Journal of Pharmacological Sciences 152 (2023) 61e67

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Peptides obtained by enzymatic decomposition of mackerel induce
recovery from physical fatigue by enhancing the SIRT1-mediatedantioxidant effect in the soleus muscle of mice
Osamu Nakagawasai a, *, Kohei Takahashi a, b, Wakana Sakuma a, Wataru Nemoto a,
Ruka Kobayashi a, Tomohiro Hoshi a, Satoshi Matsumoto c, Takeshi Tadano a, d,Koichi Tan-No aaDivision of Pharmacology, Faculty of Pharmaceutical Sciences, Tohoku Medical and Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai,
Miyagi 981-8558, Japanb
Department of Pharmacology, School of Pharmacy, International University of Health and Welfare, Ohtawara, Tochigi 324-8501, Japanc
LS Corporation Co. Ltd., 13-4 Kodenmacho-Nihonbashi, Chuo-ku, Tokyo 103-0001, JapandComplementary and Alternative Medicine Clinical Research and Development, Graduate School of Medicine Sciences, Kanazawa University, 13-1
Takaramachi, Kanazawa, Ishikawa 920-8640, Japana r t i c l e i n f o

a b s t r a c t

Article history:
Received 12 October 2022
Received in revised form
14 February 2023
Accepted 13 March 2023
Available online 15 March 2023

Fatigue is a serious health problem, and long-term fatigue can lead to mental illnesses and accelerated
aging. Oxidative stress, which causes excessive production of reactive oxygen species, is generally
thought to increase during exercise and is an indicator of fatigue. Peptides obtained by enzymatic
decomposition of mackerel (EMP) contain selenoneine, a strong antioxidant. Although antioxidants increase endurance, the effects of EMP on physical fatigue are unknown. The present study aimed to clarify
this aspect. We investigated the effects of EMP on changes in locomotor activity, expression levels ofsilent mating type information regulation 2 homolog peroxisome 1 (SIRT1), proliferator-activated receptor-g coactivator-1a (PGC1a), and antioxidative-related proteins including superoxide dismutase 1(SOD1), SOD2, glutathione peroxidase 1, and catalase in the soleus muscle following EMP treatment
before and/or after forced walking. Treatment with EMP before and after forced walking, and not only at
one or another time point, improved the subsequent decrease in the locomotor activity and enhanced thelevels of SIRT1, PGC1a, SOD1, and catalase expression in the soleus muscle of mice. Moreover, EX-527, a
SIRT1 inhibitor, abolished these effects of EMP. Thus, we suggest that EMP combats fatigue by modulating
the SIRT1/PGC1a/SOD1-catalase pathway.
© 2023 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese PharmacologicalSociety. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Keywords:
Antioxidant
Anti-Fatigue effect
SIRT1

1. Introduction

Abbreviations: AMPK, adenosine monophosphate-activated protein kinase;
ANOVA, analysis of variance; EMP, Peptides obtained by enzymatic decompositionof mackerel; FOXO, forkhead box O; FW, forced walking; GPx1, glutathione
peroxidase 1; i.p., intraperitoneally; LH, liver hydrolysate; p, phospho; p.o., per os;
PGC1a, peroxisome proliferator-activated receptor-g coactivator-1a; ROS, reactive
oxygen species; SOD, superoxide dismutase; SIRT1, silent mating type information
regulation 2 homolog 1; t, total.
* Corresponding author.
E-mail address: osamun@tohoku-mpu.ac.jp (O. Nakagawasai).
Peer review under responsibility of Japanese Pharmacological Society.

