Glucosamine Hydrochloride but Not Chondroitin Sulfate Prevents Cartilage Degradation and Inflammation Induced by Interleukin-1α in Bovine Cartilage Explants

Abstract

Objective: Glucosamine hydrochloride (GH) and chondroitin sulfate (CS) are commonly used for the treatment of osteoarthritis (OA). The aim of this study was to assess their effects, alone and in combination, on preventing aggrecan degradation and inflammation in an in vitro model of OA.

Design: To test the effects of GH and/or CS as a preventative treatment, cartilage explants were pretreated with the compound(s) using concentrations that showed no detrimental effect on chondrocyte viability. Interleukin-1α (IL-1α) was added to induce cartilage degradation, supernatant and explants were analyzed for proteoglycan degradation products, aggrecanase mRNA expression and activity, and for the release of inflammatory markers.

Results: Following treatment with IL-1α, 2 mg/mL dose of GH pretreatment was associated with a reduction of glycosaminoglycan release, reduced generation of the pathological interglobular domain aggrecan catabolites, decreased mRNA levels of ADAMTS-4 and -5 and reduced activity of ADAMTS-4. In contrast, CS alone did not have a significant effect on IL-1α-induced cartilage degradation and the addition of 0.4 mg/mL CS to 2 mg/mL GH did not further inhibit IL-1α-induced activity. Pretreatment with 2 mg/mL GH also reduced the release of inflammatory markers, prostaglandin E2 and nitric oxide induced by IL-1α while CS did not have a significant effect.

Conclusions: The results suggest that GH prevents cartilage degradation mediated by aggrecanases ADAMTS-4 and -5, and may also reduce inflammation. This could be part of the mechanisms by which GH is effective in maintaining joint integrity and function, and preventing or delaying early symptoms of OA.

Keywords: aggrecanase; chondroitin; glucosamine; osteoarthritis.

Figure 1.

Cellular metabolic activity of chondrocytes with chondroitin sulfate (A) and glucosamine hydrochloride (B), assessed individually and in combination (C) using MTT assay. Values represent the mean relative absorbance to the control without treatment ± SD, n = 3. One-sample Student t test was used. * indicates P < 0.05 for a comparison with the control without glucosamine hydrochloride (GH) and/or chondroitin sulfate (CS).

Figure 2.

Sulfated glycosaminoglycan (s-GAG) release measured using DMMB assay in tissue culture supernatant at 72 hours post-stimulation with interleukin-1α (IL-1α), in the presence of chondroitin sulfate (A) and glucosamine hydrochloride (B) individually, and with a combination (C). Values represent the mean GAG release in μg per mg of tissue ± SEM, n = 3. One-sample Student t test was used. ^§Indicates P < 0.05 for a comparison with the control without IL-1α stimulation. ^# indicates P < 0.05 for a comparison with the control with IL-1α stimulation.

Figure 3.

Western bolt analysis of tissue culture supernatant using BC-3 antibody to assess the effect of chondroitin sulfate (A) and glucosamine hydrochloride (B) individually and in combination (C) on the level of aggrecanases products at 72 hours post-stimulation with interleukin-1α (IL-1α). (1) Densitometry was used to measure the overall density of the bands. Values represent the mean area under the curve (AUC) in arbitrary units ± SEM, n = 9. ^# indicates P < 0.05 for a 1-sample Student t test comparison with the control with IL-1α stimulation. (2) Representative BC-3 Western blots. M: Molecular mass marker.

Figure 4.

ADAMTS-4 mRNA expression (A) and ADAMTS-5 mRNA expression (B) at 24 hours post-stimulation with interleukin-1α (IL-1α), with chondroitin sulfate (CS) and glucosamine hydrochloride (GH) pretreatment. Values represent the mean fold change in expression relative to the control without IL-1α ± SEM, n = 3. One-sample Student t test. ^§ indicates P < 0.05 for a comparison with the control without IL-1α stimulation. ^# indicates P < 0.05 for a comparison with the control with IL-1α stimulation.

Figure 5.

Effect of glucosamine hydrochloride (GH) and chondroitin sulfate (CS) on ADAMTS-4 activity at 72 hours post-stimulation with interleukin-1α (IL-1α). Values represent the mean relative fluorescence per mg tissue ± SEM, n = 3. One-sample Student t test was used. & indicates P < 0.05 for comparison between no-IL-1α and IL-1α control samples.

Figure 6.

Effect of glucosamine hydrochloride (GH) and chondroitin sulfate (CS) on prostaglandin E₂ (PGE₂) production (A) and on nitric oxide (NO) production (B) at 72 hours post-stimulation with interleukin-1α (IL-1α). (A) Values represent the mean PGE₂ level expressed in pg per mg tissue ± SEM, n = 3. (B) Values represent the mean NO level expressed in nanomoles per mg tissue ± SEM, n = 3. One-sample Student t test was used & indicates P < 0.05 for comparison between no-IL-1α and IL-1α samples with the same treatment. ^# indicates P < 0.05 for a comparison with the control with IL-1α stimulation.