Inhibition of NF-κB activation by 5-lipoxygenase inhibitors protects brain against injury in a rat model of focal cerebral ischemia
Journal of Neuroinflammation
Inhibition of NF-B activation by 5-lipoxygenase inhibitors protects brain against injury in a rat model of focal cerebral ischemia
Manu Jatana firstname.lastname@example.org 1
Shailendra Giri email@example.com 1
Mubeen A Ansari firstname.lastname@example.org 1
Chinnasamy Elango email@example.com 1
Avtar K Singh 0
Inderjit Singh 1
Mushfiquddin Khan firstname.lastname@example.org 1
0 Department of Pathology and Laboratory medicine, Ralph H. Johnson VA Medical Center Charleston , SC 29425 , USA
1 Department of Pediatrics, Medical University of South Carolina , Charleston, SC 29425 , USA
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 5lipoxygenase (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-B) 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 IRmediated increased expression of 5-LOX in the neurons and microglia. BW-B 70C down-regulated 5-LOX and inhibited iNOS expression by preventing NF-B activation. Two other structurally different 5-LOX inhibitors were also administered post IR: caffeic acid and 2, 3, 5-trimethyl-6- [12hydroxy-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-B 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-B from cytosol to nucleus. Conclusion: The study demonstrates the neuroprotective potential of 5-LOX inhibition through down-regulation of NF-B in a rat model of experimental stroke.
Cerebral ischemia-reperfusion (IR) triggers lipid
peroxidation and inflammation, which exacerbate injury.
Recognition of inflammatory components involved in stroke has
expanded the list of potential targets for therapy . They
include inducible nitric oxide synthase (iNOS),
cyclooxygenase-2 (COX-2), nuclear factor kappa B (NF-B) and
5lipoxygeanse (5-LOX) [2,3]. 5-LOX is the key enzyme in
leukotriene biosynthesis . It translocates to the nuclear
membrane upon stimulation, where it co-localizes with
5LOX activating protein (FLAP) and cytosolic
phospholipase A2 (cPLA2) . This event converts arachidonic acid
to leukotrienes. Emerging data implicate both 5-LOX and
FLAP in the disease process of cerebral ischemia .
Increased leukotriene levels and 5-LOX expression have
been documented in stroke patients . Also, FLAP has
recently been identified as the first common gene
associated with higher risk in atherosclerosis and stroke .
5-LOX-mediated reactive oxygen species (ROS)
generation has been implicated in the activation of NF-B [9,10].
Recently, we have demonstrated that PLA2 and 5-LOX are
involved in lipopolysaccharide (LPS)-induced iNOS gene
expression via dependent and independent NF-B
pathways in glial cells . NF-B is an important
transcription factor that plays a pivotal role in mediating
inflammatory response to pro-inflammatory cytokines
and ROS in animal models of experimental stroke
[10,12]. In unstimulated cells, p50:p65 is sequestered in
the cytoplasm by inhibitory proteins known as NF-B
inhibitors (IBs). Upon stimulation, IB is
phosphorylated by an upstream IB kinase (IKK), which leads to its
ubiquitination and proteosomal degradation. This
process liberates p50:p65, which translocates to the nucleus
and induces transcription of several genes, including
iNOS. In ischemia, the p65 subunit is recognized to play
an important role in regulation of inflammation . It
has also been shown that P65 interaction with 5-LOX
activates NF-B .
In the present study, we used a 5-LOX inhibitor, N-
phenyl]-1-methyl-2-propenyl]-Nhydroxyurea (BW-B 70C), in a rat model of focal cerebral
IR. BW-B 70C demonstrated a neuroprotective role
through inhibition of both 5-LOX and NF-B. It is a
potent and a selective inhibitor of 5-LOX in vitro and in
vivo with a long half-life and high oral bioavailability.
Other potent 5-LOX inhibitors, caffeic acid and 2, 3,
5-trimethyl-6- [12-hydroxy-5, 10-dodecadiynyl]-1,
4-benzoquinone (AA-861) confirmed the neuroprotective efficacy
of 5-LOX inhibition. A similar protective effect of AA-861
has been reported in gerbils after transient ischemia .
Our observations document that 5-LOX inhibition
protects against IR injury in rats via down-regulation of the
inflammatory mediators NF-B and iNOS. Thus,
inhibiting the 5-LOX/NF-B pathway holds therapeutic potential
to attenuate inflammation-mediated brain injury after an
Reagents and cell culture
Dulbecco's Modified Eagle's Medium (DMEM) with
glucose, L-glutamine and sodium pyruvate was purchased
from Mediatech Inc. (Herndon, VA), Fetal Bovine Serum
(FBS) and Hank's balanced salt solution were obtained
from Life Technologies (Carlsbad, CA).
