Inhibitory Effects and the Mechanisms of Jangkanghwan (Korean Traditional Health Food) on Colon Inflammation = 장강환(한국 전통건강식품)이 대장에서의 염증 억제효과 및 작용기전|RISS 상세보기

국문 초록 (Abstract)

본 논문은 in vivo및 in vitro를 통해 한국 전통건강식품인 장강환이 대장에 미치는 보호 효과를 검증하였으며, 최종적으로 장강환이 대장에서 염증 억제 효과와 그의 기전을 확인하였다. 본 논문은 3 부분 (part)으로 구성되었다.
Part 1은chapter 1과 2로 나누어져 있으며, 장강환의 성분분석과 동물체내에서의 작용에 대하여 연구를 하였다. 장강환은 대장건강식품으로서 Atractylodes macrocephala koidzumi (백출), radish leaves (무청), Viscum album var. coloratum (겨우살이), dried Zingiber officinale Roscoe (건강)성분 등 12가지로 구성된 혼합물이다. UPLC-Q-TOF MS분석에 의하면 장강환은 주요하게 pheophorbide A, nabumetone alcohol, dehydrocostus lactone, plantamajoside, kaempferol 3, 7-dirhamnoside, quercetin 3-D-glucuronide 및 viscumneoside III등의 활성물질이 있는 것으로 나타났다. Chapter 1의 실험결과 마우스 모델에서 dextran sulfate sodium (DSS)이 유도하는 궤양성 대장염 (UC)에 대한 장강환의 예방 효과가 연구되었으며 장강환이 마우스에서 DSS로 인한 염증의 손상을 감소시킬 수 있음을 발견하였다. 장강환은 마우스의 체중감소를 완화시키고, 회복기에 체중증가를 촉진시켜주며, 대장길이 단축량 및 대장무게를 감소시킬 수 있을뿐만 아니라 장강환은 마우스의 disease activity index (DAI) 도 조절하였다. 장강환은 전염증인자(interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), cyclooxygenase-2 (COX-2), and nuclear factor kappa-B (NF-κB)가 혈청에서의 함량과 조직에서의 발현량을 감소시켰으며, 반대로, 혈청과 조직에서IL-10및 inhibitor of nuclear factor kappa-B kinase-α（IκB-α）의 함량과 발현량을 증가시켰다. 그리고 대장조직에서 대장염증 바이오마커인 monocyte chemoattractant protein-1 (MCP-1)과 macrophage inflammatory protein-3α (MIP-3α)의 mRNA 발현량을 감소시켰다. NGS분석에 의하면 장강환 그룹에서 Bacteroidetes 문 (phylum)은 감소시키고Firmicutes 문 은 증가하였으며 유익한 박테리아인 Bifidobacterium, Lactobacillus 및 Akkermansia속 등도 증가시키는 것으로 나타났다.
Chapter 2에서는 lipopolysaccharide (LPS)가 유도하는 대장상피기능 장애가 있는 BALB/c 마우스에게 장강환을 투여하여 마우스의 체중과 음식섭취량을 측정하였다. Colonic paracellular permeability, 혈청 염증사이토카인, bacterial translocation등 지표를 이용하여 장강환이 마우스 대장 상피기능에 대한 작용을 평가하였으며, 대장상피 조직에서 발현하는 tight junction (TJ) 관련 유전자occluding, claudin, zonula occludens (ZOs) proteins과 junction adhesion molecules (JAM)의 발현량을 qPCR과 western blot을 이용하여 확인하였다. 실험 결과 장강환은 마우스의 간장, 비장, 및 장막림프절 조직의 부종을 완화하고, LPS주사로 인한 마우스의 식욕부진과 설사를 감소시키는 것으로 나타났다. 장강환은 마우스의 음식 섭취량은 LPS그룹의 3.7 ± 0.15 g/day로부터 4.7 ± 0.21 g/day로 증가하고 마우스의 대변 수분의 함량을 LPS 그룹 마우스보다13.8 ± 1.23% 감소시켰다. 장강환은 LPS로 인한 마우스의 염증반응을 감소하고, 대장의 상피세포를 보호하고, Fluorescent macromolecules의 투과성은 LPS그룹의 4분의1이며, 대장조직에서 TJ와 연관된 유전자의 mRNA와 단백질의 발현량을 높이는 효과를 나타내었다.
