Korean J Physiol Pharmacol.  2017 Jan;21(1):19-26. 10.4196/kjpp.2017.21.1.19.

Betulin suppressed interleukin-1β-induced gene expression, secretion and proteolytic activity of matrix metalloproteinase in cultured articular chondrocytes and production of matrix metalloproteinase in the knee joint of rat

• Ra HJ
• Lee HJ
• Jo HS
• Nam DC
• Lee YB
• Kang BH
• Moon DK
• Kim DH
• Lee CJ
• Hwang SC

• KMID: 2371064
• DOI: http://doi.org/10.4196/kjpp.2017.21.1.19

Abstract

We investigated whether betulin affects the gene expression, secretion and proteolytic activity 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 effect of betulin. Rabbit articular chondrocytes were cultured and reverse transcription-polymerase chain reaction (RT-PCR) was used to measure interleukin-1β (IL-1β)-induced gene expression of MMP-3, MMP-1, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4), ADAMTS-5 and type II collagen. Effect of betulin on IL-1β-induced secretion and proteolytic activity of MMP-3 was investigated using western blot analysis and casein zymography, respectively. Effect of betulin on MMP-3 protein production was also examined in vivo. The results were as follows: (1) betulin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5, but increased the gene expression of type II collagen; (2) betulin inhibited the secretion and proteolytic activity of MMP-3; (3) betulin suppressed the production of MMP-3 protein in vivo. These results suggest that betulin can regulate the gene expression, secretion, and proteolytic activity of MMP-3, by directly acting on articular chondrocytes.

Keyword

Betulin; Chondrocyte; Metalloproteinase; Osteoarthritis

MeSH Terms

Animals
Blotting, Western
Caseins
Chondrocytes*
Collagen Type II
Gene Expression*
Knee Joint*
Knee*
Osteoarthritis
Rats*
Thrombospondins
Caseins
Collagen Type II
Thrombospondins

Figure

• 

  Fig. 1 Effect of betulin 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 betulin 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 upper figure is a representative image data. The signal intensity of each band in images was analyzed by GelQuant software and means of individual group from the three independent experiments were converted to percent control and expressed as mean±S.E.M. Each bar in the lower figure (graph) represents a mean±S.E.M. of three independent experiments in comparison with that of the control set at 100% (Fig. 1, 3, 4 and 5). *significantly different from control (p<0.05). †significantly different from IL-1β alone (p<0.05) (cont: control, concentration unit is µM).

• 

  Fig. 2 Effect of betulin on proliferation of rabbit chondrocytes. Chondrocytes were incubated for 72 h in the presence of varying concentrations of betulin. 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. 3 Effect of betulin on the gene expression of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5, or collagen type II in rabbit chondrocytes.Primary cultured rabbit articular chondrocytes were pretreated with varying concentrations (1, 10, 50, and 100 µM) of betulin 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, ADAMTS-5, or collagen type II was measured by RT-PCR.

• 

  Fig. 4 Effects of betulin on IL-1β-induced secretion of MM P-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 betulin 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.

• 

  Fig. 5 Effect of betulin on production of MMP-3 in vivo.The knee joint of rats were pretreated with 50 or 100 µM of betulin 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. Equal protein loading was evaluated by β-actin levels.

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