고려대학교

목차

ABSTRACT i
국문 초록 vi
PREFACE xiii
ACKNOWLEDGMENTS xiv
TABLE OF CONTENTS xv
LIST OF TABLES xx
LIST OF FIGURES xxi
CHAPTER 1. Review of literature 1
1.1 Liver injury 1
1.1.1 Impairment of bile flow 3
1.1.2 NLRP3 inflammasome activation and Pyroptosis 5
1.1.3 Hepatic fibrosis and epithelial-to-mesenchymal transition (EMT) 8
1.2 Per-and poly-fluoroalkyl substances (PFAS) 10
1.3 Hexafluoropropylene oxide-dimer acid (HFPO-DA) 19
1.3.1 HFPO-DA 19
1.3.2 Liver injury induced by HFPO-DA exposure 21
CHAPTER 2. Hexafluoropropylene oxide-dimer acid (GenX) exposure induces apoptosis in HepG2 cells 24
Abstract 24
2.1 Introduction 26
2.2 Materials and Methods 29
2.2.1 Materials 29
2.2.2 Cell culture 30
2.2.3 Cell morphology 30
2.2.4 Cell viability assay 31
2.2.5 Isolation and culture of primary rat hepatocytes 31
2.2.6 Intracellular reactive oxygen species (ROS) formation 32
2.2.7 Flow cytometric analysis 32
2.2.8 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay 33
2.2.9 Western blot analysis 33
2.2.10 Statistical analysis 34
2.3 Results 38
2.3.1 Cell morphology induced by GenX 38
2.3.2 Assessment of cell viability 40
2.3.3 Assessment of ROS 42
2.3.4 Assessment of apoptotic rate by flow cytometric analysis 44
2.3.5 Assessment of apoptosis-related gene expression by qRT-PCR 47
2.3.6 Western blot analysis 50
2.4 Discussion 53
2.5 Conclusion 60
CHAPTER 3. Perfluorooctanoic acid and hexafluoropropylene oxide-dimer acid: Hepatic stress and bile acid metabolism with different pathways 62
Abstract 62
3.1 Introduction 64
3.2 Materials and Methods 69
3.2.1 Materials 69
3.2.2 Animals and Treatments 69
3.2.3 Biochemical analysis 70
3.2.4 Histological analysis 70
3.2.5 RNA sequencing 71
3.2.6 RNA-seq Data Analysis 72
3.2.7 Real-time quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) assay 73
3.2.8 Western blot analysis 74
3.2.9 Statistical analysis 75
3.3 Results 76
3.3.1 Exposure to HFPO-DA induces hepatotoxicity in mice, liver weight, liver index, and biochemical parameters in serum 76
3.3.2 Liver histopathological analysis after HFPO-DA Exposure 78
3.3.3 RNA Sequencing and hepatic gene expression after HFPO-DA Exposure 82
3.3.4 Exposure to PFOA induces hepatotoxicity in mice, liver weight, liver index, and biochemical parameters in serum 96
3.3.5 Liver histopathological analysis after PFOA Exposure 97
3.3.6 RNA Sequencing and hepatic gene expression after PFOA Exposure 100
3.4 Discussion 112
3.5 Conclusion 118
CHAPTER 4. Hexafluoropropylene oxide-dimer acid induces hepatotoxicity and NLRP3 inflammasome activation-mediated pyroptosis in vitro and in vivo 120
Abstract 120
4.1 Introduction 122
4.2 Materials and Methods 126
4.2.1 Materials 126
4.2.2 Cell culture 126
4.2.3 Cell viability assay 127
4.2.4 Intracellular reactive oxygen species (ROS) formation 128
4.2.5 Flow cytometric analysis 128
4.2.6 Animals and Treatments 129
4.2.7 Biochemical analysis 129
4.2.8 Histological analysis 130
4.2.9 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay 131
4.2.10 Protein Extraction and western blot analysis 134
4.2.11 Statistical analysis 136
4.3 Results 137
4.3.1 HFPO-DA induces cytotoxicity in liver cells 137
4.3.2 HFPO-DA triggers NLRP3 inflammasome activation and pyroptosis in liver cells 141
4.3.3 HFPO-DA induces hepatotoxicity in mice 144
4.3.4 HFPO-DA induces activation of NLRP3 in inflammasome and pyroptosis in mice 149
4.4 Discussion 152
4.5 Conclusion 155
CHAPTER 5. Hexafluoropropylene oxide-dimer acid (HFPO-DA) induces epithelial-to-mesenchymal transition and hepatic fibrosis through the Wnt/β-catenin pathway in vitro and in vivo 157
Abstract 157
5.1 Introduction 159
5.2 Materials and Methods 163
5.2.1 Materials 163
5.2.2 Cell culture 163
5.2.3 Cell viability assay 164
5.2.4 Immunofluorescence staining of LX-2 cells 164
5.2.5 Animals and Administration of HFPO-DA 165
5.2.6 Masson’s trichrome staining and immunofluorescence staining of mouse liver tissue 166
5.2.7 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay 167
5.2.8 Protein Extraction and western blot analysis 170
5.2.9 Small interfering RNA transfection 172
5.2.10 Statistical analysis 172
5.3 Results 173
5.3.1 HFPO-DA induces EMT and liver fibrosis in LX-2 cells 173
5.3.2 HFPO-DA induces liver fibrosis through the Wnt/β-catenin signaling pathway in LX-2 cells 185
5.3.3 HFPO-DA induces EMT and liver fibrosis in vivo 187
5.3.4 HFPO-DA induces liver fibrosis through the Wnt/β-catenin signaling pathway in vivo 192
5.4 Discussion 193
5.5 Conclusion 200
CHAPTER 6. Conclusion and future directions 202
REFERENCES 204