VITAMIN E ISOFORM γ-TOCOTRIENOL ALLEVIATES ASTHMA AND COPD PEH HONG YONG (B.Sc. (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2017 Supervisor: Associate Professor Wong Wai-Shiu Fred Examiners: Professor Lim Tow Keang Assistant Professor Thai Tran Professor Clive Page, King’s College London DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ____________________________________________________________ Peh Hong Yong Matric No.: A0035464H 17 July 2017 ACKNOWLEDGEMENTS First and foremost, I would like to extend my deepest appreciation to my PhD supervisor, A/Prof Fred Wong Wai-Shiu, for his guidance, encouragement and invaluable advice throughout these five years. I would also like to thank my thesis advisor Prof Ong Choon Nam and Dr Fong Chee Wai for their endless support, recommendation and enriching research collaboration. I sincerely thank the both of them for their support in my research endeavors and the constant inspiration to excel in my PhD journey. I also wish to acknowledge the President Graduate Fellowship and the National University of Singapore Industry Relevant Project (IRP) research scholarship with Davos LifeSciences, which provided research, educational and financial support for my PhD education. I am also truly honored to be the recipient of National University of Singapore Overseas Postdoctoral Fellowship, for the opportunity to begin my next milestone at Harvard Medical School. I wish to express my appreciation to Prof Benny Tan, Dr Deron Herr and Dr Judy Sng for their guidance, motivation and support. I would like to thank my mentors Eugene Ho and Cheng Chang. I am truly grateful for all the help they have provided and the skills they have imparted to me, making my experience a painless and pleasant one. I would also like to express my gratitude to other members of various laboratories (FW: Yong Loo Lin School of Medicine Pharmacology, OCN: Saw Swee Hock School of Public Health, and FCW: Davos LifeSciences) – Chan Tze Khee, Daniel Tan Wan Shun, Alan Koh Hock Meng, Lee Bee Lan, Su Jin, Winston Liao Wupeng, Lee Suet Hoay, Dong Jin Rui, Zhou Shuo, Jonathan Lim, Fera Goh, Guan Shou Ping, Lah Lin Chin, Khaing Nwe Win, Genevieve Seow, Pow Chen Wei, Amy Yong and Eunice Peh; for providing me with listening ears. I will always remember the good times we shared. I wish to also thank the Yong Loo Lin School of Medicine, Department of Pharmacology staff – Ho Lai Har, Alan Koh Hock Meng, Khoo Yok Moi, Ratnasari Bte Mohamed Basri, Cheong Yoke Ping, Fan Lu, Ho Woon Fei, Tan Yen Ling and Ting Wee Lee, for their kind assistance in making my lectures to life sciences students such a pleasant and fulfilling experience. I further thank all professors from the National University of Singapore whom I have encountered. I truly learnt a lot during my four years of PhD education. Last but not least, I would like to thank my life partner, family and friends for their boundless support and companionship through the harsh and good times. It would not have been possible without their countless encouragement and motivation. i TABLE OF CONTENTS ACKNOWLEGEMENTS i TABLE OF CONTENTS ii Ph.D. SUMMARY Ix LIST OF TABLES xii LIST OF FIGURES xiii LIST OF ABBREVIATIONS xvi HONORS AND AWARDS xxi LIST OF PUBLICATIONS xxii CONFERENCE ABSTRACTS xxiv CHAPTER 1: INTRODUCTION 1.1 MOTIVATION AND GOAL ............................................................................................ 1 1.2 SCOPE ..................................................................................................................... 2 1.3 VITAMIN E – TOCOPHEROLS AND TOCOTRIENOLS ....................................................... 5 1.3.1 Introduction ...................................................................................................... 5 1.3.1.1 History .................................................................................................. 5 1.3.1.2 Biochemical and Physical Properties of Vitamin E Isoforms .................. 6 1.3.1.3 Sources of Vitamin E ............................................................................ 9 1.3.2 Bioavailability and Pharmacokinetics of Vitamin E .......................................... 12 1.3.3 Vitamin E as an Antioxidant ........................................................................... 15 1.3.4 Vitamin E Therapy in Inflammatory Diseases ................................................. 17 1.3.5 Summary and Outlook of Vitamin E ............................................................... 20 1.4 ASTHMA ................................................................................................................. 25 ii 1.4.1 Epidemiology of Asthma................................................................................. 27 1.4.2 Development of Asthma ................................................................................. 29 1.4.3 Immunopathology of Allergic Asthma ............................................................. 30 1.4.3.1 Classical Hallmarks of Asthma ............................................................ 33 1.4.3.2 Eosinophils ......................................................................................... 35 1.4.3.3 Neutrophils .......................................................................................... 37 1.4.4 Oxidative Stress ............................................................................................. 39 1.4.5 Oxidative Stress in Asthma ............................................................................ 41 1.4.5.1 Impaired Antioxidant Defenses in Asthma ........................................... 44 1.4.5.1.1 Superoxide Dismutase (SOD) ............................................................... 45 1.4.5.1.2 Catalase (CAT) ........................................................................................ 45 1.4.5.1.3 Glutathione Peroxidase (GPx) .............................................................. 47 1.4.5.1.4 Nuclear Erythroid 2-Related Factor 2 (Nrf2) ....................................... 47 1.4.5.2 Increased Oxidative Damage in Asthma ................................................ 48 1.4.5.2.1 Lipid Peroxidation (8-Isoprostane) ....................................................... 