International Journal of

Molecular Sciences
ArticleAnti-Inflammatory Effects of Miyako Bidens pilosa in a Mouse
Model of Amyotrophic Lateral Sclerosis andLipopolysaccharide-Stimulated BV-2 Microglia
Komugi Tsuruta 1 , Takato Shidara 1 , Hiroko Miyagishi 1 , Hiroshi Nango 1 , Yoshihiko Nakatani 2 ,Naoto Suzuki 3 , Taku Amano 4 , Toyofumi Suzuki 3 and Yasuhiro Kosuge 1, *
1

2

3

4

*

Citation: Tsuruta, K.; Shidara, T.;
Miyagishi, H.; Nango, H.; Nakatani,Y.; Suzuki, N.; Amano, T.; Suzuki, T.;
Kosuge, Y. Anti-Inflammatory Effectsof Miyako Bidens pilosa in a Mouse
Model of Amyotrophic Lateral Sclerosis
and Lipopolysaccharide-Stimulated
BV-2 Microglia. Int. J. Mol. Sci. 2023,
24, 13698. https://doi.org/10.3390/
ijms241813698
Academic Editors: Terézia Kisková
and Beňadik Šmajda

Laboratory of Pharmacology, School of Pharmacy, Nihon University, 7-7-1 Narashinodai,
Funabashi 274-8555, Chiba, Japan; phko22004@g.nihon-u.ac.jp (K.T.); phta14114@g.nihon-u.ac.jp (T.S.);
miyagishi.hiroko@nihon-u.ac.jp (H.M.); phhi16003@g.nihon-u.ac.jp (H.N.)
Department of Pharmacotherapeutics, School of Pharmacy, International University of Health and Welfare,
2600-1 Kitakanemaru, Ohtawara 324-8501, Tochigi, Japan; ynakatani@iuhw.ac.jp
Laboratory of Pharmaceutics, School of Pharmacy, Nihon University, 7-7-1 Narashinodai,
Funabashi 274-8555, Chiba, Japan; suzuki.naoto65@nihon-u.ac.jp (N.S.); suzuki.toyofumi@nihon-u.ac.jp (T.S.)
Tochigi Prefectural Okamotodai Hospital, 2162 Shimookamotomachi, Utsunomiya 329-1104, Tochigi, Japan;
tamano01@okamotodai.jp
Correspondence: kosuge.yasuhiro@nihon-u.ac.jp; Tel.: +81-47-465-4027

Abstract: Neuroinflammation is a fundamental feature in the pathogenesis of amyotrophic lateralsclerosis (ALS) and arises from the activation of astrocytes and microglial cells. Previously, we
reported that Miyako Bidens pilosa extract (MBP) inhibited microglial activation and prolonged the life
span in a human ALS-linked mutant superoxide dismutase-1 (SOD1G93A ) transgenic mouse model of
ALS (G93A mice). Herein, we evaluated the effect of MBP on microglial activation in the spinal cordof G93A mice and lipopolysaccharide-stimulated BV-2 microglial cells. The administration of MBP
inhibited the upregulation of the M1-microglia/macrophage marker (interferon-γ receptor (IFN-γR))
and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) in
G93A mice. However, MBP did not affect the increase in the M2-microglia/macrophage marker(IL-13R) and anti-inflammatory cytokines (transforming growth factor (TGF)-β and IL-10) in G93A
mice. BV-2 cell exposure to MBP resulted in a decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium (MTT) reduction activity and bromodeoxyuridine incorporation, without an increase in
the number of ethidium homodimer-1-stained dead cells. Moreover, MBP suppressed the productionof lipopolysaccharide-induced pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in BV-2 cells.
These results suggest that the selective suppression of M1-related pro-inflammatory cytokines isinvolved in the therapeutic potential of MBP in ALS model mice.
Keywords: amyotrophic lateral sclerosis; Bidens pilosa; pro-inflammatory cytokines; spinal cord;M1-microglia/macrophages

Received: 18 August 2023
Revised: 2 September 2023
Accepted: 4 September 2023
Published: 5 September 2023

Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/

1. Introduction
Amyotrophic lateral sclerosis (ALS) is a fast-progressing and devastating neurodegenerative disease characterized by motor neuron death in the spinal cord, brain stem,
and motor cortex. Most cases of ALS are of unknown etiology, classified as sporadic ALS,
while approximately 5–10% of the cases are classified as familial ALS. Although more than
20 ALS genes have been identified, mutations of the copper-zinc (Cu/Zn) superoxide dismutase(SOD1) gene were the first shown to cause familial ALS [1]. In ALS research, transgenic
mice overexpressing high levels of a mutant human SOD1 containing a glycine-to-alanine
substitution at position 93 (SOD1G93A ) (G93A mice) are used as the standard model [2].
Several studies using G93A mice have revealed some mechanisms of pathogenesis in motor

4.0/).

Int. J. Mol. Sci. 2023, 24, 13698. https://doi.org/10.3390/ijms241813698

https://www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2023, 24, 13698

2 of 14neuron death such as oxidative stress [3], protein aggregation [4], and neuroinflammation [5]. Recently, attention has been focused on the direct contribution of non-neuronalcells, such as astrocytes and microglia, to motor neuron death in ALS [6]. Indeed, theselective reduction in mutant SOD1 from microglia [7] or astrocytes [8] in mice using the
Cre–Lox system slowed disease progression in SOD1G37R ALS model mice. Thus, the importance of microglia and astrocytes as non-cell autonomous players is widely recognizedin the pathological study of ALS.
Microglia are the resident macrophages in the central nervous system (CNS) and are
gaining recognition for their critical role in CNS immune surveillance. Microglia-associatedneuroinflammation is a pathological feature in both patients with ALS and mouse modelsof the disease [9], and positron emission tomography studies have shown that activatedmicroglia are existent in the brains of living patients with ALS [10,11]. Autopsies of patientswith ALS revealed that activated microglia exist around the injury sites of motor neurons
in spinal cord tissue [12]. Furthermore, extracellular mutant SOD1 from G93A mice does
not cause direct disability in cultured motor neurons, but it activates microglia, whichthen enhance motor neuron death through producing neurotoxic factors including proinflammatory molecules and free radicals [13,14]. Previous studies showed that treatment
with minocycline or Hirsutella sinensis reduced the level of activated microglia and thedegeneration of motor neurons and also prolonged the lifespan in G93A mice, suggestingthat microglial regulation may represent an effective treatment for ALS [15–17]. Interestingly, replacing wild-type microglia/macrophage via bone marrow transplantation slowed
disease progression in G93A mice [18]. These results suggest that microglia/macrophages
may be a more useful target than astrocytes in the regulation of ALS neuroinflammation.
Increasing evidence shows that microglia/macrophages in the CNS are categorized by two
opposite types, the M1 and M2 phenotypes. Generally, M1 microglia/macrophages induce
inflammation and neurotoxicity through releasing inflammatory mediators, while M2
microglia/macrophage induce anti-inflammation and neuroprotectivity via the productionof anti-inflammatory mediators [19,20]. The microglia/macrophage phenotype changes
depending on the disease severity and stage [19,20]. Therefore, a therapeutic approach
to modulate microglia/macrophage phenotypes, either enhancing anti-inflammatory M2phenotypic properties or reducing the M1 phenotype depending on toxicity, could be apotential treatment strategy for ALS.
Bidens pilosa L. var. radiata SCHERFF (BP) is an annual herb, belonging to the Asteraceae family that originated from South America and can now be found in tropical andsubtropical regions around the world [21]. Miyako BP extract (MBP) is cultivated with
only green manure in the Miyako Islands of Okinawa Prefecture, Japan, and is used asan herbal tea ingredient and in various folk medicines. MBP has been reported to havebeneficial effects including anti-inflammatory [22], antioxidant [23], and antiallergy properties [22]. More recently, we reported that MBP is capable of prolonging the life span of
G93A mice when administered at an oral gavage of 2 g/kg/day from the post-symptomaticstage to the end stage of the disease [24]. Importantly, MBP attenuated the activation or
induction of microglia in the spinal cord of G93A mice [24]. In this study, we examined
whether MBP could regulate microglial polarization in the spinal cord of G93A mice andlipopolysaccharide (LPS)-stimulated BV-2 microglial cells.
2. Results
2.1. MBP Selectively Reduced the Production of M1-Related Pro-Inflammatory Cytokines in the
Lumbar Spinal Cord of G93A Mice
M1-phenotype microglia/macrophage represent pro-inflammatory activity and secretevarious pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin1β (IL-1β), and interleukin-6 (IL-6), whereas M2-phenotype microglia/macrophage promote anti-inflammation and tissue repair and produce anti-inflammatory cytokines suchas transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) [19]. We performed
real-time PCR to evaluate the mRNA level of pro-inflammatory and anti-inflammatory

