Glucosamine prevents in vitro collagen degradation in chondrocytes by inhibiting advanced lipoxidation reactions and protein oxidation
© Tiku et al.; licensee BioMed Central Ltd. 2007
Received: 2 November 2006
Accepted: 8 August 2007
Published: 8 August 2007
Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction. Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.
Osteoarthritis (OA) is characterized by the progressive degradation and loss of articular cartilage . OA is the most common arthritic disease and its incidence increases with age. As population demographics changes to include more elderly individuals, this disease will have a serious impact in multiple ways. Along with the cost for health care and lost work time, individuals with OA suffer from pain and disability . Currently, there is no specific treatment to prevent or retard the cartilage degradation in OA. Present treatments used for OA provide only symptomatic relief from the pain. Glucosamine sulfate, which has received attention as a putative agent that may retard cartilage structural degradation in OA, has been investigated in several OA trials [3–5]. The result on applicability of glucosamine in the clinical setting is still controversial [6–8]. Glucosamine in its various salt formulations with or without chondroitin sulfate is available over-the-counter as a nutritional supplement and is consumed by large numbers of osteoarthritic patients.
The mechanism of retardation of cartilage degradation by glucosamine is not known. Glucosamine has been shown to have a number of effects in in vitro chondrocyte and explant cultures [9–13]. These effects include stimulation of proteoglycan synthesis, inhibition of the degradation of proteoglycans, and inhibition of matrix metalloproteinase-3 synthesis [14–16]. Glucosamine inhibits aggrecanase activity via suppression of glycosylphosphatidylinositol-linked proteins . Furthermore, glucosamine has been shown to inhibit cytokine (interleukin-1 [IL-1])-induced activation of chondrocytes and nuclear factor-kappa-B activity and to upregulate type II IL-l decoy receptor [18, 19]. In vivo, glucosamine helps enhance healing of cartilage injury [20–23]. Glucosamine has been demonstrated to have immunosuppressive and tumor-inhibiting activity [24, 25]. All these pleiotropic effects of glucosamine may individually or collectively have a chondroprotective effect.
Does the ability of glucosamine sulfate to retard cartilage structural degradation observed in OA clinical studies [3–5] involve the protection of collagen degradation? We tested the effect of glucosamine in an in vitro model of chondrocyte-dependent collagen degradation  in which collagen degradation is mediated mostly by the activation of chondrocyte lipid peroxidation resulting in aldehydic oxidation and fragmentation of cartilage collagen.
Materials and methods
Calcium ionophore A23187, vitamin E, butylated hydroxytoluene, tetramethoxypropane, glucose oxidase, glucosamine hydrochloride (interchangeably described as glucosamine), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rotta Research Laboratorium (Monza, Italy) provided glucosamine sulfate. Hydrogen peroxide of reagent grade was obtained from Fisher Scientific (part of Thermo Fisher Scientific Inc., Waltham, MA, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), Hanks' balanced salt solution (HBSS), Earl's balanced salt solution (EBSS), L-glutamine, gentamicin, HEPES buffer, penicillin, and streptomycin were purchased from Gibco-BRL (now part of Invitrogen Corporation, Carlsbad, CA, USA). Proline, L [2,3,4,5-H] with specific activity of 90 curies per millimole was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA).
Isolation of rabbit articular chondrocytes
NZW rabbits (2.2 to 2.9 kg) of either gender were killed by intravenous injection of Beuthanasia-D special (Schering-Plough Corporation, Kenilworth, NJ, USA). The chondrocytes were isolated as described previously . The viability of chondrocytes was confirmed by trypan blue exclusion. Primary chondrocytes were suspended in 10% FBS in DMEM containing antibiotics (1%) and HEPES buffer (10 mM, pH 7.4) (complete media).
Primary rabbit articular chondrocytes were distributed into 24-well plates at a concentration of 1 to 2 × 105 cells per well in 1 ml of complete media. Chondrocytes were allowed to attach for 3 to 5 days, and media were changed every 3 days. Confluent cells in multiwell plates were labeled with 1 to 2 μC/well with [3H]-proline during the last 24 to 48 hours of cell culture. The cell monolayer was washed at least four to five times with warm HBSS by flipping the plates to remove unincorporated proline from the matrix. Albumin- or serum-free EBSS was added to wells. Experiments were carried out in triplicate wells. The test reagents were added, and the total volume was adjusted to 0.5 ml with EBSS. The cultures were incubated at 37°C in a humidified 5% CO2 incubator for 4 to 24 hours. [3H]-proline release was measured in cell supernatant and cell lysates. A 100-μl aliquot was removed and processed for scintillation counting. The plastic-bound [3H]-proline-labeled matrix (that is, residuum) was solubilized with 0.5 M NaOH and counted. Percentage release of total [3H]-proline-labeled collagen was calculated.
