The tiliroside derivative, 3-O-[(E)-(2-oxo-4-(p-tolyl) but–3–en–1–yl] kaempferol produced inhibition of neuroinflammation and activation of AMPK and Nrf2/HO-1 pathways in BV-2 microglia

Article abstract of DOI:10.1016/j.intimp.2019.105951

Neuroinflammation is now widely accepted as an important pathophysiological mechanism in neurodegenerative disorders, thus providing a critical target for novel compounds. In this study, 3-O-[(E)-(2-oxo-4-(p-tolyl)but-3-en-1-yl] kaempferol (OTBK) prevente

Full text of DOI:10.1016/j.intimp.2019.105951

InternationalImmunopharmacology77(2019)105951
Contents lists available at ScienceDirect
International Immunopharmacology
journal homepage: www.elsevier.com/locate/intimp
The tiliroside derivative, 3-O-[(E)-(2-oxo-4-(p-tolyl) but–3–en–1–yl]
kaempferol produced inhibition of neuroinflammation and activation of
AMPK and Nrf2/HO-1 pathways in BV-2 microglia
Ravikanth Velagapudia,c, Faisal Jamshaidb,d, Izabela Lepiarza, Folashade O. Katolaa,
Karl Hemmingb, Olumayokun A. Olajidea,⁎
a Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom
b Department of Chemical Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, United Kingdom
c Center for Translational Pain Medicine, Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA1
d 1018 Liaohe Road, Xinbei Zone, Changzhou, Jiangsu, China1
A R T I C L E I N F O
A B S T R A C T
Keywords:
Neuroinflammation is now widely accepted as an important pathophysiological mechanism in neurodegenera-
tive disorders, thus providing a critical target for novel compounds. In this study, 3-O-[(E)-(2-oxo-4-(p-tolyl)but-
3-en-1-yl] kaempferol (OTBK) prevented the production of pro-inflammatory mediators TNFα, IL-6, PGE2 and
nitrite from BV-2 microglia activated with LPS and IFNγ. These effects were accompanied by reduction in the
levels of pro-inflammatory proteins COX-2 and iNOS. Involvement of NF-κB in the anti-inflammatory activity of
OTBK was evaluated in experiments showing that the compound prevented phosphorylation, nuclear accumu-
lation and DNA binding of p65 sub-unit induced by stimulation of BV-2 microglia with LPS and IFNγ. Exposure
of mouse hippocampal HT22 neurons to conditioned media from LPS + IFNγ-stimulated BV-2 cells resulted in
reduced cell viability and generation of cellular reactive oxygen species. Interestingly, conditioned media from
LPS/IFNγ–stimulated BV-2 cells which were treated with OTBK did not induce neuronal damage or oxidative
stress. OTBK was shown to increase protein levels of phospho-AMPKα, Nrf2 and HO-1 in BV-2 microglia. It was
further revealed that OTBK treatment increased Nrf2 DNA binding in BV-2 microglia. The actions of the com-
pound on AMPKα and Nrf2 were shown to contribute to its anti-inflammatory activity as demonstrated by
diminished activity in the presence of the AMPK antagonist dorsomorphin and Nrf2 inhibitor trigonelline. These
results suggest that OTBK inhibits neuroinflammation through mechanisms that may involve activation of
AMPKα and Nrf2 in BV-2 microglia.
3-O-[(E)-(2-oxo-4-(p-tolyl)-but-3-en-1-yl]
kaempferol
Anti-inflammatory
Microglia
AMPKα
Nrf2
Neuroprotection
1. Introduction
protein kinase that plays a critical role in regulating energy metabolism.
Studies have suggested that activating AMPK produces anti-in-
Neuroinflammation by the immune system in the brain is an im-
portant process in the understanding of neurobiology and therapeutics
of neurodegenerative disorders such as Alzheimer’s disease (AD) [1,2]
and Parkinson’s disease (PD) [3]. Neuroinflammation involves a sus-
tained activation of macrophages in the brain, known as microglia re-
sulting in the secretion of neurotoxic factors, including pro-in-
flammatory cytokines, chemokines and reactive oxygen species (ROS).