such as transforming growth factor-β (TGF-β) and interleukin-10 (IL-10) [19]. We performed real-time PCR to evaluate the mRNA level of pro-inflammatory and anti-inflammatory cytokines in the lumbar spinal cord of G93A mice. Our real-time PCR results inInt. J. Mol. Sci. 2023, 24, 13698
3 of 14
dicated that the mRNA levels of M1-related pro-inflammatory cytokines (TNF-α, IL-1β,
and IL-6) were significantly increased in G93A mice compared with that of vehicle-treated
WT mice (Figure 1a).cytokines
The upregulationinflammatory
in G93A
wasparin the lumbarof
spinalcord of G93A cytokines
mice. Our real-time
PCRmiceresults
indicatedthe mRNAlevelsM1-related
pro-inflammatory
cytokines(TNF-α,IL-1β,and IL-6)
tially but significantlythat
reversedvia
theoforal
administrationof MBP(Figure1a).
Additionwere significantly increased in G93A mice compared with that of vehicle-treated WT mice
ally, the mRNA level(Figureof M2-associated-anti-inflammatory
cytokines (TGF-β and IL-10)
1a). The upregulation of inflammatory cytokines in G93A mice was partially but sigwas increased in the nificantly
spinal cordof viavehicle-treated
G93Aof micecomparedwith thatvereversed
the oral administration
MBP (Figure1a). Additionally,
theof
mRNA
level
of
M2-associated-anti-inflammatory
cytokines(TGF-βandIL-10)wasincreased
inthehicle-treated WT mice (Figure 1b). Unlike M1-related pro-inflammatory cytokines, thecord of vehicle-treated G93A mice compared with that of vehicle-treated WT micetreatment of MBP didspinal
not inhibit
an increase in the supply of anti-inflammatory cytokines(Figure 1b). Unlike M1-related pro-inflammatory cytokines, the treatment of MBP did not
in G93A mice (Figureinhibit1b). an increase in the supply of anti-inflammatory cytokines in G93A mice (Figure 1b).

1. (MBP)
Miyako Bidens
pilosa (MBP)
selectively
the inductionof pro-inflammatory
cyFigure 1. Miyako BidensFigure
pilosaselectively
inhibitedthe inhibited
inductionof pro-inflammatory
cytotokines
inthelumberspinal
cord
of
G93A
mice.
Mice
were
orallyadministeredinjectionwaterkines in the lumber spinal cord of G93A mice. Mice were orally administered injection water (vehi(vehicle) or MBP (2 g/kg/day), starting at 15 weeks old (asymptomatic stage). One week after thecle) or MBP (2 g/kg/day), starting at 15 weeks old (asymptomatic stage). One week after the start ofstart of treatment, the lumbar spinal cords were analyzed via real-time polymerase chain reaction
treatment, the lumbar spinal
cords
were
via real-timepolymerase
chain
reaction(PCR). The
graphsshow analyzed
the mRNA expression
profiles ofpro-inflammatory
cytokines(tumor(PCR).
necrosis
The graphs show the mRNA
expression
profilesofpro-inflammatory
cytokines(tumornecrosisfactor (TNF)-α, interleukin (IL)-1β, and IL-6 (a)) and anti-inflammatory cytokines (TGF-β and facIL-10
tor (TNF)-α, interleukin(IL)-1β,
IL-6
(a))
and ±
anti-inflammatory
cytokines
and
IL-10
(b)).
Data areandshown
as theaveragestandard error, calculated
from 4–6 (TGF-β
independent
biologicaleach with
2 technical replicates.
(b)). Data are shown asreplicates,
the average± standard
error, calculated from 4–6 independent biologicalreplicates, each with 2 technicalreplicates.
2.2. MBP Selectively
Inhibited M1-Microglia/Macrophage Polarization in the Lumbar Spinal Cordof G93A Mice
The activatedphenotype of microglia/macrophages
is classified
by the production
2.2. MBP Selectively Inhibited
M1-Microglia/Macrophage
Polarizationin the Lumbar
Spinal pattern of cytokines: the pro-inflammatory-related M1-phenotype and the anti-inflammatoryCord of G93A Mice activated M2-phenotype microglia/macrophages [19]. To investigate whether MBP affected the phenotype
of microglia/macrophages,
PCRby
wastheperformed
and the
The activated phenotype
of microglia/macrophages
isreal-timeclassified
production
expression level of the main polarization markers was analyzed for M1 (interferon-γ repattern of cytokines: thepro-inflammatory-related M1-phenotype and the anti-inflammaceptor; IFN-γR) and M2 (interleukin-13 receptor; IL-13R) microglia/macrophages in the
tory-activated M2-phenotype
microglia/macrophages
To investigate
whether MBP
lumbar spinal
cord of G93A and WT mice.[19].
The mRNAlevel of the M1-phenotype
miaﬀected the phenotype of microglia/macrophages, real-time PCR was performed and theexpression level of the main polarization markers was analyzed for M1 (interferon-γ receptor; IFN-γR) and M2 (interleukin-13 receptor; IL-13R) microglia/macrophages in the
lumbar spinal cord of G93A and WT mice. The mRNA level of the M1-phenotype micro-

Int. J. Mol. Sci. 2023, 24, 13698

4 of 14

OR PEER REVIEW

4 of 14croglia/macrophage marker IFN-γR was significantly increased in the vehicle-treated G93A
mice compared with that of the vehicle-treated WT mice, while it was clearly reduced in
the MBP-treated G93A mice (Figure 2a). Although the mRNA level of the M2-phenotype
microglia/macrophage
IL-13R in the vehicle-treatedmice
was also
than that in the vehicle-treated
WT mice,marker
the upregulationof IL-13R inG93A
G93A
mice
didhigher
notthan that in the vehicle-treated WT mice, the upregulation of IL-13R in G93A mice did notchange after the treatment
of MBP (Figure 2b). Moreover, MBP had no marked eﬀect onchange after the treatment of MBP (Figure 2b). Moreover, MBP had no marked effect on
both M1- and M2-phenotypic
microglia/macrophage
markers in
WT mice2). 2).
both M1- andM2-phenotypic microglia/macrophage
markers
in WT(Figure
mice (Figure

Miyako Bidens
pilosa (MBP)
regulated microglia/macrophage
polarization
theFigure 2. Miyako BidensFigure
pilosa2. (MBP)
regulated
microglia/macrophage
M1–M2M1–M2
polarization
in in
the
lumberspinal
cord
of
WT
and
G93A
mice.
Mice
were
orallyadministeredinjectionwater(vehicle)lumber spinal cord of WT and G93A mice. Mice were orally administered injection water (vehicle)
or MBP (2 g/kg/day), starting at 15 weeks old (asymptomatic stage). One week after the start ofor MBP (2 g/kg/day), startingat 15 weeks old (asymptomatic stage). One week after the start oftreatment, the lumbar spinal cords were analyzed via real-time polymerase chain reaction (PCR). The
treatment, the lumbar spinal
cords were analyzed via real-time polymerase chain reaction (PCR).
graphs show the mRNA expression profiles of the M1-like microglia/macrophage marker (interferon
The graphs show the mRNA
expression
profilesof the M1-likemicroglia/macrophage(IFN)-γR (a) and M2-likemicroglia/macrophage
marker(interleukin (IL)-13R (b)) inmarker
the spinal(incord.
terferon (IFN)-γR (a) andmarker(interleukin(IL)-13R(b)) replicates,in theDataM2-likeare shownmicroglia/macrophageas the average ± standard error,
calculated
from 4–6 independent
biologicaleach with
2 technicalreplicates.
spinal cord. Data are shownas theaverage± standard error, calculated from 4–6 independent biological replicates, each with
2 technicalreplicates.
2.3. MBP
Suppressed
the Cell Proliferation of BV-2 Microglial Cells

We further examined the effect of exogenously applied MBP on the cell proliferation

2.3. MBP Suppressed the
Cell Proliferationof BV-2
Microglialof microglia
using the mousemicroglial
cell lineCells
BV-2. As shown in Figure 3, the exposureof BV-2 cells to 2–1000 µg/mL of MBP for 24 h resulted in a concentration-dependent

