Molecules 2014, 19, 8820-8839; doi:10.3390/molecules19078820
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Regulatory Effects of Fisetin on Microglial Activation
Jing-Yuan Chuang 1,†, Pei-Chun Chang 2,†, Yi-Chun Shen 1, Chingju Lin 3, Cheng-Fang Tsai 4,
Jia-Hong Chen 5, Wei-Lan Yeh 6, Ling-Hsuan Wu 7, Hsiao-Yun Lin 8, Yu-Shu Liu 7 and
Dah-Yuu Lu 9,10,*
1
2
3
4
5
6
7
8
9
10
†
Department of Medical Laboratory Science and Biotechnology, China Medical University, Taichung
40402, Taiwan; E-Mails: jychuang@mail.cmu.edu.tw (J.-Y.C.); syciac@gmail.com (Y.-C.S.)
Department of Bioinformatics, Asia University, Taichung 41354, Taiwan;
E-Mail: pcchang@asia.edu.tw
Department of Physiology, School of Medicine, China Medical University, Taichung 40402,
Taiwan; E-Mail: clin33@mail.cmu.edu.tw
Department of Biotechnology, Asia University, Taichung 41354, Taiwan;
E-Mail: tsaicf@asia.edu.tw
Department of General Surgery, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation,
Taichung 42743, Taiwan; E-Mail: guns5150@ms27.hinet.net
Department of Cell and Tissue Engineering, Changhua Christian Hospital, Changhua 500, Taiwan;
E-Mail: ibizayeh0816@hotmail.com
Graduate Institute of Basic Medical Science, China Medical University, Taichung 40402, Taiwan;
E-Mails: lulu80620@yahoo.com.tw (L.-H.W.); yushuliu220@gmail.com (Y.-S.L.)
Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan;
E-Mail: lingirl831@hotmail.com
Graduate Institute of Neural and Cognitive Sciences, China Medical University,
Taichung 40402, Taiwan
Department of Photonics and Communication Engineering at Asia University, Taichung 41354, Taiwan
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: dahyuu@mail.cmu.edu.tw;
Tel.: +886-4-2205-3366 (ext. 8206); Fax: +886-4-2207-1507.
Received: 30 April 2014; in revised form: 13 June 2014 / Accepted: 18 June 2014 /
Published: 26 June 2014
Abstract: Increasing evidence suggests that inflammatory processes in the central nervous
system that are mediated by microglial activation play a key role in neurodegeneration.
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Fisetin, a plant flavonol commonly found in fruits and vegetables, is frequently added to
nutritional supplements due to its antioxidant properties. In the present study, treatment
with fisetin inhibited microglial cell migration and ROS (reactive oxygen species)
production. Treatment with fisetin also effectively inhibited LPS plus IFN-γ-induced nitric
oxide (NO) production, and inducible nitric oxide synthase (iNOS) expression in microglial
cells. Furthermore, fisetin also reduced expressions of iNOS and NO by stimulation of
peptidoglycan, the major component of the Gram-positive bacterium cell wall. Fisetin also
inhibited the enhancement of LPS/IFN-γ- or peptidoglycan-induced inflammatory mediator
IL (interlukin)-1 β expression. Besides the antioxidative and anti-inflammatory effects of
fisetin, our study also elucidates the manner in fisetin-induced an endogenous anti-oxidative
enzyme HO (heme oxygenase)-1 expression. Moreover, the regulatory molecular mechanism
of fisetin-induced HO-1 expression operates through the PI-3 kinase/AKT and p38 signaling
pathways in microglia. Notably, fisetin also significantly attenuated inflammation-related
microglial activation and coordination deficit in mice in vivo. These findings suggest that
fisetin may be a candidate agent for the development of therapies for inflammation-related
neurodegenerative diseases.
