Alteration of Gut Microbiota and Inflammatory Cytokine/Chemokine Profiles in 5-Fluorouracil Induced Intestinal Mucositis

Abstract

Disturbed homeostasis of gut microbiota has been suggested to be closely associated with 5-fluorouracil (5-Fu) induced mucositis. However, current knowledge of the overall profiles of 5-Fu-disturbed gut microbiota is limited, and so far there is no direct convincing evidence proving the causality between 5-Fu-disturbed microbiota and colonic mucositis. In mice, in agreement with previous reports, 5-Fu resulted in severe colonic mucositis indicated by weight loss, diarrhea, bloody stool, shortened colon, and infiltration of inflammatory cells. It significantly changed the profiles of inflammatory cytokines/chemokines in serum and colon. Adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and VE-Cadherin were increased. While tight junction protein occludin was reduced, however, zonula occludens-1 (ZO-1) and junctional adhesion molecule-A (JAM-A) were increased in colonic tissues of 5-Fu treated mice. Meanwhile, inflammation related signaling pathways including NF-κB and mitogen activated protein kinase (MAPKs) in the colon were activated. Further study disclosed that 5-Fu diminished bacterial community richness and diversity, leading to the relative lower abundance of Firmicutes and decreased Firmicutes/Bacteroidetes (F/B) ratio in feces and cecum contents. 5-Fu also reduced the proportion of Proteobacteria, Tenericutes, Cyanobacteria, and Candidate division TM7, but increased that of Verrucomicrobia and Actinobacteria in feces and/or cecum contents. The fecal transplant from healthy mice prevented body weight loss and colon shortening of 5-Fu treated mice. In addition, the fecal transplant from 5-Fu treated mice reduced body weight and colon length of vancomycin-pretreated mice. Taken together, our study demonstrated that gut microbiota was actively involved in the pathological process of 5-Fu induced intestinal mucositis, suggesting potential attenuation of 5-Fu induced intestinal mucositis by manipulating gut microbiota homeostasis.

Keywords: 5-fluorouracil; fecal transplantation; gut microbiota; inflammatory chemokines/cytokines; intestinal mucositis.

Figure 1

5-Fu induced mucositis and colon shortening in mice. (A) 5-Fu induced body weight changes. Data were plotted as percentage of initial body weight. (B) The occurrence of diarrhea. Data represented the evaluation scores of diarrhea. (C) The bloody stool events measured by BASO testing paper. (D) The small intestine length. (E,F) The colon length. (G) HE staining of colonic sections. (H) MPO staining of colonic sections. Values were expressed as mean ± S.E.M (n = 10/group). Data were analyzed by t-test. ^*P < 0.05, ^***P < 0.001 vs. control group.

Figure 2

5-Fu induced the alteration of inflammatory chemokines/cytokines in colonic tissues or serum at protein and gene expression levels. (A) The alteration of inflammatory chemokines/cytokines in colonic tissure measured by protein chip analysis. (B–D) The inflammatory chemokines/cytokines gene expression measured by qPCR assay. (E,F) 5-Fu induced the alteration of inflammatory chemokines/cytokines in serum. (G,H) 5-Fu induced the alteration of inflammatory chemokines/cytokines in colonic tissues. Values were expressed as mean ± S.E.M (n = 10/group). Data were analyzed by t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. control group.

Figure 3

5-Fu regulated the expression of tight junction and adhesion molecules in colonic tissues. (A) 5-Fu regulated gene expression of tight junction and adhesion molecule measured by qPCR analysis (n = 6/group). (B,C) 5-Fu regulated protein expression of occludin, VCAM-1, ICAM-1, JAM-A, and ZO-1(n = 3-4/group). β-actin was used as the endogenous reference. Values were expressed as mean ± S.E.M. Data were analyzed by t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. control group.

Figure 4

5-Fu activated NF-κB and MAPK signaling pathways in colonic tissues. (A,B) 5-Fu treatment enhanced the protein expression of p-ERK1/2, p-JNK, and p-p38 MAPK. (C,D) 5-Fu treatment elevated the protein expression of iNOS, p-NF-κB, and p-I-κB. (E) 5-Fu treatment increased the expression of activated NF-κB in the colonic epithelial cells. Values were expressed as mean ± S.E.M (n = 3/group). Data were analyzed by t-test. ^**P < 0.01, ^***P < 0.001 vs. control group.

Figure 5

5-Fu decreased the richness and diversity of gut microbiota in feces and cecum contents of mice. (A) 5-Fu decreased the richness of gut microbiota in feces and cecum contents. Chao is an estimator of the community richness. (B,C) 5-Fu decreased the diversity of gut microbiota in feces (B, Shannon index curves and Shannon diversity histogram) and cecum contents (C, Shannon index curves and Shannon diversity histogram). (D,E) The unweighted UniFrac PCoA results of feces (D) and cecum contents (E) for beta-diversity at OTUs level. Values were expressed as mean ± S.E.M (n = 5/group, feces; n = 10/group, cecum contents). Data were analyzed by t-test. ^**P < 0.01, ^***P < 0.001 vs. control group.

Figure 6

5-Fu treatment changed the gut microflora community composition at phylum level in feces and cecum contents of mice. (A) The bar diagram of bacterial community distribution in cecum contents (n = 10/group) and feces (n = 5/group) at phylum level, respectively. (B) The pie charts depicting the mean relative abundance for feces and cecum contents at phylum level, respectively. (C) 5-Fu treatment down-regulated the ratio of F/B in cecum contents and feces. (D) The correlation between body weight changes and F/B ratio in cecum contents. (E) The correlation between body weight changes and F/B ratio in feces. Values were expressed as mean ± S.E.M (n = 5/group, feces; n = 10/group, cecum contents). Data were analyzed by t-test. ^*P < 0.05, ^***P < 0.001 vs. control group.

Figure 7

Disturbed gut microbiota resulted in body weight loss and colon shortening in 5-Fu treated mice. (A) Normal fecal microbiota transplantation inhibited 5-Fu induced body weight loss. Data plotted as percentage of initial body weight. (B,C) The effect of normal fecal microbiota transplantation on colon length. Values were expressed as mean ± S.E.M (n = 7/group). Data were analyzed by t-test. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. 5-Fu group. (D) Fecal microbiota transplantation from 5-Fu treated mice induced body weight loss in mice pretreated with vancomycin. Data were plotted as percentage of initial body weight. (E,F) The effect of fecal microbiota transplantation from 5-Fu treated mice on colon length in vancomycin pretreated mice. Values were expressed as mean ± S.E.M (n = 10/group). Data were analyzed by t-test. ^*P < 0.05, ^**P < 0.01 vs. control group.