HYDROGEN SULFIDE AND NEUROGENIC INFLAMMATION IN A MURINE MODEL OF POLYMICROBIAL SEPSIS ANG SEAH FANG (B.Sc. (Hons.), National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS It is a pleasure to thank the many people who made this dissertation possible. First and foremost, I would like to express my deepest gratitude to my supervisor, Professor Madhav Bhatia, for giving me confidence and support to begin my Ph.D studies. Professor Bhatia offered me so much advice, patiently supervising me, and always guiding me in the right direction. His passion for research, his stringent scientific attitude, and his perseverant spirits have been of great value to me. Special thanks are also given to my other supervisor, Associate Professor Paul A. MacAry. He is the one who accepted me as his student without any hesitation during the critical period of my Ph.D studies. He helped me immensely by giving me encouragement, guidance, supervison, and understanding throughout this work. It is not sufficient to express my gratitude with only a few words. I would also like to thank my co-supervisor, Associate Professor Shabbir M. Moochhala, for his engaging and proactive guidance. He has shared with me his invaluable insights on the animal model of polymicrobial sepsis. I appreciate all his contributions of time and ideas to make my Ph.D experience productive and stimulating. NUS Graduate School for Integrative Sciences and Engineering (NGS) has extended a great deal of support and ensured that I received a quality graduate education that will put me on the forefront of global competition. For that, I am truly thankful to NGS for i providing me with a generous scholarship as well as ample opportunities to shine and learn as a graduate student. My heartfelt appreciation also goes to Associate Professor Khanna Sanjay. I would like to thank him for his unselfish and unfailing support as my dissertation adviser. I am very grateful to Shoon Mei Leng, our laboratory officer, for her willingness to always go the extra mile to help me in my experiments and for excellent help in technical procedures. I would also like to thank the staff of Department of Pharmacology, especially Ting Wee Lee and Koh Yoke Moi, who have extended their warmest help to facilitate my Ph.D project. Special thanks also go to my fellow laboratory mates, Dr. Akhil Hegde, Dr. He Min, Dr. Jenab Nooruddinbhai Sidhapuriwala, Dr. Pratima Shrivastava, Dr. Raina Devi Ramnath, Dr. Ramasamy Tamizhselvi, Dr. Sun Jia, Dr. Zhang Huili, Dr. Zhang Jing, Dr. Muthu Kumaraswamy Shanmugam, Dr. Zhi Liang, Dr. Lee Jan Hau, Andy Yeo Yee, Koh Yung Hua, Sagiraju Sowmya, Yada Swathi, Yeo Ai Ling, and Abel Damien Ang for insightful discussions, moral support, and encouragement. Last but not least, I am greatly indebted to my family members for their support, love, and understanding so that I could come so far in life. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS iii SUMMARY ix LIST OF TABLES xii LIST OF FIGURES xiii LIST OF ABBREVIATIONS xvi PUBLICATIONS xx CHAPTER I INTRODUCTION 1.1 General overview 1.2 H2S 1.2.1 Chemical properties of H2S 1.2.2 H2S Toxicity 1.2.3 Biosynthesis of H2S 1.2.4 Metabolism of H2S 1.2.5 Biological roles of H2S 1.2.5.1 Roles of H2S in CNS 10 1.2.5.1.1 Physiological roles of H2S in CNS 10 1.2.5.1.2 H2S in CNS diseases 12 1.2.5.2 Roles of H2S in cardiovascular system 15 1.2.5.2.1 Physiological roles of H2S in cardiovascular system 15 1.2.5.2.2 H2S in cardiovascular diseases 18 1.2.5.3 Roles of