Bifidobacterium lactis ameliorates AOM/DSS-induced inflammation, dysbiosis, and colonic precancerous lesions

Abstract

Bowel cancer is the third most common malignancy of tumors and one of the major causes of cancer-related death. Bowel precancerous conditions can develop without any symptoms, which either makes it difficult for early diagnosis or poses a poor prognosis/gloomy relapse. This study aimed to investigate the effects of Bifidobacterium animalis subsp. lactis TCI604 (B. lactis) on inflammatory responses, gut microbiome, and protectiveness against azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colonic precancerous lesions. The AOM/DSS-induced colonic precancerous lesion murine model was studied with 24 female C57BL/6 J mice assigned to the control group, AOM/DSS-induced colonic precancerous lesion group (AOM/DSS), AOM/DSS treated with B. lactis probiotic group (B. lactis P), and AOM/DSS treated with B. lactis cell-free supernatant group (B. lactis S). The results showed that both B. lactis P and B. lactis S could attenuate AOM/DSS-induced body weight loss and intestine damage, reduce aberrant crypt foci (ACF) and the formation of colonic polyps, and significantly inhibit pro-inflammatory cytokines and the NF-κB signaling pathway, in which the B. lactis S group outperformed others. Further analysis using 16S rDNA sequencing suggested that both B. lactis P and B. lactis S optimize gut microbiota. Several bacteria, including Muribaculaceae, Prevotellaceae_UCG-001, Anaerostipes, Ruminococcaceae, Mucispirillum, Clostridia_UCG-014, and Clostridia_vadinBB60 that were known in close relation to colonic precancerous lesions, were sequenced at taxonomic level. Our results indicated that both B. lactis P and B. lactis S improved AOM/DSS-induced colonic precancerous lesions by regulating inflammation as well as optimizing gut microbiota, thereby establishing reciprocally cooperative net benefits between probiotics/postbiotics and mice with colonic precancerous lesions. KEY POINTS: • Prophylactic administration of probiotic and postbiotic of B. lactis is capable of alleviating the AOM/DSS-induced body weight loss and colon shortening, as well as diminishing the development of colonic precancerous lesions, such as the formation of ACF and colonic polyps, in an AOM/DSS mouse model • Either probiotic or postbiotic of B. lactis has a positive role in mediating immune imbalance and colonic inflammation via suppression of inflammatory immune cells, pro-inflammatory cytokines, and the NF-κB signaling pathway • AOM/DSS-induced dysbiosis can be reversed with the probiotic and postbiotic of B. lactis supplementation.

Keywords: Bifidobacterium animalis subsp. Lactis; Colonic precancerous lesions; Gut microbiota; Postbiotics; Probiotics.

Fig. 1

Schematic representation of multistep carcinogenesis progression in the AOM/DSS murine model. C57BL/6 J mice were given a single i.p. injection of AOM (10 mg/kg) on the first day and provided drinking water ad libitum containing 2% (w/v) DSS for 5 days, followed by 2 weeks of sterile water. Mice were subjected to three cycles of 2% DSS (5 days per cycle) treatment and regular water (14 days per cycle). The total experimental period was 79 days. B. lactis P (1 × 10⁸ CFUs/day) or B. lactis S (80 μg/day) were orally inoculated 7 days before AOM treatment and continued supplement for 79 days

Fig. 2

Weight changes of mice among four groups. C57BL/6 J mice were given a single i.p. injection of AOM (10 mg/kg) on the first day and provided drinking water ad libitum containing 2% (w/v) DSS for 5 days, followed by 2 weeks of sterile water. Mice were subjected to three cycles of 2% DSS (5 days per cycle) treatment and regular water (14 days per cycle). The total experimental period was 79 days. B. lactis P (1 × 10⁸ CFUs/day) or B. lactis S (80 μg/day) were orally inoculated 7 days before AOM treatment and continued supplement for 79 days. Data are expressed as the means ± SD. *p < 0.05 significantly different from the control group

