Xanthone의 in vitro 및 in vivo 모델 내 알레르기 접촉성 피부염 조절 효능 연구

다국어 초록 (Multilingual Abstract)

Xanthone is a phenolic compound found in a few higher plant families such as gentian root. Xanthone has been known as having a variety of biological properties, such as anti-oxidant, anti-bacterial, anti-inflammatory and anti-cancer activities. However, the molecular and cellular mechanisms underlying the activity of xanthone in allergic contact dermatitis (ACD) remain to be explored. Therefore, the aim of this study was to investigate the regulatory effect of xanthone on allergic contact dermatitis in human keratinocytes (HaCaT), human mast cells (HMC-1) in vitro and in an experimental murine model in vivo. In the present study, treatment with xanthone reduced the production of pro-inflammatory cytokines and chemokine including interleukin (IL)-1β, IL-6, IL-8, and expression of chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC) in tumor necrosis factor (TNF)-α and interferon (IFN)-γ-stimulated HaCaT cells. Xanthone treatment also suppressed the production of pro-inflammatory cytokines, chemokine such as TNF-α, IL-6, IL-8, histamine and expressions of thymic stromal lymphopoietin (TSLP) in phorbol myristate acetate and calcium ionophore A23187 (PMACI)-stimulated HMC-1 cells. Furthermore, xanthone significantly suppressed the phosphorylation of mitogen-activated protein kinases (MAPKs), nuclear factor-kappa B (NF-κB) signaling pathway and activation of caspase-1 in stimulated keratinocytes and mast cells. Additionally, xanthone administration alleviated epidermal hyperplasia in the murine model of 2,4-dinitrofluorobenzene (DNFB)-induced dermatitis by reducing the serum levels of immunoglobulin E (IgE), histamine, and pro-inflammatory cytokines and suppressing MAPKs phosphorylation. Xanthone administration also inhibited mortality due to compound 48/80-induced anaphylactic shock and suppressed the passive cutaneous anaphylaxis (PCA) reaction mediated by IgE. Collectively, these findings suggest that xanthone has a potential for use in the treatment of allergic inflammatory diseases.

국문 초록 (Abstract)

잔톤(xanthone)은 용담초의 뿌리 등에서 발견되는 페놀성 화합물로 독특한 화학구조를 가지고 있다. 이것은 다양한 생리활성도 가지고 있어, 항산화, 항균, 항염증, 항암 효능 등이 보고되어 있다. 하지만, 잔톤의 알러지성 접촉성 피부염에 대한 효능 및 분자생물학적 기전은 거의 밝혀지지 않았다. 그래서, 본 연구에서는 인간유래 각질세포인 HaCaT, 인간유래 비만세포인 HMC-1와 동물모델을 이용하여 잔톤의 조절작용을 검토하였다. 잔톤은 tumor necrosis factor (TNF)-α, interferon (IFN)-γ로 자극된 HaCaT세포에서 interleukin (IL)-1β, IL-6, IL-8과 같은 전염증성 사이토카인의 생산을 감소시켰고, chemokines thymus and activation-regulated chemokine (TARC)과 macrophage-derived chemokine (MDC)의 발현을 억제하였다. 또한, 잔톤은 phorbol myristate acetate, calcium ionophore A23187 (PMACI)로 자극된 HMC-1세포에서 histamine과 TNF-α, IL-6, IL-8과 같은 전염증성 사이토카인의 분비를 억제하였고, thymic stromal lymphopoietin (TSLP)의 발현도 감소시켰다. 잔톤은 두 세포에서 모두 mitogen-activated protein kinase (MAPKs)의 인산화, nuclear factor-kappa B (NF-κB) 신호전달경로과 caspase-1의 활성화를 억제하였다. 2,4-Dinitrofluorobenzene (DNFB)으로 피부염을 유도한 동물모델에서 잔톤의 경구 투여는 혈청 immunoglobulin E (IgE), histamine, 전염증성 사이토카인의 감소를 통해 표피과형성을 억제하였다. 또한, 같은 모델에서 MAPKs의 인산화를 억제하였다. 더욱이, compound 48/80으로 유도된 anaphylactic shock 사망률과 IgE로 매개된 passive cutaneous anaphylaxis (PCA) 반응이 잔톤의 투여에 의해 감소되었다. 이러한 결과는 잔톤이 알러지성 염증 질환에 생리활성물질로 활용될 수 있다는 것을 보여주는 것이다.

목차 (Table of Contents)