Part 2 (chapter 3)에서는 bioinformatic 기술을 이용하여 network pharmacology 및 장강환이 궤양성 대장염에 미치는 영향에 대한 세포 실험을 기반으로 pathway 및 기전을 연구하였다. 본 연구에서는 장강환의 활성성분을 수집하고 Gene expression omnibus (GEO) 데이터베이스를 통해 건강한 사람과 UC를 가진 사람 사이에 차별적으로 발현되는 유전자를 분석하고 UC targets을 얻었다. 마지막으로, 교차 targets 유전자를 얻기 위해 장강환과 질병 targets 유전자들을 배치하였다. 교차 유전자는 Gene ontology (GO) 과 Kyoto Encyclopedia of Genes and Genomes (KEGG)경로의 밀집 분석을 수행했으며 그 결과를 세포실험을 통해 검증되었다. 참고문헌검색, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP)데이터 검색을 통하여 장강환의 79개의 활성성분 및 264개의 활성 성분 targets을 확인하고, UC에서 발현의 차이를 보이는 유전자 910개(P value<0.005, |log2(fold change)| >1)를 선별하여 39개의 교차 유전자를 선정하였다. 삼백초 중의 Quercetin은 24개의 교차 유전자와 잠재적 작용이 있으며, PTGS2은 35개 유효성분과 상호작용이 있었다. 교차유전자의 functional annotation은 biological regulation，cellular process와 binding등과 연관이 있었다. 152개의 밀집경로 결과 중 TNF 신호전달 경로에서 IL-6, chemokine (C-C motif) ligand 2 (CCL2), matrix metallopeptidase 9 (MMP9), IL-1β, intercellular adhesion molecule 1 (ICAM1), PTGS2, vascular cell adhesion molecule 1 (VCAM1) 및FOS가 장강환이 항 대장염 억제 효과를 발휘하는 주요 경로 및 targets 유전자일 수 있다. 그리고 In vitro실험을 통하여 CCD841CoN cell 항염증 targets에 대한 장강환의 효과를 검증하였다.
Part 3은 chapter 4부분이며, 여기서는 장강환 중 함량이 제일 많게 검출된 활성화합물 pheophorbide A(phA)가LPS로 유도된 RAW264.7 cell의 염증을 완화시키는 것을 증명하였다. 이 실험에서는 laser stimulation 이 없는 세 농도의 phA를 사용하여 RAW264.7 cell 에서 LPS로 인한 염증의 완화 효과를 연구하였다.
세포 형태, cell viability, 세포 상층액중 사이토카인 함량 및 NO함량 측정, 세포 내 염증 관련 유전자들의 mRNA 발현량의 측정을 통하여 10 nM, 15 nM, 20 nM 농도의 phA가 LPS로 인한 세포손상을 억제하는 것을 발견하였고, 세포에서 NO 및 전염증인자 인IL-1β，IL-6，IFN-γ과 TNF-α의 분비를 감소시켰으며, 항염증인자 IL-10의 함량을 증가시켰다. 한편 phA는 RAW264.7 cell에서 항염인자 유전자인IκB-α의 발현을 증가시키고，NF-κB경로의 활성화를 억제하여 염증을 억제하였다. 전반적으로, phA가 laser에 의해 자극되지는 않았지만, 여전히 우수한 항염증 효과를 가지며 NF-κB 경로로 인한 염증 손상을 억제하여 phA의 향후 개발 및 활용에 대한 새로운 아이디어를 제공했다.
결론적으로, 장강환은 한국 전통건강식품으로서 마우스의 대장에 대해 현저한 보호효과가 있을 뿐만 아니라 인체 대장세포 및 RAW264.7 cell에 대해서도 좋은 항염증 효과 및 염증성 cytokine을 감소하고 항염증성을 높여주며 NF-κB활성을 낮춰주었다. 이러한 연구는 장강환의 응용과 보급에 이론적 근거를 제공한다고 하겠다.