Keywords: microglia; neuroinflammation; neurodegeneration; fisetin; cytokine
1. Introduction
Microglial activation leads to neuroinflammatory responses that exert both beneficial and
detrimental effects, including host defense and tissue repair processes [1,2]. During neuroinflammatory
periods, activated microglia are able to clear debris or invading pathogens, and produce neurotrophic
factors which modulate the microenvironment [3]. When sensing ATP leaks from an injury site,
microglia transform to a more motile state and migrate to the site of damage [4], which causes
neuroinflammation and subsequent neurodegeneration. Excessive inflammatory mediators and
oxidative stress in the CNS are associated with the pathogenesis of neurodegeneration [5,6]. In
pathological neuroinflammatory conditions, microglial activation results in expression of new proteins
like inducible nitric oxide synthase (iNOS) which have been shown to cause neuronal damage [7–10].
It has been reported that iNOS triggers NO production by activated microglia, which further aggravate
the pathological processes in neurodegeneration [11]. The expressions of the inflammation-related
enzymes iNOS have also been reported in the striatum of Parkinson disease patients [12]. Although
inflammatory mediators are necessary for normal neuronal cell functions, the microglial response must
be tightly regulated to avoid over-activation and neurotoxic consequences [13]. Interleukin-1 beta
(IL-1β) is a major pro-inflammatory cytokine that increases expression of adhesion molecules and
synthesis of cytotoxins [14,15]. Microglia activation and neuronal degeneration are co-presence of IL-1β
in Alzheimer’s disease [16]. Moreover, IL-1β was observed in cerebrospinal fluid of multiple sclerosis
patients, suggesting a possible link between inflammation and neurodegeneration [17].
Many natural products, such as flavonoids, are potential therapeutic agents recognized for their
antioxidant activity [18]. Flavonoids are low-molecular-weight natural polyphenolic compounds found
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in many fruits and vegetables. It has been reported that flavonoids exert many biological functions [19,20].
Fisetin (3,3',4',7-tetrahydroxyflavone; Figure 1A), a high Trolox-equivalent antioxidant, is found in
strawberry, persimmon, grape and cucumber [21]. Due to it being a hydrophobic compound, fisetin
easily penetrates cell membranes accumulating in cells to exert its antioxidative effects [22], with
wide-ranging biological activities including antiaging, anti-inflammatory, neuroprotection and
anticancer effects [23,24]. Previous reports have demonstrated the protective effects of fisetin on
microglial activation and subsequent neurotoxicity [25]. After oral administration of fisetin could also
pass the blood-brain barrier to exert neuroprotective and cognition-enhancing effects in animal model of
CNS disorders [26] Importantly, fisetin attenuates postischemic immune cell infiltration and reduces
infarct size after transient cerebral middle artery occlusion in mice [27]. Recent study also revealed
that orally administered fisetin exerts neuroprotection against reactive gliosis and behavioral deficits in
aluminum chloride-induced brain pathology model [28].
Figure 1. Effect of fisetin on microglial activation. (A) Chemical structure of fisetin.
(B) Cell viability following fisetin treatment in BV-2 microglia. Cells were incubated with
concentrations ranging from 1 to 5 μM of fisetin for 60 min followed by treated with LPS
(10 ng/mL) plus IFN-γ (10 ng/mL) 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.
(C) Cells were pretreated with various concentrations of fisetin for 60 min followed by
stimulation with H2O2 for 120 min. ROS generation was determined using the fluorescence
probes H2DCFH-DA and examined by flow cytometry. The results are expressed as mean
± S.E.M. from 3 to 4 independent experiments. * p < 0.05 compared with the control
group; # p < 0.05 compared with the H2O2 alone. (D) Cells were pre-incubated with various
concentrations of fisetin (1–5 μM) for 60 min followed by a 24-h treatment with ATP
(300 μM). In vitro migratory activities were examined using a cell culture insert system.
The results are expressed as mean ± S.E.M. from 3 independent experiments.
* p < 0.05 compared with the control group; # p < 0.05 compared with the ATP alone. The
migrated cells were visualized by phase-contrast imaging (Lower panel).
