臺灣博碩士論文加值系統

骨關節炎 (Osteoarthritis，OA) 的特徵是軟骨細胞發炎和軟骨退化，主要導致
膝關節和髖關節疼痛和功能喪失，是老年人中最普遍的關節炎。 KMUP-1 是黃嘌
呤的一種衍生化學合成物，已被證明具有抗炎和抗氧化特性，在最近的一份報告中，
KMUP-1 被證明可以抑制 RAW 264.7（巨噬細胞系）中的細胞毒性、炎性細胞因子
產生和氧化壓力。 然而，KMUP-1 對軟骨細胞炎症的治療作用仍然未知。 我們的
研究旨在探討 KMUP-1 在抗炎和抗氧化應激方面對 OA 的影響。培養 SW1353 人軟
骨細胞並用 KMUP-1 (10 μM) 預處理。 通過 KMUP-1 預處理 IL-1ß 和 H2O2 誘導
的 SW1353 細胞炎症，TNF-α、IL-6、MCP-1 和一氧化氮的產生減少。 KMUP-1
預處理 1 小時後 iNOS、COX-2、MMP-1、MMP-13 和 MMP-9 的表達也受到抑制。
此外，KMUP-1 抑制了 MAPK 信號通路中 ERK1/2、JNK 和 p38 的激活。 KMUP-1
預處理還降低了壞死性凋亡（MLKL、RIP1、RIP3）和細 胞焦亡（NLRP3、
caspase-1、ASC、GSDMD）的蛋白質標誌性表達。 這是導致 OA 的 2 種細胞炎症，
在最近的許多論文中都有報導。 此外，KMUP-1 還抑制 SW1353 人軟骨細胞中
H2O2 誘導的細胞凋亡、此外，KMUP-1 還抑制 IL-1β 誘導的人軟骨細胞中 p-p65
的核轉位，並恢復由 IL-1B 調節的上游因子 SIRT 1，後者調節參與 OA 過程的下游
因子。 在活體動物研究中，我們研究了 DMM 模型和 CAIA 模型來模擬 C57BL/6 雄
性小鼠的 OA 和關節炎，以比較 KMUP-1 的保護作用氧化應激和炎症介質的產生。
結果證實 KMUP-1 對 DMM 和 CAIA 小鼠具有顯著的正向作用，從而證明 KMUP-1
2
是保護 OA 患者的有前途的活性成分。我們的結果表明 KMUP-1 在體內抑制炎症反
應和氧化應激軟骨破壞，並且對保護 DMM 和 CAIA 模型小鼠具有積極的顯著作用，
KMUP-1 可能是 OA 的潛在治療劑。

Osteoarthritis (OA), characterized by inflammation of the chondrocyte and cartilage degradation that leads to pain and loss of function primarily in the knees and hips, is the most prevalent arthritis in elderly aged individuals. KMUP-1, a derived chemical synthetic of xanthine, has been shown to have anti-inflammatory and antioxidant properties. In a recent report, KMUP-1 was demonstrated to inhibit cytotoxicity, inflammatory cytokines production, and oxidative stress in RAW 264.7 (macrophage cell line). However, the therapeutic effects of KMUP-1 on chondrocyte inflammation remain unknown. Our study aimed to explore the impact of KMUP-1 on OA both in anti-inflammation and antioxidative stress. SW1353 human chondrocytes were cultured and pretreated with KMUP-1. The productions of TNF-α, IL-6, MCP-1, and nitric oxide (NO) were reduced by KMUP-1 pretreatment in IL-1ß- and H2O2-induced inflammation of SW1353 cells. The expressions of iNOS, COX-2, MMP-1, MMP-13, and MMP-9 were also inhibited by KMUP-1 pretreatment for 1 hour. In addition, KMUP-1 suppressed the activations of ERK, JNK, and p38 in the MAPK signaling pathway. Pretreatment with KMUP-1 also reduced the protein hallmark expression of necroptosis (MLKL, RIP1, RIP3) and pyroptosis (NLRP3, caspase-1, ASC, GSDMD)- 2 kinds of cell inflammation that lead to OA have been reported in recent years. Besides, KMUP-1 also inhibited H2O2-induced cell apoptosis, oxidative stress, and inflammatory mediator production in SW1353 human chondrocytes. Furthermore, KMUP-1 also inhibits IL-1ß-induced nuclear translocation of p-p65 in human chondrocytes and restores the upstream factor SIRT 1 moderated by IL-1ß that regulated the downstream factor involved in the OA process. In vivo study, we investigate the DMM and CAIA models to imitate OA, arthritis in C57BL/6 male mice to compare the protective effect of KMUP-1. The results show that KMUP-1 has a significant positive effect on mice in the DMM and CAIA models. Thereby proving that KMUP-1 is a promising active ingredient in protecting OA patients. Our results demonstrated that KMUP-1 inhibited the inflammatory responses and oxidative stress cartilage destruction in vivo and had a positive significant effect in protecting mice from the DMM and CAIA models that KMUP-1 might be a potential therapeutic agent for OA.

