New frontiers in osteoarthritis pathogenesis : emerging environmental pollutants and their pathogenic effects

목차 (Table of Contents)

• Abstract vii
• Abbreviations x
• List of tables xi
• List of figures xii
• Conclusion 220
• References 222
• Acknowledgment 239
• Abstract (in Korean). 240
• I. PART ONE. 1
• 1.1 Environmental factors and human health 2
• 1.2 The Impact of environmental pollution on human diseases 3
• 1.3 Heavy metals as environmental pollutants 6
• 1.3.1 Definition and characteristics of heavy metals 6
• 1.3.2 Sources and routes of exposure 7
• 1.3.3 Persistence and bioaccumulation 9
• 1.3.4 Mechanisms of heavy metal toxicity 10
• 1.3.5 Heavy metals and musculoskeletal health 11
• 1.4 Introduction to osteoarthritis 12
• 1.4.1 Anatomy and physiology of joints 12
• 1.4.2 Pathophysiology of osteoarthritis 14
• 1.4.3 Animal models for studying osteoarthritis 15
• 1.4.4 Rationale for investigating heavy metals in osteoarthritis 17
• 1.4.5 Aim of this study 18
• 2. PART TWO 21
• 2.1 Materials and methods 22
• 2.1.1 Reagents and chemicals 22
• 2.1.2 Cell culture and treatment 22
• 2.1.3 Measurement of cell viability 22
• 2.1.4 Animal experiments 23
• 2.1.5 Inhibitor and siRNA experiments 23
• 2.1.6 Reverse transcription-polymerase chain reaction (RT-PCR) 24
• 2.1.7 Immunohistochemistry. 24
• 2.1.8 Sample preparation for ICP-MS-MS total metal analysis 25
• 2.1.9 Quantification of elemental content by ICP-MS 25
• 2.1.10 Statistical analysis 26
• 2.2 Results 29
• 2.2.1 Cr (VI) exposed articular chondrocytes alter catabolic and anabolic gene expression 29
• 2.2.2 Cr (VI) induces cartilage degeneration and OA pathogenesis 32
• 2.2.3 Orally delivered Cr (VI) induces OA pathogenesis 35
• 2.2.4 Cr (VI) increases MMP3 and MMP13 in articular cartilage 39
• 2.2.5 HIF-2α and ZIP8 were highly expressed in the articular cartilage of Cr (VI)-
• exposed mice 42
• 2.2.6 Blockage of Cr (VI) importers inhibit Cr (VI)-induced alteration of anabolic and catabolic factors 45
• 2.2.7 HIF-2α/ZIP8 mediated NF-κB signaling pathways are the main cause of cartilage destruction by Cr (VI) 47
• 2.2.8 Genetic knockdown of HIF-2α and ZIP8 abolished the Cr (VI)-induced alteration of anabolic and catabolic factors 49
• 2.3 Discussion 51
• 3. PART THREE 55
• 3.1 Materials and Methods 56
• 3.1.1 Cell culture conditions 56
• 3.1.2 Cell viability analysis 56
• 3.1.3 Detection of ROS 56
• 3.1.4 Detection of the mitochondrial membrane potential 57
• 3.1.5 Measurement of OCR 57
• 3.1.6 Animal studies 58
• 3.1.7 Chemical inhibitor and siRNA experiments 58
• 3.1.8 qRT-PCR and RT-PCR 59
• 3.1.9 Histological analysis and immunohistochemistry 59
• 3.1.10 Quantification of As via ICP-MS 59
• 3.1.11 Western blot analyses 60
• 3.1.12 Statistical analysis 60
• 3.2 Results 61
• 3.2.1 The relationship between arsenic and OA illness 61
• 3.2.2 As induces redox homeostasis disruption in chondrocytes Cr (VI) 63
• 3.2.3 Cartilage is a target tissue of As 68
• 3.2.4 Chondrocyte metabolic dysregulation 71
• 3.2.5 Expression of Hif-2α and Zip8 in As-exposed articular cartilage 77
• 3.2.6 Hif-2α and Zip8 expression upregulation in the destabilization of medial meniscus (DMM) mouse model 82
• 3.2.7 Akt/NF-kB induces the Hif-2α/Zip8 axis 85
