종양괴사인자 TNF-α에 의하여 유도된 사람 피부 섬유아세포의 세포사에 미치는 아이소멘톤의 영향
Effect of Isomenthone on Cell Death Induced by TNF-α in Human Dermal Fibroblasts
- 주제(키워드) Isomenthone; Fibroblast; TNF-α; Apoptosis; c-Jun N-terminal kinase; p38
- 발행기관 아주대학교
- 발행년도 2010
- 학위수여년월 2010. 8
- 학위명 박사
- 학과 및 전공 일반대학원 응용생명공학과정
- 실제URI http://www.dcollection.net/handler/ajou/000000010987
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The tumor necrosis factor alpha (TNF-α) is a pleiotropic cytokine that triggers either cell survival and cell death. NF-κB plays a critical role in protection from TNF-induced cell death in survival pathway through upregulation of antiapoptotic genes and suppression of reactive oxygen species (ROS) or proapoptotic genes. Cell death evoked by TNF-α is regulated by TNF-α receptor-associated death domain containing protein (TRADD) that interacts with and activates the apoptotic proteases thus triggering cell death. TRADD pathway activates the c-Jun amino-terminal kinases/stress-activated protein kinases (JNKs/SAPKs) that induce apoptotic program. JNK is an indispensible early element in stress-induced apoptosis which controls the release of cytochrome c. JNK activation is an important mediators of apoptosis induced by TNF-α. Also the p38 MAPK pathways as a key regulator of apoptosis in cells stimulated with TNF-α, has been reported to cause inactivation of antiapoptotic proteins and activation of proapoptotic proteins, thus promote apoptosis. Isomenthone is a constituent of the essential oils of Mentha arvensis L which has been widely used as a cooling agent, a counterirritant for pain relief, and for the treatment of pruritus in Asian. Isomenthone is widely used as fragrances and flavors in the cosmetic, perfume, drug and food industries but data concerning their biological effects are known for only some. Also the effects of isomenthone on skin have remained unknown. In this study, we demonstrate that isomenthone has a potential to protect fibroblast against TNF-α induced cell death in serum free condition through blocking activation of the JNKs/SAPKs and p38 MAPK pathway and downstream apoptotic events
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CONTENTS
ABSTRACT…………………………………………………………i
CONTENTS…………………………………………………………iii
LIST OF FIGURE……………………………………………….vii
LIST OF TABLE……………………………………………………x
1. INTRODUCTION
1.1 Apotosis…………………………………………………………..1
1.2 Apoptosis and Human disease…………………………………………….3
1.3 TNFα-induced Apoptosis……………………………………………………….6
1.4 Essential oil……………………………………………………………………….10
1.5 Isomenthone……………………………………………………………………….12
1.6 Purpose of the Study………………………………………………………….14
2. MATERIALS AND METHODS
2.1 Materials……………………………………………………….15
2.2 Cell culture………………………………………………………………………….15
2.3 Cell viability assay…………………………………………………………….15
2.4 In situ apoptosis detection by TUNEL staining ……………………….16
2.5 Annexin V/PI stainning ……………………………………………………….17
2.6 Western blot analysis……………………………………………17
2.7 Luciferase reporter assay …………………………………………………….17
2.8 siRNA silencing………………………………………………….18
2.9 Caspase and PARP activity assay………………………………………….18
2.10 Cytochrom c Elisa assay …………………………………………………….19
2.11 Immunofluorescence staining …………………………………………….19
2.12 Measurement of Intracellular ROS …………………………………….20
2.13 DPPH assay …………………………………………………………………….20
2.14 Quantification of MMP-1 activity……………………………………….20
2.15 Determination of Isomenthone by High-Performance Liquid Chromatography ……………………………………………………………………….21
2.16 Statistical analysis …………………………………………………………….21
3. RESULTS
3.1 Isomenthone protects fibroblasts from TNF α-induced cell death ……………………………………………………………………..22
3.2 Isomenthone inhibits TNF α-induced apoptosis /Annexin V staining…………………………………………………………………………………….25
3.2 Isomenthone inhibits TNF α-induced apoptosis /TUNEL staining…………………………………………………………………………………….28
3.3 Isomenthone inhibits JNKs/SAPKs and p38 MAPK pathway induced by TNF α ………………………………………………….30
3.4 Isomenthone protective effect is independent of NF-κB signaling ……………………………………………………………………….32
3.5 Isomenthone suppresses caspase activity and release of cytochrome c from mitochondria. ……………………………………………….32
3.5 Isomenthone suppresses parp activity …………………………..34
3.6 Stability of Isomenthone …………………………………………………….35
4. DISCUSSION………………………………………………… 48
5. CONCLUSION…………………………………………..48
6. REFERENCES……………………………………….......50
ABSTRACT IN KOREAN………….………………….....62
