林峯輝Lin, Feng-Huei臺灣大學:醫學工程學研究所羅朝棟Loo, Siow-TungSiow-TungLoo2010-05-182018-06-292010-05-182018-06-292009U0001-2507200901053000http://ntur.lib.ntu.edu.tw//handle/246246/183726關節內注射玻尿酸是目前臨床上針對退化性關節炎常用的一種治療。降血脂藥物施德丁近年來被發現具有多效作用，例如：抗發炎反應與減低基質降解酶之表現。本論文的目的為探討玻尿酸與施德丁於體外退化性關節炎模式之治療作用。我們將 0.1mg/ml 的玻尿酸(臨床等級:雅節)混合不同濃度(5μM, 10μM, and 50μM)的新伐他丁(脂溶性)或普伐他丁(水溶性)，然後作用於受脂多醣體(內毒素)誘導發炎及細胞凋亡反應的猪軟骨細胞。我們所進行的檢測分析包含有：施德丁之細胞毒性、細胞增殖、NF-κB 轉錄因子活性、一氧化氮釋放量、DNA 片斷化以及IL-1β、TNF-α、IL-6、iNOS、Caspase 3、MMP-1、MMP-13、COL2A1 的基因表現量。我們發現雖然玻尿酸混合50μM 之新伐他丁(H50S)可以有效降低IL-1β、 IL-6、iNOS、caspase 3、MMP-1、MMP-13 的基因表現。但它卻具有細胞毒性以及有抑制細胞增殖的作用。綜合所有結果，我們認為玻尿酸混合50μM 之普伐他丁(H50P)為最具有治療效果的組合。經結果分析得知它能有效的抑制NF-κB 轉錄因子的活性、降低一氧化氮的產生量、降低IL-1β、TNF-α、IL-6、iNOS、caspase 3、MMP-1、MMP-13 的基因表現量。因此，我們認為玻尿酸混合施德丁作為關節內注射劑有可能可以應用於減輕退化性關節炎之症狀。Intra-articular therapy with hyaluronic acid (HA) is commonly used in osteoarthritis treatment. Lipid-lowering drug statins have been shown to have pleiotropic effects such as anti-inflammatory and reduces matrix-degrading enzymes expression. The objective of this study is to examine the therapeutic effects of statin and hyaluronic acid within an in vitro osteoarthritis model. 0.1mg/ml of HA (ARTZ Dispo®) in combination with different doses (5μM, 10μM, and 50μM) of simvastatin (lipophilic) or pravastatinhydrophilic) were treated to LPS-induced inflammation and apoptosis in porcine articular chondrocyes. We evaluated cytotoxicity, cell proliferation, NF-κB activity, NOroduction, DNA fragmentation, and gene expression of IL-1β, TNF-α, IL-6, iNOS, caspase 3, MMP-1, MMP-13, and COL2A1. Although results showed that the expressionf IL-1β, IL-6, iNOS, caspase 3, MMP-1, and MMP-13 were found significantly decreased when treated with HA in combination with 50μM simvastatin (H50S). However, simvastatin shown to have cytotoxicity effect and inhibits cell proliferation. Base on our findings, HA in combination with 50μM of pravastatin (H50P) appear to have the greatest therapeutics effects. It had shown to have suppress NF-κB activity,reduce NO production, and attenuate IL-1β, TNF-α, IL-6, iNOS, caspase 3, MMP-1, andMP-13mRNA expression. Therefore, we suggest that intra articular injection of HA in combination with statin may be feasible for relieve symptoms of knee osteoarthritis.TABLE OF CONTENTS試委員會審定書.........................................................I謝.................................................................................................................................... II文摘要............................................................................................................................IIIbstract ............................................................................................................................. IVable of Contents .............................................................................................................. VIist of Figures ................................................................................................................... IXist of Tables ..................................................................................................................... XIhapter 1: Introduction ........................................................................................................1.1 Foreword ....................................................................................................................1.2 Pathogenesis of Osteoarthritis ....................................................................................3.3 Current Treatments ....................................................................................................7.4 Purpose of Study ......................................................................................................10hapter 2: Theorectical Basis .......................................................................................... 11.1 Articular Cartilage .................................................................................................... 11.1.1 Structure ............................................................................................................ 11.1.2 Compositions ....................................................................................................13.2 Anabolic and Catabolic Signal of Cartilage Homeostasis .......................................18.2.1 Anabolic Signals ...............................................................................................18.2.2 Catabolic Signals ..............................................................................................21.2.2.1 Proinflammatory cytokines ............................................................................21.2.2.2 Matrix degradation enzymes ..........................................................................22.3 Signal Transduction of Osteoarthritis – NF-κB pathway ........................................25.4 Statin ........................................................................................................................27hapter 3: Materials and Methods .....................................................................................31.1 Experimental Setup ..................................................................................................31.2 List of Materials and Devices ..................................................................................32.3 Primary Culture of Porcine Articular Cartilage .......................................................34.4 Assessment of Cytotoxicity of SImvastatin and Pravastatin – Lactateehydrogenase (LDH) assay ....................................................................................34.5 Determination of Cell Proliferation .........................................................................35.6 LPS-induced Cytotoxicty and Treatment of HA in Combination with Simvastatin orravastatin ................................................................................................................37.7 Real-time Quantitative Polymerase Chain Reaction (PCR) ....................................38.8 Nitric Oxide Release Assay .....................................................................................39.9 Nuclear Protein Extraction .......................................................................................39.10 Quantification of Nuclear Protein ..........................................................................40.11 