Consequently, attention is now focused on the synthesis of novel anti-
inflammatory agents with neuroprotective potential for AD ther-
apeutics.
flammatory activity through indirect inhibition of NF–κB. In neuroin-
flammation, AMPK activation has been proposed to have beneficial
effects [4]. For example, ENERGI-F704 a known AMPK activator was
shown to inhibit neuroinflammation in LPS-stimulated BV-2 microglia
by preventing nuclear translocation and protein levels of NF-κB with
the subsequent reduction in IL-6, TNFα, iNOS and COX-2 [5].
The Nrf2 activation pathway is an emerging drug target for the
treatment of neurodegenerative disorders such as AD and PD due to its
ability to suppress neuroinflammation. Reports have shown that Nrf2
activation results in inhibition of neuroinflammation through tran-
scriptional repression of pro-inflammatory cytokines such as TNFα,
Adenosine monophosphate-activated protein kinase (AMPK) is a
⁎ Corresponding author at: Department of Pharmacy, School of Applied Sciences, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, United Kingdom.
E-mail address: o.a.olajide@hud.ac.uk (O.A. Olajide).
1 Current Address.
https://doi.org/10.1016/j.intimp.2019.105951
Received 23 April 2019; Received in revised form 24 September 2019; Accepted 30 September 2019
1567-5769/©2019ElsevierB.V.Allrightsreserved.

R. Velagapudi, et al.
InternationalImmunopharmacology77(2019)105951
Fig. 1. Structure of OTBK.
IL‐1, and IL‐6 in microglia [6]. Furthermore, the Nrf2 activator sul-
phoraphane has been reported to enhance Nrf2 DNA-binding activity as
well as inducing upregulation of Nrf2 target genes in the microglia,
while inhibiting LPS-induced interleukin IL-1β, IL-6, and iNOS pro-
duction [7].
2.3. Determination of pro-inflammatory cytokines, nitrite and PGE2 in
LPS + IFNγ-activated BV-2 microglia
This was carried out as described earlier [13,14]. BV-2 microglia
were treated with OTBK (2.5, 5 and 10 µM) 30 min prior to stimulation
with LPS (100 ng/ml) and IFNγ (5 ng/ml) for a further 24 h. Levels of
TNFα and IL-6 in culture supernatants were determined using mouse
TNFα and IL-6 ELISA kits (Biolegend, UK), while nitrite production was
measured using the Griess assay kit (Promega, UK). Levels of PGE2
were measured using PGE2 EIA kit (Arbor Assays, USA).
Culture medium in HT22 hippocampal neurons cells were com-
pletely removed and replaced with conditioned media (200 µl) from
LPS + IFNγ-stimulated BV-2 cells This was incubated for a further 24 h
in 5% CO2 at 37 °C. Neuronal viability was determined using MTT cell
viability assay, while cellular ROS generation in the neurons was
measured using the fluorescence DCFDA method assay kit (Abcam).
Previously we reported that tiliroside, a dietary glycosidic flavonoid
inhibited neuroinflammation in LPS-activated BV-2 microglia through
multiple mechanisms involving NF-κB, p38 MAPK and Nrf2 activation
pathways [8,9]. Interestingly, a novel tiliroside derivative, 3-O-[(E)-(2-
oxo-4-(p-tolyl)but-3-en-1-yl] kaempferol (OTBK) (Fig. 1) has been
shown to enhance glucose consumption by insulin-resistant HepG2 cells
[10]. In experiments reported by Shi et al. (2011), a closely related
compound to OTBK increased GLUT4 translocation in muscle cells
through a mechanism linked to AMPK activation [11]. In this study we
have investigated effects of OTBK in BV-2 microglia to determine
whether it would inhibit neuroinflammation. We have also evaluated
the roles of Nrf2 and AMPK activation in the anti-inflammatory activity
of the compound.
2.4. Immunoblotting
Following treatment, microglia cell lysates were prepared by
washing cells with PBS, followed by addition of lysis buffer and phe-
nylmethylsulfonyl fluoride (PMSF), and centrifugation for 10 min.
Nuclear extracts were prepared using EpiSeeker Nuclear Extraction Kit
(Abcam) according to the manufacturer's instructions.