Lipoprotein and lipoprotein oxidation
The very-low-density lipoprotein and low-density lipoprotein (LDL) fractions were isolated from serum by ultracentrifugation at a density of 1.063 g/ml and were kindly provided by Vincent A. Rifici and Avedis K. Khachadurian from the Department of Medicine of our medical school . Lipoproteins were tested for susceptibility for oxidation in incubation with or without glucosamine. Lipoprotein (0.25 to 0.5 mg/ml) was incubated at 30°C in phosphate-buffered saline (PBS) for 4 hours in the absence or presence of 5 μM Cu2+ (copper ion) or 5 μM Cu2+ and 50, 5, or 0.5 mM glucosamine. Data are expressed as malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) equivalents in nanometers.
Thiobarbituric acid-reactive substances
Two-hundred-microliter samples of TBARS that contained 50 μg of lipoprotein proteins were assayed by incubation with 1 ml of 1% thiobarbituric acid for 40 minutes at 90°C. The reaction tubes were cooled and centrifuged at 500 g for 10 minutes at 25°C, and the absorbencies of the supernatants were measured in a spectrophotometer at 532 nm. TBARS are expressed as nanomoles of malondialdehyde equivalents of lipoprotein protein compared with tetramethoxypropane standard .
Conjugated diene formation
A washed monolayer of primary articular chondrocytes in a 60-mm Petri dish was stimulated in the presence or absence of calcium ionophore A23187 (20 μM) with or without glucosamine or vitamin E (250 μM) in phenol-free EBSS. The media were monitored for conjugated diene formation at 234 nm at different time points . Delta absorbance was expressed as absorbance at different time points minus the absorbance at 0 hour. Conjugated diene in lipoproteins was determined directly by measuring the change in absorbance at 234 nm of the lipoprotein samples after incubation with Cu. Samples that contained 50 μg of protein were diluted 1:5 with PBS before measurement, and results were expressed as difference in absorbance at 234 nm.
Preparation of cell matrix extracts
Primary articular chondrocytes in high density (1 × 106/ml) were cultured in 60-mm Petri dishes to confluence, washed three times with HBSS, and set in EBSS, with or without agonist, in a total volume of 1.5 ml for variable durations. The medium and cell matrix were harvested with a cell scraper in the presence of a cocktail of protease inhibitors with EDTA (ethylenediaminetetraacetic acid), and the material was transferred to microcentrifuge tubes. One hundred fifty microliters of saturated trichloroacetic acid solution was added, and the tubes were incubated for 30 minutes on ice and microcentrifuged at 12,500 rpm for 10 minutes. The supernatants were discarded, and pellets were washed with 50 μl of ethanol and then suspended in 100 μl of sample buffer (29) and frozen at -70°C. The samples were thawed and boiled for 5 minutes with 5 μl of β-mercaptoethanol and later cooled on ice, vortexed, spun, and boiled as necessary. A total of 30 μl of each sample was loaded onto a 4% stacking gel and separated in 10% resolving SDS-PAGE gel in a mini-PROTEAN II electrophoresis cell (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Electrophoresis was carried out under the reducing condition of Laemmli . Proteins were stained with Coomassie Brilliant Blue.
Immunodetection of aldehyde-protein adducts
Proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane with Trans-Blot electrophoretic transfer. The blots were incubated with 50 ml of 5% bovine serum albumin (BSA) with Tris-buffered saline (TBS) (20 mM Tris/500 mM NaCl, pH 7.5) containing 0.1% Tween-20 and then were washed three times for 15 minutes with 0.5% BSA with TBS. For immunodetection, blots were incubated with antibodies diluted in 1% BSA/TBS overnight. The MDA2 mouse monoclonal antibodies, specific for malondialdehyde-modified lysine, were kindly provided by Wulf Palinski, of the University of California, San Diego (CA, USA) . The monoclonal antibodies were used at dilutions of 1:2,500. The primary antibody was removed, and the blots were washed three times (15 minutes each) with TBS-containing Tween-20. The blots were then incubated in horseradish peroxidase (HRP)-labeled goat anti-mouse immunoglobulin G in 1% BSA/TBS (diluted 1:2,500) for 1 hour at room temperature. Blots were again washed with TBS (15 minutes each), and proteins were visualized as outlined in the enhanced chemiluminescence (ECL) Western blotting protocol (Amersham, now part of GE Healthcare, Little Chalfont, Buckinghamshire, UK).
