Water-soluble C60-(OH)24 fullerene hydroxide as a therapeutic agent against the degeneration of articular cartilage in osteoarthritis

Abstract

Purpose: During the development of osteoarthritis (OA), mechanical stress on articular cartilage downregulates the stable cellular activities of chondrocytes and induce the production of catabolic factors, such as proinflammatory cytokines and chemokines. In addition, it is well known that chondrocytes produce excess amounts of reactive oxygen species (ROS; superoxide, nitric oxide, hydrogen peroxide and peroxynitrite) in response to mechanical stress. Recent reports have demonstrated that mechanical stress to articular cartilage stimulates excess production of ROS from chondrocytes, consequently resulting in the depolymerization of hyaluronic acid and chondrocyte death. It has been suggested that the generation of ROS and the depletion of cellular antioxidants may be involved in the pathogenesis of OA. C60 fullerene is a molecule composed completely of carbon in a unique spherical structure that has a high reactivity with oxygen free radicals and can potentially act as a free radical scavenger. It has been reported that the antioxidant level of C60 is several hundred-fold higher than that of other antioxidants. Recently, C60 fullerene has shown to inhibit neuronal apoptosis by scavenging oxygen reactive species and protects human skin keratinocytes from ROS-induced cell death after UV stress, suggesting that C60 is a useful agent to protect against the oxygen free radical-induced pathological features in a variety of diseases. Use of C60 could lead to the development of novel therapeutic strategies in the prevention of both chondrocyte aging and cartilage degeneration. To prevent the degeneration of articular cartilage in OA, we have newly developed on water-soluble C60(OH)24 fullerene hydroxide, a strong free radical scavenger, as an anti-oxidative agent. In this study, we examined the therapeutic effects of water-soluble C60(OH)24 on chondrocyte activity and energy metabolism, which were monitored by the production of cartilage component (proteoglycan), matrix degrading enzyme MMP-3, and the uptake of glucose in the cells, respectively. Methods: In the presence or absence of C60(OH)24 (1.0 nM, 10.0 nM, 100.0 nM), human OA chondrocytes were incubated with IL-1β (1.0, 10.0 ng/ml). After the 24-hour incubation period, chondrocyte activities (production of proteoglycan and MMP-3, glucose metabolism) were analyzed. Our previous study revealed that expression of DNA repair enzyme, apurinic/apyrimidinic endonuclease (Apex) 2, in chondrocytes was associated with the degeneration of articular cartilages and was induced by OA-relating catabolic factors. Thus, we also studied effects of C60(OH)24 on the expression of Apex 2 in osteoarthritic chondrocytes. Results: IL-1β significantly inhibited the production of proteoglycan and the glucose uptake in chondrocytes, and accelerated the secretion of MMP-3 from chondrocytes. Even in the presence of IL-1β (1.0 ng/ml, but not 10.0 ng/ml), C60(OH)24 inhibited the IL-1β-induced production of MMP-3, the IL-1β-induced down-regulation of proteoglycan production and glucose metabolism in OA chondrocytes. C60(OH)24 fullerene hydroxide also reduced the OA-relating catabolic factor-induced up-regulation of DNA repair enzyme, Apex 2, in OA chondrocytes. Conclusions: Our data indicated that nM order of water-soluble C60(OH)24 fullerene hydroxide may have a potential to protect against the catabolic factor-induced down-regulation of chondrocyte activities in OA. Our findings provide a novel pathogenic mechanism linking oxidant-mediated degenerative process of articular cartilage in OA.Figure 2. Effect of C60(OH)24 on proteoglycan production in chondrocytesView Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3. Effect of C60(OH)24 on glucose uptake into chondrocytesView Large Image Figure ViewerDownload Hi-res image Download (PPT)