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Tetracyclines inhibit nitrosothiol production by cytokine-stimulated osteoarthritic synovial cells
D Borderie1, A Hernvann, P Hilliquin
1Laboratoire de Biochimie A, Hôpital Cochin, Paris, France. didier.borderie@cch.ap-hop-paris.fr
Objective And Design:
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.
Conclusion:
This study provides new information on the mechanism by which tetracyclines exert anti-inflammatory effects, via inhibiting nitrosothiols.