Clostridium butyricum inhibits the progression of colorectal cancer and alleviates intestinal inflammation via the myeloid differentiation factor 88 (MyD88)-nuclear factor-kappa B (NF-κB) signaling pathway

Abstract

Background: Clostridium butyricum (C. butyricum, CB) is a probiotic to modulate the intestinal disorders and CB supplement has been found to have a great impact on inflammation and cancer treatment. However, the effects and mechanisms of CB on colorectal cancer (CRC) are not clear. We performed this study to investigate the influence of CB on the progression of CRC and the potential mechanisms in vivo and in vitro.

Methods: We established azoxymethane (AOM)/dextran sulfate sodium salt (DSS) model mice (male, 6-week-old C57BL/6J) and divided them into the control (Ctrl) and CB groups at the end of the second DSS cycle. Mice in the CB group received treatment with CB [1×10⁸ colony forming unit (CFU) in 100 µL phosphate buffered saline (PBS)] 3 times a week for 40 days by gavage administration. The apoptotic cells in tumor tissues were assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. IL-6 and IL-10 were detected using enzyme linked immunosorbent assay (ELISA) assayes. Microbiota was analyzed through 16S rDNA sequencing. The location of CB was detected by the fluorescence in situ hybridization (FISH) assay. The function of CB on the proliferation of cell lines, HT-29 and CT-26, was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assayes. The expression of myeloid differentiation factor 88 (MyD88) and nuclear factor-kappa B (NF-κB) in cells and tissues was evaluated by real time quantitative PCR (RT-qPCR) and western blot.

Results: Mice in the CB group showed a lower incidence and total volume of CRC, more apoptotic cells in the tumor tissue, a lower level of IL-6, and a higher level of IL-10 compared with those in the Ctrl group. CB altered the composition of the gut microbiota and was enriched in the small intestine and tumor tissue. Moreover, CB restrained the proliferation and the expression of MyD88 and NF-κB in CRC cell lines and colon tissue.

Conclusions: CB restrained the progression of CRC, improved the inflammation of AOM/DSS mice, altered the composition of their gut microbiota, and regulated the expression of MyD88 and NF-κB. We concluded that CB plays its role in CRC via MyD88 and the NF-κB signaling pathway.

Keywords: Clostridium butyricum (CB); colorectal cancer (CRC); gut microbiota; myeloid differentiation factor 88 (MyD88); nuclear factor-kappa B/p65 (NF-κB/p65).

Figure 1

CB inhibited the development of CRC in vivo. (A) A schematic diagram of the AOM/DSS-induced CRC model in C57BL/6J mice with CB treatment. (B) Images of colon tumors in mice before grouping. (C) Representative images of colon tumors in mice under different conditions. (D,E) Statistical analysis of tumor number and volume in different groups, n=5/group by Student’s t test. Error bars represent mean ± SEM; **P<0.01. (F) Representative histological appearance of the tumors in the 2 groups. The section of tissue was stained by hematoxylin and eosin. Scale bar, 50 µm. AOM, azoxymethane; DSS, dextran sulfate sodium salt; Ctrl, control group; CB, Clostridium butyricum; CRC, colorectal cancer.

Figure 2

CB facilitated the apoptosis of cancer cells in the colon. (A,B) Representative images of TUNEL staining (A) and statistical analysis (B) of TUNEL assessment. Scale bar, 150 µm. Error bars represent mean ± SD; **P<0.01. DAPI, 4',6-diamidine-2-phenylindole dihydrochloride; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; Ctrl, control group; CB, Clostridium butyricum.

Figure 3

CB improved intestinal inflammation in AOM/DSS mice. (A) Effect of CB on body weight after grouping. Error bars represent mean ± SD; **P<0.01. (B) Statistical analysis of colon length. Error bars represent mean ± SEM; ns, no significant difference. (C) Representative histological appearance of gut inflammation in the 2 groups. The section of tissue was stained by hematoxylin and eosin. Scale bar, 50 µm. (D,E) Comparison of IL-6 (D) and IL-10 (E) in plasma. Error bars represent mean ± SD; **P<0.01. Ctrl, control group; CB, Clostridium butyricum; AOM/DSS, azoxymethane/dextran sulfate sodium salt.

Figure 4

CB rebuilt the structure of gut microbiota. (A) Venn diagrams of bacterial OTUs. The y-axis represents the OTU number. (B) Alpha diversity estimation of gut microbiota by the Simpson index. (C) Student’s t test for the Simpson index. (D) Community bar plot analysis at the phylum level. (E) Firmicutes/Bacteroidetes ratio of the 2 groups by Student’s t test. Error bars represent mean ± SD; *P<0.05; ns, no significant difference. (F) Community bar plot analysis at the species level. (G) Analysis of beta diversity by PCoA. Ctrl, control group; CB, Clostridium butyricum; PCoA, principal co-ordinates analysis; OTU, operational taxonomic unit.

Figure 5

CB was enriched in small intestinal crypts and colon tumors. (A) Location and enrichment of CB in the small intestine and colon. The images on the left are the section of tissue stained by hematoxylin and eosin, scale bar, 50 µm, and the black circles mean the locations of FISH in small intestine (the top) and the colon tumor (the bottom). The images on the right are the section of tissue with FISH, scale bar, 150 µm. (B) Statistical analysis of FISH assessment. Error bars represent mean ± SD; **P<0.01. Ctrl, control group; CB, Clostridium butyricum; DAPI, 4',6-diamidine-2-phenylindole dihydrochloride; FITC, fluorescein isothiocyanate; OTU, operational taxonomic unit; FISH, fluorescence in situ hybridization.

Figure 6

CB restrained the proliferation of cancer cells in vitro. (A,B) Effects of CB on the proliferation of HT-29 cells (A) and CT26 cells (B). (C,D) Effects of NaB on the proliferation of HT-29 cells (C) and CT26 cells (D). (E,F) Effects of CB supernatant on the proliferation of HT-29 cells (E) and CT26 cells (F). Error bars represent mean ± SD; **P<0.01. Ctrl, control group; MOI, multiplicity of infection; BHI, Brain Heart Infusion Medium; CB, Clostridium butyricum.

Figure 7

CB restrained the proliferation of cancer cells and the expression of MyD88 and NF-κB/p65 in vitro. (A) MyD88 and NF-κB/p65 expression was detected by western blot in HT-29 cells and CT26 cells. (B,C) Comparison of the protein expression of MyD88 and NF-κB/p65 in HT-29 cells (B) and CT26 cells (C). (D,E) Effects of CB on MyD88 and NF-κB/p65 expression in HT-29 cells and CT26 cells were detected by RT-qPCR. Error bars represent mean ± SD; **P<0.01. MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor-kappa B; Ctrl, control group; MOI, multiplicity of infection; CB, Clostridium butyricum; RT-qPCR, real time quantitative PCR.

Figure 8

CB restrained the proliferation of colon cells and the expression of MyD88 and NF-κB/p65 in vivo. (A) Representative images of the colon tumors’ sections with immunohistochemistry staining for Ki-67. Scale bar, 150 µm. (B) Statistical analysis of Ki-67-positive cells in the colon. (C) Comparison of the relative expression of MyD88 and NF-κB/p65 in the 2 groups by RT-qPCR. (D) MyD88 and NF-κB/p65 expression was detected by western blot in the 2 groups. (E) Comparison of the relative expression of MyD88 and NF-κB/p65 in the 2 groups. Error bars represent mean ± SD; **P<0.01. Ctrl, control group; CB, Clostridium butyricum; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor-kappa B; RT-qPCR, real time quantitative PCR.