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PHARMACEUTICAL POTENTIAL OF FISETIN

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Abstract
피세틴은 사과, 딸기, 감, 양파, 연근 및 오이와 같은 일부 과일 및 채소에서 다량 함유되어 있는 식물 2차 대사산물이다. 강력한 항산화 특성을 보유하고 있으며, 전자분산 능력으로 인해 광범위한 UV 스펙트럼을 흡수 할 수 있는 저분자량 생리활성 플라보노이드이다. 피세틴의 약리학적인 잠재력은 항암활성을 중심으로 광범위하게 연구되고 있으며, 특히, 전립선암, 골육종, 폐암 및 백혈병과 같은 광범위한 암에 대해 효과적이다. 세포사멸유도 이외에도 포식능 증가, 암세포 전이 억제, 신생 혈관형성 저해, 세포주기 정지 및 전사 인자 조절과 같은 다른 세포 신호 전달 메커니즘은 암에서 피 세틴의 작용 방식과 관련있다.. 그러나 PM2.5 유발 조건에서 멜라닌 생성 특성, 항염증 효과, 골형성 효과 및 항산화 특성은 광범위하게 확인된 바가 없다. 따라서 현재 연구에서 우리는 wnt/β-catenin 세포신호 전달경로를 표적으로 하여 앞서 언급한 다양한 연구모델에서의 효과를 검증하과자 한다.
첫째, 피세틴은 비 ATP 경쟁 결합 부위에서 GSK-3β에 결합하고 MITF 매개 티로시나아제의 활성화를 촉진하는 β-카테닌의 방출을 통해 B16F10 세포와 제브라피쉬 유충의 멜라닌 생성을 촉진 할 수 있음을 확인 할 수 있었다. 피세틴이 멜라닌 생성을 예상치 않게 증가 시켰지만, 피세틴은 백반증과 같은 다양한 질병의 치료에 유용 할 수 있으며, GSK-3β에 대한 억제를 통한 다양한 질환의 치료제로도 사용이 가능 할 것으로 판단된다. 둘째, 피세틴은 β-카테닌 매개 NF-κB 신호 전달 경로를 억제하여 LPS 유발 염증 및 내독성 쇼크를 약화시켜 전신 염증에 대한 강력한 항염증제로 사용이 가능할 것으로 판단된다.
다음으로, 피세틴은 앞서 언급한 두 가지 주요 세포 신호 전달 경로를 통해 inflammasome 형성을 억제하였다. 첫째, 피세틴은 MD2의 소수성 포켓에 경쟁적으로 결합하여 TLR4의 LPS 인식을 길항하여, NF-κB 세포 신호 전달 경로의 억제를 통하여, IL-1β의 전사를 억제함을 확인하였다. 둘째, 피세틴은 p62 의존적방식으로 손상된 미토콘드리아의 제거를 촉진함으로써 미토콘드리아내의 활성산소 형성을 억제한다. 미토콘드리아내의 활성산소 생성의 억제는 활성 IL-1β 로의 pro-IL-1β의 절단을 연속적으로 억제하며, 이는NLRP3 inflammasome 형성의 하향 조절과 연관되어 있다.
또한, 피세틴이 MC3T3-E1 마우스 조골세포 및 제브라피쉬 유충에서 각각 조골 세포 분화 및 척추 형성을 촉진한다는 것을 입증하였다. 연구에서 우리는 피세틴이 GSK-3β ser9 인산화를 자극하여, 파괴적인 복합체에서 β-catenin을 방출시키게 되고, 더불어서β-catenin의 핵내 이동을 촉진하여 결과적으로 골아 세포 분화와 뼈 형성을 상향 조절한다는 것을 발견하였다. 또한 피 세틴은 MC3T3-E1 세포와 제브라피쉬 유충에서도 파골세포 분화를 억제함으로써 프레드니솔론 매개 골다공증을 완화 할 수 있음을 규명하였다. 마지막으로, 우리는 피 세틴이 HaCaT 인간 각질세포에서 PM2.5로 유도된 세포사멸을 강력하게 억제한다는 것을 증명하였다. 이전 연구에 따르면 PM2.5는 AhR 의존 경로를 통해 활성산소를 유도하는 반면 상향 조절 된 활성산소는 PERK-ATF4 축을 통한 소포체 스트레스의 시작과 관련이 있다. ER 스트레스 반응의 시작으로 세포질 구획에 칼슘이 축적되어 미토콘드리아 막 전위가 손실되었으며, 이는 Bcl-2와 Bax 사이의 균형을 깨뜨려 서 세포 사멸을 실행하는 caspase-8, caspase-9 및 caspase-3과 PARP의 절단을 촉진하였다. 결론적으로 피세틴이 활성산소의 형성, 소포체 스트레스 반응의 시작을 효과적으로 차단하고 결과적으로 HaCaT 인간 각질 세포에서 PM2.5 유도 세포 사멸을 억제한다는 것을 확인하였다.
