Figure 1

Cp and Cp-ox potentiate lipopolysaccharide (LPS)-induced nitric oxide (NO) production in the absence of additional inducible nitric oxide synthase (iNOS) induction. (A) Nitrite production assessed by Griess assay in culture medium of microglial cells after 24 hours of treatment with medium alone (Ctrl), LPS (10 ng/ml), ceruloplasmin (Cp), oxidized-ceruloplasmin (Cp-ox), heat-denatured ceruloplasmin (Cp-heated) and BSA alone (all at 20 μg/ml) or in combined treatment with LPS. Results are expressed as μM of nitrite present in culture medium that reflects the NO-production. (B) Western blot analysis of inducible nitric oxide synthase expression in microglial cells after treatments as described in (A). Densitometric optical density (OD) for iNOS bands were normalized with α-tubulin expression and are reported as ratio of the OD of specific treatments versus OD of LPS treatment. Bottom panels are representative of one experiment. (C) Dose-dependent analysis of Cp co-treatment in the potentiation of the LPS-induced nitrite production. Microglial cells were treated with a steady amount of LPS (10 ng/ml) plus increasing concentrations of Cp (1, 2.5, 5, 10 and 20 μg/ml). The nitrite production was reported as ratio of nitrite production in specific treatment versus LPS treatment. (D) Western blot analysis for iNOS expression in microglial cells upon LPS treatment combined with dose-dependent increase of Cp. Densitometric analysis was reported as described in (B). Bottom panels are representative of one experiment. Three/four independent experiments (as indicated n =) were performed and mean values, calculated using pooled data from different experiments, with standard error are reported. Statistical P-values were evaluated by non-parametric Mann-Whitney test. In all analyses, P < 0.05 was considered to be statistically significant.