We further examinedthe eﬀect of exogenously applied MBP on the cell proliferation
decrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) reduction activityof microglia using the(Figure
mouse
microglial
cell
line BV-2.
Asinshownin Figure3, the3b).
A statisticallysignificant
decrease
MTT reduction
activitywas exposure
observed at aconcentration
of
20
µg/mL
(80.1
±
2.2%,
p
<
0.001),
100
µg/mL
(64.8
±
4.5%,
p < 0.0001),
of BV-2 cells to 2–1000 µg/mL of MBP for 24 h resulted in a concentration-dependent
de200 µg/mL (48.9 ± 3.1%, p < 0.0001), and 1000 µg/mL (33.8 ± 2.3%, p < 0.0001) comparedcrease in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium (MTT) reduction activity
to that of vehicle-treated cells (101.4 ± 1.5%). On the other hand, the bromodeoxyuridine(Figure 3b). A statisticallyin MTT
reduction
activity wasobservedat(BrdU)significant
incorporationdecrease
assay showedthat only
the bath applicationof 1000µg/mL MBP
decrease
BrdU incorporation
into
BV-2±cells
comparedwith thata concentration of 20 causedµg/mLa significant(80.1 ± 2.2%,
p <in0.001),
100 µg/mL
(64.8
4.5%,
p < 0.0001),of vehicle-treated cells (Figure 3c). Moreover, observation via phase contrast microscopy
200 µg/mL (48.9 ± 3.1%,
p < 0.0001), and 1000 µg/mL (33.8 ± 2.3%, p < 0.0001) compared todemonstrated that treatment with MBP for 24 h at any tested concentration did not lead tothat of vehicle-treatedancells(101.4± 1.5%).
Oncelltheother hand,the bromodeoxyuridine
increasein the cell
displaying
death-related
morphologicalcharacteristics including
blebbing,
cell
shrinkage,anddetachmentoftheadherentcells
from
the plate(Figure3d).
(BrdU) incorporation assay showed that only the bath application of 1000
µg/mL
MBP
caused a significant decrease in BrdU incorporation into BV-2 cells compared with that ofvehicle-treated cells (Figure 3c). Moreover, observation via phase contrast microscopy
demonstrated that treatment with MBP for 24 h at any tested concentration did not leadto an increase in the cell displaying cell death-related morphological characteristics including blebbing, cell shrinkage, and detachment of the adherent cells from the plate (Figure 3d).

x FOR PEER REVIEW
Int. J. Mol. Sci. 2023, 24, 13698

5 of 145 of 14

Figure3. Miyako
pilosa (MBP)
the proliferationof BV-2 microglia(a) TheFigure 3. Miyako Bidens
pilosa(MBP)Bidens
suppressed
thesuppressed
proliferationof BV-2 microglia
cells.cells.
(a) Theresearch
protocolconducted
in
this
study
is
shown.
BV-2
cells
were
treatedwiththeindicatedresearch protocol conducted in this study is shown. BV-2 cells were treated with the indicated conof MBP for 24 h, and cell proliferation was determined via 3-(4,5-dimethylthiazol-2centrations of MBP forconcentrations
24 h, and cellproliferation was determined via 3-(4,5-dimethylthiazol-2-yl)yl)-2,5-diphenyl tetrazolium (MTT) reduction assay and bromodeoxyuridine (BrdU) incorporation
2,5-diphenyl tetrazolium (MTT) reduction assay and bromodeoxyuridine (BrdU) incorporation asassay. (b) The graph shows the effect of MBP on MTT reduction activity in the cells. Values aresay. (b) The graph showsthe eﬀect
of MBPrelativeon MTT
reduction
activityinshown
the cells.
Valuesindiindicatedas percentages
to untreatedcells.
Data areas theaverageare± standard
cated as percentages relative
tocalculated
untreated
cells.
Data are shown
as replicates,
the average
standard
errorreplicates.
(SE),error (SE),fromfour independent
biologicaleach ±withfour technicalcalculated from four independent
biologicalreplicates,
each
with
four
technical
replicates.
(c)
The of(c) The graph shows the effect of MBP on BrdU incorporation into newly synthesized DNA
Valuesare indicated asinto
percentages
to untreatedcells.
Data are shown asgraph shows the eﬀectproliferatingof MBP oncells.
BrdU
incorporation
newly relativesynthesizedDNAof proliferating
theaverage±SE,
calculated
from
four
independent
biologicalreplicates,
each
with
technical
cells. Values are indicated as percentages relative to untreated cells. Data are shown as thefour
average
replicates.
(d)Cellmorphology
wasobserved
withanopticalmicroscope;
photographs
show± SE, calculated from four independent biological replicates, each with four technical replicates. typical(d)phase-contrast microscopy images of BV-2 cells treated with indicated concentrations of MBP. The
Cell morphology was observed with an optical microscope; photographs show typical phase-conscale bar represents 100 µm.
trast microscopy images of BV-2 cells treated with indicated concentrations of MBP. The scale bar
represents 100 µm. 2.4. MBP Did Not Induce Cell Death in BV-2 Cells

Next, we investigated cell mortality after treatment with MBP in BV-2 cells with theLive/Deadcell staining
kit using
2.4. MBP Did Not Induce Cell Deathin BV-2
Cellsthe time schedule as shown in Figure 4a. The exposureof BV-2 cells to 10–1000 µg/mL MBP for 24 h did not change the percentage of dead cells
Next, we investigatedmortality
after treatment
with MBP
cells
thestained bycell
EthD-1.
The percentage
of EthD-1-positive
cells inin
theBV-2
10, 100,
andwith1000 µg/mL
MBP-treated
cells
was
2.7
±
0.5%,
3.1
±
0.8%,
and
8.6
±
2.6%,
respectively.
The
total
Live/Dead cell staining kit using the time schedule as shown in Figure 4a. The exposurecell
number of 1000 µg/mL MBP-treated cells was significantly reduced to approximately 75%
of BV-2 cells to 10–1000µg/mL MBP for 24 h did not change the percentage of dead cellsof that in the vehicle-treated cells (Figure 4b). Taken together, these results suggest thatstained by EthD-1. The
percentage
ofreduction
EthD-1-positive
cells in10, 100,
and 1000µg/mL
the decreasein MTT
activity inducedby thethe highconcentrationof MBP
is not
MBP-treated cells was
2.7 ±in0.5%,
3.1 ± 0.8%,
and
2.6%, respectively. The total cellinvolved
the cytotoxicity
of MBP
on 8.6
BV-2±cells.

number of 1000 µg/mL MBP-treated cells was significantly reduced to approximately 75%of that in the vehicle-treated cells (Figure 4b). Taken together, these results suggest thatthe decrease in MTT reduction activity induced by the high concentration of MBP is not
involved in the cytotoxicity of MBP on BV-2 cells.

Int. J. Mol. Sci. 2023, 24, x13698
FOR PEER REVIEW

of 14
66of

Figure 4. Miyako
Miyako Bidens
Bidens pilosa
pilosa (MBP)(MBP) did
did not
not induce
induce cell
cell death
death inin BV-2
BV-2 cells. (a)(a) The
The researchresearch protocol
protocolconducted
BV-2
cells
were
treatedwithwith
indicatedconcentrationsof MBP
24
conducted ininthisthisstudy
studyisisshown.
shown.
BV-2
cells
were
treatedindicatedconcentrationsof for
MBP
h.
(b)
The
photographs
show
typicalfluorescence
imagesofcalcein-AM(green,live
cells)
andethfor 24 h. (b) The photographs show typical fluorescence images of calcein-AM (green, live cells)
idium
homodimer-1(EthD-1)(EthD-1)(red, deadcells)
doublein each treatmentgroup. The
scaleThebar
and ethidium
homodimer-1
(red,
dead
cells)staining
double staining
in each treatmentgroup.
indicates 100 µm. The left graph shows the percentage of EthD-1-positive dead cells in these cells. The
scale bar indicates 100 µm. The left graph shows the percentage of EthD-1-positive dead cells inright graph shows the number of EthD-1-positive dead cells (red bar) and calcein-AM-positive live
these cells. The right graph shows the number of EthD-1-positive dead cells (red bar) and calceincells (pale green bar) in these cells. n.s.: no significance. Data are shown as the average ± standard
AM-positive
livefrom
cellsfour(palegreen bar) biologicalin these cells.
n.s.: nosignificance.
Data arereplicates.
shown as theerror, calculated
independent
replicates,
eachwith eight technicalaverage ± standard error, calculated from four independent biological replicates, each with eighttechnical
2.5.
MBPreplicates.
Inhibited LPS-Induced Pro-Inflammatory Cytokine Production in BV-2 Cells

In orderto investigate
thePro-Inflammatory
potential role ofCytokine
MBP inProduction
the LPS-inducedof
2.5. MBP
Inhibited
LPS-Induced
in BV-2 production
Cells
pro-inflammatory
cytokinesthe(TNF-α,IL-1β,
IL-6),inBV-2
cells were treatedwith 100
In order to investigate
potential
roleandof MBP
the LPS-induced
production
of
and
1000
µg/mL
MBP
with
or
without
1
µg/mL
LPS
for
4
h.
Real-time
PCR
analysispro-inflammatory cytokines (TNF-α, IL-1β, and IL-6), BV-2 cells were treated with100
demonstrated
thatMBPthe level
pro-inflammatory
cytokines
upregulated
and 1000 µg/mL
withoforwithout 1 µg/mL
LPS forwas
4 h.dramatically
Real-time PCR
analysisbyLPS
stimulation,asevidencedbytheincrease
in
the
mRNA
levelsof
TNF-α,
IL-1β, anddemonstrated that the level of pro-inflammatory cytokines was dramatically upregulated
IL-6
comparedto thatasofevidenced
untreated by
cells5). inAstheshown
in Figuretreatment
by LPSstimulation,the(Figure
increasemRNAlevels5,ofMBP
TNF-α,
IL-1β,
significantly
suppressedtheLPS-induced
productionofpro-inflammatory
cytokines.
Adand IL-6 compared to that of untreated cells (Figure 5). As shown in Figure 5, MBP
ditionally,
althoughnostatisticalsignificancewasfound,
themRNAlevelsofpro-inflamtreatment significantly suppressed the LPS-induced production of pro-inflammatory cymatory
pretended
to increasein the MBP-alone-treated
BV-2 cells(Figurelevels
5).
tokines.cytokines
Additionally,
althoughno statisticalsignificance was found,the mRNAof pro-inflammatory cytokines pretended to increase in the MBP-alone-treated BV-2 cells(Figure 5).

