Abstract
INTRODUCTION
Many patients suffering from osteoarthritis (OA) take daily glucosamine (GlcN) in the hope of slowing down disease progression and ameliorating pain. However, the physiological basis of this effect is not known. We previously presented preliminary data suggesting that GlcN prevented the increase in interleukin-1beta (IL-1b) expression caused by addition of inflammatory cytokines to cultures of healthy human articular chondrocytes. Previous studies had also shown that, in OA, epigenetic DNA methylation loss at specific CpG sites in relevant promoters ‘unsilences’ the genes and that this DNA de-methylation underlies the aberrant gene expression of proteases (Arthritis Rheum 52;3110-24). Furthermore, exogenous inflammatory cytokines have the capacity to cause DNA de-methylation in the IL-1b promoter (Arthritis Rheum. 2009, 60, 3303-3313). The aims of the present study were to investigate whether GlcN not only prevents the increased IL-1b expression, but also inhibits epigenetic unsilencing by preventing the cytokine-induced loss of DNA methylation.
METHODS
Healthy chondrocytes were isolated from the articular cartilage of four femoral heads, after operations following femoral neck fracture (ethic permission was obtained). The chondrocytes were cultured for 5 weeks in four treatment groups: no treatment (control); with IL-1b and oncostatin M (IL1b+OSM); with 2.0mM GlcN; and with IL1b+OSM+GlcN. Total RNA and genomic DNA were extracted. The % DNA methylation at the CpG site at -299bp (previously identifies as the crucial CpG site) was determined after bisulphite modification with a pyrosequencer. Gene expression of IL-1B was quantified by SybrGreen-based qRT-PCR.
RESULTS
The chondrocytes from one patient grew too slowly to obtain results. In two patients (aged 80 and 85), exogenous IL-1b increased expression of IL-1b several 100-fold and reduced the % DNA methylation from 60% to 40%. GlcN alone showed no significant difference to the control group. When GlcN was present together with IL1b, the increase in expression of IL1B was only 1/3 of that cause by IL-1b and the loss of DNA methylation was prevented. The final sample (patient aged 94), showed a low % DNA methylation (46%) even in the control culture and glucosamine had no effect. This may be an anomaly due to the great age of the patient.
DISCUSSION
The results suggest that GlcN is capable of ameliorating the cytokine-induced loss of DNA methylation. However, patient numbers are too low to have statistical significance. Further work is clearly needed confirm the hypothesis that DNA de-methylation of the IL-1b promoter caused by exogenous IL-1b is reversed by GlcN.
