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Rheumatology 2008 47(1):31-35; doi:10.1093/rheumatology/kem289
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© The Author 2007. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Influence of glucosamine sulphate on oxidative stress in human osteoarthritic chondrocytes: effects on HO-1, p22Phox and iNOS expression

C. Valvason1, E. Musacchio2, A. Pozzuoli3, R. Ramonda1, R. Aldegheri3 and L. Punzi1

1Rheumatology Unit, Department of Clinical and Experimental Medicine, 2Clinica Medica I, Department of Medical and Surgical Sciences and 3Department of Medical and Surgical Specialties, Orthopaedic Clinic, University of Padova, Italy.

Correspondence to: L. Punzi, Rheumatology Unit, Department of Clinical and Experimental Medicine, University of Padova, Via Giustiniani, 2-35128 Padova, Italy. E-mail: punzireu{at}unipd.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgement
 References
 
Objective. Reactive oxygen species (ROS) are major determinants in the alteration of articular cartilage. Among protective cellular mechanisms, the inducible isoform of haem oxygenase (HO-1) plays a particularly relevant role. On the other hand, the enzymatic activity of the Nicotinamide adenine dinucleotide phosphate (NADPH) system could contribute to the generation of ROS. Glucosamine sulphate (GS) is one of the drugs used in the treatment of osteoarthritis; however, its mechanism of action is still largely unknown. The aim of the present study was to investigate the effects of GS on primary human chondrocytes in vitro, in particular with regard to HO-1, p22Phox (a subunit of NADPH complex) and inducible nitric oxide synthase (iNOS) expression.

Methods. Primary human chondrocytes were treated with different concentrations of GS; gene expression of HO-1, p22Phox and iNOS was assessed by the reverse transcriptase–polymerase chain reaction method. In a separate set of experiments, the cells were stimulated with human recombinant interleukin (IL)-1β and simultaneously treated with GS. Moreover, HO-1 protein and total nitrite production were evaluated.

Results. HO-1 gene expression was up-regulated (+40% with respect to the controls, P < 0.001) by 10 mmol/l GS at 24 h, while p22Phox gene expression was down-regulated by 10 mmol/l GS with a maximum inhibitory effect observed after 48 h treatment. IL-1β stimulation induced expression of iNOS reverted by 1 and 10 mmol/l GS. Moreover, HO-1 gene expression was down-regulated by IL-1β and 10 mmol/l GS restored baseline values. These data were confirmed by evaluating HO-1 protein level and nitrite production.

Conclusions. The influence of GS on oxidative stress observed in this study discloses a possible new mechanism of action and seems to be in keeping with a potential protective effect on chondrocyte population.

KEY WORDS: Glucosamine sulphate, Osteoarthritis, Chondrocytes, Haemoxygenase, p22Phox


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgement
 References
 
Osteoarthritis (OA) is the most common joint disease with a strong socio-economic impact. It is characterized by quantitative and qualitative changes in the architecture and composition of all joint structures [1–3] and accompanied by an excess production of pro-inflammatory cytokines [4–6]. Among these, in chondrocytes, interleukin (IL)-1β induces a cascade of catabolic events including the up-regulation of metalloproteinases (MMPs), inducible nitric oxide synthase (iNOS), cyclo-oxygenase-2 (COX-2) and IL-6 genes [7–10]. In particular, the expression of iNOS has been associated with chondrocytes during the pathogenesis of OA [11] and the overproduction of nitric oxide (NO) was detected in synovial tissue and articular cartilage [12].

Mechanical and chemical stress is thought to increase the production of local free radicals, thereby leading to oxidative damage [13–16] that in the long run alters the joint structure.

Some symptomatic slow-acting drugs, such as glucosamine sulphate (GS), have proved effective in relieving the symptoms of OA [17–19]. Although GS has been proposed as a scavenger of free oxygen radicals, the mechanism of action of this drug remains to be fully elucidated.

Moreover, several studies have demonstrated that oxidative damage due to overproduction of NO and other reactive oxygen species (ROS) derived from superoxide may be involved in the pathogenesis of OA [7, 20–22] and the main ROS produced by chondrocytes are NO and superoxide anion (O2–) that generate derivative radicals, such as peroxynitrite (ONOO–) and hydrogen peroxide (H2O2).

Nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases are a group of plasma membrane-associated enzymes present in a variety of cells of mesodermal origin. The most thoroughly studied of these enzymes is the NADPH oxidase found in phagocytes that catalyses the production of O2–. This complex plays a vital role in the non-specific host defence against pathogens by generating large amounts of O2–. The biochemical activity of non-phagocytes’ NAD(P)H oxidases differs substantially from that of the phagocytes’ (e.g. neutrophils). NADPH oxidase contains the plasma membrane-associated low-potential cytochrome b558 made of two subunits: p22Phox and gp91Phox (the membrane-bound proteins of 22 and 91 kDa, respectively) [23–25]. Moulton and co-workers [26] have demonstrated that p22Phox is expressed by the immortalized chondrocyte cell line C-20/A4.

Haem oxygenase-1 (HO-1) is a rate-limiting enzyme in the oxidative degradation of haem to biliverdin, free iron and carbon monoxide (CO). It is induced by a variety of stimuli involved in cellular stress, including cytokines, mitogens, metals, ROS, heat shock, radiations, hypoxia or hyperoxia. Several lines of evidence support the hypothesis that HO-1 is induced as an adaptive mechanism against injury elicited by these factors [27]; nevertheless, possible protective mechanisms in cartilage are largely unknown, and in particular the role of HO-1 has not been thoroughly investigated.

The aim of this study was to investigate the effects of GS on primary human chondrocytes in vitro.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgement
 References
 
Materials
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, L-glutamine and human IL-1β were purchased from Euroclone, Milano, Italy. Phosphate buffered saline (PBS) and trypsin/ethylene diamine tetra-acetic acid (EDTA) were from Gibco, Milano, Italy. RNAzolB was purchased from Biotecx, Houston, USA, the GeneAmp RNA PCR kit components were from Applied BioSystems, Monza, Italy. The specific primers used for HO-1, p22Phox, iNOS and 18S rRNA were obtained from Primm, Milano, Italy. Collagenase type IA and Bradford Reagent for protein determination were from Sigma, Milano, Italy as well as all the other reagents unless otherwise specified. All chemicals were of the highest grade available. HO-1 determination enzyme-linked immunosorbent assay (ELISA) kit was from Assay Designs Inc., USA (limit of detection 0.78 ng/ml). Griess reagent for NO assessment was from Cayman Chemical, USA. The sulphate salt of glucosamine was kindly provided by the Rotta Research Laboratorium, Monza, Italy.

Cell culture and treatment
Osteoarthritic cartilage was obtained from patients undergoing total hip arthroplasty (n = 5) after informed consent. Chondrocytes were isolated from femoral heads following sequential digestions with trypsin 0.25%–EDTA 0.02% for 15 min and collagenase type IA 1 mg/ml with 5% FBS overnight. Cells were grown in DMEM low-glucose medium supplemented with 10% (v/v) HI-FBS (heat-inactivated FBS) and 100 U/ml penicillin, 100 µg/ml streptomycin. Cultures were maintained in T25 flasks in a humidified 5% CO2 atmosphere at 37°C and the cell viability was tested by trypan blue exclusion. For the experiments, cells were used between passages 2 and 4. Chondrocytes were seeded into six-well plates at 80 000 cells/well and treated at subconfluency for the experiments. For the time-dependent studies, the cells were cultured under conditions of 0.5% (v/v) HI-FBS with GS at concentrations of 0.1, 1, 10 and 50 mmol/l for 6, 24 and 48 h.

A separate set of experiments was performed to determine the effect of GS in oxidative stress conditions induced by IL-1β treatment; the cells were stimulated with human recombinant IL-1β at 200 U/ml and simultaneously treated with GS 1 mmol/l and 10 mmol/l for 24 h, since preliminary experiments demonstrated that IL-1β effect reached its maximum effect at this time point.