Fisetin is a plant secondary metabolite which is ubiquitously expressed in some of the fruits and vegetables such as apple, strawberry, persimmon, onion, lotus root and cucumber. It is a low molecular weight bioactive polyphenolic flavonoid which could absorb broad range of UV spectrum due to the electron decentralizing ability that enables it to possess strong anti-oxidative properties. Pharmaceutical potentials of fisetin have been extensively studied focusing its anti-cancer activities. The compound itself shown to be effective against wide range of cancers such as prostate cancer, osteosarcoma, lung cancer and leukemia by triggering apoptosis. In spite of the apoptosis other cell signaling mechanisms such as autophagy, metastasis, angiogenesis, cell cycle arrest and transcription factor regulation are also associated with the mode of action of fisetin in cancers. However, melanogenic properties, anti-inflammatory effects, anti-inflammasome effect, osteogenic effect and anti-oxidative properties under the PM2.5-induced conditions have not been extensively identified. Therefore, during the current study, we were able to identify the aforementioned properties specifically targeting wnt/β-catenin cell signaling pathway.
Firstly, we investigated that fisetin-mediated could promote the melanogenesis in B16F10 cells and zebrafish larvae through binding to GSK-3β at a non-ATP-competitive binding site, and the subsequent release of β-catenin, which promotes MITF-mediated tyrosinase activation. Although fisetin caused an unexpected increase in melanogenesis, fisetin may be useful for the treatment of many different diseases such as vitiligo and the inhibitory effect on GSK-3β is also paramount important. Secondly, fisetin attenuates LPS-induced inflammation and endotoxic shock by suppressing the β-catenin-mediated NF-κB signaling pathway indicating the possibility of using it as a potent anti-inflammatory drug for systemic inflammation.
Then, fisetin inhibited the inflammasome formation via two main cell signaling pathways. Firstly, Fisetin antagonizes the LPS recognition of TLR4 via competitively binding to the hydrophobic pockets of MD2 which consequently prevent the stimulation of canonical NF-κB cell signaling pathway to inhibit the transcription of IL-1β. Secondly, fisetin inhibits the formation of mtROS by promoting the elimination of damaged mitochondria in a p62 dependent manner. Inhibition of mtROS generation associate with the downregulation of NLRP3 inflammasome formation which will subsequently inhibits the cleavage of pro-IL-1β in to active IL-1β.
Next we demonstrated that fisetin promote osteoblast differentiation and vertebrae formation in MC3T3-E1 mouse osteoblast and zebrafish larvae respectively. During the study we noticed that fisetin stimulate the GSK-3β ser9 phosphorylation to liberate β-catenin from the destructive complex and thereby promote the nuclear translocation of β-catenin consequently upregulate the osteoblast differentiation and bone formation. Furthermore, fisetin could alleviate prednisolone mediated osteoporosis by inhibiting osteoclast differentiation in MC3T3-E1 cells and zebrafish larvae as well. Finally, we proved that fisetin potently inhibits PM2.5-induced apoptosis in HaCaT human keratinocytes. Previous studies showed that PM2.5 induced ROS levels via AhR dependent pathway while upregulated ROS levels are associated with initiation of ER stress via PERK-ATF4 axis. Initiation of ER stress responses resulted calcium accumulation in the cytosolic compartment that lead to the loss of mitochondrial membrane potential. Loss of mitochondrial membrane potential broke the balance between Bcl2 to Bax and thereby promoted the cleavage of caspase8, 9 and 3 and PARP which execute apoptosis. We investigated that fisetin effectively blocked the ROS formation, initiation of ER stress responses and consequently inhibited the PM2.5-induced apoptosis in HaCaT human keratinocytes.
In conclusion, fisetin possess broad range of pharmaceutical potentials targeting various cells signaling pathways in different cells. Therefore, our results imply that fisetin could be a potent drug target for several diseases with minimum side effects. However, further studies are needed to warrant to show the fisetin-mediated pharmaceutical potentials in detailed in-vivo studies.