Int. J.J. Mol.
Mol. Sci.
Sci. 2023,
2023, 24,
24, x13698
Int.
FOR PEER REVIEW

14
7 7ofof 14

Figurethelipopolysaccharide(LPS)-induced
productionofFigure 5.
5. Miyako
MiyakoBidens
Bidenspilosa
pilosa(MBP)(MBP)inhibited
inhibitedthelipopolysaccharide(LPS)-induced
production
pro-inflammatory
cytokinesin
BV-2
cells.
Cells
incubated
atdiﬀerent
concentrations
of
MBP
with
of pro-inflammatory cytokines in BV-2 cells. Cells incubated at different concentrations of MBP
orwithout
LPS (1LPSµg/mL)
for 4 h.forThe
expression
level oflevelpro-inflammatory
cytokines(tumor (tumor
necrowithor without(1 µg/mL)
4 h.
The expressionof pro-inflammatory
cytokines
sis
factor(TNF)-α,interleukin(IL)-1β,
and
IL-6)
was
analyzedvia
real-timepolymerase
chainreacnecrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) was analyzed via real-time polymerase chain
tion (PCR). The graphs show the relative expression of mRNA of LPS-induced pro-inflammatory
reaction (PCR). The graphs show the relative expression of mRNA of LPS-induced pro-inflammatory
cytokines (TNF-α, IL-1β, and IL-6). Data are shown as the average ± standard error, calculated fromcytokines (TNF-α, IL-1β, and IL-6). Data are shown as the average ± standard error, calculated fromsix independent biological replicates, each with two technical replicates.
six independent biological replicates, each with two technical replicates.

3.
3. Discussion
Discussion
ALS
ALS isis aa progressive
progressive andand lethal
lethal neurodegenerative
neurodegenerative diseasedisease ofof thethe motor
motor neurons
neurons with
with
noeﬀective
treatment
or
cure
to
date.
Therefore,
there
is
a
need
to
createnovel
no effective treatment or cure to date. Therefore, there is a need to create novel diseasediseasemodifying
modifying strategies
strategies toto suppress
suppress diseasedisease progression
progression andand extend
extend thethe lifespanlifespan ofof patientspatients
with
ALS.
Previously,
wereported
that
the
oral
administration
of
MBP
after
the
onset
of
with ALS. Previously, we reported that the oral administration of MBP after the
onset
ALS-like
symptoms
extendedsurvival
duration,
improved
motor
performance,and proof ALS-like
symptoms
extendedsurvival
duration,
improved
motor
performance,andtectedmotor
neurons
throughthe inhibitionof astrocyte
and microglia
activationin G93A
protected
motor
neurons
throughthe inhibitionof astrocyte
and microglia
activationinmice
In[24].
this study,
demonstrated
for the first
timefirst
thattime
MBPthatselectively
reducedG93A[24].
mice
In thiswestudy,
we demonstrated
for theMBP selectively
theproductionof pro-inflammatory
cytokines
throughthroughinhibiting
the polarizationof M1
reducedthe productionof pro-inflammatory
cytokines
inhibiting
the polarization
microglia/macrophages
inthelumbarspinal
cord
of
G93A
mice.
We
also
observed
thatof M1 microglia/macrophages in the lumbar spinal cord of G93A mice. We also observed
exposureof BV-2
cells cells
to MBP
suppressed
LPS-induced
pro-inflammatory
cytokine
prothat exposureof BV-2
to MBP
suppressed
LPS-induced
pro-inflammatory
cytokine
ductionandandcell proliferation
withoutaﬀecting
the numberof EthD-1-positive
dead cells.
production
cell proliferation
without
affectingthe numberof EthD-1-positive
dead
Overall,the results
show
thatthat
MBP
restored
microglia/macrophagecells. Overall,
the resultsshow
MBP
restored
microglia/macrophagebalancebalancenot
not only
only
throughmicroglia/macrophages tothrough inhibiting
inhibiting the polarization of microglia/macrophages
to thethe M1-like
M1-like phenotype
phenotypebut also
also through regulating cell proliferation.
but
Neuroinflammation caused
moststriking
Neuroinflammation
caused bybypro-inflammatory
pro-inflammatorycytokines
cytokinesisisone
oneofofthethemoststrikpathologicalhallmarksof ALS
[25].
InIn
the
microglialing
pathologicalhallmarksof ALS[25].
thelumbar
lumbarspinal
spinalcord
cordofofG93A
G93A mice, microglial
activationwas
wasdetected
detectedatat1515
weeks
(105
days)
of ageolder
whichalso correactivation
weeks
(105
days)
of ageandandolder[26],[26],which
also correlated
lated transcriptionalwith transcriptionalchangesof genes
involved
in inflammation
M1-phenotype
withchangesof genes
involved
in inflammation[27]. [27].
M1-phenotype
mimicroglia/macrophage
producea violent
inflammatory
responsecharacterized
characterizedby
bythe
theproprocroglia/macrophage
producea violent
inflammatory
response
duction of pro-inflammatory
pro-inflammatory cytokines (TNF-α, IL-1β,
IL-1β, andand IL-6)
IL-6) andand increased
increased expression
expression
ductionof
IFN-γR[16,28–30].
On
the
other
hand,
the
M2
microglia/macrophage
phenotype
leadsof IFN-γR [16,28–30]. On the other hand, the M2 microglia/macrophage phenotype leads
tothereleaseofanti-inflammatory
cytokines(TGF-βandIL-10)
andincreased
expressionofto the release of anti-inflammatory cytokines (TGF-β and IL-10) and increased expression
IL-13R[16,28,31].
Ourpresent
study
found
thatthat
bothbothM1- M1and andM2-phenotype
markers
wereof
IL-13R[16,28,31].
Ourpresentstudy
found
M2-phenotype
markersincreasedin theinspinalcord ofcord
G93A
mice atmice
an early
stage (16
weeks
age).
were
increased
the spinalof G93A
at ansymptomatic
early symptomaticstage(16 ofweeks
IFN-γR
andIL-13
receptors
are
not
only
expressedonmicroglia,
but
also
on
B
cells
[32]
of age). IFN-γR and IL-13 receptors are not only expressed on microglia, but also on
Banda
numberofimmune
cells
[33].
In
periods
ofneuroinflammation,
theblood–spinal
cells [32] and a number of immune cells [33]. In periods of neuroinflammation, the blood–barrierbarrier
becomespermeable,facilitating
the entry
peripheral
macrophages,
monocytes,
spinalbecomespermeable,facilitating
the ofentry
of peripheral
macrophages,
monT
cells,
and
B
cells
into
the
spinal
cord
of
both
ALS
mice
and
patients[34,35].
In theseocytes, T cells, and B cells into the spinal cord of both ALS mice and patients [34,35].
In
cases,cases,
microglia
and macrophages
are indistinguishable;
therefore,
we usecollective
thesemicroglia
and macrophages
are indistinguishable;
therefore,
wethe
use
the col-

