D. Borderie et al., Tetracyclines inhibit nitrosothiol production by cytokine-stimulated osteoarthritic synovial cells, INFLAMM RES, 50(8), 2001, pp. 409-414
Objective and design: To evaluate the capacity of doxycycline and minocycli
ne 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-1 beta (1ng
/ml), TNF-alpha (500 pg/ml), IFN-gamma (10(4) U/ml) plus minocycline or dox
ycycline (10(-4) to 10(-6) M), diclofenac (10(-5) M), or cortisol (10(-5) M
). Nitrosothiols were determined by fluorimetry, nitrite by the Griess reac
tion, nitrate by a spectrophotometric assay using oxidation by nitrate redu
ctase 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 nitrosothi
ols, 5.2 times higher for nitrate and about 3.5 times higher for nitrite, D
oxycycline and minocycline induced a dose-dependent decrease in the product
ion of nitrosothiols, nitrate and nitrite, and inhibited the synthesis of t
he iNOS protein. Doxycycline and minocycline inhibited the N-nitrosation re
action of DAN effectively, with IC50 values close to 100 muM. Diclofenac an
d cortisol had no effect.
Conclusion: This study provides new information on the mechanism by which t
etracyclines exert anti-inflammatory effects, via inhibiting nitrosothiols.