Title Page
ABSTRACT
국문 초록
Contents
NOMENCLATURE 14
1. INTRODUCTION 15
2. LITERATURE REVIEW 17
2.1. Stress 17
2.1.1. Overview of Stress 17
2.1.2. Treatment for Stress 19
2.2. HPA Axis and MGB Axis 21
2.2.1. HPA Axis 21
2.2.2. MGB Axis 23
2.3. Gut Microbiome 26
3. MATERIALS AND METHODS 27
3.1. Preparation and Evaluation of Lactobacillus Strains 27
3.1.1. Evaluation of Acid and Bile Tolerance 27
3.1.2. Evaluation of Antioxidant Ability with DPPH scavenging test 28
3.1.3. Evaluation of Antibiotics Resistance with E-test 29
3.1.4. Evaluation of Adhesion ability in HT-29 29
3.2. Cell Culture 30
3.3. Cytotoxicity and Lactate Dehydrogenase Production 31
3.3.1. CCK-8 assay 31
3.3.2. LDH assay 32
3.4. In vitro Experiment and Treatment 33
3.5. Animal Experiment and Treatment 34
3.6. Sample Collection 37
3.7. Histological Analysis 37
3.8. Serum Analysis 38
3.9. Western Blot Evaluation of Protein Expression 39
3.10. Quantitative Reverse Transcription PCR (qRT-PCR) 41
3.11. Fecal Short Chain Fatty Acid Analysis 44
3.12. Gut Microbiome Analysis 45
3.13. Statistical Analysis 45
4. RESULT 46
4.1. Selection of Lactobacillus Strains 46
4.2. Cytotoxicity of Lactobacillus Strains in HT-29 and SH-SY5Y 52
4.3. The Effects of L. fermentum on Inflammation and Tight Junction Proteins in HT-29 55
4.4. The Effects of L. fermentum on Stress and Neurological Biomarkers in SH-SY5Y 57
4.5. Histological Analysis in Hippocampus and Colon 60
4.6. The mRNA Expression Level in Ileum and Hippocampus. 63
4.7. The Proteins Expression of Neurological Biomarkers in Hippocampus 69
4.8. The Concentration of Stress-related Hormones in Serum 72
4.9. Stress-induced Alterations in Fecal Microbiome Composition 74
5. DISCUSSION 76
REFERENCES 79
Table 1. Treatment groups 36
Table 2. Antibodies used in Western Blot 40
Table 3. Primer sequences used in qRT-PCR 42
Table 4. Antibiotic resistance of Lactobacillus strains in E-test 48
Figure 1. The Microbiome-Gut-Brain axis 25
Figure 2. Experimental Design for unpredictable chronic mild stress 35
Figure 3. Acid tolerance and bile tolerance of Lactobacillus strains 47
Figure 4. Antioxidant ability of Lactobacillus strains in DPPH scavenging assay 50
Figure 5. Adhesion ability of Lactobacillus strains in HT-29 51
Figure 6. The cell viability of Lactobacillus strains in HT-29 cells 53
Figure 7. The cell viability of Lactobacillus strains in SH-SY5Y cells 54
Figure 8. Anti-inflammatory effect of Lactobacillus strains in HT-29 cell line 56
Figure 9. Anti-inflammatory effect of Lactobacillus strains in SH-SY5Y 58
Figure 10. Hematoxylin and Eosin (H&E)-stained sections of each group in the mouse hippocampus 61
Figure 11. H&E stained sections of each group in the mouse colon 62
Figure 12. The mRNA expression level of inflammatory cytokines in ileum 64
Figure 13. The mRNA expression level of tight junction proteins in ileum 65
Figure 14. The mRNA expression level of inflammatory cytokines and tight junction proteins in hippocampus 67
Figure 15. The mRNA expression level of neurological biomarkers in hippocampus 68
Figure 16. The protein expression level in hippocampus 70
Figure 17. Relative protein expression level in hippocampus 71
Figure 18. The concentration of stress-related hormones in serum 73
Figure 19. Fecal microbiome composition of 0 weeks and 8 weeks 75