許多證據顯示氧化壓力在類風濕性關節炎致病機轉中扮演了重要的角色。發炎關節內反覆進行的缺氧與復氧循環可產生活性氧化物質。這些活性氧化物質本身可作為一種次級傳訊者而活化NF-κB，NF-κB又統合了一系列與發炎反應相關的基因表現。本研究中，我們使用水溶性富勒烯（碳六十）作為自由基清除劑，研究其對大鼠之膠原誘導關節炎的可能療效。我們將牛的第二型膠原蛋白加入佛氏佐劑加以乳化後，以間隔一週皮內注射兩次的方式使雄性Sprague-Dawley大鼠免疫。第一次免疫後之第七日，於大鼠的踝關節以關節內注射方式給予水溶性富勒烯（碳六十，作為實驗組）或磷酸鹽緩衝溶液（作為對照組）以治療關節炎。我們藉由關節腫脹指數、關節X光影像分數及組織病理分數評估動物對療程的反應，並測量踝關節勻漿內之介白素1及血管內皮細胞生長因子之濃度。經由免疫組織化學染色，我們也計算實驗組與對照組動物發炎關節內的微血管數以確認療效。實驗結果發現，以水溶性碳六十治療的實驗組大鼠比對照組大鼠有顯著較低的關節腫脹指數；於第二十天犧牲大鼠後，其踝關節狀態評估顯示，碳六十治療可顯著改善關節的X光影像分數及組織病理分數，踝關節勻漿內之介白素1濃度也顯著的減低。實驗組大鼠踝關節滑膜組織內的微血管數亦比對照組顯著地減少。這項研究顯示於關節內注射水溶性碳六十可有效治療並改善大鼠的膠原誘導關節炎，而此治療效果與抗發炎及抗血管新生機轉有關。

Several lines of evidence suggest a role for oxidative stress in the pathogenesis of rheumatoid arthritis (RA). Repetitive cycles of hypoxia and reoxygenation within arthritic joints can generate reactive oxygen species (ROS). ROS can function as a second messenger to activate nuclear factor kappa-B, which orchestrates the expression of a spectrum of genes involved in the inflammatory responses. In this study, we investigated the potential therapeutic effect of a free-radical scavenger, water-soluble fullerene (C60), on collagen-induced arthritis (CIA) of rats, an experimental animal model of human RA. Male Sprague-Dawley rats were immunized by two intradermal injections one week apart with bovine type II collagen emulsified in Freund’s adjuvant. The water-soluble C60 or phosphate buffered saline (PBS) was administered by intraarticular injection into ankle joints of rats on day 7 after the first immunization. The response to treatments was determined by measuring the articular index and radiographic and histological scores. Levels of interleukin-1β (IL-1β) and vascular endothelial growth factor (VEGF) in homogenate extracts of ankle joints were analyzed. Microvessel numbers in the arthritic joints were also measured after immunohistochemical staining. Significant reduction in articular index was observed in C60-treated rats as compared with their control counterparts. C60 injection significantly ameliorated radiological and histological scores in ankle joints as well. There were significantly reduced concentrations of IL-1β but not VEGF in homogenate extracts of ankle joints. Marked decreased microvessel numbers were observed in the ankle joints of C60-treated rats as compared with PBS-injected animals. In conclusion, this study demonstrates that intraarticular administration of C60 ameliorates CIA of rats through anti-inflammatory and anti-angiogenic mechanisms.

1. McInnes IB, and Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011; 365(23): 2205-19.
2. Firestein GS. Evolving concepts of rheumatoid arthritis. Nature. 2003; 423(6937): 356-61.
3. Chen SY, Shiau AL, Shieh GS, Su CH, Lee CH, Lee HL, Wang CR, and Wu CL. Amelioration of experimental arthritis by a telomerase- dependent conditionally replicating adenovirus that targets synovial fibroblasts. Arthritis Rheum. 2009; 60(11): 3290-302.
4. Li YT, Chen SY, Wang CR, Liu MF, Lin CC, Jou IM, Shiau AL, and Wu CL. Brief Report: Amelioration of collagen-induced arthritis in mice by lentivirus-mediated silencing of microRNA-223. Arthritis Rheum. 2012; 64(10): 3240-5.