Contents
中文摘要 1
Abstract 3
I. Introduction: 8
Background of the study 8
1. Osteoarthritis（OA） 10
2. Articular cartilage and OA 13
3. Pyroptosis 16
4. Necroptosis 20
5. Interleukin-1ß (IL-1ß) 22
6. Hydrogen peroxide 23
7. KMUP-1 24
8. SIRT 1 25
9. Specific aims 28
II. Material and method: 29
1. Study design cell culture 29
2. Chemicals and material 30
3. Method 33
3.1 Cell culture: 33
3.2 Cell viability assay 33
3.3 Measurement of nitrite oxide 33
3.4 ELISA quantification of TNF-α, IL-6 and MCP-1 34
3.5 Intracellular reactive oxygen species (ROS) measurement 34
3.6 Western blotting analysis: 35
3.7 Micro-computed tomography (μ CT) 36
3.8 Animal study design 37
3.9 ELISAs serum level of mice 40
3.10 Histological analysis 40
3.11 Statistical analysis 41
III. Results 42
Part 1: in vitro study 42
1. Impact of KMUP-1 on the viability of SW1353 human chondrocyte. 42
2. KMUP-1 inhibits IL-1ß-induced inflammatory cytokines and NO production. 43
3. KMUP-1 prevents IL-1ß-induced inflammatory protein expressions and cytokines response in human chondrocyte. 45
4. KMUP-1 suppresses necroptosis and the MAPK pathway in human chondrocytes. 47
5. KMUP-1 inhibits Il-1ß-induced nuclear translocation of p-p65. 49
6. KMUP-1 inhibits IL-1ß-induced pyroptosis in OA human chondrocytes. 50
7. KMUP-1 attenuates IL-1ß-induced ROS or suppressed H2O2-induced ROS leading to inflammation in human chondrocytes. 52
8. KMUP-1 prevents H2O2-induced inflammatory protein expressions in human chondrocytes. 55
9. Inhibition of SIRT 1 weakened the protective effect of KMUP-1 on chondrocytes. 57
Part 2: in vivo study 59
1. KMUP-1 significantly reduces the development of arthritis 59
2. KMUP-1 restores the DMM-induced OA and CAIA in articular cartilage 60
3. KMUP-1 protects the periarticular bones of the knees induced damage by CAIA and DMM. 64
4. Serum level of DMM and CAIA models mice 66
III. Discussion 68
IV. Conclusion 74

References
1.Woolf, A.D. and B. Pfleger, Burden of major musculoskeletal conditions. Bull World Health Organ, 2003. 81(9): p. 646-56.
2.Davis, M.A., et al., Body fat distribution and osteoarthritis. Am J Epidemiol, 1990. 132(4): p. 701-7.
3.Petersson, I.F. and L.T. Jacobsson, Osteoarthritis of the peripheral joints. Best Pract Res Clin Rheumatol, 2002. 16(5): p. 741-60.
4.Croft, P., et al., Osteoarthritis of the hip: an occupational disease in farmers. BMJ, 1992. 304(6837): p. 1269-72.
5.Kellgren, J.H. and J.S. Lawrence, Osteo-arthrosis and disk degeneration in an urban population. Ann Rheum Dis, 1958. 17(4): p. 388-97.
6.Murray, C.J. and A.D. Lopez, Global mortality, disability, and the contribution of risk factors: Global Burden of Disease Study. Lancet, 1997. 349(9063): p. 1436-42.
7.Gilbert, J.E., Current treatment options for the restoration of articular cartilage. Am J Knee Surg, 1998. 11(1): p. 42-6.
8.Hunziker, E.B., Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage, 2002. 10(6): p. 432-63.
9.Venn, M. and A. Maroudas, Chemical composition and swelling of normal and osteoarthrotic femoral head cartilage. I. Chemical composition. Ann Rheum Dis, 1977. 36(2): p. 121-9.
10.Brocklehurst, R., et al., The composition of normal and osteoarthritic articular cartilage from human knee joints. With special reference to unicompartmental replacement and osteotomy of the knee. J Bone Joint Surg Am, 1984. 66(1): p. 95-106.
11.Sandell, L.J. and T. Aigner, Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis Res, 2001. 3(2): p. 107-13.
