Discovery and Validation of Therapeutic Targets for the Treatment of Osteoarthritis
- 주제(키워드) Osteoarthritis , Cartilage , Safflower seed , Schisandra , BRD4 inhibitor
- 주제(DDC) 570
- 발행기관 아주대학교
- 지도교수 모정순
- 발행년도 2023
- 학위수여년월 2023. 8
- 학위명 박사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032921
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Osteoarthritis (OA) is a disease characterized by chronic inflammation and pain due to deterioration of the joint. Current OA treatment strategies mainly involve the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for pain relief or surgery. However, NSAIDs cause certain side effects, such as gastrointestinal disorders, renal dysfunction, and short duration, while surgery is disadvantageous due to high treatment costs and uncomfortable for life activities. Therefore, this study aimed to find a natural functional ingredient that could play a role in cartilage protection with fewer side effects and is relatively easily available. I screened several natural products that are expected to affect OA by inhibiting OA-related pathways, such as MAPK or NF-κB signaling. As a result, two natural compounds, Schisandra extract and safflower seed extract, were selected. Therefore, this study investigated the anti-OA ability of safflower seed and Schisandra extracts and evaluate the effects of their constituents. To simulate in vitro OA conditions, chondrocytes obtained from mouse joints were treated with IL-1β, resulting in elevated expression of catabolic factors, such as matrix metalloproteinases (MMPs), collagenases, and Cox-2. Schisandra extract and Schisandrol A prevented the elevated expression of catabolic factors, including MMPs and Cox-2. Safflower seed extract and its main components, N-(p-coumaroyl) serotonin and N-feruloyl serotonin, reduced MMPs. For animal studies, safflower seed and Schisandra extracts were orally administered to a mouse model representing the destabilization of the medial meniscus (DMM). Safranin O and immunohistochemical staining analysis showed that cartilage decay was blocked in mice administrated with Schisandra and safflower seed extracts. In addition, Schisandra and Safflower seed extracts suppressed the expression of catabolic factors in mouse cartilage by suppressing NF-κB signaling. Epigenetic changes are known to be involved in OA and, Bromodomain and extra-terminal domain (BET) protein family can regulate the expression of molecules involved in inflammatory diseases. BRD4, a major member of the BET family, regulates transcription through epigenetic mechanisms. Although BRD4 inhibitors, such as JQ1, have been identified as potential therapeutic agents for OA, they have side effects. Thus, for the second part of this study, I investigated the OA inhibitory effect of compound ABC, a BRD4 inhibitor. Chondrocytes were treated with IL-1β and then with ABC, and the expression of catabolic factors appeared to be reduced. Moreover, intra-articular injection of ABC into DMM mice resulted in suppressed cartilage destruction, as indicated by Safranin O and immunohistochemical staining. ABC effectively suppressed MAPK and NF-κB signaling and lowered the expression of catabolic factors. In conclusion, these results suggest that natural functional ingredients, Schisandra and Safflower seed extracts, may be helpful for the treatment of OA and that ABC may play a key role in suppressing OA development.
more목차
I. Introduction 1
1. Osteoarthritis 1
2. Cartilage and chondrocytes in OA 2
3. Treatment of OA 3
4. Natural product extracts and single compounds in OA 5
5. BRD4 inhibitors and OA 7
6. Aim of this study 8
II. Material and Methods 11
1. Primary articular mouse chondrocyte culture 11
2. Reagents and Treatments 11
3. Cell viability analysis 12
4. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription PCR (qRT-PCR) 13
5. Western blot analysis 14
6. Prostaglandin E2 (PGE2), collagenase assay and luciferase reporter gene assay 15
7. Animal study in OA mice 16
8. Cartilage tissue histology and immunohistochemistry 17
9. Culture of cartilage explants and Alcian blue staining 17
10. High‐performance liquid chromatography (HPLC) analysis 18
11. Protein structural homology modeling 18
12. RNA-sequencing (RNA-seq) analysis and Ingenuity Pathway Analysis (IPA) 19
13. Statistical analysis 20
III. Results 21
PART I. Inhibitory effect of natural product extracts and single compounds on OA 21
1. Schisandra extract and Schisandrol A suppress OA progression 21
1.1. Schisandra extract regulates catabolic factors induced by IL-1β in mouse articular chondrocytes 21
1.2. Oral gavage of Schisandra extract prevents cartilage destruction in the DMM mouse OA model 26
1.3. Schisandrol A controls catabolic factors in chondrocytes stimulated with IL-1β 30
1.4. Schisandrol A modulates catabolic factors and attenuates cartilage degradation in an experimental OA mouse model 34
1.5. Schisandra extract regulates NF-κB and MAPK signaling pathways 37
2. Safflower seed extract represses OA pathogenesis 43
2.1. Safflower seed extract reduces Mmp3 and Mmp13 levels, and collagenase activity in chondrocytes stimulated by IL-1β 43
2.2. Safflower seed extract attenuates cartilage degradation in a DMM mouse model 47
2.3. N-feruloyl serotonin and N-(p-coumaroyl) serotonin reduce catabolic factors in IL-1β-stimulated chondrocytes 51
2.4. Safflower seed extract, N-feruloyl serotonin and N-(p-coumaroyl) serotonin suppress IL-1β-induced NF-κB signaling 55
PART Ⅱ. Roles of a BRD4 inhibitor, ABC in OA 59
ABC modulates OA progression by controlling catabolic factors 59
1. ABC reduces the expression of IL-1β-induced catabolic factors in mouse articular chondrocytes 59
2. ABC modulates the decay of cartilage in the DMM mouse model 63
3. ABC modulates the IL-1β-induced NF-κB and MAPK signaling pathways in articular chondrocytes 67
IV. Discussion 69
V. Conclusions 82
VI. References 83
국문 초록 104
