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研究生:阮吳黎明英
研究生(外文):NGUYEN NGO LE MINH ANH
論文名稱:Therapeutic effects of Puerarin in Retinal and Optic Nerve Degeneration in Ischemia
論文名稱(外文):Therapeutic effects of Puerarin in Retinal and Optic Nerve Degeneration in Ischemia
指導教授:蔡榮坤
指導教授(外文):Tsai Rong-Kung
口試委員:陳建同孫銘輝黃舜平何宗融蔡榮坤
口試委員(外文):Chen Jiann-ThorngSun Ming-HuiHuang Shun-PingHo Tsung-JungTsai Rong-Kung
口試日期:2019-09-18
學位類別:博士
校院名稱:慈濟大學
系所名稱:醫學科學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2019
畢業學年度:108
語文別:英文
論文頁數:98
中文關鍵詞:PuerarinARPE-19 cellsrAION modelanti-apoptoticanti-inflammationM2 microglia and macrophage polarization
外文關鍵詞:PuerarinARPE-19 cellsrAION modelanti-apoptoticanti-inflammationM2 microglia and macrophage polarization
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ABSTRACT
ACKNOWLEDGEMENTS
LIST OF FIGURES
1 CHAPTER 1: GENERAL INFORMATION 1
1.1 Puerariae lobata 1
1.1.1 History and Traditional Uses 1
1.1.2 Chemical components 2
1.2 Puerarin (PR) 3
1.2.1 Pharmacology activities and pharmacokinetics 3
1.2.2 PR in the protection of liver injuries 5
1.2.3 PR in the protection of cardiomyocytes 5
1.2.4 PR in the protection of hyperlipidemia 6
1.2.5 PR in the protection of diabetes 6
1.2.6 PR in protection in Alzheimer’s disease models 7
1.2.8 PR in the protection of ischemia-reperfusion and other injuries 9
1.2.9 Summary 10
1.3 Overview of thesis rationale 11
2 CHAPTER 2: PUERARIN PROTECTS RETINAL PIGMENT EPITHELIUM (ARPE-19) CELLS AGAINST HYPOXIA-INDUCED BLOOD-RETINAL BARRIER BREAKDOWN AND AKT-DEPENDENT APOPTOSIS 13
2.1 Introduction 13
2.1.1 Retinal cell death and age-related macular degeneration (AMD) disease 13
2.1.2 Apoptosis and retinal degeneration 14
2.1.3 PR and cellular apoptosis 16
2.1.4 Aim of this study 17
2.2 Materials and Methods 18
2.2.1 ARPE-19 cell culture 18
2.2.2 ARPE-19 cells hypoxic conditions 18
2.2.3 Cell Viability Assay 18
2.2.4 Detection of apoptosis 19
2.2.5 Measurement of Transepithelial Electrical Resistance (TER) 19
2.2.6 Immunofluorescence staning of tight junction proteins 19
2.2.7 Western Blot analysis 20
2.2.8 Statistical Analysis 20
2.3 Results 20
2.3.1 Cytotoxicity of PR in the ARPE-19 cells 20
2.3.2 PR reduced the hypoxia-induced apoptosis of ARPE-19 cell 21
2.3.3 PR inhibited the hypoxia-induced barrier disruption in ARPE-19 cells 23
2.3.4 PR reduced the levels of apoptosis-associated factors 25
2.3.5 Puerarin induced Akt activation and reduced Akt1-dependent apoptosis 26
2.4 Discussion 29
3 CHAPTER 3: NEUROPROTECTIVE EFFECTS OF PUERARIN IN rAION MODEL 31
3.1 introduction 31
3.1.1 Ischemic optic neuropathy 31
3.1.2 Non-anterior ischemic optic neuropathy (NAION) 32
3.1.3 A rat model of Nonarteritic anterior ischemic optic neuropathy (rAION) 35
3.1.4 Microglia activation and its polarization 36
3.1.5 Microglia-mediated neuroinflammation to optic neuropathy diseases 38
3.1.6 PI3K/Akt signaling pathway in M1/M2 microglia-macrophage polarization 40
3.1.7 PR with cellular inflammation 42
3.1.8 Aim of this study 43
3.2 Materials and Methods 44
3.2.1 Study design 44
3.2.2 Experimental animals 46
3.2.3 Administration and formulation of PR 46
3.2.4 Intravitreal injections of the Pi3k/Akt pathway inhibitor LY294002 46
3.2.5 rAION induction model 47
3.2.6 Flash visual evoked-potentials (FVEP) recordings 47
3.2.7 Retrograde labeling of RGCs by Fluorogold 48
3.2.8 Optic nerve (ON) and retinal sample preparation 49
3.2.9 Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay 49
3.2.10 Immunohistochemistry (IHC) on optic nerve (ON) tissues 50
3.2.11 Optical Coherence Tomography (OCT) imaging of optic nerve head (ONH) 50
3.2.12 Western blotting 51
3.2.13 Statistical Analysis 52
3.3 Results 52
3.3.1 Treatment with PR preserved visual function 52
3.3.2 Treatment with PR preserved RGC survival 53
3.3.3 Treatment with PR inhibited RGC apoptosis 53
3.3.4 Treatment with PR reduced ONH edema 54
3.3.5 PR treatment prevented macrophage infiltration in the ON tissue 55
3.3.6 PR treatment reduced proinflammatory cytokine production and enhanced anti-inflammatory cytokine production in rAION 56
3.3.7 PR treatment enhanced M2 microglia and macrophage polarization 57
3.3.8 PR treatment promoted Akt1 activation to induce M2 polarization 58
3.4 Discussion 60
4 CHAPTER 4: GENERAL DISCUSSION 62
4.1 Summary 62
4.2 Implications 63
4.3 Conclusions 64
5 References 65

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