5. Filippin LI, Vercelino R, Marroni NP, and Xavier RM. Redox signalling and the inflammatory response in rheumatoid arthritis. Clin Exp Immunol. 2008; 152(3): 415-22.
6. Afonso V, Champy R, Mitrovic D, Collin P, and Lomri A. Reactive oxygen species and superoxide dismutases: role in joint diseases. Joint Bone Spine. 2007; 74(4): 324-9.
7. Volk T, Gerst J, Faust-Belbe G, Stroehmann A, and Kox WJ. Monocyte stimulation by reactive oxygen species: role of superoxide and intracellular Ca2+. Inflamm Res. 1999; 48(10): 544-9.
8. Hitchon CA, and El-Gabalawy HS. Oxidation in rheumatoid arthritis. Arthritis Res Ther. 2004; 6(6): 265-78.
9. Muz B, Khan MN, Kiriakidis S, and Paleolog EM. Hypoxia. The role of hypoxia and HIF-dependent signalling events in rheumatoid arthritis. Arthritis Res Ther. 2009; 11(1): 201.
10. Sandhu JK, Robertson S, Birnboim HC, and Goldstein R. Distribution of protein nitrotyrosine in synovial tissues of patients with rheumatoid arthritis and osteoarthritis. J Rheumatol. 2003; 30(6): 1173-81.
11. Mathy-Hartert M, Martin G, Devel P, Deby-Dupont G, Pujol JP, Reginster JY, and Henrotin Y. Reactive oxygen species downregulate the expression of pro-inflammatory genes by human chondrocytes. Inflamm Res. 2003; 52(3): 111-8.
12. Henrotin YE, Bruckner P, and Pujol JP. The role of reactive oxygen species in homeostasis and degradation of cartilage. Osteoarthritis Cartilage. 2003; 11(10): 747-55.
13. Gelderman KA, Hultqvist M, Pizzolla A, Zhao M, Nandakumar KS, Mattsson R, and Holmdahl R. Macrophages suppress T cell responses and arthritis development in mice by producing reactive oxygen species. J Clin Invest. 2007; 117(10): 3020-8.
14. Goebel KM, Storck U, and Neurath F. Intrasynovial orgotein therapy in rheumatoid arthritis. Lancet. 1981; 1(8228): 1015-7.
15. Edmonds SE, Winyard PG, Guo R, Kidd B, Merry P, Langrish-Smith A, Hansen C, Ramm S, and Blake DR. Putative analgesic activity of repeated oral doses of vitamin E in the treatment of rheumatoid arthritis. Results of a prospective placebo controlled double blind trial. Ann Rheum Dis. 1997; 56(11): 649-55.
16. Afonso V, Santos G, Collin P, Khatib AM, Mitrovic DR, Lomri N, Leitman DC, and Lomri A. Tumor necrosis factor-alpha down-regulates human Cu/Zn superoxide dismutase 1 promoter via JNK/AP-1 signaling pathway. Free Radic Biol Med. 2006; 41(5): 709-21.
17. Marklund SL. Human copper-containing superoxide dismutase of high molecular weight. Proc Natl Acad Sci U S A. 1982; 79(24): 7634-8.
18. Adachi T, Toishi T, Takashima E, and Hara H. Infliximab neutralizes the suppressive effect of TNF-alpha on expression of extracellular- superoxide dismutase in vitro. Biol Pharm Bull. 2006; 29(10): 2095-8.
19. Wikipedia contributors. Antioxidant. http://en.wikipedia.org/w/index.php?title=Antioxidant&oldid=610160625. Updated 26 May 2014 04:32 UTC Accessed 2 June 2014 06:50 UTC.
20. Wikipedia contributors. Adsorption. http://en.wikipedia.org/w/index.php?title=Adsorption&oldid=610313305. Updated 27 May 2014 07:15 UTC Accessed 2 June 2014 06:57 UTC.
21. Krusic PJ, Wasserman E, Keizer PN, Morton JR, and Preston KF. Radical reactions of c60. Science. 1991; 254(5035): 1183-5.
22. Tsai CY, Shiau AL, Chen SY, Chen YH, Cheng PC, Chang MY, Chen DH, Chou CH, Wang CR, and Wu CL. Amelioration of collagen-induced arthritis in rats by nanogold. Arthritis Rheum. 2007; 56(2): 544-54.
