Aim: To investigate the effect of epigallocatechin gallate (EGCG) on structural changes of gut microbiota in colorectal carcinogenesis.
Methods: An azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis mouse model was established. Forty-two female FVB/N mice were randomly divided into the following three groups: group 1 (10 mice, negative control) was treated with vehicle, group 2 (16 mice, positive control) was treated with AOM plus vehicle, and group 3 (16 mice, EG) was treated with AOM plus EGCG. For aberrant crypt foci (ACF) evaluation, the colons were rapidly took out after sacrifice, rinsed with saline, opened longitudinally, laid flat on a polystyrene board, and fixed with 10% buffered formaldehyde solution before being stained with 0.2% methylene blue in saline. For tumor evaluation, the colon was macroscopically inspected and photographed, then the total number of tumors was enumerated and tumor size measured. For histological examination, the fixed tissues were paraffin-embedded and sectioned at 5 mm thickness. Microbial genomic DNA was extracted from fecal and intestinal content samples using a commercial kit. The V4 hypervariable regions of 16S rRNA were PCR-amplified with the barcoded fusion primers. Using the best hit classification option, the sequences from each sample were aligned to the RDP 16S rRNA training set to classify the taxonomic abundance in QIIME. Statistical analyses were then performed.
Results: Treatment of mice with 1% EGCG caused a significant decrease in the mean number of ACF per mouse, when compared with the model mice treated with AOM/DSS (5.38 ± 4.24 vs 13.13 ± 3.02, P < 0.01). Compared with the positive control group, 1% EGCG treatment dependently decreased tumor load per mouse by 85% (33.96 ± 6.10 vs 2.96 ± 2.86, respectively, P < 0.01). All revealed that EGCG could inhibit colon carcinogenesis by decreasing the number of precancerous lesions as well as solid tumors, with reduced tumor load and delayed histological progression of CRC. During the cancerization, the diversity of gut microbiota increased, potential carcinogenic bacteria such as Bacteroides were enriched, and the abundance of butyrate-producing bacteria (Clostridiaceae, Ruminococcus, etc.) decreased continuously. In contrast, the structure of gut microbiota was relatively stable during the intervention of EGCG on colon carcinogenesis. Enrichment of probiotics (Bifidobacterium, Lactobacillu, etc.) might be a potential mechanism for EGCG's effects on tumor suppression. Via bioinformatics analysis, principal coordinate analysis and cluster analysis of the tumor formation process, we found that the diversity of gut microbiota increased in the tumor model group while that in the EGCG interfered group (EG) remained relatively stable.
Conclusion: Gut microbiota imbalance might be a potential mechanism for the prevention of malignant transformation by EGCG, which is significant for diagnosis, treatment, prognosis evaluation, and prevention of colorectal cancer.
Keywords: Animal experiment; Chemoprevention; Colorectal cancer; Epigallocatechin gallate; Gut microbiota; High throughput sequencing.