Author(s)
ILANDARAGE MENU NEELAKA MOLAGODA
Issued Date
2021
Awarded Date
2021. 2
Type
Dissertation
URI
https://oak.jejunu.ac.kr/handle/2020.oak/23500
Alternative Author(s)
일랑다라게 메누 닐라카 몰라고다
Affiliation
제주대학교 대학원
Department
대학원 해양생명과학과
Advisor
김기영
Table Of Contents
Chapter1 1
GSK-3β-Targeting Fisetin Promotes Melanogenesis in B16F10 Melanoma Cells and Zebrafish Larvae through β-Catenin Activation. 1
1.1 Introduction 3
1.2 Materials and Methods 7
1.2.1 Reagents and Antibodies 7
1.2.2 Cell Culture and Viability Assay 7
1.2.3 In Vitro Mushroom Tyrosinase Activity 8
1.2.4 Flow Cytometry Analysis 8
1.2.5 Intracellular and Extracellular Melanin Content 8
1.2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 9
1.2.7 Protein Extraction and Western Blotting Analysis 10
1.2.8 In Vivo Melanogenic Effect in Zebrafish Larvae 10
1.2.9 Determination of Cardiotoxicity in Zebrafish 11
1.2.10 Molecular Docking Prediction 11
1.2.11 Statistical Analysis 11
1.3 Results 13
1.3.1 Fisetin is a Non-Specific Inhibitor of Mushroom Tyrosinase Activity in Vitro 13
1.3.2 High Concentrations of Fisetin Decrease Relative Viability of B16F10 Melanoma Cells 15
1.3.3 Fisetin Increases Intracellular and Extracellular Melanin Content of B16F10 Melanoma Cells 17
1.3.4 Fisetin Upregulates MITF and Tyrosinase Expression 19
1.3.5 Fisetin Inhibits Melanogenesis in Zebrafish Larvae but Did not Affect Heart Rate 21
1.3.6 Fisetin Possibly Binds to GSK-3β 23
1.3.7 Activation of β-Catenin Positively Stimulates Fisetin-Mediated Melanogenesis 26
1.4 Discussion 29
1.5 Conclusions 35
Chapter2 36
Fisetin-mediated β-catenin Activation Inhibits Lipopolysaccharide-induced Inflammatory Response by Suppressing NF-κB Activation, Leading to a Decrease in Endotoxic Shock 36
Abstract 37
2.1 Introduction 39
2.2 Material and Methods 42
2.2.1 Reagents and Antibodies 42
2.2.2 Cell Culture and Viability 42
2.2.3 Flow Cytometry Analysis 43
2.2.4 Isolation of Total Cellular RNA from RAW 264.7 Macrophages and RT-PCR 43
2.2.5 Western Blot Analysis 44
2.2.6 NO Assay 44
2.2.7 Measurement of IL-6, TNF-α and PGE2 45
2.2.8 Immunostaining 45
2.2.9 Maintenance of Zebrafish Embryo and Larvae 46
2.2.10 LPS Microinjection and Cardiac Toxicity Evaluation 46
2.2.11 Neutral Red Staining 46
2.2.12 Sudan Black Staining 47
2.2.13 Isolation of Total Zebrafish mRNA and RT-PCR 47
2.2.14 Statistical Analysis 48
2.3 Results 49
2.3.1 High Concentrations of Fisetin Decrease the Viability of RAW 264.7 Macrophages 49
2.3.2 Fisetin Inhibits LPS-induced Proinflammatory Mediators and Cytokines in RAW 264.7 Macrophages 52
2.3.3 Fisetin Attenuates Mortality, Abnormality, and Lowered Heart Rate in LPS-microinjected Zebrafish Larvae 55
2.3.4 Fisetin Inhibits LPS-induced Proinflammatory Gene Expression and Concomitantly Decreases Macrophage and Neutrophil Recruitment to the Inflammatory Sites in Zebrafish Larvae 58
2.3.5 Fisetin Inhibits LPS-induced NF-κB Activity in RAW 264.7 Macrophages 61
2.3.6 Fisetin Enhances Phosphorylation of GSK-3β at Ser9 and Subsequent Activation of β-catenin in RAW 264.7 Macrophages 63
2.3.7 Fisetin Inhibits β-Catenin-mediated NF-κB Activity, Causing a Significant Decrease in LPS-induced IL-6 and TNF-α Release 65
2.3.8 Fisetin-induced Anti-inflammatory Response Is Related to Activation of β-Catenin in an Endotoxic Shock Model of Zebrafish Larvae 68
2.4 Discussion 72
2.5 Conclusions 76
Chapter3 77
Fisetin inhibits NLRP3 inflammasome by suppressing mitochondrial ROS production, resulting from the inhibition of the TLR4-MD2 signaling pathway 77
3.1 Introduction 79
3.2 Materials and methods 81
3.2.1 Reagents and antibody 81
3.2.2 Cell culture and viability assay 81
