PURIFIED HERBA LEONURI AND LEONURINE PROTECT MIDDLE CEREBRAL ARTERY OCCLUDED-RATS FROM BRAIN INJURY THROUGH ANTIOXIDATIVE MECHANISM AND MITOCHONDRIAL PROTECTION

In particular, I am deeply grateful to my supervisor, Professor Zhu Yi-Zhun, M.B.B.S., Ph.D., Associate Professor of Pharmacology, Yong Loo Lin School of Medicine, National University of

PURIFIED HERBA LEONURI AND LEONURINE

PROTECT MIDDLE CEREBRAL ARTERY

OCCLUDED-RATS FROM BRAIN INJURY THROUGH

ANTIOXIDATIVE MECHANISM AND MITOCHONDRIAL

PROTECTION

LOH KOK POH

B.Sc (Hons.), NUS

A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

DEPARTMENT OF PHARMACOLOGY

NATIONAL UNIVERSITY OF SINGAPORE

2009

Once we accept our limits,

we go beyond them

Albert Einstein (1879 – 1955)

In particular, I am deeply grateful to my supervisor, Professor Zhu Yi-Zhun, M.B.B.S.,

Ph.D., Associate Professor of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, who gave me an invaluable opportunity to work with him, introduced me to the field of natural products and cerebral ischemia His extensive discussions during my course of research and untiring help during my difficult moments have been very helpful His understanding, encouraging and personal guidance have provided a good basis for the present thesis

I would like to express my deep and sincere gratitude to my supervisor, Associate

Professor Tan Kwong Huat, Benny, M.B.B.S., Ph.D., Department of Pharmacology,

Yong Loo Lin School of Medicine, National University of Singapore, for his wide knowledge and his detailed and constructive comments, which have been of great value for me

I express my warm and sincere thanks to Dr Wang Hong for her valuable advice and

friendly help Her extremely valuable experiences support and insights have been of great

value in this study I warmly thank Dr Wang Zhong Jing, for introducing me the field

Annette Shoba, and Ms Lim Hwee Ying from Professor Sit Kim Ping’s lab, for their

excellent guidance on mitochondrial studies

My sincere thanks are due to my thesis advisory committe, Associate Professor

Charanjit Kaur, Associate Professor Ng Yee Kong and Associate Professor Tan Kwong Huat, Benny, for their detailed review, constructive criticism and excellent

advice during the preparation of this thesis

I wish also to extend my appreciation to Animal Holding Unit (AHU), National

University of Singapore, which provides excellent research facilities for animal study I

am thankful to AHU laboratory staff Low Wai Mun James, Loo Eee Yong Jeremy, and the rest for their assistance, friendship and extremely positive attitude towards me

I am grateful for the scholarship from Yong Loo Lin School of Medicine, National

University of Singapore, without which writing this thesis might not be possible The

financial support of the Herbatis is gratefully acknowledged

Acknowledgement

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Without friends, life as a graduate student would not be the same Ms Ler Lian Dee, Ms

Irene Lee Cheng Jie, Mr Ling Moh Lung, special friends to me, and the many

discussions we had, be they research-related or not, were often the occasion for new

discoveries and always truly agreeable moments Ms Ning Li, Ms Chuah Shin Chet, Ms

Wong Wan Hui, Ms Low Lishan, their friendships are valuable to me and I want to

thank them for their pragmatic approach to problem solving and their honesty It is, however, not possible to list all of them here Their support in this research, to be directly,

or indirectly, is greatly appreciated

I owe my loving thanks to my family Without their encouragement and understanding for the past 27 years, it would have been impossible for me to be whom I am today My special gratitude is due to my brother, for his never-ending loving support I thank my

husband, Mr Yeong Sai Hooi His love to me is always a powerful source of inspiration and energy

