Anti-inflammatory and anti-mitophagy dependent apoptotic cell death activities of Quercetogetin isolated from Citrus unshiu peel : 귤피로부터 분리된 쿼세토제틴의 항염증 및 항마이토파지 의존성 세포사멸 효과에 관한 연구

다국어 초록 (Multilingual Abstract)

Inflammation is a complex response of a body to harmful stimuli and plays important pathological roles in the progress of chronic diseases such as chronic obstructive pulmonary disease, cancers, Alzheimer’s diseases cardiovascular disease, asthma, rheumatoid arthritis, multiple sclerosis etc. Controlling chronic inflammation is crucial to human health and a key future preventative and therapeutic target.
The citrus unshiu peel (CUP) has been used in Asian traditional medicine for treatment of cough, asthma, bronchial disorders. The CUP is a rich source of flavanones, as well as many polymethoxylflavones (PMFs), which are very little amounts in other plants.
Thus, the present study, the isolation and identification of the active compounds from the extracts of CUP as inhibitors of NO production in LPS-induced. And we investigated the anti-inflammatory effect and the underlying molecular mechanisms of active compound in LPS-induced RAW264.7 cells. Using the screening of NO production assay from CUP extracts, we isolated type PMFs compounds such as nobiletin (NOB), tangeretin(TAN) and quercetogetin (QUE), isolated from the chloroform layers. These compounds inhibited the NO production in LPS-induced RAW264.7 cells, in a dose-dependent manner. One of compounds, QUE has not been previously reported the biological activities for human health including an anti-inflammatory effect. Therefore, we attempted to unravel the mechanisms of the anti-inflammatory effects of QUE.
We investigated the anti-inflammatory effect and the molecular mechanisms of QUE in LPS-induced RAW264.7 cells. The results, QUE inhibited the production of NO and PGE2 by suppressing the LPS-induced expression of inducible iNOS and COX-2 at both mRNA and protein levels. In addition, QUE suppressed the production of pro-inflammatory cytokines, such as IL-1β, IL-6 and TNF-α. We also evaluated the effects of QUE on the activation of NF-κB. QUE inhibited the translocation of the NF-κB subunit p65 into the nucleus by interrupting the phosphorylation of IκB-α in LPS-induced RAW 264.7 cells. Moreover, we confirmed that the suppression of the inflammatory process by QUE was mediated through the MAPKs pathway based on the fact that QUE significantly decreased p-ERK protein expression in LPS-induced RAW264.7 cells. Taken together, the anti-inflammatory effects of QUE were mediated by the inhibition of the NF-κB and MAPK pathways.
Chronic obstructive pulmonary disease (COPD) is contributed the fourth leading cause of mortality worldwide with cigarette smoke (CS) as the main cause. This disease includes two main phonotypes of chronic bronchitis and emphysema with different physiopathology and symptoms. Emphysema is mainly caused by CS and is characterized by the increase alveolar wall cell death and/or failure of the alveolar wall maintenance. Recent data suggest that chronic inflammation and increased oxidative stress contribute to increased destruction and/or impaired lung maintenance and repair in the pathogenesis of emphysema. There are no therapies available that can either prevent or cure the progression of COPD-emphysema.
Accordingly, the aim of this study is investigate whether mitophagic cell death in CSE-exposed lung bronchial epithelial cells (BEAS-2B), and to confirmed whether or not QUE has inhibitory effects on mitophagy dependent apoptosis in CSE-induced. Firstly, our results demonstrated that CSE induce mitophagy that is associated with a mitochondrial dysfunction and an increase of mitophagy regulator protein expression. The CSE treatment significantly caused the induction of apoptotic indicator and the increase of apoptosis related protein expression levels. To examine whether p-DRP-1 or PINK1, has a role in CSE-induced apoptotic cell death, we knocked down DRP-1 or PINK1 expression by transfecting targeted siRNA in BEAS-2B cells. Consequently, we founded that DRP-1 regulates the activity of caspase-3 by regulating of apoptosis related protein expressions, and subsequently regulates apoptotic indicator such as cell cytotoxicity and viability. Based on these findings, DRP-1 is an essential factor for CSE-induced mitophagy, and it affects the efficiency of apoptosis, suggesting that mitophagy dependent apoptotic pathway may be a therapeutic target for emphysema. Secondly, we tested the effect of QUE on mitophagy dependent apoptotic cell death in CSE–induced BEAS-2B cells. QUE has never been studied in pulmonary diseases. As results that QUE protected against CSE-induced mitochondrial dysfunction and cell death in vitro. QUE inhibited the mitophagy by suppressing the expression levels of p-DRP-1 and PINK1 proteins in CSE-induced BEAS-2B cells. In additional, QUE decreased the expression of apoptosis related proteins, such as cleaved cas-3, -8 and -9. We confirmed that the suppression of the mitophagic cell death by QUE was mediated through the regulation of caspase activity based on the fact that QUE significantly decreased p-DRP1 and PINK1 protein expression in CSE-induced human bronchial epithelial cells.
These findings show that anti-inflammatory and anti-mitophagy dependent apoptotic cell death activities of QUE in vitro, suggesting that QUE may be a therapeutic potential agent in the inflammatory diseases such as COPD-emphysema.

