Apoptosis of the pancreatic acinar cells and acute pancreatitits

APOPTOSIS OF THE PANCREATIC ACINAR CELLS AND ACUTE PANCREATITIS CAO YANG NATIONAL UNIVERSITY OF SINGAPORE 2007 APOPTOSIS OF THE PANCREATIC ACINAR CELLS AND ACUTE PANCREATITIS CAO YANG (M.B.B.S., Wuhan University, PRC) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2007 ACKNOWLEDGEMENTS I would like to express my great gratitude to my supervisor, Associate Professor Bhatia Madhav for his invaluable guidance, advice and patience in guiding me into the field of medical research I would like to thank National University of Singapore for its favorable scientific environment and generous research scholarship I would also like to thank the laboratory officers Mei Leng, Wee Lee, Xiao Guang, and Annie for their unfailing assistance Moreover, my appreciation is extended to all my friends in National University of Singapore Last but not least, I would like to express my great appreciation to my family for their support, patience, understanding and love i TABLE OF CONTENTS PAGE ACKNOWLEDGEMENTS I TABLE OF CONTENTS II LIST OF ORIGINAL REPORTS V LIST OF ABSTRACTS PUBLISHED IN JOURNALS VI LIST OF INTERNATIONAL CONFERENCE PRESENTATIONS .VIII LIST OF FIGURES IX LIST OF TABLES XII LIST OF ABBREVIATIONS .XIII LIST OF ABBREVIATIONS .XIII SUMMARY CHAPTER 1: GENERAL INTRODUCTION 1.1 GENERAL OVERVIEW 1.2 PATHOPHYSIOLOGY OF CLINICAL ACUTE PANCREATITIS 1.3 PATHOPHYSIOLOGY OF EXPERIMENTAL ACUTE PANCREATITIS 1.3.1 INDUCTION OF ACUTE PANCREATITIS IN MICE BY CAERULEIN INJECTIONS 1.3.2 PRINCIPLE OF CAERULEIN-INDUCED EXPERIMENTAL ACUTE PANCREATITIS IN MICE 1.3.3 CHARACTERISTICS OF CAERULEIN-INDUCED PANCREATITIS 10 1.3.3.1 INCREASE IN LEVELS OF PANCREATIC ENZYMES IN THE SERUM 10 1.3.3.2 PANCREATIC EDEMA 10 1.3.3.3 NEUTROPHIL INFILTRATION 11 1.3.3.4 PANCREATIC ACINAR CELLS INJURY 12 1.4 ACINAR CELL DEATH IN ACUTE PANCREATITIS 15 1.4.1 TWO ALTERNATIVE FORMS OF CELL DEATH 16 1.4.2 MECHANISMS OF APOPTOSIS 21 1.4.2.1 CASPASE ACTIVATION 21 1.4.2.2 TWO CASPASE-DEPENDENT SIGNALING PATHWAYS OF APOPTOSIS 24 1.4.2.2.1 DEATH RECEPTOR-MEDIATED APOPTOTIC PATHWAY 25 1.4.2.2.2 MITOCHONDRION-MEDIATED APOPTOTIC PATHWAY 26 1.4.2.3 PHAGOCYTOSIS OF APOPTOTIC CELLS 29 1.4.2.3.1 SURFACE SIGNALS OF APOPTOTIC CELLS 30 1.4.2.3.2 PHAGOCYTIC RECEPTORS 31 1.4.2.3.3 THE ANTI-INFLAMMATORY EFFECT OF APOPTOTIC-CELLS CLEARANCE 33 1.4.3 MECHANISMS OF APOPTOSIS IN ACUTE PANCREATITIS 37 1.4.3.1 MECHANISMS OF APOPTOSIS ARISING FROM ACUTE PANCREATITIS 37 1.4.3.1.1 CASPASE ACTIVATION 37 1.4.3.1.2 APOPTOSIS ASSOCIATED GENES 39 1.4.3.1.3 NEUTROPHILS 39 1.4.3.1.4 CYTOKINES 40 ii 1.4.3.2 INDUCTION OF APOPTOSIS IN PANCREATIC ACINAR CELLS BY COMPOUNDS THAT DO NOT CAUSE ACUTE PANCREATITIS 41 1.4.3.2.1 INDUCTION OF APOPTOSIS IN PANCREATIC ACINAR CELL BY MENADIONE 41 1.4.3.2.2 INDUCTION OF APOPTOSIS IN PANCREATIC ACINAR CELL BY CRAMBENE 42 1.5 RESEARCH QUESTIONS AND AIMS 44 CHAPTER 2: MECHANISM OF CRAMBENE INDUCED APOPTOSIS IN ISOLATED PANCREATIC ACINAR CELLS 47 2.1 MATERIALS AND METHODS 47 2.1.1 ANIMALS 47 2.1.2 CRAMBENE 47 2.1.3 PREPARATION OF PANCREATIC ACINI 48 2.1.4 INDUCTION OF APOPTOSIS IN PANCREATIC ACINAR CELLS 48 2.1.5 ANNEXIN V-FITC/PROPIDIUM IODIDE STAINING