Fig. 3

B. lactis P and B. lactis S attenuated the AOM/DSS-induced colon shortening. Assessment of the small intestine and the colon length in AOM/DSS murine model on day 79. (A) Macroscopic images of the small intestine are shown. (B) The small intestine length of each mouse was measured. (C) The small intestine weight of each mouse was measured. (D) Macroscopic images of the colon are shown. (E) The colon length of each mouse was measured. (F) The colon weight of each mouse was measured. Data are expressed as the means ± SD. *p < 0.05 significantly different from the compared group

Fig. 4

B. lactis P and B. lactis S prevented AOM/DSS-induced aberrant crypt foci and the formation of colonic polyps. A Representative gross macroscopic image of the colon. Red arrows indicate polyps. B Representative pictures of opened specimens by cutting along the bowel. Yellow arrows indicate polyps. C The number of polyps per mouse in different parts of small intestines. Data are expressed as the means ± SD. *p < 0.05 significantly different from the compared group. D Correlation between colon weight and polys number in colons. E Representative H&E sections of colons

Fig. 5

Effects of B. lactis P and B. lactis S on the hematological and spleen immunological parameters in AOM/DSS-treated mice. A Red blood cell indices. B White blood cell indices. C Spleenocyte parameter. Data are expressed as the means ± SD. *p < 0.05 significantly different from the compared group

Fig. 6

Effects of B. lactis P and B. lactis S on expression of inflammatory cytokine and NF-κB signaling in AOM/DSS-treated mice. A The protein levels of TNF-α, IL-1β, IL-6, IFN-γ, and IL-10 in the plasma of mice were detected by ELISA. B Semiquantitative analysis of colonic tissue protein levels of IκB-α, IKK-β, NF-κB, and GAPDH levels. GAPDH served as an internal control for equal loading. C The intensities of western blot bands were determined by the ImageJ. The intensity (mean ± SD) was normalized to the control group that was set to 1. Protein levels differed significantly among the groups, * indicates p < 0.05 vs. the compared group

Fig. 7

Effects of B. lactis P and B. lactis S on the structural and functional composition of gut microbiota. A Changes in the relative phylum level abundances of gut microbiota components in AOM/DSS-treated mice. B The ratio of Firmicutes to Bacteroidetes (F/B) boxplot showing gut microbiota in AOM/DSS-treated mice. Boxes contain 50% of all values and whiskers represent the 25th and 75th percentiles. *p < 0.05 significantly different from the compared group. C PCA plot based on the covariance matrix of genus-level relative abundance of gut microbiota components in AOM/DSS-treated mice. D Box plots showing species richness estimator (Chao1 and observed features) and species evenness estimator (Shannon and Simpson) of alpha diversity. *p < 0.05 significantly different from the compared group. E, F Heatmap depicting the relative abundance of the most abundant family (E) and genera (F) (> 0.1%) of gut microbiota from different treatments. The color intensity in each sample is normalized to represent its relative ratio in AOM/DSS-treated mice. A range of colors, from green to red, indicates the relative values of microbiota (0–1). G The relative abundance of Muribaculaceae, Prevotellaceae_UCG-001, Anaerostipes, Ruminococcaceae, Mucispirillum, Clostridia_UCG-014, and Clostridia_vadinBB60. Data are expressed as the means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 significantly different from the compared group

Fig. 8

Schematic of the representative mechanisms for the action of B. lactis on AOM/DSS-induced colonic precancerous lesions. In AOM/DSS-treated mice, the body weight, inflammations, ACF, polyps, and gut dysbiosis were enhanced, while the B. lactis P or B. lactis S administration significantly reversed the carcinogenesis process and gut dysbiosis, as well as mitigated weight losing, colon shorting, and inflammatory response. The present study provided information regarding the B. lactis P or B. lactis S administration in AOM/DSS-induced colonic precancerous lesions and the regulation of inflammation and gut dysbiosis