Expression Of Inducible Nitric Oxide Synthase And Apoptosis Induced By Activating Nuclear Factor κB In Mouse Macrophages Stimulated With Ureaplasma Urealyticum Lipid-associated Membrane Proteins

Objectives: The present study was undertaken to investigate the potential pathogenicity of ureaplasma urealyticum and the molecular mechanisms that is responsible for the induction of inducible nitric oxide synthase(iNOS) gene expression and cell apoptosis in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from Ureaplasma urealyticum (Uu).LAMPs were utilized to study the expression of iNOS and the production of nitric oxide (NO); The activation of nuclear factor kappa B (NF-kB) and cell apoptosis in mouse macrophages stimulated by LAMPs were also detected.Methods: Mouse macrophages were stimulated with Uu LAMPs to assay NO with Griess Reagents,the expression of iNOS were detected by RT-PCR and western blot. The activation of NF-kB was examined in mouse macrophages treated with LAMPs by indirect immuno- fluorescence (IFA) , unmunocytochemistry and western blot.the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kB and of cycloheximide (CHX), a protein synthase inhibitor, on the expression of iNOS and on the activation of NF-kB were also investivated in mouse macrophages treated with LAMPs by RT-PCR and western blotCell apoptosis was detected in mouse macrophages treated with LAMPs by Annexin-FITC Apoptosis Detection Kit.Results: Uu LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose-and time-dependent manner. There was an optimal concentration of LAMPs at 3μg/mL in the induction of NO (13.56±0.54μmol/L), when the concentration of LAMPs added from 3μg/mL to 5μg/mL,whereas the production of NO decreased. The NO could be detected in the conditioned media after 4h by stimu-...