목차 (Table of Contents)

• List of Tables vii
• List of Figures viii
• Abstract x
• Chapter 1 General introduction 1
• 1.1. Inflammation and anti-inflammation 1
• 1.2. Chronic obstructive pulmonary disease (COPD) 9
• 1.2.1. Emphysema 13
• 1.3. Citrus unshiu 17
• 1.3.1. Citrus unshiu peel (CUP) 18
• 1.3.2. Polymethoxyflavonoids (PMFs) 20
• 1.4. Purpose of this study 27
• Chapter 2 Anti-inflammatory activity of quercetogetin Isolated from Citrus unshiu peel in LPS-induced RAW264.7 murine macrophage cells; dawn-regulation of iNOS and COX-2 through suppression NF-κB and ERK pathways 28
• 2.1. Introduction 28
• 2.2. Materials and Methods 30
• 2.2.1. Materials 30
• 2.2.2. Extraction and isolation of active compounds 30
• 2.2.3. High performance liquid chromatography analysis 31
• 2.2.4. Nuclear magnetic resonance spectroscopy (NMR) analysis 31
• 2.2.5. Electrospray ionization-mass spectrometry (ESI-MS) analysis 32
• 2.2.6. Cell culture 32
• 2.2.7. Cell viability 32
• 2.2.8. Nitrite oxide measurement 33
• 2.2.9. Prostaglandin E2, IL-1β, IL-6 and TNF-α assay 33
• 2.2.10. Preparation of cytosolic and nuclear extracts 34
• 2.2.11. Western blotting analysis 34
• 2.2.12. Reverse transcriptase-polymerase chain reaction (RT-PCR) 35
• 2.2.13. Statistical analysis 35
• 2.3. Results 36
• 2.3.1. Isolation and purification of active compounds from CUP 36
• 2.3.2. Isolation and characterization of 5,6,7,8,3',4'-hexamethoxyflavone (Compound 1, Nobiletin (NOB)), 4',5,6,7,8-Pentamethoxyflavone (Compound 2, Tangeretin (TAN)) and 3,5,6,7,3',4'-hexamethoxyflavone (Compound 3, Quercetogetin (QUE)) 39
• 2.3.3. Effect of active compounds on NO production in LPS-induced of RAW264.7 cells. 48
• 2.3.4. Anti-inflammatory activity of quercetogetin from CUP in LPS-induced RAW264.7 murine macrophage cells 48
• 2.3.4.1. Effect of QUE on cell viability in LPS-induced RAW264.7 cells 48
• 2.3.4.2. QUE inhibits the production of NO by suppressing iNOS expression in LPS-induced RAW264.7 cells 50
• 2.3.4.3. QUE inhibits the production of PGE2 by suppressing COX-2 expression in LPS-induced RAW264.7 cells 52
• 2.3.4.4. QUE inhibits the production and mRNA expression of pro-inflammatory cytokines in LPS-induced RAW264.7 cells 54
• 2.3.4.5. QUE inhibits the degradation of IκB-α and the nuclear translocation of NF-κB in LPS-induced RAW264.7 cells 56
• 2.3.4.6. QUE inhibits the phosphorylation of ERK MAPK in LPS-induced RAW264.7 cells 58
• 2.4. Discussion 61
• Chapter 3 Anti-mitophagy dependent apoptotic cell death activity of quercetogetin in CSE (Cigarette Smoke Extract)-induced BEAS-2B human bronchial epithelial cells 65
• 3.1. Introduction 65
• 3.2. Materials and Methods 68
• 3.2.1. Chemicals and reagents 68
• 3.2.2. Cell culture 68
• 3.2.3. Preparation of cigarette smoke extract (CSE) 69
• 3.2.4. Cytotoxicity and viability assays 69
• 3.2.5. Flow cytometry analysis 70
• 3.2.6. Confocal microscopy 70
• 3.2.7. Caspase activity assays 71
• 3.2.8. Western blotting analysis 71
• 3.2.9. Small interfering RNA transfection 72
• 3.2.10. Statistical analysis 73
• 3.3. Results 74
• 3.3.1. CSE causes mitochondrial dysfunction and mitophagy in BEAS-2B epithelial cells 74
• 3.3.2. Mitophagy regulated apoptotic cell death through the mitochondria fission regulator DRP-1 and the mitophagy regulator PINK1 in CSE-induced BEAS-2B epithelial cells 77
• 3.3.3. QUE inhibited cell death in CSE-induced BEAS-2B epithelial cells 79
• 3.3.4. QUE inhibited mitochondrial dysfunction and mitophagy in CSE-induced BEAS-2B epithelial cells 81
• 3.3.5. QUE inhibited apoptotic cell death in CSE-induced BEAS-2B epithelial cells 83
• 3.3.6. QUE inhibited mitophagy dependent apoptotic cell death in CSE-induced BEAS-2B epithelial cells 85
• 3.4. Discussion 88
• References 92
• Abstract (in Korean) 116