Title Page
Contents
I. Introduction 10
II. Materials and Methods 12
1. Patients 12
2. Isolation of human peripheral blood mononuclear cell (PBMCs) 13
3. Animal chronic-plus-binge alcohol feeding model (NIAAA model) 13
4. Quantitative real time polymerase chain reaction 14
5. Western blot 16
6. Isolation of hepatic immune cells (liver mononuclear cell isolation) and NK cell 17
7. Flow cytometry analyzes and fluorescence-activated single cell sorting (FACS) 17
8. Serum endotoxin level analysis 18
9. QuantSeq 3'mRNA-sequencing 18
10. Statistical analysis 19
III. Results 20
1. Function and activity of NK cells change depending on the degree of progression of alcoholic liver disease 20
2. Alterations of human gut microbiome composition in alcoholic liver disease 24
3. Supplementation with P. dorei and L. helveticus ameliorates the progression of alcoholic liver disease 29
4. Gut microbiome composition is altered by strain supplementation in the chronic-plus-binge alcohol feeding model (NIAAA model) 32
5. P. dorei and L. helveticus attenuate ethanol-induced liver damage by modulating inflammatory and immune responses 35
6. P. dorei and L. helveticus can improve intestinal mucosa damage caused by ethanol 39
7. P. dorei and L. helveticus promote NK cell activation 41
8. Involvement of a probiotic strain in immune response is confirmed through hepatic RNA gene expression analysis 45
IV. Discussion 49
V. References 55
Abstract Korean 61
Abstract English 63
Table 1. Primers used in this study 14
Table 2. Antibodies used in this study 17
Table 3. Flow diagram of study. 26
Table 4. Baseline characteristics of participants. 26
Figure 1. NK cell frequency and function change with the progression of alcoholic liver disease. 22
Figure 2. Comparisons of relative abundances of gut microbiome and functional biomarkers between healthy control and ALD patients. 27
Figure 3. Results of histopathological changes in liver supplemented with P. dorei and L. helveticus in the chronic-plus-binge alcohol feeding model (NIAAA model). 30
Figure 4. Mouse stool analysis in the chronic-plus-binge alcohol feeding model (NIAAA model). 33
Figure 5. Effects of strains on expression of genes related to inflammation and immune response in chronic-plus-binge alcohol feeding model (NIAAA model)... 37
Figure 6. Effect of strains on recovery of impaired intestinal barrier caused by ethanol feeding in chronic-plus-binge alcohol feeding model (NIAAA model). 40
Figure 7. Effects of strains that increase the activity and function of NK cells that have been reduced by ethanol intake. 43
Figure 8. Results of QuantSeq mRNA-Seq analysis in chronic-plus-binge alcohol feeding model (NIAAA model). 47
Supplementary Figure 1. Effect of strains administration on liver inflammation in an acute alcoholic steatohepatitis model. 65
Supplementary Figure 2. Changes in liver NK cells caused by acute ethanol intake. 67
Supplementary Figure 3. Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) intake for 21 weeks in mice induced liver fibrosis and nodules. 68
Supplementary Figure 4. Insignificant effect of strain administration on the development of liver cirrhosis in choline-deficient, L-amino acid-defined, high-... 69