Immunodetection of protein-bound 2,4-dinitrophenylhydrazones
Derivatization with dinitrophenylhydrazones was performed as published . Proteins separated by SDS-PAGE were transferred as above. For immunodetection, anti-dinitrophenyl (DNP) antibody was supplied by DAKO (Dako North America, Inc., Carpinteria, CA, USA') (V401) and used at a dilution of 1:4,000. The secondary antibody was goat anti-rabbit antibody conjugated with HRP as outlined above in the ECL Western blotting protocol (GE Healthcare).
Results are expressed as means ± standard error of the mean. There was a 10% coefficient of variation between the mean and highest and lowest counts in random wells of each experiment. The differences of the means between groups in the same experiment were evaluated by Student t test (Statview® program; SAS Institute Inc: Cary, NC USA). P values less than or equal to 0.05 were considered statistically significant.
Glucosamine hydrochloride and glucosamine sulfate inhibit calcium ionophore-induced chondrocyte-dependent collagen degradation
Dose and time effect of glucosamine hydrochloride and glucosamine sulfate on collagen degradation
Glucosamine hydrochloride does not inhibit conjugated diene formation by activated chondrocytes and lipoprotein oxidation
Glucosamine hydrochloride inhibits TBARS formation by copper-induced lipoprotein oxidation
Immunoblot analysis of the effect of glucosamine hydrochloride on aldehyde-protein adduct in chondrocyte matrix extracts
Western blot analysis of effect of glucosamine on protein oxidation
Using this in vitro model of chondrocyte activation-dependent collagen degradation, we show that glucosamine specifically and significantly inhibited collagen degradation. Inhibition of collagen degradation by glucosamine was not mediated by inhibiting the chondrocyte lipid peroxidation process but by inhibiting advanced lipoxidation reactions. Specifically, glucosamine inhibited purified lipoprotein protein oxidation and aldehydic oxidation of chondrocyte matrix.
Using this in vitro model, we had previously shown [26, 35, 36] that chondrocyte-derived lipid radicals specifically mediate degradation of cartilage collagen [26, 35]. This model therefore is a fair representation of cartilage collagen degradation. The relevance of this in vitro model to human OA pathogenesis was demonstrated by detection of in vivo molecular imprints of lipid peroxidation in which OA and normal cartilage tissue sections were studied . We also demonstrated the presence of OA disease-specific malondialdehyde and hydroxynonenal adducts in human OA cartilage tissue sections, suggesting the in vivo role of lipid peroxidation in the OA pathogenesis [36, 37]. Collectively, these observations indicate that lipid peroxidation may play a larger role in the pathogenesis OA than has previously been recognized.
We investigated the effect of glucosamine in our assay system. As shown, only glucosamine hydrochloride or glucosamine sulfate specifically and significantly inhibited collagen degradation by activated chondrocytes and the effect was dose-dependent. Similar effects by both agents (glucosamine hydrochloride and glucosamine sulfate) excluded the possibility that the inhibition observed was mediated by the sulfate moiety in the latter compound. Glucosamine hydrochloride had little or variable effect on hydrogen peroxide-induced collagen degradation, suggesting that it did not inhibit oxygen radical/hydrogen peroxide-mediated collagen degradation (data not shown).
Since the mechanism of collagen degradation in this model appears to involve the activation of lipid peroxidation in chondrocytes, it raises the possibility that glucosamine was acting like a chain-breaking antioxidant similar to vitamin E. However, glucosamine had no discernable effect on conjugated diene formation by activated chondrocytes, suggesting that its mechanism of action was not due to chain-breaking antioxidant activity. As expected, vitamin E inhibited conjugated diene formation by chondrocytes. To further confirm these findings, we tested the effect of glucosamine in a purified lipoprotein oxidation model system, a commonly used in vitro model for studies on lipoxidative modification of proteins . Again, glucosamine hydrochloride had no discernable effect on Cu-induced conjugated diene formation in lipoproteins. Furthermore, glucosamine did not cause an increase in the lag phase of LDL oxidation or a decrease in absorbance at 234 nm during the later plateau phase of the reaction. Together, these observations indicate that glucosamine does not interfere with initiation or propagation of lipid peroxidation reaction.
