Nitric oxide에 의한 세포손상에 대한 항산화 효소의 방어기작 및 isocitrate dehydrogenase와 질병과의 연관성 = Protective role of antioxidant cnayme in nitric oxide-induced cellular damage and implication of isocitrate dehydrogenase in diseases

다국어 초록 (Multilingual Abstract)

Nitric oxide (NO) is a messenger molecule with multiple biological functions. NO or its derivatives also interact with the thiol groups of proteins and glutathione to form nitrosothiols. The interaction of NO with sulfhydryl-containing molecules and enzymes have gained considerable importance. The isocitrate dehydrogenases (ICDHs; EC 1.1.1.41 and EC 1.1.1.42) catalyze oxidative decarboxylation of isocitrate to a-ketoglutarate and require either NAD^(+) or NADP^(+), producing NADH and NADPH. NADPH is an essential reducing equivalent for the regeneration of reduced glutathione (GSH) by glutathione reductase and for the activity of the NADPH-dependent thioredoxin system, both being important in the protection of cells from oxidative damages. Therefore, NADP^(+)-dependent ICDH may play important antioxidant roles during oxidative stress. In the present study, we investigated the role of SOD, catalase, and ICDH in cellular defense system against the nitric oxide-mediated oxidative damage and apoptosis. U937 cells were pre-treated with 1 mM DETC (SOD inhibitor), 20 mM ATZ (catalase inhibitor) or 3 mM oxalomalate (ICDH inhibitor) and exposed to nitric oxide. The activities of antioxidant enzymes were decreased upon exposure to nitric oxide. The cellular redox status in inhibitor-treated cells, reflected by an increased level of reactive oxygen species as well as a decrease in the ratio of [NADPH]/[NADPH + NADP^(+)] and the efficiency of glutathione turnover, was shifted to prooxidant state compared to control cells. The cellular damage in inhibitor-treated cells was significantly increased compared to control cells. Furthermore, apoptotic cellular damages like chromatin condensation, mitochondrial membrane potential transition and caspase-3 activation were observed in inhibitor-treated cells. The nitric oxide mediated damage to antioxidant enzymes may result in the perturbation of the cellular antioxidant defense mechanism and subsequently lead to a prooxidant condition. Modification of ICDH by peroxynitrite which induced with ethanol will likely have biological and medicinal significance. When HepG2 cells were incubated with 100 mM ethanol, a significant decrease in both cytosolic and mitochondrial ICDH activities were observed. Using immunoprecipitaion, we were also able to isolate and positive identify S-nitrosylated and nitrated mitochondrial ICDH from ethanol treated HepG2 cells. Intracellular ROS and nitrite levels were increased with ethanol treatment, and mitochondrial dysfunction was observed. We observed a decrease in mitochondrial ICDH activity and the modulation of the cellular redox status in the liver from the ethanol-fed rats. S-nitrosocysteine and nitrotyrosine adducts of mitochondrial ICDH purified by immunoprecipitation were also detected. It has been reported that chronic ethanol administration increases peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. The present results show that the accumulation of NO and O_(2)^(-) in liver from the ethanol-fed rats was associated with significant reduction of Mn-SOD and CuZn-SOD activities and increased iNOS activity and induction of iNOS protein. Therefore, it is tempting to speculate that inactivation of ICDH by peroxynitrite is, presumably, at least in part responsible for the perturbation of cellular redox status and oxidative damage in chronic alcoholism. The possibility that inactivation of ICDH by peroxynitrite in neurodegenerative diseases, diabetes, and chronic inflammation is worthy of further consideration.

목차 (Table of Contents)

