The flavonoid fisetin attenuates postischemic immune cell infiltration, activation and infarct size after transient cerebral middle artery occlusion in mice.

Abstract

The development of the brain tissue damage in ischemic stroke is composed of an immediate component followed by an inflammatory response with secondary tissue damage after reperfusion. Fisetin, a flavonoid, has multiple biological effects, including neuroprotective and antiinflammatory properties. We analyzed the effects of fisetin on infarct size and the inflammatory response in a mouse model of stroke, temporary middle cerebral artery occlusion, and on the activation of immune cells, murine primary and N9 microglial and Raw264.7 macrophage cells and human macrophages, in an in vitro model of inflammatory immune cell activation by lipopolysaccharide (LPS). Fisetin not only protected brain tissue against ischemic reperfusion injury when given before ischemia but also when applied 3 hours after ischemia. Fisetin also prominently inhibited the infiltration of macrophages and dendritic cells into the ischemic hemisphere and suppressed the intracerebral immune cell activation as measured by intracellular tumor necrosis factor α (TNFα) production. Fisetin also inhibited LPS-induced TNFα production and neurotoxicity of macrophages and microglia in vitro by suppressing nuclear factor κB activation and JNK/Jun phosphorylation. Our findings strongly suggest that the fisetin-mediated inhibition of the inflammatory response after stroke is part of the mechanism through which fisetin is neuroprotective in cerebral ischemia.

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The flavonoid fisetin attenuates postischemic
immune cell infiltration, activation and infarct size
after transient cerebral middle artery occlusion in mice
Mathias Gelderblom1,6, Frank Leypoldt1,6, Jan Lewerenz2, Gabriel Birkenmayer1,
Denise Orozco3, Peter Ludewig1, John Thundyil4, Thiruma V Arumugam4,
Christian Gerloff1, Eva Tolosa3, Pamela Maher5,7and Tim Magnus1,7
1Department of Neurology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;2Department
of Neurology, University of Ulm, Ulm, Germany;3Department of Immunology, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany;4School of Biomedical Sciences, The University of Queensland, St
Lucia, Australia;5Cellular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla,
California, USA
The development of the brain tissue damage in ischemic stroke is composed of an immediate
component followed by an inflammatory response with secondary tissue damage after reperfusion.
Fisetin, a flavonoid, has multiple biological effects, including neuroprotective and antiinflammatory
properties. We analyzed the effects of fisetin on infarct size and the inflammatory response in a
mouse model of stroke, temporary middle cerebral artery occlusion, and on the activation of immune
cells, murine primary and N9 microglial and Raw264.7 macrophage cells and human macrophages,
in an in vitro model of inflammatory immune cell activation by lipopolysaccharide (LPS). Fisetin not
only protected brain tissue against ischemic reperfusion injury when given before ischemia but also
when applied 3 hours after ischemia. Fisetin also prominently inhibited the infiltration of
macrophages and dendritic cells into the ischemic hemisphere and suppressed the intracerebral
immune cell activation as measured by intracellular tumor necrosis factor a (TNFa) production.
Fisetin also inhibited LPS-induced TNFa production and neurotoxicity of macrophages and
microglia in vitro by suppressing nuclear factor jB activation and JNK/Jun phosphorylation. Our
findings strongly suggest that the fisetin-mediated inhibition of the inflammatory response after
stroke is part of the mechanism through which fisetin is neuroprotective in cerebral ischemia.
Journal of Cerebral Blood Flow & Metabolism advance online publication, 11 January 2012; doi:10.1038/jcbfm.2011.189
Keywords: fisetin; flavonoids; macrophages; microglia; stroke
Introduction
Ischemic stroke is a devastating disease representing
the second leading cause of death in the Western
world and the leading cause of disability in adults
(WHO, 2011). Ischemic stroke is believed to evolve in
distinct phases (Dirnagl et al, 1999). The initial
ischemia leads to apoptosis and necrotic cell death,
followed by inflammation triggered and sustained by
reperfusion of ischemic brain tissue, which is
ultimately replaced by regeneration. We among
others have provided evidence in a rodent model of
ischemic stroke that poststroke inflammation indeed
contributes to secondary tissue damage (Gelderblom
et al, 2009).