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본 논문은 in vivo및 in vitro를 통해 한국 전통건강식품인 장강환이 대장에 미치는 보호 효과를 검증하였으며, 최종적으로 장강환이 대장에서 염증 억제 효과와 그의 기전을 확인하였다. 본 논...

다국어 초록 (Multilingual Abstract)

This thesis is to verify the protective effect of Jangkanghwan (JKH), a traditional Korean food, on the colon through in vivo and in vitro experiments. Finally, the way in which JKH exerts its effects and the functional effects of the main active ingredients are confirmed. The overall thesis contains three studies.
The first study contains chapter 1 and chapter 2, which focus on the composition analysis of JKH and its anti-inflammation effect in animals. JKH is a mixture of colonic healthy foods composed of radish leaves, Atractylodes macrocephala Koidzumi, Viscum album var. coloratum, dried Zingiber officinale Roscoe, etc. According to UPLC-Q-TOF MS analysis, JKH consists mainly of 17 active substances, such as pheophorbide A (phA), Nabumetone alcohol, Dehydrocostus lactone, Plantamajoside, kaempferol 3, 7-dirhamnoside, Quercetin 3-D-glucuronide, and Viscumneoside III. In chapter 1, the first animal experiment, we investigated the preventive effects of JKH on dextran sulfate sodium (DSS)-induced ulcerative colitis in a murine model. It was found that JKH can delay weight loss in mice, promote weight gain during recovery, and reduce colonic shortening and colon weight. In addition, the murine disease activity index was controlled after treatment using JKH. It can reduce the content of pro-inflammatory factors in serum and expression in tissues, such as interleukin (IL)-6, IL-1β, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), cyclooxygenase-2 (COX-2), and nuclear factor kappa-B (NF-κB). In contrast, the content and expression of IL-10 and the inhibitor of nuclear factor kappa-B kinase-α (IκB-α) in the serum and tissues were increased. The mRNA expression of the colitis characteristic biomarker monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-3α (MIP-3α) were reduced in colon tissues. Using next-generation sequencing technology, the Bacteroidetes phylum in the JKH group decreased, while the Firmicutes phylum increased, and the number of beneficial bacteria—Bifidobacterium, Lactobacillus, and Akkermansia—increased.
In the second animal experiment (chapter 2), JKH was administered by gavage to BALB/c mice with lipopolysaccharide (LPS)-induced colonic epithelial dysfunction, and mouse body weight and food intake were recorded. Indexes such as colonic paracellular permeability, serum inflammatory cytokines, and bacterial translocation were used to comprehensively evaluate the regulatory effect of JKH on mouse colonic epithelial function, and qPCR and Western blot were also used to analyze the expression of tight junction (TJ)-related genes, such as occludin, claudin, zonula occludens (ZOs) proteins, and junction adhesion molecules (JAM) in the colonic epithelial tissue. The experimental results indicated that JKH relieved the edema of the liver, spleen, and mesenteric lymph node tissues, and reduced the loss of appetite and diarrhea caused by LPS injection in mice. It increased the amount of mice food intake from 3.7 ± 0.15 g/day in the LPS group to 4.7 ± 0.21 g/day; the water content in the feces of mice in the JKH group was 13.86 ± 1.23% less than that in the LPS group. JKH reduced the inflammatory response in mice caused by LPS, protected the integrity of the colon, the permeability of fluorescent macromolecules was one-fourth of the LPS group, and enhanced the mRNA and protein expression of TJ-related proteins in colon tissue.
