Inhibitory effects of Clostridium butyricum culture and supernatant on inflammatory colorectal cancer in mice.

Clostridium butyricum (CB) is a spore-forming, gram-positive and obligate anaerobic rod bacterium. CB can modulate the composition of the gut microbiome and promote the growth of beneficial microbes in the intestine by generating short-chain fatty acids (SCFAs), which in turn protect against colitis and prevents the formation of inflammatory-associated colorectal cancer (CRC) by ameliorating colon inflammatory processes. Yet, it remains unclear whether the culture and supernatant of CB could directly influence inflammatory CRC in mice. In this study, azoxymethane (AOM)+dextran sodium sulphate (DSS) was used to induce CRC model in C57BL/6 mice. Next, the serum levels of inflammatory cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), and cytokines TNF-α, were measured and the pathohistological examination of the large intestine was performed. Both CB culture and supernatant were found to have anti-inflammatory properties. Subsequently, Western blot and Real-Time Quantitative PCR (RT-qPCR) revealed that CB and supernatant regulate the NF-κB/p65 pathway to inhibit the development and progression of inflammatory CRC in AOM+DSS-treated mice, which could be due to the high levels of butyric acid in the supernatant.

Pu W ，Zhang H ，Zhang T ，Guo X ，Wang X ，Tang S ... -  《Frontiers in Immunology》

Clostridium butyricum modulates gut microbiota and reduces colitis associated colon cancer in mice.

We investigate the effects of Clostridium butyricum(CB) on gut microbiota and colitis associated colon cancer(CAC) in mice.6-8 weeks old C57BL/6 mice were randomly divided into control, azoxymethane (AOM) + dextran sodium sulphate (DSS) and AOM + DSS + CB groups. Mice in the latter two groups received an intraperitoneal injection of AOM (12.5 mg/kg), followed by three cycles of DSS diluted in water (2.5% w/v). Mice in treatment group received CB (2 × 108 CFU in 200 ul normal saline) by gavage administration three times one week. Microbiota composition was assessed by 16S rRNA high-throughput sequencing. Colon samples were collected to examine severity of colitis and tumorigenesis. Cytokines including TNF-a, IL-6 and Cyclo-oxygenase-2 (COX-2) were detected by RT-qPCR. Expression of Bcl-2, Bax and the state of components of NF-κB signaling pathway were detected by western blot. The results revealed that CB regulated structure of intestinal flora and changed the microbial composition; decreased Firmicutes/ Bacteroidetes ratio in phylum level and increased the relative abundance of probiotics; decreased colitis, decreased incidence and size of colorectal cancer(CRC) and increased apoptosis of tumor cells; decreased cytokines including TNF-a and IL-6; decreased level of COX-2; decreased phosphorylation of NF-κB; decreased level of Bcl-2 and increased expression of Bax. In conclusion, CB could regulate structure and composition of gut microbiota and reduces colitis associated colon cancer in mice, the mechanism may be inhibiting NF-κB pathway and promoting apoptosis.

Liu M ，Xie W ，Wan X ，Deng T ... -  《-》

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.

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. 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. 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. 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.

Zhou M ，Yuan W ，Yang B ，Pei W ，Ma J ，Feng Q ... -  《-》

Short-chain fatty acids administration is protective in colitis-associated colorectal cancer development.

Reduced short-chain fatty acids (SCFAs) have been reported in patients with ulcerative colitis, and increased intake of dietary fiber has shown to be clinically beneficial for colitis. Whether SCFAs suppress tumorigenesis in colitis-associated colorectal cancer remains unknown. The chemopreventive effect of SCFAs in colitis-associated colorectal cancer was evaluated in this study. Model of colitis-associated colorectal cancer in male BALB/c mice was induced by azoxymethane (AOM) and dextran sodium sulfate (DSS). SCFAs mix (67.5 mM acetate, 40 mM butyrate, 25.9 mM propionate) was administered in drink water during the study period. Macroscopic and histological studies were performed to examine the colorectal inflammation and tumorigenesis in AOM/DSS-induced mice treated with or without SCFA mix. The effects of SCFAs mix on colonic epithelial cellular proliferation were also assessed using Ki67 immunohistochemistry and TUNEL staining. The administration of SCFAs mix significantly reduced the tumor incidence and size in mice with AOM/DSS-induced colitis associated colorectal cancer. SCFAs mix protected from AOM/DSS-induced colorectal cancer by improving colon inflammation and disease activity index score as well as suppressing the expression of proinflammatory cytokines including IL-6, TNF-α and IL-17. A decrease in cell proliferation markers and an increase in TUNEL-positive tumor epithelial cells were also demonstrated in AOM/DSS mice treated with SCFAs mix. SCFAs mix administration prevented development of tumor and attenuated the colonic inflammation in a mouse model of colitis-associated colorectal cancer. SCFAs mix may be a potential agent in the prevention and treatment of colitis-associated colorectal cancer.

Tian Y ，Xu Q ，Sun L ，Ye Y ，Ji G ... -  《-》

Berberine inhibits intestinal carcinogenesis by suppressing intestinal pro-inflammatory genes and oncogenic factors through modulating gut microbiota.

The role of Berberine (BBR) in colorectal cancer (CRC) and gut microbiota has begun to appreciate. However, there was no direct evidence confirm that the gut microbiota regulated by BBR could inhibit CRC. This report investigated the effect of stool from BBR treated subjects and its effect on CRC. A mouse model for CRC was developed using azoxymethane (AOM) and dextran sulfate sodium (DSS). Intestinal tissue from affected mice were used to determine the efficacy of BBR against CRC. Stool samples were collected for the 16s rRNA gene sequencing and fecal microbiota transplantation (FMT). Finally, the mechanism of gut microbiota from BBR treated mice on CRC was explored using immunohistochemistry, RNA-Sequencing, quantitative RT-PCR, and western blot analyses. BBR significantly reduced intestinal tumor development. The richness of gut microbiota were notably decreased by BBR. Specifically, the relative abundance of beneficial bacteria (Roseburia, Eubacterium, Ruminococcaceae, and Firmicutes_unclassified) was increased while the level of bacteria (Odoribacter, Muribaculum, Mucispirillum, and Parasutterella) was decreased by BBR treatment. FMT experiment determined that the mice fed with stool from BBR treated AOM/DSS mice demonstrated a relatively lower abundance of macroscopic polyps and a significantly lower expression of β-catenin, and PCNA in intestinal tissue than mice fed with stool from AOM/DSS mice. Mechanistically, intestinal tissue obtained from mice fed with stool from BBR treated AOM/DSS mice demonstrated a decreased expression of inflammatory cytokines including interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), C-C motif chemokine 1 (Ccl1), Ccl6, and C-X-C motif ligand (Cxcl9). In addition, the NF-κB expression was greatly suppressed in mice fed with stool from BBR treated AOM/DSS mice. Real-time PCR arrays revealed a down-regulation of genes involved in cell proliferation, angiogenesis, invasiveness, and metastasis in mice fed with stool from BBR treated AOM/DSS mice. Stool obtained from BBR treated AOM/DSS mice was able to increase colon length while simultaneously decreasing the density of macroscopic polyps, cell proliferation, inflammatory modulators and the expression of NF-κB. Therefore, it was concluded that suppression of pro-inflammatory genes and carcinogens factors by modulating gut microbiota was an important pathway for BBR to inhibit tumor growth in conventional mice.

Chen H ，Ye C ，Cai B ，Zhang F ，Wang X ，Zhang J ，Zhang Z ，Guo Y ，Yao Q ... -  《BMC CANCER》