Objective—To evaluate inflammatory responses induced via intra-articular recombinant human interleukin (IL)-1β treatment in horses receiving a dietary nutraceutical (DN; composed of mussel, shark cartilage, abalone, and Biota orientalis lipid extract) and assess the clinical effects of long-term DN administration.
Animals—22 healthy horses.
Procedures—12 horses were fed 0, 15, 45, or 75 mg of DN (3 horses/treatment) daily for 84 days. General health and clinicopathologic variables were monitored at intervals. Ten other horses received 0 or 15 g of DN/d (5 horses/treatment) for 29 days (beginning day −14). One intercarpal joint in each horse was injected twice with IL-1β (10 and 100 ng on days 0 and 1, respectively), and the contralateral joint was similarly injected with saline (0.9% NaCl) solution. Synovial fluid prostaglandin E2 (PGE2), sulfated glycosaminoglycan (GAG), nitric oxide (NO), and protein concentrations and leukocyte counts were analyzed before and at intervals after injections.
Results—Administration of the DN (up to 75 g/d) to horses for 84 days did not induce any adverse effects. In the other experiment, synovial fluid PGE2, GAG, and protein concentrations and leukocyte count increased after intra-articular injections of IL-1β (compared with effects of saline solution injections) in horses that received no DN; NO concentration was not affected. In horses that were fed the DN, intra-articular IL-1β injections did not induce significant increases in synovial fluid PGE2 and GAG concentrations.
Conclusions and Clinical Relevance—Results suggested that administration of the DN may be useful in preventing inflammation associated with arthritis and degenerative joint disease in horses.
Objective—To determine effects of glucosamine (GLN) and chondroitin sulfate (CS) on expression of genes encoding putative mediators of osteoarthritis in bovine cartilage explants cultured for 2 weeks.
Sample Population—Articular cartilage explants harvested from carpal joints of 4 Holstein steers after slaughter.
Procedures—Cartilage disks were treated as follows: fetal bovine serum only (control treatment), human recombinant interleukin (IL)-1β (50 ng/mL; IL-1 treatment), GLN (5 μg/mL) with addition of CS (20 μg/mL; GLN-CS treatment), and human recombinant IL-1β (50 ng/mL) with addition of GLN and CS (IL-1–GLN-CS treatment). Media were analyzed for nitric oxide and prostaglandin E2 (PGE2) release. Explants were subjected to quantitative real-time PCR analysis; expressions of mRNA for inducible nitric oxide synthase, cyclooxygenase-2, microsomal prostaglandin E synthase 1, matrix metalloproteinase (MMP)-3 and -13, aggrecanase-1 and -2, tissue inhibitor of metalloproteinase (TIMP)-3, type II collagen, and aggrecan were assessed.
Results—IL-1–GLN-CS and GLN-CS treatments decreased nitrite release, compared with IL-1 treatment; IL-1–GLN-CS treatment decreased IL-1–induced PGE2 release. Expressions of inducible nitric oxide synthase, cyclooxygenase-2, and microsomal prostaglandin E synthase 1 mRNA were abrogated by GLN-CS and IL-1–GLN-CS treatments. Interleukin-1–induced mRNA expressions of proteolytic enzymes were diminished by IL-1–GLN-CS treatment. Compared with control treatment, GLN-CS treatment decreased MMP-3 and aggrecanase-2 mRNA expression. Transcripts of TIMP-3 were increased by IL-1–GLN-CS treatment, compared with IL-1 treatment. Genes encoding type II collagen and aggrecan on day 14 were upregulated by GLN-CS and IL-1–GLN-CS treatments, compared with control treatment.
Conclusions and Clinical Relevance—Treatment with GLN and CS consistently downregulated mRNA expression for inflammatory mediators and matrix degrading enzymes while increasing TIMP-3 transcripts.
Objective—To determine the effects of glucosamine
(GLN) and chondroitin sulfate (CS), at concentrations
attainable in vivo, on expression of genes encoding
proteolytic enzymes, enzyme inhibitors, and macromolecules
of articular cartilage in interleukin-1(IL-
1)–challenged bovine cartilage explants.
Sample Population—Articular cartilage explants harvested
from 9 steers.
Procedures—Cartilage explants were exposed to
media containing 10% fetal bovine serum (FBS) only, IL-
1 (50 ng/mL), IL-1 with GLN (5 µg/mL), IL-1 with CS (20
µg/mL), or IL-1 with GLN and CS for 24 and 48 hours.
Cartilage was frozen, and RNA was extracted. Gene
expression of matrix metalloproteinases (MMPs)-2, -3,
-9, -13, and -14; aggrecanases (Aggs)-1 and -2; tissue
inhibitors of metalloproteinases (TIMPs)-1, -2, and -3;
and type II collagen and aggrecan were assessed with
quantitative real-time polymerase chain reaction.
