Title Page
Contents
Abstract 16
CHAPTER 1. Inhibitory effect of Jangkanghwan (Korean traditional health food) on experimental ulcerative colitis in mice 21
Ⅰ. INTRODUCTION 22
Ⅱ. MATERIALS AND METHODS 24
1. Source of Jangkanghwan 25
2. UPLC-Q-TOF MS conditions of Jangkanghwan 25
3. Animal experiments 26
4. Evaluation of disease activity index (DAI) 27
5. Hematoxylin and eosin (H&E) sections analysis of colon tissue 27
6. Measurements of serum IL-6, IL-1β, TNF-α, and IFN-γ cytokine levels in mice 30
7. mRNA expression determination using RT-qPCR 30
8. Protein expression determination by Western blotting 30
9. Next-generation sequencing (NGS) analysis of murine feces 32
10. Statistical analysis 33
Ⅲ. RESULTS 33
1. UPLC-Q-TOF MS analysis of Jangkanghwan 33
2. Body weight analysis of mice 36
3. Colon length and weight analysis of mice 36
4. Liver, spleen, kidney, and testis index analysis of mice 39
5. Colon H&E section analysis of mice 39
6. Disease activity index data analysis of mice 42
7. Cytokine level of TNF-α, IFN-γ, IL-1β, and IL-6 in the serum of mice 42
8. qPCR analysis of murine colon tissue 44
9. Colon tissue protein expression analysis of mice 47
10. NGS analysis of mice 49
Ⅳ. DISCUSSION 52
Ⅴ. CONCLUSION 57
CHAPTER 2. Protective effect of Jangkanghwan (Korean traditional health food) on lipopolysaccharide-induced disruption of the colonic epithelial barrier 58
Ⅰ. INTRODUCTION 59
Ⅱ. MATERIALS AND METHODS 61
1. Introduction of Jangkanghwan 61
2. Animal experimental design 62
3. Diarrhea after LPS administration 64
4. Bacterial translocation assay 64
5. Determination of the intestinal mucosa permeability 64
6. Serum inflammatory cytokine test 65
7. H&E staining of colon tissue 65
8. mRNA expression determination using RT-qPCR 65
9. Protein expression determination by Western blotting 67
10. Statistical analysis 68
Ⅲ. RESULTS 68
1. Food intake analysis 68
2. Organ index analysis 69
3. Diarrhea after LPS administration 69
4. Serum inflammatory cytokine analysis 73
5. Colon bacteria translocation analysis 73
6. Colonic permeability analysis 75
7. Colon histopathological observation 75
8. mRNA and protein expression analysis of colon tissue 79
Ⅳ. DISCUSSION 79
Ⅴ. CONCLUSION 85
CHAPTER 3. Based on network pharmacology and cell experiment to explore the molecular mechanism of anti-ulcerative colitis properties of Jangkanghwan (Korean traditional health food) 87
Ⅰ. INTRODUCTION 88
Ⅱ. MATERIALS AND METHODS 90
1. Chemical composition and target collection 90
2. Identification of differentially expressed genes in ulcerative colitis 91
3. Screening and analysis of Jangkanghwan and UC intersection targets 91
4. Bioinformatic analysis 92
5. Cell culture 92
6. Jangkanghwan's cytotoxicity test 92
7. Live/dead cell staining 93
8. Cell enzyme-linked immunosorbent assay (ELISA) 93
9. mRNA expression determination using RT-qPCR 94
10. Protein expression determination by Western blotting 94
11. Statistical analysis 96
Ⅲ. RESULTS 96
1. Compound-Target Network analysis 96
2. Identification of candidate Targets for Jangkanghwan against UC 102
3. Analysis of GO enrichment of Jangkanghwan target genes for alleviating colitis 107
4. Gene-pathway network analysis 109
5. Effects of Jangkanghwan on cell viability 109
6. Morphological changes and live/dead cell staining of CCD841CoN cells 111
7. Effects of Jangkanghwan on MPO, TNF-α, IFN-γ, IL-6, IL-1β, and, IL-10 levels of cell 111
8. Effects of Jangkanghwan on mRNA and protein expression in the CCD841CoN cell 115
Ⅳ. DISCUSSION 115
Ⅴ. CONCLUSION 123
CHAPTER 4. Inhibitory effect of pheophorbide A on Lipopolysaccharide-induced inflammation in RAW264.7 cells via NF-κB pathway 124
Ⅰ. INTRODUCTION 125
Ⅱ. MATERIALS AND METHODS 127
1. Sample preparation 128
2. Cells cultures 128
3. Cell viability 128
4. Cell morphology observation 129
5. Measurement of NO production 129
6. Enzyme-linked immunosorbent assay (ELISA) 130
7. mRNA expression determination using RT-qPCR 130
8. Statistical analysis 131
Ⅲ. RESULTS 131
1. The effect of pheophorbide A on the viability of RAW264.7 cells 133