• 3.3 Discussion 91
• 4. PART FOUR 98
• 4.1 Materials and Methods 99
• 4.1.1 Cell culture condition 99
• 4.1.2 Cell viability analysis 99
• 4.1.3 Chemical inhibitor experiment 100
• 4.1.4 RNA Isolation, cDNA synthesis, and quantitative RT-PCR 100
• 4.1.5 Animal studies 101
• 4.1.6 Histological analysis and immunohistochemistry 102
• 4.1.7 Immunofluorescence staining 103
• 4.1.8 Cd accumulation in organ tissues 104
• 4.1.9 Western blot analysis 104
• 4.1.10 Biochemical analyses 105
• 4.1.11 Microbial profiling of proximal small intestine content using 16S rRNA
• sequencing and bioinformatics analysis 106
• 4.1.12 LEfSe analysis 108
• 4.1.13 Functional profiling using PICRUSt2 108
• 4.1.14 KEGG Othology (KO) abundance for PPAR signaling 109
• 4.1.15 Correlation analysis between microbiome and metabolite data 109
• 4.1.16 Sample preparation for metabolomics analysis 110
• 4.1.17 Metabolite analysis using ultra performance liquid chromatography
• quadrupole time of flight mass spectrometry (UPLC-Q-TOF MS) 111
• 4.1.18 Microarray data acquisition and analysis 111
• 4.1.19 Statistical analysis 112
• 4.2 Results 113
• 4.2.1 Cadmium disrupts chondrocyte equilibrium: enhancing catabolic pathways while suppressing anabolic genes 113
• 4.2.2 Murine joint degeneration induced by localized cadmium exposure via intra articular injection 116
• 4.2.3. Cadmium detection and localization in primary culture articular chondrocytes and cartilage tissues 120
• 4.2.4 Articular cartilage as a key site for cadmium accumulation: insights from ICP-MS analysis 123
• 4.2.5 Cadmium induced structural and functional disruptions in the small intestine and inflammatory response 125
• 4.2.6 Cadmium exposure drives gut microbiota dysbiosis: implications for diversity and host microbe interactions 131
• 4.2.6.1 Firmicutes/Bacteroidetes ratio 133
• 4.2.6.2 Lactobacillus/Clostridium ratio 133
• 4.2.7 Cadmium induced perturbation of gut microbiota drives dysbiosis and disrupts metabolic homeostasis: insights from LEfSe profiling 141
• 4.2.8 Impact of gut microbial genera on the PPAR signaling pathway: functional disruption by cadmium exposure 144
• 4.2.9 Cadmium induced disruption of PPAR signaling impairs hepatic lipid metabolism and promotes inflammation 155
• 4.2.10 Metabolomic profiling reveals cadmium-induced disruptions in serum, fecal, and cartilage metabolites associated with OA pathology 163
• 4.2.11 Impact of oral cadmium on liver enzymes and serum lipid profile: implications for metabolic health 177
• 4.2.12 Cadmium exposure via drinking water exacerbates osteoarthritic changes in murine knee joints 179
• 4.2.13 Blocking cadmium importer, HIF-2α, or NF-κb pathways reverses cadmium induced alterations in anabolic and catabolic factor expression in primary
• chondrocytes 183
• 4.2.14 HIF-2α overexpression modulates gene expression in chondrocytes: insights from microarray analysis 188
• 4.3 Discussion 192
• New Frontiers in Osteoarthritis Pathogenesis: Emerging Environmental
• Pollutants and Their Pathogenic Effects
• Godagama Gamaarachchige Dinesh Suminda
• Interdisciplinary Graduate Program in Advanced Convergence Technology and
• Science, The Graduate School, Jeju National University, Korea
• Supervised by Professor Young-ok Son