Inhibition of the nitrosothiol production of cultured osteoarthritic chondrocytes by rhein, cortisol and diclofenac.
OBJECTIVE
Nitric oxide (NO degrees ) is a free molecule produced by NO synthases which acts as a mediator in inflammatory processes. NO degrees can react with thiol groups of proteins to produce nitrosothiols. Increased concentrations of these bioactive compounds have been found in sera and synovial fluids from patients with osteoarthritis (OA). The aim of this study was to assess the ability of human osteoarthritic chondrocytes to synthesize nitrosothiols and to compare the in vitro effects of rhein, cortisol and diclofenac on nitrosothiol and nitrite production.


METHODS
Osteoarthritic chondrocytes were incubated for 24 h with 1 ng/ml of recombinant human interleukin-1beta (IL-1beta) in the presence or absence of rhein (1.3x10(-5) M, 6.5x10(-6) M, or 1.3x10(-6) M), cortisol (10(-5) M) or diclofenac (10(-5) M or 10(-6) M). Nitrite levels were measured in cell supernatants by the Griess method; nitrosothiol levels were determined in supernatants and cellular lysates by fluorimetry.


RESULTS
At the basal level, nitrosothiols represented 80% of the total of nitrite and nitrosothiol production. After IL-1beta stimulation, NO degrees production was highly increased in the supernatants (45-fold increase in nitrite, 60-fold increase in nitrosothiols) as well as in cell lysates (35-fold increase in nitrosothiols). Rhein caused a dose-dependent decrease in nitrosothiol and nitrite production. In comparison, diclofenac (10(-5) M) moderately decreased nitrite and nitrosothiol levels in the supernatants but had no effect on lysate nitrosothiol. Cortisol had no significant effect on NO degrees production.


CONCLUSIONS
The IL-1beta stimulation increased nitrosothiol production by osteoarthritic chondrocytes. These results demonstrate the need to measure nitrosothiol as well as nitrite production. Rhein inhibited the IL-1beta induced NO degrees production, and may be a suitable treatment for osteoarthritis.
Borderie
D
D
Laboratoire de Biochimie A, Hôpital Cochin, 27 rue du Faubourg Saint Jacques, Paris, 75014, France.
Hernvann
A
A
Lemarechal
H
H
Menkes
C J
CJ
Ekindjian
O
O
D000368:Aged; D000880:Anthraquinones / Q000494:pharmacology*; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D019902:Chondrocytes / Q000502:physiology; D004008:Diclofenac / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D008623:Mercaptoethanol; D009569:Nitric Oxide / Q000037:antagonists & inhibitors*; D009603:Nitroso Compounds / Q000037:antagonists & inhibitors*; D010003:Osteoarthritis / Q000378:metabolism; D026403:S-Nitrosothiols
D000880:Anthraquinones; D000893:Anti-Inflammatory Agents; D000894:Anti-Inflammatory Agents, Non-Steroidal; D004791:Enzyme Inhibitors; D009603:Nitroso Compounds; D026403:S-Nitrosothiols; D004008:Diclofenac; D009569:Nitric Oxide; D008623:Mercaptoethanol; C032635:S-nitrosomercaptoethanol; D006854:Hydrocortisone; C020491:rhein