• 목차 = Ⅰ
• 약어 = Ⅵ
• 표 목록 = Ⅶ
• 그림 목록 = Ⅸ
• 연구배경 = 1
• 1. 활성 산소종 (reactive oxygen species) = 1
• 2. 활성 질소종 (reactive nitrogen species) = 4
• 2-1. Nitric oxide의 성질 = 4
• 2-2. Nitric oxide의 생성 = 5
• 3. 항산화 효소 및 항산화 방어계 = 8
• 3-1. 항산화 방어계 8
• 3-2. Superoxide dismutase (SOD) = 8
• 3-3. Catalase = 9
• 3-4. Isocitrate dehydrogenase (ICDH) = 11
• 4. 연구 목적 = 12
• 제1장 Nitric Oxide에 의한 세포손상에 대한 항산화 효소의 방어기작 = 15
• 서론 = 16
• 재료 및 방법 = 20
• 1. 재료 = 20
• 2. 세포 배양 및 생존율 측정 = 21
• 3. RSNO (S-nitrosothiol)의 합성 = 22
• 4. 항산화 효소의 활성 측정 = 22
• 5. 세포내 GSSG 함량 측정 = 24
• 6. 세포내 NADPH 함량 측정 = 24
• 7. 단백질의 산화 손상 측정 = 25
• 8. 지질과산화 측정 = 25
• 1) Thiobarbituric acid-reaction substance (TBARS) assay를 이용한 지질과산화 측정. = 25
• 2) DPPP fluorescent dye를 이용한 지질과산화 측정 = 26
• 9. 세포내 hydrogen peroxide 생성량 측정 = 26
• 1) Xylenol orange를 이용한 H₂O₂ 생성량 측정 = 26
• 2) DCFHDA fluorescent dye를 이용한 H₂O₂ 생성량 측정 = 27
• 10. DNA 손상 측정 = 27
• 11. DAPI 염색법 = 27
• 12. Western blot analysis = 28
• 결과 = 29
• 1. 세포 생존율 변화 = 29
• 2. 세포내 항산화 효소의 활성 변화 = 31
• 3. 세포 형태 변화 관찰 = 31
• 4. 손상된 세포내의 hydrogen peroxide 생성량 확인 = 34
• 5. 세포내 지질 과산화 확인 = 36
• 6. 단백질의 산화 손상 확인 = 38
• 7. 세포내 NADPH 및 GSSG 함량 비교 = 40
• 8. DNA 손상 확인 = 42
• 9. DNA 절단 및 응축 확인 = 42
• 10. Caspase-3 활성과 PARP, lamin-B의 절단 확인 = 45
• 11. 미토콘드리아 Bcl-x protein family의 발현 확인 = 47
• 12. MAP kinase의 변화 확인 = 47
• 고찰 = 50
• 제2장 Isocitrate Dehydrogenase와 항산화 관련 질병인 alcoholism과의 연관성 = 54
• 서론 = 55
• 1. 알코올과 ROS 및 RNS에 의한 세포손상 = 55
• 2. 알코올에 의한 항산화계의 손상 = 57
• 재료 및 방법 = 59
• 1. 재료 = 59
• 2. 세포 배양 및 생존율 측정 = 60
• 3. 시약처리 및 동물실험 = 60
• 4. 세포질 및 미토콘드리아 분획 제조 = 61
• 5. 미토콘드리아 ROS 측정 = 62
• 6. 미토콘드리아 막전위 변화 측정 = 62
• 7. 면역 침전 반응 (Immunoprecipitation) = 62
• 8. 세포내 superoxide anion (O₂^(-)) 발생량 측정 = 63
• 9. Griess reaction를 이용한 세포내 nitrite 발생량 측정 = 63
• 10. iNOS 효소 활성 측정 = 64
• 11. 미토콘드리아 ATP 생성량 측정 = 64
• 결과 = 65
• 1. 세포 생존율 변화 = 65
• 2. 세포내 ICDH의 효소 활성 변화 = 67
• 3. Superoxide dismutase (SOD) 의 활성 변화 = 69
• 4. 세포내 hydrogen peroxide (H₂O₂) 발생량 = 71
• 5. 미토콘드리아 ROS 발생량 = 73
• 6. 세포내 superoxide anion (O₂^(-)) radical 정량 = 73
• 7. 세포내 Nitrite 함량 조사 = 75
• 8. Peroxynitrite에 의한 직접적인 세포 손상 = 77
• 9. 세포내 NADPH 함량 조사 = 77
• 10. 세포내 GSSG 함량 조사 = 80
• 11. 지질 과산화 확인 = 82
• 12. 단백질 손상 정도 확인 = 84
• 13. Nitrotyrosine 및 S-nitrosocysteine의 확인 = 86
• 14. 형광 dye를 이용한 세포손상 확인 = 88
• 15. 미토콘드리아의 막전위 변화 = 90
• 16. 미토콘드리아 내 ATP 생성량 비교 = 90
• 17. Ethanol 식이쥐의 간조직에서 미토콘드리아 ICDH의 활성변화 = 92
• 18. Ethanol 식이쥐의 간조직에서 SOD 활성 변화 = 94
• 19. Ethanol 식이쥐의 간조직에서 iNOS 활성 변화 = 94
• 20. Ethanol 식이쥐의 간조직에서 H₂O₂의 발생량 확인 = 97
• 21. Ethanol 식이쥐의 간조직에서 S-nitrosocysteine 및 nitrotyrosine의 확인 = 97
• 22. Ethanol 식이쥐의 간조직에서 NADPH 및 GSSG 함량 = 100
• 고찰 = 102
• 참고문헌 = 109
• Abstract = 123