The inhibition of collagen degradation by glucosamine was manifested even when the addition of glucosamine was delayed in activated chondrocyte cultures, indicating that its mechanism of action involved downstream events of chondrocyte activation rather than interfering with or blocking the early events of chondrocyte activation by calcium ionophore. We tested the effect of glucosamine on TBARS formation by Cu-induced oxidation of purified lipoproteins. Glucosamine in a dose-dependent manner inhibited malondialdehyde formation by oxidized lipoprotein. The data suggest that glucosamine either inhibited or scavenged aldehydic products of lipid peroxidation. However, glucosamine did not interfere in the detection of control malondialdehyde in TBARS assay, suggesting that most likely glucosamine inhibited advanced lipoxidation reactions rather than scavenging aldehydic products.
The identification of aldehydic adducts provides a molecular clue of chondrocyte matrix damage mediated by lipid-free radicals . On immunoblot analysis of the effect of glucosamine, we identified activation-dependent low-molecular-weight MDA adduct in chondrocyte matrix extracts; the intensity of higher-molecular-weight aldehydic adducts increased in activated chondrocyte extracts as compared with extracts from control chondrocyte matrix. In the presence of glucosamine, the low-molecular-weight aldehydic adducts in activated extracts disappeared whereas the intensity of high-molecular-weight adducts decreased, indicating that glucosamine prevented oxidation and/or fragmentation of chondrocyte matrix components. These observations are consistent with the finding that glucosamine inhibited malondialdehyde (TBARS) formation in Cu-induced oxidation of lipoprotein. Together, these observations suggest that glucosamine inhibits advanced lipoxidation reactions. By preventing advanced lipid-free radical production, glucosamine perhaps inhibits collagen degradation observed in the in vitro model system.
Inhibitors of advanced lipoxidation reactions such as aminoguanidine and pyridoxamine have been evaluated in animal models of diseases such as diabetes [38, 39]. These compounds are being evaluated in clinical trials for the treatment of diabetic nephropathy . Aminoguanidine inhibits chemical modification of proteins during lipid peroxidation reactions and inhibits metal-catalyzed oxidation of LDLs and uptake of oxidized LDL into macrophages via the scavenger receptor [41, 42]. Pyridoxamine has also been shown to have potent advanced lipoxidation inhibitory activities in a variety of tests [38, 39]. In addition to showing advanced lipoxidation inhibitory activity, these compounds show inhibitory activity against advanced glycation reactions (AGEs) [38, 43]. AGE products formed during autoxidation of carbohydrates and lipid peroxidation reactions produce reactive carbonyl species that cause a carbonyl modification reaction in protein structure and function and cause the formation of high-molecular-weight protein aggregates . Osteoarthritic cartilage shows increased levels of insoluble protein aggregates and AGE-modified products [44–46]. Identification of carbonyl modification of proteins provides a powerful tool to monitor the development of a number of pathologies mediated by a condition commonly described as 'carbonyl stress' [33, 34, 47]. As shown, glucosamine inhibited Cu-induced carbonyl modification of lipoproteins, indicating that glucosamine also traps reactive carbonyl compounds. In addition to aminoguanidine and pyridoxamine, therapeutic agents such as L-arginine, OPB-9195, tenilsetam, and metformin have been proposed to trap reactive carbonyl compounds [48–53].
The pharmacokinetics of oral administration of glucosamine sulfate show that plasma levels increase more than 30-fold from baseline and peak at approximately 10 μM with the standard 1,500-mg once-daily dosage . We postulate that because in vivo tissue levels of glycosaminoglycans in cartilage are hundreds perhaps thousands of folds higher than in serum or joint fluids, glucosamine, which is a structural component of aggrecan, may locally provide an antioxidant environment that may protect cartilage collagen from oxidative damage.
Our data suggest that the decrease in collagen degradation by glucosamine observed in this in vitro model system may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.
In an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction, glucosamine decreases collagen degradation by inhibiting advanced lipoxidation reaction and thus prevents the oxidation and loss of collagen matrix from labeled chondrocyte matrix.
= advanced glycation reaction
= bovine serum albumin
= Dulbecco's modified Eagle's medium
= Earl's balanced salt solution
= enhanced chemiluminescence
= fetal bovine serum
= Hanks' balanced salt solution
= horseradish peroxidase
= low-density lipoprotein
= phosphate-buffered saline
= thiobarbituric acid-reactive substances
= Tris-buffered saline.
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