방사선 치료에 의해 유도되는 구내염에 대한 Epicatechin의 치료효과와 보호기전연구
Therapeutic effects and protective mechanism of Epicatechin on radiation-induced oral mucositis
- 주제(키워드) radiation , mucositis , head and neck cancer , epicatechin , radioprotector , HaCaT cell , fibroblast , rat , zebrafish
- 발행기관 아주대학교
- 지도교수 김철호
- 발행년도 2014
- 학위수여년월 2014. 2
- 학위명 박사
- 학과 및 전공 일반대학원 의학과
- 실제URI http://www.dcollection.net/handler/ajou/000000016024
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Radiotherapy has become increasingly important for treatment of head and neck squamous cell carcinoma. Oral mucositis, associated with both radiation and chemotherapy, is a very common, painful, and dose-limiting toxicity, with an incidence that can be as high as 90%. Exposure of normal tissue to radiation can cause both acute and chronic toxicities including dermatitis, xerostomia or mucositis, and this can result in not only a cessation of the intended therapy, but also a decrease in quality of life for the patient. There has been a lot of effort to reduce the radiation toxicities, mainly by focusing on technological improvements in radiation delivery. Also, many radioprotective agents have been attempted to prevent radiation-induced mucositis. Despite this, no intervention has yet been completely successful in preventing radiation-induced mucositis. Green tea consumed in a balanced and controlled diet improves anti-oxidative status and can protect against oxidative damage. Beneficial activities attributed to green tea extracts and/or constituents include antibacterial, antiviral, antioxidative, antitumor, and antimutagenic activities. Furthermore, green tea extract can scavenge nitric oxide (NO) and superoxide anions (O2 −) very effectively. Epicatechin (EC), a component of green tea, prevents cisplatin and radiation-induced, ROS-mediated ototoxicity and prevents changes in mitochondrial membrane potential. EC is among the important constituents responsible for the protective and antioxidant effects exhibited by green tea and is also active in lessening ionizing radiation-induced damage to DNA. However, the effect of EC as a radioprotective agent has not been investigated. In this study, the therapeutic effects and protective mechanism of epicatechin on radiation-induced oral mucositis were investigated in HaCaT human keratinocyte line, primary cultured human fibroblasts, and in vivo in rat and zebrafish model. EC significantly inhibited radiation-induced apoptosis, change of MMP, and intracellular ROS generation in HaCaT cells and fibroblasts. EC treatment markedly attenuated the expression of p-JNK, p-38, and cleaved caspase-3 after irradiation in the HaCaT cells and fibroblasts. EC represents an effective means of reducing cellular damage and facilitating wound healing after radiation exposure. Rats with radiation-induced oral mucositis showed decreased oral intake, weight and survival rate, but oral administration of EC significantly restored all three parameters. Histopathologic changes were significantly decreased in the EC-treated irradiated rats. TUNEL staining of rat oral mucosa revealed that EC treatment significantly decreased radiation-induced apoptotic cells. Also EC attenuated the radiation-induced embryotoxicity in a zebrafish model. Although larger numbers of animals and clinically relevant fractionation schemes are necessary to confirm the effect of EC, these results suggest the possibility of EC to inhibit radiation-induced oral mucositis, a common complication in radiotherapy of head and neck cancers. EC may be a safe and effective candidate treatment for the prevention of radiation-induced mucositis.
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TABLE OF CONTENTS
ABSTRACT i
TABLE OF CONTENTS iii
LIST OF FIGURES vi
LIST OF TABLES vii
PART ONE : Effect of Epicatechin against Radiation-Induced Oral Mucositis : In Vitro and In Vivo Study
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 3
A. Cell lines and irradiation conditions 3
B. Cell viability assay 3
C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end
labeling (TUNEL) assay 4
D. Annexin V-fluorescein isothiocynate (FITC)/propidium iodide (PI) double
staining 4
E. Mitochondrial membrane potential assay 4
F. Measurement of intracellular ROS production 5
G. Western blot assay 5
H. Animal study 6
I. Assessment of radiation damage in rats 7
J. TUNEL staining in rats 8
K. Immunohistochemical analysis in rats 8
L. Statistical analyses 9
Ⅲ. RESULTS
A. Pretreatment with EC increased viability of irradiated HaCaT cells 10
B. EC protected HaCaT cells against radiation-induced apoptosis 12
C. EC inhibited radiation-induced changes in the mitochondrial membrane
potential 15
D. EC inhibited intracellular ROS generated by radiation 17
E. Inhibition of MAPK activity by epicatechin rescued the HaCaT cells from
radiation induced cytotoxicity 20
F. EC restored the oral intake, weight loss, and survival rate of irradiated rats 22
G. EC inhibited histopathologic changes of oral mucosa by irradiation in rats 26
H. EC reduced radiation-induced apoptosis in rat oral mucosa 29
IV. DISCUSSION 32
V. CONCLUSION 37
PART TWO : Radioprotective effect of epicatechin in cultured human fibroblasts and zebrafish
Ⅰ. INTRODUCTION 38
Ⅱ. MATERIALS AND METHODS 40
A. Cell lines and irradiation conditions 40
B. Cell viability assay 40
C. Colony-forming assay 41
D. Wound-healing assay 41
E. Mitochondrial membrane potential assay 42
F. Measurement of intracellular ROS production 42
G. Western blot assay 43
H. Radioprotective effects on zebrafish 43
I. Statistical analyses 44
Ⅲ. RESULTS
A. Epicatechin increased the viability and migration capability of irradiated primary
cultured fibroblasts 45
B. Epicatechin inhibited radiation-induced decrease of mitochondrial membrane
potential (MMP) 49
C. Epicatechin inhibited intracellular ROS generated by radiation 51
D. Inhibition of MAPK activity by epicatechin rescued the primary cultured
fibroblasts from radiation-induced cytotoxicity 53
E. Epicatechin protects zebrafish embryos from radiation cytotoxicity 55
IV. DISCUSSION 57
V. CONCLUSION 62
REFERENCES 63
국문요약 72