H2S in gastrointestinal system 21 1.2.5.3.1 Physiological roles of H2S in gastrointestinal system 21 1.2.5.3.2 H2S in gastrointestinal diseases 25 1.2.5.4 Roles of H2S in endocrine system 26 iii 1.2.5.5 Roles of H2S in reproductive system 28 1.2.5.6 Roles of H2S in inflammation 28 1.2.5.6.1 Pro-inflammatory roles of H2S 31 1.2.5.6.2 Anti-inflammatory roles of H2S 42 1.2.5.6.3 H2S and neurogenic inflammation 45 1.3 Sepsis 55 1.3.1 Definition of sepsis 55 1.3.2 Epidemiology of sepsis 57 1.3.3 Pathophysiology of sepsis 58 1.3.4 Animal models of sepsis 61 1.3.4.1 Toxemia models 61 1.3.4.2 Bacterial infection models 62 1.3.4.3 Host-barrier disruption models 63 CHAPTER II RESEARCH RATIONALE AND OBJECTIVES 66 2.1 Question of interest 66 2.2 Approach 68 2.3 Objectives 69 CHAPTER III H2S PROMOTES TRPV1-MEDIATED NEUROGENIC INFLAMMATION IN POLYMICROBIAL SEPSIS 70 3.1 Introduction 70 3.2 Materials and methods 71 3.2.1 Induction of sepsis 71 3.2.2 Measurement of MPO activity 73 3.2.3 Measurement of plasma H2S 73 3.2.4 Assay of liver CSE activity 74 3.2.5 Histopathological examination 74 3.2.6 Survival studies 75 iv 3.2.7 Statistics 3.3 Results 75 75 3.3.1 Capsazepine attenuates systemic inflammation and multiple organ damage in sepsis 75 3.3.2 Capsazepine has no effect on endogenous generation of H2S in sepsis 77 3.3.3 Capsazepine protects against mortality in CLP-induced sepsis 77 3.3.4 Capsazepine reverses the pro-inflammatory effects of NaHS in sepsis 78 3.3.5 Capsazepine protects against NaHS-augmented mortality in CLPinduced sepsis 79 3.3.6 Capsazepine has no effect on blockade of endogenous generation of H2S in sepsis 79 3.3.7 Capsazepine has no effect on PAG-mediated attenuation of proinflammatory effects of H2S in sepsis 80 3.3.8 Capsazepine has no effect on PAG-mediated protection of mortality in CLP-induced sepsis 81 3.4 Discussion 81 CHAPTER IV H2S PROMOTES TRPV1-MEDIATED NEUROGENIC INFLAMMATION IN POLYMICROBIAL SEPSIS THROUGH ENHANCEMENT OF SP PRODUCTION 98 4.1 Introduction 98 4.2 Materials and methods 100 4.2.1 Induction of sepsis 100 4.2.2 Measurement of SP levels 100 4.2.3 Cytokines, chemokines, and adhesion molecules analysis 101 4.2.4 Reverse transcriptase-polymerase chain reaction analysis 101 4.2.5 Measurement of pulmonary edema 102 4.2.6 Alanine aminotransferase and aspartate aminotransferase assay 102 4.2.7 Statistics 102 4.3 Results 4.3.1 Capsazepine attenuates endogenous SP concentrations in both septic and septic mice administrated with NaHS 103 103 v 4.3.2 The attenuated SP concentration correlates with reduced production of pro-inflammatory molecules in both septic and septic mice administrated with NaHS 104 4.3.3 Capsazepine protects against MODS in both septic and septic mice administrated with NaHS 105 4.3.4 Capsazepine has no effect on PAG-mediated attenuation of SP levels in sepsis 106 4.3.5 Inhibition of H2S formation impaired pro-inflammatory molecules production after septic injury, but capsazepine has no effect on them 106 4.3.6 Beneficial effects of capsazepine and PAG are not additive in protection against MODS in sepsis 107 4.4 Discussion 107 CHAPTER V H2S PROMOTES TRPV1-MEDIATED NEUROGENIC INFLAMMATION IN POLYMICROBIAL SEPSIS BY ACTIVATING ERK1/2 AND NF-ΚB SIGNALING PATHWAYS 136 5.1 Introduction 136 5.2 Materials and methods 138 5.2.1 Induction of sepsis 138 5.2.2 Nuclear extraction and measurement of NF-κB activation 138 5.2.3 Western immunoblot 138 5.2.4 Statistics 139 5.3 Results 140 5.3.1 Effect of capsazepine on ERK1/2 