Int. J. Mol. Sci. 2023, 24, 13698

8 of 14term “microglia/macrophage” in this study. It has been reported that the suppressionof M1-polarized microglia might be beneficial to the protection of motor neurons andprevention of disease progression in G93A mice. Indeed, treatment with minocycline
before the motor dysfunction of G93A mice significantly prolonged survival via the selective inhibition of M1 microglia polarization without affecting the neuroprotective M2
microglia [16]. Moreover, treatment with Hirsutella sinensis suppressed M1 microglial polarization and motor neuron death in the spinal cord of G93A mice [17]. Our results clearly
demonstrated that MBP selectively downregulated M1 microglia/macrophage polarization
without affecting the increased state of M2 microglia/macrophage polarization in G93A
mice even after post-onset administration (Figure 2). Furthermore, it has been reported
that M2 microglia/macrophages from transplanted donor marrow delayed onset motor
dysfunction in G93A mice [36]. These results suggest that the ability of MBP to not only
maintain the neuroprotective M2 microglia/macrophage polarization but also to preventthe neurotoxic M1 microglia/macrophage polarization could be involved in improving the
motor neuron loss and disease progression of G93A mice.
It has been reported that the levels of pro-inflammatory cytokines such as IFN-γ, TNFα, IL-1β, C-C motif chemokine ligand-2 (also known as monocyte chemotactic protein-1),
C-C motif chemokine ligand-5 (also called RANTES), C-X-C motif chemokine ligand 10(also known as interferon γ-induced protein 10), and IL-17A increased with aging anddisease progression in G93A mice, whereas anti-inflammatory cytokines including IL-4,
IL-10, and TGF-β reached peak levels at an early symptomatic stage (90 days of age) and
rapidly decreased until 120 days of age (at the symptomatic stage) [37]. On the other
hand, previous studies have shown that the increased expression of pro-inflammatory
cytokines such as TNF-α, IL-1β, and IL-6 [16,17,38,39] was accompanied by the increased
expression of anti-inflammatory cytokines including TGF-β and IL-10 [16,37] at the symptomatic stage in G93A mice. Consistent with previously reported results, we demonstrated
that the mRNA levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and antiinflammatory cytokines (TGF-β and IL-10) were increased in the spinal cord of G93A miceat an early symptomatic stage (Figure 1). Erythropoietin dramatically decreased the level ofpro-inflammatory cytokines and highly maintained the upregulation of anti-inflammatory
cytokines in the spinal cord of G93A mice until the symptomatic stage [37]. Recombinanthuman erythropoietin has also been reported to prolong the life span and protect the motor
neurons of G93A mice [40]. Moreover, rofecoxib, an anti-inflammatory drug, has beenreported to decrease the expression of TNF-α and IL-1β in the spinal cord and to prolong
survival in G93A mice [41], suggesting that it may be important to reduce pro-inflammatory
cytokines in ALS therapy. Consistent with the effects of erythropoietin and rofecoxib, wefound that MBP selectively suppressed the upregulation of pro-inflammatory cytokines
with no influence on anti-inflammatory cytokine expression in the spinal cord of G93A
mice at an early symptomatic stage (Figure 1). Our findings demonstrate that LPS induced
the overproduction of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) in BV-2 cells(Figure 5). This suggests that LPS-treated BV-2 cells are a cytotoxic M1-type microglia
model rather than an M2-type model. In the present study, MBP inhibited LPS-induced
pro-inflammatory cytokine gene expression in BV-2 cells (Figure 5). Hirsutella sinensis selectively suppressed M1 microglial polarization and M1-related pro-inflammatory cytokine
expression (TNF-α, IL-6, and IL-1β) in the spinal cord of G93A mice [17]. Minocycline also
possessed a selective inhibitory effect on the increase in M1-polarized microglia as well as
the production of pro-inflammatory cytokines (IL-1β, TNF-α, and IFN-γ) in G93A mice [16].
Taken together, these results suggest that the suppression of pro-inflammatory cytokines
in G93A mice may be due to the inhibitory effect of MBP on M1 microglial polarization.
Additionally, we found that MBP tended to possess weak pro-inflammatory effects in BV-2
cells under basal conditions without LPS treatment (Figure 5), but not in the vehicle-treated
WT mice (Figure 1). Future studies are required to determine whether the MBP-induced
activation of pro-inflammatory cytokines contributes to the therapeutic mechanism of MBP
for ALS.

Int. J.J. Mol.
Mol. Sci.
Sci. 2023,
2023, 24,
24, x13698
Int.
FOR PEER REVIEW

14
9 9ofof 14

Generally, an
an increase
increase inin microglial
microglial proliferation
proliferation has
has been
been shown
shown toto be
be associatedassociatedGenerally,withinflammatory
processes
in
ALS
[39].
We
demonstrated
that
theexposureof BV-2
with inflammatory processes in ALS [39]. We demonstrated that the exposure of BV-2
cells
cells to MBP decreased the MTT reduction activity and uptake of BrdU incorporation,
to MBP decreased the MTT reduction activity and uptake of BrdU incorporation, suggestsuggesting that MBP had an inhibitory effect on cell proliferation activity with no impact on
ing that MBP had an inhibitory eﬀect on cell proliferation activity with no impact on thethe cell death of BV-2 cells (Figures 3 and 4). These findings suggest that the inflammationcell death of BV-2 cells (Figures 3 and 4). These findings suggest that the inflammationregulating effect of MBP on pro-inflammatory cytokines might be involved in not only theregulating eﬀect of MBP on pro-inflammatory cytokines might be involved in not only the
inhibition of M1 microglia/macrophage polarization but also the suppression of microglial
inhibition of M1 microglia/macrophage polarization but also the suppression of microglial
proliferation. Moreover, it has been reported that cell cycle inhibitors, including roscovitine
proliferation. Moreover, it has been reported that cell cycle inhibitors, including roscovitand flavopiridol, inhibited microglial proliferation and protected the neuron in animal modine and flavopiridol, inhibited microglial proliferation and protected the neuron in animalels of neuroinflammation-related diseases, such as in a rat model of spinal cord injury [42],models of neuroinflammation-related diseases, such as in a rat model of spinal cord injury
a mouse model of multiple sclerosis [43], and a rat model of status epilepticus [44]. Because[42], a mouse model of multiple sclerosis [43], and a rat model of status epilepticus [44].
BV-2 microglial cell lines differ from primary microglia in morphology and activation state,
BecauseBV-2
microglial
cell lines
diﬀer frommicroglia
morphology
and actiadditional
studiesare required
to confirm
the primary
specificity
of MBP’sineffecton microglia
andvationstate,
additional
studiesarerequiredtoconfirmthespecificity
of
MBP
s
eﬀect
on
macrophages, as well as the mechanism regulating this effect, using more biologically
microglia
and macrophages,
welltheseas themechanism
regulatingeﬀect,
usingofmore
relevant models.
The resultsas
fromprevious
modelsand ourthis
mouse
model
ALS
biologically
relevantmodels.
The
resultsfromtheseprevious
models
and
our
mouse
suggest that the suppression of microglial proliferation might be involved in thedesirable
modelof ALSsuggestof microglial
proliferation might be involved
protective
potentialof that
MBPtheon suppression
motor neurons
in G93A mice.
in theOne
desirable
protective
potential
of
MBP
on
motor
neurons
in G93A
mice. any activeof the limitations of our present study is that we could
not identify
One
of
the
limitationsofourpresent
study
is
that
we
could
not
identify
any active
compound associated with the anti-inflammatory effect of MBP. MBP has been reported
tocompoundassociatedwithchlorogenic
the anti-inflammatoryof MBP. MBPacid
has beenreported tocontain caffeic
acid, someacids sucheffectas neochlorogenic
and chlorogenic
containcaffeicacid,
some chlorogenic
as neochlorogenic
acid and hyperin,
chlorogenic
acid, andvariousflavonoids
including acids
rutin,such
quercetin,
quercetin derivatives,
isoacid,andvariousflavonoids
includingrutin,
quercetin,
quercetin
derivatives,
hyperin,quercitrin, centaurein, and jacein [45,46]. We speculate that the various active compounds
isoquercitrin,
centaurein,jacein[45,46]. We
speculate that the various activeneuroinflamcompounds
in MBP interact
with eachandotherto regulate
microglia/macrophage-mediatedin
MBP
interact
with
each
other
to
regulate
microglia/macrophage-mediatedneuroinflammation in the spinal cord of G93A mice. Although further studies will be necessary tomationthespecificspinal cord
G93A mice. present
Althoughstudies
will be necessary
to idenidentifyinthe
activeofcompounds
in further
MBP, thedevelopmentof pharmacologitify
the specificactivecompoundsassociatedpresent inwith
MBP,
the developmentof pharmacologicalcal agentstargetinginflammation
M1-phenotype
microglia/macrophages
agentstargeting inflammation
associatedM1-phenotype
ususing ethnomedicine
and functional
foodswithcould
be a potentialmicroglia/macrophages
strategy for treating ALS.
ing ethnomedicine
foods
could
be a potential
strategytreating ALS.
In conclusion,andthe functional
therapeutic
effectsof MBPagainst ALS
may forbe mediatedvia antiIn conclusion,
the therapeutic
effectsofofmicroglia/macrophage
MBP against ALS may be
mediated via
inflammation
throughthe suppression
polarization
intoantiM1
inflammation
through the(Figure
suppressionof microglia/macrophage
polarization
into
M1 mimicroglia/macrophages
6). Morestudies are required to
identify themolecular
croglia/macrophages(Figure6). Moreareto identify
targets
targets of these effects
by MBP.
The studies
findings
ofrequired
this study
indicatethe
themolecularpromisingtherapeuticpotential
MBP
forfindings
neurodegenerative
activated
microglia,
ofthese effects
by of
MBP.
The
of this studydiseases
indicate involving
the promisingtherapeuticpoincluding
ALS.for neurodegenerative diseases involving activated microglia, including ALS.
tentialof MBP

Figure 6.
6. A
Aproposed
proposedmechanism
mechanismofoftreatment
treatmentwith
withMiyako
MiyakoBidenspilosa(MBP)
spinalcord
FigureBidenspilosa(MBP)
in inthethespinal
cord
of
G93A
mice.
Red Redarrowsindicateincreaseof cells
proliferation
or upregulationof cytokines.
Redof G93A
mice.
arrowsindicateincreaseof cells
proliferation
or upregulationof cytokines.
triangles
and skyindicate
pro-inflammatory
cytokines
and anti-inflammatory
cytoRed triangles
andbluesky squares
blue squares
indicate
pro-inflammatory
cytokines
and anti-inflammatory
kines
respectively.
cytokines respectively.