The study 2 (chapter 3), investigated the pathway and mechanism based on network pharmacology and cell experiment of the effect of JKH on ulcerative colitis (UC). In this study, we collected the active ingredients of JKH and identified the targets, obtained UC targets by analyzing the differentially expressed genes between healthy people and people with UC through the (gene expression omnibus) GEO database. Finally, the JKH’s targets and the disease targets were mapped to obtain the intersection target gene. The intersection genes were subject to the enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and the results were verified by cell experiments. A search of the existing literature, Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and other databases helped screen for the 79 active ingredient and 264 active ingredient targets in JKH and 910 differentially expressed genes in UC (p-value < 0.05 and |log2(fold change)|> 1), and finally got 39 intersection genes. Quercetin from Sambaekcho has potential effects with 24 intersection genes and prostaglandin-endoperoxide synthase 2 (PTGS2) gene interacts with 35 active ingredients. The functional annotation of the intersection gene is related to biological regulation, cellular processes, and binging. Among 152 enriched pathways and all intersecting genes, IL-6, chemokine (C-C motif) ligand 2 (CCL2), matrix metallopeptidase 9 (MMP9), IL-1β, intercellular adhesion molecule 1 (ICAM1), PTGS2, vascular cell adhesion molecule 1 (VCAM1), and FOS in the TNF signaling pathway may be the key pathways and target genes for JKH to exert anti-colitis effects. It also uses in vitro experiments to determine the mechanism of action of JKH against colitis. This study demonstrates the application of network pharmacology in clarifying the mechanism of action and molecular target of JKH against colitis. Cellular experiments validated the effect of JKH on CCD841CoN cell anti-inflammatory targets.
The last study is the chapter 4, three concentrations of phA without laser stimulation were used to study the alleviating effect of lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells. Through the observation of cell morphology, examination of cell viability, quantitation of cytokines and nitric oxide (NO) in the cell supernatant medium, and determination of the expression of relevant mRNA in cells, it was found that phA at concentrations of 10 nM, 15 nM, and 20 nM all inhibited the inflammatory cellular damage caused by LPS to different degrees, decreased cell-secreted NO and pro-inflammatory factors such as IL-1β, IL-6, IFN-γ, and TNF-α, and increased the anti-inflammatory factor IL-10. Additionally, phA increased the expression of anti-inflammatory genes such as IκB-α in RAW264.7 cells and inhibited the activation of the NF-κB pathway, thus suppressing inflammation. Overall, although phA was not stimulated by laser, it still possessed satisfactory anti-inflammatory effects and inhibited inflammatory damage caused by the NF-κB pathway, providing a new idea for the future development and utilization of phA.
In conclusion, JKH, as a Korean traditional food, not only has good protective effect on the colon of mice, but also has good anti-inflammatory effect on human colon cells and RAW264.7 cells, which provides a theoretical basis for the application and promotion to JKH.

