Effects of glucosamine hydrochloride on the productionof prostaglandin E2, nitric oxide and metalloproteases bychondrocytes and synoviocytes in osteoarthritis
H. Nakamura, A. Shibakawa, M. Tanaka, T. Kato, K. Nishioka
Department of Bioregulation, St. Marianna University School of Medicine, Kawasaki,
Kanagawa, Japan
Abstract
Objectives
To determine the response of glucosamine hydrochloride on chondrocytes and synoviocytes in terms of
prostaglandin E2 (PGE2), nitric oxide (NO) and matrix metalloproteases (MMPs).
Methods
Chondrocytes and synoviocytes were prepared from joint specimens of patients who underwent total knee
arthroplasty for osteoarthritis (OA). Chondrocytes from patients with femoral neck fracture were served as anormal control. Culture cells were stimulated by 5 ng/ml of IL-1 and treated with various concentration of
glucosamine hydrochloride (from 1 g/ml to 500 g/ml). PGE2, NO, MMP-1, MMP-3, and MMP-13 levels
were evaluated in the culture supernatant. Further, the expression of COX-2 mRNA was studied by semiquantitative PCR.
Results
With IL-1 stimulation, the levels of these mediators increased dramatically, except for NO fromsynoviocytes. After stimulation, levels of these mediators in OA chondrocytes were higher than synoviocytes
and normal chondrocytes, and the level of MMP-3 was higher than those of MMP-1 and MMP-13. Glucosamine hydrochloride at a concentration of 100 g/ml suppressed PGE2 production, and partly suppressed
NO production. It also suppressed the production of MMPs from normal chondrocytes and synoviocytes but
not from OA chondrocytes.
Conclusion
Glucosamine modulates the metabolism of chondrocytes and synoviocytes and its mode of action differs
between cells and conditions.
Key words
Glucosamine, osteoarthritis, chondrocytes, synovial cells, MMP, PGE2, NO, COX-2.

Clinical and Experimental Rheumatology 2004; 22: 293-299.

Glucosamine forPGE2, NO, MMPproduction in OA/ H. Nakamura et al.
Hiroshi Nakamura, MD, PhD; Atsuyuki
Shibakawa, MD; Michiaki Tanaka, DVM;
Tomohiro Kato, MD, PhD; Kusuki
Nishioka, MD, PhD.
Supported in part by grants from the Japanese Ministry of Education, Culture,
Sports, Science and Technology.
Please address reprint requests andcorrespondence to: Hiroshi Nakamura,
MD, Institute of Medical Science,
St. Marianna University, 2-16-1, Sugao,
Miyame-ku, Kawasaki, 216-8512, Japan.
E-mail nakamura@marianna-u.ac.jp
Received on August 25, 2003; accepted inrevised form on February 6, 2004.
© Copyright CLINICAL AND EXPERIMENTAL RHEUMATOLOGY 2004.

Introduction
Glucosamine is aminosugar that isformed from fructose in vivo by glutamin-fructose-6-phosphate amidotransferase and composes various glycosaminoglycans, the major component of
extracellular matrix such as articularcartilage (1). Hyaluronic acid and keratan sulfate are gigantic polymers ofthe disaccharides found in cartilage,
both of which consist of N-acetyl glucosamine and galactose or glucuronic
acid. Metabolism of glycosaminoglycan together with that of collagen fibers
is important in maintaining matrix ofarticular cartilage and the disruption ofbalance between synthesis and breakdown causes osteoarthritis (OA).
Biological and chemical factors as wellas mechanical factors are involved in
the pathogenesis of OA. Among various chemical mediators, interleukin-1(IL-1) has been revealed to play a central role in degradation of articular cartilage (2); it is produced by mononuclear cells in arthritic synovium (3) andchondrocytes (4). IL-1β stimulates theproliferation of synoviocytes (5) and enhances the production of various chemical mediators such as IL-6 (6), prostaglandin E2 (PGE2) (4) and nitric oxide(NO) (7) from synoviocytes. IL-1β also
stimulates the production of PGE2 (8),NO (9) and metalloproteases (10) fromchondrocytes.
Matrix metalloproteases (MMPs) are
important molecules that induce cartilage depletion in OA. MMP-1 andMMP-13 cleave predominantly type II
collagen (11), and MMP-3, also known
as stromelysin-1, degrades proteoglycans (12). Activated chondrocytes in
OA produce these MMPs and degrade
extracellular matrix surrounding them.
PGE2, an inflammatory mediator, isinvolved in the homeostasis of chondrocytes (13) and enhances pain (14).
Chondrocyte-derived NO directly breaks
down cartilage matrix and induces
chondrocyte apoptosis (15,16).
Nutraceutical glucosamine has beenused as a supplement for the treatmentof osteoarthritis for decades, possibly
because the extracellular matrix was believed to wear out in OA cartilage.
Many clinical results were reported (reviewed in 17) and some in vitro studies
have been published on the effects of294