Lipopolysaccharide (LPS; 0111:B4)) from Escherichia coli, and MTT (3-(4,
bromide) were obtained from Sigma-Aldrich Chemical
Corporation (St. Louis, MO). Antibody against 5-LOX was
purchased from Cayman Chemical (Ann Arbor,
Michigan). Antibody against p65, p50, iNOS, NSE and -actin
were purchased from Santa Cruz Biotechnology, Inc.
(Santa Cruz, CA), and RCA-1, (ricinus communis
agglutinin-1) was purchased from Vector Laboratories,
(Burlingame, CA). Anti-cow GFAP was purchased from
DakoCytomation California Inc. (Carpinteria, CA). BW-B
70C was purchased from Tocris (Ellisville, MO). Caffeic
acid and AA-861 were purchased from Biomol (Plymouth
Meeting, PA). The enhanced chemiluminescence (ECL)
detecting reagent was from Amersham Pharmacia Biotech
(Arlington Heights, IL), and the luciferase assay system
was from Promega (Madison, WI,). NF-B-luciferase
chemiluminescence kit was purchased from Clontech
(Palo Alto, CA). IQ Sybr Green Supermix was purchased
from Bio-Rad (Hercules, CA).
Male Sprague-Dawley rats weighing 240260 g (Harlan
Laboratories, Wilmington, MA) were used in this study.
All animal procedures were approved by the Medical
University of South Carolina (MUSC) Animal Review
Committee and animals received humane care in compliance
with MUSC's experimental guidelines and the National
Research Council's criteria for humane care (Guide for the
Care and Use of Laboratory Animals).
The animals were divided into three groups: i) Control
(sham), ii) Ischemia/reperfusion (Vehicle) and (iii)
Treated (treated with 5-LOX inhibitors). In the treatment
group, the rats were given 5-LOX inhibitor, dissolved in
sterile DMSO (15 l) intravenously (IV) in the jugular
vein either before ischemia or at reperfusion. Three
structurally different 5-LOX inhibitors used were: BW-B 70C,
2.0 mg/kg 30 minutes before ischemia or 3.0 mg/kg at
reperfusion; AA-861, 3.0 mg/kg at reperfusion; caffeic
acid, 3.0 mg/kg of body weight at reperfusion. The rats in
Measurements were performed before MCAO (base) and at 30 min of reperfusion after drug administration as described in Methods. All
measurements other than temperature were performed in blood. Data are presented as mean SD for n = 3 in each group. Measurements were
also performed for sham group. No significant differences were observed among the groups. Basal, 30 min before MCAO; MBP, mean blood
pressure; Rep, reperfusion; Temp, temperature
the vehicle and sham groups were administered the same
volume of DMSO alone.
Transient focal cerebral ischemia model
Rats were subjected to middle cerebral artery occlusion
(MCAO) as described previously [16,17] with slight
modification. Briefly, rats were anesthetized with
intraperitoneal injection of xylazine (10 mg/kg body weight) and
ketamine hydrochloride (100 mg/kg). With the aid of a
dissecting microscope, the right common, internal and
external carotid arteries were exposed and the vagus nerve
separated carefully. Next the external carotid artery (ECA)
was isolated and ligated. A 4-0-monofilament nylon
suture (Harvard Apparatus, MA) was inserted through the
ECA into the internal carotid artery until a mild resistance
was felt, to occlude the middle cerebral artery (MCA) .
Animals were kept under constant conditions for 20
minutes of ischemia. At the end of the ischemic period, the
monofilament was withdrawn and the common carotid
artery clamp was removed. The animals were then
allowed to recover from anesthesia. During surgery, the
whole body temperature was maintained at 37.0 0.5C
using a heating pad, and monitored using a rectal
Measurement of regional cerebral blood flow (CBF)
The occlusion of MCA and reperfusion were monitored by
measuring the regional CBF using laser Doppler flow
meter (Oxyflo, Oxford Optronics, England and Periflux
system 5000; Perimed Inc., Sweden). For measurement of
the blood flow, a needle-shaped laser probe was placed
over the skull (on right side) at 1 mm posterior and 4.0
mm lateral to the bregma. Baseline of CBF was obtained
before MCAO. CBF was monitored continuously during
ischemia (20 min) with a criterion of < 20% of baseline
blood flow remaining after MCAO. Reperfusion was
confirmed by laser Doppler readings.