12.Khan, I.M., R. Williams, and C.W. Archer, One flew over the progenitor's nest: migratory cells find a home in osteoarthritic cartilage. Cell Stem Cell, 2009. 4(4): p. 282-4.
13.Del Carlo, M., Jr. and R.F. Loeser, Cell death in osteoarthritis. Curr Rheumatol Rep, 2008. 10(1): p. 37-42.
14.van den Berg, W.B., Osteoarthritis year 2010 in review: pathomechanisms. Osteoarthritis Cartilage, 2011. 19(4): p. 338-41.
15.Dreier, R., Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Res Ther, 2010. 12(5): p. 216.
16.Xu, L., et al., Induction of high temperature requirement A1, a serine protease, by TGF-beta1 in articular chondrocytes of mouse models of OA. Histol Histopathol, 2014. 29(5): p. 609-18.
17.Scanzello, C.R. and S.R. Goldring, The role of synovitis in osteoarthritis pathogenesis. Bone, 2012. 51(2): p. 249-57.
18.Hayami, T., et al., Characterization of articular cartilage and subchondral bone changes in the rat anterior cruciate ligament transection and meniscectomized models of osteoarthritis. Bone, 2006. 38(2): p. 234-43.
19.Dixon, A.S., et al., Clinical trial of intra-articular injection of sodium hyaluronate in patients with osteoarthritis of the knee. Curr Med Res Opin, 1988. 11(4): p. 205-13.
20.Benito, M.J., et al., Synovial tissue inflammation in early and late osteoarthritis. Ann Rheum Dis, 2005. 64(9): p. 1263-7.
21.Martel-Pelletier, J., et al., Osteoarthritis. Nat Rev Dis Primers, 2016. 2: p. 16072.
22.Loeser, R.F., et al., Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum, 2012. 64(6): p. 1697-707.
23.Kapoor, M., et al., Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol, 2011. 7(1): p. 33-42.
24.Zhang, W., et al., Current research on pharmacologic and regenerative therapies for osteoarthritis. Bone Res, 2016. 4: p. 15040.
25.Cookson, B.T. and M.A. Brennan, Pro-inflammatory programmed cell death. Trends Microbiol, 2001. 9(3): p. 113-4.
26.Feng, S., D. Fox, and S.M. Man, Mechanisms of Gasdermin Family Members in Inflammasome Signaling and Cell Death. J Mol Biol, 2018. 430(18 Pt B): p. 3068-3080.
27.Zhao, L.R., et al., NLRP1 and NLRP3 inflammasomes mediate LPS/ATP‑induced pyroptosis in knee osteoarthritis. Mol Med Rep, 2018. 17(4): p. 5463-5469.
28.Evavold, C.L., et al., The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages. Immunity, 2018. 48(1): p. 35-44 e6.
29.von Moltke, J., et al., Recognition of bacteria by inflammasomes. Annu Rev Immunol, 2013. 31: p. 73-106.
30.Kawai, T. and S. Akira, TLR signaling. Semin Immunol, 2007. 19(1): p. 24-32.
31.Kufer, T.A. and P.J. Sansonetti, Sensing of bacteria: NOD a lonely job. Curr Opin Microbiol, 2007. 10(1): p. 62-9.
32.Han, J., C.Q. Zhong, and D.W. Zhang, Programmed necrosis: backup to and competitor with apoptosis in the immune system. Nat Immunol, 2011. 12(12): p. 1143-9.
33.Chen, A.L., et al., Expression of bone morphogenetic proteins, receptors, and tissue inhibitors in human fetal, adult, and osteoarthritic articular cartilage. J Orthop Res, 2004. 22(6): p. 1188-92.
34.Wang, H., et al., Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3. Mol Cell, 2014. 54(1): p. 133-146.
35.Stolberg-Stolberg, J., et al., Cartilage Trauma Induces Necroptotic Chondrocyte Death and Expulsion of Cellular Contents. Int J Mol Sci, 2020. 21(12).
36.Degterev, A., D. Ofengeim, and J. Yuan, Targeting RIPK1 for the treatment of human diseases. Proc Natl Acad Sci U S A, 2019. 116(20): p. 9714-9722.
37.Yang, J., et al., Targeting Cell Death: Pyroptosis, Ferroptosis, Apoptosis and Necroptosis in Osteoarthritis. Front Cell Dev Biol, 2021. 9: p. 789948.
38.Maldonado, M. and J. Nam, The role of changes in extracellular matrix of cartilage in the presence of inflammation on the pathology of osteoarthritis. Biomed Res Int, 2013. 2013: p. 284873.
