Carbon monoxide (co) downregulates hepatic inducible nitric oxide synthase (inos) by a mitogen-activated protein kinase (mapk) dependent mechanism

Abstract

Background: Low dose CO is non-toxic and has been shown to possess potent anti-inflammatory properties. We hypothesize that CO would decrease cytokine-stimulated iNOS induction in rat hepatocytes. Methods: Primary hepatocytes from Sprague-Dawley rats were exposed to combinations of cytokines (TNFα, IL-1, IFγ) in the presence or absence of CO pre- and co- treatment at 250 ppm. Protein was harvested for Western blotting at 0, 1 and 6 hours after treatment. Greiss reaction was used to quantify NO production and crystal violet staining was used to measure cell viability. Experiments were performed 2–3 times each and t-test was used to assess significance between groups. Results: CO exposure at 250 ppm did not decrease hepatocyte viability measured by crystal violet staining at 24 hours. Pretreatment with CO resulted in a statistically significant decrease in cytokine mixture (TNFα 500 U/ml, IL-1 200 U/ml, IFγ 100 U/ml) stimulated hepatocyte NO production measured by Greiss reaction (37.8 ± 7.4 to 20.8 ± 5.7 μM, p < 0.05). Western blot 6 hours after stimulation resulted in a similar decrease in iNOS protein in the CO exposed hepatocytes. Pretreatment with CO up-regulated mitogen activated protein kinase phosphatase-1 (MKP-1) protein and decreased TNFα induced c-Jun N-terminal kinase (JNK) activation measured by Western blot. Conclusions: CO pretreatment decreased cytokine stimulated hepatocyte iNOS protein synthesis and NO production. A possible mechanism for this effect is the up-regulation of MKP-1 protein and subsequent decrease in cytokine stimulated JNK activation. The study provides a new mechanism for the anti-inflammatory effects of CO and validates the importance of JNK activation in pro-inflammatory signaling.