Measurement of physiologic parameters
The physiological variables were measured before and
after 30 min of reperfusion and are presented in Table 1.
The rectal temperature was monitored and maintained at
about 37 to 37.8C. Body temperature was monitored by
a rectal probe and maintained at about 37 0.5 C by a
homeothermic blanket control unit (Harvard Apparatus,
Holliston, MA). Cranial temperature was measured by
HSE Plugsys TAM-D (Harvard Apparatus). Blood gases
and blood pH were measured by pH/blood gas analyzer
iSTAT (Heska, Fort Collins, CO). Mean blood pressure
(MBP) was measured using a XBP1000 NIBP system (Kent
Scientific, Torrington, CT). It is non-invasive
computerized tail-cuff system and uses automated
inflation/deflation pump. Blood glucose levels were measured in plasma
using Quantichrom glucose assay kit (Bioassay systems,
Neurological deficit in the animals was determined by an
observer blinded to the identity of the groups, and was
assessed at 24 h of reperfusion. The scoring was based on
method of Huang et al.  as follows: 0, no observable
neurological deficit (normal); 1, failure to extend left
forepaw on lifting the whole body by tail (mild); 2, circling to
the contralateral side but normal posture at rest
(moderate); 3, leaning to the contralateral side at rest (severe); 4,
no spontaneous motor activity (very severe).
Measurement of ischemic infarct and image acquisition
Infarct volume was evaluated as previously described .
Briefly, after 24 h of reperfusion, the brains were quickly
removed and placed in ice-cold saline for 5 min and then
coronal sections were obtained at 2-mm intervals from
the frontal pole. The slices were incubated in 2% 2, 3,
5triphenyltetrazolium chloride (TTC) (Sigma, MO)
dissolved in saline for 15 min at 37C. The brain sections
were fixed by immersion in 10% formalin. The image of
infarct area was acquired in Photoshop 7.0 (Adobe
Systems) and quantified using Scion image image-analysis
software (Scion Corporation). The volume of infarction
was obtained from the product of average slice thickness
(2 mm) and sum of infarction area in all brain slices.
Infarct values were corrected for edema as described by
Swanson et al . Edema in this model contributed less than
10% of total infarction.
Protein expression was detected by
immunohistochemical analysis. Paraffin embedded sections of brain tissues
were stained for 5-LOX, RCA-1 (Ricinus communis
agglutinin-1), GFAP (Glial fibrillary acidic protein), iNOS, p65
and NSE (Neuron-specific enolase). BV2 cells and rat
primary microglia were stained for p65 protein expression.
In brief, the brain tissue sections were deparafinised and
rehydrated in sequential gradations of alcohol. After
antigen unmasking in unmasking solution (Vector Labs, CA),
sections were cooled and washed three times for two
minutes each in PBS. Sections were immersed for 10 min in
3% hydrogen peroxide to eliminate endogenous
peroxidase activity and blocked in 1% bovine serum albumin for
1 hour. Sections were incubated overnight with respective
primary antibody (1:100 dilution in blocking buffer).
After washing in PBS containing 0.1% Tween-20, sections
were incubated with the appropriate fluorophore tagged
secondary antibody (1:100 dilution in blocking buffer)
(Vector Labs, CA). Fluorescence was visualized under the
microscope. All the sections were analyzed using a Zeiss
Olympus Microscope and images were captured using a
Kontron Digital Camera. At least ten different fields were
recorded for each measurement and a representative
image was presented in figures. Images were captured and
processed in Adobe Photoshop 7.0 (Adobe Systems, CA)
and were adjusted by using the brightness and contrast
level and unmasking tools to enhance image clarity.
Maintenance of cell lines and preparation of rat primary
BV2 cells were maintained in DMEM (4.5 g glucose/L)
supplemented with 10% fetal FBS plus antibiotics and
induced with stimuli as indicated. The cell line was kindly
provided by Dr Michael McKinney of Mayo Clinic
(Jacksonville, FL, USA). Primary microglia were prepared from
rat cerebral tissue as described previously . Briefly,
after 10 days of culture, astrocytes were separated from
microglia and oligodendrocytes by shaking for 24 h in an
orbital shaker at 240 rpm. The microglia were plated onto
poly-lysine-coated plates for one hour, subsequently the
unattached cells were removed. For induction of nitric
oxide (NO), cells were stimulated with LPS under
Assay for NO synthesis
NO production was determined in cell culture
supernatants by measurement of nitrite, a stable reaction product
formed from released NO and molecular oxygen. Briefly,
100 l of culture supernatant was made to react with 100
l of Griess reagent and incubated at room temperature
for 15 minutes for optimal reaction product formation.
