Inhibitory effect of nicardipine on LPS/IFN-γ or peptidoglycan-stimulated nitric oxide production.

<p>BV-2 microglial cells were pretreated with different concentrations of nicardipine (1, 3, 5, or 10 μM) for 60 min before application of LPS (10 ng/ml) plus IFN-γ (10 ng/ml) (A) or peptidoglycan (10 μg/ml; B) for another 24 h. The culture media were collected and analyzed by a Griess reaction. Nitric oxide production is significantly different between the LPS/IFN-γ (or peptidoglycan) treatment alone and the LPS/IFN-γ (or peptidoglycan) treatment with nicardipine groups (one-way ANOVA followed by Bonferroni's <i>post hoc</i> test). The results are expressed as mean ± S.E.M. from 3 to 4 independent experiments. *, <i>p</i><0.05 compared with the control group; #, <i>p</i><0.05 compared with the LPS/IFN-γ or peptidoglycan treatment.</p

Inhibitory effect of nicardipine on LPS/IFN-γ- or peptidoglycan-stimulated iNOS and COX-2 expressions.

<p>(A and B) BV-2 microglial cells were pretreated with different concentrations of nicardipine (1, 3, 5, or 10 μM) for 60 min before application of LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 24 h. (C and D) Cells were pretreated with different concentrations of nicardipine (1, 3, 5, or 10 μM) for 60 min before application of peptidoglycan (10 μg/ml) for another 24 h. Western blot analysis for iNOS (A and C) and COX-2 (B and D) expression was performed on whole cell lysates. The quantitative results are shown in the bottom panels. iNOS expression was significantly different between the LPS/IFN-γ (or peptidoglycan) treated-group and the group treated LPS/IFN-γ (or peptidoglycan) with nicardipine (one-way ANOVA followed by Bonferroni's post hoc test). COX-2 expression was significantly different between the LPS/IFN-γ (or peptidoglycan) treated- group and the LPS/IFN-γ (or peptidoglycan) with nicardipine treated- group (one-way ANOVA followed by Bonferroni's <i>post hoc</i> test). The results are expressed as mean ± S.E.M. from 4 to 5 independent experiments. *, <i>p</i><0.05 compared with the LPS/IFN-γ or peptidoglycan treatment.</p

Nicardipine prevents LPS-induced microglial activation.

<p><b><i>Mice received</i></b> intraperitoneal injections of nicardipine at concentrations of either 5/kg or 50 mg/kg, <b><i>once per day</i></b>, for <b><i>3 consecutive days. On the third day</i></b>, nicardipine treatment was followed with a single <b><i>intraperitoneal injection</i></b> of LPS (20 mg/kg). Microglial morphology was visualized by anti-Iba-1 immunolabeling and DAB (n = 5 each group).</p

Effect of nicardipine on ATP-induced microglial cell migration.

<p>(A) Cell viability following nicardipine treatment in BV-2 microglia. Cells were treated with concentrations ranging from 1 to 10 μM of nicardipine for 24 h, and cell viability was measured by the MTT assay. The results are expressed as mean ± S.E.M. of three independent experiments. (B) Cells were pre-incubated with or without nicardipine (10 μM) for 60 min followed by a 24-h treatment with ATP (100 or 300 μM). <i>In vitro</i> migratory activities were examined using a cell culture insert system. The results are expressed as mean ± S.E.M. from 4 to 5 independent experiments. *, <i>p</i><0.05 compared with the control group; #, <i>p</i><0.05 compared with the ATP (300 μM) alone. (C) The migrated cells were visualized by phase-contrast imaging.</p

Nicardipine suppresses LPS/IFN-γ-induced signaling pathways.

<p>BV-2 microglial cells were pretreated with nicardipine (10 μM) for 60 min, then exposed to LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 60 min. Western blot analysis was performed on whole cell lysates, and the signal intensities were normalized to total protein expression. The results are expressed as mean ± S.E.M. from 3 to 4 independent experiments. *, <i>p</i><0.05 compared with the LPS/IFN-γ treatment alone group.</p

LPS-induced intracellular IL-6 and TNF-α expression on microglia is reduced by nicardipine.

<p>Mice received intraperitoneal injections of saline or nicardipine (5 mg/kg) for 3 consecutive days. On the third day, 2 h after injection of saline or nicardipine, mice were injected with LPS (5 mg/kg) and housed for another 24 h. (A) Representative bivariate dot plots of Percoll-isolated microglial cells stained with anti-CD11b-FITC and anti-CD45-PerCP-Cy5.5. Microglia were identified by CD11b⁺/CD45^low staining. Representative histograms of intracellular IL-6 (B) and TNF-α (C) expression in isolated microglia. Mean fluorescence intensity (M.F.I.) of intracellular IL-6 and TNF-α expressed by CD11b⁺/CD45^low microglia following experimental treatments.</p