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

PURIFIED HERBA LEONURI AND LEONURINE PROTECT MIDDLE CEREBRAL ARTERY OCCLUDEDRATS 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) Acknowledgement Acknowledgement I would like to acknowledge my debt to those who have helped along the way and influenced the formation of this thesis. 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 Department of Pharmacology, YLL School of Medicine i Acknowledgement of mitochondria and for sharing me his experiences about the problem issues involved. His ideals and concepts have had a remarkable influence on my research path. I wish to extend my appreciation to Dr. Huang Shan Hong for her expert GC-MS analysis of the samples and drug used in this study, Tan Tong San, Ms. Vincent 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. Department of Pharmacology, YLL School of Medicine ii Acknowledgement 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. Department of Pharmacology, YLL School of Medicine iii Table of Contents 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 General Overview…………………………………………………. 1.1 Overview……………………………………………………………… . 1.2 Objectives………………………………………………………………… 1.3 Structure of thesis………………………………………………………… Chapter Introduction: Ischemic Stroke, CNS mitochondria and Therapeutic Potential of Traditional Chinese Medicine……… . 2.1 11 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……………………………………… . 2.1.4 Rodent ischemic stroke models………………………………… 30 Department of Pharmacology, YLL School of Medicine 25 iv Table of Contents 2.2 CNS mitochondria…………………………………………………………. 35 2.2.1 Protective physiological roles of CNS mitochondria……………. 35 2.2.2 Mitochondria and apoptosis…………………………………… . 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 Mitochondrial involvement in stroke……………………………. 44 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 2.2.4 2.3 Chapter 3.1 37 Materials and Methods……………………………………………. 59 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 62 Experimental protocol I…………………………………………. 3.4.1.1 Objectives……………………………………………… 62 3.4.1.2 Experimental design…………………………………… 62 Department of Pharmacology, YLL School of Medicine v Table of Contents 3.4.2 Experimental protocol II………………………………………… 64 3.4.2.1 Objectives……………………………………………… 64 3.4.3 3.4.2.2 In vitro mitochondrial studies…………………………. 64 3.4.2.3 In vivo mitochondrial studies………………………… 65 Experimental protocol III……………………………………… . 67 3.4.3.1 Objectives……………………………………………… 67 3.5 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 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 Department of Pharmacology, YLL School of Medicine vi Table of Contents 3.6 3.5.14 Mitochondrial respiration measurement…………………………. 85 3.5.15 Glutathione level in isolated mitochondria……………………… 87 Statistical analysis…………………………………………………………. 88 Chapter 4.1 Results……………………………………………………………… 89 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- 4.1.2 induced rats……………………………………………. 92 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 . Department of Pharmacology, YLL School of Medicine 105 vii Table of Contents 4.3 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 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………………………… 4.3.1 119 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 4.3.4 Effect of Leonurine on ATP biosynthesis in isolated mitochondria…………………………………………………… 123 126 4.3.5 Effect of Leonurine on mitochondrial respiration……………… 129 4.3.6 Effect of Leonurine on mitochondrial GSH in vivo…………… . 134 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 Chapter Conclusion and Future Perspectives…………………………… . 174 6.1 Conclusion…………………………………………………………………. 175 6.2 Limitation of study………………………………………………………… Department of Pharmacology, YLL School of Medicine 179 viii List of Figures 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 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 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) Department of Pharmacology, YLL School of Medicine xv List of Figures Figure 4-13 GSH concentration from each treatment group in vivo. GSH concentration was enhanced 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. Department of Pharmacology, YLL School of Medicine xvi Summary Summary Ischemic stroke refers to the condition caused by the occlusion of blood vessels, causing the injury of the territory of the supplied brain region. With ample evidences showed that free radicals play a role in ischemic cascade, studies focusing on traditional Chinese medicines (TCMs) have shown its promising effects in neuroprotection due to their antioxidant properties. In this research, therapeutic potentials of purified Herba leonuri (pHL) and one of its active ingredients Leonurine on ischemic stroke were investigated. Rodent model of permanent focal ischemia by left middle cerebral artery occlusion (MCAO) in Wistar rats was employed in this research. Left MCAO model resulted in infarct spanning left cortical and striatal tissues with neurological impairment, oxidative stress, apoptosis and mitochondrial dysfunction. In experiment I, MCAO-induced rats pretreated with pHL for 14 days prior to the induction of stroke, followed by another days of post-operation treatment was demonstrated to have reduction in infarct volume from 20.75 ± 0.03% to 15.19 ± 0.02% (p[...]... B.K.H Tan, V Chou, Y.Z Zhu Mechanisms of cerebral protection of Chinese herbal extract-Braintone on middle cerebral occluded rats Journal of Chinese Pharmaceutical Sciences, 18(2), 106- 113, 2009 5 K.P Loh, J Qi, B.K.H Tan, V Chou, Y.Z Zhu Leonurine (4-guanidino-n-butyl Syringate) Protects the Middle Cerebral Artery Occluded Rats through Antioxidant effect and Regulation of Mitochondrial Function (manuscript... study of cerebral protection using Ginkgo biloba extract and Losartan on stroked rats Neuroscience Letters, 398, 28-33, 2006 2 K.P Loh, S.H Huang, R.D Silva, B.K.H Tan and Y.Z Zhu Oxidative Stress: Apoptosis in Neuronal Injury Current Alzheimer Research, 3, 327-337, 2006 3 K.P Loh, S.H Huang, B.K.H Tan, V Chou, Y.Z Zhu Cerebral Protection of Purified Herba Leonuri Extract on Middle Cerebral Artery Occluded... the therapeutic potential of Leonurine, a key and unique compound found in both Herba leonuri (HL) and pHL, on ischemic stroke model Animal was pretreated with Leonurine orally for 7 days before the middle cerebral artery occlusion (MCAO) was done This study demonstrated that one day after surgery, Leonurine treatment at 60mg/kg/day could significantly reduce infarct volume from 22.45%±2.88% to 13.5%±1.74%... et al, 2009) through antioxidative mechanisms and apoptosis intervention However, the understanding of scientific principles of Chinese herbs is unsatisfactory, resulting in the limitation of their widespread use in patients This thesis aims to demonstrate the therapeutic potential of Chinese Herbs purified Herba leonuri (pHL) and Leonurine on ischemic stroke It is suggested that pHL and Leonurine exert... Experimental protocol II: Modulation of Mitochondrial ROS Generation and Function by Purified Herba leonuri Extract Department of Pharmacology, YLL School of Medicine 9 Chapter 1: General Overview Experimental protocol III: Leonurine (4-guanidino-n-butyl Syringate) Protects the Middle Cerebral Artery Occluded Rats through Antioxidant effect and Regulation of Mitochondrial Function Experimental techniques... p