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Table of Contents

Acknowledgement……… i

Table of Contents……… iv

List of Abbreviations……… x

List of Tables……… xiii

List of Figures……… xiv

Summary……… xvii

List of Publications……… xx

Chapter 1 General Overview……… 1

1.1 Overview……… 2

1.2 Objectives……… 7

1.3 Structure of thesis……… 9

Chapter 2 Introduction: Ischemic Stroke, CNS mitochondria and Therapeutic Potential of Traditional Chinese Medicine………

11 2.1 Pathophysiology of stroke……… 12

2.1.1 Ischemic stroke……… 13

2.1.2 Cell death in stroke……… 16

2.1.2.1 Ischemic cascade……… 16

2.1.2.2 Apoptosis……… 19

2.1.3 Oxidative stress of stroke……… 25

2.1.4 Rodent ischemic stroke models……… 30

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2.2 CNS mitochondria……… 35

2.2.1 Protective physiological roles of CNS mitochondria……… 35

2.2.2 Mitochondria and apoptosis……… 37

2.2.3 Mitochondria and ROS……… 39

2.2.3.1 Mitochondria source of ROS……… 39

2.2.3.2 Mitochondria target of ROS……… 42

2.2.4 Mitochondrial involvement in stroke……… 44

2.3 Traditional Chinese medicines (TCM)……… 48

2.3.1 Gingko biloba 49

2.3.2 Braintone……… 52

2.3.3 Herba leonuri (HL) and purified Herba leonuri (pHL)………… 54

2.3.4 Leonurine……… 57

Chapter 3 Materials and Methods……… 59

3.1 Drug preparations……… 60

3.1.1 Purified Herba leonuri (pHL)……… 60

3.1.2 Leonurine……… 60

3.2 Animals……… 61

3.3 Middle cerebral artery occlusion (MCAO)……… 61

3.4 Experimental protocols……… 62

3.4.1 Experimental protocol I……… 62

3.4.1.1 Objectives……… 62

3.4.1.2 Experimental design……… 62

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3.4.2 Experimental protocol II……… 64

3.4.2.1 Objectives……… 64

3.4.2.2 In vitro mitochondrial studies……… 64

3.4.2.3 In vivo mitochondrial studies……… 65

3.4.3 Experimental protocol III……… 67

3.4.3.1 Objectives……… 67

3.4.3.2 In vivo studies – animal treatment and MCAO……… 67

3.4.3.3 In vitro mitochondrial studies……… 68

3.4.3.4 In vivo mitochondrial studies……… 69

3.5 Experimental techniques……… 71

3.5.1 Infarct volume measurement……… 71

3.5.2 Evaluation of neurological deficit……… 71

3.5.3 Total antioxidant assay……… 72

3.5.4 DNA oxidative damage analysis using GC/MS……… 73

3.5.5 TUNEL (TdT-mediated dUTP Nick-End Labeling) assay……… 75

3.5.6 Immunohistochemical staining……… 77

3.5.7 Superoxide dismutase (SOD) activity assay……… 79

3.5.8 Glutathione peroxidase (GPx) activity assay……… 80

3.5.9 Lipid peroxidation product measurement……… 81

3.5.10 Preparation of intact rat brain mitochondria……… 81

3.5.11 Measurement of mitochondrial membrane potential – JC-1 assay 82 3.5.12 Measurement of ROS in isolated mitochondria……… 83

3.5.13 ATP biosynthesis in isolated mitochondria……… 84

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3.5.14 Mitochondrial respiration measurement……… 85

3.5.15 Glutathione level in isolated mitochondria……… 87

3.6 Statistical analysis……… 88

Chapter 4 Results……… 89

4.1 Results of experiment I: Cerebral Protection of Purified Herba Leonuri Extract on Middle Cerebral Artery Occluded-Rats………