activation in H2S-induced neurogenic inflammation in sepsis 140 5.3.2 Effect of capsazepine on IκBα phosphorylation and degradation levels in H2S-induced neurogenic inflammation in sepsis 140 5.3.3 Effect of capsazepine on NF-κB activation in H2S-induced neurogenic inflammation in sepsis 141 5.4 Discussion 142 vi CHAPTER VI H2S AUGMENTS COX-2 AND PROSTAGLANDIN E2 METABOLITE PRODUCTION IN SEPSIS-EVOKED ACUTE LUNG INJURY BY A TRPV1 CHANNEL-DEPENDENT MECHANISM 152 6.1 Introduction 152 6.2 Materials and methods 153 6.2.1 Induction of sepsis 153 6.2.2 Measurement of COX-2 activity 154 6.2.3 Measurement of PGE2 metabolite levels 154 6.2.4 Western immunoblot 154 6.2.5 Measurement of MPO activity 154 6.2.6 Histopathogical examination 155 6.2.7 Cytokines, chemokines, and adhesion molecules analysis 155 6.2.8 Measurement of pulmonary edema 155 6.2.9 Survival studies 155 6.2.10 Statistics 155 6.3 Results 155 6.3.1 H2S regulates COX-2 levels in septic lungs in a TRPV1 channeldependent manner 155 6.3.2 The H2S-augmented, TRPV1-dependent COX-2 response correlates with concurrent PGE2 metabolite production following septic injury 156 6.3.3 COX-2 inhibition prevents H2S from aggravating ALI in sepsis 157 6.3.4 Blockade of H2S-mediated activation of COX-2 impaired proinflammatory cytokines, chemokines and adhesion molecules production in sepsis-induced ALI 158 6.3.5 Inhibition of COX-2 attenuates H2S-augmented PGE2 metabolite production in septic lungs 159 6.3.6 Inhibition of COX-2 protects against H2S-augmented, CLP-induced lethality, but has no effect on PAG-mediated protection of mortality in sepsis 159 6.4 Discussion 160 vii CHAPTER VII GENERAL DISCUSSION AND CONCLUSION 180 7.1 Significance of findings 180 7.2 Limitations of the study 184 7.3 Conclusions and future perspectives 185 REFERENCES 189 viii SUMMARY Hydrogen sulfide (H2S), a malodorous gas with the characteristic odor of rotten eggs, has been recognized as an important endogenous gaseous signaling molecule of the cardiovascular, gastrointestinal, genitourinary, and nervous systems. 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Cystathionine gamma-Lyase-deficient mice require dietary cysteine to protect against acute lethal myopathy and oxidative injury. J Biol Chem 2010; 285(34): 26358-68. 221 [...]... increasingly being established as a novel mediator of inflammation It has been demonstrated to play a pro-inflammatory role in animal models of local and systemic inflammation, including carrageenan-induced hindpaw edema [7], burn-induced acute lung injury (ALI) [8], caerulein-induced acute pancreatitis [9], lipopolysaccharide (LPS)-evoked endotoxemia [10], and cecal ligation and puncture (CLP)-induced... sequelae remain an important global healthcare problem for both scientists and clinicians and are a leading cause of morbidity and mortality in medical and surgical intensive care units (ICUs) Therefore, intense research on elucidating the cascades of mechanisms associated with pathogenesis of sepsis and potential preventive and therapeutic strategies are of great interest Nitric oxide (NO), carbon... Effect of PAG and capsazepine on protein expression and activity of COX-2 in septic lungs 168 Figure 6.3 Effect of NaHS or PAG and capsazepine on PGE2 metabolite production in septic lungs 170 Figure 6.4 Effect of NaHS and parecoxib on lung MPO activity, histopathological evaluation (hematoxylin and eosin staining) of lung polymorphonuclear leukocyte infiltration and injury, and pulmonary edema in