48 1.4.5.2.2 Protein Nitration (3-Nitrotyrosine) ......................................................... 50 1.4.5.2.3 Oxidative DNA Damage (8-OHdG) ...................................................... 51 1.4.5.2.4 Relief of Asthma by targeting Oxidative Stress in the Lungs ........... 52 1.4.6 Current Therapies of Asthma ......................................................................... 52 1.4.7 Corticosteroids in Asthma: Prednisolone ........................................................ 57 1.4.7 Vitamin E as a Potential Therapy in Asthma ................................................... 60 1.4.8 Hypotheses & Objectives ............................................................................... 61 1.5 CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD) ........................................... 64 1.5.1 Epidemiology of COPD .................................................................................. 67 1.5.2 Etiology of COPD ........................................................................................... 68 iii 1.5.3 Inflammation in COPD ................................................................................... 70 1.5.3.1 Neutrophils .......................................................................................... 72 1.5.3.2 Macrophages ...................................................................................... 75 1.5.3.3 STAT3 and NF-κB ............................................................................... 76 1.5.4 Oxidative Stress in COPD .............................................................................. 79 1.5.4.1 Impaired Antioxidants in COPD ........................................................... 82 1.5.4.1.1 Superoxide Dismutase (SOD) ............................................................... 83 1.5.4.1.2 Catalase ................................................................................................... 83 1.5.4.1.3 Glutathione Peroxidase (GPx) .............................................................. 84 1.5.4.1.4 Nuclear Erythroid 2-Related Factor 2 (Nrf2) ....................................... 85 1.5.4.2 Heightened Oxidative Damage in COPD ............................................ 86 1.5.4.2.1 Lipid Peroxidation (8-Isoprostane) ....................................................... 87 1.5.4.2.2 Protein Nitration (3-Nitrotyrosine) ......................................................... 88 1.5.4.2.3 Protein Oxidation (AOPP) ...................................................................... 89 1.5.4.2.4 DNA Oxidative Damage (8-OHdG) ...................................................... 90 1.5.4.2.5 Targeting Oxidative Stress with Antioxidants in COPD .................... 90 1.5.5 Protease-Antiprotease Imbalance in COPD ................................................... 91 1.5.6 Emphysema in COPD .................................................................................... 94 1.5.7 Current Therapies in COPD ........................................................................... 95 1.5.8 Prednisolone and Vitamin E Isoform γ-Tocotrienol ....................................... 100 1.5.9 Hypotheses and Objectives .......................................................................... 101 1.6 CONCLUSIONS ...................................................................................................... 102 CHAPTER 2: METHODS 2.1 REAGENTS ........................................................................................................... 105 2.2 MURINE ASTHMA MODEL ...................................................................................... 106 iv 2.2.1 Mice ............................................................................................................. 106 2.2.2 House dust mite (HDM) Airway Challenge ................................................... 106 2.2.3 Treatment Groups for HDM-development .................................................... 107 2.2.4 Treatment Groups for screening of vitamin E isoforms ................................. 108 2.2.5 Treatment Groups for vitamin E isoform γ-tocotrienol in HDM-induced asthma mouse model ............................................................................................... 110 2.3 MURINE COPD MODEL ......................................................................................... 112 2.3.1 Mice ............................................................................................................. 112 2.3.2 Cigarette smoke (CS) exposure ................................................................... 113 2.3.3 Treatment groups for vitamin E isoform γ-tocotrienol in acute 2-weeks CS- induced COPD mouse model ....................................................................... 115 2.3.4 Treatment groups for vitamin E isoform γ-tocotrienol in chronic 2-months CS- induced COPD mouse model ....................................................................... 117 2.4 BRONCHOALVEOLAR LAVAGE ................................................................................ 118 2.5 TOTAL CELL COUNT ............................................................................................. 119 2.6 DIFFERENTIAL CELL COUNT OF BAL FLUID ............................................................ 119 2.7 2,7-DICHLORODIHYDROFLUORESCIN DIACETATE (DCFH-DA) ASSAY ....................... 120 2.8 HISTOLOGICAL EXAMINATION ................................................................................ 121 2.8.1 Hematoxylin and Eosin (H&E) Staining .......................................................... 121 2.8.2 Periodic Acid-Fluorescence Schiff (PAFS) Staining ....................................... 123 2.9 FREEZE DRY OF LUNG TISSUE .............................................................................. 124 2.10 ENZYMATIC ASSAY ............................................................................................... 125 2.10.1 Total Antioxidant Capacity (TAC) Assay ....................................................... 125 2.10.2 Superoxide Dismutase (SOD) Assay ............................................................ 125 2.10.3 Catalase (CAT) Assay .................................................................................. 