Fig 1.
Reagents and conditions: (a) Acetyl oxide, oil bath, 120°C (b) SOCl2, oil bath, 80°C (c) Sulfamethoxazole, THF, Pyridine, ice bath (d) HCl, THF, 60°C.
Table 1.
Primers for RT-PCR performance.
Fig 2.
Cytotoxicity of JEZ-C(A), GA (B) and SMZ(C) on chondrocytes after 3 d (mean ± SD, n = 5).
*p<0.05, **p<0.01, ***p<0.001.
Fig 3.
Quantitative comparison of ECM-related gene expression of MMP-1 (A), MMP-13 (B) and TIMP-1 (C) by qRT-PCR.
The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (without IL-1β), Model (with IL-1β), JEZ-C (J-1: 6.25×10−7 μg/ml; J-2: 6.25×10−6 μg/ml; J-3: 6.25×10−5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SMZ (S-1: 6.25×10−6 μg/ml; S-2: 6.25×10−5μg/ml; S-3: 6.25×10−4 μg/ml) on the induction by IL-1β for 6 days (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analysed by the 2-ΔΔCT method using β-actin as the internal control. The data represent the mean±SD of three independent culture experiments. Bars with different letters are significantly different from each other at P﹤0.05.
Fig 4.
Immunohistochemical staining of MMP-1 (a) and TIMP-1 (b) of chondrocytes cultured in vitro with different concentrations of JEZ-C, GA and SMZ on the induction by IL-1β for 6 d: JEZ-C (A. 6.25×10−7 μg/ml; B. 6.25×10−6 μg/ml; C. 6.25×10−5 μg/ml); Control (D. without IL-1β); GA (E. 0.078 μg/ml; F. 0.125 μg/ml; G. 0.156 μg/ml); Model (H. with IL-1β); SMZ (I. 6.25×10−6 μg/ml; J. 6.25×10−5 μg/ml; K. 6.25×10−4 μg/ml), cell seeding density: 2×104/ml (original magnification ×100).
Scale bar = 200 μm.
Fig 5.
Quantification of cell proliferation (DNA) and matrix production (glycosaminoglycan (GAG)) of cells by biochemical assays: (A) the proliferation of chondrocytes cultured with different concentrations of JEZ-C, GA and SMZ: Control (K: 0 μg/ml), JEZ-C (J-1: 6.25×10−7 μg/ml; J-2: 6.25×10−6 μg/ml; J-3: 6.25×10−5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SDM (S-1: 6.25×10−6 μg/ml; S-2: 6.25×10−5μg/ml; S-3: 6.25×10−4 μg/ml) in vitro for 6 d; (B) GAG (mg) normalized to DNA (mg).
Data from six independent experiments were evaluated and mean ±SD was showed, *P<0.05, **P<0.01, ***P<0.001.
Fig 6.
Hematoxylin-eosin staining (a) and Safranin O/fast green staining (b) images respectively showing the GAG production and the morphology of chondrocytes and cultured in vitro with different concentrations of JEZ-C, GA and SMZ for 6 d: JEZ-C (A. 6.25×10−7 μg/ml; B. 6.25×10−6 μg/ml; C. 6.25×10−5 μg/ml), Control (D. without IL-1β), GA (E. 0.078 μg/ml; F. 0.125 μg/ml; G. 0.156 μg/ml), SMZ (H. 6.25×10−6 μg/ml; I. 6.25×10−5 μg/ml; J. 6.25×10−4 μg/ml); cell seeding density: 2×104/ml (original magnification ×100).
Scale bar = 200 μm.
Fig 7.
Confocal laser scanning microscopy images showing the viability (a) and distribution of the actin cytoskeleton (b) of chondrocytes cultured in vitro with different concentrations of JEZ-C, GA and SMZ for 6 d: JEZ-C (A. 6.25×10−7 μg/ml; B. 6.25×10−6 μg/ml; C. 6.25×10−5 μg/ml), Control (D. without IL-1β), GA (E. 0.078 μg/ml; F. 0.125 μg/ml; G. 0.156 μg/ml), SMZ (H. 6.25×10−6 μg/ml; I. 6.25×10−5 μg/ml; J. 6.25×10−4 μg/ml); cell seeding density: 2×104/ml (original magnification ×100).
Scale bar = 200 μm.
Fig 8.
Immunohistochemical staining images revealed the presence of type I (a) and type II (b) collagen.
Chondrocytes cultured in vitro with different concentrations of JEZ-C, GA and SMZ for 6 d: JEZ-C (A. 6.25×10−7 μg/ml; B. 6.25×10−6 μg/ml; C. 6.25×10−5 μg/ml), Control (D. without IL-1β), GA (E. 0.078 μg/ml; F. 0.125 μg/ml; G. 0.156 μg/ml), SMZ (H. 6.25×10−6 μg/ml; I. 6.25×10−5 μg/ml; J. 6.25×10−4 μg/ml); cell seeding density: 2×104/ml (original magnification ×100). Scale bar = 200 μm.
Fig 9.
Quantitative comparison of ECM-related gene expression of aggrecan (a), collagen II (b), Sox9 (c) collagen I (d) and by qRT-PCR.
The chondrocytes were cultured with different concentrations of JEZ-C, GA and SMZ: Control (K: 0 μg/ml), JEZ-C (J-1: 6.25×10−7 μg/ml; J-2: 6.25×10−6 μg/ml; J-3: 6.25×10−5 μg/ml), GA (G-1: 0.078 μg/ml; G-2: 0.125 μg/ml; G-3: 0.156μg/ml) and SDM (S-1: 6.25×10−6 μg/ml; S-2: 6.25×10−5μg/ml; S-3: 6.25×10−4 μg/ml) for 6 d (n = 3 for each experiment). The gene expression levels in JEZ-C, GA and SMZ media relative to the control group were analyzed by the 2-ΔΔCT method using β-actin as the internal control. The data represent the means ± SD of three independent culture experiments. *p<0.05, **p<0.01, ***P<0.001.