Nicardipine is a dihydropyridine-type calcium channel blocker (CCB) with a peculiar cerebrovascular profile developed approximately 30 years ago and the most frequent indication is antihypertensive agent for acute brain injury (Degoute, 2007;
Gianino and Afuwape, 2012; Kim et al., 2012; Narotam et al., 2008; Qureshi et al., 2006; Sato et al., 2012). Nicardipine is widely administrated in the patients of intensive care because it shows effective antihypertensive effect and more safety. In comparison with other antihypertensive agents, their effects are similar to or even better than those exerted by other drugs. In clinical practice, nicardipine is superior to other antihypertensive agents in less cardiopulmonary suppression.
Some studies show nicardipine may also play a role as neuroprotectants (Amenta et al., 1996; Amenta and Tomassoni, 2004; Amenta et al., 2008; Inzitari and Poggesi, 2005; Sato et al., 2012) but the definite mechanisms between nicardipine and glia in unknown.
Acute brain injuries include ischemia stroke, spontaneous intracerebral hemorrhage and traumatic brain injury. All of these conditions often carried the crisis hypertensive due to autoregulation in CNS. The injuries within brain parenchyma trigger a series of adverse events causing secondary insults and severe neurological deficits. The neuroinflammatory response is the key mechanism in secondary insults and microglia play the most important role in neuroinflammation (Aloisi, 1999;
Aronowski and Zhao, 2011; Block and Hong, 2005; Kettenmann et al., 2011).
We design a series study including in vivo and in vitro method and the result demonstrate that nicardipine could suppress neuroinflammatory response through MAPK/Akt pathway and inhibit the activation of microglia. These results maybe explain the neuroprotective effect in clinical practice of nicardipine.
Figures
Figure 4 Chemical structure and the viability on nicardipine treatment. (A) Chemical structures of (±)-2-(benzyl-methyl amino) ethyl methyl 1,4-dihydro-2,6-dimethyl- 4-(m-nitrophenyl)-3,5-pyridinedicarboxylate monohydrochloride. (B) Cell viability on nicardipine treatment in BV-2 microglia. Cells were treated with various concentrations of niardipine for 24 h, and cell viability was measured by the MTT assay. The results are expressed as means ± S.E.M. of three independent experiments.
Figure 5 Effect of nicardipine on ATP-cellular migration in microglia. (A) BV-2 microglia were pre-incubated with or without nicardipine for 60 mins followed by incubation with ATP (100 or 300 µM) for another 24 h. In vitro migratory activities were examined using cell culture inserts system. The results are expressed as means ± S.E.M. of three independent experiments. The migrated cells were visualized by phase-contrast imaging (B). * means p value <0.05 compared with control group. # means p< 0.05 compared with the ATP-treatment group.
Figure 6 Inhibitory effect of cytokine expressions in LPS/IFN-γ-stimulated BV-2 microglia. (A) Cells were pretreated with various concentrations of nicardipine (1, 3, 5, or 10 µM) for 60 mins before application of LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 24 h. (B) Cells were pretreated with various concentrations of nicardipine (1, 3, 5, or 10 µM) for 60 mins before application of peptidoglycan (10 µg/ml) for another 24 h. The culture media were collected and analyzed by NO production. The results are expressed as means ± S.E.M. of three independent experiments. * means p<0.05 compared with control group.
Figure 7 Inhibitory effect of iNOS and COX-2 expressions in LPS/IFN-γ-stimulated BV-2 microglia. (A and B) Cells were pretreated with various concentrations of nicardipine (1, 3, 5, or 10 µM) for 60 mins before application of LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 24 h. (C and D) Cells were pretreated with various concentrations of nicardipine (1, 3, 5, or 10 µM) for 60 mins before application of peptidoglycan (10 µg/ml) for another 24 h. Whole cell lysis protein was extracted and subjected to western blot analysis for iNOS (A and C) and COX-2 (B and D) expression, respectively. The results are expressed as means ± S.E.M. of three independent experiments. The results are representative of three independent experiments.
Figure 8 Nicardipine suppresses expression of inflammatory mediators in microglial cells. BV-2 microglia were pretreated with various concentrations of nicardipine (1, 5, or 10 µM) for 60 mins followed by stimulation with LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 6 h. The expressions of iNOS, COX-2, IL-6, and IL-1β were determined by real-time PCR. The results are expressed as means ± S.E.M. from at least three independent experiments. * means p<0.05 compared with control group. # means p<0.05 compared with the LPS/IFN-γ-treatment group.
Figure 9 Nicardipine suppresses LPS plus IFN-γ-induced MAPK and Akt signaling pathways. BV-2 microglia were pretreated with nicardipine (10 µM) for 60 mins followed by stimulation with LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 60 mins. Whole cell lysis protein was extracted and subjected to western blot analysis.
The results are representative of three independent experiments.
Figure 10 Nicardipine suppresses LPS plus IFN-γ-induced NF-κB and AP-1 signaling pathways. BV-2 microglia were pretreated with nicardipine (10 µM) for 60 mins followed by stimulation with LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 90 mins. Whole cell lysis protein was extracted and subjected to western blot analysis.
The results are representative of three independent experiments.
Figure 11 Inhibitory effect of nicardipine prevents LPS-induced microglial activation.
Mice were treated with 5 or 50 mg/kg nicardipine intraperitoneally once daily for three consecutive days before a single intraperitoneal injection of 20 mg/kg LPS.
Microglial morphology was visualized by DAB immunohistostaining with the anti-Iba-1 antibody. Each group n= 3.
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