Short-term gene expression changes in cartilage explants stimulated with interleukin 1β plus glucosamine and chondroitin sulfate

Pooi See Chan, John P. Caron, Michael W. Orth

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Abstract

Objective. To determine the short-term effects of glucosamine (GLN) and chondroitin sulfate (CS) on expression of genes encoding inflammatory mediators and matrix enzymes in bovine cartilage explants stimulated with interleukin 1 (IL-1). Methods. Dose-response experiments were conducted for IL-1, GLN, and CS to select concentrations of each optimized for detecting treatment effects on cartilage explants. Based on the dose-response experiments, treatments included fetal bovine serum (FBS) control, 15 ng/ml IL-1, and 15 ng/ml IL-1 with the addition of 10 μg/ml GLN and 20 μg/ml CS. Media were measured for nitric oxide (NO) and prostaglandin E 2 (PGE 2) while explants were frozen for RNA extraction at 8, 16, and 24 hours. Gene expression relative to FBS control for inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), microsomal PGE synthase-1 (mPGEs1), nuclear factor-κB p65 subunit (NF-κB), matrix metalloproteinase (MMP)-3 and 13, aggrecanase (Agg)-1 and 2, and tissue inhibitor of metalloproteinase-3 (TIMP-3) were assessed by quantitative real-time polymerase chain reaction (RT-PCR). In a separate study using incubation of explants with the same treatments for 48 hours, proteoglycan release was measured with dimethylmethylene blue assay and TIMP-3 protein was evaluated with Western blots. Results. The GLN and CS combination abrogated IL-1-induced gene expression of iNOS, COX-2, mPGEs1, and NF-κB at all timepoints. NO, PGE 2, and proteoglycan release were reduced with the combination. The abundance of stimulated MMP-13, Agg-1, and Agg-2 mRNA was repressed, whereas TIMP-3 was upregulated by the combination at all timepoints. The abundance of TIMP-3 protein was increased by the combination relative to IL-1 at 48 hours. Conclusion. GLN and CS in combination suppress synthesis and expression of genes encoding inflammatory mediators and proteolytic enzymes while upregulating TIMP-3. This provides a plausible mechanism for the purported mild antiinflammatory and chondroprotective properties of GLN and CS.

Original languageEnglish
Pages (from-to)1329-1340
Number of pages12
JournalJournal of Rheumatology
Volume33
Issue number7
StatePublished - Jul 1 2006

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Keywords

  • Arthritis
  • Chondroitin sulfate
  • Gene expression
  • Glucosamine
  • Inflammatory mediators
  • Matrix metalloproteinase

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