The absorbance of the assay samples was measured
spectrophotometrically at 570 nm using SpectraMax 190
(Molecular Devices, CA). NO concentrations were
calculated from a standard curve derived from the reaction of
NaNO2 (sodium nitrite) used as a standard in the assay.
Preparation of nuclear extracts
Nuclear extracts were prepared from treated and untreated
cells as described previously  based on modified
method of Dignam and coworkers . At stipulated time
points after treatment, cells were harvested, washed twice
with ice-cold PBS. Cells were then lysed in 400 l of buffer
A (containing: 10 mM HEPES, pH 7.9, 10 mM KCl, 2 mM
MgCl2, 0.5 mM dithiothreitol, 1 mM PMSF, 5 g/ml
aprotinin, 5 g/ml pepstatin A, and 5 g/ml leupeptin
containing 0.1% Nonidet P-40) for 15 minutes on ice. Cells were
vortexed vigorously for 15 seconds, and then centrifuged
at 20,000 g for 30 seconds. The pelleted nuclear fraction
was then resuspended in 40 l of buffer B (20 mM HEPES,
pH 7.9, 25% (v/v) glycerol, 0.42 M NaCl, 1.5 mM MgCl2,
0.2 mM EDTA, 0.5 dithiothreitol, 1 mM PMSF, 5 g/ml
aprotinin, 5 g/ml pepstatin A, and 5 g/ml leupeptin)
and kept on ice for 30 minutes. Lysates were centrifuged
at 20,000 g for 10 minutes. Supernatants containing the
nuclear proteins were diluted with 20 l of modified
buffer C (20 mM HEPES, pH 7.9, 20% (v/v) glycerol, 0.05
M KCl, 0.2 mM EDTA, 0.5 mM dithiothreitol, and 0.5 mM
PMSF) and were processed for immunoblotting
immediately or stored at -70C until further use.
For immunoblotting, after incubation in the presence and
absence of stimuli, the cells were scraped off, washed with
Hank's buffer, and sonicated in 50 mM Tris-HCl (pH 7.4)
containing protease inhibitors (1 mM PMSF, 5 g/ml
aprotinin, 5 g/ml antipain, 5 g/ml pepstatin A, and 5
g/ml leupeptin). Proteins were resolved by SDS-PAGE
and transferred onto nitrocellulose membranes. The
membranes were blocked for 1 h in 5% nonfat dry milk
TTBS (20 mM Tris, 500 mM NaCl, and 0.1% Tween 20,
pH 7.5) and incubated overnight at 4C in primary
antibody containing 5% nonfat dry milk. The blots were then
washed four times with TTBS (5 min/wash) and incubated
for 45 minutes at room temperature with HRP-conjugated
secondary antibody at a dilution of 1:5000. The blots were
then washed three times in TTBS and once in 0.1 M PBS
(pH 7.4) at room temperature; the desired protein was
detected with ECL, per the manufacturer's specifications
(Amersham Pharmacia Biotech).
Transfection of cells was done as described previously .
Plasmids were purified using the endotoxin-free plasmid
midi-prep kit (Qiagen, Santa Clarita, CA, USA). For
transient transfections, BV2 cells were seeded in 6-well plates
and grown to 6080% confluence in DMEM medium with
5% FBS (without antibiotics), and were transfected using
FuGene reagent (Promega) with 1.5 g of NF-B-luc
reporter construct vector or insertless expression vector
(pBluescript). 24 h after transfection, cells were
maintained in serum-free medium overnight and then treated
with LPS and/or BW-B 70C. Finally, 24 h later, luciferase
activity was measured in cell lysates prepared using lysis
buffer (Promega) as per the manufacture's protocol.
cDNA synthesis and real time PCR analysis
cDNA Synthesis and real time PCR analysis was carried
out with certain modifications of method described
earlier . Total RNA from brain tissue was isolated using
Trizol reagent (Gibco BRL, Carlsbad, CA) as per
manufacturer's instructions. Single-stranded cDNA was
synthesized from pooled RNA samples of rat brains from three
similarly treated rats by using the superscript
preamplification system (Life Technologies, Carlsbad, CA).