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Figure 1. Cont.
Heme oxygenase (HO), a cytoprotective enzyme, degrades heme to bilirubin, carbon monoxide, and
iron [29,30]. Induction of HO-1 expression and related signal pathways exert anti-inflammatory effects
in macrophages [31–33]. Recently, we have reported that neuroinflammatory responses can be repressed
by HO-1 induction in microglia [34,35] and astrocytes [36], and that increased HO-1 expression
protects neurons against neurotoxin-induced cell death [37,38]. Previous report shown that fisetin
protects cells from oxidative-stress-induced death and induces HO-1 in human retinal pigment epithelial
cells [39]. Fisetin also been reported to protect against hydrogen peroxide-induced oxidative stress
through induction of HO-1 expression in human umbilical vein endothelial cells [40]. A recent study
also reported that fisetin up-regulates HO-1 expression and interferes with reactive oxygen species
production in macrophage-differentiated osteoclasts [41].
Although the beneficial effects of fisetin in brain have been investigated, the mechanism of regulation
of microglia polarity has not yet been determined. In the present study, we addressed whether, in
addition to inhibiting cytokine production, HO-1 expression also contributes to fisetin-regulated
anti-inflammatory responses in microglial cells.
2. Results
2.1. Fisetin Suppresses Neuroinflammatory Responses in Microglial Cells
We used BV-2 microglia to study the effects of fisetin on neuroinflammatory responses. Concentrations
ranging from 1 to 5 µM fisetin were used in the current study. A colorimetric cell viability assay (MTT
assay) confirmed that these concentrations did not affect cell viability (Figure 1B). H2O2 induced an
increase in intracellular ROS levels, as shown by H2DCF-DA staining which were analyzed by FACS
detection assay (Figure 1C). Treatment with fisetin reduced H2O2-induced ROS productions (Figure 1C).
Fisetin inhibited an ATP-induced increase in BV-2 microglial migratory activity (Figure 1D; upper
panel). Representative micrographs of migrating cells are shown in Figure 1D (lower panel). To
determine the effect of fisetin on iNOS/NO expression, cells were treated with different concentrations
of fisetin (1 to 10 µM) and were stimulated with LPS plus IFN-γ. The supernatant of cell culture was
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then collected to determine NO production. Previously, we have demonstrated that peptidoglycan a
major component of the Gram-positive bacterium cell wall, induces neuroinflammatory responses in
microglial cells [42,43]. Hence, to further determine the effect of fisetin on nitric oxide production,
BV-2 microglia were also stimulated with peptidoglycan. As shown in Figure 2A,B, fisetin effectively
inhibited iNOS expression in a concentration-dependent manner following exposure to either LPS
(10 ng/mL) plus IFN-γ (10 ng/mL) or peptidoglycan (10 μg/mL). Furthermore, fisetin also
reduced LPS/IFN-γ- and peptidoglycan-induced NO production (Figure 2C,D, respectively) in a
concentration-dependent manner.
Figure 2. Inhibitory effect of fisetin on LPS/IFN-γ or peptidoglycan-stimulated iNOS/NO
expression. (A,C) BV-2 microglial cells were pretreated with different concentrations of
fisetin (1, 3, or 5 μM) for 60 min before application of LPS (10 ng/mL) plus IFN-γ
(10 ng/mL) for another 24 h. (B,D) Cells were pretreated with different concentrations of
fisetin (1, 3, or 5 μM) for 60 min before application of peptidoglycan (10 μg/mL) for
another 24 h. Western blot analysis for iNOS (A,B) expression was performed on whole
cell lysates. The quantitative results are shown in the bottom panels. The culture media
were collected and analyzed NO production by a Griess reaction (C,D). iNOS or
NO expression was significantly different between the LPS/IFN-γ (or peptidoglycan)
treated-group and the group treated LPS/IFN-γ (or peptidoglycan) with fisetin. The results
are expressed as mean ± S.E.M. from 3 to 4 independent experiments. * p < 0.05 compared
with the control group; # p < 0.05 compared with the LPS/IFN-γ or peptidoglycan treatment.
