Tetracyclines inhibit nitrosothiol production by cytokine-stimulated osteoarthritic synovial cells.
OBJECTIVE
To evaluate the capacity of doxycycline and minocycline to inhibit NO production and N-nitrosation reactions in vitro.


METHODS
Synovial cells obtained from 6 patients with osteoarthritic joint disease were incubated for 24 hours with (i) or without (ii) IL-1beta (1 ng/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(4) U/ml) plus minocycline or doxycycline (10(-4) to 10(-6) M), diclofenac (10(-5) M), or cortisol (10(-5) M). Nitrosothiols were determined by fluorimetry, nitrite by the Griess reaction, nitrate by a spectrophotometric assay using oxidation by nitrate reductase and iNOS by immunoblotting.


RESULTS
After 24 hours of stimulation, the level of NO production was much higher than that in untreated cells: about 5.5 times higher for nitrosothiols, 5.2 times higher for nitrate and about 3.5 times higher for nitrite. Doxycycline and minocycline induced a dose-dependent decrease in the production of nitrosothiols, nitrate and nitrite, and inhibited the synthesis of the iNOS protein. Doxycycline and minocycline inhibited the N-nitrosation reaction of DAN effectively, with IC50 values close to 100 microM. Diclofenac and cortisol had no effect.


CONCLUSIONS
This study provides new information on the mechanism by which tetracyclines exert anti-inflammatory effects, via inhibiting nitrosothiols.
Borderie
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D
Laboratoire de Biochimie A, Hôpital Cochin, Paris, France. didier.borderie@cch.ap-hop-paris.fr
Hernvann
A
A
Hilliquin
P
P
Lemarchal
H
H
Kahan
A
A
Ekindjian
O G
OG
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology*; D004318:Doxycycline / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D008911:Minocycline / Q000494:pharmacology; D009566:Nitrates / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D010003:Osteoarthritis / Q000473:pathology*; D010047:Ovalbumin / Q000494:pharmacology; D026403:S-Nitrosothiols / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013256:Steroids; D013583:Synovial Membrane / Q000166:cytology / Q000378:metabolism*; D013754:Tetracyclines / Q000494:pharmacology*
D000900:Anti-Bacterial Agents; D000893:Anti-Inflammatory Agents; D000894:Anti-Inflammatory Agents, Non-Steroidal; D016207:Cytokines; D009566:Nitrates; D020030:Nitric Oxide Donors; D009573:Nitrites; D026403:S-Nitrosothiols; D015842:Serine Proteinase Inhibitors; D013256:Steroids; D013754:Tetracyclines; D010047:Ovalbumin; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D008911:Minocycline; D004318:Doxycycline