The flavonoid fisetin (3,30,40,7-tetrahydroxyxfla-
vone) has direct intrinsic antioxidant activity and
indirect antioxidant effects by increasing levels of
glutathione, thus improving survival of neuronal
cells upon chemical ischemia in vitro using iodoa-
cetic acid (Maher et al, 2007). Moreover, fisetin has
been shown to suppress microglial activation follow-
ing lipopolysaccharide (LPS) treatment (Zheng et al,
2008). Fisetin not only improves the clinical out-
come in a rabbit model of ischemic stroke after 24
hours when given 5 minutes after onset of ischemia
(Maher et al, 2007) but has also been reported to
Received 20 July 2011; revised 6 November 2011; accepted 27
November 2011
Correspondence: Dr P Maher, The Salk Institute for Biological
Studies, Cellular Neurobiology Laboratory, Post Office Box 85800,
San Diego, CA 92186-580, USA or Dr T Magnus, Department
of Neurology, University Medical Center Hamburg-Eppendorf,
Martinistr. 52; 20246 Hamburg, Germany.
E-mail: pmaher@salk.edu or tmagnus@uke.de
6These authors are equally contributing first authors.
7These authors are equally contributing last authors.
This work was supported by AHA Grant 075514Y to PM,
Landesexzellenzinitiative (LEXI) Hamburg and DFG HO 3312/1-1
to TM.
Journal of Cerebral Blood Flow & Metabolism (2012), 1–9
& 2012 ISCBFM All rights reserved 0271-678X/12 $32.00
www.jcbfm.com

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reduce infarct size after 24 hours in a permanent
middle cerebral artery occlusion (MCAO) model in
rats when given 30 minutes after induction of
ischemia (Rivera et al, 2004).
Whether fisetin is protective even when given at a
clinically relevant time point 3 hours after ischemia
and whether the protective effect persists over a
longer time course is unknown. Moreover, the
mechanisms through which fisetin protects against
ischemia have not been analyzed.
We hypothesized that an important aspect of the
neuroprotective effect of fisetin in stroke could be
due to modulation of the poststroke inflammatory
response. Since secondary inflammation is initiated
hours after the initial ischemia (Gelderblom et al,
2009), we assumed that delayed treatment with
fisetin 3 hours after ischemia should still be similarly
protective as pretreatment. To provide evidence that
the observed changes in the inflammatory response
are indeed due to antiinflammatory effects of fisetin,
we also tested whether immune cells respond to fisetin
in an in vitro paradigm of immune cell activation by LPS.
Materials and methods
In Vivo Stroke Model (Middle Cerebral Artery
Occlusion)
A total of 90 mice were used in this study. All experiments
were approved by and conducted in accordance with the
laws and regulations of the regulatory authorities for
animal care and use in Hamburg (Beho ¨rde fuer Lebensmit-
telsicherheit und Veterina ¨rwesen—26/07). The investiga-
tion conforms to the Guide for the Care and Use of
Laboratory Animals published by the US National Insti-
tutes of Health (NIH Publication No. 83-123, revised 1996)
and was performed in accordance with the ARRIVE
guidelines (http://www.nc3rs.org/ARRIVE). In all, 12-
week-old C57Bl/6 wild-type mice (Charles River, Sulzfeld,
Germany) were used. The weight of mice ranged between
18 and 24g. Mice were randomized and the scientists were
blinded to group. Sample size calculation was performed
(stroke size from pilot experiments, significance level 0.05,
power 90%) and resulted in 12 or 8 animals per group to
see a difference of 21% or 27% in stroke size, respectively.
Temporary MCAO (tMCAO) was performed for 60 minutes
as previously described (Gelderblom et al, 2009). Earlier
experiments have shown fisetin doses of 30mg/kg body
weight to be effective and doses up to 90mg/kg body
weight (bw) to be safe in rats (Rivera et al, 2004). Animals
were injected 20 minutes before or 180 minutes after the
onset of ischemia with fisetin (Indofine, Hillsborough, NJ,
USA, final concentration high-dose 50mg/kg body weight,
low-dose 25mg/kg body weight in 15mL DMSO (dimethyl
sulfoxide), diluted immediately before injection in 150mL
10% cyclodextran; Sigma, St Louis, MO, USA) or placebo
(15mL DMSO in 150mL 10% cyclodextran; Sigma). Mice
were monitored using transcranial temporal laser Doppler
and showed a 90% drop in blood flow on placement of the
filament (Gelderblom et al, 2009). Every mouse was
repeatedly scored on a scale from 0 to 5 (0 no deficit, 1
preferential turning, 2 circling, 3 longitudinal rolling, 4 no
movement, 5 death) after reawakening and every day until
sacrifice. Only mice with a score greater or equal to one
after reawakening were included for stroke size analysis.
