Comparison of glucose derivatives effects on cartilage degradation

Abstract

Background: Glucosamine (GlcN) is a well-recognized candidate for treatment of osteoarthritis. However, it is currently used in derivative forms, such as glucosamine-hydrochloride (GlcN-HCl) or glucosamine sulfate (GlcN-S). However, the molecular mode of action remains unclear. In this study, we compared the effects of Glucose (Glc), Glucuronic acid (GlcA), Glucosamine hydrochloride (GlcN-HCl) and Glucosamine sulfate (GlcN-S) on cartilage degradation.

Methods: Porcine cartilage explants were co-cultured with recombinant human IL-1beta and each tested substance for 3 days. HA, s-GAG and MMP-2 releases to media were measured using ELISA, dye-binding assay and gelatin zymography, respectively. Similar studies were performed in a human articular chondrocytes (HAC) monolayer culture, where cells were co-treated with IL-1beta and each reagent for 24 hours. Subsequently, cells were harvested and gene expression measured using RT-PCR. All experiments were carried out in triplicate. Student's t-tests were used for statistical analysis.

Results: In cartilage explants treated with IL-1beta, GlcN-S had the highest chondroprotective activity of all four chemicals as shown by the inhibition of HA, s-GAG and MMP-2 released from cartilage. The anabolic (aggrecan core protein; AGG, SOX9) and catabolic (MMP-3, -13) genes in HACs treated with IL-1beta and with/without chemicals were studied using RT-PCR. It was found that, GlcN-HCl and GlcN-S could reduce the expression of both MMP-3 and -13 genes. The IL-1beta induced-MMP-13 gene expression was decreased maximally by GlcN-S, while the reduction of induced-MMP-3 gene expression was greatest with GlcN-HCl. Glc and GlcA reversed the effect of IL-1beta on the expression of AGG and SOX9, but other substances had no effect.

Conclusion: This study shows that glucosamine derivatives can alter anabolic and catabolic processes in HACs induced by IL-1beta. GlcN-S and GluN-HCl decreased induced MMP-3 and -13 expressions, while Glc and GlcA increased reduced-AGG and SOX9 expression. The chondroprotective study using porcine cartilage explant showed that GlcN-S had the strongest effect.

Figure 1

The effects of Glc, GlcN-S, GlcA and GlcN-HCl: release of s-GAG, HA from porcine cartilage tissues to the media, the uronic acid remaining in the cartilage tissue. Porcine cartilage explants were cultured with IL-1β (25 ng/ml) in absence and presence of each chemical (at varying concentrations of 20, 40, 80 mM) for 3 days. In the media, the s-GAG release was measured by using a dye-binding assay, and HA release was measured by ELISA. Cartilage discs were digested with papain and then the uronic acid content was measured. *, ** Denotes: a value that is significantly different (p < 0.05 and p < 0.01, respectively) from the IL-1β control.

Figure 2

Effects of Glc, GlcN-S, GlcA and GlcN-HCl on the production of MMP-2. Porcine cartilage explants were cultured with IL-1β (25 ng/ml) in the absence and presence of each chemical (at varying concentrations of 20, 40, 80 mM) for 3 days. Media were collected and were then analyzed by gelatin zymography as described in the text. Three experiments were carried out independently and they were reproducible. *, ** Denotes a value that is significantly different (p < 0.05 and p < 0.01, respectively) from the IL-1β control.

Figure 3

The cytotoxic effects of Glc, GlcN-S, GlcA and GlcN-HCl. The cytotoxic effect of all reagents at concentrations 5, 10 and 20 mM in human articular chondrocytes were studied by the MTT assay.

Figure 4

Effects of Glc, GlcN-S, GlcA and GlcN-HCl on the release of HA (A), s-GAG (B) and MMP-2 (C) from chondrocytes. Chondrocytes were co-treated with 10 ng/ml IL-1β and various concentrations of each chemical (5, 10, 20 mM) for 24 hours. The conditioned media were analyzed for HA, s-GAG and MMP-2 activity as described in the Experimental section. *,** Denotes a value that is significantly different (p<0.05 and p<0.01, respectively) from the IL-1β control.

Figure 5

The mRNA expression of MMP-3, MMP-13, AGG and SOX9 in fresh isolated chondrocytes (P0) and in the fourth cultured passage chondrocytes (P4). Passage 0 and P4 confluent human chondrocytes in 25-cm³ flasks were cultured in serum free-DMEM for 24 hours. Cells were harvested and gene expression was analyzed. MMP, matrix metalloproteinase, AGG, aggrecan; SOX9, SRY-type HMG box.

Figure 6

Effect of Glc, GlcN-S, GlcA and GlcN-HCl on the mRNA expression of proteinases [MMP-3 (A), -13 (B)]. Confluent human chondrocytes in 25-cm³ flasks were cultured with IL-1β (10 ng/ml) in the presence and absence of each chemical for 24 hours. Cells were harvested and gene expression was analyzed. MMP, matrix metalloproteinase. *, ** Denotes a value that is significantly different (p < 0.05 and p < 0.01, respectively) from the IL-1β control.

Figure 7

Effects of Glc, GlcN-S, GlcA and GlcN-HCl on the mRNA expression of cartilage genes [AGG (A), SOX9 (B)]. Confluent human chondrocytes in 25-cm³ flasks were cultured with IL-1β (10 ng/ml) in the presence and absence of each chemical for 24 hours. Cells were harvested and gene expression was analyzed. AGG, aggrecan; SOX9, SRY-type HMG box. *, ** Denotes a value that is significantly different (p < 0.05 and p < 0.01, respectively) from the IL-1β control.