The Anti-Inflammatory Effect and Mucosal Barrier Protection of Clostridium butyricum RH2 in Ceftriaxone-Induced Intestinal Dysbacteriosis

Abstract

This study aimed at determining the beneficial effect of Clostridium butyricum (CB) RH2 on ceftriaxone-induced dysbacteriosis. To this purpose, BALB/c mice were exposed to ceftriaxone (400 mg/ml) or not (control) for 7 days, and administered a daily oral gavage of low-, and high-dose CB RH2 (10⁸ and 10¹⁰ CFU/ml, respectively) for 2 weeks. CB RH2 altered the diversity of gut microbiota, changed the composition of gut microbiota in phylum and genus level, decreased the F/B ratio, and decreased the pro-inflammatory bacteria (Deferribacteres, Oscillibacter, Desulfovibrio, Mucispirillum and Parabacteroides) in ceftriaxone-treated mice. Additionally, CB RH2 improved colonic architecture and intestinal integrity by improving the mucous layer and the tight junction barrier. Furthermore, CB RH2 also mitigated intestinal inflammation through decreasing proinflammatory factors (TNF-α and COX-2) and increasing anti-inflammatory factors (IL-10). CB RH2 had direct effects on the expansion of CD4⁺ T cells in Peyer's patches (PPs) in vitro, which in turn affected their immune response upon challenge with ceftriaxone. All these data suggested that CB RH2 possessed the ability to modulate the intestinal mucosal and systemic immune system in limiting intestinal alterations to relieve ceftriaxone-induced dysbacteriosis.

Keywords: CB RH2 in Intestinal Dysbiosis; Clostridium butyricum; gut microbiota; immune response; mucosal barrier function.

Figure 1

Schematic overview of ceftriaxone treatment and CB RH2 administration. (A) Experimental protocol for ceftriaxone treatment with administration of CB RH2 as intervention; (B) Survival rate; (C) Colon length (cm), data are represented as mean ± SEM of eight mice in each group.

Figure 2

Evaluation of illumina HiSeq sequencing data showing that CB RH2 could modulate the structure and composition of gut microbiota. (A) Venn diagram of shared and independent bacterial OTUs in different experimental groups (n >6); (B–D) Analysis of α-diversity of gut microbiota by Observed species, Chao1 and Shannon index; (E) Principal coordinate analysis (PCoA) based on weighted Unifrac distances among different samples. PC1 and PC2 account for 63.65% of the variation; (F) Multivariate analysis of variance from PCoA matrix scores using UPGMA method based on weighted Unifrac distances; (G) The average abundance of microbial community in different mice groups at phylum level; (H) The ratio of Firmicutes to Bacteroidetes; (I, J) Statistical analysis for Bacteroidetes and Deferribacteres at the phylum level; (K) Bar charts at genus level of gut microbiota in the four groups; (L) Relative abundance of Bacteroides, Oscillibacter, Desulfovibrio, Mucispirillum and Parabacteroides are significantly manipulated by CB RH2 at genus level. Statistical analysis was performed using the t tests method. All values are mean ± SEM (n >6). *p < 0.05, **p < 0.01.

Figure 3

CB RH2 changes the mechanical barriers in intestinal mucosa of ceftriaxone-treated mice. (A) H&E-stained results for the sections of mouse colon; (B) Histopathological analysis of the H&E-stained sections; (C) Transmission electron microscopy (TEM) analysis of colon; (D) LPS of serum were detected by ELISA following the manufacturer’s protocol; (E, F) The concentrations of α-defensin and β-defensin in colon of mice. All data were evaluated as mean ± SEM (n = 5), *p <0.05, **p <0.01, ***p <0.001.

Figure 4

CB RH2 enhances intestinal barrier function of ceftriaxone-treated mice. (A–E) The relative RNA expression of genes encoding zonula occludens (ZO-1), Occludin, Claudin-1, Claudin-4, and mucin-2 (MUC-2) in colon tissues of mice, detected by qPCR; (F, G) Representative blots and comparison of protein expression of ZO-1, Occludin, Claudin-1, Claudin-4, and MUC-2 by western blot with β-actin as internal control. All data were evaluated as mean ± SEM (n = 5), *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 5

Anti-inflammatory effect of CB RH2 on mice with ceftriaxone-induced intestinal dysbacteriosis. (A, B) The relative RNA expression of genes encoding tumor necrosis factor alpha (TNF-α) and cyclooxygenase-2 (COX-2) in colon tissues of mice (n = 5); (C) IL-10 of serum were detected by ELISA following the manufacturer’s protocol (n = 5); (D) Representative flow cytometry plots of CD4⁺ and CD8⁺ T cells identified (n = 3); (E, F) Percentage of CD4⁺ and CD8⁺ T cells in spleen; (G) The proportion of CD4⁺/CD8⁺. All data were evaluated as mean ± SEM, *p <0.05, **p <0.01, ***p <0.001.

Figure 6

CB RH2 and its metabolites promote expansion of CD4⁺ T cells. (A) Representative flow cytometry plots; (B, C) Percentage of CD4⁺ and CD8⁺ T cells in Payer’s patches (PPs) of mice post stimulation of the supernatant and lysate of CB RH2; (D) The proportion of CD4⁺/CD8⁺. The MRS medium was used as control. All data were evaluated as mean ± SEM (n = 3), *p <0.05, **p <0.01.