glucosamine on chondrocytes or articular cartilage (18-21). Here, we evaluated the effects of glucosamine hydrochloride on cultured cells derived fromnormal cartilage, OA cartilage and OA
synovium in terms of production of
PGE2, NO, MMP-1, MMP-3 and
MMP-13.
Materials and methods
Chondrocyte and synoviocyte culture
Chondrocytes and synovial cells wereprepared from surgical specimens obtained during arthroplasty for OA andfemoral neck fracture. As articular cartilage from the latter samples was macroscopically and microscopically normal, they were used as the normal controls. OApatients (n =7) had an averageage of 70.1 (range 50 - 77 yrs) and werediagnosed as having primary OA. Articular changes were advanced and thepatients were being treated with analgesics. Fracture patients (n = 6) had an
average age of 76.6 yrs (range 70- 83).
Cartilage was carefully cut from thesubchondral bone.
Fragments of cartilage and synovial tissue were minced to fine pieces followed by digestion in Dulbecco’s modified
Eagle’s medium (DMEM; Gibco BRL,
NY) with 1 mg/ml of bacterial collagenase (Sigma, MO). The cells were filtered, washed, and seeded into 75 cm2
flasks (Becton Dickinson Labware,
Bedford, UK). The cells were cultured
in DMEM containing 10% fetal calf
serum (FCS; Gibco BRL,NY), 100
units/ml penicillin (Gibco BRL), and100 µg/ml streptomycin (Gibco BRL)
and cells from the primary cultures
were used for the experiments.
After the cells became confluent in 24well plates, medium was changed to2.5% FCS/DMEM and the cells were
stimulated with 5 ng/ml of IL-1β (Rosch,Mannheim, Germany) in the presenceof 1, 10 or 100 µg/ml of glucosaminehydrochloride (Yaizu Marine Products,
Japan) for 24 hours. For synoviocytes,
5, 50 and 500 µg/ml of glucosaminehydrochloride were used as they wereconsidered to be less responsive to glucosamine. Supernatants were then collected to measure the levels of PGE2,
NO, MMP-1, -3, and -13. The cells were
harvested to detect COX-2 by semiquantitative RT-PCR and Western blot.

Glucosamine for PGE2, NO, MMPproduction in OA/ H. Nakamura et al.