Int. J. Mol. Sci. 2023, 24, 13698

10 of 144. Materials and Methods
4.1. Animals
All experimental protocols were performed according to the guidelines for animal
experiments at Nihon University, accredited by the Ministry of Education, Culture, Sports,
Science, and Technology, Japan. Every attempt was made to minimize animal suffering,
reduce the number of animals used, and utilize alternatives to in vivo techniques. Transgenic G93A mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) andmaintained as hemizygotes through mating transgenic males with non-transgenic females.
The offspring were genotyped using a polymerase chain reaction (PCR) with DNA fromvenous blood samples. The non-transgenic littermates were used as the control group (WTmice). We used a total of 20 mice divided into the following four groups: vehicle-treated
WT mice (n = 6), MBP-treated WT mice (n = 5), vehicle-treated G93A mice (n = 4), andMBP-treated G93A mice (n = 5). G93A mice show disease onset at approximately 15 weeks
of age and die approximately 4–5 weeks post-onset [26]. These mice were housed understandard conditions (temperature 22 ◦ C, 12 h light/dark cycle) with free access to foodand water in the animal center at the School of Pharmacy, Nihon University. Animal
experiments were carried out after obtaining approval from the Nihon University Animal
Care and Use Committee (AP19PHA026-2 and 2004YAKU003-4).
4.2. Cell Culture
The immortalized murine microglial cell line, BV-2, was provided by Professor Eui-Ju
Choi (Department of Life Science, Korea University, Seoul, Republic of Korea). Briefly, BV-2
cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Life Technologies Corporation, Carlsbad, CA, USA) containing 1% penicillin–streptomycin solution (100×) (LifeTechnologies Corporation) and 10% fetal bovine serum (Life Technologies Corporation),as described previously [47]. BV-2 cells were seeded at a density of 1.2 × 104 cells/cm2 ina culture dish or plate (Asahi Glass Co., Ltd., Shizuoka, Japan). The cells were routinely
passaged every three days.
4.3. MBP Treatment
MBP (Musashino Miyako BP® ) was provided free of charge from the Musashino
Research Institute for Immunity (Miyako Island, Okinawa, Japan). MBP was dissolved in
injection water (Otsuka, Tokyo, Japan). MBP (2 g/kg/day) or a vehicle was administeredorally once per day for 1 week at 15 weeks of age in WT and G93A mice, as described
previously [24].
4.4. Real-Time PCR
Real-time PCR was performed as described previously [48]. BV-2 cells were treatedwith MBP and then immediately incubated for 4 h with or without 1 µg/mL of LPS (SigmaAldrich, St. Louis, MO, USA). Briefly, total RNA was extracted from BV-2 cells and mice
lumbar spinal cords using the High Pure RNA Isolation Kit (Roche, Basel, Switzerland).
cDNA was synthesized from 0.5 µg of total RNA using reverse transcriptase (Roche). Realtime PCR was performed using TB Green® Premix Ex Taq™ II (TaKaRa, Shiga, Japan).
Relative mRNA expression levels were determined using the ∆∆CT method, normalized
to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers used for real-time
PCR are indicated in Table 1.

Int. J. Mol. Sci. 2023, 24, 13698

11 of 14

Table 1. Primer sequences.
Sequence (50 to 30 )PrimerIFN-γR

IL-13R

TNF-α
IL-1β

IL-6
TGF-β

IL-10

GAPDH

ForwardTGACGGGAGCACCTGTTACAC

ReverseTTTCGACCGTATGTTTCGTATGTAG

ForwardCAGTCTTGCAGCATGGGAACA

ReverseTGAGTCCCTAAGGCCTGGAGATTAC

Forward

AGCCCACGTCGTAGCAAACCAC

ReverseAGGTACAACCCATCGGCTGGCA

ForwardGCAACTGTTCCTGAACTCAACT

ReverseATCTTTTGGGGTCCGTCAACT

ForwardGAGGATACCACTCCCAACAGACC

ReverseAAGTGCATCATCGTTGTTCATACA

ForwardTGACGTCACTGGAGTTGTACGG

ReverseGGTTCATGTCATGGATGGTGC

ForwardGCATGGCCCAGAAATCAAGG

ReverseGAGAAATCGATGACAGCGCC

ForwardTGTGTCCGTCGTGGATCTGA

ReverseTTGCTGTTGAAGTCGCAGGAG

4.5. MTT Reduction Assay and BrdU Incorporation Assay
The proliferation of BV-2 cells was determined by means of a MTT reduction assay
and BrdU incorporation assay. BV-2 cells were treated with MBP in the concentration range
of 2–1000 µg/mL for 24 h.
For the MTT assay, BV-2 cells were incubated with MTT reagent (0.25 mg/mL) for
3 h at 37 ◦ C, and the reaction was quenched using a solution containing 50% dimethyl
formamide and 20% sodium dodecyl sulfate (pH 4.7) as described previously [49]. The
next day, the amount of MTT formazan product in the cells was measured at 570 nm(reference wavelength: 655 nm) with a microplate reader (SH-1000Lab; Corona Electric,
Ibaraki, Japan).
The BrdU incorporation assay was performed using the Cell Proliferation ELISA, BrdU
Kit (Roche), according to the instruction manual. After labelling the BV-2 cells with BrdU
for 24 h, the cells were fixed, and then anti-BrdU-peroxidase was added to the wells to
combine with the incorporated BrdU. Following a wash to remove unbound antibody,
chromogenic substrate and tetramethyl benzidine was added. The stop solution, 1 M
H2 SO4 , was added to each well, and the absorbance at 450 nm (reference wavelength:
690 nm) was measured using a microplate reader (SH-1000Lab; Corona Electric).
4.6. LIVE/DEAD Assay
The amount of viable and dead cells was evaluated with a LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells (Thermo Fisher Scientific, San Diego, CA, USA)using 8-well glass plates (Thermo Fisher Scientific) as described previously [50]. Twentyfour hours after MBP treatment, the BV-2 cells were stained with calcein AM (2 µM) andethidium homodimer-1 (EthD-1; 4 µM) for 40 min. Fluorescence images were collectedwith a confocal laser microscope (LSM-710; Zeiss, Oberkochen, Germany). The numbersof calcein-positive live cells and EthD-1-positive dead cells were counted to calculate cellmortality. The cell mortality value indicates the percentage of EthD-1-positive cells to thesum of calcein-positive live cells and EthD-1-positive dead cells (the total number of cells)
as described previously [50].

Int. J. Mol. Sci. 2023, 24, 13698

12 of 144.7. Statistics
Data analyses were evaluated using one-way repeated measure ANOVA followed by
Tukey’s test using GraphPad PRISM 9 software (GraphPad Software Inc., San Diego, CA, USA).
Results are expressed as the mean ± standard error (SE) or standard deviation (SD).
Author Contributions: Conceptualization, K.T. and Y.K.; methodology, K.T., T.S. (Takato Shidara),
H.N. and Y.N.; validation, K.T., H.M., T.S. (Toyofumi Suzuki) and Y.K.; formal analysis, K.T., H.N.,
H.M. and N.S.; investigation, K.T., T.S. (Takato Shidara), H.N. and N.S.; resources, Y.N. and T.A.; data
curation, K.T., H.M., H.N. and Y.K.; writing—original draft preparation, K.T. and H.M.; writing—review and editing, H.N., T.S. (Toyofumi Suzuki) and Y.K.; visualization, K.T., T.S. (Takato Shidara)and H.M.; supervision, Y.K.; project administration, Y.K.; funding acquisition, H.M. and Y.K. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was funded in part by the Research Foundation for Pharmaceutical Sciences(Y.K.) and Shorei Foundation for Science and Technology (Y.K.), a Nihon University Research Grant
for 2022–2023 (Y.K.), and JSPS KAKENHI Grant Numbers 22K06654 (Y.K.) and 21K06620 (H.M. andY.K.). The funding bodies had no role in the design of the study or in the writing of the manuscript.
Institutional Review Board Statement: All animal procedures were approved by and performed inaccordance with the guidelines of the Nihon University Animal Care and Use Committee (AP19PHA0262 (18 March 2000) and 2004YAKU003-4 (20 August 2004)).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data used to support the findings of this study are available fromthe corresponding author upon request.
Acknowledgments: We thank all the members of our laboratories, especially R. Murata and R.
Shiobara for their excellent technical help.
Conflicts of Interest: Yasuhiro Kosuge had grants from Refine Holdings Co., Ltd., and the otherauthors declare no competing financial interest.

References
1.

2.

3.

4.

5.
6.
7.
8.

9.
10.

