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dc.contributor.advisor하헌주-
dc.contributor.author박미영-
dc.creator박미영-
dc.date.accessioned2016-08-26T04:08:03Z-
dc.date.available2016-08-26T04:08:03Z-
dc.date.issued2014-
dc.identifier.otherOAK-000000085070-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/210869-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000085070-
dc.description.abstractNatural products and their derivatives have traditionally been invaluable as a source of therapeutic agents. As an attempt to identify bioactive natural products, an anti-obesity effects by 18β-glycyrrhetinic acid (18β-GA) and anti-photoaging by carnosic acid (CA) were evaluated in vitro and/or in vivo, respectively. In addition, possible mechanisms for biological activities of 18β-GA and CA were investigated. In PART I, the effects of licorice extract and its active ingredients including GA on adipocyte differentiation and fat accumulation were examined. Previous reports suggest that licorice extract has various metabolically beneficial effects and may help to alleviate adiposity and hyperlipidemia. However, underlying anti-obesity mechanisms still remain elusive. Moreover, it is unknown which single ingredient in licorice extracts would mediate such effects. I aimed to demonstrate that licorice extract and its active ingredients can inhibit adipocyte differentiation and fat accumulation. 18β-GA inhibited CB1R activation induced by CB1R agonist, anandamide (AEA), in a concentration-dependent manner. Consistently, 18β-GA suppressed AEA-induced adipocyte differentiation in 3T3-L1 cells through the down-regulation of AEA-induced MAPK activation and expression of adipogenic genes including C/EBPα and PPARγ. The protein levels of FAS and SCD-1 were also decreased and the phosphorylation of ACC was increased in 18β-GA pretreated cells. The supplementation of 18β-GA significantly lowered body weight, fat weight, and plasma lipids levels in obese animal models. These data demonstrate that: (i) 18β-GA is able to block the activation of CB1R; (ii) 18β-GA significantly inhibits the AEA-mediated adipocyte differentiation in mature adipocytes; (iii) 18β-GA decreases body weight and adiposity in diet-induced obese models. This suggests that 18β-GA is a novel CB1R antagonist that would exert therapeutic effects in obesity-related metabolic disorders. In PART II, the effects of CA, a phenolic diterpene from rosemary on UV-induced MMPs expression in human skin cells were examined. Exposure of the skin to ultraviolet (UV) irradiation induces photoaging through the upregulation of matrix metalloproteinases (MMPs) and subsequent breakdown of extracellular matrices. Reactive oxygen species (ROS) and epidermal growth factor receptor (EGFR) activation play central roles in UV-induced MMPs expression through initiating extracellular signal-regulated kinase (ERK) mediated AP-1 signaling. This study was aimed to explore the effects of CA on UV-induced MMPs expression in human skin cells. I then investigated the molecular mechanism underlying the effects of CA in the