UNIVERSITI TEKNOLOGI MARA

CELLULAR MECHANISMS OF ACTION OF RESVERATROL IN

REGULATION OF AQUEOUS HUMOUR DYNAMICS

NORHAFIZA BINTI RAZALI

Thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy

Faculty of Medicine

October 2016

CONFIRMATION BY PANEL OF EXAMINERS

I certify that a panel of examiners has met on 15 July 2016 to conduct the final examination of Norhafiza Binti Razali on her Doctor of Philosophy thesis entitled "Cellular Mechanisms of Action of Resveratrol in Regulation of Aqueous Humour Dynamics" in accordance with Universiti Teknologi MARA Act 1976 (Akta 173). The Panel of Examniners recommends that the student be awarded the relevant degree. The panel of Examiners was as follows:

Azian Abd. Latiff, PhD Professor Faculty of Medicine Universiti Teknologi MARA (Chairlady)

Harbindar Jeet Singh, PhD Professor Faculty of Medicine Universiti Teknologi MARA (Internal Examiner)

Nor Azizan Abdullah, PhD Professor Department of Pharmacology Faculty of Medicine University of Malaya (External Examiner)

Rohit Saxena, MBBS, MD (Ophthalmology) Associate Professor Faculty of Medicine All India Institute of Medical Sciences (AIIMS) (External Examiner)

DR MOHAMMAD NAWAWI DATO' HAJI SEROJI Dean Institute of Graduate Studies Universiti Teknologi MARA Date: 19 October 2016

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AUTHOR'S DECLARATION

I declare that the work in this dissertation was carried out in accordance with the

regulations of Universiti Teknologi MARA. It is original and is the results of my own

work, unless otherwise indicated or acknowledged as referenced work. This topic has

not been submitted to any other academic institution or non-academic institution for

any degree or qualification.

I hereby acknowledge that I have been supplied with the Academic Rules and

Regulations for Post Graduate, Universiti Teknologi MARA, regulating the conduct

of my study and research.

Name of Student

Student I.D. No.

Programme

Faculty

Thesis Title

Signature of Student

Date

Norhafiza Bmti Razah

2011271998

Doctor of Philosopy (Pharmacology) - MD990

Faculty of Medicine

Cellular Mechanisms of Action

of Resveratrol in Regulation of Aqueous

Humour Dynamics

: - < & • • •

October 2016

iii

ABSTRACT

Glaucoma, a common cause of optic neuropathy, is associated with elevated intraocular pressure (IOP) and is the leading cause of irreversible visual disability. Steroid-induced glaucoma, a common type of secondary glaucoma, is also associated with elevated IOP (steroid-induced ocular hypertension (SIOH)). SIOH and glaucoma are currently treated with antiglaucoma agents, which often have suboptimal efficacy and are associated with adverse effects. The objective of this study was to determine if topical application of /nms-resveratrol reduces IOP in rats with SIOH and to investigate its mechanisms of action. This study was divided into in vivo and in vitro studies. In the in vivo study, we evaluated the oculohypotensive effects of topical toms-resveratrol in normal and SIOH rats and investigated the role of adenosine receptors (ARs) and transforming growth factor-P (TGF-p) signalling in the IOP lowering effect of fnms-resveratrol. Involvement of AR was studied by observing the IOP changes in response to /rara-resveratrol after pre-treating SIOH animals with AR subtype-specific antagonists. The study also investigated phospholipase C (PLC) activation, extracellular regulated kinase 1/2 (ERK1/2) phosphorylation and increased matrix metalloproteinases (MMPs) secretion in the aqueous humour (AH) as mechanism of resveratrol-induced oculohypotension in SIOH rats. In vitro studies evaluated the effect of frans-resveratrol on cellular signalling pathways of ARs and TGF-p in primary human trabecular meshwork cells (HTMCs). Single drop of all concentrations of toms-resveratrol produced significant oculohypotension in normotensive rats and 0.2% concentration produced maximum IOP reduction. Twice-daily topical application of fnms-resveratrol 0.2% for 21-day in SIOH rats resulted in significant and sustained IOP reduction. This was associated with significantly higher AH MMP-2 level; significantly reduced trabecular meshwork (TM) thickness and increased number of TM cells. Treatment with /raws-resveratrol also significantly increased the ganglion cell survival and reduced retinal oxidative stress. Pretreatment with adenosine Ai receptor antagonist inhibited the oculohypotensive effect of resveratrol. The use of Ai AR, PLC and ERK 1/2 inhibitors also reduced resveratrol-induced MMP-2 secretion. These results were further supported by in vitro study that demonstrated that ERK1/2, PLC and MMP-2 secretion by HTMC is stimulated after resveratrol treatment and these effects are associated with upregulation of Ai AR gene expression. Topical /r<ms-resveratrol also produced significantly raised plasminogen activator levels and combined TGF-p2+resveratrol treatment caused significant upregulation of inhibitory SMAD7 when compared to TGF-P2-only treated group. Hence, it could be concluded that frvms-resveratrol-induced oculohypotension in SIOH rats involves its agonistic activity at the AiAR leading to PLC activation, ERK 1/2 phosphorylation and increased MMP-2 secretion. Increased MMP-2 secretion seems to cause changes in TM favourable for AH outflow leading to reduced IOP. 7><ms-resveratrol-induced oculohypotension could also be attributed to increased level of plasminogen activators, which seems to result from increased expression of inhibitory SMAD7, a TGF-p2 signalling molecule. Although current study, for the first time, has clearly demonstrated the significant effects of topical fratts-resveratrol on IOP in rats with SIOH and some of the underlying mechanisms; further investigations are needed to fully understand the mechanisms of action of trans-resveratrol and to explore its potential as a future antiglaucoma agent.

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TABLE OF CONTENTS

Page

CONFIRMATION BY PANEL OF EXAMINERS ii

AUTHOR'S DECLARATION iii

ABSTRACT iv

ACKNOWLEDGEMENT v

TABLE OF CONTENTS vi

LIST OF TABLES xi

LIST OF FIGURES xiv

LIST OF ABBREVIATIONS xix

CHAPTER ONE: INTRODUCTION 1

1.1 Background of Study 1

1.2 Problem Statement 6

1.3 Aims of Study 7

1.4 Study Objectives 7

1.5 Research Questions 7

1.6 Research Hypotheses 8

1.7 Significance of Study 8

1.8 Scope and Limitations of Study 9

CHAPTER TWO: LITERATURE REVIEW 10

2.1 Glaucoma: Definition and Prevalence 10

2.2 Glaucoma: Classification 10

2.2.1 Primary Glaucomas 10

2.2.2 Secondary Glaucomas 11

2.3 POAG 12

2.3.1 Risk Factors Involved In The Development And

Progression Of POAG 13

2.3.1.1 Ocular Hypertension 13

2.3.1.2 Age 13

2.3.1.3 Ethnicity 14

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