Anti-osteoporosis effect of sulforaphane through suppression of NOX2 expression = NOX2 활성 억제를 통한 sulforaphane의 항 골다공증 효과

국문 초록 (Abstract)

Sulforaphane은 glucoraphanin이 myrosinase에 의해 가수분해 되어 항산화, 항염증, 항암성의 특성을 갖는 유기 황 화합물로 알려져 있다. Nrf2의 발현 증가를 통한 sulforaphane의 RANKL에 유도된 파골세포 분화 억제에 대한 선행연구가 있다. 그러나 RAW 264.7 세포에서 NADPH oxidase 발현의 감소를 통한 파골세포 분화에 대한 sulforaphane의 억제 효과는 보고되지 않았다. 본 연구에서는 RANKL에 의해 유도된 파골세포 분화에 대한 sulforaphane의 억제 효과와 NADPH oxidase 발현 조절을 통한 파골세포 분화 억제 기전을 연구하였다. Sulforaphane의 처리는 RANKL에 유도된 파골세포 분화에서 RANKL 처리 군에 비해 0.5-1.0 μM 및 0.1-1.0 μM에서 TRAP 양성 다핵 세포의 수와 TRAP 활성을 감소시켰다. 0.1-1.0 μM sulforaphane의 처리는 cathepsin K와 NFATc1 발현을 억제하였다. ROS 및 O2-에 대한 억제 효과는 각각 0.5-1.0 μM 및 0.1-1.0 μM 에서 관찰되었다. sulforaphane의 Nrf2 및 Keap1 발현 억제 효과는 각각 0.5-1.0 μM 및 0.1-1.0 μM 에서 나타났다. 0.1-1.0 μM sulforaphane의 처리는 RANKL에 의해 유도된 파골세포 분화에서 NQO1과 GST 발현을 강력하게 억제하였다. 0.1-1.0 μM sulforaphane의 처리는 NOX1 발현에 영향을 미치지 않지만 NOX2 발현을 농도 의존적으로 약화시켰다. 0.1-1.0 μM sulforaphane의 처리에 의한 강한 억제효과는 p47phox의 발현이 아닌 p22phox의 발현에서 관찰되었다. RANKL에 의해 유도된 파골세포 분화에서 sulforaphane의 처리는 0.5-1.0 μM에서 NF-κB의 발현을 약하게 감소시켰고, 핵에 함유된 인산화 된 NF-κB를 0.1-1.0 μM에서 농도 의존적으로 감소시켰다. 본 연구는 sulforaphane의 항 파골세포 억제 효과가 RANKL에 유도된 파골세포 분화의 초기 단계에서 O2-의 감소에 의존할 수 있음을 나타낸다. 결론적으로 sulforaphane으로 인한 O2-의 감소는 NQO1 및 GST와 같은 Nrf2 관련 phase II 항산화 효소의 상향 조절뿐만 아니라 NF-κB의 핵 안으로의 감소된 이동으로 말미암은 NOX2와 p22phox의 하향 조절을 통한 활성 NOX2 복합체 형성의 억제에 달려 있다.

다국어 초록 (Multilingual Abstract)

