Synergistic chondroprotective effect of alpha-tocopherol, ascorbic acid, and selenium as well as glucosamine and chondroitin on oxidant induced cell death and inhibition of matrix metalloproteinase-3--studies in cultured chondrocytes

Abstract

Overproduction of reactive oxygen species and impaired antioxidant defence accompanied by chronic inflammatory processes may impair joint health. Pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) stimulate the expression of metalloproteinases which degrade the extracellular matrix. Little is known regarding the potential synergistic effects of natural compounds such as alpha-tocopherol (alpha-toc), ascorbic acid (AA) and selenium (Se) on oxidant induced cell death. Furthermore studies regarding the metalloproteinase-3 inhibitory activity of glucosamine sulfate (GS) and chondroitin sulfate (CS) are scarce. Therefore we have studied the effect of alpha-toc (0.1-2.5 micromol/L), AA (10-50 micromol/L) and Se (1-50 nmol/L) on t-butyl hydroperoxide (t-BHP, 100-500 micromol/L)-induced cell death in SW1353 chondrocytes. Furthermore we have determined the effect of GS and CS alone (100-500 micromol/L each) and in combination on MMP3 mRNA levels and MMP3 secretion in IL-1beta stimulated chondrocytes. A combination of alpha-toc, AA, and Se was more potent in counteracting t-BHP-induced cytotoxicity as compared to the single compounds. Similarly a combination of CS and GS was more effective in inhibiting MMP3 gene expression and secretion than the single components. The inhibition of MMP3 secretion due to GS plus CS was accompanied by a decrease in TNF-alpha production. Combining natural compounds such as alpha-toc, AA, and Se as well as GS and CS seems to be a promising strategy to combat oxidative stress and cytokine induced matrix degradation in chondrocytes.

Figure 1

Effect of glucosamine and chondroitin on cell viability in SW1353 cells. Cells were treated with increasing concentrations of test substances for 24 h. The test compounds did not impair cell viability at any of the concentrations tested. Cytotoxicity was determined by the neutral red assay and cell viability is expressed as percentage of control (untreated cells).

Figure 2

Effect of α-tocopherol, ascorbic acid, selenium, and their combination on cell viability in SW1353 cells after challenge with tBHP. Cells were treated with α-tocopherol (0.1 µmol/L), ascorbic acid (10 mmol/L), selenium (1 nmol/L), and a combination of all three test compounds (0.1 µmol/L α‑tocopherol, 10 µmol/L ascorbic acid, and 1 nmol/L selenium) for 24 h. After 3 h of challenge with 200 µmol/L t-BHP the neutral red assay was performed. Data are means + SEM of two independent experiments performed in duplicate. # p < 0.01; compared to control.

Figure 3

MMP3 mRNA levels in SW1353 cells following 6, 12, and 24 h of incubation with IL-1β (10 ng/mL). The highest induction of MMP3 was observed after 24 h of incubation. Results are calculated in relation to β-actin and expressed as means + SEM of three independent experiments performed in duplicate.

Figure 4

Effect of glucosamine and chondroitin on MMP3 mRNA levels (A) and protein secretion (B) in IL-1β stimulated SW1353 cells. Cells were stimulated with IL-1β (10 ng/mL) in the presence of different concentrations of glucosamine, chondroitin and the combination of both test compounds for 24 h. Cell culture supernatants were collected and the amount of MMP3 secreted by SW1353 cells was measured by specific ELISA. Results for MMP3 mRNA are calculated in relation to β-actin and compared to IL-1β-treated cells. Data are means + SEM of three independent experiments performed in duplicate. * p < 0.05; # p < 0.01; § p < 0.001; compared to IL-1β.

Figure 5

Effect of glucosamine and chondroitin on TNFα protein secretion in IL-1β stimulated SW1353 cells. Cells were stimulated with IL-1β (10 ng/mL) in the presence of 500 µmol/L of glucosamine, chondroitin and the combination of both test compounds for 24 h. Cell culture supernatants were collected and the amount of TNFα secreted by SW1353 cells was measured by specific ELISA. Data are means + SEM of three independent experiments performed in duplicate. * p < 0.05; compared to IL-1β.