aspect of MMPs expression, ERK/AP-1 pathway, ROS generation and EGFR-activation. Human dermal fibroblast cell line(Hs68), primary normal human dermal fibroblasts (HDFs), and normal human epidermal keratinocytes (HEKs) were employed and anti-photoaging effects of CA were assessed by western blotting, quantitative real-time RT-PCR and enzyme assays. CA significantly inhibited UVA and UVB-induced MMPs expression in a concentration-dependent manner in Hs68 cells. UVB-induced ERK-activation and the formation of transcription factor, AP-1 were significantly suppressed by CA. Among the upstream events of MMPs expression, UVB-induced ROS generation was attenuated by CA while EGFR activation was not affected. Confirming the anti-photoaging effects of CA through the suppression of UV-induced ROS, UVB-upregulated GADD45 expression, a marker for oxidative DNA damages was significantly reduced. Inhibitory effects of CA on UVB-induced MMPs expression could be also seen in HDFs and NHEKs. Collectively, our study demonstrates the anti-photoaging effects of CA in human skin cells that may be mediated by the inhibition of ROS production and ERK/AP-1 activation.;천연물과 천연물 유래 물질은 전통적으로 치료제의 원천으로 사용되어 왔다. 생리활성을 가진 천연물의 작용을 규명하고자 carnosic acid (CA)의 피부 항노화 작용과 18β-glycyrrhetinic acid (18β-GA)의 항비만 작용을 평가하였다. 또한 CA와 GA의 생물학적 활성에 대한 작용 메커니즘을 규명하였다. Part I 에서는 감초 추출물과 그 유효 성분이 지방세포 분화 및 지방 축적에 미치는 영향을 연구하였다. 감초 추출물은 각종 대사에 유용한 효과를 나타내며 특히 비만과 고지혈증을 완화하는 작용이 알려져 왔다. 그러나 감초 추출물의 항비만 작용에 관한 메커니즘은 규명된 바 없으며 감초 추출물의 작용을 나타내는 유효 성분이 알려지지 않았다. 본 연구에서는 감초 추출물과 그 유효 성분이 지방세포 분화 및 지방 축적에 미치는 영향을 조사하고 작용 메커니즘 규명을 목적으로 하였다. 18β-GA는 CB1R 촉진제인 anandamide (AEA)에 의한 CB1R 활성화를 농도의존적으로 억제하였다. 또한 18β-GA는 3T3-L1 세포에서 AEA-유도 MAPK 활성화 및 C/EBPα와 PPARγ 등의 비만유전자 발현을 억제함으로써 지방세포 분화를 억제하였다. 18β-GA는 또한 FAS와 SCD-1의 단백질 수준을 감소시켰으며 ACC 인산화를 증가시켰다. 동물실험에서는 18β-GA를 비만 모델 동물에 경구투여 하였을 때, 체중, 지방 무게, 혈중 지질 수준이 감소하였다. 연구 결과를 종합하면, (1) 18β-GA는 CB1R 활성화를 억제하였고 (2) 지방세포에서 AEA로 유도된 지방세포 분화를 억제하였으며 (3) 식이로 유도된 비만 동물에서 체중과 지방 축적을 감소시켰다. 이러한 결과는 18β-GA가 CB1R의 길항제로서 비만 관련 대사 이상증에 치료제로 사용될 수 있음을 의미한다. Part II 에서는 인간 피부 세포에서 자외선으로 유도된 MMPs 발현에 미치는 CA의 작용을 조사하였다. 피부에 자외선을 조사하면 MMPS의 발현이 증가되어 광노화가 유도되고 이에 따라 세포외 기질이 분해된다. 활성산소종(ROS) 및 상피성장인자수용체(EGFR)의 활성화는 ERK가 매개하는 AP-1 신호전달을 유도함으로써 자외선으로 유도된 MMPs 발현에 중심 역할을 한다. 본 연구는 인간 피부 세포에서 자외선으로 유도된 MMPs의 발현에 미치는 CA의 작용을 평가함을 목적으로 하였으며 MMPs 발현, ERK/AP-1 경로, ROS 생성 및 EGFR 활성화에 작용하는 CA의 작용 메커니즘을 규명하고자 하였다. 본 실험에는 human dermal fibroblast cell line (Hs68), human dermal fibroblasts (HDFs), human epidermal keratinocytes (HEKs) 등의 세포주가 사용되었으며 western blotting, 정량 실시간 RT-PCR, 효소 반응 실험 등을 통하여 CA의 피부 항노화 작용을 평가하였다. CA는 Hs68 세포에서 농도의존적으로 자외선-유도 MMPs 발현을 억제하였다. UAB로 유도된 ERK 활성화, 전사인자인 AP-1의 생성이 CA에 의하여 억제되었다. MMPs 발현 조절 경로에 있어서는 CA에 의하여 EGFR 활성화는 영향을 받지 않은데 비하여, 자외선-유도 ROS 생성은 CA에 의하여 억제되었다. 자외선-유도 ROS 생성 억제를 통한 CA의 피부 항노화 작용을 확인하고자 산화적 DNA 손상의 지표인 GADD45의 발현이 CA에 의하여 감소함을 확인하였다. Hs68 세포에서 나타난 CA에 의한 MMPs 발현 감소는 HDFs 및 HEKs 에서도 관찰되었다. 결론적으로 본 연구를 통하여 인간 피부 세포에서 나타나는 CA의 피부 항노화 작용은 ROS 생성 및 ERK/AP-1 활성화를 억제함으로써 나타남을 규명하였다.-
