Macrophages arise as an interesting target for modulation of inflammatory disease. Inflammatory mediators derived from these cells have a critical role during synovial inflammation and bone destruction in some patients with RA [7, 8]. Obtained using various approaches, our results clearly indicate that RU, a molecule already used in vascular diseases, inhibited the activation of human macrophages and the subsequent secretion of proinflammatory mediators from these cells. RU was shown to inhibit the transcription of more than 20 genes encoding critical proinflammatory factors, including TNF-α, IL-1, IL-8, TNF-α, MIF, and chemoattracting factors. This effect was confirmed by decreased concentrations of IL-1β, TNF-α, and IL-6 observed in cell supernatants.
NO has been identified as another proinflammatory mediator in human arthritis and experimental animal studies [3, 31]. Increased concentrations of nitrites, stable metabolites of NO, have been observed in the serum and the synovial fluid of patients with RA and osteoarthritis [3, 32]. Increased iNOS activity and NO production have also been detected in the blood mononuclear cells of patients with RA and correlated with the tender and swollen joint counts . Here, we show that RU decreased the production of iNOS-mediated NO by human macrophages in a dose-dependent manner. Of particular interest, and by contrast to the above mediators, RU induced a slight but significant increase of IL-10 mRNA. However, we failed to detect an increase of IL-10 protein level. This may be due to the absorption of IL-10 by macrophages, as yet observed in CD23-activated human epithelial cells . This Th2 type cytokine is well known as a downregulator of the above-mentioned Th1 mediators in human macrophages . These preliminary data suggest that RU preferentially lowers Th1-like cytokine generation from human macrophages.
To support the therapeutic interest of RU, we investigated its effects in a rat model of adjuvant-induced chronic arthritis, well known to mimic Th1 type pathogenic signs of RA . RU significantly reversed growth delay and severe AIA development in rats with persistent partial or complete long-term recovery, not observed in rats treated with HC. These data confirm early observations in experimental acute inflammation models [18–20, 36] and further revealed the preventive property of RU in arthritic rats. Ex vivo analysis of rat sera and macrophages confirmed RU-mediated inhibition of critical proarthritic factors such as TNF-α, IL-1, MCP-1, and NO in vivo. This property correlated with RU-mediated inhibition of murine macrophage activation and inflammatory mediator release in vitro (Figures 3 and 4). In contrast to quercetin , RU did not mediate the inhibition of the PGE2 pathway.
Together, the above data provide new insight into the possible mechanism of anti-inflammatory effects of RU in macrophages. Human and murine analyses of cytokine expression clearly show that RU suppresses the major inflammatory and proarthritic mediators of macrophages. The ability of RU to decrease MCP-1 levels in vivo and in vitro may add to its beneficial effects because this cytokine is a potent stimulator of monocyte recruitment into the site of inflammation . We have previously shown that the nonglycosylated derivative of RU, quercetin, inhibits the production of TNF-α and NO from activated human macrophages . These flavonols inhibit the phosphorylation and activation of Jun N-terminal kinase/stress-activated protein kinase, leading to the suppression of AP-1 activation. They also decrease the activation of NF-κB in both human and experimental models [12, 40]. These observations may explain the anti-inflammatory property of RU because both nuclear factors are necessary for the generation of most inflammatory mediators analyzed in the present study [41–43].
However, the exact mechanism underlying the improvement in the arthritis model requires more experimental clarifications. Despite the direct relationship between arthritis signs and macrophage inflammatory markers in the AIA rat model, we must not exclude the simultaneous effect of RU on other inflammatory partners (such as lymphocytes) that may directly or indirectly reduce arthritis manifestations. In addition, AIA is a good model for Th1-mediated and macrophage inflammatory response but is a poor model for Th2-mediated immune reactions. Further analysis of RU activities in the presence of various human lymphocyte populations is required to clarify these points.
Finally, in vivo use of quercetin as medicine suffers from the lack of approved formulation despite a first preliminary assay as adjunct treatment of prostatitis/chronic pelvic pain in humans . In contrast, formulations containing either RU or its derivatives are currently used in the treatment and prevention of venous circulation disorders [11, 45].