Results—Upregulated MMP-3, MMP-13, and Agg-1
transcripts at 24 hours were repressed by the GLN
and CS combination by at least approximately 6-fold.
Glucosamine was effective in suppressing IL-1–induced mRNA expression of MMP-13, Agg-1, and
Agg-2, whereas CS was effective in decreasing IL-1–induced MMP-13 transcript at 24 hours. At 48
hours, GLN and CS added separately and in combination
significantly abrogated Agg-1 and Agg-2 gene
induction. The combination also decreased IL-1–stimulated
Conclusions and Clinical Relevance—GLN and CS,
at concentrations that are within the range measured
in synovial fluid and blood after oral administration,
may regulate expression of matrix degrading
enzymes and their inhibitors at the transcriptional
level, providing a plausible mechanism for their purported
chondroprotective properties. (Am J Vet Res
Objective—To test the hypothesis that simulated digests of Biota orientalis (BO) and a dietary nutraceutical (DN; composed of mussel, shark cartilage, abalone, and BO seed lipid extract) inhibit prostaglandin E2 (PGE2), nitric oxide (NO), and glycosaminoglycan (GAG) production in interleukin (IL)-1–stimulated cartilage explants.
Sample Population—Cartilage tissue from 12 pigs.
Procedures—Articular cartilage explants were conditioned with a simulated digest of BO (BOsim) or DN (DNsim) at concentrations of 0, 0.06, or 0.18 mg/mL or indomethacin (INDOsim; 0 or 0.02 mg/mL) for 72 hours. Control explants received digest vehicle only. Explants were or were not stimulated with recombinant human-IL-1β (10 or 0 ng/mL) during the final 48 hours of culture. Concentrations of PGE2, GAG, and NO in media samples (mPGE2,mGAG, and mNO concentrations, respectively) were analyzed, and explant tissue was stained fluorochromatically to determine chondrocyte viability. Treatment effects during the final 48-hour culture period were analyzed.
Results—IL-1 increased mPGE2, mGAG, and mNO concentrations in control explants without adversely affecting cell viability. Treatment with INDOsim blocked PGE2 production and increased mNO concentration in IL-1–stimulated and unstimulated explants and increased mGAG concentration in unstimulated explants. Treatment with DNsim (0.06 and 0.18 mg/mL) reduced mPGE2 concentration in IL-1–stimulated and unstimulated explants, reduced mNO concentration in IL-1–stimulated explants, and increased mNO concentration in unstimulated explants. Treatment with 0.18 mg of DNsim/mL increased cell viability in the presence of IL-1. In IL-1–stimulated explants, BOsim (0.06 and 0.18 mg/mL) reduced mPGE2 concentration, but 0.18 mg of BOsim/mL increased cell viability.
Conclusions and Clinical Relevance—Effects of IL-1 on cartilage explants in vitro were modulated by DNsim and BOsim.
Objective—To characterize potential mechanisms of
action of glucosamine inhibition of matrix metalloproteinase
(MMP) expression and activity in lipopolysaccharide
(LPS)-stimulated equine chondrocytes.
Sample Population—Chondrocytes cultured from
samples of metacarpophalangeal articular cartilage
collected from cadaveric limbs of horses.
Procedure—The effect of glucosamine on MMP activity
in conditioned medium from LPS-stimulated cartilage
explants was determined by a colorimetric assay
with azocoll substrate. Treatments consisted of negative
and positive controls, glucose (50mM), and glucosamine
(50, 25, 6.25, 3, and 1.5mM). The influence
of glucosamine on MMP synthesis was determined in
chondrocytes in pellet culture incubated with LPS (20
µg/mL). Concentration of MMP-13 was quantified in
spent medium via ELISA; nonspecific MMP activity
was determined via azocoll digestion in organomercurial-
activated medium. Effects of glucosamine on
MMP mRNA concentration in similarly treated chondrocytes
were determined by northern blot hybridization
with MMP-1, -3, and -13 probes. Statistical analyses
were performed with 2-way ANOVA.
Results—Glucosamine had no effect on activated
MMP activity but inhibited MMP protein expression,
as determined by azocoll digestion (glucosamine, 3 to
50mM) and MMP-13 ELISA (glucosamine, 1.5 to
50mM). Resting mRNA concentrations for MMP-1,
-3, and -13 mRNA were significantly lower in cultures
exposed to glucosamine at concentrations of 50 and
25mM than those of positive controls.
Conclusions and Clinical Relevance—Glucosamine
appears capable of pretranslational, and possibly also
translational, regulation of MMP expression; data
suggest a potential mechanism of action for chondroprotective
effects of this aminomonosaccharide.
( Am J Vet Res 2003;64:666–671)
Objective—To determine the effects of orally administered
glucosamine on concentrations of markers of
bone and cartilage metabolism in Standardbred horses
during race training.
