Abstract
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. 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. 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.
Highlights
The supernatants from chondrocyte cultures pretreated with Diphenyleneiodonium chloride (DPI), in concentrations ranging from 0.001 to 10 m M, were used to evaluate nitric oxide (NO) production, whereas the adherent cells were used to assess the effect of DPI on cell viability
The amount of formazan produced by cells treated with 1 m M (0.358 ± 0.02 O.D.) or 10 m M (0.361 ± 0.01 O.D.) of DPI did not change relatively to that obtained in control cells
The results presented show that DPI effectively inhibited NO production induced by IL-1 in primary cultures of articular chondrocytes (Fig. 1A,B)
Summary
Interleukin-1b (IL-1), a potent pro-inflammatory cytokine, has been detected at elevated levels in the synovial fluid of patients with various forms of arthritis.[1,2] This cytokine has been shown to inhibit the synthesis of cartilage matrix proteins,[3,4] as well as to induce chondrocytes to produce matrix metalloproteinases[5] and inflammatory mediators, such as prostaglandins[6] and nitric oxide (NO).[7]The large amounts of NO produced by chondrocytes in response to IL-1 result from activation of the gene expression and synthesis of the inducible isoform of the enzyme NO synthase (iNOS).[7,8] The iNOS protein synthesis is mainly regulated at the transcriptional level and the promoter region of the iNOS gene from different species has been reported to contain binding sites for several transcription factors, including nuclear factor-kappa B (NF-k B).[9,10] In most cell types, this transcription factor seems to be an essential requirement for iNOS induction in response to pro-inflammatory cytokines, such as IL-1 and tumor necrosis factor-a (TNF-a ).[11,12,13] Inhibitors of NF-k B activation have been used to prevent iNOS expression and the subsequent NO production in response to a variety of stimuli.[10,14].
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