Clinical scores 1 hour after reawakening were not sig-
nificantly different between the groups (Supplementary
Figure 1). Recovery was defined as the time until mice
reached score 1 (mice stopped circling). Mice were killed 3
or 7 days after reperfusion using isoflurane and decapita-
tion. In the pretreatment group, a total of 13 placebo-
treated, 12 low-dose fisetin-treated and 8 high-dose fisetin-
treated mice were included. In the posttreatment group, 10
placebo-treated and 9 high-dose fisetin-treated mice were
included. Mortality was not different between the groups.
The brains were harvested, cut into 1mm standardized
slices (Braintree Scientific, Braintree, MA, USA; 1mm) and
vital stained using 2% (w/v) TTC (2,3,5-triphenyl-2-
hydroxy-tetrazolium chloride; Sigma) in phosphate buffer.
Slices were scanned on a flat bed scanner and infarct
volume determined by blinded examiners using NIH
ImageJ (rsbweb.nih.gov/ij/) and statistics (analysis of
variance, post hoc Tukey–Cramer test, Graph Pad Prism
(La Jolla, CA, USA). Fisetin (50mg/kg bw) did not influence
blood pressure after intraperitoneal injection 15 minutes,
1 hour, and 3 hours after intraperitoneal injection compared
with placebo (Figure 1F).
Flow Cytometry
Flow cytometry cell-type analyses were performed as
previously described (Gelderblom et al, 2009) (Supple-
mentary Materials and methods). Only brain hemispheres
with visible cortical infarcts and mice with scores Z2
(circling) at 60 minutes after stroke were included in the
flow cytometric analysis. For each experiment, three to
four hemispheres were pooled. For intracellular cytokine
staining, cells were stained with Ly6g (BD Biosciences,
San Jose, CA, USA), CD11c (eBioscience, San Diego, CA,
USA), CD11b (BD Biosciences), CD45 (eBioscience) for
surface staining. Intracellular staining was performed
according to the manufacturer’s instruction (BD Bios-
ciences), with anti-tumor necrosis factor a (TNFa) mono-
clonal antibody (BD Bioscience) or the corresponding
isotype control (rat IgG1; eBioscience).
In Vitro Assays
Mouse N9 microglial cells or mouse Raw264.7 macro-
phages were grown in standard medium (Supplementary
Materials and methods), treated with LPS (10ng/mL) alone
or following a 30-minute pretreatment with varying doses
of fisetin. For analysis of nitric oxide (NO) production, we
used the Griess assay (Sigma). Tumor necrosis factor a
concentration was measured using a TNFa-kit (R&D Research,
Minneapolis, MN, USA) according to the manufacturer’s
instructions. For western blot antibodies the following were
used: phospho-stress-activated protein kinase/c-Jun NH2-
terminal kinase (SAPK/JNK), phospho-c-Jun, phospho-IkBa,
horseradish peroxidase (HRP)-actin, total SAPK/JNK (Cell
Fisetin protects from ischemic stroke
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Signaling, Danvers, MA, USA); total c-Jun/AP-1 (Santa Cruz,
Santa Cruz, CA, USA) and inducible nitric oxide synthase
(iNOS) (BD Bioscience), for details see Supplementary
Materials and Figures.
For coculture experiments, primary murine microglia
cells were treated with LPS (1mg/mL; InvivoGen, San
Diego, CA, USA) in the presence or absence of fisetin
(2.0mg/mL) for 24 hours. Next, microglia cells were washed
and transferred to primary cortical neuronal cultures.
Neuronal survival was measured after 24 hours of
cocultivation by trypan blue exclusion (for details see
Supplementary Materials and methods).
Human Monocyte-Derived Macrophages In Vitro
Human monocyte-derived macrophages were isolated from
leukapheresis samples (written permission, anonymous,
healthy donors, Eppendorf Blood Bank). Macrophages
were stimulated with LPS (1mg/mL; InvivoGen) in the
presence or absence of fisetin. Macrophages were stained
for fluorescence activated cell sorting (FACS) with CD80
(BD Bioscience), CD86 (BD Bioscience), and CD11b
(Invitrogen, Carlsbad, CA, USA). Tumor necrosis factor a
concentrations were measured using human TNFa-kit
(BioLegend, San Diego, CA, USA) (for details see Supple-
mentary Materials and methods).
