Curcumin의 감광활성 기전 및 광활성화에 의한 대식세포 염증반응 조절 효과 = Investigating photosensitizing mechanisms and modulating effects of curcumin on photo-inflammatory responses in macrophages

다국어 초록 (Multilingual Abstract)

Singlet oxygen is a type of reactive oxygen species and generated from the reaction of Type II photosensitization. Since 1,3-Diphenylisobenzofuran (DPBF) is used as a chromogenic probe with a peak absorbance at 410 nm to detect singlet oxygen, there is severe interference with photosensitizing pigments through absorption of blue light. In this study, the level of singlet oxygen was quantified through the analysis of the decrease in DPBF fluorescence, a indicative of the reaction with singlet oxygen. The fluorescence-based analysis was more sensitive and accurate than the one based on absorbance measurement. The fluorescence detection of singlet oxygen levels generated by various photosensitizers, including riboflavin, flavin mononucleotide, flavin adenine dinucleotide, lumichrome and lumiflavin, under blue light irradiation also permitted more effective measurement of their activity without interference from color. The results suggest that singlet oxygen detection based on the fluorescence of DPBF is a more effective approach than colorimetric analysis, and it can be utilized to assess the Type II photosensitizing property of diverse compounds.
Curcumin exhibits both photosensitizing and antioxidant activities, suggesting a dualistic behavior that may involve a non-classical mechanism distinct from conventional photosensitizers. 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan assays under vacuum confirmed that curcumin's photosensitizing activity is oxygen-independent. Moreover, 9,10-Anthracenediyl-bis(methylene dimalonic acid), DPBF, 2',7'-Dichlorodihydrofluorescein and ferrous oxidation−xylenol orangebassays showed no detectable ROS generation upon light exposure, indicating a oxygen-independent photosensitivity. In linoleic acid oil in water (O/W) systems, a biphasic pattern was observed depending on emulsifier concentration, with increased photosensitivity below the critical micelle concentration and suppression above it, suggesting contact-dependent lipid interaction. A strong correlation between curcumin decolorization and lipid hydroperoxide formation indicates that lipid oxidation is accompanied by chromophore disruption, consistent with a Type I photosensitization pathway. But sodium linoleate formed self-micelles, and lipid peroxidation was not induced. In edible oil O/W models, lipid oxidation was more pronounced at low emulsifier concentrations, supporting the role of direct lipid contact in curcumin's activity. Collectively, these results identify curcumin as oxygen-independent, contact-dependent photosensitizer, offering a novel perspective on photosensitization mechanisms.
To further investigate this mechanism and to enhance its photosensitizing activity, the interaction of curcumin with bovine serum albumin (BSA), a protein with electron-rich residues that create an electron-donating environment, were investigated. A complex formation between curcumin and BSA was confirmed by the observation of a decrease in the fluorescence of BSA and an increase of light absorption and the fluorescence of curcumin. The photostability of curcumin decreased with the concentration of BSA, while the fluorescence of BSA was not affected under blue LED. Curcumin did not produce H2O2 under the blue LED; the level of H2O2 by photosensitized curcumin increased in a BSA concentration-dependent manner. The curcumin-BSA complex exhibited a lower oxidation potential than free curcumin, indicating an enhanced ability to act as an electron donor. The induction of lipid peroxidation by blue LED-sensitized curcumin was also enhanced with increasing BSA concentrations. These findings indicate that the electron-rich environment provided by BSA enhances curcumin's photosensitizing activity and protein-assisted photodynamic therapy could be a novel approach for improving the performance of a Type I photosensitizer.
In addition, the photosensitizing activity of curcumin was investigated for its potential application in photodynamic therapy (PDT) aimed at regulating inflammation. Light-activated curcumin was more effective at reducing the production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated macrophages. The present results suggest that photoactivation could enhance the anti-inflammatory potential of curcumin.

