Diacerhein and rhein are anthraquinone compounds that ameliorate the course of osteoarthritis. Recent reports also suggest that these compounds may have antiinflammatory properties, but the cellular mechanisms by which they exert antiosteoarthritic and possibly antiinflammatory effects are still incompletely understood. The purpose of this study was to investigate the ability of diacerhein and rhein to inhibit the activation of the transcription factor nuclear factorkB, induced by the proinflammatory cytokine interleukin-1b, in primary monolayer cultures of bovine articular chondrocytes. We also studied the ability of diacerhein and rhein to prevent the expression of the inducible nitric oxide synthase gene, which is driven by nuclear factor-kB. We observed that interleukin-1b induced the degradation of the inhibitor kBa protein and the translocation of the protein p65 (a member of the nuclear factor-kB family) to the nucleus, which were inhibited by diacerhein and rhein, in a dose-dependent manner. Interleukin-1b-induced nuclear factor-kB binding to a specific (g-32 P)-labelled oligonucleotide probe was also inhibited by treatment of chondrocytes with diacerhein or rhein, as revealed by electrophoretic mobility shift assay. Inducible nitric oxide synthase mRNA and protein synthesis and nitric oxide production were also inhibited by diacerhein and rhein, in a dose-dependent manner. The half-maximal inhibitory concentrations of diacerhein and rhein, relative to nitric oxide production, were 8.2 mM and 7.7 mM, respectively. These results suggest that diacerhein and rhein inhibit nuclear factor-kB activation and, consequently, the expression of nuclear factor-kB-dependent genes, such as the inducible nitric oxide synthase gene, which can explain their antiosteoarthritic and antiinflammatory effects.

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Our previous studies showed that reactive oxygen species (ROS) are required for the pro-inflammatory cytokine interleukin-1 beta (IL-1) to induce the activity of the Nuclear transcription Factor-kappa B (NF-kappa B) and the expression of the inducible isoform of the nitric oxide synthase (iNOS) in bovine articular chondrocytes. This study aimed at elucidating the role of hydrogen peroxide (H(2)O(2)) and the superoxide radical, two major ROS, in mediating those IL-1-induced responses. The results obtained show that chondrocytes produce both H(2)O(2) and superoxide radical in response to IL-1. Treatment of the chondrocyte cultures with H(2)O(2) alone did not induce NF-kappa B activation or iNOS expression. Addition of H(2)O(2) simultaneously with IL-1 did neither enhance nor inhibit NF-kappa B activation and iNOS expression, relatively to treatment with IL-1 alone. Accordingly, treatment with catalase did not inhibit those IL-1-induced responses. Treatment with superoxide dismutase, however, effectively prevented IL-1-induced I kappa B-alpha degradation and iNOS expression. Taken together, the results obtained indicate that superoxide mediates IL-1-induced I kappa B-alpha degradation and the consequent NF-kappa B activation and iNOS expression in chondrocytes, whereas H(2)O(2) does not seem to participate in those IL-1-induced responses. In conclusion, the present study identifies the superoxide radical as the ROS involved in mediating the IL-1-induced signaling pathway that leads to NF-kappa B activation and to the expression of NF-kappa B-dependent genes in bovine articular chondrocytes.

AIMS: In this work, we studied the mechanisms by which diphenyleneiodonium chloride (DPI) inhibits nitric oxide (NO) synthesis induced by the proinflammatory cytokine interleukin-1beta (IL-1) in bovine articular chondrocytes. To achieve this, we evaluated the ability of DPI to inhibit the expression and activity of the inducible isoform of the NO synthase (iNOS) induced by IL-1. We also studied the ability of DPI to prevent IL-1-induced NF-kappaB activation and reactive oxygen species (ROS) production. RESULTS: Northern and Western blot analysis, respectively, showed that DPI dose-dependently inhibited IL-1-induced iNOS mRNA and protein synthesis in primary cultures of bovine articular chondrocytes. DPI effectively inhibited NO production (IC50=0.03+/-0.004 microM), as evaluated by the method of Griess. Nuclear factor-kappa B (NF-kappaB) activation, as evaluated by electrophoretic mobility shift assay, was inhibited by DPI (1-10 microM) in a dose-dependent manner. IL-1-induced ROS production, as evaluated by measurement of dichlorofluorescein fluorescence, was inhibited by DPI at concentrations that also prevented NF-kappaB activation and iNOS expression. CONCLUSIONS: DPI inhibits IL-1-induced NO production in chondrocytes by two distinct mechanisms: (i) by inhibiting NOS activity, and (ii) by preventing iNOS expression through the blockade of NF-kappaB activation. These results also support the involvement of reactive oxygen species in IL-1-induced NF-kappaB activation and expression of NF-kappaB-dependent genes, such as iNOS.

The content of dopamine and noradrenaline in the mucosa-submucosa and muscular layers of different gastrointestinal areas of the dog, and its modification by 6-hydroxydopamine or pargyline plus 6-hydroxydopamine was studied by means of high pressure liquid chromatography with electrochemical detection. The amounts of dopamine and noradrenaline show a cephalocaudal increase but their physiological levels were rather low when compared with those reported in other tissues. This finding was consistent with the sparse noradrenergic innervation classically described for the gut with histochemical methodologies. On the basis that noradrenergic neurones are considered more susceptible to 6-hydroxydopamine than dopaminergic neurones, a difference abolished by previous treatment with pargyline, our findings did not suggest the existence of dopaminergic neurones in the gastrointestinal tract of the dog. In the muscular layer of the duodenum, jejunum and ileum it was observed that catecholamine depletion by both treatments was smaller than that obtained in the mucosa-submucosa. Due to the resistance to both kinds of chemical sympathectomy exhibited by the dopamine content, the existence of dopamine-containing enterochromaffin cells is proposed in the mucosa-submucosa of different portions of the stomach and small intestine.