Results
Fisetin Pretreatment and Posttreatment Reduces
Infarct Volume 72 Hours After Temporary Middle
Cerebral Artery Occlusion
Fisetin has previously been shown in other models
to improve stroke severity after 24 hours when given
5 minutes (Maher et al, 2007) or 30 minutes after
induction of ischemia (Rivera et al, 2004). To
substantiate these findings in a model more prone
to poststroke inflammation, we used a reperfusion
stroke model, tMCAO mice. When given 20 minutes
before onset of 1 hour of ischemia, treatment with
25mg/kg bw resulted in a trend to a smaller infarct
size by 13%, whereas 50mg/kg bw significantly
reduced infarct size by 46% (mean/s.d.: vehicle
56mL/22mL, 25mg/kg fisetin 49mL/23mL, 50mg/kg
fisetin 30mL/23mL, analysis of variance with Bonfer-
roni post hoc test: vehicle versus 25mg/kg bw: n.s.,
vehicle versus 50mg/kg bw: P<0.05; Figures 1A and
1B). Thus, we tested whether delayed treatment still
protects against cerebral ischemia. Even when given
3 hours after onset of ischemia, 50mg/kg bw reduced
infarct size similarly by 35% (mean/s.d. vehicle
42mL/17mL, fisetin 27mL/19mL, one-sided Student’s
t-test, P<0.05; Figure 1C). Of note, stroke severity at
the time of inclusion did not differ between groups
(Supplementary Figure 1) and fisetin did not influ-
ence systemic blood pressure (Figure 1F). Either with
fisetin pretreatment or posttreatment we found a
strong trend toward earlier recovery in fisetin-treated
animals compared with vehicle (days until recovery;
Figure 1 Dose-dependent protection from ischemic stroke
by fisetin. (A) Representative vital-stained (2,3,5-triphenyl-2-
hydroxy-tetrazolium chloride (TTC)) brain sections of placebo
and fisetin (50mg/kg bw posttreatment) treated mice 3 days
after ischemia. (B, C) Fisetin treatment 20 minutes before (A) or
3 hours after onset of ischemia (B). Infarct size as edema
corrected infarct volume in mL. Scatter plot with mean. (D, E)
Time until animals stopped circling (reached score 1) in days in
fisetin pretreatment (D) and fisetin posttreatment (E). Error bars
indicate s.d. Significance by one-way analysis of variance
(ANOVA) and Bonferroni post hoc test (A, C) or one-sided
Student’s t-test (B, D). *P<0.05. (F) Systemic blood pressure
is not influenced by fisetin. Tail cuff measurements after
intraperitoneal injection show no significant changes (two-way
ANOVA).
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mean/s.d. pretreatment; Figure 1D: placebo 1.7/1.5;
fisetin 25mg/kg bw 1.1/1.1; fisetin 50mg/kg bw 1.2/
1.5; P>0.05; posttreatment; Figure 1E: placebo 1.2/
0.6; 50mg/kg bw 0.9/0.2; P>0.05).
Fisetin Treatment Reduces Infiltration of the Ischemic
Hemisphere With Macrophages, Dendritic Cells, and
Lymphocytes
Next, we compared the number and subtype of
immune cells in the ischemic hemisphere at day 3
after ischemia in animals pretreated with 50mg/kg
bw fisetin or vehicle using FACS analysis after
visualization of subtype-specific surface markers by
fluorophore-labeled antibodies (Gelderblom et al,
2009). Fisetin did not change the number of
microglia identified as intermediate CD45-positive
cells in the ischemic hemisphere (Figure 2A). In
contrast, the ischemic hemispheres of fisetin-treated
mice contained significantly less highly CD45-
positive cells, a group comprised of lymphocytes,
macrophages, and dendritic cells (DCs) (cells per
ischemichemisphere;
179.252±23.389; fisetin 93.045±46.963; P=0.045;
Figure 2B). For further analysis, highly CD45-
positive cells were subdivided into lymphocytes,
macrophages, and DCs based on the expression of
CD3, CD11b, and CD11c. Since we found similar
absolute numbers of microglia in vehicle- and fisetin-
treated mice, we used the number of microglia as an
internal reference. Using this approach, we observed
a significant decrease in percentage of both subtypes
of antigen presenting cells, macrophages (percentage
of macrophages relative to microglia; mean±s.d.;
vehicle 49%±5.2%; fisetin 12%±3.3%; P=0.037;
Figure 2C) and DCs (percentage of DC relative to
microglia; mean±s.d.; vehicle 23%±0.7%; fisetin
mean±s.d.; vehicle
14%±2.3%; P=0.038; Figure 2C), as well as lympho-
cytes (percentage of lymphocytes relative to microglia;
mean±s.d.; vehicle 5.7%±0.1%; fisetin 3.3%±0.2%;
P=0.038; Figure 2D) in the ischemic hemisphere of
fisetin-treated animals compared with vehicle.