Rosen, D.R.; Siddique, T.; Patterson, D.; Figlewicz, D.A.; Sapp, P.; Hentati, A.; Donaldson, D.; Goto, J.; O’Regan, J.P.; Deng, H.X.;
et al. Mutations in Cu/Zn Superoxide Dismutase Gene Are Associated with Familial Amyotrophic Lateral Sclerosis. Nature 1993,
362, 59–62. [CrossRef]
Gurney, M.E.; Pu, H.; Chiu, A.Y.; Dal Canto, M.C.; Polchow, C.Y.; Alexander, D.D.; Caliendo, J.; Hentati, A.; Kwon, Y.W.; Deng,
H.-X.; et al. Motor Neuron Degeneration in Mice That Express a Human Cu, Zn Superoxide Dismutase Mutation. Science 1994,
264, 1772–1775. [CrossRef] [PubMed]
Miana-Mena, F.J.; González-Mingot, C.; Larrodé, P.; Muñoz, M.J.; Oliván, S.; Fuentes-Broto, L.; Martínez-Ballarín, E.; Reiter, R.J.;
Osta, R.; García, J.J. Monitoring Systemic Oxidative Stress in an Animal Model of Amyotrophic Lateral Sclerosis. J. Neurol. 2011,
258, 762–769. [CrossRef] [PubMed]
Kalmar, B.; Novoselov, S.; Gray, A.; Cheetham, M.E.; Margulis, B.; Greensmith, L. Late Stage Treatment with Arimoclomol Delays
Disease Progression and Prevents Protein Aggregation in the SOD1G93A Mouse Model of ALS. J. Neurochem. 2008, 107, 339–350.
[CrossRef]
Cai, M.; Choi, S.-M.; Song, B.K.; Son, I.; Kim, S.; Yang, E.J. Scolopendra Subspinipes Mutilans Attenuates Neuroinflammation in
Symptomatic hSOD1G93A mice. J. Neuroinflamm. 2013, 10, 131.
Lee, J.; Hyeon, S.J.; Im, H.; Ryu, H.; Kim, Y.; Ryu, H. Astrocytes and Microglia as Non-Cell Autonomous Players in the
Pathogenesis of ALS. Exp. Neurobiol. 2016, 25, 233–240. [CrossRef] [PubMed]
Boillée, S.; Yamanaka, K.; Lobsiger, C.S.; Copeland, N.G.; Jenkins, N.A.; Kassiotis, G.; Kollias, G.; Cleveland, D.W. Onset andProgression in Inherited ALS Determined by Motor Neurons and Microglia. Science 2006, 312, 1389–1392. [CrossRef] [PubMed]
Yamanaka, K.; Chun, S.J.; Boillee, S.; Fujimori-Tonou, N.; Yamashita, H.; Gutmann, D.H.; Takahashi, R.; Misawa, H.; Cleveland,
D.W. Astrocytes as Determinants of Disease Progression in Inherited Amyotrophic Lateral Sclerosis. Nat. Neurosci. 2008, 11,
251–253.
Hooten, K.G.; Beers, D.R.; Zhao, W.; Appel, S.H. Protective and Toxic Neuroinflammation in Amyotrophic Lateral Sclerosis.
Neurotherapeutics 2015, 12, 364. [CrossRef]
Turner, M.R.; Cagnin, A.; Turkheimer, F.E.; Miller, C.C.J.; Shaw, C.E.; Brooks, D.J.; Leigh, P.N.; Banati, R.B. Evidence of Widespread
Cerebral Microglial Activation in Amyotrophic Lateral Sclerosis: An [11 C](R)-PK11195 Positron Emission Tomography Study.
Neurobiol. Dis. 2004, 15, 601–609. [CrossRef]

Int. J. Mol. Sci. 2023, 24, 13698

11.
12.

13.
14.

15.

16.

17.

18.

19.
20.
21.
22.
23.
24.

25.
26.
27.
28.
29.

30.

31.

32.
33.
34.

13 of 14

Corcia, P.; Tauber, C.; Vercoullie, J.; Arlicot, N.; Prunier, C.; Praline, J.; Nicolas, G.; Venel, Y.; Hommet, C.; Baulieu, J.L.; et al.
Molecular Imaging of Microglial Activation in Amyotrophic Lateral Sclerosis. PLoS ONE 2012, 7, e52941. [CrossRef] [PubMed]
Henkel, J.S.; Engelhardt, J.I.; Siklós, L.; Simpson, E.P.; Kim, S.H.; Pan, T.; Goodman, J.C.; Siddique, T.; Beers, D.R.; Appel, S.H.
Presence of Dendritic Cells, MCP-1, and Activated Microglia/Macrophages in Amyotrophic Lateral Sclerosis Spinal Cord Tissue.
Ann. Neurol. 2004, 55, 221–235. [CrossRef] [PubMed]
Zhao, W.; Beers, D.R.; Henkel, J.S.; Zhang, W.; Urushitani, M.; Julien, J.P.; Appel, S.H. Extracellular Mutant SOD1 Induces
Microglial-Mediated Motoneuron Injury. Glia 2010, 58, 231–243. [CrossRef] [PubMed]
Xu, X.; Zhang, J.; Li, S.; Al-Nusaif, M.; Zhou, Q.; Chen, S.; Le, W. Bone Marrow Stromal Cell Antigen 2: Is a Potential
Neuroinflammation Biomarker of SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis in Pre-Symptomatic Stage. Front.
Neurosci. 2022, 15, 788730. [CrossRef]
Zhu, S.; Stavrovskaya, I.G.; Drozda, M.; Kim, B.Y.S.; Ona, V.; Li, M.; Sarang, S.; Liu, A.S.; Hartley, D.M.; Wu, D.C.; et al.
Minocycline Inhibits Cytochrome c Release and Delays Progression of Amyotrophic Lateral Sclerosis in Mice. Nature 2002, 417,
74–78. [CrossRef]
Kobayashi, K.; Imagama, S.; Ohgomori, T.; Hirano, K.; Uchimura, K.; Sakamoto, K.; Hirakawa, A.; Takeuchi, H.; Suzumura,A.; Ishiguro, N.; et al. Minocycline Selectively Inhibits M1 Polarization of Microglia. Cell Death Dis. 2013, 4, e525. [CrossRef][PubMed]Shang, H.Y.; Zhang, J.J.; Fu, Z.F.; Liu, Y.F.; Li, S.; Chen, S.; Le, W.D. Therapeutic Effects of Hirsutella Sinensis on the Disease Onset
and Progression of Amyotrophic Lateral Sclerosis in SOD1G93A Transgenic Mouse Model. CNS Neurosci. Ther. 2020, 26, 90–100.
[CrossRef]
Beers, D.R.; Henkel, J.S.; Xiao, Q.; Zhao, W.; Wang, J.; Yen, A.A.; Siklos, L.; McKercher, S.R.; Appel, S.H. Wild-Type Microglia
Extend Survival in PU.1 Knockout Mice with Familial Amyotrophic Lateral Sclerosis. Proc. Natl. Acad. Sci. USA 2006, 103,
16021–16026. [CrossRef] [PubMed]
Tang, Y.; Le, W. Differential Roles of M1 and M2 Microglia in Neurodegenerative Diseases. Mol. Neurobiol. 2016, 53, 1181–1194.
[CrossRef] [PubMed]
Guo, S.; Wang, H.; Yin, Y. Microglia Polarization From M1 to M2 in Neurodegenerative Diseases. Front. Aging Neurosci. 2022,
14, 75. [CrossRef]
Lee, W.C.; Peng, C.C.; Chang, C.H.; Huang, S.H.; Chyau, C.C. Extraction of Antioxidant Components from Bidens pilosa Flowers
and Their Uptake by Human Intestinal Caco-2 Cells. Molecules 2013, 18, 1582–1601. [CrossRef] [PubMed]
Horiuchi, M.; Seyama, Y. Antiinflammatory and Antiallergic Activity of Bidens pilosa L. Var. Radiata SCHERFF. J. Health Sci. 2006,
52, 711–717. [CrossRef]
Horiuchi, M.; Wachi, H.; Seyama, Y. Effects of Bidens pilosa L. var. Radiata Scherff on Experimental Gastric Lesion. J. Nat. Med.
2010, 64, 430–435. [CrossRef] [PubMed]
Kosuge, Y.; Kaneko, E.; Nango, H.; Miyagishi, H.; Ishige, K.; Ito, Y. Bidens pilosa Extract Administered after Symptom Onset
Attenuates Glial Activation, Improves Motor Performance, and Prolongs Survival in a Mouse Model of Amyotrophic Lateral
Sclerosis. Oxid. Med. Cell. Longev. 2020, 2020, 1020673. [CrossRef] [PubMed]
Cai, M.D.; Yang, E.J. Gamisoyo-San Ameliorates Neuroinflammation in the Spinal Cord of hSOD1G93A Transgenic Mice. Mediators
Inflamm. 2018, 2018, 5897817. [CrossRef]
Miyagishi, H.; Kosuge, Y.; Ishige, K.; Ito, Y. Expression of Microsomal Prostaglandin E Synthase-1 in the Spinal Cord in a
Transgenic Mouse Model of Amyotrophic Lateral Sclerosis. J. Pharmacol. Sci. 2012, 118, 225–236. [CrossRef]
Olsen, M.K.; Roberds, S.L.; Ellerbrock, B.R.; Fleck, T.J.; McKinley, D.K.; Gurney, M.E. Disease Mechanisms Revealed by Transcription Profiling in SOD1-G93A Transgenic Mouse Spinal Cord. Ann. Neurol. 2001, 50, 730–740. [CrossRef]
Tada, S.; Okuno, T.; Yasui, T.; Nakatsuji, Y.; Sugimoto, T.; Kikutani, H.; Sakoda, S. Deleterious Effects of Lymphocytes at the Early
Stage of Neurodegeneration in an Animal Model of Amyotrophic Lateral Sclerosis. J. Neuroinflamm. 2011, 8, 19. [CrossRef]
Tada, S.; Okuno, T.; Hitoshi, Y.; Yasui, T.; Honorat, J.A.; Takata, K.; Koda, T.; Shimagami, H.; Chi-Jing, C.; Namba, A.; et al. Partial
Suppression of M1 Microglia by Janus Kinase 2 Inhibitor Does Not Protect against Neurodegeneration in Animal Models of
Amyotrophic Lateral Sclerosis. J. Neuroinflamm. 2014, 11, 179. [CrossRef]
Cunha, C.; Santos, C.; Gomes, C.; Fernandes, A.; Correia, A.M.; Sebastião, A.M.; Vaz, A.R.; Brites, D. Downregulated Glia
Interplay and Increased miRNA-155 as Promising Markers to Track ALS at an Early Stage. Mol. Neurobiol. 2018, 55, 4207–4224.
[CrossRef]
Xu, M.; Wang, J.; Zhang, X.; Yan, T.; Wu, B.; Bi, K.; Jia, Y. Polysaccharide from Schisandra Chinensis Acts via LRP-1 to Reverse
Microglia Activation through Suppression of the NF-κB and MAPK Signaling. J. Ethnopharmacol. 2020, 256, 112798. [CrossRef][PubMed]Valente, G.; Ozmen, L.; Novelli, F.; Geuna, M.; Palestro, G.; Forni, G.; Garotta, G. Distribution of Interferon-γ Receptor in Human
Tissues. Eur. J. Immunol. 1992, 22, 2403–2412. [CrossRef]
Khurana Hershey, G.K. IL-13 Receptors and Signaling Pathways: An Evolving Web. J. Allergy Clin. Immunol. 2003, 111, 677–690.
[CrossRef] [PubMed]
Zhong, Z.; Deane, R.; Ali, Z.; Parisi, M.; Shapovalov, Y.; O’Banion, M.K.; Stojanovic, K.; Sagare, A.; Boillee, S.; Cleveland, D.W.;
et al. ALS-Causing SOD1 Mutants Generate Vascular Changes Prior to Motor Neuron Degeneration. Nat. Neurosci. 2008, 11,
420–422. [CrossRef] [PubMed]