목차 (Table of Contents)

• Part I. 1,3–Diphenylisobenzofuran을 이용한 일중항 산소 검출 민감도 향상 및 Type II 감광활성 평가에 응용 1
• 1. 서론 2
• 2. 재료 및 방법 5
• 2.1. 실험재료 5
• 2.2. Light emitting diode 조사 장치 5
• 2.3. DPBF 및 감광제의 흡광 및 형광 특성 5
• 2.4. DPBF의 광안정성 6
• 2.5. 감광활성에 의해 생성된 일중항 산소 검출 6
• 2.6. 통계처리 7
• 3. 결과 및 고찰 11
• 3.1. 다양한 광원 하에서 DPBF의 안정성 평가 11
• 3.2. DPBF를 이용해 빛 조사 하에서 감광제에 의해 형성되는 일중항 산소 검출 환경 조성 16
• 3.3. DPBF를 이용한 RF와 유도체의 일중항 산소 생성 검출 20
• 4. 결론 38
• Part II. 비산소성 기질 접촉 의존 메커니즘에 따른 커큐민의 감광활성 규명 40
• 1. 서론 41
• 2. 재료 및 방법 44
• 2.1. 실험재료 44
• 2.2. Light emitting diode 조사 장치 44
• 2.3. 세포 주 및 세포 배양 44
• 2.4. 진공 환경에서 감광활성 평가 45
• 2.5. ROS (Reactive Oxygen Species) 생성 수준 평가 45
• 2.6. 지질과산화물 생성량 측정 47
• 2.7. Free fatty acid에 따른 용액의 pH 측정 48
• 2.8. 광안정성 평가 48
• 2.9. Zeta 입자 크기 측정 49
• 2.10. 통계처리 49
• 3. 결과 및 고찰 53
• 3.1. 산소와의 상호작용을 통한 커큐민 감광활성 분석 53
• 3.1.1. 산소 비의존성 광활성화 전자전달 양상 53
• 3.1.2. ROS 생성능 평가 56
• 3.2. 유리지방산 함유 Oil–in–Water (O/W) 시스템에서의 광활성 동태 분석 64
• 3.2.1. 커큐민의 광감작 활성에 의한 linoleic acid 산화 64
• 3.2.2. Linoleic acid 기반 Oil–in–Water (O/W) 시스템에서 커큐민의 광안정성 평가 74
• 3.2.3. 커큐민의 광감작 활성에 의한 sodium linoleate 산화 평가 78
• 3.3. 식용유지 함유 O/W system에서 커큐민에 의한 광산화 유도 83
• 4. 결론 93
• Part III. BSA가 커큐민의 감광활성에 미치는 영향과 염증 조절을 위한 광역학적 활용 95
• 1. 서론 96
• 2. 재료 및 방법 98
• 2.1. 실험재료 98
• 2.2. Light emitting diode 조사 장치 99
• 2.3. 세포 주 및 세포 배양 99
• 2.4. 커큐민과 BSA 상호작용의 분광학적 평가 99
• 2.5. H₂O₂ 생성량 평가 100
• 2.6. 지질과산화물 생성량 측정 100
• 2.7. 산화 전위 측정 101
• 2.8. 세포독성 평가 102
• 2.9. NO 분비량 측정 102
• 2.10. Western blot 103
• 2.11. qRT–PCR 104
• 2.12. 세포 내·외 ROS 측정 104
• 2.13. 통계처리 105
• 3. 결과 및 고찰 107
• 3.1. BSA에 의한 커큐민의 감광활성 강화 107
• 3.1.1. 광학적 특성 분석 및 광안정성 평가 107
• 3.1.2. 커큐민–BSA 복합체의 감광활성 평가 115
• 3.1.3. 커큐민–BSA 복합체의 기저 상태 산화 특성 및 광여기 시 전자 공여 활성 분석 120
• 3.2. 커큐민의 감광활성에 의한 염증조절활성 평가 124
• 4. 결론 133
• 참고문헌 135
• ABSTRACT 143