Fisetin Modifies the Activation of Infiltrating
Macrophages and Resident Microglia in the Ischemic
Hemisphere
The activation status of cells infiltrating the brain is
more relevant for secondary brain damage than their
number. Therefore, we tested whether pretreatment
or posttreatment with 50mg/kg bw fisetin changes
the cytokine profile of the brain resident microglia,
infiltrating macrophages and DC compared with
vehicle using flow cytometry labeling intracellular
cytokines of the brain- and spleen-derived cells 3
days after stroke.
Indeed, pretreatment with 50mg/kg bw fisetin
before tMCAO strongly reduced the relative propor-
tion of TNFa producing microglia (3 days after
ischemia; vehicle 30.4%; fisetin 17.3%; Figure 3A)
and macrophages (3 days after ischemia; vehicle
24.6%; fisetin 2.9%; Figure 3B) found in the brain.
No change was seen in the brain invading DCs
(Supplementary Figure 2A). Similarly, fisetin post-
treatment 3 hours after ischemia still resulted in
suppression of TNFa production in the brain-derived
microglia and macrophages although to a slightly
lesser degree (3 days after ischemia; microglia
vehicle 36.9%; fisetin 22.6%; Figure 3A; macro-
phages vehicle 31.2%; fisetin 19.1%; Figure 3B).
Fisetin treatment did not alter TNFa expression of
spleen-derived macrophages after stroke indepen-
dent of pretreatment or posttreatment with fisetin
(pretreatment 3 and 7 days after ischemia; vehicle
Figure 2 Decreased inflammatory cells in postischemic brains of fisetin pretreated mice. (A) Absolute numbers of microglia per
hemisphere by flow cytometry 3 days after ischemia are not different between fisetin and vehicle pretreated mice. (B–D) Significant
reduction of ischemic brain infiltrating CD45highleukocytes, macrophages, dendritic cell (DC), and lymphocytes relative to numbers
of microglia 3 days after stroke (n=3). Error bars indicate s.d. Significance by two-sided Student’s t-test. n.s., not significant,
*P<0.05, **P<0.05.
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4.2%/4.7%; fisetin 4.4%/4.5%; Figure 4A; posttreat-
ment vehicle 5.5%, fisetin 5.8%; Figure 4B) or DCs
(3 days after ischemia; vehicle 0.2%; fisetin 0.5%;
Supplementary Figure 2B). Tumor necrosis factor a
production in spleen-derived macrophages was not
influenced by fisetin alone or by poststroke systemic
immunosuppression, since similar percentages were
observed in sham-operated mice (Figure 4C).
To examine the stability of these changes to the
activation profile of the brain-derived immune cells
induced by a single fisetin injection (50mg/kg bw)
before ischemia, we examined mice 7 days after the
Figure 3 Fisetin pretreatment and posttreatment reduces tumor necrosis factor a (TNFa) production in the brain. Flow cytometry
of microglia (A) and macrophages (B) isolated from ipsilesional brain hemispheres 3 days and 7 days after stroke
following pretreatment or posttreatment with fisetin 50mg/kg body weight. Staining for intracellular TNFa shows attenuation of
TNFa production in fisetin pretreated mice in the brain-derived microglia and macrophages at day 3 and macrophages at day 7 after
stroke. The same effect is observed in mice treated with fisetin 3 hours after ischemia. Percentages relative to gated cells.
Representative experiments with pooled hemispheres from three mice are shown. TNFa-positive cells are defined by isotype control
(not shown).
Figure 4 Fisetin does not exhibit systemic effects on tumor necrosis factor a (TNFa) production. Flow cytometry of macrophages
isolated from spleen following stroke (A, B) or sham-operated mice and pretreatment (A) or posttreatment (B) with fisetin or placebo
at day 3 or 7 (fisetin 50mg/kg body weight). Staining for intracellular TNFa does not show differences in TNFa production in spleen
macrophages between fisetin and placebo. Of note, no difference in TNFa production between stroke and sham-operated mice is
seen (A–C). Percentages relative to gated cells. TNFa-positive cells are defined by isotype control (not shown). Representative
experiments with pooled spleens from three mice are shown.