39.Diaz-Gallego, L., et al., Apoptosis and nitric oxide in an experimental model of osteoarthritis in rabbit after hyaluronic acid treatment. J Orthop Res, 2005. 23(6): p. 1370-6.
40.Bates, E.J., C.C. Johnson, and D.A. Lowther, Inhibition of proteoglycan synthesis by hydrogen peroxide in cultured bovine articular cartilage. Biochim Biophys Acta, 1985. 838(2): p. 221-8.
41.Zhuang, C., et al., Polysaccharide from Angelica sinensis protects chondrocytes from H2O2-induced apoptosis through its antioxidant effects in vitro. Int J Biol Macromol, 2016. 87: p. 322-8.
42.Yeh, J.L., et al., KMUP-1 attenuates isoprenaline-induced cardiac hypertrophy in rats through NO/cGMP/PKG and ERK1/2/calcineurin A pathways. Br J Pharmacol, 2010. 159(5): p. 1151-60.
43.Wu, B.N., et al., Inhibition of proinflammatory tumor necrosis factor-alpha-induced inducible nitric-oxide synthase by xanthine-based 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine (KMUP-1) and 7-[2-[4-(4-nitrobenzene)piperazinyl]ethyl]-1, 3-dimethylxanthine (KMUP-3) in rat trachea: The involvement of soluble guanylate cyclase and protein kinase G. Mol Pharmacol, 2006. 70(3): p. 977-85.
44.Hsu, Y.Y., et al., KMUP-1 attenuates serum deprivation-induced neurotoxicity in SH-SY5Y cells: roles of PKG, PI3K/Akt and Bcl-2/Bax pathways. Toxicology, 2010. 268(1-2): p. 46-54.
45.Liou, S.F., et al., KMUP-1 suppresses RANKL-induced osteoclastogenesis and prevents ovariectomy-induced bone loss: roles of MAPKs, Akt, NF-kappaB and calcium/calcineurin/NFATc1 pathways. PLoS One, 2013. 8(7): p. e69468.
46.Huang, S.E., et al., In Vitro Evaluation of the Anti-Inflammatory Effect of KMUP-1 and In Vivo Analysis of Its Therapeutic Potential in Osteoarthritis. Biomedicines, 2021. 9(6).
47.Coryell, P.R., B.O. Diekman, and R.F. Loeser, Mechanisms and therapeutic implications of cellular senescence in osteoarthritis. Nat Rev Rheumatol, 2021. 17(1): p. 47-57.
48.Xu, M., et al., Epigenetic regulation of chondrocyte hypertrophy and apoptosis through Sirt1/P53/P21 pathway in surgery-induced osteoarthritis. Biochem Biophys Res Commun, 2020. 528(1): p. 179-185.
49.Glasson, S.S., T.J. Blanchet, and E.A. Morris, The surgical destabilization of the medial meniscus (DMM) model of osteoarthritis in the 129/SvEv mouse. Osteoarthritis Cartilage, 2007. 15(9): p. 1061-9.
50.Maleitzke, T., et al., Standardized protocol and outcome measurements for the collagen antibody-induced arthritis mouse model. STAR Protoc, 2022. 3(4): p. 101718.
51.Yan, Z., et al., Activating Nrf2 signalling alleviates osteoarthritis development by inhibiting inflammasome activation. J Cell Mol Med, 2020. 24(22): p. 13046-13057.
52.Goldring, M.B. and K.B. Marcu, Cartilage homeostasis in health and rheumatic diseases. Arthritis Res Ther, 2009. 11(3): p. 224.
53.Ulivi, V., et al., p38/NF-kB-dependent expression of COX-2 during differentiation and inflammatory response of chondrocytes. J Cell Biochem, 2008. 104(4): p. 1393-406.
54.Rigoglou, S. and A.G. Papavassiliou, The NF-kappaB signalling pathway in osteoarthritis. Int J Biochem Cell Biol, 2013. 45(11): p. 2580-4.
55.Nandakumar, K.S. and R. Holmdahl, Efficient promotion of collagen antibody induced arthritis (CAIA) using four monoclonal antibodies specific for the major epitopes recognized in both collagen induced arthritis and rheumatoid arthritis. J Immunol Methods, 2005. 304(1-2): p. 126-36.
56.Wang, C., et al., Puerarin attenuates inflammation and oxidation in mice with collagen antibody-induced arthritis via TLR4/NF-kappaB signaling. Mol Med Rep, 2016. 14(2): p. 1365-70.
57.Moore, A.R., et al., Collagen II antibody-induced arthritis in Tg1278TNFko mice: optimization of a novel model to assess treatments targeting human TNFalpha in rheumatoid arthritis. J Transl Med, 2014. 12: p. 285.