Short-term gene expression changes in cartilage explants stimulated with interleukin beta plus glucosamine and chondroitin sulfate.
OBJECTIVE
To determine the short-term effects of glucosamine (GLN) and chondroitin sulfate (CS) on expression of genes encoding inflammatory mediators and matrix enzymes in bovine cartilage explants stimulated with interleukin 1 (IL-1).


METHODS
Dose-response experiments were conducted for IL-1, GLN, and CS to select concentrations of each optimized for detecting treatment effects on cartilage explants. Based on the dose-response experiments, treatments included fetal bovine serum (FBS) control, 15 ng/ml IL-1, and 15 ng/ml IL-1 with the addition of 10 microg/ml GLN and 20 microg/ml CS. Media were measured for nitric oxide (NO) and prostaglandin E2 (PGE2) while explants were frozen for RNA extraction at 8, 16, and 24 hours. Gene expression relative to FBS control for inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), microsomal PGE synthase-1 (mPGEs1), nuclear factor-kB p65 subunit (NF-kB), matrix metalloproteinase (MMP)-3 and 13, aggrecanase (Agg)-1 and 2, and tissue inhibitor of metalloproteinase-3 (TIMP-3) were assessed by quantitative real-time polymerase chain reaction (RT-PCR). In a separate study using incubation of explants with the same treatments for 48 hours, proteoglycan release was measured with dimethylmethylene blue assay and TIMP-3 protein was evaluated with Western blots.


RESULTS
The GLN and CS combination abrogated IL-1-induced gene expression of iNOS, COX-2, mPGEs1, and NF-kB at all timepoints. NO, PGE2, and proteoglycan release were reduced with the combination. The abundance of stimulated MMP-13, Agg-1, and Agg-2 mRNA was repressed, whereas TIMP-3 was upregulated by the combination at all timepoints. The abundance of TIMP-3 protein was increased by the combination relative to IL-1 at 48 hours.


CONCLUSIONS
GLN and CS in combination suppress synthesis and expression of genes encoding inflammatory mediators and proteolytic enzymes while upregulating TIMP-3. This provides a plausible mechanism for the purported mild antiinflammatory and chondroprotective properties of GLN and CS.
Chan
Pooi-See
PS
Bone and Joint Center, Henry Ford Hospital, Michigan, USA.
Caron
John P
JP
Orth
Michael W
MW
D000818:Animals; D015153:Blotting, Western; D002358:Cartilage, Articular / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D002417:Cattle; D002809:Chondroitin Sulfates / Q000494:pharmacology*; D017077:Culture Media, Conditioned / Q000737:chemistry; D015232:Dinoprostone / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004338:Drug Combinations; D004798:Enzymes / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D005944:Glucosamine / Q000494:pharmacology*; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology*; D009569:Nitric Oxide / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000378:metabolism
D017077:Culture Media, Conditioned; D004338:Drug Combinations; D004798:Enzymes; D007375:Interleukin-1; D011509:Proteoglycans; D012333:RNA, Messenger; D019717:Tissue Inhibitor of Metalloproteinase-3; D009569:Nitric Oxide; D002809:Chondroitin Sulfates; D015232:Dinoprostone; D005944:Glucosamine