Quantitative real-time PCR was performed with the Bio-Rad
(Hercules, CA) iCycler iQ Real-Time PCR Detection
System as per conditions described previously . Briefly,
primer sets were designed and synthesized from
Integrated DNA Technologies (IDT, Coralville, IA). The
primer sequences were: GAPDH, forward primer,
5'-cctacccccaatgtatccgttgtg-3', reverse primer,
5'-ggaggaatgggagttgctgttgaa-3'; iNOS, forward primer,
5'ggaagaggaacaactactgctggt-3', reverse primer,
5'-gaactgagggtacatgctggagc-3'. Thermal cycling conditions were as
follows: activation of iTaq DNA polymerase at 95C for 10
minutes, followed by 40 cycles of amplification at 95 C
for 30 seconds and 5557.5C for 1 minute. The
normalized expression of target gene with respect to GAPDH was
computed for all samples by using Microsoft Excel data
Statistical analysis was performed using software
Graphpad Prism 3.0, unless stated otherwise. Values are
expressed as mean SD of n determinations or as
mentioned. Comparisons among means of groups were made
with a two-tailed Student's t-test for unpaired variables.
Multiple comparisons were performed using one-way
ANOVA followed by Bonferroni test. p values less than
0.05 were considered significant.
Treatment with 5-LOX inhibitors improves brain infarction
and neurological score after IR injury
Pretreatment with 5-LOX inhibitor BW-B 70C (2 mg/kg)
reduced infarct volume and improved neurological deficit
score (Fig. 1AC) recorded at 24 h reperfusion after 20 min
MCAO in rats. The animals were monitored for changes in
regional CBF before during and after occlusion (Fig. 1D).
Changes in the CBF were not significantly different after
ischemia between the untreated (vehicle) and BW-B 70C
treated groups. CBF measurements indicated that all rats
were subjected to a similar degree of ischemia (>80% drop
in CBF compared to baseline). Fig. 1A shows representative
TTC stained sections from sham; vehicle and BW-B 70C
treated animals, showing that the treatment reduced
infarction. As seen in Fig. 1B, BW-B 70C treated animals had
reduced infarct volume 215.0 35.0 mm3 compared with
vehicle group (512.2 30.5 mm3). Furthermore, significant
neuroprotection was observed even when BW-B 70C (3 mg/
kg) was administered at the time of reperfusion after
ischemia (infarct volume: 205.2 8.9 mm3) as shown in
Table 2A. Other structurally different 5-LOX inhibitors,
caffeic acid and AA-861, provided similar degrees of protection
when administered at the time of reperfusion. They reduced
the infarct volumes to 229.5 18.5 and 210.4 20.5,
respectively (Table 2A). Neurological deficit scores were
evaluated at 24 h after reperfusion (Fig. 1C and Table 2B),
and were consistent with the changes observed in infarct
volume. Pre-treatment with BW-B 70C improved the
neurological score to median1.0 compared to the vehicle group
median 3.0, (Fig. 1C). Even after ischemia, treatment with
5-LOX inhibitors reduced neurological score to median1.0
compared to the vehicle group (median 3.0) as shown in
Table 2B. The use of an effective dose of 5-LOX inhibitors is
based on maximal brain protection (infarct volume) at
lowest dose determined from a study carried out separately for
each inhibitor. Administration of either 2 mg/kg or 3 mg/kg
of BW-B 70C prior to ischemia had similar effect on
reduction of infarctions. However, the treatment with 2 mg/kg
BW-B 70C after ischemia was less effective compared to 3
mg/kg. The selected dose had no significant effects on
physiologic parameters (blood gases, cranial temperature, mean
blood pressure, blood glucose and pH) as shown in Table 1.
Postmortem studies have shown that brain sections from
stroke patients are positive for 5-LOX expression . To
investigate the increased expression of the 5-LOX enzyme
after IR injury, and whether administration of BW-B 70C
reduces the 5-LOX expression, we subjected rat brain
tissue sections to immunohistochemistry (Fig. 1E, ac).
There was increased expression of 5-LOX in the ipsilateral
hemisphere of brain tissue sections at 24 h after
reperfusion (Fig. 1E, b), which was reduced by the
administration of BW-B 70C (Fig. 1E, c). The mechanisms of 5-LOX
PFrigeturreeat1ment with BW-B 70C protects the brain from infarction and improves neurological score
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
BWB 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.