Measurement of PGE2, NO, MMP-1,
MMP-3 and MMP-13
PGE2 and NO were assessed by a PGE2
EIA kit (Cayman Chemical Co., MI) anda Nitrite colorimetric assay kit (COXISInternational, Inc., OR), respectively.
MMP-1, MMP-3, and MMP-13 were
determined by ELISA or EIA kits
(MMP-1 and MMP-13; Amersham Pharmacia Bioteck, UK, MMP-3, Daiichi
Kagaku Yakuhin, Japan). Precursor andactive MMP as well as active complex
with tissue inhibitor of metalloprotease-1 (TIMP-1) are measured with thesesystems.
Preparation of mRNAand semiquantitative PCR
COX-2 mRNA was quantified using a
Gene Specific Relative RT-PCR kit(Ambion,Inc, UK) following the manufacture’s instruction. Briefly, mRNAwas extracted from 106 chondrocytes
using Isogen (Nippon Gene, Japan).
cDNA was synthesized in a 20 µl reaction mixture containing 5 µg of total
RNA, 2.5 mM of each dNTP, 1 mM ofrandom hexamer primers, 40 units ofribonuclease inhibitor (RNasin; Toyobo, Japan), and 200 units of Superscript
II RT (Gibco BRL) at 42°C for 2 hours.
The resulting cDNAs were amplified in
50 µl reaction mixture containing COX2 and 18S primer pairs (included in thekit), 2.5 mM of each dNTPand 0.3ul of
Taq polymerase (Takara, Japan) at 94°
C for 30 seconds, 59°C for 30 seconds,
and 72°C for 30 seconds for 24 cycles
in a PCR thermal cycler (Perkin-Elmer,
CT). PCR products were electrophoresed on 2% agarose gels, stained withethidium bromide (10 mg/ml), and theintensity of the bands were quantified
by a fluoro-image analyzer, FLA-2000(Fuji Film, Japan). The result was expressed as the intensity of COX-2 PCR
products against those of 18S.
Western blot for COX-2
Normal chondrocytes in 24-well dishwere stimulated by 5 ng/ml of IL-1β, inthe presence of 100 µg/ml glucosaminehydrochloride or 1 mM diclofenac. After 24 hours, cells were dissolved inlysis buffer (20 mM Tris, 250 mM NaCl,
10 1%NP-40, 1mM DTT, 10 mM NaF,
2mM Na3VO4, 10 mM sodium pyro-

phosphate) and sonicated for 3 seconds
on ice following centrifugation at 3000
rpm for 5 minutes. Supernatant was dissolved in the same amount of sample
buffer (0.1M Tris, 4% SDS, 12 mM
DTT, 20% glycerol, 1% BPB) and boiled for 5 minutes. Electrophoreticallyanalyzed proteins (15 µg/lane) on
12.5% SDS-PAGE gel were transferred
onto Nitrocellulose Membrane (BioRAD, CA). The membrane was blocked in 5% skim milk for 1 hour at room
temperature and subsequently immunoblotted with anti-COX-2 antibody(Cayman Chemical Co.) at a 1000 dilution and then incubated with anti-rabbit-IgG antibody conjugated with HRP(Nichirei, Japan) at a 10,000 dilution
for 1 hour. The membrane was washed
3 times between each step. Then themembrane was developed using an
ECLsystem (Amersham, IL).
Statistical analysis
Each value was from individual samples.
Values were expressed as the mean ±SE. Statistical analysis included the

Wilcoxon signed rank test for comparisons of paired values, and the ManWhitney U test for inter-group comparisons.
Results
IL-1 stimulated production of PGE2,
NO, MMP-1, MMP-3, and MMP-13
With IL-1β stimulation, the level of all
mediators from chondrocytes and synoviocytes increased dramatically, except
for NO from synoviocytes. These levels
and the response to IL-1β were higher in
OA chondrocytes than in normal chondrocytes and synoviocytes. It is worth
noting that the net level of MMP-3 instimulated OA chondrocytes was extremely high compared to those of
MMP-1 and MMP-13 (Fig. 1).
Effects of glucosamine on PGE2
production
Prior to assay of the effects of glucosamine hydrochloride on the productionof various mediators, its effect on cell
viability was evaluated by MTT assay(22). Cell viability was not affected at

Fig. 1. PGE2, NO, MMP-1, MMP-3 and MMP-13 values in the supernatant of normal chondrocytes (n= 6), OAchondrocytes (n = 7) and OAsynoviocytes (n = 5) at baseline and after stimulation with IL1β. Except for NO in normal chondrocytes and synoviocytes, IL-1β dramatically stimulated the production of these mediators. Blank bars indicate the baseline and dotted bars indicate the stimulation.