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Notably, fisetin treatment alone did not affect iNOS or nitric oxide expression. We further analyzed
the expression of inflammatory mediator using real-time PCR. BV-2 microglia were treated with
different concentrations of fisetin (1 to 5 µM) and stimulated with LPS plus IFN-γ, or peptidoglycan
for 6 h. Fisetin potentiates a concentration-dependent suppression of iNOS when stimulating cells with
LPS/IFN-γ or peptidoglycan (Figure 3A,C, respectively). Similarly, treatment with fisetin also
inhibited LPS/IFN-γ- or peptidoglycan-induced IL-1β expression in a concentration-dependent manner
(Figure 3B,D, respectively).
Figure 3. Inhibitory effect of fisetin on LPS/IFN-γ- or peptidoglycan-stimulated iNOS and
IL-1β expressions. BV-2 microglial cells were pretreated with different concentrations of
fisetin (1, 3, or 5 μM) for 60 min, then challenged with LPS plus IFN-γ (A,B), or
peptidoglycan (C,D) for another 6 h. The expression of iNOS and IL-1β were determined
by real-time PCR. Cytokine expression was significantly different between the LPS/IFN-γ
(or peptidoglycan) alone and the LPS/IFN-γ (or peptidoglycan) with fisetin groups. The
results are expressed as mean ± S.E.M. from 3 to 4 independent experiments. * p < 0.05
compared with the control group; # p < 0.05 compared with the LPS/IFN-γ treatment
alone group.
Next, we investigated regulation of STAT-1 protein on activatied effects in microglia. LPS plus
IFN-γ treatment resulted in STAT1 (Tyr701) phosphorylation (Figure 4A). Treatment with different
concentrations of fisetin (1 to 5 µM) attenuated LPS/IFN-γ-induced STAT1 (Tyr701) phosphorylation
(Figure 4A,B). In addition, fisetin reduced LPS/IFN-γ-induced JAK1 and JAK2 phosphorylation as
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well (Figure 4C,D, respectively). These results indicate that fisetin exerts anti-inflammatory effects in
microglial cells.
Figure 4. Fisetin suppresses LPS/IFN-γ-induced STAT signaling pathways. BV-2
microglial cells were pretreated with fisetin (1, 3, or 5 μM) for 60 min, then exposed to
LPS (10 ng/mL) plus IFN-γ (10 ng/mL) for another 60 min. The phosphorylated levels of
STAT-1, JAK1 or JAK2 were determined by western blot analysis, and the signal intensities
were normalized to total protein expression. The results are expressed as mean ± S.E.M.
from 3 independent experiments. * p < 0.05 compared with the vehicle control treatment
group. # p < 0.05 compared with the LPS/IFN-γ treatment alone group.
2.2. Fisetin Induces HO-1 Up-Regulation in Microglia Cells
Since HO-1 induction is known to participate in regulation of inflammatory responses, we investigated
whether fisetin might lead to HO-1 induction. When BV-2 microglia were treated with fisetin for 24 h,
HO-1 levels increased in a concentration-dependent manner (Figure 5B). In addition, the HO-1 expression
was further enhanced under fisetin treatment after LPS/IFN-γ administration (Figure 5A). We further
analyzed HO-1 mRNA expression induced by various concentrations of fisetin (Figure 5C).
