3

Antibiotic pretreatment improves reconstruction of gut microbiota after radiation for 3 months. (A) Venn diagram illustrating overlap of gut microbiota OTUs for Post.Con14, Post.MDE14, and Post.Abx14 groups. (B) Alpha-diversity of the gut microbiota community for Post.Con14, Post.MDE14, and Post.Abx14 groups. (C) PCA of gut microbiota for Post.Con14, Post.MDE14, and Post.Abx14 groups. (D) Heat map analysis of gut microbiota for Post.Con14, Post.MDE14, and Post.Abx14 groups. The results were expressed as mean ± SEM. n = 3. *P < 0.05.

Figure 4

Antibiotic pretreatment elevates the survival rate of mice.

Figure 5

Antibiotic pretreatment promotes ileum apoptosis and proliferation in mice. (A) Protein expression of PCNA and cleaved caspase3 of the ileum in preradiation mice. (B) Protein expression of PCNA and cleaved caspase3 of the ileum of mice 1 month after radiation. (C) Protein expression of PCNA and cleaved caspase3 of the ileum of mice 3 months after radiation. The results were expressed as mean ± SEM. n = 5.*P < 0.05, **P < 0.01.

Figure 6

Antibiotic pretreatment reduces ileal fibrosis in mice. (A) Mason staining of the ileum (×100). (B) Sirius red staining of the ileum (×100). The results were expressed as mean ± SEM. n = 5. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 7

Antibiotic pretreatment inhibits the TLR4/MyD88/NF-κB p65 signaling pathway in the ileum of mice. (A) LPS content of the ileum of mice before radiation, 12 h after radiation, and 3d after radiation. (B) Protein expression of TLR4/MyD88/NF-κB p65 of the ileum in preradiation mice. (C) Protein expression of TLR4/MyD88/NF-κB p65 in the ileum of mice 12 h after radiation. (D) Protein expression of TLR4/MyD88/NF-κB p65 in the ileum of mice 3d after radiation. The results were expressed as mean ± SEM. n = 5. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 8

Antibiotic pretreatment reduces iNOS and CD163 protein expression in the ileum of mice. (A) Protein expression of iNOS and CD163 of the ileum in preradiation mice. (B) Protein expression of iNOS and CD163 in the ileum of mice 1 month after radiation. (C) Protein expression of iNOS and CD163 in the ileum of mice 3 months after radiation. The results were expressed as mean ± SEM. n = 3. **P < 0.01,***P < 0.001, ****P < 0.0001.

Figure 9

Antibiotic pretreatment regulates the TGF-β1/Smad-3/α-SMA/E-cadherin signaling pathway in the ileum of mice. (A) Protein expression of TGF-β1, Smad-3, α-SMA, and E-cadherin in the ileum of mice 1 month after radiation. (B) Protein expression of TGF-β1, Smad-3, α-SMA, and E-cadherin in the ileum of mice 3 months after radiation. The results were expressed as mean ± SEM. n = 3.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.