295

Glucosamine forPGE2, NO, MMPproduction in OA/ H. Nakamura et al.

concentrations ranging from 1 to 500µg/ml (data not shown).
PGE2 production was enhanced by IL-1
stimulation in normal chondrocytes and
OA synoviocytes as well as OA chondrocytes. The PGE2 level in IL-1 stimulated OA chondrocyte cultures was 10times higher than that in unstimulated
ones. PGE 2 production was suppressedby 100 µg/ml glucosamine in normal
and OAchondrocytes, whereas dexamethasone and diclofenac, which were
tested as positive controls, almost completely inhibited stimulated PGE2 production. IL-1β enhanced the expressionof COX-2 mRNA, but glucosamine
had no effect on this response. Dexamethasone suppressed the expression of
COX-2 mRNA, whereas diclofenac enhanced its expression in OA chondrocytes, which may reflect a feedback regulation caused by diclofenac-mediated COX-2 inhibition (Fig.2). This result was also supported by Western blot
analysis which showed that COX-2
expression was enhanced by IL-1β, andthat addition of 100 µg/ml glucosamine
had no effect whereas the addition of
diclofenac slightly increased the expression (Fig. 3).

Fig. 2. Production of PGE2 and expression of COX-2 mRNA in normal chondrocytes (n = 6), OA
chondrocytes (n = 7) and OA synoviocytes (n = 5). Cells were stimulated with IL-1β (5 ng/ml) andtreated with glucosamine hydrochloride (1-100 µg/ml), dexamethasone (0.1 mM) or diclofenac (1mM). Glucosamine (100 µg/ml) suppressed the production of PGE2 in normal and OAchondrocytes. In
synoviocytes, 500 mg/ml of glucosamine showed suppressed PGE2 production but this change was not
significant. The expression of COX2 mRNAwas not affected by glucosamine, whereas dexamethasone
suppressed it and diclofenac enhanced its expression in normal chondrocytes. Statistical analysis was
performed using the Wilcoxon signed rank test. *p < 0.05. N: normal chondrocytes, OA: OAchondrocytes, S: OAsynoviocytes. Gluco: Glucosamine, Dex: Dexamethasone, Diclo: Diclofenac

Effects of glucosamine on NO
production
Glucosamine slightly suppressed the
production of NO from IL-1 treatednormal chondrocytes, whereas it did
not affect OA chondrocytes and OA
synoviocytes (Fig. 4).
Effects of glucosamine on MMP
production
The production of MMP-1, MMP-3
and MMP-13 was measured in the
supernatants of chondrocytes and synoviocytes. The net level of MMP-3 was
the highest among the MMPs tested
and the level of MMP13 was the lowest. All MMP levels were enhanced by
IL-1β. After stimulation with IL-β, theproduction of MMP-1, -3 and -13 waspartly suppressed by 100 µg/ml glucosamine in normal chondrocytes. In
OA synoviocytes, 500 µg/ml glucosamine suppressed the production of
MMP-1, -3 and -13. Constant suppression by dexamethasone was shown in
all cell types. MMP production was not

Fig. 3. The effect of glucosamine hydrochloride and diclofenac on IL-1β stimulated COX-2 protein
expression was analyzed by Western immunoblot. COX-2 expression was enhanced by IL-1β stimulation; 100 µg/ml of glucosamine had no effect on the expression and 1 mM dicloefenac slightly
enhanced the expression. Repeated experiments showed the same results.

suppressed by glucosamine at the concentrations to 100 µg/ml in OA chondrocytes (Fig. 5).
Discussion
Degradation of articular cartilage is driven by an imbalance of catabolic andanabolic factors in OA(23). The former

296

include proinflammatory cytokines suchas IL-1 and TNF, proteases such as
MMPs and cathepsins, and NO that induces chondrocyte apoptosis as well as
matrix damage. IL-1 also enhanced the
production of PGE2. In this experiment,
the level of MMP-3 in the supernatantof stimulated OA chondrocytes was 4

Glucosamine forPGE2, NO, MMPproduction in OA/ H. Nakamura et al.