Gene expression analysis
Semi-quantitative comparative kinetic reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers was employed for the evaluation of gene expression, using 18S rRNA as the housekeeping gene. Total cellular RNA was extracted using the commercial kit RNAzolB, according to Chomczynski and Sacchi [28], as described elsewhere. Total RNA (300 ng) was used as template for cDNA synthesis in a 20 µl reaction volume containing 2.5 U/µl M-MuLV (Moloney murine leukaemia virus) RT, 2.5 µmol/l random hexamers, 1 U/µl RNasin, 1 mmol/l of each dNTP, 5 mmol/l MgCl2 in 50 mmol/l KCl and 10 mmol/l Tris/HCl buffer (pH 8.3). Following subsequent incubations at room temperature for 10 min, at 42°C for 30 min, at 99°C for 5 min and at 4°C for 5 min, the reaction was stopped and cDNAs were stored at –20°C until used. PCR analysis was performed amplifying 1 µl of the RT reaction product in 25 µl of PCR mixture containing 1 x PCR buffer, 0.2 mmol/l of each dNTP, 1.2 mmol/l MgCl2 and 0.4 µmmol/l of primers using AmpliTaq Gold polymerase (0.2 U/µl) to increase the specificity and the efficiency of the PCR reaction (‘hot start’ procedure). The primer sequences are reported in Table 1.


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  		TABLE 1. PCR primers for gene expression analysis

 
The amplification profile was carried out in a programmable thermal cycler (MJ Research, USA) employing the following conditions for amplification: 95°C for 10 min, 60°C for 1 min and 72°C for 1 min for 23 cycles (HO-1), 28 cycles (p22Phox) and 16 cycles (18S). For iNOS selective amplification, the touchdown procedure was applied consisting, after an initial 10 min denaturation at 95°C, of 1 min at 94°C, annealing for 1 min at temperatures decreasing from 64 to 59°C, the final annealing temperature (with 1°C decremental steps every two cycles), and ending with an extension step at 72°C for 1 min (34 cycles). The number of cycles used for the amplifications of HO-1, p22Phox, iNOS and 18S was obtained from the analysis of a kinetic curve, set for each gene using an increasing number of cycles from 10 to 40 to determine the number of cycles corresponding to the exponential phase of the amplification. PCR products were electrophoresed on a 7% (w/v) polyacrylamide gel (3% cross-linker; 5% glycerol). Silver staining was used to visualize the gel bands and to quantify the PCR products directly on the gel. The HO-1, p22Phox, iNOS and 18S gene expressions were quantified employing a PCR-based densitometric semiquantitative analysis using NIH image software. The ratio of HO-1, p22Phox and iNOS to 18S PCR products, expressed as pixel density (densitometric units), was used as index of gene expression. The amount of HO-1, p22Phox and iNOS was measured and expressed as the ratio between the absorbance of the target gene and the standard gene PCR products.

HO-1 protein determination
HO-1 production was quantified using a commercially available competitive ELISA kit. Briefly, the assay procedure consisted of preparing cell lysates with a mixture of the extraction buffer and protease inhibitors (0.1 mmol/l PMSF, 1 µg/ml leupeptin, 1 µg/ml aproptin, 1 µg/ml pepstatin) in accordance with the supplier's protocol. The samples were added to the coated plates and incubated for 1 h at room temperature; the plates were washed six times and incubated with 100 µl of previously diluted anti-human HO-1 at room temperature for 1 h. After repeated washings, the plates were incubated with 100 µl of anti-rabbit IgG : HRP (horseradish peroxidase) conjugate for 30 min at room temperature and then treated with TMB (tetramethylbenzidine) substrate for peroxidase. The reaction was stopped after 15 min and the optical density (OD) was read at 450 nm on a microplate reader (Labsystems Multiskan MS) and the results expressed as nanograms per millilitre, corrected by the protein content of each lysate sample.

Nitric oxide assay
NO was measured as the concentration of its stable breakdown product, nitrite (NO2–), by the Griess reaction. At the end of the treatment period, the conditioned media were collected, centrifuged, aliquoted and stored at –80°C. The samples were plated in 96-well plates and the reaction was carried out in the presence of nitrate reductase mixture and its enzyme cofactor according to the manufacturer's instructions. Absorbance at 540 nm was determined on a microplate reader (Labsystems Multiskan MS) and the results expressed as micromoles of nitrite/106 cells.

