Water-soluble fullerene (C60) inhibits the development of arthritis in the rat model of arthritis

Abstract

Recently, it has been demonstrated that oxygen free radicals have an important role as a signaling messenger in the development of inflammation and osteoclastogenesis, suggesting the implication of oxygen free radicals in the pathogenesis of arthritis. The aim of this study was to examine the potential of a strong free-radical scavenger, water-soluble fullerene (C60), as a protective agent against synovitis in arthritis, both in vitro and in vivo. In the presence or absence of C60 (0.1, 1.0, 10.0 muM), human synovial fibroblasts, synovial infiltrating lymphocytes or macrophages were incubated with tumor necrosis factor-alpha (TNF-alpha) (10.0 ng/mL), and the production of proinflammatory cytokines by the individual cells were analyzed. C60 significantly suppressed the TNF-alpha-induced production of proinflammatory cytokines in synovial fibroblasts, synovial infiltrating lymphocytes and macrophages in vitro. Adjuvant induced arthritic rats were used as an animal model of arthritis. Rats were divided into two subgroups: control and treatment with C60 at 10.0 muM. The left ankle joint was injected intraarticularly with water-soluble C60 (20 mul) in the C60-treated group, while, as a control, the left ankle joint in the control rats received phosphate-buffered saline (20 mul), once weekly for eight weeks. Ankle joint tissues were prepared for histological analysis. In adjuvant-induced arthritic rats, intra-articular treatment with C60 in vivo reduced synovitis and alleviated bone resorption and destruction in the joints, while control ankle joints showed progression of synovitis and joint destruction with time. These findings indicate that C60 is a potential therapeutic agent for inhibition of arthritis.

Keywords: arthritis; bone resorption; fullerene; inflammation; synovitis.

Figure 1

Effects of C60 on the production of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in synovial fibroblasts, synovial infiltrating lymphocytes and macrophages. Synovial fibroblasts, synovial infiltrating lymphocytes or macrophages were incubated with catabolic factor, TNF-α (10.0 ng /ml) in the presence or absence of water-soluble C60 (0.1, 1.0, or 10.0 μM). After 24-hour incubation, the concentrations of proinflammatory cytokines were analyzed by ELISA (A: TNF-α, B: IL-1β). TNF-α stimulated the production of TNF-α and IL-1β by individual cells (*P < 0.05, **P < 0.01 compared to each control). C60 significantly decreased the TNF-α-induced excess production of TNF-α A) and IL-1β B) from individual cells (*P < 0.05, **P < 0.01). Data from four independent experiments were analyzed.

Figure 2

Intraarticular treatment of C60 inhibits the arthritis score in the arthritis model rats. In each rat after immunization, the left ankle joint was treated by intra-articular injection of the appropriate solution (20.0 μl, Control: phosphate-buffered saline, C60-treated group: 10.0 μM C60) by micro-needle syringe once a week during the observation periods. In the control joint group, the arthritis score gradually increased with the advance of time. The score in the left ankles of rats that had been treated with C60 (10.0 μM) showed lower at four and eight weeks after immunization in contrast to the control ankle joints (four rats per time point).

Figure 3

Intraarticular treatment of C60 inhibits the pathological score in the arthritis model rats. The degree of arthritis was histologically evaluated in each ankle joint sample (four rats per time point). Although infiltration of inflammatory cells into the synovium and joint destruction progressed with time in both control and C60-treated groups, the C60-treated group showed significantly lower pathological tissue scores at four weeks and later compared to the respective control group.

Figure 4

Representative images of the joint damage at each time point in arthritis model rats. A) After four weeks of treatment, there was a tendency in the group treated with phosphate-buffered saline (PBS) towards a more severe degree of synovitis than in the group treated with C60 (10.0 μM). B) At six weeks after immunization, intraarticular treatment with C60 inhibited the infiltration of inflammatory cells, synovial hyperplasia, and bone resorption by multinucleated osteoclastic cells in the ankle joints of arthritis model rats, whereas the control joints that was treated with PBS showed more severe damage. C) At the eight-week time point, all ankle joints that had been treated with PBS showed severe joint destruction and bony ankylosis. In contrast, inhibitory effects were observed following administration of C60 in the C60-treated group.

Figure 4

Representative images of the joint damage at each time point in arthritis model rats. A) After four weeks of treatment, there was a tendency in the group treated with phosphate-buffered saline (PBS) towards a more severe degree of synovitis than in the group treated with C60 (10.0 μM). B) At six weeks after immunization, intraarticular treatment with C60 inhibited the infiltration of inflammatory cells, synovial hyperplasia, and bone resorption by multinucleated osteoclastic cells in the ankle joints of arthritis model rats, whereas the control joints that was treated with PBS showed more severe damage. C) At the eight-week time point, all ankle joints that had been treated with PBS showed severe joint destruction and bony ankylosis. In contrast, inhibitory effects were observed following administration of C60 in the C60-treated group.