Fig. 4. Production of NO in normal chondrocytes (n = 6), OAchondrocytes (n = 7) and OAsynoviocytes (n = 5). Cells were stimulated with L-1β (5 ng/ml) and treated with glucosamine hydrochloride(1-100 µg/ml) or dexamethasone (0.1 mM). Glucosamine (100 µg/ml) suppressed the production of
NO only in normal chondrocytes. Statistical analysis was performed using the Wilcoxon signed-rank
test. *p < 0.05. N: normal chondrocytes, OA: OA chondrocytes, S: OA synoviocytes. Gluco: Glucosamine, Dex: Dexamethasone.

Fig. 5. MMP-1, -3 and -13 levels in the normal chondrocytes (n = 6), OAchondrocytes (n = 7) andOAsynoviocytes (n = 5). After stimulation with IL-1β (5 ng/ml), cells were treated with various concentration (1-100 µg/ml) of glucosamine hydrochloride or dexamethasone (0.1 mM). Glucosamine(100 µg/ml) showed a suppressive effect on MMPproduction from normal chondrocytes and OAsynoviocytes. The Wilcoxon signed-rank test was used for the statistical analysis. *p < 0.05. N: normalchondrocytes, OA: OAchondrocytes, S: OAsynoviocytes. Gluco: Glucosamine, Dex: Dexamethasone.

297µg/ml whereas its concentration in OA
synovial fluid has been reported to be alittle higher (from 13 µg/ml to 15 µg/ml) (24, 25). On the contrary, MMP-1
and MMP-13 levels in the supernatantof OA chondrocytes (551.6 ng/ml and12.8 ng/ml, respectively) were higher
than those in OA synovial fluid (356.1
ng/ml and 5.27 ng/ml, respectively)(25, 26). Overall, OA chondrocytes
showed a higher sensitivity to the stimulation of MMP, PGE2 and NO by IL-1
than normal chondrocytes did. This
could be attributed to the fact that OA
chondrocytes had a higher (about 2fold) density of IL-1 receptor compared with normal chondrocytes (27).
As the cartilage samples of OA were
derived from an advanced stage of thedisease in which arthroplasty is needed,
the chondrocytes are supposed to havebeen exposed to the specific conditionin the OA joint for a long time, which
may have altered their characteristics.
PGE2 modulates cartilage degradationin the presence of IL-1 (28) and seems
to have a bivalent effect on chondrocytes (29). PGE2 also induces inflammation and augments pain (14). The
latter function is important with regard
to the clinical effects of glucosamine
on OA, as one of the major short-term
effects of glucosamine is pain relief(30). PGE2 is spontaneously released
from OA chondrocytes and its production is enhanced by IL-1 (27). In thisstudy, the production of PGE2 from IL1-stimulated chondrocytes, especiallychondrocytes derived from OA patients, was higher than that from synoviocytes. Thus, it is speculated that thesource of PGE2 in the OA joint waschondrocytes as well as synoviocytes.
The suppressive effect of glucosamineon PGE2 production might reflect its
beneficial role in the treatment of OA.
PGE2 synthesis is regulated by the coordination of phospholipase A2 (PLA2),
COX and PGE synthase (PGES) activities (31,32). Nonsteroidal anti-inflammatory drugs inhibit COX activity,
whereas glucocorticoids suppress its
expression at the transcriptional level.
Shikhman et al. reported that 10 mM
N-acetyl glucosamine suppressed COX1 and COX-2, but did not observe suppressive effects on the expression of

Glucosamine forPGE2, NO, MMPproduction in OA/ H. Nakamura et al.