90 4.1.1 Pharmacological and functional outcome studies……… 90

4.1.1.1 pHL reduced infarct volume resulted from MCAO…… 90

4.1.1.2 pHL ameliorated the neurological outcome of MCAO-induced rats………

92 4.1.2 Biochemical, cellular and molecular approaches……… 95

4.1.2.1 MCAO decreased plasma antioxidant level and protection of pHL on the plasma antioxidant level……

95 4.1.2.2 Increased oxidative stress by MCAO and prevention by pHL………

96 4.1.2.3 Enhanced TUNEL nuclear green by MCAO and prevention by pHL………

97 4.1.2.4 Apoptosis involvement of stroke and protection of pHL 99 4.2 Results of experiment II: Modulation of Mitochondrial ROS Generation and Function by Purified Herba Leonuri Extract………

103 4.2.1 Quality of isolated cortical mitochondria preparation……… 103

4.2.2 Mitochondrial ROS production and prevention by pHL in vitro 105

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4.2.3 Effect of pHL on ATP biosynthesis in isolated mitochondria… 108

4.2.4 Effect of pHL on mitochondrial respiration and RCR value…… 110

4.2.5 Effect of pHL on mitochondrial GSH in vivo……… 117

4.3 Results of experiment III: Leonurine (4-guanidino-n-butyl Syringate) Protects the Middle Cerebral Artery Occluded-Rats through Antioxidant effect and Regulation of Mitochondrial Function………

119 4.3.1 Effect of Leonurine on functional outcome of MCAO-induced rats………

119 4.3.2 Effect of Leonurine on oxidative stress in MCAO-induced rats… 122 4.3.3 Effect of Leonurine on ROS production in isolated mitochondria 123 4.3.4 Effect of Leonurine on ATP biosynthesis in isolated mitochondria………

126 4.3.5 Effect of Leonurine on mitochondrial respiration……… 129

4.3.6 Effect of Leonurine on mitochondrial GSH in vivo……… 134

Chapter 5 Discussion……… 135

5.1 Discussion on experiment I……… 137

5.2 Discussion on experiment II……… 146

5.3 Discussion on experiment III……… 159

5.4 General discussion……… 166

Chapter 6 Conclusion and Future Perspectives……… 174

6.1 Conclusion……… 175

6.2 Limitation of study……… 179

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References……… 184

ADP adenosine diphosphate

AHA American Heart Association

ANT adenine nucleotide translocator

Apaf-1 apoptotic protease activating factor 1

AIF apoptosis inducing factor

AMP adenosine monophosphate

ATP adenosine triphosphate

BBB blood brain barrier

BNIP3 Bcl-2/adenovirus E1B 19kDa-interacting protein

BID Bcl-2 interacting domain

BSA bovine serum albumin

CAD caspase-activated deoxyribonuclease

CBF cerebral blood flow

CBV cerebral blood volume

CMRO2 cerebral metabolic rate of oxygen

CoQ coenzyme Q

DD death domain

DED death effector domain

DISC death-inducing signaling complex

DNA deoxyribonucleic acid

DTNB 5, 5’-dithiobis-2-nitrobenzoic acid

List of Abbreviations

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ESR electron spin resonance

ETC electron transport chain

FADD Fas-associated death domain protein

FasL Fas ligand

FDA Food and Drug Administration

IAP inhibitor of apoptosis

IHC immunohistochemical staining

LC-ESI-MS liquid chromatograph electrospray ionization mass spectrometry MCAO middle cerebral artery occlusion

MDA malondialdehyde

MI myocardial infarction

mitoKATP mitochondrial ATP-sensitive K+ channel

MPTP mitochondrial permeability transition pore

NFkB nuclear factor-kappa B

NMDA N-methyl-D-aspartate

NO nitric oxide

NOS nitric oxide synthase

PARP poly (ADP-ribose) polymerase

List of Abbreviations

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PCD programmed cell death

PET Positron emission tomography

pHL purified Herba leonuri

ROS reactive oxygen species

rt-PA recombinant tissue type plasminogen activator

SOD superoxide dismutase

tBID truncated form of BID

TBA thiobarbituric acid

TCA tricarboxylic acid cycle

TCM Traditional Chinese medicine

Table 3-1 Groups for studies of effect of pHL on isolated mitochondria

Table 3-2 Grouping for studies of effect of Leonurine on isolated mitochondria

Table 4-1 Neurological deficit grading system

Table 4-2 Levels of SOD, GPx and MDA in each treatment group

Figure 2-1 a) Ischemic stroke; and b) hemorrhagic stroke (arrow)