126 2.10.4 Glutathione Peroxidase (GPx) Assay ........................................................... 127 v 2.11 ENZYME IMMUNOASSAY (EIA) ............................................................................... 127 2.11.1 Multiplex ....................................................................................................... 128 2.11.2 8-Isoprostane and 8-hydroxy-2-deoxyguanosine (8-OHdG) EIA ................... 129 2.11.3 3-Nitrotyrosine (3-NT) EIA ............................................................................ 129 2.11.4 Advanced Oxidation of Protein Products (AOPP) EIA .................................. 130 2.12 AIRWAY HYPERRESPONSIVENESS (AHR) ............................................................... 131 2.13 PULMONARY FUNCTION TEST (PFT) ...................................................................... 131 2.14 IMMUNOBLOTTING (WESTERN BLOTTING) ............................................................... 132 2.15 NF-ΚB, NRF2 AND STAT3 TRANSACTIVATION ASSAY ............................................. 133 2.16 REAL-TIME POLYMERASE CHAIN REACTION ........................................................... 134 2.16.1 RNA Isolation ............................................................................................... 134 2.16.2 Reverse Transcription .................................................................................. 136 2.16.3 Real-time Polymerase Chain Reaction ......................................................... 136 2.17 PHARMACOKINETICS OF γ-TOCOTRIENOL ............................................................... 139 2.18 1,1-DIPHENYL-2-PICRYLHYDRAZYL (DPPH) ASSAY ................................................. 140 2.19 STATISTICAL ANALYSIS ......................................................................................... 140 CHAPTER 3: TIME COURSE DEVELOPMENT OF INFLAMMATION AND OXIDATIVE STRESS IN HOUSE DUST MITE-INDUCED ASTHMA, AND SELECTION OF VITAMIN E ISOFORM 3.1 ABSTRACT ........................................................................................................... 141 3.2 INTRODUCTION ..................................................................................................... 143 3.3 MOUSE MODELS AND STATISTICAL ANALYSIS .......................................................... 145 3.4 RESULTS ............................................................................................................. 146 3.4.1 Increased Inflammation and Mucus Hypersecretion in Asthma .................... 146 3.4.2 Augmented Oxidative Stress in Asthma ....................................................... 154 vi 3.4.3 Upregulated NF-κB and Downregulated Nrf2 Levels Contribute to Inflammation and Oxidative Stress in Asthma ................................................................... 156 3.4.4 γ-Tocotrienol is Selected Amongst the Vitamin E Isoforms ........................... 158 3.5 DISCUSSION ......................................................................................................... 165 3.6 SUPPLEMENTARY ................................................................................................. 172 CHAPTER 4: VITAMIN E ISOFORM γ-TOCOTRIENOL DOWN REGULATES HOUSE DUST MITE-INDUCED ASTHMA 4.1 ABSTRACT ........................................................................................................... 173 4.2 INTRODUCTION ..................................................................................................... 175 4.3 RESULTS ............................................................................................................. 178 4.3.1 γ-Tocotrienol Attenuates HDM-Induced Airway Inflammation ....................... 178 4.3.2 γ-Tocotrienol Ameliorates HDM-Induced Oxidative Stress in the Airways ..... 182 4.3.3 γ-Tocotrienol Abrogates HDM-Induced AHR ................................................ 186 4.3.4 γ-Tocotrienol Reduces Nuclear NF-κB and Promotes Nuclear Nrf2 .............. 188 4.4 DISCUSSION ......................................................................................................... 190 4.5 SUPPLEMENTARY ................................................................................................. 196 CHAPTER 5: VITAMIN E ISOFORM γ-TOCOTRIENOL PROTECTS AGAINST EMPHYSEMA IN CIGARETTE SMOKE-INDUCED COPD 5.1 ABSTRACT ........................................................................................................... 197 5.2 INTRODUCTION ..................................................................................................... 199 5.3 RESULTS ............................................................................................................. 202 5.3.1 γ-Tocotrienol Attenuates CS-Induced Airway Inflammation .......................... 202 5.3.2 γ-Tocotrienol Abates Inflammation Via Downregulation of Nuclear STAT3 and NF-κB ........................................................................................................... 206 5.3.3 γ-Tocotrienol Suppresses CS-Induced Oxidative Stress in the Airways ......... 208 5.3.4 γ-Tocotrienol Strengthens Antioxidant Defence Promoting Nuclear Nrf2 ........ 211 vii 5.3.5 γ-Tocotrienol Ameliorates 2-month CS-Induced Emphysema ...................... 214 5.3.6 γ-Tocotrienol Abrogates 2-month CS-Induced Lung Function Impairment ..... 216 5.4 DISCUSSION ......................................................................................................... 218 CHAPTER 6: SUMMARY, LIMITATIONS AND FUTURE WORK 6.1 SUMMARY AND CONCLUSION ................................................................................. 225 6.2 LIMITATIONS AND FUTURE WORK ........................................................................... 231 6.2.1 Modifications to Improve Bioavailability ........................................................ 231 6.2.2 In Vitro Analyses .......................................................................................... 232 6.2.3 Clinical Testing ............................................................................................. 232 6.2.4 Complement Corticosteroids and/or Steroid-Resensitization ........................ 233 REFERENCES ................................................................................................................... 235 viii