MAP kinases or translocation of NFκB21). On the other hand, Largo et al.
demonstrated that glucosamine sulfate
inhibited IL-1 induced NFκB activity
in a dose-dependent manner and suppressed COX-2 expression at the concentration of 1 mg/ml (33). In these reports, authors used different glucosamine derivatives with a higher concentration than those used in this study.
Glucosamine hydrochloride did not affect the expression of COX-2 mRNAand protein at 100 µg/ml, thus the suppressive effect of glucosamine hydrochloride on PGE2 in this study should
be investigated further. Phospholipase
activity might be involved in this process as Piperno et al. clearly showedthat glucosamine suppressed PLA 2
activity (18).
Recently, Dodge et al. reported that 25µM glucosamine sulfate suppressed
MMP-3 from IL-1 stimulated OA chondrocytes (34) and Byron et al. demonstrated that glucosamine suppressed
MMP-1, -3 and -13 from LPS stimulated horse chondrocytes (35). In thisstudy, glucosamine was shown to suppress MMP-1, -3 and -13 in normalchondrocytes and OA synoviocytes.
However, OA chondrocytes showed no
response to glucosamine at 100 µg/ml.
As OA chondrocytes intensely express
IL-1 receptor and are sensitive to IL-1
stimulation (27), they are supposed tobe less responsive to counteracting glucosamine.
Nitric oxide is also a catabolic factor
for extracellular matrix in articular cartilage and glucosamine hydrochloride
slightly suppressed IL-1 induced NO,
while N-acetyl glucosamine dramatically suppressed it (21). In other positive reports, the concentrations of glucosamine derivatives were considerably higher than those used in this study;
thus it is possible that a higher concentration of glucosamine hydrochloride
might suppress the mediators that weinvestigated. However it should be noted that high-dose glucosamine (0.25 25 mg/ml) impairs the metabolic activity of chondrocytes in terms of proteoglycan synthesis, cell viability and cell
death (36). Though there have been noreports describing the optimal clinicaldosage of glucosamine for the treat-ment of OA, excess administration
could be harmful and concentrations of
up of 100 µg/ml are supposed to berational to investigate clinical effects of
glucosamine. Moreover, as our data
originated from human samples, theresults are considered to reflect thepractical clinical response of glucosamine.
Our data show that glucosamine hydrochloride had pharmacological effects
on chondrocytes and synoviocytes by
inhibition of IL-1 stimulated production of PGE2, NO, MMP-1, MMP-3
and MMP-13. Glucosamine hydrochloride (100 µg/ml) suppressed the MMP
production of normal chondrocytes andsynoviocytes but not of OA chondrocytes. It was revealed that glucosamine
modulates the metabolism of chondrocytes and synoviocytes, and that its
mode of action differs between cells
and experimental conditions. These results may reflect the mechanism of action of glucosamine on pain relief andthe prevention of articular destruction.
References
1. HUGENBERG ST, BRANDT KD, COLE CA :Effect of sodium salicylate, aspirin, andibuprofen on enzymes required by the chondrocyte for synthesis of chondroitin sulfate. J
Rheumatol 1993; 20: 2128-33.
2. VAN DE LOO FA, JOOSTEN LA, VAN LENT PL,
ARNTZ OJ, VAN DEN BERG WB: IL-1/NO/
chondrocyte Role of interleukin-1, tumor
necrosis factor alpha, and interleukin-6 in
cartilage proteoglycan metabolism and destruction. Effect of in situ blocking in murine
antigen- and zymosan-induced arthritis.
Arthritis Rheum 1995; 38: 164-72.
3. FARAHAT MN, YANNI G, POSTON R, PANAYI GS: Cytokine expression in synovial
membranes of patients with rheumatoid arthritis and osteoarthritis. Ann Rheum Dis 1993;
52: 870-5.
4. MOOS V, FICKERT S, MULLER B, WEBER
U, SIEPER J : Immunohistological analysis ofcytokine expression in human osteoarthritic
and healthy cartilage. J Rheumatol 1999; 26:
870-9.
5. KUMKUMIAN GK, LAFYATIS R, REMMERS
EF, CASE JP, KIM SJ, WILDER RL: Plateletderived growth factor and IL-1 interactionsin rheumatoid arthritis. Regulation of synoviocyte proliferation, prostaglandin production, and collagenase transcription. J Im munol 1989; 143: 833-7.
6. GUERNE PA, ZURAW BL, VAUGHAN JH,CARSON DA, LOTZ M: Synovium as a sourceof interleukin 6 in vitro. Contribution to local
and systemic manifestations of arthritis. J
Clin Invest 1989; 83: 585-92.
7. STEFANOVIC-RACIC M, STADLER J, GEORGESCU HI, EVANS CH: Nitric oxide synthesis