We then used a HO-1 activator CoPP IX to examine whether HO-1 expression participated in the
anti-inflammatory pathways. As shown in Figure 5D, treatment with HO-1 activator increased HO-1
expression and abrogated LPS/IFN-γ-induced iNOS expression. Furthermore, treatment with CoPP IX
also abrogated LPS/IFN-γ-induced nitric oxide production (Figure 5E). We further studied the
signaling pathways involved in regulatory effects of fisetin on HO-1 in microglia. Treatment with p38
(SB203580) and PI3 kinase/Akt (LY294002) inhibitors, but not ERK (PD98059) and JNK (SP600125)
inhibitors effectively antagonized fisetin-induced HO-1 expression (Figure 6A). As shown in
Figure 6B,C, incubation with p38 or PI3 kinase/Akt inhibitors reduced fisetin-induced HO-1
expression concentration-dependently. As shown in Figure 6D,E, fisetin increased p38 and Akt
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activation in a time-dependent manner as well. These results demonstrate that fisetin-induced
anti-neuroinflammation may be regulated by HO-1 signaling pathway.
Figure 5. Effect of fisetin on HO-1 expression in microglia cells. (A) Cells were treated
with fisetin, LPS (10 ng/mL) plus IFN-γ (10 ng/mL), or fisetin with LPS/IFN-γ for 24 h.
Cells were treated with different concentrations of fisetin (1, 3, or 5 μM) for 24 h (B) or 6 h
(C). Whole cell lysates were subjected to western blot analysis for HO-1 protein
expression (A,B). HO-1 mRNA expressions were analyzed by real time-PCR (C). Cells
were treated with Copp IX for 60 min, then challenged with LPS plus IFN-γ for another 24 h.
The expression of iNOS and HO-1 were determined by western blot analysis (D). NO
production was analyzed by Griess reaction (E). The results are expressed as mean ±
S.E.M. from 3 to 4 independent experiments. * p < 0.05 compared with the control group;
#
p < 0.05 compared with the LPS/IFN-γ treatment alone group.
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Figure 6. Involvement of p38 and Akt signaling pathways in fisetin-stimulated HO-1
up-regulation in BV-2 microglia. (A) Cells were pretreated with various inhibitors
(PD98059, SB203580, SP600125 or LY294002) for 60 min, followed by stimulation with
fisetin for 24 h. Cells were pretreated with various concentrations of SB203580 (B) or
LY294002 (C) for 60 min, followed by stimulation with fisetin for 24 h. Whole cell lysis
proteins was extracted and subjected to western blot analysis for HO-1 expression. Cells
were incubated with fisetin for indicated periods (5, 10, 30, 60 or 120 min). Whole cell
lysates were subjected to western blot analysis by using antibodies against the
phosphorylated p38 (D) or Akt (E). Similar results were obtained from at least three
independent experiments. * p < 0.05 compared with the control group; # p < 0.05 compared
with the fisetin treatment group.
2.3. Fisetin Inhibits Microglial Activation in a Mouse Model
To determine the improvements induced by fisetin treatment on neuroinflammatory responses in vivo,
we performed a motor behavior test and an immunohistochemical analysis. Mice were continuously
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administered fisetin for three consecutive days, and were then injected or not with LPS (Figure 7A).
Motor balance and coordination function were analyzed using an accelerating rotarod test [34]. As
shown in Figure 7B, LPS-treated mice showed a reduced latency in the rotarod test, thus demonstrating
motor impairments compared to the control group. However, administration with fisetin significantly
ameliorated the motor-impaired effects of LPS-injected mice (Figure 7B).
Figure 7. Fisetin prevents LPS-induced microglial activation. Mice received intraperitoneal
injections of fisetin at concentrations of either 10 or 20 mg/kg once per day for 3 consecutive
days. On the third day, fisetin treatment was followed with a single intraperitoneal injection
of LPS (20 mg/kg). Motor balance and coordination function were analyzed using an
accelerating rotarod test (B). Microglial morphology was visualized by anti-Iba-1
immunolabeling in cortical (C) and hippocampal (D) regions.
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The activation of microglia was analyzed morphologically Iba-1 immunoreactivity by
immunohistochemical analysis. After LPS injection for 24 h, activated microglial cells were distributed
throughout whole mouse brains. The Iba-1 positive immunoreactivity was much stronger in LPS-treated
mice, microglial cells with pronounced hypertrophy and enlarger cell bodies compared with the control
groups in cortical and hippocampal regions (Figure 7C,D). Similar to the motor behavioral results,
microglial activation in the mice brains were effectively attenuated by administration of fisetin
(Figure 7C,D).