2. Materials and methods
2.1. Synthesis of 3-O-[(E)-(2-oxo-4-(p-tolyl) but-3-en-1-yl] kaempferol
(OTBK)
Twenty-five micrograms of protein was subjected to sodium dodecyl
sulfate polyacrylamide (SDS) gel electrophoresis. Proteins were then
transferred to polyvinylidene fluoride (PVDF) membranes (Millipore,
Bedford, MA, USA) for 2 h. Membranes were then blocked at room
temperature for 1 h and then incubated with primary antibodies over-
night at 4 °C. Primary antibodies used in the experiments were rabbit
anti-HO-1 (Santa Cruz; 1:500), rabbit anti-COX-2 (Santa Cruz; 1:500),
rabbit anti-iNOS (Santa Cruz; 1:500), rabbit anti-phospho-AMPKα
(Santa Cruz; 1:500), rabbit anti-AMPKα (Santa Cruz; 1:500), rabbit
anti-lamin B1 (Santa Cruz; 1:500) and rabbit anti-Nrf2 (Santa Cruz;
1:500). Blots were detected with Alexa Fluor® 680 goat anti-rabbit IgG
(Life technologies, UK) using Licor Odyssey. Equal protein loading was
assessed using rabbit anti-actin antibody (Sigma, 1:1000).
OTBK was synthesised from the reaction of kaempferol with freshly
prepared (E)-1-bromo-4-(p-tolyl) but-3-en-2-one [12], in the presence
of potassium carbonate in boiling dioxane (Supplementary Data 1).
After 48 h, silica gel chromatographic work-up gave a 34% yield of the
pure desired product compared to the 13.4% yield reported previously
[10]. Lipopolysaccharide (LPS) derived from Salmonella enterica ser-
otype typhimurium SL1181 (Sigma) was used in these experiments at a
concentration of 100 ng/ml. Interferon (IFNγ) derived from E. coli (R
and D Systems) was used at a concentration of 5 ng/ml. In all cases,
cells were treated with OTBK 30 min prior to stimulation with LPS and
IFNγ.
2.2. Cell culture
2.5. Immunofluorescence microscopy
BV-2 mouse microglia cell line ICLC ATL03001 (Interlab Cell Line
Collection, Banca Biologica e Cell Factory, Italy) were cultured in
RPMI1640 medium (Gibco). HT22 mouse hippocampal neurons were a
kind gift from Dr Jeff Davis and were cultured in DMEM (Gibco). All
culture media were supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 mg/
ml streptomycin.
This was carried out as described elsewhere [9]. Immuno-
fluorescence detection of rabbit anti-NF-κB p65 antibody (Santa Cruz;
1:100) was carried with Alexa Fluor 488–conjugated donkey anti rabbit
IgG secondary antibody (Life Technologies; 1:500) and images obtained
using EVOS® FLoid® cell imaging station.
2.6. DNA binding assays
DNA binding assays were used to determine the effects of OTBK on
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R. Velagapudi, et al.
InternationalImmunopharmacology77(2019)105951
DNA binding of NF-κB and Nrf2 transcription factors in BV-2 microglia.
Following cell treatment, nuclear extracts were prepared and assayed
using the TransAM NF-κB and Nrf2 transcription factor EMSA kits
(Activ Motif, Belgium) according to the manufacturer’s instructions.
The TransAM transcription factor assay kits employed a 96-well plate to
which oligonucleotides containing the NF-κB consensus site (5′-GGGA
CTTTCC-3′) or the ARE consensus binding site (5′-GTCACAGTGACTC
AGCAGAATCTG-3′) have been immobilised.
10 µM.
3.2. Protective effect of OTBK in microglia conditioned media-induced
neurotoxicity
Having observed that OTBK inhibited inflammatory mediator re-
lease from LPS + IFNγ-activated BV-2 microglia, we were interested in
determining whether this compound would prevent microglia-induced
neurotoxicity in HT22 mouse hippocampal neurons. Results in Fig. 5A
show that exposure of HT22 cells to microglia conditioned media from
cells stimulated with a combination of LPS and IFNγ resulted in sig-
nificant (p < 0.001) reduction in neuronal viability. Interestingly, in-
cubation of conditioned media from BV-2 cells treated with OTBK
(2.5–10 µM) prior to LPS and IFNγ stimulation resulted in significantly
increased (p < 0.05) number of viable neurons. In addition, there was
a corresponding significant (p < 0.01) increase in the generation of
cellular ROS in neurons incubated with conditioned media from LPS
and IFNγ only treated BV-2 microglia, while there was a reduction in
ROS production when neurons were exposed to conditioned media from
microglia treated with OTBK prior to LPS and IFNγ (Fig. 5B). These
results have confirmed that inhibition of neuroinflammation involving
reactive microglia is a critical target in achieving neuroprotection in the
treatment of neurodegenerative diseases [16]. Furthermore the activity
of OTBK in preventing microglia-mediated neurotoxicity has been de-
monstrated.