Fatigue has become a serious health concern that requires urgent attention. It is deﬁned as the inability in sustaining or initiating voluntary activities, resulting from hard physical or mental
work and severe stress.1,2 Long-term fatigue increases the risk ofpsychiatric disorders, Parkinson's disease, and cancer and promotes
aging.3 Fatigue is a complex and encompassing physiological phenomenon without clear etiology and may require long-term
medication. Anti-fatigue drugs, such as modaﬁnil and caffeine,
often have several deleterious effects.4 Thus, it is important to ﬁnd
novel anti-fatigue agents or formulations with clear efﬁcacy andfewer side effects.

https://doi.org/10.1016/j.jphs.2023.03.001
1347-8613/© 2023 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

O. Nakagawasai, K. Takahashi, W. Sakuma et al.

Journal of Pharmacological Sciences 152 (2023) 61e67

administered before and/or after forced walking. Following forced
walking for 3 h, mice were individually placed in a multichannel
Supermex activity box (Muromachi Kikai, Co., Tokyo) and allowed
to adapt for 15 min before injection of EMP. The locomotor activity
was measured every 15 min for 90 min. The protocol for themeasurement of locomotor activity has been described previously.19e24 This instrument can monitor movements per minutein all three planes of motion (sagittal, coronal, and horizontal) as
one movement, owing to its infrared sensor with multiple Fresnel
lenses that can be moved close enough to the cage to capture
multidirectional locomotor alterations. The Supermex instrument
was connected to a behavioral analyzing system (CompACT AMS,
Muromachi Kikai), which could interpret each movement as one
count. Therefore, vertical movements such as jumping, as well as
horizontal movements such as walking and running, were counted.
Measurements were performed between 12:00 h and 16:00 h,
during the light phase.

Physical exercise-induced fatigue is associated with the production of reactive oxygen species (ROS). Antioxidants delay orpromote recovery from fatigue by preventing the accumulation of
ROS.5 Furthermore, activated silent mating type information
regulation 2 homolog 1 (SIRT1) increases the levels of antioxidantrelated proteins like glutathione peroxidase 1 (GPx1), superoxide
dismutase (SOD) 1, SOD2, and catalase by enhancing peroxisome
proliferator-activated receptor-g coactivator-1a (PGC1a), resulting
in anti-fatigue effects.6e8 These ﬁndings suggest that components
with antioxidant properties exert anti-fatigue effects.
Selenoneine, the selenium isolog of the natural antioxidant
ergothioneine, has strong antioxidative effects.9,10 Moreover, theantioxidant effects of ergothioneine are associated with SIRT1
activation.11 Peptides obtained by enzymatic decomposition ofmackerel (EMP) are abundant in selenoneine. However, it is unknown whether EMP has an anti-fatigue effect.
Here, we examined the effects of EMP on fatigue in mice, byperforming the forced walking test, which is useful for evaluating
physical fatigue.12 Furthermore, we investigated the underlying
molecular mechanisms of this effect.

2.4. Western immunoblotting study
Twenty-four mice were divided into four groups [forced walking
3 h ()/water, forced walking 3 h ()/EMP (250 mg/kg), forced
walking 3 h (þ)/water, and forced walking 3 h (þ)/EMP (250 mg/kg)] or two groups [vehicle/forced walking 3 h (þ)/EMP (250 mg/kg) and EX-527/forced walking 3 h (þ)/EMP (250 mg/kg)]. Micethat had not undergone any behavioral tests were sacriﬁced bydecapitation 15 min after the last administration of the vehicle or
EMP. Protein extraction from soleus muscle and Western blot
analysis were performed as previously described.17,25e28 Afterelectrophoresis, proteins were transferred to polyvinylidene
diﬂuoride membranes and incubated with blocking solution[10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 0.01% Tween 20, and 5%
skim milk]. The membranes were treated overnight at 4  C with
antibodies against phospho-(p-) SIRT1 (1:1000; Bioss Antibodies,
Boston, USA, bs-3393R), total (t) -SIRT1 (1:10000; Millipore, Burlington, USA, #07-131), p-adenosine monophosphate-activatedprotein kinase (AMPK; 1:1000; Cell Signaling Technology, Danvers, USA, #2531), t-AMPK (1:1000; Cell Signaling Technology,
Danvers, USA, #2532), PGC1a (1:2000; Abcam Ltd., Cambridge, UK,
ab54481), SOD1 (1:5000; Abcam, ab13498), SOD2 (1:5000; Abcam,
ab13533), GPx1 (1:000; Abcam, ab22604), catalase (1:2000;
Abcam, ab16731) and b-actin (1:1000; Cell Signaling Technology,#4967). The membranes were then washed with blocking solution
without 5% skim milk, incubated with horseradish peroxidaseconjugated secondary antibody [1:5000 except for SOD1
(1:2000), SOD2 (1:20000), and GPx1 (1:50000); Cell Signaling
Technology, #7074)] for 2 h, and visualized using an enhanced
chemiluminescence western blotting detection reagent (Amersham Life Science, Amersham, UK). The band density was measured
using densitometry (Image-J 1.43m, National Institute of Health).