23. Bosi S, Da Ros T, Spalluto G, and Prato M. Fullerene derivatives: an attractive tool for biological applications. Eur J Med Chem. 2003; 38(11-12): 913-23.
24. Satoh M, and Takayanagi I. Pharmacological Studies on Fullerene (C60), a Novel Carbon Allotrope, and Its Derivatives. J Pharmacol Sci. 2006; 100(5): 513-8.
25. Kroto HW, Heath JR, O'Brien SC, Curl RF, and Smalley RE. C60: Buckminsterfullerene. Nature. 1985; 318(6042): 162-3.
26. Sijbesma R, Srdanov G, Wudl F, Castoro JA, Wilkins C, Friedman SH, DeCamp DL, and Kenyon GL. Synthesis of a fullerene derivative for the inhibition of HIV enzymes. J Am Chem Soc. 1993; 115(15): 6510-2.
27. Bosi S, Da Ros T, Castellano S, Banfi E, and Prato M. Antimycobacterial activity of ionic fullerene derivatives. Bioorg Med Chem Lett. 2000; 10(10): 1043-5.
28. Dugan LL, Turetsky DM, Du C, Lobner D, Wheeler M, Almli CR, Shen CK, Luh TY, Choi DW, and Lin TS. Carboxyfullerenes as neuroprotective agents. Proc Natl Acad Sci U S A. 1997; 94(17): 9434-9.
29. Satoh M, Mashino T, Nagano T, Hirobe M, Takayanagi I, and Koike K. Inhibitory effects of fullerene C60 derivatives on endothelium-derived relaxation in rabbit thoracic aorta. Fullerene Sci Tech. 2001; 9(2): 141-51.
30. Chiang LY, Wang LY, Swirczewski JW, Soled S, and Cameron S. Efficient Synthesis of Polyhydroxylated fullerene derivatives via hydrolysis of polycyclosulfated precursors. J Org Chem. 1994; 59(14): 3960-8.
31. Görlach A, and Kietzmann T. In: Helmut S, and Bernhard B eds. Methods in Enzymology. Academic Press; 2007:421-46.
32. Woods JM, Katschke KJ, Volin MV, Ruth JH, Woodruff DC, Amin MA, Connors MA, Kurata H, Arai KI, Haines GK, et al. IL-4 adenoviral gene therapy reduces inflammation, proinflammatory cytokines, vascularization, and bony destruction in rat adjuvant-induced arthritis. J Immunol. 2001; 166(2): 1214-22.
33. Rasband WS. ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2014.
34. Yudoh K, Karasawa R, Masuko K, and Kato T. Water-soluble fullerene (C60) inhibits the development of arthritis in the rat model of arthritis. Int J Nanomedicine. 2009; 4: 217-25.
35. Klareskog L, Catrina AI, and Paget S. Rheumatoid arthritis. Lancet. 2009; 373(9664): 659-72.
36. Ha H, Kwak HB, Lee SW, Jin HM, Kim HM, Kim HH, and Lee ZH. Reactive oxygen species mediate RANK signaling in osteoclasts. Exp Cell Res. 2004; 301(2): 119-27.
37. Bai XC, Lu D, Liu AL, Zhang ZM, Li XM, Zou ZP, Zeng WS, Cheng BL, and Luo SQ. Reactive oxygen species stimulates receptor activator of NF-κB ligand expression in osteoblast. J Biol Chem. 2005; 280(17): 17497-506.
38. Lee NK, Choi YG, Baik JY, Han SY, Jeong DW, Bae YS, Kim N, and Lee SY. A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood. 2005; 106(3): 852-9.
39. Yudoh K, Karasawa R, Masuko K, and Kato T. Water-soluble fullerene (C60) inhibits the osteoclast differentiation and bone destruction in arthritis. Int J Nanomedicine. 2009; 4: 233-9.
40. Konisti S, Kiriakidis S, and Paleolog EM. Hypoxia—a key regulator of angiogenesis and inflammation in rheumatoid arthritis. Nat Rev Rheumatol. 2012; 8(3): 153-62.
41. Murugesan S, Mousa SA, O’Connor L J, Lincoln DW, 2nd, and Linhardt RJ. Carbon inhibits vascular endothelial growth factor- and fibroblast growth factor-promoted angiogenesis. FEBS Lett. 2007; 581(6): 1157-60.