2. The effect of pheophorbide A on the morphology of RAW264.7 cells 133
3. The effect of pheophorbide A on NO production in RAW264.7 cells 135
4. Effect of pheophorbide A on IL-10, IL-1β, IL-6, TNF-α, and IFN-γ levels 138
5. Effect of pheophorbide A on IL-1β, IL-6, IL-10, iNOS, TNF-α, COX-2, NF-κB, and IκB-α mRNA expression 138
Ⅳ. DISCUSSION 140
Ⅴ. CONCLUSION 145
REFERENCES 147
ABSTRACT IN KOREAN 169
CHAPTER 1 11
Table 1. Disease activity index assessment standards 29
Table 2. Primer sequences list 31
Table 3. Components of Jangkanghwan 35
Table 4. Index of liver, kidney, testis, and spleen in mice 40
Table 5. Phylum level intestinal microbiota of DSS-induced mice 50
Table 6. Bifidobacterium, Lactobacillus, and Akkermansia genus level intestinal microbiota of DSS-induced mice 51
CHAPTER 2 11
Table 7. Primer sequence list of colon tight junctions genes in BALB/c mice that underwent LPS-induced colonic epithelial injury 66
Table 8. Organ indexes of BALB/c mice that underwent LPS-induced colonic epithelial injury 71
Table 9. Bacterial translocation in the liver, spleen, and mesenteric lymph node 76
CHAPTER 3 11
Table 10. Primer sequences list 95
Table 11. The active pharmaceutical ingredients in Jangkanghwan selected for analysis 97
Table 12. IL-6, CCL2, MMP9, IL-1β, ICAM1, PTGS2, VCAM1, and c-fos mRNA expression in the CCD841CoN cell 116
CHAPTER 4 12
Table 13. Primer sequences list 132
Table 14. NO production of LPS-induced RAW264.7 cell 137
Table 15. IL-1β, IL-6, IL-10, TNF-α, and IFN-γ expression level in LPS-induced RAW264.7 cell 139
CHAPTER 1 13
Fig. 1. Experimental test treatments. 28
Fig. 2. Jangkanghwan UPLC-Q-TOF MS analysis chromatogram. 34
Fig. 3. Body weight of mice during the experiment. 37
Fig. 4. Colon length and colon index of mice. 38
Fig. 5. H&E section of colon tissue in mice. 41
Fig. 6. Disease activity index of mice. 43
Fig. 7. ELISA analysis in serum of mice. 45
Fig. 8. qPCR analysis of colon tissue in mice. 46
Fig. 9. Colon tissue Western blot analysis of mice. 48
CHAPTER 2 13
Fig. 10. Animal experimental design of LPS-induced colonic epithelial injury in BALB/c mice. 63
Fig. 11. Daily food intake of BALB/c mice that underwent LPS-induced colonic epithelial injury. 70
Fig. 12. Fecal moisture content of BALB/c mice that underwent LPS-induced colonic epithelial injury. 72
Fig. 13. Cytokine indexes in the serum of BALB/c mice that underwent LPS-induced colonic epithelial injury. 74
Fig. 14. Colon FITC-D permeability in BALB/c mice that underwent LPS-induced colonic epithelial injury. 77
Fig. 15. Pathological observation by H&E staining of colon tissue from BALB/c mice that were subject to LPS-induced colonic epithelial injury. 78
Fig. 16. Tight junctions mRNA expression in the colon tissue of BALB/c mice that underwent LPS- induced colonic epithelial injury. 80
Fig. 17. Tight junctions protein expression in the colon tissue of BALB/c mice that underwent LPS-induced colonic epithelial injury. 81
CHAPTER 3 14
Fig. 18. UC differentially expressed genes heat map (a) and volcano map (b). 103
Fig. 19. Venn diagram of UC differentially expressed genes and the Jangkanghwan target gene. 104
Fig. 20. Ingredient-compound-target network. 105
Fig. 21. Protein-protein interaction network diagram of 39 intersecting genes. 106
Fig. 22. GO enrichment analysis. 108
Fig. 23. Enrichment analysis of KEGG pathway. 110
Fig. 24. Effect of Jangkanghwan on CCD841CoN cell viability. 112
Fig. 25. (a) Microscopic morphological observation of CCD841CoN cells and (b) results of live/dead double staining cell experiments. 113
Fig. 26. The medium concentration of MPO, TNF-α, IFN-γ, IL-6, IL-10, and IL-1β in Jangkanghwan and/or TNF-α treatment with CCD841CoN cells. 114
Fig. 27. IL-6, CCL2, MMP9, IL-1β, ICAM1, PTGS2, VCAM1, and c-fos protein expression in CCD841CoN cells. 117
CHAPTER 4 15
Fig. 28. Effect of pherphorbide A on RAW264.7 cell viability. 134
Fig. 29. Cell morphology observation in LPS-induced RAW264.7 cell. 136
Fig. 30. Cytokine indexes in the medium in LPS-induced RAW264.7 cell. 141