2. Materials and methods
All experiments were performed with the approval of the Ethics
Committee of Animal Experiments in Tohoku Medical and Pharmaceutical University (approval numbers: 20051-cn, 21035-cn, and22061-cn) and according to the provisions of the National Institutesof Health Guide for the Care and Use of Laboratory Animals. Efforts
were made to minimize suffering and reduce the number of usedanimals.
2.1. Animals
We used male ddY mice (age: 6e7 weeks, weight: 26e28 g;
Japan SLC, Shizuoka, Japan) for all experiments (total: n ¼ 221;
behavioral test: n ¼ 197; western blotting analysis: n ¼ 24). The
mice were housed 5e6 per cage, under controlled environmental
conditions (23 ± 1  C, 55 ± 5%, 12/12 h light-dark cycle with lights
on at 7:00 a.m.), and provided free access to food or water. The
behavioral tests were performed between 09:00 h and 17:00 h. All
mice were earmarked for individual identiﬁcation and randomly
divided into four groups using a table of random numbers.
2.2. Compounds
EMP, supplied by LS Corporation Co. Ltd., Tokyo, was dissolved indrinking water, and doses of 100, 200, 250, and 500 mg/kg wereadministered per os (p.o.) in a volume of 0.1 mL/10 g mouse bodyweight using a 1-mL syringe with an oral probe. EX-527 (Tokyo
Chemical Industry Co., Ltd., Tokyo, Japan), a SIRT1 inhibitor, wasdissolved in 0.5% Tween 80 and a dose of 10 mg/kg was administered intraperitoneally (i.p.) 1 h before the ﬁrst administration of
EMP. The dose for EX-527 used was chosen based on a previous
report.13 EX-527 is a potent and highly selective SIRT1 inhibitor andexhibits much lower or no inhibitory activity against other
SIRTs.14e16

2.5. Statistical analysis
The results of experiments are expressed as mean ± standard
error of the mean. The statistical signiﬁcance of differences wasdetermined using Student's t-test for two-group comparisons. The
statistical signiﬁcance of the differences was determined using
one- or two-way analysis of variance (ANOVA), followed by the
TukeyeKramer post hoc test for multiple group comparisons using
GraphPad Prism 7 (GraphPad Software, California, USA). The criterion of signiﬁcance was set at p < 0.05. In some cases, when amain effect was signiﬁcant without an interaction effect, an
exploratory and limited pairwise post hoc comparison consistent
with our a priori hypothesis was performed. All statistical analyses

2.3. Forced walking test
The protocol for forced walking has been described previously.17,18 Brieﬂy, forced walking was imposed on 10 mice in a cylindrical cage (diameter: 37 cm) at 23 ± 1  C, which was rotated on
the horizontal axis at 2.0 rpm by an electric motor, giving a walking
speed of 2.3 m/min for 3 h. Control mice (n ¼ 10) were placed in acylindrical cage without rotation. EMP was dissolved in water and62

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Journal of Pharmacological Sciences 152 (2023) 61e67

As shown in Fig. 2, EMP administration signiﬁcantly increased
the phosphorylation level of SIRT1 folded by b-actin and the level oft-SIRT1 expression in the soleus muscle compared to the vehicle
treatment in the forced walking groups [Fig. 2 (C and D)], whilephosphorylation level of SIRT1 folded by t-SIRT1 was unchanged
among all groups [Fig. 2 (B)].

were performed by investigators other than the experimenters to
avoid bias and to ensure blinding.
3. Results
3.1. EMP showed an anti-fatigue effect in the forced walking test
Mice showed signiﬁcantly reduced locomotor activity after theforced walking test, while treatment with EMP before and after theforced walking test improved this change [One-way ANOVA, F (4,42) ¼ 10.65, p < 0.0001, Fig. 1 (D)]. However, this effect was notobserved when administered either before or after the forced
walking test [One-way ANOVA, F (2, 27) ¼ 7.715, p ¼ 0.0022, Fig. 1(B); and F (3, 66) ¼ 7.677, p ¼ 0.0002, Fig. 1 (C)].