3. Discussion
There were eight major findings of current study: (1) When microglia were exposed to H2O2, the
intracellular ROS was highly increased, which was mitigated by fisetin. (2) Fisetin effectively inhibited
ATP-induced increase microglial migratory activity. (3) LPS/IFN-γ- or peptidoglycan-induced
NO production were suppressed by treating fisetin to microglia. (4) Fisetin is nontoxic at 1–5 μM
and dose-dependently reduced iNOS and IL-1β productions. (5) LPS plus IFN-γ enhanced STAT
signaling activation was diminished by treatment with fisetin. (6) HO-1 levels were up-regulated by
fisetin treatment dose-dependently. Treatment with HO-1 activator increased HO-1 expression and
abrogated LPS/IFN-γ-induced iNOS/NO expressions. (7) Administration with fisetin significantly
ameliorated the motor-impaired effects of LPS-injected mice. (8) Microglial activation in the mouse
brain was effectively attenuated by administration of fisetin. Accordingly, fisetin was effective in
retarding microglial activation. Fisetin potently inhibits oxidative reactions and proinflammatory responses
in microglia, probably via promoting up-regulation of an endogenous antioxidant HO-1 expression.
Cytokines are important mediators involved in immune, inflammatory, and immunomodulatory
functions [15]. Although inflammatory responses regulate normal functions of neuronal cells, microglial
activation must be tightly control to avoid exaggerated nenurotoxicity [6,13]. Bacterial meningitis is
the most frequently fatal infection in CNS, which results in significant neurological sequelae [44,45].
In Gram-negative infections, LPS is a well-known activator of microglia. Peptidoglycan, a major
component of the Gram-positive bacterium cell wall, activates microglia and induces productions of
chemokines and cytokines [42,43,46]. STAT1 is a key inflammatory signaling molecule to regulate
iNOS expression in microglial cells and macrophages [47,48]. Activated microglial cells migrate to the
neuronal injury sites and express inflammatory cytokines which aggravate neuronal damage [7–10]. In
the present study, fisetin effectively inhibited microglial cell migration induced by ATP, and inflammatory
cytokine expressions induced by both LPS/IFN-γ and peptidoglycan stimulations.
Classical activation of macrophages/microglia by microbial compounds or pro-inflammatory
cytokines yields a phenotype, which linked to neuroinflammation and neurotoxicity [49,50]. Otherwise,
alternative activation of macrophages/microglia leads to anti-inflammatory phenotype [51–54], which
generates neuronal growth factors as well as anti-inflammatory cytokines, thus contributing to
neuroprotection [55–58]. Therefore, induction of alternative activation of macrophages/microglia and
increases in anti-inflammatory cytokines may provide a novel strategy for anti-neuroinflammation and
neuroprotection. HO-1 is known to be a potent anti-oxidative enzyme, the key mediators participating
in signal transduction mechanisms remain to be fully identified. It has been reported that HO-1 may as
a therapeutic target in neurodegenerative diseases and brain infections [59]. Our results support
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previous reports showing that HO-1 is a critical regulator of NO production in numerous cells [60,61].
Specifically, recent reports have shown that PI-3 kinase/Akt and p38 signaling pathways regulate
HO-1 expression in various cells [62,63]. Again, our results from the microglial cells support the
role of these pathways in fisetin-induced HO-1 expression. Specifically, fisetin activates Akt and p38
phosphorylation and the blockade of PI-3 kinase/AKT and p38 pathways antagonizes fisetin-induced
HO-1 expression.
Intraperitoneal injection of LPS is a useful method for investigating inflammation-caused motor
and coordination function deficits in rodent model. Peripheral inflammation exacerbates brain cytokine
expression, leading to neurodegeneration [64], motor impairment [65], and cognitive impairment [66].