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목차 (Table of Contents)

TABLE OF CONTENTS
ACKNOWLEDGEMENTS i
TABLE OF CONTENTS ii
LIST OF TABLES vii
LIST OF FIGURES ix

TABLE OF CONTENTS
ACKNOWLEDGEMENTS i
TABLE OF CONTENTS ii
LIST OF TABLES vii
LIST OF FIGURES ix
Abstract xii
CHAPTER 1.
Inhibitory effect of Jangkanghwan (Korean traditional health food) on experimental ulcerative colitis in mice
Ⅰ. INTRODUCTION 2
Ⅱ. MATERIALS AND METHODS 4
1. Source of Jangkanghwan 5
2. UPLC-Q-TOF MS conditions of Jangkanghwan 5
3. Animal experiments 6
4. Evaluation of disease activity index (DAI) 7
5. Hematoxylin and eosin (H&E) sections analysis of colon tissue 7
6. Measurements of serum IL-6, IL-1β, TNF-α, and IFN-γ cytokine levels in mice 10
7. mRNA expression determination using RT-qPCR 10
8. Protein expression determination by Western blotting 10
9. Next-generation sequencing (NGS) analysis of murine feces 12
10. Statistical analysis 13
Ⅲ. RESULTS 13
1. UPLC-Q-TOF MS analysis of Jangkanghwan 13
2. Body weight analysis of mice 16
3. Colon length and weight analysis of mice 16
4. Liver, spleen, kidney, and testis index analysis of mice 19
5. Colon H&E section analysis of mice 19
6. Disease activity index data analysis of mice 22
7. Cytokine level of TNF-α, IFN-γ, IL-1β, and IL-6 in the serum of mice 22
8. qPCR analysis of murine colon tissue 24
9. Colon tissue protein expression analysis of mice 27
10. NGS analysis of mice 29
Ⅳ.DISCUSSION 32
Ⅴ. CONCLUSION 37
CHAPTER 2.
Protective effect of Jangkanghwan (Korean traditional health food) on lipopolysaccharide-induced disruption of the colonic epithelial barrier
Ⅰ. INTRODUCTION 39
Ⅱ. MATERIALS AND METHODS 41
1. Introduction of Jangkanghwan 41
2. Animal experimental design 42
3. Diarrhea after LPS administration 44
4. Bacterial translocation assay 44
5. Determination of the intestinal mucosa permeability 44
6. Serum inflammatory cytokine test 45
7. H&E staining of colon tissue 45
8. mRNA expression determination using RT-qPCR 45
9. Protein expression determination by Western blotting 47
10. Statistical analysis 48
Ⅲ. RESULTS 48
1. Food intake analysis 48
2. Organ index analysis 49
3. Diarrhea after LPS administration 49
4. Serum inflammatory cytokine analysis 53
5. Colon bacteria translocation analysis 53
6. Colonic permeability analysis 55
7. Colon histopathological observation 55
8. mRNA and protein expression analysis of colon tissue 59
Ⅳ. DISCUSSION 59
Ⅴ. CONCLUSION 65
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)
Ⅰ. INTRODUCTION 68
Ⅱ. MATERIALS AND METHODS 70
1. Chemical composition and target collection 70
2. Identification of differentially expressed genes in ulcerative colitis 71
3. Screening and analysis of Jangkanghwan and UC intersection targets 71
4. Bioinformatic analysis 72
5. Cell culture 72
6. Jangkanghwan's cytotoxicity test 72
7. Live/dead cell staining 73
8. Cell enzyme-linked immunosorbent assay (ELISA) 73
9. mRNA expression determination using RT-qPCR 74
10. Protein expression determination by Western blotting 74
11. Statistical analysis 76
Ⅲ. RESULTS 76
1. Compound-Target Network analysis 76
2. Identification of candidate Targets for Jangkanghwan against UC 82
3. Analysis of GO enrichment of Jangkanghwan target genes for alleviating colitis 87
4. Gene-pathway network analysis 89
5. Effects of Jangkanghwan on cell viability 89
6. Morphological changes and live/dead cell staining of CCD841CoN cells 91
7. Effects of Jangkanghwan on MPO, TNF-α, IFN-γ, IL-6, IL-1β, and, IL-10 levels of cell 91
8. Effects of Jangkanghwan on mRNA and protein expression in the CCD841CoN cell 95
Ⅳ. DISCUSSION 95
Ⅴ. CONCLUSION 103
CHAPTER 4.
Inhibitory effect of pheophorbide A on Lipopolysaccharide-induced inflammation in RAW264.7 cells via NF-κB pathway
Ⅰ. INTRODUCTION 105
Ⅱ. MATERIALS AND METHODS 107
1. Sample preparation 108
2. Cells cultures 108
3. Cell viability 108
4. Cell morphology observation 109
5. Measurement of NO production 109
6. Enzyme-linked immunosorbent assay (ELISA) 110
7. mRNA expression determination using RT-qPCR 110
8. Statistical analysis 111
Ⅲ. RESULTS 111
1. The effect of pheophorbide A on the viability of RAW264.7 cells 113
2. The effect of pheophorbide A on the morphology of RAW264.7 cells 113
3. The effect of pheophorbide A on NO production in RAW264.7 cells 115
4. Effect of pheophorbide A on IL-10, IL-1β, IL-6, TNF-α, and IFN-γ levels 116
5. Effect of pheophorbide A on IL-1β, IL-6, IL-10, iNOS, TNF-α, COX-2, NF-κB, and IκB-α mRNA expression 118
Ⅳ. DISCUSSION 120
Ⅴ. CONCLUSION 125
REFFERENCES 127
[ABSTRACT IN KOREAN] 149

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