Sulforaphane is known to be an organic sulfur compound that has antioxidant, anti-inflammatory, and anti-cancer property as glucoraphanin is hydrolyzed by myrosinase. There have been previous studies on the inhibition of sulforaphane on RANKL-induced osteoclast differentiation through Nrf2 up-regulation. However, the inhibitory effect of sulforaphane on osteoclast differentiation through down-regulation of NADPH oxidase expression in RAW 264.7 cells has not been reported. In this study, the inhibitory effect of sulforaphane on RANKL-induced osteoclast differentiation and its underlying mechanism via control of NADPH oxidase expression. The sulforaphane treatment attenuated the number of TRAP-positive multi-nucleated cells and the TRAP activity compared to the RANKL-treated group in RANKL-induced osteoclast differentiation between 0.5 and 1.0 μM and between 0.1 and 1.0 μM, respectively. The 0.1 to 1.0 μM sulforaphane treatment obviously down-regulated the cathepsin K and NFATc1 expression. The suppressive effect of sulforaphane on ROS and superoxide anions was observed at 0.5 to 1.0 μM and 0.1 to 1.0 μM, respectively. The inhibitory effect of sulforaphane on Nrf2 and Keap1 expression was showed at 0.5 to 1.0 and 0.1 to 1.0 μM, respectively. The 0.1 to 1.0 μM sulforaphane treatment potently suppressed NQO1 and GST expression in RANKL-induced osteoclast differentiation. The 0.1 to 1.0 μM sulforaphane treatment do not affect NOX1 expression, but dose-dependently attenuated NOX2 expression. The strong suppressive effect of 0.1 to 1.0 μM sulforaphane treatment was observed in p22phox expression, not in p47phox expression. The whole translated NF-κB was slightly attenuated by 0.5 to 1.0 μM sulforaphane in RANKL-induced osteoclast differentiation and the nuclear phosphorylated NF-κB was dose-dependently diminished by 0.1 to 1.0 sulforaphane. This study indicates that the anti-osteoclastic effect of sulforaphane may rely on the attenuation of superoxide anion level in early stage of RANKL-induced osteoclast differentiation. Taken together, the attenuation of superoxide anion level by sulforaphane depends upon the up-regulation of Nrf2-mediated phase II antioxidant enzyme such as NQO1 and GST as well as the interruption of active NOX2 complex formation through down-regulation of NOX2 and p22phox translation resulting from attenuated translocation of NF-κB into the nucleus.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Literature Review
• 2.1. Sulforaphane 3
• 2.2. A role of bone cell in bone remodeling 6
• 2.2.1. Bone and bone cells 6
• 2.2.2. Bone remodeling 9
• 2.3. Reactive oxygen species (ROS) 12
• 2.3.1. Role of reactive oxygen species in bone 12
• 2.3.2. Signaling pathway RANK-RANKL in osteoclastgenesis 14
• 2.3.3. Inhibition of ROS via Nrf2 in osteoclast differentiation 16
• 2.3.4. Role of ROS via NADPH oxidase 2 (NOX2) in osteoclast differentiation 19
• 3. Material and method
• 3.1. Chemical reagents and materials 22
• 3.2. Oxygen radical absorbance capacity (ORAC) 23
• 3.3. Reducing capacity 24
• 3.4. RAW 264.7 cell culture and osteoclast differentiation 24
• 3.5. TRAP activity and TRAP staining 25
• 3.6. Measurement of intracellular ROS Level 26
• 3.7. Measurement of intracellular O2- Level 26
• 3.8. Western blot analysis 27
• 3.8.1. Total protein extraction 27
• 3.8.2. Nuclear protein extraction 28
• 3.9. Statistical analysis 29
• 4. Result and Discussion
• 4.1. Peroxyl-radical scavenging activity and reducing capacity of sulforaphane derivatives 30
• 4.2. Inhibitory effect of sulforaphane on RANKL-induced osteoclast differentiation 32
• 4.3. Inhibitory effect of sulforaphane on RANKL-induced NFATc1 and cathepsin K expression 35
• 4.4. Suppressive effect of sulforaphane on ROS and O2- level in RANKL-induced osteoclast differentiation 38
• 4.5. Regulating effect of sulforaphane on Nrf2 and related proteins expression in RANKL-induced osteoclast differentiation 41
• 4.6. Inhibitory effect of sulforaphane on NOX2 expression in RANKL-induced osteoclast differentiation 46
• 4.7. Inhibitory effect of sulforaphane on NF-κB expression in RANKL-induced osteoclast differentiation 50
• 5. Conclusion 52
• REFERENCES 54
• ABSTRACT 71
• ABSTRACT IN KOREAN 74
• LIST OF ABBREVIATIONS 76