Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Nov 15;11(11):5338-5357.
eCollection 2021.

YYFZBJS inhibits colorectal tumorigenesis by remodeling gut microbiota and influence on M2 macrophage polarization in vivo and in vitro

Ni Chai  1 , Yibai Xiong  2 , Yuli Zhang  3   4 , Yuelei Cheng  5 , Wenfei Shi  6 , Yiqing Yao  1 , Hua Sui  3 , Huirong Zhu  7
Affiliations

YYFZBJS inhibits colorectal tumorigenesis by remodeling gut microbiota and influence on M2 macrophage polarization in vivo and in vitro

Ni Chai et al. Am J Cancer Res. .

Abstract

Our previous studies indicated that the extract of Yi-Yi-Fu-Zi-Bai-Jiang-San (YYFZBJS) had potent anticancer activities by significantly inhibiting intestinal tumor development in ApcMin/+ mice. However, knowledge regarding the mechanism and effect of YYFZBJS in the prevention of colorectal cancer is limited. In this study, we aim to investigate the preventive effects of YYFZBJS in enterotoxigenic Bacteroides fragilis (ETBF)-colonized mice with azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced tumorigenesis. First, the colonic tissues of the AOM/DSS mouse models were collected for biomedical analysis, and gut microbiota profiling was detected post YYFZBJS treatment using a 16S rRNA gene sequencing. Then, antibiotic solution (Abx) mice were acclimated with AOM/DSS treatment and then fed with ETBF with or without YYFZBJS for three cycles. As expected, the intragastric administration of YYFZBJS in the AOM/DSS mouse model significantly decreased the tumor load, the severity of disease activity index (DAI) scores, and the level of M2 macrophage markers such as CD206, Arg-1 and IL-10. Notably, the reverse of polarized macrophages induced by YYFZBJS could suppress CRC cell proliferation and infiltration, as demonstrated by the decrease of some tumor proliferation-related proteins in a dose-dependent manner. Importantly, ETBF dysbiosis can contribute to colon tumor development by stimulating p-STAT3 mediated M2 macrophages polarization to promote chronic inflammation and adenoma malignant transformation, which YYFZBJS can effectively limit. Altogether, we demonstrate that ETBF dysbiosis may contribute to M2 macrophages-promoted colon carcinogenesis and progression of CRC cells, while YYFZBJS could be a promising protective agent against ETBF-mediated colorectal cancer.

Keywords: AOM/DSS-induced colitis-associated colorectal cancer; Colorectal cancer; ETBF; macrophage; signal pathway.