Infarct volume (mm3)
B. Neurological Score
Infarct volume and infarct area (A) and neurological deficit score (B) were measured at 24 h of reperfusion after 20 min MCAO. Animals were
treated either with 5-LOX inhibitors dissolved in DMSO alone (vehicle) at reperfusion as described in Methods. Data in Table B represent number
of animals showing corresponding neurological score (ranging from 0 to 4 as detailed in Methods). Data are expressed as mean SD for infarct area
and infarct volume and as range and median for neurological deficit score. *p < 0.001 vs. vehicle.
Co-localization of 5-LOX expression in neurons and
microglia/macrophage in brain after IR injury.As inferred
from the data above (Fig. 1E, ac), there is an increase in
the expression of 5-LOX enzyme after IR injury. To
determine the cellular localization of 5-LOX expression,
sections from ischemic brain after 24 h of reperfusion were
subjected to immunohistochemistry with antibody
against 5-LOX. 5-LOX expression co-localized with NSE, a
neuron specific marker, implying that 5-LOX protein
expression was increased in neurons after IR (Fig. 2c). The
expression also co-localized in the microglia/macrophage,
as seen by merging the 5-LOX and microglia/macrophage
antigen RCA-1 (Fig. 2i). RCA-1 is not a widely used
antigen for microglia. However, Rezaie et. al. have lately
described this antigen as specific to microglia, detecting
both developing and resting adult microglia . A
colocalization study of 5-LOX and GFAP, a marker for
activated astrocytes, showed a few 5-LOX/GFAP positive cells
(Fig. 2f). These observations indicate that 5-LOX protein
is up regulated mainly in microglia/macrophage and
neurons in the ipsilateral hemisphere after IR injury.
Inhibition by BW-B 70C of iNOS protein expression and
p65 translocation in brain after IR injury
As concluded from the data above (Figs. 1E and 2), there
is an increased expression of 5-LOX enzyme in the brain
after IR injury. Growing evidence suggests that 5-LOX and
iNOS communicate and regulate the signaling cascade of
inflammatory gene expression [11,26]. BW-B 70C
treatment reduced the IR injury-induced inflammatory
5F-iLgOurXe i2s expressed in neurons and microglia/macrophages
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
5LOX/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
response by down-regulating iNOS expression (Fig. 3A iii)
via inhibition of iNOS gene expression (Fig. 3B). This
gene expression was quantified by real time PCR analysis
of mRNA 3 h after reperfusion.
Activation of NF-B is involved in iNOS gene expression
and is associated with the translocation of p50:p65
heterodimer into the nucleus. BW-B 70C prevented nuclear
translocation of p65 subunit in vivo, as demonstrated by
immunohistochemistry (Fig. 3C, iii). Taken together,
these data suggest that BW-B 70C leads to
down-regulaBFWigu-Bre703C inhibits expression of iNOS and p65 in vivo
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-B (i-iii); the
vehicletreated 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).
tion of iNOS gene expression by inhibiting p65
translocation to the nucleus.
BW-B 70C attenuates LPS-mediated expression of iNOS
and levels of NO in BV2 microglial cell line
It has been documented that iNOS-derived NO from
microglia/macrophages contributes to the pathobiology
of cerebral IR injury . Earlier we showed that glial
cells, specifically microglia, produce NO in response to
induction of iNOS by LPS and cytokines . NO
produced by iNOS has been shown to contribute to neuronal
death in neurogenerative diseases . Furthermore,
growing evidence suggests the role of 5-LOX in induction
of inflammatory genes . Therefore, we examined the
role of 5-LOX in the regulation of iNOS expression in glial
cells in vitro.
BW-B 70C was not toxic up to 75 M concentration as
assessed in vitro by MTT assay (data not shown). The other
selective 5-LOX inhibitor, AA-861 was found to be toxic at
5075 M concentrations and caused cell death. MTT
assays were not performed on caffeic acid-treatment
experiments because, while it is a potent 5-LOX inhibitor,
it is not selective. Hence, we used BW-B 70C in cell culture
Treatment with BW-B 70C (up to 75 M) attenuated NO
production measured as nitrite (Fig. 4A) and iNOS
protein expression (Fig. 4B) quantified by immunoblot
analysis in LPS-treated cells. These data suggest that 5-LOX
inhibition lead to the down-regulation of iNOS
BW-B 70C attenuates LPS-mediated expression of NF-B
luciferase activity in BV2 microglial cells
Because the activation of NF-B is important for the
induction of iNOS in glial cells , the effect of BW-B
70C on the activation of NF-B was examined in BV2
cells. BW-B 70C inhibited the LPS-mediated
NF-Bdependent luciferase activity in a dose-dependent manner
To confirm further the inhibition of NF-B by BW-B 70C,
we examined the effect of BW-B 70C on nuclear
translocation of p50/p65 complex in LPS-treated primary
microglial cells. LPS stimulated the nuclear translocation of p65
and p50 subunits to the nucleus, and the translocation
was prevented by BW-B 70C treatment (Fig. 5BC). The
observation was confirmed by immunocytochemical
analysis (Fig. 5D). These results indicate that inhibition of
5-LOX prevents nuclear translocation of p50/p65
complex, which may cause down-regulation of NF-B activity.