298

and its regulation by rabbit synoviocytes. J
Rheumatol 1994; 21: 1892-8.
8. CAMPBELL IK, PICCOLI DS, HAMILTON JA :Stimulation of human chondrocyte prostaglandin E2 production by recombinant human interleukin-1 and tumour necrosis factor. Biochim Biophys Acta 1990; 1051: 310-8
9. STADLER J, STEFANOVIC-RACIC M, BILLIAR TR et al.: Articular chondrocytes synthesize nitric oxide in response to cytokines
and lipopolysaccharide. J Immunol 1991;
147: 3915-20.
10. TETLOW LC, ADLAM DJ, WOOLLEY DE:
Matrix metalloproteinase and proinflammatory cytokine production by chondrocytes ofhuman osteoarthritic cartilage: associations
with degenerative changes. Arthritis Rheum
2001; 44: 585-94.
11. BILLINGHURST RC, DAHLBERG L, IONESCU M et al.: Enhanced cleavage of type II
collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest 1997; 99:
1534-45.
12. OKADA Y, SHINMEI M, TANAKA O et al.:Localization of matrix metalloproteinase 3(stromelysin) in osteoarthritic cartilage andsynovium. Lab Invest 1992; 66: 680-90.
13. FULKERSON JP, DAMIANO P: Effect of prostaglandin E2 on adult pig articular cartilage
slices in culture. Clin Orthop 1983; 179: 2669.
14. FERREIRA SH, NAKAMURA M, CASTRO
MSA: The hyperalgesic effects of prostacyclin and prostaglandin E2. Prostaglandins
1978; 16: 31-7.
15. HASHIMOTO S, TAKAHASHI K, AMIEL D,
COUTTS RD, LOTZ M: Chondrocyte apoptosis and nitric oxide production during experimentally induced osteoarthritis. Arthritis
Rheum 1998 ; 41: 1266-74.
16. PELLETIER JP, JOVANOVIC DV, LASCAUCOMAN V et al.: Selective inhibition of inducible nitric oxide synthase reduces progression of experimental osteoarthritis invivo: possible link with the reduction in chondrocyte apoptosis and caspase 3 level. Arthri tis Rheum 2000; 43: 1290-9.
17. MCALINDON TE, LAVALLEY MP, GULIN JP,FELSON DT : Glucosamine and chondroitin
for treatment of osteoarthritis: a systematic
quality assessment and meta-analysis. JAMA
2000; 283: 1469-75.
18. PIPERNO M, REBOUL P, HELLIO LE GRAVERAND MP et al.: Glucosamine sulfate modulates dysregulated activities of human osteoarthritic chondrocytes in vitro. Osteoar thritis Cartilage 2000; 8: 207-12.
19. BASSLEER C, ROVATI L, FRANCHIMONT P:
Stimulation of proteoglycan production by
glucosamine sulfate in chondrocytes isolated
from human osteoarthritic articular cartilage
in vitro. Osteoarthritis Cartilage 1998; 6:
427-34.
20. GOUZE JN, BORDJI K, GULBERTI S et al.:Interleukin-1beta down-regulates the expression of glucuronosyltransferase I, a key enzyme priming glycosaminoglycan biosynthesis: influence of glucosamine on interleukin1beta-mediated effects in rat chondrocytes.
Arthritis Rheum 2001; 44: 351-60.
21. SHIKHMAN AR, KUHN K, ALAAEDDINE N,
LOTZ M: N-acetylglucosamine prevents IL-1