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ischemic insult. Even at that time point, TNFa-
positive cerebral macrophages were still reduced by
the single dose of fisetin before ischemia (7 days after
ischemia; vehicle 33.8%; fisetin 13.9%; Figure 3B),
whereas the fisetin effect on microglial TNFa
production had almost disappeared at day 7 (7 days
after ischemia; vehicle 31.9%; fisetin 26.8%; Figure
3A). The abating difference supports the idea that the
suppression of TNFa expression is a direct result of
fisetin treatment rather than an indirect effect of the
smaller infarct size.
Fisetin Suppresses Activation and Neurotoxicity of
Murine Macrophage and Microglial Cell Lines and
Human Primary Macrophages in an In Vitro Model
of Inflammatory Activation by Lipopolysaccharide
To substantiate the idea that the fisetin-induced
reduction of postischemic activation of microglia
and infiltrating macrophages demonstrated by sup-
pressed TNFa expression is a direct effect of fisetin
on immune cells, we next tested whether fisetin
modulates the activation of a murine microglial and a
macrophage cell line as well as human primary
macrophages on LPS treatment. Fisetin dose depen-
dently reduced TNFa secretion in both murine
Raw264.7 macrophage and N9 microglial cells
compared
macrophages 29%±3.4%; P<0.001; N9 microglia
56%±8.8%; P<0.001 at 2.9mg/mL fisetin; Figures
5A and 5B). Fisetin treatment of human monocyte-
derived macrophages led to a similar significant,
dose-dependent reduction of TNFa secretion on LPS
treatment (2.0mg/mL fisetin 62%±13.4%; P<0.05;
Figure 5C). In addition, fisetin also reduced NO
production and expression of inducible NO synthase
as an alternative marker of inflammatory activation
in murine immune cell lines (Supplementary Figure 3).
Next, we examined the functional relevance of this
activation on neurotoxicity in a murine primary
microglia/neuroncoculture
observed that fisetin reduced the neurotoxicity of
LPS-stimulated primary microglia (percentage cell
death mean±s.d.; DMSO 46%±17% n=13; 2.0mg/mL
fisetin only 47%±11% n=14; LPS+DMSO 69%±
12%n=25;2.0mg/mL
n=51; P<0.001; Figure 5D).
with vehicle (mean±s.d.;Raw264.7
model.Indeed, we
fisetin+LPS 56%±13%
Fisetin Inhibits Lipopolysaccharide-Induced
Activation of Proinflammatory Signaling via Nuclear
Factor jB and JNK Suppression
To elucidate the mechanism underlying the anti-
inflammatory actions of fisetin, we looked at the
Figure 5 Fisetin attenuates lipopolysaccharide (LPS)-stimulated tumor necrosis factor a (TNFa) production and LPS-induced
neurotoxicity of murine macrophage and microglia as well as human macrophages. Reduced TNFa production in murine macrophage
cell lines (A; Raw264.7, n=3), murine microglia cell lines (B; N9, n=3) and human monocyte-derived macrophages (C; n=3) on
fisetin treatment. Percentage of TNFa production relative to LPS-stimulated cells. (D) Reduced microglial neurotoxicity after fisetin
pretreatment of microglia. Murine primary microglia pretreated with fisetin or dimethyl sulfoxide (DMSO) together with or in the
absence of LPS and incubated with primary cortical neurons. Percentage of neuronal death by trypan blue exclusion. Error bars
indicate s.d. Significance by one-way analysis of variance (ANOVA) and Bonferroni post hoc test. *P<0.05, **P<0.01,
***P<0.001. LPS=10ng/mL for (A, B) and 1mg/mL for (C). Fisetin dose in mg/mL final concentration.
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effect of fisetin on several intracellular signal
transduction pathways implicated in inflammation
including IkB, JNK, and its target c-Jun. Fisetin
treatment of LPS-stimulated murine N9 microglial
and Raw264.7 macrophage cells significantly re-
duced both phosphorylated IkB (Figures 6A and
6B) and JNK phosphorylation regardless of JNK
isoforms (Figures 6A and 6B). In addition, the
phosphorylation of the target of JNK c-Jun was
reduced (Figures 6A and 6B) suggesting that fisetin
directly inhibits these proinflammatory signaling
pathways. The c-Jun signaling pathway has a
relevant role in vivo, since immunohistochemically
colocalization of c-Jun and phospho-c-Jun with
monocytes (CD11+) was observed 3 days after stroke
(Supplementary Figure 4).
Discussion
The flavonoid fisetin has direct glutathione-inducing
and antiinflammatory properties (Maher et al, 2007).