sepsis. .. prophylactic PAG, and septic mice received therapeutic PAG 178 Figure 7.1 Flowchart summarizing the pro-neuroinflammatory role of H2S in polymicrobial sepsis 188 xv LIST OF ABBREVIATIONS 3-MST 3-mercaptopyruvate sulfurtransferase AC adenylyl cyclase AECOPD Acute exarcebation of chronic obsructive pulmonary disease ALI Acute lung injury ALT Alanine aminotransferase ANOVA Analysis of variance ARDS Acute... of PAG and capsazepine on lung and liver MPO activity in septic mice 95 Figure 3.10 Effect of PAG and capsazepine on lung and liver injury in septic mice 96 Figure 3.11 Effect of PAG and capsazepine on CLP-induced mortality in septic mice 97 xiii Figure 4.1 Effect of NaHS and capsazepine on lung and plasma SP levels, and lung PPT -A mRNA expression in septic mice 112 Figure 4.2 Effect of NaHS and capsazepine... SWS, Ang SF, Lu J, Moochhala SM, Bhatia M Substance P Upregulates Cyclooxygenase-2 and Prostaglandin E Metabolite by Activating ERK1/2 and NF-κB in a Mouse Model of Burn-Induced Remote Acute Lung Injury Journal of Immunology 2010; 185(10):6265-6276 ABSTRACT Ang SF, Moochhala SM, MacAry PA, Bhatia M Hydrogen Sulfide Regulates Transient Receptor Potential Vanilloid 1-Mediated Neurogenic Inflammation in Sepsis- Associated... hypothesized that H2S may modulate neuroinflammation in sepsis However, there has been little progress in understanding the potential interaction and involvement of both H2S and TRPV1 in the setting of sepsis Therefore, in the present study, we have investigated the potential role of H2S in instigating TRPV1-mediated neurogenic inflammation in a mouse model of polymicrobial sepsis Additionally, we have identified... MacAry PA, Bhatia M 2010 A Key Role of Substance P in Hydrogen Sulfide- Induced Neurogenic Inflammation in Sepsis- Associated Lung Injury 10th Annual Meeting of the Federation of Clinical Immunology Societies (FOCIS 2010), June 24-27, Boston, Massachusetts, USA Ang SF, Moochhala SM, MacAry PA, Bhatia M 2011 Hydrogen Sulfide Regulates Transient Receptor Potential Vanilloid 1-Mediated Neurogenic Inflammation. .. expression of adhesion molecules in the lung and liver of septic mice 120 Figure 4.6 Effect of NaHS and capsazepine on plasma ALT and AST activities, and pulmonary edema (measured as lung wet-to-dry weight ratio) in septic mice 122 Figure 4.7 Effect of PAG and capsazepine on lung and plasma SP levels, and lung PPT -A mRNA expression in septic mice 124 Figure 4.8 Effect of PAG and capsazepine on protein levels... distributions are not homogenous CBS appears to be the main H2S-synthesizing enzyme in the CNS and is highly expressed in liver, kidney, and the hippocampus and cerebellum in mammalian brain CSE is primarily responsible for H2S formation in the cardiovascular system and is predominantly found in the liver and in vascular and non-vascular smooth muscle, and at much lower levels, in small intestine and stomach of . gastrointestinal, genitourinary, and nervous systems. Besides acting as a potent vasodilator and an atypical neuromodulator, H 2 S is increasingly being established as a novel mediator of inflammation. . Effect of NaHS and capsazepine on mRNA expression of adhesion molecules in the lung and liver of septic mice 120 Figure 4.6 Effect of NaHS and capsazepine on plasma ALT and AST activities, and. Effect of PAG and capsazepine on mRNA expression of adhesion molecules in the lung and liver of septic mice 132 Figure 4.12 Effect of PAG and capsazepine on plasma ALT and AST activities, and