Statistical analysis
Data represent means ± S.D. of five different experiments performed in duplicate. Statistical analysis was carried out by analysis of variance with a between-within design and by Student's t-test, as specified. A P-value less than 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgement
 References
 
Figure 1 shows the time- and dose-dependent effects of GS on HO-1 gene expression in primary human chondrocytes. After 6 h cell culture incubation, 10 mmol/l GS induced up-regulation of 30%, and the peak of expression was reached after 24 h (+40% vs control, P < 0.001). The stimulation was still present after 48 h, though at a lower level. A lower up-regulation was observed at 6 and 24 h with 50 mmol/l GS (26 and 32%, respectively, vs control, P < 0.001, data not shown). This concentration was not used in longer lasting (48 h) experiments as it was found to be cytotoxic in preliminary assays.


Figure 1
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  		FIG. 1. Effect of different concentrations of GS on HO-1 gene expression in human chondrocytes: 10 mmol/l GS, after 6 h cell culture incubation, induced up-regulation of HO-1 reaching the peak after 24 h. The stimulation was still present after 48 h, though at a lower extent. Data are expressed as percentage with respect to basal conditions and represent the mean ± S.D. of five different experiments performed in duplicate (*P < 0.001 vs control). One representative of five experiments is shown at 24 h. Ctr, control.

 
As shown in Fig. 2, a down-regulation of p22Phox mRNA by GS treatment over time was observed; the maximum inhibitory effect was reached after 48 h with both 1 and 10 mmol/l GS (–20 and –25%, respectively, P < 0.005 vs control) and it was already significant after 24 h (P < 0.005 vs control).


Figure 2
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  		FIG. 2. Effect of GS on p22Phox gene expression in human chondrocytes: 1 and 10 mmol/l GS, after 24 and 48 h (maximum inhibitory effect) cell culture incubation, induced down-regulation of p22Phox. Data are expressed as percentage with respect to basal conditions and represent the mean ± S.D. of five different experiments performed in duplicate (*P < 0.005 vs control). One representative of five experiments is shown at 24 h.

 
At 200 U/ml IL-1β induced iNOS expression that was absent in the control. Simultaneous GS treatment at 1 and 10 mmol/l reduced gene expression by 14 and 30%, respectively (P < 0.001) (Fig. 3A). Moreover, HO-1 gene expression was down-regulated by IL-1β (–30% vs control) and GS at 10 mmol/l restored baseline values (P < 0.001) (Fig. 3B).


Figure 3
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  		FIG. 3. GS effect on IL-β-induced iNOS up-regulation (A) and HO-1 gene expression (B). At 24 h, 200 U/ml IL-β-induced iNOS gene expression (a), absent in control cells; simultaneous GS treatment at 1 mmol/l (b) and 10 mmol/l (c) reduced gene expression (*P < 0.001 vs IL-1β) (A). Moreover, HO-1 gene expression was down-regulated by IL-1β (a) and 10 mmol/l GS treatment (c) restored baseline values, while 1 mmol/l GS (b) was ineffective (*P < 0.001 vs IL-1β and vs IL-1β+GS 1 mmol/l) (B). One representative of five experiments is shown at 24 h.

 
Data concerning HO-1 expression were confirmed at the protein level. The analysis of HO-1 protein in cell lysates, reported in Table 2, indicated that it was not significantly modulated by GS after 6 h of treatment, while it was significantly increased (P < 0.001) by GS 1 and 10 mmol/l after 24 and 48 h. Interestingly, in the presence of IL-1β, HO-1 protein level showed a 40% decrease with respect to controls (P < 0.001), but it returned to basal levels after GS 10 mmol/l treatment.


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  		TABLE 2. HO-1 protein and NO production by human chondrocytes treated with GS and IL-1β

 
The modulation of iNOS activity was further investigated by means of the determination of NO2–, the end products of NO breakdown. As shown in Table 2, consistent with the effect observed at molecular level, IL-1-induced nitrite production was significantly decreased by GS 10 mmol/l treatment (P < 0.05).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgement
 References
 
In this study, we have demonstrated that GS stimulates HO-1 gene expression and protein production in human chondrocytes in a dose-dependent manner. The effective concentrations used were 0.1, 1, 10 and 50 mmol/l at 6, 24 and 48 h time points. The most relevant effect was observed using 10 mmol/l for 24 h.

