Modulation of Apoptosis and Differentiation by the Treatment of Sulfasalazine in Rabbit Articular Chondrocytes

Abstract

This study was conducted to examine the cellular regulatory mechanisms of sulfasalazine (SSZ) in rabbit articular chondrocytes treated with sodium nitroprusside (SNP). Cell phenotype was determined, and the MTT assay, Western blot analysis and immunofluorescence staining of type II collagen was performed in control, SNP-treated and SNP plus SSZ (50~200 μg/mL) rabbit articular chondrocytes. Cellular proliferation was decreased significantly in the SNP-treated group compared with that in the control (p < 0.01). SSZ treatment clearly increased the SNP-reduced proliferation levels in a concentration-dependent manner (p < 0.01). SNP treatment induced significant dedifferentiation and inflammation compared with control chondrocytes (p < 0.01). Type II collagen expression levels increased in a concentration-dependent manner in response to SSZ treatment but were unaltered in SNP-treated chondrocytes (p < 0.05 and < 0.01, respectively). Cylooxygenase-2 (COX-2) expression increased in a concentration-dependent manner in response to SSZ treatment but was unaltered in SNP-treated chondrocytes (p < 0.05). Immunofluorescence staining showed that SSZ treatment increased type II collagen expression compared with that in SNP-treated chondrocytes. Furthermore, phosphorylated extracellular regulated kinase (pERK) expression levels were decreased significantly in the SNP-treated group compared with those in control chondrocytes (p < 0.01). Expression levels of pERK increased in a concentration-dependent manner by SSZ but were unaltered in SNP-treated chondrocytes. pp38 kinase expression levels increased in a concentration-dependent manner by SSZ but were unaltered in control chondrocytes (p < 0.01). In summary, SSZ significantly inhibited nitric oxide-induced cell death and dedifferentiation, and regulated extracellular regulated kinases 1 and 2 and p38 kinase in rabbit articular chondrocytes.

Keywords: Apoptosis; Chondrocyte; Differentiation; Nitric oxide; Sulfasalazine.

Fig. 1

Effects of sulfasalazine (SSZ) on the morphological change and proliferation in chondrocyte cells. Rabbit articular chondrocytes untreated (control) or treated with 298 μg/mL SNP and 50~200 μg/mL of SSZ plus 298 μg/mL of SNP at 24 hr. (A) The apoptotic cells death was determined by phase-contrast microscope (magnification, 200×). (B) Primary chondrocytes apoptosis were determined by MTT assay. ^**Significantly different compared from control; ^##Significantly different compared from SNP treatment (p< 0.01); Values were presented as the mean ± SD.

Fig. 2

Effects of sulfasalazine (SSZ) on SNP-induced dedifferentiation and inflammation in chondrocyte cells. (A) Rabbit articular chondrocytes treated 298 μg/mL of SNP and 50~200 μg/mL of SSZ plus SNP at 24 hr and subjects to western blot analysis with antibodies type II collagen, COX-2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B, C) It was determined that the protein levels of type II collagen and COX-2 were subsequently quantified by densitometry analysis. Expression of GAPDH was used as loading control. The data represented a typical experiment, whereby similar results obtained from three. ^{*, **}Significantly different compared from control (p < 0.05 and p < 0.01, respectively); ^{#, ##}Significantly different compared from SNP treatment (p < 0.05 and p < 0.01, respectively); Values were presented as the mean ± SD.

Fig. 3

Expression of type II collagen in chondrocyte by immunofluorescence staining. Rabbit articular chondrocytes untreated (control) or treated 298 μg/mL of SNP and 200 μg/mL of sulfasalazine (SSZ) plus SNP at 24 hr. Expression of type II collagen (green) and DAPI (blue) identify by immunofluorescence microscopy (200×).

Fig. 4

Expression of pERK and pp38 kinase protein in chondrocyte cell by western blot analyses. (A) Rabbit articular chondrocytes treated 298 μg/mL of SNP and 50~200 μg/mL of sulfasalazine (SSZ) plus SNP at 24 hr and subjects to western blot analysis with antibodies pERK, pp38 kinase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). (B, C) It was determined that the protein levels of pERK and pp38 kinase were subsequently quantified by densitometry analysis. Expression of GAPDH was used as loading control. The data represented a typical experiment, whereby similar results obtained from three. ^{*, **}Significantly different compared from control (p < 0.05 and p < 0.01, respectively); ^{#, ##}Significantly different compared from SNP treatment (p < 0.05 and p < 0.01, respectively); Values were presented as the mean ± SD.