Glucosamine forPGE2, NO, MMPproduction in OA/ H. Nakamura et al.
beta-mediated activation of human chondrocytes. J Immunol 2001; 166: 5155-60.
22. DENIZOT F, LANG R : Rapid colorimetricassay for cell growth and survival. J Immunol
Methods 1986; 89: 271–7.
23. GOLDRING MB: The role of the chondrocytes in osteoarthritis. Arthritis Rheum 2000;
43: 1916-26.
24. RIBBENS C, ANDRE B, KAYE O et al.: Synovial fluid matrix metalloproteinase-3 levels
are increased in inflammatory arthritides whether erosive or not. Rheumatology (Oxford)
2000; 39: 1357-65.
25. YOSHIHARA Y, NAKAMURA H, OBATA K etal.: Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial
fluids from patients with rheumatoid arthritis
or osteoarthritis. Ann Rheum Dis 2000; 59:455-61.
26. ITO T, UZUKI M, SHIMAMURA T, SAWAI T:
Dynamics of matrix metalloproteinase(MMP)-13 in the patients with rheumatoid
arthritis. Ryumachi 2002; 42: 60-9.
27. MARTEL-PELLETIER J, M CCOLLUM R, D I-

BATTISTA J et al.: The interleukin-1 receptor

in normal and osteoarthritic human articularchondrocytes. Identification as the type I
receptor and analysis of binding kinetics andbiologic function. Arthritis Rheum 1992; 35:
530-40.
28. HARDYMM, SEIBERT K, MANNING PT et al.:Cyclooxygenase 2-dependent prostaglandin
E2 modulates cartilage proteoglycan degradation in human osteoarthritis explants. Arth ritis Rheum 2002; 46: 1789-803.
29. ABRAMSON SB: The role of COX-2 produced by cartilage in arthritis. Osteoarthritis
Cartilage 1999; 7: 380-1.
30. NAKAMURAH, NISHIOKA K: Effects of glucosamine/chondroitin supplement on osteoarthritis: Involvement of PGE2 and YKL-40.
Jpn J Rheum Surg 2002, 21: 175-84.
31. LESLIE CC : Properties and regulation of cytosolic phopholipase A2. J Biol Chem 1997;
27: 16709-12.
32. JAKOBSSON P-J, THOREN S, MORGENSTERN R, SAMUELSSON B: Identification ofhuman prostaglandin E synthase: a microso-299

mal glutathione-dependent, inducible enzyme, constituting a potential novel drug target. Proc Natl Acad Sci USA 1999; 96: 7220-5.
33. LARGO R, ALVAREZ-SORIA MA, DIEZ-ORTEGO I et al.: Glucosamine inhibits IL-1betainduced NFkappaB activation in human osteoarthritic chondrocytes. Osteoarthritis Carti lage 2003; 11: 290-8.
34 DODGE GR, JIMENEZ SA: Glucosamine sulfate modulates the levels of aggrecan andmatrix metalloproteinase-3 synthesized bycultured human osteoarthritis articular chondrocytes. Osteoarthritis Cartilage 2003; 11:424-32.
35 BYRON CR, ORTH MW, VENTA PJ et al.:Influence of glucosamine on matrix metalloproteinase expression and activity in lipopolysaccharide-stimulated equine chondrocytes. Am J Vet Res 2003; 64: 666-71.
36. DE MATTEI M, PELLATI A, PASELLO M etal.: High doses of glucosamine-HCl havedetrimental effects on bovine articular cartilage explants cultured in vitro. Osteoarthritis
Cartilage 2002; 10: 816-25.