Effect of oleanolic acid on the activity, secretion and gene expression of matrix metalloproteinase-3 in articular chondrocytes in vitro and the production of matrix metalloproteinase-3 in vivo

Abstract

In the present study, we tried to examine whether oleanolic acid regulates the activity, secretion and gene expression of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as the production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effect of oleanolic acid. Rabbit articular chondrocytes were cultured in a monolayer, 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. In rabbit articular chondrocytes, the effects of oleanolic acid on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of oleanolic acid on in vivo MMP-3 protein production was also examined, after intra-articular injection to the knee joint of rat. The results were as follows: (1) oleanolic acid 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) oleanolic acid reduced the secretion and proteolytic activity of MMP-3; (3) oleanolic acid suppressed the production of MMP-3 protein in vivo. These results suggest that oleanolic acid can regulate the activity, secretion and gene expression of MMP-3, by directly acting on articular chondrocytes.

Keywords: Chondrocytes; MMPs; Oleanolic acid; Osteoarthritis.

Fig. 1. Effect of oleanolic acid 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 oleanolic acid 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% (Figs. 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 oleanolic acid on proliferation of rabbit chondrocytes.

Chondrocytes were incubated for 72 h in the presence of varying concentrations of oleanolic acid. 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 oleanolic acid 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 oleanolic acid 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 oleanolic acid 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 oleanolic acid for 2 h and then stimulated with IL-1β (10 ng/mL) for 24h. 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 oleanolic acid on production of MMP-3 in vivo.

The knee joint of rats were pretreated with 50 or 100 µM of oleanolic acid 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.