A recent study has shown that intraperitoneal injection of LPS induced progressive motor impairment
from 4 to 24 h [65]. It has also been reported that peripheral LPS injection induced microglial activation
and motor impairment [67]. In current study, we also performed an intraperitoneally injection of LPS,
which caused a shorter latency on the rotarod test and microglia activation significantly in cortex and
hippocampus; however, treatment with fisetin significantly alleviated these motor-impaired effects and
microglia activation.
4. Experimental
4.1. Reagents and Antibodies
Recombinant murine IFN-γ was purchased from PeproTech (Rocky Hill, NJ, USA). LPS from
Escherichia coli Serotype 055:B5, SB203580, PD98059, SP600125 and LY294002 were obtained
from Sigma-Aldrich (St. Louis, MO, USA). Peptidoglycan from Staphylococcus aureus was purchased
from Fluka (Buchs, Switzerland). The antibody against ionized calcium binding adaptor molecule 1
(Iba 1) was purchased from Wako Pure Chemical Industries (Osaka, Japan). Primary antibodies against
Akt, β-actin, phosphorylated Akt, STAT1 and p38 were purchased from Santa Cruz Biotechnology
(Santa Cruz, CA, USA). HO-1 antibody was purchased from StressGen Biotechnologies (San Diego,
CA, USA). Primary antibodies against phosphorylated p38 and STAT-1 phosphorylated at Tyr701 were
purchased from Cell Signaling and Neuroscience (Danvers, MA, USA). The primary antibody against
iNOS was purchased from BD Transduction Lab (Lexington, KY, USA).
4.2. Cell Culture
The murine microglial cell BV-2 was generated from infecting primary microglial cell culture with
a v-raf/v-myc oncogene carrying a retrovirus, and the cells retain the morphological, phenotypical, and
functional properties of primary microglial cells [68]. Cells were cultured in DMEM (Gibco, Grand
Island, NY, USA) with 10% FBS at 37 °C, incubated in a humidified atmosphere consisting of 5%
CO2 and 95% air.
4.3. Animals
The animal experiments were accordance with the Animal Care and Use Guidelines of the China
Medical University (Taichung, Taiwan). Eight-week-old male ICR mice were purchased from the
National Laboratory Animal Center (Taipei, Taiwan). The mice were housed in a temperature- and
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humidity-controlled environment, and given free access to foods and water, and acclimated to their
environment for at least 7 days before conducting the experiments.
4.4. Tissue Preparation and Immunohistochemistry
Mice received an intraperitoneal injection of saline or 20 mg/kg LPS. Twenty-four hours later, mice
were deeply anesthetized with chloral hydrate and transcardially perfused with 10% formaldehyde.
These brains were sliced coronal serial sections (30 μM) using a freezing sliding microtome
cryostat (CM305S, Leica, Microsystems; Wetzlar, Germany). Brain sections were quenched by
endogenous peroxidases with hydrogen peroxide, blocked by nonspecific binding with goat serum,
permeabilized with Triton X-100, and then incubated with a primary antibody against Iba-1. Following
incubation with a biotin-conjugated secondary antibody, the sections were incubated with an
avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA), and labeling was visualized with
diaminobenzidine. The cerebral cortex and hippocampus were digitally captured at 200× magnification
using a light microscope.
4.5. Reactive Oxygen Species (ROS) Assay
The procedure of ROS assay was according to our previous studies. Production of ROS was
assessed oxidation of specific probes with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) by
using flow cytometry. Cells were incubated to H2DCFDA (10 μM) for 30 min at 37 °C, and then
stimulated with hydrogen peroxide. The fluorescence intensity was measured with an excitation filter
of 488 and 525 nm emission wavelengths.
4.6. Western Blot Analysis
Western blotting was performed according to our previous report [69,70]. Briefly, cells were lysed
in a homogenization buffer and equal amounts of the samples were loaded in a SDS-PAGE membrane.