Int. J. Mol. Sci. 2023, 24, 13698

35.
36.

37.
38.
39.

40.

41.
42.
43.
44.
45.
46.

47.

48.

49.
50.

14 of 14

Winkler, E.A.; Sengillo, J.D.; Sullivan, J.S.; Henkel, J.S.; Appel, S.H.; Zlokovic, B.V. Blood–Spinal Cord Barrier Breakdown and
Pericyte Reductions in Amyotrophic Lateral Sclerosis. Acta Neuropathol. 2013, 125, 111. [CrossRef]
Epperly, M.W.; Fisher, R.; Rigatti, L.; Watkins, S.; Zhang, X.; Hou, W.; Shields, D.; Franicola, D.; Bayir, H.; Wang, H.; et al.
Amelioration of Amyotrophic Lateral Sclerosis in SOD1G93A Mice by M2 Microglia from Transplanted Marrow. In Vivo 2019, 33,
675–688. [CrossRef]
Noh, M.Y.; Cho, K.A.; Kim, H.; Kim, S.M.; Kim, S.H. Erythropoietin Modulates the Immune-Inflammatory Response of a
SOD1G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis (ALS). Neurosci. Lett. 2014, 574, 53–58. [CrossRef]
Audet, J.N.; Gowing, G.; Paradis, R.; Soucy, G.; Julien, J.P. Ablation of Proliferating Cells in the CNS Exacerbates Motor Neuron
Disease Caused by Mutant Superoxide Dismutase. PLoS ONE 2012, 7, e34932. [CrossRef]
Chiarotto, G.B.; Cartarozzi, L.P.; Perez, M.; Biscola, N.P.; Spejo, A.B.; Gubert, F.; Franca˛ Junior, M.; Mendez-Otero, R.; De
Oliveira, A.L.R. Tempol Improves Neuroinflammation and Delays Motor Dysfunction in a Mouse Model (SOD1G93A ) of ALS.
J. Neuroinflamm. 2019, 16, 218. [CrossRef]
Koh, S.H.; Kim, Y.; Kim, H.Y.; Won Cho, G.; Kim, K.S.; Kim, S.H. Recombinant Human Erythropoietin Suppresses Symptom Onset
and Progression of G93A-SOD1 Mouse Model of ALS by Preventing Motor Neuron Death and Inflammation. Eur. J. Neurosci.
2007, 25, 1923–1930. [CrossRef]
Zou, Y.H.; Guan, P.P.; Zhang, S.Q.; Guo, Y.S.; Wang, P. Rofecoxib Attenuates the Pathogenesis of Amyotrophic Lateral Sclerosis byAlleviating Cyclooxygenase-2-Mediated Mechanisms. Front. Neurosci. 2020, 14, 817. [CrossRef] [PubMed]
Byrnes, K.R.; Stoica, B.A.; Fricke, S.; Di Giovanni, S.; Faden, A.I. Cell Cycle Activation Contributes to Post-Mitotic Cell Death andSecondary Damage after Spinal Cord Injury. Brain 2007, 130, 2977–2992. [CrossRef] [PubMed]
Mi, G.; Gao, Y.; Liu, S.; Ye, E.; Li, Y.; Jin, X.; Yang, H.; Yang, Z. Cyclin-Dependent Kinase Inhibitor Flavopiridol Promotes
Remyelination in a Cuprizone Induced Demyelination Model. Cell Cycle 2016, 15, 2780–2791. [CrossRef]
Kim, J.E.; Park, H.; Choi, S.H.; Kong, M.J.; Kang, T.C. Roscovitine Attenuates Microglia Activation and Monocyte Infiltration via
P38 MAPK Inhibition in the Rat Frontoparietal Cortex Following Status Epilepticus. Cells 2019, 8, 746. [CrossRef] [PubMed]
Akiko, K.; Yoshiyuki, S.; Eiji, U.; Takanori, K.; Makio, S.; Daisuke, T.; Genjiro, K. Studies on the Antioxidant Active Constituentsof the Dried Powder from Bidens pilosa L. var. radiata SCH. Nat. Med. 2003, 57, 100–104.
Matsumoto, T.; Horiuchi, M.; Kamata, K.; Seyama, Y. Effects of Bidens pilosa L. Var. radiata SCHERFF Treated with Enzyme on
Histamine-Induced Contraction of Guinea Pig Ileum and on Histamine Release from Mast Cells. J. Smooth Muscle Res. 2009, 45,
75–86. [CrossRef]
Nakatani, Y.; Yaguchi, M.; Ogino, K.; Noguchi, R.; Yamamoto, N.; Amano, T. Duloxetine Ameliorates Lipopolysaccharide-Induced
Microglial Activation by Suppressing INOS Expression in BV-2 Microglial Cells. Psychopharmacology 2022, 239, 3133–3143.
[CrossRef]
Kosuge, Y.; Miyagishi, H.; Shinomiya, T.; Nishiyama, K.; Suzuki, S.; Osada, N.; Ishige, K.; Okubo, M.; Kawaguchi, M.; Ito, Y.
Characterization of Motor Neuron Prostaglandin E2 EP3 Receptor Isoform in a Mouse Model of Amyotrophic Lateral Sclerosis.
Biol. Pharm. Bull. 2015, 38, 1964–1968. [CrossRef]
Nango, H.; Kosuge, Y.; Miyagishi, H.; Sugawa, K.; Ito, Y.; Ishige, K. Prostaglandin E2 Facilitates Neurite Outgrowth in a Motor
Neuron-like Cell Line, NSC-34. J. Pharmacol. Sci. 2017, 135, 64–71. [CrossRef]
Kosuge, Y.; Nango, H.; Kasai, H.; Yanagi, T.; Mawatari, T.; Nishiyama, K.; Miyagishi, H.; Ishige, K.; Ito, Y. Generation of Cellular
Reactive Oxygen Species by Activation of the EP2 Receptor Contributes to Prostaglandin E2-Induced Cytotoxicity in Motor
Neuron-Like NSC-34 Cells. Oxid. Med. Cell. Longev. 2020, 2020, 6101838. [CrossRef]

Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury topeople or property resulting from any ideas, methods, instructions or products referred to in the content.