It also has been shown that fisetin increases ATP
levels in an in vitro model of ischemic neuronal cell
death, chemical ischemia on treatment with iodo-
acetic acid (Maher et al, 2007). Thus, fisetin is a
promising candidate substance for treatment of
ischemic stroke, where oxidative stress, ATP deple-
tion and secondary inflammation are pathophysiolo-
gically relevant (recently reviewed in Macrez et al,
2011). In line with these assumptions, fisetin has been
shown to be beneficial in a rabbit small clot embolism
model and in a rat permanent MCAO model 24 hours
after stroke when given up to 30 minutes after onset of
ischemia (Maher et al, 2007; Rivera et al, 2004). In
contrast, we examined later time points and our
animal model of stroke, the occlusion reperfusion
model of tMCAO in mice, is a much more potent
initiator of poststroke inflammation than permanent
ischemia (reviewed in Tuttolomondo et al, 2009). We
show that in this model, fisetin is effective when
given up to 3 hours after onset of ischemia.
Efficiency of delayed treatment is not only relevant
for putative future therapeutic purposes in humans
but also indicates that a late stage of secondary
infarct growth is modified by fisetin. Thus, we
analyzed whether fisetin modifies the inflammatory
response after cerebral ischemia. Indeed, fisetin
robustly reduced the infiltration of the ischemic
hemisphere with macrophages, DCs, and lympho-
cytes. The reduced postischemic inflammation was
not merely due to a difference in chemotaxis as we
could demonstrate that fisetin also suppressed the
activation of infiltrating macrophages and DCs as
well as resident microglia as shown by reduced
expression of the proinflammatory cytokine TNFa.
However, an additional indirect effect of fisetin on
poststroke inflammation via its antiapoptotic effects
and consecutive reduction on stroke size cannot be
completely excluded. Poststroke inflammation is
known to be influenced by infarct size (Hug et al,
2009). Nevertheless, our data indicate that fisetin
exhibits direct effects on inflammatory cells, since
fisetin suppressed TNFa secretion of murine N9
microglial and macrophage Raw264.7 cells and
human primary monocyte-derived macrophages in
an in vitro model of inflammatory activation by
stimulation with LPS, which is in line with earlier
reports (Zheng et al, 2008; Lyu and Park, 2005). In
addition, fisetin attenuates LPS-induced neurotoxic
effects of microglial cells on primary cortical neurons.
Activation of resident microglia is accepted as a
main component of the postischemic periinfarct
inflammation and microlia cells are among the main
sources of proinflammatory cytokines such as TNFa
(Gregersen et al, 2000). Microglial activation is a
cause of secondary tissue damage, as is demonstrated
by an improved outcome in stroke models through
microglia-directedtreatment
Additionally, microglial activation results in the
attraction of inflammatory cells from the circulation,
(Liuetal,2007).
Figure 6 Fisetin blocks phosphorylation of JNK, c-Jun, and IkB. (A) Representative western blots of fisetin inhibition of
lipopolysaccharide (LPS)-induced phosphorylation of IkB, c-Jun, and JNK isoforms in microglial (N9) and macrophage (Raw264.7)
cell lines. Fisetin 2.9mg/mL (10mmol). (B) Quantification of the reduction of phosphorylation (in percentage; LPS=100%); Fisetin
2.9mg/mL (10mmol). Error bars indicate s.d. Significance by one-way analysis of variance (ANOVA) and Bonferroni post hoc test.
**P=0.002, ***P<0.0001; n=3. LPS=10ng/mL.
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which further enhances the inflammatory damage
(Yilmaz et al, 2006). Thus, the fisetin-induced
reduction of postischemic microglial TNFa expres-
sion might explain diminished invasion of macro-
phages and DCs observed by us on fisetin treatment.
Remarkably, the fisetin-induced attenuation of TNFa
production in microglia and macrophages in the
brain can still be observed when fisetin is applied 3
hours after ischemia. At this time point, inflamma-
tory mechanisms are gaining momentum (Gelderblom
et al, 2009), further indicating that antiinflammatory
properties of fisetin have a role in this setting.