PubMed Disclaimer

Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Effect of YYFZBJS on the occurrence and progression of CAC. (A) Experimental protocol used for the induction of the CAC model in C57BL/6J mice. (B, D-F) Effects of the model and treatment on mice body weight (B), colon length (D), number of intestinal polyps (E) and survival (F) in mice. (C) Microscopic view of the colon in mice. (G) The tumor size distribution in the intestine was listed and compared with the control. Data are presented as means ± SD of 8 animals per experimental group, with Welch’s correction, two-tailed t-test. #P<0.05, ##P<0.01; *P<0.05, **P<0.01 vs. control.
Figure 3
Figure 3
ETBF infection enhances colonic tumorigenesis and reduces the inflammatory response in the AOM/DSS mouse model. (A) Experimental design indicating the timing of the intragastric administration and organization of groups. Mice were treated with Abx from W1 (age at week 4) to W4, then injected with AOM (12.5 mg/kg, i.p.) and provided drinking water for 1 week, and three cycles of DSS and drinking water for 3 weeks as described in the Methods section. During the treatment, ETBF and Vehicle were orally administered at a dosage of 108 CFU/mice. (B, C) Effects of ETBF on colon length (B) and the number of tumors (C) in the GF/AOM/DSS mouse model. (D) Typical adenomatous intestinal polyp with an early invasion of neoplastic glands into the muscular layers was often observed in ETBF infected GF/AOM/DSS mice. This typical regressive intestinal cancer morphology is observed throughout the intestine in the mice. Blue arrows indicated adenocarcinoma cells. Magnification bars, 100 μM. (E) Histology evaluation of colon tumors: quantitatively represented as polyp, adenoma, and adenocarcinoma. (F) Immunohistochemical staining using an antibody against Ki67 and PCNA in the vehicle group and ETBF infection group. Magnification bars, 100 μM. Data are presented as the means ± SD of 5 regions per slice with Welch’s correction, two-tailed t-test. **P<0.05, vs. Vehicle. (G) The difference between the two groups in inflammatory cytokines as assessed by cytokine antibody array. (H) The mRNA expression levels of inflammatory factors, such as IL-12, TNF-α, and IL-10 in the PB of mice in different groups. The data are presented as the mean ± SD from at least three experiments. **P<0.01 vs. Vehicle.
Figure 2
Figure 2
YYFZBJS modulated the composition of the gut microbiota in AOM/DSS mice. A. Cladogram generated from the linear discriminant analysis effect size (LEfSe) between YYFZBJS group and Control group (n = 8 for each group). The analyses were performed at the end of the experiment. B. α Diversity of gut microbiota for mice in four different groups. C. Bar plot of compositional differences at the genus level in the gut microbiome of mice in the combination YYFZBJS group vs. the control group by the Wilcoxon rank-sum test. Data are expressed as mean ± SD. *0.01<P≤0.05, **0.001<P≤0.01, ***P≤0.001, Two-sided Hypotheses. D. Heat map of the genus with relative abundances significantly different from their relative abundances at the time of YYFZBJS administration. The differentially enriched bacterial genus in C57BL/6J mice receiving N.S and YYFZBJS. The relative abundance between control and treatment mice for the genus was calculated for each time point. E. Gut microbiota level was analyzed for mRNA expression of Lactobacillus rhamnosus (LGG), Akkermansia, ETBF, and Clostridium butyricum by quantitative RT-PCR. Data are presented as means ± SD of 8 animals per experimental group, with Welch’s correction, two-tailed t-test. *P<0.05, **P<0.01, vs. Control.
Figure 4
Figure 4
Effect of ETBF on CRC cells through TAMs in vitro. (A) After TAMs were co-cultured with MC-38 cells, ETBF was added into the mixture for 6 h. The ratio of cell to bacterial was 1:10. (B) The mRNA of MC-38 cells at the bottom of the cell plate was analyzed by RNA-seq as described in the Methods section. (C) Experimental flow chart of (A). (D) Western blot analysis of JNK, NFκ-B and STAT3 expression in RAW 264.7 cells after infection with ETBF and co-culture with MC-38 cells. Quantifications of migrated cells from three independent experiments are shown as the mean ± S.D. *P<0.05, **P<0.01, vs. RAW 264.7 cells. (E) Immunohistochemical staining with an antibody against Arg-1 in the different groups. Scale bars, 100 μM.
Figure 5
