Korean J Physiol Pharmacol.  2016 Mar;20(2):221-228. 10.4196/kjpp.2016.20.2.221.

Effects of prunetin on the proteolytic activity, secretion and gene expression of MMP-3 in vitro and production of MMP-3 in vivo

• Nam DC
• Kim BK
• Lee HJ
• Shin HD
• Lee CJ
• Hwang SC

• KMID: 2388679
• DOI: http://doi.org/10.4196/kjpp.2016.20.2.221

Abstract

We investigated whether prunetin affects the proteolytic activity, secretion, and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the rat knee joint to evaluate the potential chondroprotective eff ect of prunetin. Rabbit articular chondrocytes were cultured in a monolayer, and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1beta (IL-1beta)-induced expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), and ADAMTS-5. In rabbit articular chondrocytes, the effects of prunetin on IL-1beta-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The eff ect of prunetin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) prunetin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5; (2) prunetin inhibited the secretion and proteolytic activity of MMP-3; (3) prunetin suppressed the production of MMP-3 protein in vivo. These results suggest that prunetin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.

Keyword

Chondrocyte; Metalloproteinase; Osteoarthritis; Prunetin

MeSH Terms

Animals
Blotting, Western
Caseins
Chondrocytes
Gene Expression*
Interleukin-1beta
Knee Joint
Osteoarthritis
Rats
Thrombospondins
Caseins
Interleukin-1beta
Thrombospondins

Figure

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  Fig. 1 Chemical structure of glycyrrhizin, quercitrin and prunetin.

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  Fig. 2 Effect of glycyrrhizin, quercitrin or prunetin on MMP-3 gene expression in rabbit chondrocytes.Primary cultured rabbit articular chondrocytes were pretreated with varying concentrations (1, 10, 50, and 100 µM) of glycyrrhizin, quercitrin or prunetin for 2 h and then stimulated with IL-1β (10 ng/mL) for 24 h. MMP-3 gene expression level was measured by RT-PCR. Three independent experiments were performed and the representative data were shown. The signal intensity of each band was analyzed by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). Each bar represents a mean±S. E.M. of three independent experiments in comparison with that of the control set at 100%. *significantly different from control (p<0.05), +significantly different from IL-1β alone (p<0.05) (cont: control, concentration unit is µM).

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  Fig. 3 Effect of prunetin on proliferation of rabbit chondrocytes.Chondrocytes were incubated for 72 h in the presence of varying concentrations of prunetin. Cell viability was determined using SRB assay as described in Materials and Methods. Each bar represents a mean±S.E.M. of three independent experiments in comparison with that of the control set at 100%.

• 

  Fig. 4 Effect of prunetin on the gene expression of MMP-1, MMP-13, ADAMTS-4, or ADAMTS-5 in rabbit chondrocytes.Primary cultured rabbit articular chondrocytes were pretreated with varying concentrations (1, 10, 50, and 100 µ M) of prunetin for 2 h and then stimulated with IL-1β (10 ng/mL) for 24 h. The gene expression level of MMP-1, MMP-13, ADAMTS-4, or ADAMTS-5 was measured by RT-PCR. Three independent experiments were performed and the representative data were shown. The signal intensity of each band was analyzed by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). Each bar represents a mean±S.E.M. of three independent experiments in comparison with that of the control set at 100%. *significantly different from control (p<0.05), +significantly different from IL-1β alone (p<0.05) (cont: control, concentration unit is µM).

• 

  Fig. 5 Effects of prunetin on IL-1β-induced secretion of MMP-3 and caseinolytic activity of MMP-3 in rabbit articular chondrocytes.Primary cultured rabbit articular chondrocytes were pretreated with varying concentrations (1, 10, 50, and 100 µM) of prunetin for 2 h and then stimulated with IL-1β (10 ng/mL) for 24 h. Culture supernatants were collected for measurement of both the levels of produced and secreted MMP-3 by western blot analysis and the proteolytic activity of MMP-3 by casein zymography. Three independent experiments were performed and the representative data were shown. The signal intensity of each band was analyzed by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). Each bar represents a mean±S. E.M. of three independent experiments in comparison with that of the control set at 100%. *significantly different from control (p<0.05), +significantly different from IL-1β alone (p<0.05) (cont: control, concentration unit is µM).

• 

  Fig. 6 Effect of prunetin on production of MMP-3 in vivo.The knee joint of rats were pretreated with 50 or 100 µM of prunetin for 3 h and then stimulated with IL-1β (20 ng/30 µL) for 72 h, by intraarticular injection. Tissue lysates from articular cartilage homogenates containing MMP-3 proteins were collected for measurement of the level of produced MMP-3 in vivo, by western blot analysis. The representative data were shown. Equal protein loading was evaluated by β-actin levels. The signal intensity of each band was analyzed by GelQuant software (DNR Bio-Imaging Systems Ltd., Jerusalem, Israel). Each bar represents a mean±S.E.M. of three independent experiments in comparison with that of the control set at 100%. *significantly different from control (p<0.05), +significantly different from IL-1β alone (p<0.05) (cont: control, concentration unit is µM).

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