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Abstract
OBJECTIVE To determine if a new inhibitor, esculetin (EST), can block resorption of cartilage.
METHODS Interleukin 1α (IL1α, 0.04–5 ng/ml) and oncostatin M (OSM, 0.4–50 ng/ml) were used to stimulate the release of proteoglycan and collagen from bovine nasal cartilage and human articular cartilage in explant culture. Proteoglycan and collagen loss were assessed by dimethylmethylene blue and hydroxyproline assays, respectively. Collagenase levels were measured by assay of bioactivity and by enzyme linked immunosorbent assay (ELISA). The effects of EST on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in the transformed human chondrocyte cell line T/C28a4 were assessed by northern blot analysis. TIMP-1 protein levels were assayed by ELISA. The effect of EST on the MMP-1 promoter was assessed using a promoter-luciferase construct in transient transfection studies.
RESULTS EST inhibited proteoglycan and collagen resorption in a dose dependent manner with significant decreases seen at 66 μM and 100 μM EST, respectively. Collagenolytic activity was significantly decreased in bovine nasal cartilage cultures. In human articular cartilage, EST also inhibited IL1α + OSM stimulated resorption and decreased MMP-1 levels. TIMP-1 levels were not altered compared with controls. In T/C28a4 chondrocytes the IL1α + OSM induced expression of MMP-1, MMP-3, and MMP-13 mRNA was reduced to control levels by 250 μM EST. TIMP-1 mRNA levels were unaffected by EST treatment. All cytokine stimulation of an MMP-1 luciferase-promoter construct was lost in the presence of the inhibitor.
CONCLUSION EST inhibits degradation of bovine nasal cartilage and human articular cartilage stimulated to resorb with IL1α + OSM.
- inhibitor
- cartilage
- interleukin 1
- oncostatin M