3.2.3 Analysis of viability and dead cells populations 82
3.2.4 Measurement of IL-1β by ELISA 82
3.2.5 Western blotting 82
3.2.6 Reverse transcriptase polymerase chain reactions (RT-PCR) using mouse specific primer. 82
3.2.7 Immunostaining of p65 and p62/SQSTM1 83
3.2.8 Analysis of mtROS 84
3.2.9 Mitochondrial depolarization 84
3.2.10 Transfection of p62 small interfering RNA (siRNA) 84
3.2.11 Maintenance of zebrafish embryo and larvae 84
3.2.12 Cardiac toxicity evaluation 85
3.2.13 Neutral red staining 85
3.2.14 Isolation of total zebrafish mRNA and RT-PCR 85
3.2.15 Statistical analysis 87
3.3 RESULTS 87
3.3.1 High concentrations of fisetin possess cytotoxicity in BV2 microglia cells at 87
3.3.2 Fisetin possibly binds to TLR4/MD2 complex and inhibits the LPS-induced downstream signaling pathway 89
3.3.3 Fisetin inhibits the NF-κB cell signaling pathway. 92
3.3.4 Fisetin inhibits the expression of NLRP3 inflammasome components 93
3.3.5 Fisetin inhibits mitophagy by downregulating mitochondria depolarization and mtROS production. 95
3.3.6 Silencing of p62 reverses fisetin-mediated mitophagy and NLRP3 inflammasome formation. 98
3.3.7 Fisetin inhibits NLRP3 inflammasome formation in zebrafish larvae. 100
3.4 Discussion 102
Chapter4 106
Fisetin promotes osteoblast differentiation and bone formation through GSK-3β Ser9 phosphorylation and consequent β-catenin activation 106
Abstract 107
4.1 Introduction 108
4.2 Materials and method 110
4.2.1 Reagents and antibody 110
4.2.2 Cell culture and MTT activity 110
4.2.3 Analysis of viability and dead cells populations 111
4.2.4 Alizarin red staining 111
4.2.5 ALP assay 112
4.2.6 Reverse transcription polymerase chain reaction (RT-PCR) 112
4.2.7 Western Blotting Analysis 114
4.2.8 Immunostaining 114
4.2.9 Vertebrae formation in zebrafish larvae 115
4.2.10 RT-PCR of zebrafish larvae 115
4.2.11 Statistical Analysis 117
4.3 Results 118
4.3.1 Fisetin shows no cytotoxicity at low concentrations 118
4.3.2 Fisetin induces osteoblast differentiation accompanied by osteoblast-specific gene expression 120
4.3.3 Fisetin promotes vertebrae formation in zebrafish larvae along with the upregulation of osteoblast-specific gene expression. 122
4.3.4 Fisetin mitigates PDS-induced anti-osteogenic activity 125
4.3.5 Fisetin alleviates PDS-induced delay in vertebrae formation of zebrafish larvae 127
4.3.6 Fisetin promotes osteoblast differentiation and mineralization through the phosphorylation of GSK-3β at Ser9 130
4.4 Discussion 132
Chapter5 135
Fisetin protects HaCaT human keratinocytes from fine particulate matter (PM2.5)-induced oxidative stress and apoptosis via inhibiting endoplasmic reticulum stress response 135
Abstract 136
5.1 Introduction 137
5.2 Material and Method 138
5.2.1 Regent and antibodies 138
5.2.2 Cell culture and cell viability 139
5.2.3 Annexin V staining for apoptosis detection 139
5.2.4 Protein extraction and western blotting 140
5.2.5 Caspase3/7 activity 140
5.2.6 Quantification of intracellular ROS 140
5.2.7 Intracellular ROS 140
5.2.8 Cytosolic calcium 141
5.2.9 ROS staining in live zebrafish larvae 141
5.2.10 Statistical Analysis 141
5.3 Results 142
5.3.1 Fisetin protects HaCaT keratinocytes from PM2.5induced apoptosis. 142
5.3.2 Fisetin inhibits PM2.5- induced apoptosis through modulating apoptosis-related proteins 144
5.3.3 Fisetin inhibits PM2.5-induced ROS formation. 145
5.3.4 Fisetin inhibits PM2.5-induced apoptosis by alleviating ER stress 147
5.4 Discussion 149
Bibliography 152
Degree
Doctor
Publisher
제주대학교 대학원
Citation
ILANDARAGE MENU NEELAKA MOLAGODA. (2021). PHARMACEUTICAL POTENTIAL OF FISETIN
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