Cho, Sung Won 이기명 2005 377 https://aurora.ajou.ac.kr/handle/2018.oak/16246 학위논문(박사)--아주대학교 대학원 :의학과,2005 Helicobacter pylori (HP)가 위점막에 감염되면 염증성 사이토카인를 분비하여 염증 세포를 유인하고, 산화적 스트레스를 가하여 DNA의 손상 및 세포 사멸을 유발하여 위점막 손상을 일으킨다. 녹차 catechin의 일종인 Epigallocatechin-3-gallate (EGCG)는 과거부터 항염증 및 항균 작용이 있다고 알려져 있고 최근 HP에 의한 위점막 손상을 감소시키는 효과가 있다고 보고 되었으나 구체적으로 어떤 기전으로 위점막 손상을 감소시키는지 알려져 있지 않아 그 기전을 규명하고자 본 연구를 시작하였다. 재료 및 방법: EGCG가 HP 감염된 위상피 세포의 생존에 미치는 영향을 알기 위하여 MTT법과 dye exclusion법을, DNA 손상에 대한 효과를 알기 위하여 Comet법과 DNA fragmentation 분석법을, toll-liker receptors 4 (TLR-4)에 대한 영향을 알기 위하여 RT-PCR법과 glycosylated TLR-4에 대한 western blot법을, lipooxygenase(LOX) 대사 산물의 변화를 알기 위하여 reverse phase-high performance liquid chromatography(RP-HPLC)법을 각각 이용하였다. 결과: EGCG를 전처치하였을 때 HP 감염에 의한 세포 사멸과 DNA 손상이 현저히 감소하였고 위상피 세포의 증식이 증가하였다. HP의 감염은 세균 감염에 대한 세포내 신호 전달을 담당하는 TLR-4의 glycosylation을 증가시키고 NF-κB와 ERK1/2의 발현을 증가시켰으나 EGCG로 전처치할 경우 TLR-4의 glycosylation을 완전히 억제시켰고 NF-κB와 ERK1/2의 발현을 억제시켰다. EGCG를 전처치할 경우 HP 감염에 의한 염증 매개 물질 및 hydroxyeicosatetraneoci acid(HETE)의 발현 증가를 현저히 감소시켰다. 결론: EGCG의 전처치는 TLR-4의 발현 억제릉 통하여 HP 감염에 의한 위점막 손상을 감소시켰고, HP가 감염된 경우 지속적으로 녹차를 음용할 HP에 의한 위점막 손상을 예방할 수 있으리라 생각된다. TABLE OF CONTENTS ABSTRACT = ⅰ TABLE OF CONTENTS = ⅲ LIST OF FIGURES = ⅴ ABBREVIATION = ⅵ Ⅰ. INTRODUCTION = 1 Ⅱ. MATERIALS AND METHODS = 4 A. MATERIALS = 4 1. Chemicals and reagents = 4 2. Cells, Bacterial strains, and Growth conditions = 4 B. METHODS = 5 1. MTT assay and cell growth = 5 2. Comet assay = 6 3. DNA fragmentation method = 6 4. Western blot assay = 6 5. RT-PCR = 7 6. Electrophoretic mobility shift assay (EMSA) = 7 7. RP-HPLC for identification of LOX metabolites = 8 8. Statistical analysis = 8 Ⅲ. RESULTS = 9 1. Prevention of H. pylori-induced gastric mucosal cytotoxicity with EGCG pretreatment = 9 2. DNA damage by H. pylori infection, and its attenuation by EGCG = 10 3. Blockage of H. pylori-induced TLR-4 glycosylation by EGCG = 17 4. Inactivation of MAPK ERK1/2 and NF-kB by EGCG = 20 5. EGCG inhibits the synthesis of 5S-HETE induced by H. pylori infection = 20 Ⅳ. DISCUSSION = 27 Ⅴ. CONCLUSION = 32 REFERENCES = 33 국문요약 = 42 eng The Graduate School, Ajou University 아주대학교 논문은 저작권에 의해 보호받습니다. Protective Mechanism of Epigallocatechin-3-gallate against Helicobacter pylori- induced Gastric Epithelial Cytotoxicity via Blockage of TLR-4 Signaling Helicobacter 위염에서 TLR-4 신호전달을 매개로 한 Epigallocatechin-3-gallate의 위점막 보호 효과 Thesis 아주대학교 일반대학원 Lee, Kee Myung 일반대학원 의학과 2005. 2 Master http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000377 Helicobacter pylori infection leads to gastric mucosal damage by several mechanisms including the direct effect of virulence factors produced by H. pylori, propagation of inflammation, oxidative stress, DNA damage, and induction of apoptosis. (-)-Epigallocatechin-3-gallate (EGCG), one of green tea catechins, is known to suppress H. pylori-induced gastritis through its antioxidative and anti-bacterial actions. In this study, we evaluated protective mechanism of EGCG against H. pylori-induced cytotoxicity in gastric epithelial cells. For analyzing EGCG effect on viability of gastric epithelial cells, MTT assay and dye exclusion assay were performed. The degree of DNA damage was evaluated by Comet assay and apoptotic DNA fragmentation assay. To investigate EGCG effect on H. pylori-induced the toll-like receptors 4 (TLR-4) signaling, RT-PCR and western blot analysis corresponding to glycosylated TLR-4 was done. LOX metabolites were measured with RP-HPLC. EGCG pretreatment effectively rescued gastric mucosal cells from the H. pylori?induced apoptotic cell death and DNA damage, and administration of this catechin enhanced gastric epithelial cell proliferation. H. pylori infection stimulated the glycosylation of TLR-4 which initiates intracellular signaling of infected host cell, and then pretreatment of EGCG completely blocked its glycosylation. The blockage of TLR-4 activation by EGCG resulted in inactivation of ERK1/2 and NF-kB as downstream molecules of TLR-4 signaling induced by H. pylori. This disturbance of H. pylori-induced host cell signaling by EGCG attenuated the synthesis of proinflammatory mediators, HETEs. EGCG pretreatment showed significant cytoprotective effects against H. pylori-induced gastric cytotoxicity via interference of TLR-4 signaling induced by H. pylori. Thus, our result implies that continuous intakes of green tea could prevent the deleterious consequences of H. pylori infection.