2.7. Statistical analyses
Values of all experiments were represented as a mean
SEM of at
least 3 independent experiments. Differences between values from
different treatments were compared using one-way analysis of variance
(ANOVA) followed by a post-hoc Tukey test.
3. Results and discussion
3.1. OTBK reduced the production of pro-inflammatory cytokines, NO/
iNOS and PGE2/COX-2 from LPS + IFNγ-stimulated BV-2 microglia
without affecting cell viability
Analyses of BV-2 culture supernatants revealed that OTBK produced
significant (p < 0.05) reduction in the levels of pro-inflammatory
TNFα (Fig. 2A), IL-6 (Fig. 2B), PGE2 (Fig. 2C) and nitrite (Fig. 2D) in
BV-2 microglia activated with LPS (100 ng/ml) and IFNγ (5 ng/ml).
Furthermore, results of immunoblotting experiments in Fig. 3A–C
suggest that the compound reduced PGE2 and nitrite production
through inhibition of COX-2 and iNOS protein expression, respectively.
These actions are consistent with those shown by natural compounds
such as curcumin [15], and are similar to those earlier observed with
tiliroside, which showed marked inhibition of neuroinflammation in
BV-2 microglia by the compound [8,9].
3.3. OTBK inhibited NF-κB activation
It is now widely accepted that the production of pro-inflammatory
mediators such as TNFα, IL-6 and expression of proteins like COX-2 and
iNOS during neuroinflammation is regulated by the transcription factor
NF-κB. We were therefore interested in establishing whether inhibition
of NF-κB activation is
a mechanism responsible for the anti-in-
We also observed that treatment of BV-2 cells with 2.5, 5 and 10 µM
OTBK followed by LPS + IFNγ stimulation for 24 h did not affect the
viability of these cells, whereas higher concentrations (20 and 40 µM)
of the compound produced significant (p < 0.001) reduction in cell
viability (Fig. 4A and B). This clearly suggests that the compound was
not cytototoxic at anti-inflammatory concentrations of 2.5, 5 and
flammatory activity of OTBK. Initial ELISA experiments demonstrated
that the compound prevented the phosphorylation of p65 sub-unit of
NF-κB, which is one of the critical steps in the microglia NF-κB acti-
vation pathway (Fig. 6A). This was confirmed by results of immuno-
fluorescence experiments which showed that OTBK prevented
LPS + IFNγ-induced nuclear accumulation of NF-κB p65 sub-unit in BV-
Fig. 2. OTBK reduced levels of TNFα (A), IL-6 (B), PGE2 (C) and nitrite (D) in culture supernatants of BV-2 microglia stimulated with LPS (100 ng/ml) and IFNγ
(5 ng/ml) for 24 h. All values are expressed as mean SEM for three independent experiments. Data were analysed using one-way ANOVA for multiple comparisons
with post-hoc Tukey test. *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with LPS/ IFNγ control.
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InternationalImmunopharmacology77(2019)105951
A
Fig. 3. Inhibition of LPS/IFNγ-induced increase in protein levels of COX-2 and iNOS in BV-2 microglia by OTBK. Cultured cells were treated with OTBK prior to
stimulation with LPS (100 ng/ml) and IFNγ (5 ng/ml) for 24 h. Cell lysates were immunoblotted using COX-2 and iNOS antibodies (A). Densitometric analyses of
three independent experiments are shown for COX-2 (B) and iNOS (C). Data were analysed using one-way ANOVA for multiple comparisons with post-hoc Tukey test.
*p < 0.05, **p < 0.01, ***p < 0.001 in comparison with LPS/ IFNγ control.
Fig. 4. Treatment of BV-2 microglia with OTBK (2.5, 5 and 10 µM), followed by stimulation with LPS (100 ng/ml) and IFNγ (5 ng/ml) for 24 h did not reduce cell
viability. Data were analysed using one-way ANOVA for multiple comparisons with post-hoc Tukey test. ***p < 0.001 in comparison with untreated control.
