Iksan526 Rice Callus Extract Induces Dedifferentiation of Rabbit Articular Chondrocytes via ERK1/2 and PI-3K/Akt Pathways

doi:10.1016/j.rsci.2020.09.007

摘要/Abstract

. [J]. Rice Science, 2020, 27(6): 504-514.

Fig. 1. Resveratrol content in rice callus determined Dongjin high performance liquid chromatography and effects of resveratrol-enriched rice callus extract on rabbit articular chondrocyte. A, Resveratrol content in the callus of wild-type Dongjin and IS526. Cells were treated with resveratrol, Dongjin or IS526 for 24 h. B, Cellular morphology observed using a phase-contrast microscopy (× 100) and cell proliferation determined by the methylthiazole tetrazolium assay. C, Expression levels of proteins determined by western blot analysis with β-actin as a loading control, and by densitometry measurement. Data represent Mean ± SD (n = 3). *, P < 0.05.

Fig. 2. Effects of IS526 on cell proliferation in rabbit articular chondrocytes. A, Cellular morphology observed using a phase-contrast microscopy (× 100). B, Cell proliferation determined by the methylthiazole tetrazolium assay. Data represent Mean ± SD (n = 3). *, P < 0.05. C, Chondrocytes treated with 94 nmol/L IS526 for 24 h, and cells analyzed for DNA content by a fluorescence-activated cell sorting flow cytometer.

Fig. 3. Effect of IS526 treatment on chondrocyte differentiation and MMP-13 expression. A, Expression levels of type II collagen, SOX-9 and β-actin determined by western blot analysis with β-actin as a loading control. B, Synthesis of proteoglycans determined by Alcian blue staining. C, Expression levels of MMP-13 and β-actin determined by western blot analysis with β-actin as a loading control. D, Relative amounts of MMP-13 quantified by densitometry measurements. Rabbit articular chondrocytes were treated with various concentrations of IS526 for 24 h or 94 nmol/L IS526 for the indicated times. Data represent Mean ± SD (n = 3). *, P < 0.05.

Fig. 4. Effects of matrix metalloproteinase inhibitor (MMPI) and MMP-13 on IS526-induced dedifferentiation in rabbit articular chondrocytes. A, Expression levels of type II collagen, SOX-9, pERK, pp38, pAkt and β-actin (loading control) determined by western blot analysis. B, Synthesis of proteoglycans determined by Alcian blue staining. Data represent Mean ± SD (n = 3). *, P < 0.05.

Fig. 5. Effects of IS526 on the phosphorylation of MAPKs and PI-3K/Akt detected by western blot analyses. A and B, Rabbit articular chondrocytes were treated with 94 nmol/L IS526 for the indicated times or various concentrations of IS526 for 10 min.

Fig. 6. Effects of PD98059 (PD), SB203580 (SB) and LY294002 (LY) on IS526- induced dedifferentiation in rabbit articular chondrocytes. A, Type II collagen, SOX-9, MMP-13, pERK, pp38, pAkt and β-actin (loading control) were detected by western blotting. B, Sulfated proteoglycan synthesis was detected by Alcian blue staining. Data represent Mean ± SD (n = 3), and *, P < 0.05.

Fig. 7. IS526 induces dedifferentiation via the ERK1/2 and PI-3K/Akt pathways. A, Expression of type II collagen detected by immunocytochemistry (× 400). B, Cartilage tissues at 7 days past fertilization visualized by Alcian blue staining (magnification × 50) and morphometric measurements (× 50). MK, Meckel’s cartilage; CB, Ceratobranchial cartilage; CH, Ceratohyal cartilage; PD, PD98059; SB, SB203580; LY, LY294002.Data represent Mean ± SD (n = 3). *, P < 0.05.

Fig. 8. Graphical depiction of effects of IS526 on regulation of dedifferentiation in rabbit articular chondrocytes.

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