Figure 5
YYFZBJS inhibited ETBF-induced colorectal tumor development in AOM/DSS model. A. Macroscopic view of the representative mouse large intestinal shows several polypoid and discoid colonic tumors from different groups of ETBF-infected mice administered with or without YYFZBJS as shown in Figure S1. GF/AOM/DSS mice were infected with ETBF. During ETBF infection, the YYFZBJS-treated group was orally gavaged with YYFZBJS (15.3 g/kg, p.o., once a day). B. Tumors number and colon length distribution. C, D. Immunohistochemical staining with an antibody against STAT3, CD163, and Arg-1 among Vehicle group, ETBF group and ETBF+YYFZBJS group. Magnification bars, 100 μM. Data are presented as the means ± SD of 8 animals per experimental group with Welch’s correction two-tailed t-test. *P<0.05, **P<0.01, vs. Vehicle; #P<0.05, ##P<0.01, vs. ETBF group. E. Representative gating strategy used to identify pulmonary macrophage subsets from intestinal tumor tissue in the GF/AOM/DSS mouse model is shown. Macrophages were analyzed using flow cytometry analysis of the M2 macrophage marker CD206 in the F4/80+ cell fraction. The data are presented as the mean ± SD of at least three independent experiments. F. IL-6, IL-12, TGF-β, and TNF-α levels in Tumor were evaluated using ELISA. The data are presented as the mean ± SD of at least three experiments. *P<0.05, **P<0.01, Vehicle vs. ETBF; #P<0.05, ##P<0.01, Vehicle vs. ETBF+YYFZBJS.
Figure 6
Figure 6
YYFZBJS inhibited tumor cell proliferation through regulating ETBF primed BMDMs in vitro. A. Experimental design indicating bone marrow-derived macrophages (BMDMs) were isolated from the bone marrow of C57BL/6J mice treated with or without YYFSBJS at the doses of 3.825 g/kg, 7.65 g/kg and 15.3 g/kg for 2 weeks. The BMDMs were from femurs and tibias of mice and cultured in a special medium (DMEM containing 10% FBS supplemented with 50 μg/ml penicillin/streptomycin and 10 ng/ml recombinant macrophage colony-stimulating factor [M-CSF; Thermo Fisher Scientific]). Then the primed BMDMs (TAM) were collected and were assigned to MC-38 cells in a 10:1 ratio. B. MC-38 cells proliferation was assayed at 24, 48, and 72 h after co-culture with the M2φ. The data are presented as the mean ± SD from at least three experiments. *P<0.05, **P<0.01 vs. MC-38+BMDMs (YYFZBJS-L). C, F. Western blot and quantitative assay of c-Met, cyclinD1, MMP-2, MMP-9, STAT3 and phosphorylation of STAT3 in MC-38 cells. GAPDH is the loading control. The data are presented as the mean ± SD from at least three independent experiments. **P<0.01 vs. MC-38; #P<0.05, ##P<0.01 vs. MC-38+TAM (Veh). D. Cell invasion assay result using Matrigel-coated Transwell. (left, representative pictures of invasion chambers; right, average counts from five random microscopic fields). Data are presented as mean ± SD of triplicate experiments. **P<0.01 vs. MC-38. E. mRNA expression of genes associated with cell proliferation in MC-38 cells was evaluated using quantitative RT-PCR. The data are presented as the mean ± SD from at least three experiments. *P<0.05, **P<0.01 vs. MC-38.
Figure 7
Figure 7
A schematic of the effect of YYFZBJS in host-microbiota interactions in colorectal cancer prevention.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Lee YK, Mehrabian P, Boyajian S, Wu WL, Selicha J, Vonderfecht S, Mazmanian SK. The protective role of bacteroides fragilis in a murine model of colitis-associated colorectal cancer. mSphere. 2018;3:e00587–18. - PMC - PubMed
    1. Hwang S, Jo M, Hong JE, Park CO, Lee CG, Rhee KJ. Protective effects of zerumbone on colonic tumorigenesis in enterotoxigenic bacteroides fragilis (ETBF)-colonized AOM/DSS BALB/c mice. Int J Mol Sci. 2020;21:857. - PMC - PubMed
    1. Gopalakrishnan V, Helmink BA, Spencer CN, Reuben A, Wargo JA. The influence of the gut microbiome on cancer, immunity, and cancer immunotherapy. Cancer Cell. 2018;33:570–580. - PMC - PubMed
    1. Si H, Yang Q, Hu H, Ding C, Wang H, Lin X. Colorectal cancer occurrence and treatment based on changes in intestinal flora. Semin Cancer Biol. 2021;70:3–10. - PubMed
    1. Sui H, Zhang L, Gu K, Chai N, Ji Q, Zhou L, Wang Y, Ren J, Yang L, Zhang B, Hu J, Li Q. YYFZBJS ameliorates colorectal cancer progression in Apc(Min/+) mice by remodeling gut microbiota and inhibiting regulatory T-cell generation. Cell Commun Signal. 2020;18:113. - PMC - PubMed

LinkOut - more resources