dc.description.tableofcontentsGENERAL INTRODUCTION 1 PART I: 18β-glycyrrhetinic acid prevents anandamide-induced adiposity and high fat diet-induced obesity 3 INTRODUCTION 4 1. Obesity 4 2. Obesity therapies 5 3. The endocannbinoid system 7 3.1 Cannabinoid receptors 8 3.2 Signal transduction mechanisms of CB1 receptor 11 3.2.1 Cannabinoid receptors 11 3.2.2 Regulation of adenylyl cyclase 12 3.2.3 Regulation of mitogen-activated protein (MAP) kinase 12 3.2.4 Modulation of intracellular Ca2+ concentration 13 4. Glycyrrhetinic acid 13 MATERIALS & METHODS 15 1. Materials 15 2. Ca2+ flux assay in CB1-expressing Chem-1 cell 15 3. Cell culture and differentiation 16 4. Oil Red O staining and quantification of lipid content 16 5. Protein extraction and immunoblotting 17 6. RNA extraction and real-time quantitative PCR analysis 18 7. Animal care and experimental protocol 18 8. Statistical analysis 19 RESULTS 20 1. Inhibitory effect of licorice extracts and 18β-glycyrrhetinic acid on cannabinoid receptor type 1 activation 20 2. Inhibitory effect of 18β-GA on adipocyte differentiation of 3T3-L1 preadipocytes 20 3. Inhibitory effect of 18β-GA on the expression of adipogenic markers by AEA-induced MAPK inactivation in 3T3-L1 cells 22 4. Effect of 18β-GA on body weight gain and the plasma levels of lipid parameters in obese mice 26 5. Inhibitory effect of 18β-GA on adiposity and expression of genes involved in lipid metabolism in obese mice 28 DISCUSSION 31 PART II: Carnosic acid, a phenolic diterpene from rosemary prevents UV-induced expression of matrix metalloproteinases in human skin fibroblasts and keratinocytes 36 INTRODUCTION 37 1. Structure and function of normal skin 37 1.1 Epidermis 37 1.2 Dermis 38 1.3 Hypodermis 39 2. Structural changes in aged skin 39 2.1 Skin thickness changes 40 2.2 Epidermal changes 40 2.3 Dermal changes 41 2.4 Hypodermal changes 41 3. UV radiation and skin biology 42 4. Carnosic acid 44 MATERIALS & METHODS 46 1. Materials 46 2. Cell culture and UVB irradiation 46 3. Western blot analysis 47 4. Determination of MMP-1 secretion by ELISA 47 5. RNA preparation and quantitative real-time RT-PCR 48 6. Measurement of reactive oxygen species production 49 7. Statistical analysis 49 RESULTS 50 1. Effect of carnosic acid on UV-induced MMPs expression and secretions 50 2. Effect of carnosic acid on UVB-induced ERK activation and c-Fos expression 52 3. Effect of carnosic acid on activation of EGFR by UVB 54 4. Effect of carnosic acid on ROS generation and GADD45 expression in UVB-exposed cells 57 5. Inhibition of MMP-1 secretion in primary human dermal fibroblastsand epidermal keratinocytes by carnosic acid 60 DISCUSSION 61 CONCLUSION 66 REFERENCES 67 APPENDIX 90 국문초록 92 감사의 글 95-
dc.formatapplication/pdf-
dc.format.extent3090453 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.subject.ddc600-
dc.titleMolecular mechanisms for anti-obesity and anti-photoaging effects of natural product-derived substances-
dc.typeDoctoral Thesis-
dc.format.pagexii, 97 p.-
dc.identifier.thesisdegreeDoctor-
dc.identifier.major대학원 약학과-
dc.date.awarded2014. 2-
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