Title Page
Contents
ABSTRACT 15
Ⅰ. INTRODUCTION 18
Ⅱ. LITERATURE REVIEW 21
1. Colorectal cancer 21
2. Association between gut microbiota and colorectal cancer 22
3. β-glucuronidase and colorectal cancer 24
4. Lactic acid bacteria and safety of probiotics 27
Ⅲ. MATERIALS AND METHODS 28
1. Screening of lactic acid bacteria with β-glucuronidase activity 28
1.1. Sample collection and bacterial isolation 28
1.2. Analysis of β-glucuronidase activity 29
1.3. Bacterial identification by 16s rRNA analysis 30
2. Analysis of cell adhesion of lactic acid bacteria 30
2.1. Cell culture condition 30
2.2. Bacterial culture condition 31
2.3. Cell adhesion assay 31
3. MTT assay 32
4. Correlation analysis between β-glucuronidase activity of lactic acid bacteria and colorectal cancer in AOM/DSS mouse model 33
4.1. Preparation of lactic acid bacteria inoculum 33
4.2. Animals and experimental design 34
4.3. Measurement of body and organ weights 38
4.4. Analysis of colon 38
4.5. Analysis of β-glucuronidase activity in feces 41
4.6. Quantification of protein expression levels 41
4.7. Analysis of gut microbiota 45
5. Statistical analysis 45
Ⅳ. RESULTS AND DISCUSSION 47
1. Selection of lactic acid bacteria 47
1.1. β-glucuronidase activity of isolates from food and infant feces 47
1.2. Cell adhesion ability of lactic acid bacteria 51
1.3. Cell viability of lactic acid bacteria 53
2. Effect of β-glucuronidase of lactic acid bacteria on colorectal cancer in AOM/DSS mouse model 55
2.1. Survival rate of mice treated with lactic acid bacteria according to β-glucuronidase activity 55
2.2. Changes in body and organ weights of mice 58
2.3. Histological changes in colon 61
2.4. β-glucuronidase protein expression levels in the colon and β-glucuronidase activity in feces 68
2.5. Protein expression levels related to cancer progression in the colon tissue 73
2.6. Changes in gut microbiota of AOM/DSS mouse model after a 10-week treatment with lactic acid bacteria 79
Ⅴ. SUMMARY AND CONCLUSION 109
Ⅵ. REFERENCES 113
APPENDICES 14
Appendix 1. β-glucuronidase activity (µM/h; mean ± standard deviation) of isolates from infant feces 131
Appendix 2. β-glucuronidase activity (µM/h; mean ± standard deviation) of isolates from food 133
Appendix 3. Relative gene expression level of uidA in the cecum of AOM/DSS mouse model treated with lactic acid bacteria for 10 weeks. 140
ABSTRACT IN KOREAN 141
Table 1. Treatment groups in animal experiments 36
Table 2. Criteria for scoring histopathological hyperplasia 39
Table 3. Criteria for scoring histopathological inflammation 40
Table 4. Primary antibodies used in this study 44
Table 5. Isolates obtained from infant feces and food for β-glucuronidase activity 48
Table 6. Histopathological scoring results in AOM/DSS mouse model treated with lactic acid bacteria for 10 weeks 65
Table 7. Relative abundance in gut microbiota functional profile following a 10-week lactic acid bacteria treatment 104
Figure 1. Mechanism of tumorigenesis by bacterial β-glucuronidase. 26
Figure 2. Scheme of the animal experiment of this study. 37
Figure 3. Adhesion ability of selected lactic acid bacteria to HT-29 cells. 52
Figure 4. Cell viability of selected lactic acid bacteria to HT-29 cells. 54
Figure 5. Survival rate of mice in the AOM/DSS mouse model during lactic acid bacteria treatment. 56
Figure 6. Body weight change of mice in the AOM/DSS mouse model during lactic acid bacteria treatment. 59
Figure 7. Weight of spleen (A) and spleen-weight index (B) of AOM/DSS mouse model treated with lactic acid bacteria for 10 weeks. 60
Figure 8. Colon length (A) and the number of tumors (B) in the AOM/DSS mouse model after a 10-week of lactic acid bacteria treatment. 62
Figure 9. Representative histology of the large intestine in AOM/DSS mouse model treated with lactic acid bacteria for 10 weeks. Naive (A), Con (B), LF... 66
Figure 10. The protein expression level of the β-glucuronidase in the colon of AOM/DSS mouse model treated with lactic acid bacteria for 10 weeks. 69
Figure 11. Fecal β-glucuronidase activity in AOM/DSS mouse model treated with lactic acid bacteria for 10 weeks. 71
Figure 12. The protein expression levels of TNF-α (A), NF-κB (B), IκB-α (C), Cyclin D1 (D), Bcl-2 (E), and β-catenin (F), Bax (G), and p53 (H) in the... 74
Figure 13. Correlation analysis result of β-glucuronidase protein expression level in colon with CRC related factors 78
Figure 14. α-diversity indices Shannon index (A) and Chao 1 (B) in fecal microbiota diversity of AOM/DSS mouse model after 10-week lactic acid... 80
Figure 15. Principal coordinate analysis (PCoA) plot of β-diversity in fecal microbiota of AOM/DSS mouse model after 10-week lactic acid bacteria treatment. 82
Figure 16. The phylum-level gut microbiota changes in Bacillota (A), Bacteroidota (B), Actinomycetota (C), Candidatus Melainabacteria (D),... 84
Figure 17. The family-level gut microbiota changes in Christensenellaceae (A), Rikenellaceae (B), Helicobacteraceae (C), Akkermansiaceae (D), Lactobacillaceae... 86
Figure 18. The genus-level gut microbiota changes in Acetatifactor (A), Anaerostipes (B), Anaerotaenia (C), Oscillibacter (D), Ruminococcus (E),... 88
Figure 19. The gut microbiota changes in Limosilactobacillus fermentum (A), Lactobacillus paragasseri (B), and Pediococcus pentosaceus (C) following 10-week lactic acid bacteria treatment in the AOM/DSS mouse model. 92
Figure 20. The species-level gut microbiota changes in Intestinimonas butyriciproducens (A), Christensenella massiliensis (B), Kineothrix alysoides (C),... 93
Figure 21. Cladogram of linear discriminant analysis effect size (LEfSe) comparison on the taxonomy profile of the gut microbiota after 10-week... 97
Figure 22. Histogram of linear discriminant analysis effect size (LEfSe) comparison on the taxonomy profile of the gut microbiota after 10-week... 98
Figure 23. Correlation between the relative abundance of the gut microbiota at the species level and CRC related factors. 100
Figure 24. Histogram of linear discriminant analysis effect size (LEfSe) comparison on the functional profile of gut microbiota after 10-week treatment with lactic acid bacteria. 102
Figure 25. Correlation between predicted gut microbiota pathway abundance and CRC related factors. 107