The mechanisms through which fisetin mediates
its antiinflammatory effects can be linked to the
suppression of both proinflammatory intracellular
pathways, AP-1 and nuclear factor (NF)-kB. These
intracellular pathways control the expression of
almost all genes encoding proinflammatory cyto-
kines (Dinarello, 2010). The AP-1 pathway uses
primarily phosphorylation of c-Jun by JNK for
signaling into the nucleus. Nuclear factor-kB on the
other hand is associated with the inhibitory protein
IkB. Following treatment of cells with proinflamma-
tory stimuli, IkB is phosphorylated and subsequently
degraded. The free NF-kB translocates to the nucleus
where it can initiate transcription of a diverse array
of target genes including cytokines. In our experi-
ments, this dual mode of inhibiting both AP-1 and
NF-kB signaling can explain the potent inhibition of
LPS-induced TNFa production in mouse and human
phagocytes and murine N9 microglial cells. Earlier
studies have already reported that fisetin inhibits
NF-kB activation on LPS treatment in microglial cells
(Zheng et al, 2008). However, whereas fisetin has
been shown to reduce phosphorylation of JNK and
c-Jun in a prostate cancer cell line (Chien et al, 2010),
to our knowledge the efficacy of fisetin to block JNK
and c-Jun phosphorylation on inflammatory activa-
tion in phagocytes has not been reported before. One
can speculate, that this mode of action explains the
observed lack of effect on the peripheral immune
compartment, since the TNFa expression of spleen-
derived macrophages was unaltered by treatment.
Although our data on the mechanism of fisetin
effects are highly suggestive, they are based on
association and the inhibition of other proinflamma-
tory cascades might as well be involved in the
fisetin-mediated protection from cerebral ischemia.
Fisetin decreases PGE-2 production, downregulates
cyclooxygenase-2, interleukin-1b as well as iNOS
expression and NO production on LPS stimulation of
BV2 microglial cells and primary microglia (Zheng
et al, 2008). We could also show that fisetin is
effective in suppressing the LPS-induced iNOS
expression and NO production in N9 microglia
and Raw264.7 macrophages. Especially, iNOS and
cyclooxygenase-2 are additional interesting proin-
flammatory targets of fisetin as inhibition of cycloox-
ygenase-2 after experimental stroke in rats results in
smaller infarcts and preserved blood–brain barrier
(Candelario-Jalil et al, 2007) and the role of iNOS in
exacerbating postischemic tissue damage is generally
acknowledged (Willmot et al, 2005).
For some of the discussed effects, fisetin has to be
available in the central nervous system. Fisetin
injected intraperitoneally is quickly glucuronidated
and sulfated (Maher, 2009) but biologically active
metabolites are detected systemically for >24 hours.
Fisetin also crosses the blood–brain barrier and its
concentration in the central nervous system peaks 2
hours after injection (Rivera et al, 2004). Off-target
effects that can contribute to toxicity are certainly a
concern for any potential drug. However, in the case
of fisetin, we have no evidence for either short- or
long-term toxicity. Mice fed with fisetin at 500p.p.m.
for several months showed no indications of toxicity
(Maher et al, 2011).
The concept that antiinflammatory treatments in
stroke are beneficial has received more attention over
the last few years (reviewed in Iadecola and
Anrather, 2011). For example, recent studies have
highlighted the beneficial effects on infarct size by
inhibition of microglial and macrophage activation
using the immunosuppressant FK506 (also known as
tacrolimus) or intrathecal steroids (Goericke et al,
2010; Brecht et al, 2009). Similar observations have
also been made for other antiinflammatory drugs
such as fingolimod (Hasegawa et al, 2010). However,
these treatments have the potential drawback of
aggravating the systemic immunodepression that is
observed after stroke (Klehmet et al, 2009). Unlike
these compounds, fisetin seems to have no overt
systemic immunosuppressive side effects, as we
found no difference in systemic TNFa expression. It
rather appears to act specifically under inflammatory
conditions like LPS stimulation or stroke.
Taken together, we and others have shown that
fisetin has multiple beneficial actions that reduce
ischemia-induced brain damage. Ischemic stroke is
increasingly recognized as the multiphasic process
hypothesized more than a decade ago and is initially
composed of necrosis and apoptosis evolving into an
immune-mediated process (Dirnagl et al, 1999),
leading to further lesion growth. Compounds that
modulate more than one of these aspects could
provide a very promising approach. Fisetin combines
several of these properties without inducing sys-
temic immune suppression. Thus, the experimental
evidence supports the idea that fisetin may represent
a good starting point for the development of future
therapeutic substances for ischemic stroke.
Disclosure/conflict of interest
The authors declare no conflict of interest.
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www.nature.com/jcbfm)
Fisetin protects from ischemic stroke
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Journal of Cerebral Blood Flow & Metabolism (2012), 1–9