BFWigu-Bre704C inhibits LPS-mediated iNOS expression in BV2 cells
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 g/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 -actin after 24 h of stimulation with LPS (1 g/ml). Figures are representative of 3 different
experiments. *p < 0.001 vs. LPS; **p < 0.001 vs. LPS+25 M BW-B70C; ***p < 0.001 vs. LPS+25 M BW-B70C; +p < 0.001vs
LPS + 50 M BW-B 70C.
Brain damage caused by IR is due, in part, to secondary
injury from inflammation . The degree of
inflammation is exacerbated by increased lipid peroxidation which
increases neuronal death . Leukotrienes derived from
the metabolism of arachidonic acid by the 5-LOX enzyme
are potent inflammatory mediators in IR injury .
Recently, 5-LOX has been shown to be involved in
ischemic-like injury in neuronal PC12 cells . As in
human brain following stroke , we found increased
expression of 5-LOX after IR in rat brain neurons and
microglia/macrophage (Fig. 2).
Many substituted N-hydroxyureas, including BW-B 70C
and Zileuton, are well-known potent and selective
inhibitors of 5-LOX [36,37]. BW-B 70C has a long half-life and
high oral bioavailability. Zileuton, an anti-leukotriene
drug, has been identified as an anti-inflammatory
compound  and is currently undergoing clinical trials in
patients suffering from diseases in which leukotrienes
play a pathogenic role . To further support the idea
that 5-LOX inhibition provides significant
neuroprotection in experimental stroke, we used other
well-established 5-LOX inhibitors, (caffeic acid and AA-861). The
three 5-LOX inhibitors used in this study are structurally
different. All were highly protective, reduced infarction
and improved neurological deficit score (Fig. 1AC and
Table 2). 5-LOX inhibitors are therefore neuroprotective
irrespective of their structural identities. BW-B 70C may
be safer as a therapeutic agent due to the toxicity (cell
viability assay using MTT) associated with AA-861 in vitro
(data not shown). Furthermore, BW-B 70C may show
greater potential in humans due to its structural similarity
to Zileuton, high oral bioavailability and longer half life.
In our studies, we have used a focal cerebral ischemia
animal model involving transient MCAO followed by
reperfusion . This model closely reproduces clinical
ischemic brain damage, showing the oxidative stress and
the inflammation observed in human ischemic stroke
patients . To test whether secondary inflammatory
injury can be reversed by 5-LOX inhibitors, we selected a
model with short-term (20 min) MCAO (with ~80% drop
in CBF compared to basal value Fig. 1D) followed by
reperfusion. In this model, there was more significant
inflammatory response than cell death, which is
appropriate to determine the efficacy of anti-inflammatory drugs
BFmWiagruy-Brme7i05crCoginlihaibits LPS-induced NF-B activation in microglial BV2 cells and prevents nuclear translocation of p65 in rat
priBW-B 70C inhibits LPS-induced NF-B 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 g of NF-B luciferase
reporter construct. Cells were pre-treated with BW-B 70C (2575 M) for 30 min followed by LPS (1 g/ml) stimulation for 4
h. Luciferase activity was normalized with respect to -galactosidase activity. Data are means SD of three different
experiments. Immunoblot was performed at 1 h post treatment with LPS (1 g/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 M) for 30 min before stimulation with LPS (1 g/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 M).
NF-B is one of the key regulators of inflammation. In
brain ischemia, its roles in both cell survival and cell death
have been shown . Recently, Zhang et al 
documented that activation of NF-B in neurons is deleterious
in cerebral ischemia. In addition, the NF-B pathway has
been shown to be instrumental in up-regulation of iNOS
and of other inflammatory mediators that are damaging
in stroke . Inhibition of iNOS expression in
microglia/macrophage by antioxidants has been shown to
protect the brain after IR injury . We demonstrated in this
study that IR injury caused an increased expression of
5LOX, NF-B and iNOS. The attenuation of expression of
these inflammatory mediators by 5-LOX inhibitors
resulted in neuroprotection as seen by decreased
infarction size and improved neurological deficit scores (Fig.