Figure 2-2 A diagram illustrates the ischemic cascade

Figure 2-3 A schematic diagram of apoptosis

Figure 2-4 A flow chart showing the involvement of ROS in multiple ischemic

cascades Figure 2-5 The spatial pattern of cerebral blood flow (CBF) in MCAO

Figure 2-6 A schematic model of ETC and ROS generation in the mitochondria

Figure 2-7 Fenton reaction

Figure 2-8 Gene expression level of a) AT2 receptor, b) Fas, c) Bax, and d) ratio of

Bcl-xL/Bcl-xS was measured from left cortex after 7 days of MCAO

Figure 2-9 Gene expression level of a) AT2 receptor, b) Fas, c) Bax, and d) ratio of

Bcl-xL/Bcl-xS Figure 2-10 The 5 known compounds from pHL

Figure 3-1 A Flow chart represents the experimental outline in the pilot study of pHL Figure 3-2 Flow charts represent the general outlines of experimental protocol II Figure 3-3 A Flow chart represents the experimental outline in the pilot study of

Leonurine

Figure 3-4 Flow charts represent the general outlines of mitochondrial studies for

Leonurine

List of Figures

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Figure 4-1 Infarct volume measured from each treatment group a) Images of cerebral

sections among the treatment groups b) Infarct volume was analyzed by image analyzer system (Scion image for windows)

Figure 4-2 Neurological deficit score among treatment groups (n>20)

Figure 4-3 Plasma total antioxidant concentration of each treatment group under the

influence the pHL

Figure 4-4 The DNA oxidative damage level in each treatment group

Figure 4-5 Apoptotic staining in cerebral cortex after 7 days of MCAO (20x

magnification) for each treatment group

Figure 4-6 Light photomicrographs (10x magnification) of cryostat section of the rat

left cerebral cortex

Figure 4-7 Immunohistochemical staining of pro-apoptotic protein (b: BAX and c:

FAS) and anti-apoptotic protein (d: BCL-2 and e: BCL-XL) in cerebral cortex after 7 days of MCAO (40x magnification), with a: negative control Figure 4-8 Quantitative recordings of the membrane potential from isolated cortical

mitochondria

Figure 4-9 Effects of pHL on cortical mitochondrial ROS generation determined by

oxidation of DCFDA

Figure 4-10 Effects of pHL on ATP biosynthesis of succinate treated cortical

mitochondria in the absence (a) or presence (b) of 1mM H2O2 Figure 4-11 Effects of pHL on metabolic rates of isolated mitochondria (n=4)

Figure 4-12 Oxygen consumption of left cortical mitochondria (a, b, c) and right

cortical mitochondria (e, d, f)

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Figure 4-13 GSH concentration from each treatment group in vivo GSH concentration

was enhanced 3 hours after MCAO

Figure 4-14 a) Infarct area of each treatment group was observed by TTC staining b)

Infarct volume was then analyzed by image analyzer system (Scion image for windows)

Figure 4-15 Effect of Leonurine on cortical mitochondrial ROS production determined

by changes in DCF oxidation

Figure 4-16 Effect of Leonurine on ATP biosynthesis of succinate treated cortical

mitochondria in the absence (a) or presence (b) of 1mM H2O2 Figure 4-17 Oxygen consumption (a) and respiratory control ratio (RCR) (b) of

isolated mitochondria

Figure 4-18 Oxygen consumption of left cortical mitochondria (a) and right cortical

mitochondria (c) Respiratory control ratio (RCR) of left cortical mitochondria (b) and right cortical mitochondria (d)

Figure 4-19 GSH concentration from each treatment group in vivo