Fig. 5. Protection against microglia-induced neuronal damage by OTBK. HT22 neurons were incubated with conditioned culture medium obtained from BV-2
microglia that were stimulated with LPS and IFNγ for 24 h (A). MTT assay revealed an increase in HT22 viability in cells treated with OTBK, in comparison with
control. OTBK treatment caused a reduction in cellular ROS generation in HT22 cells (B). Data were analysed using one-way ANOVA for multiple comparisons with
post-hoc Tukey test. *p < 0.05, **p < 0.01, ***p < 0.001 in comparison with LPS/ IFNγ stimulation.
2 microglia (Fig. 6B). Furthermore, DNA binding assays revealed that
OTBK prevented LPS + IFNγ-induced DNA binding of NF-κB in BV-2
microglia (Fig. 6C). Based on these results, we can conclude that OTBK
inhibits neuroinflammation by modulating cellular activities resulting
in the activation of NF-κB in a similar manner as the related compound,
tiliroside.
3.4. Activation of AMPK in BV-2 microglia contributes to anti-
inflammatory activity of OTBK
Based on reports demonstrating the effects of OTBK on glucose
metabolism, studies showing activation of AMPK by a related com-
pound [10,11], as well as reported role of AMPK activation in neu-
roinflammation [4], we used immunoblotting to determine whether
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Fig. 6. OTBK suppresses neuroinflammation by interfering with NF-κB activity through inhibition of p65 phosphorylation (A), nuclear accumulation of p65 sub-unit
of NF-κB (B) and inhibition of DNA binding of NF-κB (C) in BV-2 cells stimulated with LPS (100 ng/ml) and IFNγ (5 ng/ml). Data were analysed using one-way
ANOVA for multiple comparisons with post-hoc Tukey test. **p < 0.01, ***p < 0.001 in comparison with LPS/ IFNγ or TNFα stimulation.
this compound could activate AMPKα in BV-2 microglia. We were also
interested in evaluating the effect of OTBK on LPS + IFNγ-induced
dephosphorylation of AMPK. Our results revealed that treatment of
unstimulated BV–2 microglia with OTBK for 24 h resulted in significant
(p < 0.05) elevation in phospho-AMPKα protein in comparison with
untreated control (Fig. 7a). Separate experiments also revealed that
stimulation of BV-2 microglia with a combination of LPS (100 ng/ml)
and IFNγ (5 ng/ml) resulted in significant (p < 0.01) suppression of
phospho-AMPKα protein expression in comparison with control cells
(Fig. 7b). However, in the presence of OTBK (2.5–10 µM), there was a
significant (p < 0.001) reversal of LPS + IFNγ-induced inactivation of
AMPK. This is an interesting outcome as studies have revealed a link
between inflammation and dephosphorylation of AMPK in mice [17]. It
therefore appears that reversal of inflammation-induced depho-
sphorylation of AMPK by OTBK and its anti-inflammatory activity are
linked.
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Fig. 7a and b. OTBK directly increased levels of phospho-AMPKα in BV-2 microglia following incubation for 24 h (A), and reversed dephosphorylation of AMPKα
induced by stimulation with LPS (100 ng/ml) and IFNγ (5 ng/ml) for 24 h (B). Cell lysates were analysed using immunoblotting for phospho-AMPKα and total
AMPKα. Representative blots and densitometric analyses of three independent experiments are shown (Mean
for multiple comparisons with post-hoc Tukey test).
SEM; **p < 0.01, ***p < 0.001; one way ANOVA
Fig. 7c and d. Inhibition of neuroinflammation by OTBK was diminished in the presence of dorsomorphin. BV-2 cells were treated with either OTBK (10 µM) and LPS
(100 ng/ml) + IFNγ (5 ng/ml), dorsomorphin (10 µM) and LPS (100 ng/ml) + IFNγ (5 ng/ml), or dorsomorphin (10 µM), followed by OTBK (10 µM) and LPS
(100 ng/ml) + IFNγ (5 ng/ml) for 24 h. Cells were analysed for TNFα (C) and nitrite (D). Data were analysed using one-way ANOVA for multiple comparisons with
post-hoc Tukey test. ***p < 0.001; untreated control versus LPS/ IFNγ stimulation. θθp < 0.01; OTBK + LPS + IFNγ treatment versus LPS + IFNγ only. &p < 0.05;
dorsomorphin + OTBK + LPS/IFNγ treatment compared with OTBK + LPS/ IFNγ treatment (n = 6).