DETECTION 49 2.1.6 CASPASE ASSAY 50 2.1.7 MITOCHONDRIAL MEMBRANE POTENTIAL DETECTION 52 2.1.8 MEASUREMENT OF CYTOCHROME C RELEASE FROM MITOCHONDRIA 53 2.1.9 TNF-Α AND FAS LIGAND ASSAY 55 2.1.10 STATISTICAL ANALYSIS 55 2.2 RESULTS 56 2.2.1 CRAMBENE STIMULATES AN INCREASE OF ANNEXIN-V BINDING 56 2.2.2 CRAMBENE STIMULATES ACTIVATIONS OF CASPASES 3, 8, AND 60 2.2.3 CRAMBENE STIMULATES A DECREASE OF MITOCHONDRIAL MEMBRANE POTENTIAL 66 2.3.4 CRAMBENE STIMULATES A RELEASE OF CYTOCHROME C FROM MITOCHONDRIA 68 2.4 DISCUSSION 69 CHAPTER 3: MECHANISMS BY WHICH INDUCTION OF APOPTOSIS IN PANCREATIC ACINAR CELLS BY CRAMBENE PROTECTS MICE AGAINST ACUTE PANCREATITIS 75 3.1 MATERIALS AND METHODS 75 3.1.1 ANIMALS 75 3.1.2 IN VIVO EXPERIMENTS 75 3.1.2.1 CRAMBENE ADMINISTRATION 75 3.1.2.2 INDUCTION OF ACUTE PANCREATITIS 76 3.1.2.3 AMYLASE ESTIMATION 78 3.1.2.4 PANCREATIC MYELOPEROXIDASE ESTIMATION 78 3.1.2.5 PANCREATIC WATER CONTENT ESTIMATION 79 3.1.2.6 MORPHOLOGICAL EXAMINATION 79 3.1.2.7 IL-10, TGF-Β, MCP-1, IL-1Β, TNF-Α ASSAY 81 3.1.2.8 RT-PCR 81 3.1.2.9 WESTERN BLOTTING 83 3.1.2.10 TERMINAL DEOXYNUCLEOTIDYLTRANSFERASE-MEDIATED DUTP-BIOTIN NICK-END LABELING (TUNEL) 85 3.1.2.11 IMMUNOHISTOCHEMISTRY AND DOUBLE-IMMUNOFLUORESCENCE LABELING 86 iii 3.1.3 IN VITRO EXPERIMENTS 87 3.1.3.1 MEDIUM 87 3.1.3.2 ISOLATION OF PANCREATIC ACINAR CELLS AND INDUCTION OF APOPTOSIS 87 3.1.3.3 ISOLATION OF RESIDENT PERITONEAL MACROPHAGES 88 3.1.3.4 INTERACTION BETWEEN MACROPHAGE AND APOPTOTIC ACINAR CELLS 89 3.1.3.5 DOUBLE-IMMUNOFLUORESCENCE LABELING 89 3.1.4 ANALYSIS OF DATA 90 3.2 RESULTS 91 3.2.1 IN SITU DETECTION OF APOPTOSIS BY HEMATOXYLIN AND EOSIN STAINING 91 3.2.2 EFFECT OF CRAMBENE TREATMENT ON THE SEVERITY OF CAERULEIN-INDUCED ACUTE PANCREATITIS AS A FUNCTION OF TIME 93 3.2.3 RT-PCR ANALYSIS OF PHAGOCYTIC RECEPTORS IN ACUTE PANCREATITIS 95 3.2.4 CLEARANCE OF APOPTOTIC ACINAR CELLS VIA CD36 POSITIVE MACROPHAGES 98 3.2.5 EFFECT OF CRAMBENE TREATMENT ON LEVELS OF PRO-INFLAMMATORY MEDIATORS IN CAERULEIN-INDUCED ACUTE PANCREATITIS AS A FUNCTION OF TIME 103 3.2.6 EFFECT OF CRAMBENE TREATMENT ON LEVELS OF ANTI-INFLAMMATORY MEDIATORS IN THE PANCREAS 105 3.2.7 EFFECT OF CO-CULTURES OF EARLY APOPTOTIC ACINAR CELLS WITH PERITONEAL MACROPHAGES ON IL-10 PRODUCTION 107 3.2.8 EFFECT OF PRETREATMENT WITH NEUTRALIZING ANTI-IL-10 ANTIBODY AND CRAMBENE ON THE SEVERITY OF CAERULEIN-INDUCED ACUTE PANCREATITIS 109 3.3.9 MRNA AND PROTEIN EXPRESSION OF CD36 IN CAERULEIN-INDUCED ACUTE PANCREATITIS WITH PRETREATMENT OF NEUTRALIZING ANTI-IL-10 ANTIBODY AND CRAMBENE 113 3.3 DISCUSSION 119 CHAPTER 4: SUMMARY OF CONTRIBUTIONS 124 REFERENCE 128 iv LIST OF ORIGINAL REPORTS Bhatia M, Wong FL, Cao Y, Lau HY, Huang J, Puneet P, and Chevali L Pathophysiology of acute pancreatitis Pancreatology 2005; 5: 132-44 Cao Y, Adhikari S, Ang AD, Clement MV, Wallig M, Bhatia M Crambene induces pancreatic acinar cell apoptosis via the activation of mitochondrial pathway Am J Physiol Gastrointest Liver Physiol 2006; 291(1): G95-G101 Cao Y, Adhikari S, Ang AD, Moore PK, Bhatia M Mechanism of Induction of pancreatic acinar cell apoptosis by hydrogen sulfide Am J Physiol Cell Physiol 2006; 291(3): C503-10 Cao Y, Adhikari S, Clement MV, Wallig