Inhibition of NF-kappaB activation by 5-lipoxygenase inhibitors protects brain against injury in a rat model of focal cerebral ischemia - PubMed (original) (raw)

Inhibition of NF-kappaB activation by 5-lipoxygenase inhibitors protects brain against injury in a rat model of focal cerebral ischemia

Manu Jatana et al. J Neuroinflammation. 2006.

Abstract

Background: Stroke is one of the leading causes of death worldwide and a major cause of morbidity and mortality in the United States of America. Brain ischemia-reperfusion (IR) triggers a complex series of biochemical events including inflammation. Leukotrienes derived from 5-lipoxygenase (5-LOX) cause inflammation and are thus involved in the pathobiology of stroke injury.

Methods: To test the neuroprotective efficacy of 5-LOX inhibition in a rat model of focal cerebral IR, ischemic animals were either pre- or post-treated with a potent selective 5-LOX inhibitor, (N- [3-[3-(-fluorophenoxy) phenyl]-1-methyl-2-propenyl]-N-hydroxyurea (BW-B 70C). They were evaluated at 24 h after reperfusion for brain infarction, neurological deficit score, and the expression of 5-LOX. Furthermore, the mechanism and the anti-inflammatory potential of BW-B 70C in the regulation of nuclear factor kappa B (NF-kappaB) and inflammatory inducible nitric oxide synthase (iNOS) were investigated both in vivo and in vitro.

Results and discussion: Both pre- and post-treatment with BW-B 70C reduced infarctions and improved neurological deficit scores. Immunohistochemical study of brain sections showed IR-mediated increased expression of 5-LOX in the neurons and microglia. BW-B 70C down-regulated 5-LOX and inhibited iNOS expression by preventing NF-kappaB activation. Two other structurally different 5-LOX inhibitors were also administered post IR: caffeic acid and 2,3,5-trimethyl-6-[12-hydroxy-5,10-dodecadiynyl]-1,4-benzoquinone (AA-861). As with BW-B 70C, they provided remarkable neuroprotection. Furthermore, in vitro, BW-B 70C inhibited lipopolysaccharide (LPS) mediated nitric oxide production, iNOS induction and NF-kappaB activation in the BV2 microglial cell line. Treating rat primary microglia with BW-B70C confirmed blockage of LPS-mediated translocation of the p65 subunit of NF-kappaB from cytosol to nucleus.

Conclusion: The study demonstrates the neuroprotective potential of 5-LOX inhibition through down-regulation of NF-kappaB in a rat model of experimental stroke.

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Figures

Figure 1

Figure 1

Pretreatment with BW-B 70C protects the brain from infarction and improves neurological score. (A) Photograph showing effect of BW-B 70C on TTC-stained sections, (B) Effect of BW-B 70C on infarct volume (measured in six serial coronal sections arranged from cranial to caudal regions), (C) Effect of BW-B 70C on neurological score and (D) Effect of BW-B 70C on regional cerebral blood flow (CBF). Changes in CBF were not significantly different after ischemia between the untreated (vehicle) and treatment (BW-B 70C) groups. (E) Photomicrograph of expression of 5-LOX (n = 4) at 24 h reperfusion after 20 min MCAO (magnification X200). Data for infarct volume (n = 7) and blood flow (n = 4) are presented as means ± SD. *p < 0.001 vs. vehicle. Data for neurological deficit score (n = 7) are presented as individual data points.

Figure 2

Figure 2

5-LOX is expressed in neurons and microglia/macrophages. Co-localization of expression of (a) NSE, (d) GFAP; (g) RCA; and 5-LOX (b,e,h) at 24 h reperfusion after 20 min MCAO. Yellow fluorescence indicates co-localization of 5-LOX/NSE (c) and 5-LOX/RCA (i). 5-LOX/GFAP (f) showed very few yellow-fluorescent structures. Figures are representative of similar results obtained from three different sections of three different animals in each group (Magnification 400 ×).

Figure 3

Figure 3

BW-B 70C inhibits expression of iNOS and p65 in vivo. (A) Photomicrographs of immunohistochemistry of rat brain sections at 24 h reperfusion after 20 min MCAO showing remarkable iNOS expression in vehicle-treated (ii) but not in BW-B 70C-treated rats (iii). (B) BW-B 70C inhibited IR-induced iNOS gene expression measured as mRNA levels at 3 h of reperfusion after 20 min MCAO. The results are presented as mean ± SD of normalized expression of target gene with respect to GAPDH mRNA from three sets of animals. (C) Treatment with BW-B 70C prevented the nuclear translocation of p65 subunit of NF-κB (i-iii); the vehicle-treated animals showed nuclear translocation (ii) and treatment with BW-B 70C reversed this (iii) at 24 h reperfusion after 20 min MCAO. Figures are representative of similar results obtained from three groups of animals. A (i-iii) magnification 100 X; C (i-iii) magnification 200 X. *p < 0.01 vs. vehicle (n = 3).

Figure 4

Figure 4

BW-B 70C inhibits LPS-mediated iNOS expression in BV2 cells. (A) BV2 Cells were pretreated for 30 min with different concentrations of BW-B 70C followed by LPS (1 μg/ml) treatment. After 24 h, NO as nitrite was quantified in supernatant by Griess reagent. Data are presented as means ± SD for 3 different experiments. (B) Cell lysates were processed for western blot analysis of iNOS and β-actin after 24 h of stimulation with LPS (1 μg/ml). Figures are representative of 3 different experiments. *p < 0.001 vs. LPS; **p < 0.001 vs. LPS+25 μM BW-B70C; ***p < 0.001 vs. LPS+25 μM BW-B70C; +p < 0.001vs LPS + 50 μM BW-B 70C.

Figure 5

Figure 5

BW-B 70C inhibits LPS-induced NF-κB activation in microglial BV2 cells and prevents nuclear translocation of p65 in rat primary microglia. (A) Microglial cells (BV2) were transiently co-transfected with 1.5 μg of NF-κB luciferase reporter construct. Cells were pre-treated with BW-B 70C (25–75 μM) for 30 min followed by LPS (1 μg/ml) stimulation for 4 h. Luciferase activity was normalized with respect to β-galactosidase activity. Data are means ± SD of three different experiments. Immunoblot was performed at 1 h post treatment with LPS (1 μg/ml) for p65 (B) and p50 (C) in nuclear extract from primary microglia. A non-specific band (NS) was taken as internal standard. Blots are representative of three different experiments. (D) Immunohistochemcal analysis of rat primary microglia showing nuclear translocation of p65 1 h post LPS treatment. Cells were pretreated with BW-B 70C (75 μM) for 30 min before stimulation with LPS (1 μg/ml). Red fluorescence shows positive reaction for p65 and blue fluorescence showed nuclear staining with DAPI. LPS treatment translocated p65 to the nucleus, and treatment with BW-B 70C reversed it. Figures are representative of 3 experiments. (Magnification 200 X). #p < 0.001 vs. untreated; *p < 0.001 vs. untreated; **p < 0.001 vs. LPS; ***p < 0.001 vs. LPS+BW-B 70C (50 μM).

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