Clostridium butyricum protects intestinal barrier function via upregulation of tight junction proteins and activation of the Akt/mTOR signaling pathway in a mouse model of dextran sodium sulfate-induced colitis

Abstract

Clostridium butyricum (CB), a probiotic, is a gram-positive obligate anaerobic bacillus with acid and heat resistant properties. Previous studies have reported that CB has beneficial effects in intestinal diseases and regulates intestinal function. The aim of the present study was to investigate the protective effects and mechanisms of CB on the intestinal barrier function. Mice were randomly divided into three experimental groups (n=15 mice/group), including control, dextran sodium sulfate (DSS) and DSS + CB. In the DSS and DSS + CB groups colitis was induced with 3% DSS dissolved in drinking water for 7 days. DSS + CB group mice were co-treated daily with 200 µl (2x10⁸ CFU) CB solution via gavage. The intestinal mucosal barrier function in mice was assessed by measuring FITC-labeled 4-kDa dextran (molecular weight, 4,000 Da) flux and by analyzing the expression of tight junction (TJ)-related proteins using western blot analysis. In addition, the secretion levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, IL-10 and IL-13, and the concentration of malondialdehyde, glutathione and superoxide dismutase were detected using ELISAs to determine inflammation and oxidative stress, respectively. The activation status of the Akt/mTOR signaling pathway was also investigated using western blot analysis. The results demonstrated that, in mice with DSS-induced colitis treatment, co-treatment with CB attenuated colitis symptoms and intestinal permeability, increased the expression levels of TJ-related proteins, decreased TNF-α, IL-1β and IL-13 secretion levels but increased those of IL-10, and reduced oxidative stress. Additionally, CB elevated the phosphorylation of Akt, mTOR and p70 ribosomal protein S6 kinase. Collectively, the present results indicated that CB protected intestinal barrier function and decreased intestinal mucosal permeability via upregulating the expression levels of TJ-related proteins in a mouse model of DSS-induced colitis. Moreover, the results suggested that the effects of CB could be mediated by the Akt/mTOR signaling pathway.

Keywords: Akt/mTOR signaling pathway; Clostridium butyricum; intestinal barrier function; tight junctions.

Figure 1

CB treatment alleviates intestinal inflammation in DSS-induced colitis. Effect of CB on (A) body weight and (B) DAI score, (C) colon length, which is quantified in (D), and (E) FD-4 permeability. Data are presented as the mean ± SD. ^*P<0.05 vs. control group; ^#P<0.05 vs. DSS group. IBW, initial body weight; CB, Clostridium butyricum; DSS, dextran sodium sulfate; DAI, disease activity index; FD-4, FITC-labeled 4-kDa dextran.

Figure 2

Effects of CB on colon histology in the DSS-induced colitis mouse model. (A) Representative images of colon tissues from control, DSS and DSS+CB groups (original magnification, x200). (B) Histological scores of colon tissue. Data are presented as the mean ± SD. ^*P<0.05 vs. control group; ^#P<0.05 vs. DSS group. DSS, dextran sodium sulfate; CB, Clostridium butyricum.

Figure 3

Effect of CB on cytokines secretion levels and oxidative stress indicators. (Α) Serum levels of TNF-α, IL-1β, IL-6, IL-10 and IL-13 in different groups. Levels of oxidative stress indicators, (B) SOD, (C) MDA and (D) GSH in different groups. Data are presented as the mean ± SD. ^*P<0.05 vs. control group; ^#P<0.05 vs. DSS group. CB, Clostridium butyricum; TNF-α, tumor necrosis factor α; IL, interleukin; DSS, dextran sodium sulfate; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase.

Figure 4

Effects of CB on the expression levels of claudin-1, claudin-2, occludin and ZO-1. (A) Protein expression levels of claudin-1, claudin-2, occludin and ZO-1 in each group were measured using western blot analysis. (B) mRNA expression levels of claudin-1, claudin-2, occludin and ZO-1 were determined using reverse transcription-quantitative PCR. Data are presented as the mean ± SD. ^*P<0.05 vs. control group; ^#P<0.05 vs. DSS group. ZO-1, zona occludens protein 1; CB, Clostridium butyricum; DSS, dextran sodium sulfate.

Figure 5

Effect of CB on the Akt/mTOR signaling pathway. (A) Representative western blot analysis results of Akt, mTOR and S6K. (B) Comparison of semi-quantitative results. Data are presented as the mean ± SD. ^*P<0.05 vs. control group; ^#P<0.05 vs. DSS group. CB, Clostridium butyricum; DSS, dextran sodium sulfate; S6K, p70 ribosomal protein S6 kinase; p-, phosphorylated.