The role of AMPK activation in the anti-inflammatory activity of
OTBK was then further investigated by treating BV-2 cells with potent
and direct inhibitor of AMPK, dorsomorphin [18] (10 µM) followed by
OTBK (10 µM) prior to LPS + IFNγ stimulation for 24 h. Analyses of
culture supernatants showed that LPS + IFNγ stimulation resulted in a
significant (p < 0.05) elevation of TNFα and IL-6 levels, in comparison
with control cells. Also, OTBK produced a characteristic reduction in
the levels of these cytokines in BV-2 microglia stimulated with
LPS + IFNγ. In cells treated with dorsomorphin (10 µM) prior to sti-
mulation with LPS + IFNγ, there was no reduction in the production of
neuroinflammation [21]. An example of such a compound is thymo-
quinone found in cumin seed oil which was reported to inhibit neu-
roinflammation through mechanisms linked to AMPK and SIRT1 [22].
Similar activity has been reported for lycopene [23], hydrogen sulphide
[24], caffeic acid phenethyl ester (CAPE) [25] and resveratrol [26].
3.5. Activation of Nrf2 by OTBK contributes to its anti-inflammatory
activity in BV-2 microglia
Research reported by Zimmermann et al. [27] showed that there is a
crosstalk between the AMPK and Nrf2/HO–1 signalling pathways and
suggested that compounds like xanthohumol are able to induce dual
activation of both pathways through mechanisms involving reduced
endoplasmic reticulum stress. This led us to investigate whether OTBK
would have any effect on Nrf2 activation in BV-2 microglia. In Fig. 8A
and B, we show that treatment with OTBK induced an increase in ac-
cumulation of Nrf2 in the nucleus in both unstimulated and
LPS + IFNγ–stimulated BV-2 microglia. Results of DNA binding assays
in Fig. 8C show that there was significant (p < 0.05) increase in the
binding of Nrf2 to ARE consensus sites following treatment of BV-2 cells
with OTBK (5 and 10 µM). Interestingly, at 2.5 µM, the compound did
not produce significant effect on Nrf2 activity. Similarly, treatment of
LPS + IFNγ-stimulated BV-2 cells with 5 and 10 µM of OTBK resulted in
significant increase in DNA binding of Nrf2, while 2.5 µM of the
these pro-inflammatory mediators, suggesting
a lack of anti-in-
flammatory activity. However, inhibition of neuroinflammation as
shown by reduction in TNFα (Fig. 7c) and nitrite (Fig. 7d) production
by OTBK was diminished in the presence of dorsomorphin. The loss of
anti-inflammatory activity in the presence of the AMPK antagonist
further shows that inhibition of neuroinflammation by OTBK is related
to its ability to activate AMPK. Some studies have suggested a role for
AMPK as a negative regulator of inflammatory signalling pathways in
macrophages [19]. Taken together, our results appear to suggest that
OTBK possibly targets mechanisms involved in negative regulation of
inflammation by AMPK.
Other studies have linked anti-neuroinflammatory activity to acti-
vation of AMPK in BV-2 microglia [20], indicating that compounds
which activate this kinase in the microglia may inhibit
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Fig. 8. OTBK (5 and 10 µM) treatment significantly increased nuclear Nrf2 in the absence (A) and presence of LPS + IFNγ stimulation (B) in BV-2 microglia. Nuclear
extracts were analysed using immunoblotting for Nrf2 and lamin B. Representative blots and densitometric analyses of 3 independent experiments are shown. OTBK
(5 and 10 µM) treatment without (C) and with LPS + IFNγ stimulation (D) increased DNA binding of Nrf2 to immobilised ARE consensus binding site in BV-2
microglia. Values are mean
SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one way ANOVA for multiple comparisons with post-hoc Tukey test).
B
A
Fig. 9. OTBK (5 and 10 µM) treatment significantly increased HO-1 protein in the absence (A) and presence of LPS + IFNγ stimulation (B) in BV-2 microglia.
Representative blots and densitometric analyses of 3 independent experiments are shown (Mean
with post-hoc Tukey test).
