Activation of the inducible nitric oxide synthase (iNOS) pathway contributes to inflammation-induced osteoporosis by suppressing bone formation and causing osteoblast apoptosis. We investigated the mechanism of action by which YS-51S, a synthetic isoquinoline alkaloid, inhibits iNOS expression and nitric oxide (NO) production in ROS 17/28 osteoblast cells activated with the mixture of TNF-alpha, IFN-gamma and LPS (MIX). YS-51S, concentration- and time-dependently, increased heme oxygenase (HO-1) expression. Treatment with YS-51S 1 h prior to MIX significantly reduced MIX-induced NO production and iNOS expression with the IC50 to NO production of 47+/-3.3 microM. Electrophoretic mobility shift assay (EMSA) and western blot analysis showed that YS-51S inhibited MIX-mediated activation and translocation of NF-kappaB to nucleus by suppressing the degradation of its inhibitory protein IkappaBalpha in cytoplasm. YS-51S also reduced NF-kappaB-luciferase activity. In addition, an HO-1 inhibitor ZnPPIX, antagonized the inhibitory effect of YS-51S on iNOS expression and DNA strand break induced by MIX, indicating prevention of NO production by YS-51S is associated with HO-1 activity. Moreover, YS-51S inhibited the oxidation of cytochrome c(2+) by peroxynitrite (PN). Our results indicated that YS-51S may be beneficial in NO-mediated inflammatory conditions such as rheumatoid arthritis by alleviating iNOS expression and NO-mediated cell death of osteoblast with 1) inducing HO-1 expression, 2) interfering the activation of NF-kappaB and 3) quenching of PN.