Animals—Twenty 16- to 20-month-old Standardbreds
beginning race training.
Procedure—Horses were randomly assigned to 2
groups. One group received glucosamine hydrochloride
(4 g, PO, q 12 h), and the second (control) group
received glucose (4 g, PO, q 12 h). Serum samples
were obtained prior to onset of the study (baseline)
and at regular intervals for 48 weeks for determination
of concentrations of keratan sulfate (KS), osteocalcin
(OC), and pyridinoline crosslinks (PYD).
Results—Osteocalcin concentrations changed significantly
with time; mean serum concentrations were
significantly higher than baseline values for samples
obtained at 24 to 48 weeks after onset of the study.
Although a significant effect of time was observed for
mean concentration of KS, concentrations did not differ
significantly from baseline values at any time during
the study when groups were analyzed separately.
However, pooled analysis revealed significant increases
of mean serum KS concentration at weeks 24 and
30. Significant changes in serum PYD concentrations
were not detected. Oral administration of glucosamine
did not significantly affect serum concentrations
of any of the markers.
Conclusions and Clinical Relevance—Increased
serum OC in clinically normal Standardbreds during
race training may reflect bone formation that accompanies
adaptive remodeling of the appendicular skeleton.
For these experimental conditions, glucosamine did
not appear to exert a detectable influence on serum
concentrations of these 3 markers of connective tissue
metabolism. (Am J Vet Res 2002;63:1106–1110)
Objective—To determine whether glucosamine and
chondroitin sulfate (CS) at concentrations approximating
those achieved in plasma by oral administration
would influence gene expression of selected mediators
of osteoarthritis in cytokine-stimulated equine
Sample Population—Samples of grossly normal
articular cartilage obtained from the metacarpophalangeal
joint of 13 horses.
Procedure—Equine chondrocytes in pellet culture
were stimulated with a subsaturating dose of recombinant
equine interleukin (reIL)-1β. Effects of prior
incubation with glucosamine (2.5 to 10.0 µg/mL) and
CS (5.0 to 50.0 µg/mL) on gene expression of matrix
metalloproteinase (MMP)-1, -2, -3, -9, and -13; aggrecanase
1 and 2; inducible nitric oxide synthase (iNOS);
cyclooxygenase (COX)-2; nuclear factor κB; and c-Jun-
N-terminal kinase (JNK) were assessed by use of a
quantitative real-time polymerase chain reaction
Results—Glucosamine at a concentration of
10 µg/mL significantly reduced reIL-1β–induced
mRNA expression of MMP-13, aggrecanase 1, and
JNK. Reductions in cytokine-induced expression were
also observed for iNOS and COX-2. Chondroitin sulfate
had no effect on gene expression at the concentrations
Conclusions and Clinical Relevance—Concentrations
of glucosamine similar to those achieved in
plasma after oral administration in horses exerted pretranslational
regulation of some mediators of
osteoarthritis, an effect that may contribute to the cartilage-
sparing properties of this aminomonosaccharide.
Analysis of results of this study indicated that the
influence of CS on pretranslational regulation of these
selected genes is limited or lacking. (Am J Vet Res
Procedures—Explants were cultured in commercial
medium for 48 hours. Cartilage was exposed to medium
containing 10% fetal bovine serum, 10 µg of
lipopolysaccharide/mL, and 0.5, 1.0, 2.5, 5.0, and
10.0 mg of glucosamine or mannosamine/mL for 24
hours. Nitric oxide (NO) production (nitrite concentration)
and proteoglycan (PG) release (PG concentration)
in media were measured . Cartilage extracts
were analyzed via zymography to detect gelatinolytic
activity. At the end of the experiment, explants were
assessed for chondrocyte viability.
Results—Addition of lipopolysaccharide resulted in
increased NO production and PG release, but no
increase in gelatinolytic activity, compared with controls.
Glucosamine and mannosamine at concentrations
as low as 0.5 mg/mL inhibited NO production.
Glucosamine inhibited PG release at a minimum concentration
of 1.0 mg/mL, whereas mannosamine
inhibited PG release at a concentration of 0.5 mg/mL.
Concentrations of glucosamine ≤ 5.0 mg/mL did not
adversely affect chondrocyte viability; however, at a
concentration of 10.0 mg/mL, cell death was evident.
Mannosamine had a toxic effect at a concentration of
5.0 mg/mL and was associated with pronounced
chondrocyte death at a concentration of 10.0 mg/mL.
Conclusions and Clinical Relevance—Glucosamine
and mannosamine inhibit selected indices of bovine
articular cartilage degradation at concentrations that
do not affect chondrocyte viability. The potential for
cytotoxic effects at higher concentrations underscores
the importance of establishing appropriate
dosage regimens for these aminomonosaccharides.
(Am J Vet Res 2004;65:1440–1445)