SEM; ***p < 0.001; one way ANOVA for multiple comparisons
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Fig. 10. Inhibition of neuroinflammation by OTBK was diminished in the presence of trigonelline. BV-2 cells were treated with either OTBK (10 µM) and LPS
(100 ng/ml) + IFNγ (5 ng/ml), trigonelline (100 µM) and LPS (100 ng/ml) + IFNγ (5 ng/ml), or trigonelline (100 µM), followed by OTBK (10 µM) and LPS (100 ng/
ml) + IFNγ (5 ng/ml) for 24 h. Cells were analysed for TNFα (A) and nitrite (B). Data were analysed using one-way ANOVA for multiple comparisons with post-hoc
θθθ
Tukey test. ***p < 0.001; untreated control versus LPS/ IFNγ stimulation.
p < 0.001; OTBK + LPS + IFNγ treatment versus LPS + IFNγ only. &p < 0.05;
trigonelline + OTBK + LPS/IFNγ treatment compared with OTBK + LPS/ IFNγ treatment (n = 6).
compound did not produce a significant effect (Fig. 8D).
earlier reported similar Nrf2-mediated anti-inflammatory activity in
Western blotting experiments revealed that treatment of un-
stimulated BV-2 microglia with 2.5 µM of OTBK did not produce sig-
nificant increase in the downstream HO-1 protein (Fig. 9A). On in-
creasing the concentration of the compound to 5 and 10 µM, there was
significant (p < 0.001) increase in levels of HO-1 protein in BV-2 mi-
croglia. In LPS + IFNγ-stimulated cells however, significant increase in
HO-1 protein was observed with 2.5–10 µM of OTBK, in comparison
with either unstimulated cells or with cells stimulated with LPS + IFNγ
(Fig. 9B). These results suggest that OTBK produces Nrf2-mediated in-
crease in the levels of the antioxidant HO-1 protein.
Following results showing that OTBK activated Nrf2 in BV-2 mi-
croglia and based on our previous studies which demonstrated that
anti-inflammatory activity of compounds such as tiliroside is dependent
on Nrf2 [9,14,28], we used a known Nrf2 inhibitor, trigonelline to in-
vestigate whether OTBK would retain its anti-inflammatory activity in
the presence of the inhibitor. Treatment of LPS + IFNγ stimulated BV-2
microglia with OTBK (10 µM) resulted in suppression of both TNFα and
nitrite production, while anti-inflammatory activity was not observed
when the cells were pre-treated with trigonelline (100 µM) prior to
LPS + IFNγ stimulation. However, following treatment of LPS + IFNγ-
activated BV-2 microglia with trigonelline (100 µM) prior to OTBK
(10 µM) we observed that the anti-inflammatory effects of the com-
pound on TNFα and nitrite production were partially reduced (Fig. 10A
and B). These results appear to suggest that inhibition of Nrf2 by tri-
gonelline reduced the ability of OTBK to exert anti-inflammatory ac-
tivity in BV-2 microglia. Several studies have reported that trigonelline
is a potent inhibitor of Nrf2; experiments in cellular models have re-
vealed that trigonelline interfered with Nrf2 activation, and suppressed
induction of Nrf2/ARE-dependent gene expression [29–32]. Also, in-
hibition of Nrf2 by trigonelline in mice was shown to produce similar
effects to Nrf2 knockout in attenuating hypertension, renal injury, as
well as tubulointerstitial fibrosis [33]. Consequently, our results in BV-2
cells indicate that OTBK probably produces anti-inflammatory activity
through mechanisms requiring activation of Nrf2.
BV-2 microglia (8, 9). However, unlike OTBK it is not yet known if its
ability to inhibit neuroinflammation is related to activation of AMPK in
the microglia.
4. Conclusions
Our study has demonstrated for the first time that 3-O-[(E)-(2-oxo-4-
(p-tolyl)-but-3-en-1-yl] kaempferol inhibits neuroinflammation and
neuroinflammation-mediated neurotoxicity. Furthermore, the com-
pound produced activation of AMPKα and Nrf2 in BV-2 microglia
which possibly contribute to its anti-inflammatory activity. These data
suggest that 3-O-[(E)-(2-oxo-4-(p-tolyl) but-3-en-1-yl] kaempferol is a
potential small molecule for preventing microglia-mediated inflamma-
tion.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.intimp.2019.105951.
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