M, Bhatia M Induction of apoptosis by crambene protects mice against acute pancreatitis via anti-inflammatory pathways Am J Pathol 2007;170(5):1521-34.2006 Bhatia M, Adhikari S, Ramasamy T, and Cao Y Cell death mechanisms in pancreatitis: in focus on cell apoptosis research Boole DD Ed, Nova Science Publishers, Inc., New York, 2006 (In press) v LIST OF ABSTRACTS PUBLISHED IN JOURNALS Cao Y, Wallig M, Ang AD, and Bhatia M Effect of apoptosis-inducing agents on caspase-3 activity in isolated pancreatic acinar cells Pancreatology 2004; 4:202 Bhatia M, Wallig M, Ang AD, and Cao Y Stimulation of caspase-3 activity in isolated pancreatic acinar cells by apoptosis-inducing agents Pancreatology 2004; 4:288 Bhatia M, Wallig M, Ang AD, and Cao Y Three different apoptosis-inducing agents stimulate caspase activity in isolated pancreatic acinar cells J Gastroenterol Hepatol 2004; 19: A399 Bhatia M, Cao Y, Ang AD, and Wallig M Stimulation of caspase 3, 8, and activity and decrease in mitochondrial membrane potential in isolated pancreatic acinar cells by crambene J Gastroenterol Hepatol 2005; 20: A230-231 Cao Y, Adhikari S, Clément MV, Wallig M, and Bhatia M Neutralizing IL-10 reverses the protection against acute pancreatitis by crambene Pancreas 2006; 33: 450 Cao Y, Adhikari S, Clément MV, Wallig M, and Bhatia M Treatment with a neutralizing anti-IL-10 reverses the protection against acute pancreatitis by crambene Pancreatology 2006; 6:350 vi Adhikari S, Cao Y, Ang AD, Clément MV, Wallig M, and Bhatia M Mechanism of pancreatic acinar cell apoptosis by crambene FEBS J 2006; 273: 47 Cao Y, Adhikari S, Moore PK, and Bhatia M Pro-apoptotic effect of hydrogen sulfide (H2S) in isolated pancreatic acinar cells FEBS J 2006; 273: 111 Cao Y, Adhikari S, Moore PK, and Bhatia M Mechanism of induction of pancreatic acinar cell apoptosis by hydrogen sulfide Acta Pharmacol Sin 2006; 27: 396 Cao Y, Adhikari S, Clément MV, Wallig M, and Bhatia M Neutralizing IL-10 reverses the protection against acute pancreatitis by crambene Pancreatology 2006; 6: 564 Bhatia M, Cao Y, Adhikari S, and Wallig M Induction of apoptosis by crambene protects mice against acute pancreatitis via anti-inflammatory pathways J Gastroenterol Hepatol 2006; 21: A377 vii LIST OF INTERNATIONAL CONFERENCE PRESENTATIONS Cao Y, Ang AD, Clement MV, Wallig M, Bhatia M Treatment of isolated pancreatic acini with apoptosis-inducers causes a stimulation of caspase-3 activity 36th Meeting of the European Pancreatic Club, 2004, Padova, Italy (Poster presentation) 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Blood 89(4): 1121-32 149 ... microscopy, the cell injury exhibits (i) the presence of acinar- cell ghosts; (ii) vacuolization and swelling of acinar cells or (iii) the destruction of the histoarchitecture of the whole or parts of the. .. xv SUMMARY The mechanisms of apoptosis in pancreatic acinar cells and the mechanisms by which induction of acinar cell apoptosis protects against acute pancreatitis were investigated The results... form of cell death in acute pancreatitis However, little is known about the molecular mechanisms of pancreatic acinar cell death and how the induction of apoptosis in pancreatic acinar cells