Anti-inflammatory effect of Tonggyu-tang via interukin and NF-κB pathway in HMC-1 and HaCaT

국문 초록 (Abstract)

배경: 알레르기성 비염, 천식 및 아토피성 피부염을 비롯한 알레르기성 염증질환이 전 세계적으로 증가하는 추세이다. 현재 알레르기 반응을 치료에 주로 사용되는 약물인 항 히스타민제와 코르티코이드 스테로이드제는 지속시간이 짧다는 점뿐만 아니라 여러 부작용을 나타내고 있어 사용을 제한하고 있어 이를 보완 및 대체할 수 있는 의약품을 찾기 위해 노력하고 있다.

목적: 알레르기성 비염환자를 치료하기 위해 한의학에서 처방하는 통규탕의 항염증 기전을 확인하고자 한다.

방법: 본 연구에서는 HMC-1 (Human mast cell-1)세포와 피부각질세포인 HaCaT 세포를 RT-PCR 및 ELISA를 이용하여 염증관련 interukin(IL)의 mRNA 발현량과 cytokine 방출량을 측정하였다. 또한, 염증관련 다양한 cell signaling pathway 에서의 단백질 생성량 확인을 통하여 통규탕이 항염효과를 확인하였다.

결과: 본 연구는 통규탕이 HMC-1과 HaCaT 세포에서 IL-4, IL-6, IL-8, IL-13, TNF-α 와 같은 염증성 cytokine 의 발현 및 생성을 효과적으로 감소시킴을 확인하였다. 또한, MAPK pathway, PI3K-AKT pathway, NF-κB pathway 에서 염증반응에 주요하게 작용하는 경로를 효과적으로 억제함을 확인하였다.

결론: 통규탕은 HMC-1과 HaCaT 세포에서 염증관련 경로에 관여하여 염증성 cytokine의 발현을 억제함으로써, 알레르기성 염증 치료에 효과적임을 나타내었다.

다국어 초록 (Multilingual Abstract)

Allergic inflammatory diseases including allergic rhinitis (AR), asthma and atopic dermatitis (AD) are increasing worldwide. Current medications commonly used to treat allergies reactions are anti-histamines and corticosteroids but they have their own limitations such as short duration and severe side effects. Thus, complementary and alternative medicine is gaining an interest in the field of allergic diseases.
In traditional Korean Medicine, several herbal medicines have been used for allergic rhinitis treatment. Especially, among such medicines, 通竅湯 (Tonggyu-tang, TGT) composed of 14 herbs has shown anti allergic effects in patients with nasal disorders. HMC-1 (Human mast cell-1) and HaCaT cells are essential reaction components for allergic reactions which are located in almost all tissues, influencing inflammation by releasing histamine. Excessive activation of these inflammatory cells leads to histamine degranulation, which results in hypersensitive allergic reactions.
In my study, I identified the mechanisms for allergic reactions using TGT in these two representative immune cells, HMC-1 cells and HaCaT cells Activated HMC-1 and HaCaT cells were treated with various concentration of TGT, and I investigated pro-inflammatory cytokines and inflammation-related cell signaling pathways.

목차 (Table of Contents)

• 1. INTRODUCTION 1
• 2. MATERIALS AND METHODS 4
• 1) Chemicals and reagent 4
• 2) Cell culture and treatment 5
• 3) Cell viability measurement with MTS assay or MTT assay 5
• 4) RNA extraction and RT-PCR 5
• 5) Cytokine release measurement with ELISA 7
• 6) Western blot assay 7
• 7) Statistical analysis 8
• 3. RESULTS 9
• 1) Effect of TGT on cell viability in HaCaT cells 11
• 2) Effect of TGT on inflammatory cytokine mRNA expression level and release level changes of IL-4 in HaCaT cells 13
• 3) Effect of TGT on inflammatory cytokine mRNA expression level changes of IL-6 in HaCaT cells 15
• 4) Effect of TGT on inflammatory cytokine mRNA expression level and release level changes of IL-8 in HaCaT cells 17
• 5) Effect of TGT on inflammatory cytokine release level changes of IL-13 in HaCaT cells
• 6) Effect of TGT on inflammatory cytokine mRNA expression level and release level changes of TNF-α in HaCaT cells 19
• 7) Effect of TGT on cell viability in HMC-1 cells 21
• 8) Effect of TFT on inflammatory cytokine mRNA expression level and release level changes of IL-4 in HMC-1 cells 23
• 9) Effect of TGT on inflammatory cytokine mRNA expression level and release level changes of IL-6 in HMC-1 cells 25
• 10) Effect of TGT on inflammatory cytokine mRNA expression level and release level changes of IL-8 in HMC-1 cells 27
• 11) Effect of TGT on inflammatory cytokine mRNA expression level and release level changes of IL-13 in HMC-1 cells 29
• 12) Effect of TGT on inflammatory cytokine mRNA expression level and release level changes of TNF-α in HMC-1 cells 31
• 13) Effect of TGT on PI3K-AKT signaling pathway in HMC-1 cells 33
• 14) Effect of TGT on ERK signaling pathway in HMC-1 cells 35
• 15) Effect of TGT on NF-κB signaling pathway in HMC-1 cells 37
• 4. DISCUSSION 39
• REFERENCES 42
• 국문초록 45