• CONTENTS I
• LIST OF TABLES VIII
• LIST OF FIGURES IX
• ABSTRACT XI
• CHAPTER I: BACKGROUND 1
• 1.1. Skin immunity 2
• 1.2. Allergic contact dermatitis 2
• 1.3. Keratinocytes 5
• 1.4. Mast cells 6
• 1.5. Anaphylaxis 7
• 1.6. Treatment for allergic contact dermatitis 8
• 1.7. Natural products 9
• 1.8. Xanthone 10
• 1.9. Biological activities of xanthone 11
• 1.9.1. Anti-bacterial activity 11
• 1.9.2. Anti-oxidant activity 12
• 1.9.3. Anti-allergic activity 12
• 1.9.4. Anti-malarial activity 13
• 1.9.5. Anti-cancer activity 13
• 1.10. Aim of present research 14
• 1.11. References 15
• CHAPTER II: XANTHONE SUPPRESSES TNF-α/IFN-γ-STIMULATED PRO-INFLAMMATORY MEDIATORS IN HUMAN KERATINOCYTES 23
• 2.1. Abstract 24
• 2.2. Introduction 25
• 2.3. Materials and Methods 29
• 2.3.1. Reagents 29
• 2.3.2. Preparation of xanthone 29
• 2.3.3. Cell culture 30
• 2.3.4. Cell viability 30
• 2.3.5. Enzyme-linked immunosorbent assay (ELISA) 30
• 2.3.6. RNA isolation and reverse transcription polymerase chain reaction (RT-PCR) 31
• 2.3.7. Real-time RT-PCR 32
• 2.3.8. Preparation of total cell lysates and nuclear extracts and western blot analysis 33
• 2.3.9. Statistical analysis 34
• 2.4. Results 35
• 2.4.1. Effect of xanthone on the production of pro-inflamamtory cytokines and chemokine in TNF-α/IFN-γ-stimulated HaCaT cells 35
• 2.4.2. Effect of xanthone on the mRNA expression of pro-inflamamtory cytokines and chemokine in TNF-α/IFN-γ-stimulated HaCaT cells 38
• 2.4.3. Effect of xanthone on the mRNA expression of TARC and MDC in TNF-α/IFN-γ-stimulated HaCaT cells 41
• 2.4.4. Effect of xanthone on the phosphorylation of MAPKs in TNF-α/ IFN-γ-stimulated HaCaT cells 43
• 2.4.5. Effect of xanthone on the phosphorylation of NF-κB signaling pathway in TNF-α/IFN-γ-stimulated HaCaT cells 45
• 2.4.6. Effect of xanthone on the activation of caspase-1 in TNF-α/IFN-γ-stimulated HaCaT cells 47
• 2.5. Discussions 49
• 2.6. References 52
• CHAPTER III: XANTHONE ATTENUATES MAST CELL MEDIATED ALLERGIC INFLAMMATION 59
• 3.1. Abstract 60
• 3.2. Introduction 61
• 3.3. Materials and Methods 65
• 3.3.1. Reagents 65
• 3.3.2. Preparation of xanthone 65
• 3.3.3. Cell culture 66
• 3.3.4. Cell viability 66
• 3.3.5. Enzyme-linked immunosorbent assay (ELISA) 66
• 3.3.6. Histamine assay 67
• 3.3.7. RNA isolation and real-time RT-PCR 67
• 3.3.8. Preparation of total cell lysates and nuclear extracts and western blot analysis 68
• 3.3.9. Statistical analysis 69
• 3.4. Results 70
• 3.4.1. Effect of xanthone on the production of pro-inflamamtory cytokines and chemokine in PMACI-stimulated HMC-1 cells 70
• 3.4.2. Effect of xanthone on the mRNA expression of pro-inflamamtory cytokines and chemokine in PMACI-stimulated HMC-1 cells 73
• 3.4.3. Effect of xanthone on the mRNA expression of TSLP in PMACI-stimulated HMC-1 cells 76
• 3.4.4. Effect of xanthone on the release of histamine in PMACI-stimulated HMC-1 cell 78
• 3.4.5. Effect of xanthone on the phosphorylation of MAPKs in PMACI-stimulated HMC-1 cells 80
• 3.4.6. Effect of xanthone on the phosphorylation of NF-κB signaling pathway in PMACI-stimulated HMC-1 cells 82
• 3.4.7. Effect of xanthone on the activation of caspase-1 in PMACI-stimulated HMC-1 cells 84
• 3.5. Discussion 86
• 3.6. References 90
• CHAPTER IV: XANTHONE SUPPRESSES ALLERGIC CONTACT DERMATITIS IN MURINE MODEL 96
• 4.1. Abstract 97
• 4.2. Introduction 98
• 4.3. Materials and Methods 102
• 4.3.1. Reagents 102
• 4.3.2. Preparation of xanthone 102
• 4.3.3. Experimental animal model 102
• 4.3.4. DNFB-induced dermatitis 103
• 4.3.5. Histological analysis 104
• 4.3.6. Measurement of serum IgE, histamine and pro-inflammatory cytokines 104
• 4.3.7. Western blot analysis 104
• 4.3.8. Compound 48/80-induced anaphylactic shock 105
• 4.3.9. Passive cutaneous anaphylaxis (PCA) 105
• 4.3.10. Statistical analysis 106
• 4.4. Results 107
• 4.4.1. Effect of xanthone on DNFB-induced dermatitis in mice 107
• 4.4.2. Effect of xanthone on the levels of serum IgE, histamine and IL-6 in DNFB-induced dermatitis in mice 111
• 4.4.3. Effect of xanthone on the productoin of pro-inflamamtory cytokines in skin lesions of DNFB-induced dermatitis in mice 114
• 4.4.4. Effect of xanthone on the phosphorylation of MAPKs in skin lesions of DNFB-induced dermatitis in mice 117
• 4.4.5. Effect of xanthone on the compound 48/80-induced anaphylactic shock in mice 119
• 4.4.6. Effect of xanthone on IgE-mediated PCA reaction in mice 121
• 4.5. Discussion 123
• 4.6. References 127
• CONCLUSION 133
• ABSTRACT IN KOREAN 135
• ACKNOWLEDGEMENT 137