The membranes were probed with a primary antibody and then incubated with a peroxidase-conjugated
secondary antibody. The blots were developed by a western chemiluminescent HRP substrate
(Millipore, Billerica, MA, USA) and visualized by using Fuji medical X-ray film (Fujifilm, Tokyo,
Japan). The blots were then stripped by using a stripping buffer [71] and re-probed anti-β-actin
antibody to as a loading control.
4.7. Migration Assay
In vitro migration assay was performed using Costar Transwell inserts (pore size: 8 μm; Corning,
Albany, NY, USA) in 24-well plates as described previously [72]. Approximately 1 × 104 cells in 200 μL
of medium were placed in the upper chamber, and the same medium containing ATP was placed in the
lower chamber [34,73]. Before performing the migration assay, cells were pre-treated for 60 min with
fisetin followed by treatment with ATP during the 24-h migration assay (incubated at 37 °C in 5%
CO2). After the 24-h assay, the cells were stained with 0.05% crystal violet and 2% methanol.
Non-migratory cells on the upper surface of the filters were removed by wiping with a cotton swab.
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Cell number was counted in five random fields per well under a microscope at 200× magnification.
Images of migratory cells were observed and acquired using a digital camera and light microscope.
4.8. Quantitative Real-Time PCR
Quantitative real-time PCR was performed according to our previous report [35,38]. Briefly,
quantitative real-time PCR using SYBR Green Master Mix was performed with StepOne Plus System
(Applied Biosystems, Singapore). After incubation at 50 °C for 2 min and 95 °C for 10 min, the PCR
was performed as follows: 40 cycles at 95 °C for 10 s and 60 °C for 1 min. The threshold was set
above the non-template control background and within the linear phase of target gene amplification to
calculate the cycle number at which the transcript was detected (denoted as CT).
4.9. Nitric Oxide Assay
Nitric oxide production was analyzed by measuring nitrite content in culture media as described in
our previous report [46]. Briefly, the media were determined by a colorimetric assay with a Griess
reaction, the absorbance was determined at 550 nm using a microplate reader (Thermo Scientific,
Vantaa, Finland).
4.10. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay
Cell viability was determined using the MTT assay, the procedure was according to previous
reports [74]. After treatment with fisetin for 24 h, cell culture media were removed. MTT (0.5 mg/mL)
was added to each culture well and the mixture was incubated for 2 h at 37 °C. The MTT reagent was
removed and washed with PBS for several times. DMSO (200 μL per well) was added to dissolve
formazan crystals, absorbance was determined at 550 nm using a microplate reader (Thermo Scientific,
Vantaa, Finland).
4.11. Statistical Analyses
The values are reported as mean ± S.E.M. Statistical analyses for two groups were performed using
Student’s t-test. The difference was determined to be significant if the p value was <0.05.
5. Conclusions
The results of current study suggest that fisetin exerts both anti-oxidative and anti-inflammatory
activities may be suggested as a promising strategy in the treatment of neurodegenerative diseases.
Acknowledgments
This work is supported in part by grants from the National Science Council (NSC 101-2320-B-039048-MY2, NSC 102-2320-B-039-051-MY3, NSC 102-2314-B-303-006- and NSC 102-2320-B-039026-MY3), China Medical University (CMU101-ASIA-10), Taiwan Ministry of Health and Welfare
Clinical Trial and Research Center of Excellence (DOH102-TD-B-111-004), and Taichung Tzu Chi
General Hospital (TTCRD 102-11). The authors thank S. H. Ko for technical support.
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Author Contributions
J.-Y. Chuang, P.-C. Chang, C.-F. Tsai, and D.-Y. Lu designed the research; W.-L. Yeh, C. Lin,
and D.-Y. Lu wrote the paper; Y.-C. Shen, L.-S. Wu, H.-Y. Lin and Y.-S. Liu performed experiments;
Y.-C. Shen, P.-C. Chang, C.-F. Tsai, and J.-H. Chen analyzed results.
Conflicts of Interest
The authors declare no conflict of interest.
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