1AC and Table 2). These inhibitors did not significantly
alter physiologic parameters as shown in Table 1.
The critical aspect of 5-LOX/NF-B signaling may be its
direct interaction with the p65 subunit of NF-B . It
has been reported that NF-B activation occurs via
5-LOXmediated generation of ROS in lymphoid cells .
However, the mechanisms of NF-B activation by direct
interaction with 5-LOX remain to be investigated. The 5-LOX
inhibitor BW-B 70C used in this study inhibited NF-B
luciferase activity in vitro (Fig. 5A). It also inhibited p65
translocation to nucleus both in vitro (Fig 5B and 5D) and
in vivo (Fig. 3C, iii). Furthermore, BW-B 70C reduced
iNOS expression in vitro (Fig. 4B). We hypothesize that
5LOX action is upstream to NF-B, and its inhibition
results in reduced expression of NF-B and iNOS, thereby
The 5-LOX-leukotriene pathway has been implicated in
aneurysm formation . The present study accords with
other studies showing neuroprotective effects against
inflammation-mediated injury . Inhibition of
phospholipase A2 (PLA2), the enzyme that liberates
arachidonic acid from membrane phospholipids, provides
limited protection in stroke because the metabolism of
polyunsaturated fatty acids (PUFA) is required to generate
several anti-inflammatory eicosanoids  and epoxides
. The COX-2 pathway has been documented as a
valuable therapeutic target for ischemic brain injury [48,49].
However, it is also recognized that chronic inhibition of
this pathway produces an eicosanoid synthesis imbalance
that promotes undesirable vascular effects .
Furthermore, the inhibition of the COX-2 pathway would result
in the bioavailability of PLA2-derived PUFAs to
lipoxygenases. This may result in excessive production of
leukotrienes and the generation of ROS. Increased leukotriene
production results in depletion of GSH, thus making cells
vulnerable to ROS injury . Inhibiting the
5-LOX/NFB pathway has potential to circumvent the problems
associated with blocking PLA2 or the COX-2 pathway.
However, it has been demonstrated that 5-LOX knock out
mice do not show protection during IR . In this
context, we hypothesize that 5-LOX expression modulates
multiple events, and its role may become evident only
under pathological conditions. Further, a certain basal
activity of the enzyme may be required physiologically,
and the complete depletion of the enzyme is detrimental,
as is the case with TNF- knock out mice .
This study demonstrates the protective effect of 5-LOX
inhibitors when administered either prior to ischemic
insult or at reperfusion in a rat model of experimental
stroke. The protective effect of 5-LOX inhibitors is due in
part to down-regulation of iNOS via inhibition of NF-B
activation. Acute stroke is a multi-component
inflammatory disorder, and its treatment will involve agents
antagonizing multiple mechanisms of inflammation.
Therefore, we propose that 5-LOX inhibitors may have
therapeutic potential to treat neuroinflammation in
ischemic stroke injury.
This study is based on an original idea of Mushfiquddin
Khan (MK) and Inderjit Singh. MK and Manu Jatana (MJ)
wrote the manuscript. Shailendra Giri directed and
performed the in vitro experiments. Elango Chinnasamy,
Manu Jatana and Mubeen A Ansari carried out the animal
studies. Avtar K Singh critically examined the
The authors gratefully acknowledge Dr. G. Rawadi for providing the
NFB-luciferase promoter construct and Dr. Michael McKinney for providing
the BV2 cell line. Animal facilities were supported by NIH grant No C06
RR015455 from the Extramural Research Facilities Program of the National
Center for Research Resources. This work was supported by NIH grant
Number C06 RR018823 from the Extramural Research Facilities Program
of the National Center for Research Resources. We acknowledge the
writing assistance of Dr Tom G. Smith, MUSC writing center. Dr. Abdul Aziz
from Morgan State University, Baltimore, MD helped in statistical
evaluations and Dr. Anne G. Gilg from MUSC, helped in construction of figures.
We thank Ms. Joyce Bryan for her help in arrangement of animals and
chemicals. We also thank Ms. Carrie Barnes for providing histopathological
support. This work was supported by grants (NS-40144, NS-22576,
NS34741, NS-37766, and NS-40810) from the National Institutes of Health
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