The inducible form of HO confers protection against oxidative stress conditions in vitro and in vivo, through anti-oxidative, anti-apoptotic and anti-inflammatory actions [27, 29–34]. In the context of rheumatoid arthritis, Chae et al. [35] reported that HO-1 is required to protect against the cytotoxicity induced by tumour necrosis factor-{alpha}, an important mediator of inflammatory processes. A recent study demonstrated that there is a partial reduction in the clinical score in collagen-induced arthritis (CIA) mice treated with the HO-1 inducer cobalt protoporphyrin (CoPP) and that a prophylactic administration of this molecule partially reduced the incidence of CIA [36].

Recent evidence has demonstrated strongly contributory roles for endogenous CO generated from HO-1 activity and these potential effects involve the production of guanosine 3',5'-cyclic monophosphate (cGMP), a second messenger molecule [37–39]. In addition, HO-1-deficient mice were reported to exhibit an immune phenotype that was associated with exaggerated activation of mononuclear phagocytes [40]. In the RA model, the induction of HO-1 diminishes osteoclast number, suggesting that this molecule's activity may regulate osteoclastogenesis and consequently bone erosion in RA [41]. Among the effects of IL-1β on the cartilage in OA, an up-regulation of caspase-3 and -7 has been recently demonstrated at mRNA level, indicating that IL-1β can regulate the apoptotic pathway in human chondrocytes. In agreement with Fernandez and co-workers [42], we found that the incubation with IL-1β causes an inhibition of HO-1 gene expression with respect to controls. Moreover, our findings indicate that simultaneous treatment with 10 mmol/l GS restores the HO-1 gene expression control values and these results were confirmed by protein analysis.

Intracellular CO potentially influences the activity of other cellular haemo-proteins such as cytochrome P450, NOS, NADPH oxidase and cytochrome-c oxidase.

Another major factor involved in oxidative stress is the NADPH oxidase system complex. The 22 kDa {alpha}-subunit of cytochrome b558 included in the NADH/NADPH oxidase, p22Phox, is an integral subunit of the final electron transport from NADPH to haem and molecular oxygen in generating O2– [43]. This anion can be converted to other cytotoxic products, mainly H2O2 that oxidizes a variety of aromatic compounds and generates reactive nitrogen species from NO. In our study, GS treatment induced its maximum inhibitory effect after 48 h and a significant p22Phox inhibition was also observed after 24 h; to our knowledge, this is the first report that describes p22Phox expression and modulation in primary cultures of human chondrocytes.

The increased production of O2– and other ROS induces oxidative stress and plays an important role in cartilage erosion during OA and RA. In fact, when joint cartilage cells do not have enough antioxidant capacities, high ROS concentrations lead to a degradation not only of membrane proteins and nucleic acids but also of extracellular matrix components including proteoglycans and collagen. Thus, in the presence of oxygen, hydroxyl radicals (OH) degrade collagen and modify the amino acid composition [44].

NO, present in large amounts in OA cartilage, is responsible for inflammation and articular cartilage degeneration by enhancing cytokine production, elevating MMP and suppressing collagen and proteoglycan syntheses [45–48]. Moreover, different reports have implicated NO in cell death, affecting the mitochondrial respiratory activity [49], in particular under conditions in which other ROS are also generated [22].

Chan et al. [50] have demonstrated that glucosamine treatment has an effect on NO production, reducing IL-1β-induced iNOS gene expression in bovine articular cartilage explants. Our in vitro study on cultured human chondrocytes confirms these data since GS inhibits up to 30% of the IL-1β-induced iNOS expression.

Overall, GS was effective in counteracting IL-1β negative actions, as demonstrated by the restoration of HO-1 gene expression and protein production basal levels.

The differences between the drug concentrations used in vitro and the dose commonly available in vivo can be considered a limitation to our study. It is nevertheless interesting to know that an adequate concentration of glucosamine induces effects in vitro that could be considered useful in controlling OA progression.

In conclusion, the influence of GS on oxidative stress observed in this study discloses a possible new mechanism of action and seems to be in keeping with a potential protective effect on the chondrocyte population.

Formula


   Acknowledgement
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgement
References
 
The authors are grateful to Ms Linda Inverso for the English revision of the manuscript.

Disclosure statement: The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgement
 References
 

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Submitted 15 January 2007; revised version accepted 20 July 2007.
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