NF-κB p50 transcription factor assay ....................................................................40.12 TdT-mediated X-dUTP nick end labeling (TUNEL) assay ...................................41.13 Statistical Analysis .................................................................................................42hapter 4: Results.1 Cytotoxicity of Simvastatin and Pravastatin ............................................................43.2 Evaluation of Cell Proliferation ...............................................................................44.3 Assessment of Nitric Oxide (NO) Production .........................................................45.4 NF-κB activation ......................................................................................................46.5 Real-time Quantitative PCR ....................................................................................47.5.1 Effect of statin and HA on mRNA expression of proinflammatory cytokinesL-1β, TNF-α and IL-6 ...........................................................................................47.5.2 Effect of statin and HA on mRNA expression of iNOS and Caspase ...............50.5.3 Effect of statin and HA on mRNA expression of MMPs and COL2A1 ...........51.6 TUNEL assay ...........................................................................................................54hapter 5: Discussion ........................................................................................................57hapter 6: Conclusions ......................................................................................................60eferences ..........................................................................................................................61IST OF FIGUREShapter 1ig. 1.1 Potential mechanisms involved in the pathogenesis of osteoarthritis ....................5ig. 1.2 Molecular pathogenesis of OA ...............................................................................6ig. 1.3 Scheme of events involved in the initiation of osteoarthritis (OA) androgression to irreversible cartilage damage .........................................................6ig. 1.4 Structure of Simvastatin and Pravastatin ..............................................................10hapter 2ig. 2.1 Main zones of articular cartilage organization .....................................................12ig. 2.2 Structure of collagen .............................................................................................16ig. 2.3 Molecular conformation of a typical proteoglycan aggregate showing size of theolecule .............................................................................................................16ig. 2.4 Chemical Structure of Chondroitin-4-Sulfate (C4S), Chondroitin-6-Sulfate (C6S),ermatan Sulfate (DS) and Keratin Sulfate (KS) ..............................................17ig. 2.5 Chemical Structure of Hyaluronic Acid .............................................................17ig. 2.6 Anabolic and catabolic signaling factors affecting cartilage homeostasis ..........24ig. 2.7 Signalling pathways in chondrocytes ...................................................................25ig. 2.8 Schematic Diagram of NF-κB Activation ..........................................................29ig. 2.9 Cholesterol synthesis pathway..............................................................................29hapter 3ig. 3.1 Schematic illustration of the experimental setup ..................................................31ig. 3.2 Chemical reactions of LDH ..................................................................................35ig. 3.3 Chemical reaction of WST-1 ................................................................................36ig. 3.4. Schematic illustration of in situ end labeling ......................................................42ig. 4.1 Cytotoxicity of different concentrations of simvastatin (S) and pravastatin (P) onhondrocytes. .......................................................................................................43ig. 4.2 Cell proliferation of chondrocytes with treatment of simvastatin (S) orravastatin (P). .....................................................................................................44ig. 4.3 Effect of statin (simvastatin or pravastatin) and in combination with or withoutA (0.1mg/ml) on chondrocyte NO release ........................................................45ig. 4.4 Effect of statin (simvastatin or pravastatin) and in combination with or withoutA (0.1mg/ml) on chondrocyte NF-κB p50 transcription factor DNA bindingctivity. .................................................................................................................46ig. 4.5 Effect of statin (simvastatin or pravastatin) and in combination with or withoutA (0.1mg/ml) on chondrocyte (A) IL-1β, (B) TNF-α and (C) IL-6 mRNAxpression ............................................................................................................47ig. 4.6 Effect of statin (simvastatin or pravastatin) and in combination with or withoutA (0.1mg/ml) on chondrocyte (A) iNOS, (B) caspase 3 mRNA expression ....50ig. 4.7 Effect of statin (simvastatin or pravastatin) and in combination with or withoutA (0.1mg/ml) on chondrocyte (A) MMP-1, (B) MMP-13, and (C) COL2A1RNA expression ..............................................................................................51ig. 4.8 Effect of Statins and HA treatment on chondrocyte apoptosis .............................54IST OF TABLESable 1.1 Pharmacologic therapy for patients with osteoarthritis ........................................9able 2.1 Part list of NF-κB induced genes .......................................................................30able 2.2 Role of prenylated proteins in cellular functioning ............................................30able 3.1 Groups’ abbreviation and description ................................................................37able 3.2 List of TaqMan gene expression assays .....................................................38application/pdf2194761 bytesapplication/pdfen-US玻尿酸施德丁退化性關節炎軟骨細胞脂多醣體發炎反應Hyaluronic acid (HA)StatinOsteoarthritisChondrocytesLipopolysaccharideInflammationthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/183726/1/ntu-98-R96548051-1.pdf