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. 2016 Mar 1;24(2):163-70.
doi: 10.4062/biomolther.2015.217.

Apigenin Regulates Interleukin-1β-Induced Production of Matrix Metalloproteinase Both in the Knee Joint of Rat and in Primary Cultured Articular Chondrocytes

Jin Sung Park  1 , Dong Kyu Kim  2 , Hyun-Dae Shin  2 , Hyun Jae Lee  3 , Ho Seung Jo  1 , Jin Hoon Jeong  1 , Young Lac Choi  1 , Choong Jae Lee  3 , Sun-Chul Hwang  1
Affiliations

Apigenin Regulates Interleukin-1β-Induced Production of Matrix Metalloproteinase Both in the Knee Joint of Rat and in Primary Cultured Articular Chondrocytes

Jin Sung Park et al. Biomol Ther (Seoul). .

Abstract

We examined whether apigenin affects the gene expression, secretion and activity of matrix metalloproteinase-3 (MMP-3) in primary cultured rabbit articular chondrocytes, as well as in vivo production of MMP-3 in the knee joint of rat to evaluate the potential chondroprotective effects of apigenin. 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 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 apigenin on IL-1β-induced secretion and proteolytic activity of MMP-3 were investigated using western blot analysis and casein zymography, respectively. The effect of apigenin on MMP-3 protein production was also examined in vivo. In rabbit articular chondrocytes, apigenin inhibited the gene expression of MMP-3, MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. Furthermore, apigenin inhibited the secretion and proteolytic activity of MMP-3 in vitro, and inhibited production of MMP-3 protein in vivo. These results suggest that apigenin can regulate the gene expression, secretion, and activity of MMP-3, by directly acting on articular chondrocytes.

Keywords: Apigenin; Chondrocyte; Metalloproteinase; Osteoarthritis.

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Figures

Fig. 1.
Fig. 1.
Effect of hesperidin, hesperetin or apigenin 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 hesperidin, hesperetin or apigenin 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 (A, B and C). 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. 2.
Fig. 2.
Effect of apigenin on proliferation of rabbit chondrocytes. Chondrocytes were incubated for 72 h in the presence of varying concentrations of apigenin. 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.
Fig. 3.
Effect of apigenin 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 μM) of apigenin 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. 4.
Fig. 4.
Effect of apigenin 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 apigenin 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. The fourth panel was black-and-white version of 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. 5.
Fig. 5.
Effect of apigenin on the production of MMP-3 in vivo. The knee joint of rats were pretreated with 50 or 100 μM of apigenin 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 immunoreactive bands 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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