3.3. EMP enhanced PGC1a, SOD1, catalase expression levels in thesoleus muscle
The two-way ANOVA analysis revealed a signiﬁcant interaction
between group and treatment in Fig. 3 (B and F), while these resultsin Fig. 3 (C, D, and E) were unchanged about interaction effect[group: F (1, 20) ¼ 3.783, p ¼ 0.0660, treatment: F (1, 20) ¼ 6.941,
p ¼ 0.0159, group  treatment: F (1, 20) ¼ 4.841, p ¼ 0.0397, Fig. 3(B); group: F (1, 20) ¼ 0.0002503, p ¼ 0.9875, treatment: F (1,20) ¼ 18.88, p ¼ 0.0003, group  treatment: F (1, 20) ¼ 0.3417,
p ¼ 0.5654, Fig. 3 (C); group: F (1, 20) ¼ 0.2696, p ¼ 0.6093,
treatment: F (1, 20) ¼ 1.013, p ¼ 0.3262, group  treatment: F (1,20) ¼ 0.1131, p ¼ 0.7401, Fig. 3 (D); group: F (1, 20) ¼ 0.05706,
p ¼ 0.8136, treatment: F (1, 20) ¼ 7.529, p ¼ 0.0125,
group  treatment: F (1, 20) ¼ 0.679, p ¼ 0.4197, Fig. 3 (E); andgroup: F (1, 20) ¼ 2.947, p ¼ 0.1015, treatment: F (1, 20) ¼ 2.49,
p ¼ 0.1302, group  treatment: F (1, 20) ¼ 5.521, p ¼ 0.0292, Fig. 3(F)]. Thus, we focused on the effects of EMP in mice in Fig. 3 (C, D,
and E).
As shown in Fig. 3, the expression level of PGC1a in the soleus
muscle was reduced by forced swimming, while EMP treatment

3.2. EMP can activate SIRT1 in the soleus muscle
The two-way ANOVA revealed a signiﬁcant interaction between
group and treatment, as shown in Fig. 2 (B and C), while this result,as shown in Fig. 2 (D), was unchanged for the interaction effect[group: F (1, 19) ¼ 0.2367, p ¼ 0.6322, treatment: F (1, 19) ¼ 1.056,
p ¼ 0.3171, group  treatment: F (1, 19) ¼ 8.244, p ¼ 0.0098, Fig. 2(B); group: F (1, 19) ¼ 1.189, p ¼ 0.2891, treatment: F (1, 19) ¼ 3.396,
p ¼ 0.0810, group  treatment: F (1, 19) ¼ 5.037, p ¼ 0.0369, Fig. 2(C); group: F (1, 19) ¼ 0.3307, p ¼ 0.5720, treatment: F (1,19) ¼ 11.65, p ¼ 0.0029, group  treatment: F (1, 19) ¼ 2.317,
p ¼ 0.1445, Fig. 2 (D)]. Thus, we focused on the effects of EMP in
mice, as shown in Fig. 2 (D).

Fig. 1. Effect of EMP on locomotor activity after forced walking for 3 h. (A) Timeline of each experimental protocol. (BeD) Locomotor activity. Numbers in square brackets indicate
the number of animals in each group. Bars represent mean ± standard error mean. **: p < 0.01 vs. control group. ##: p < 0.01 vs. vehicle-treated mouse group.
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Journal of Pharmacological Sciences 152 (2023) 61e67

Fig. 2. Effect of EMP administration on the levels of p-SIRT1/t-SIRT1 (B), p-SIRT1/b-actin (C), and t-SIRT1/b-actin (D) in the soleus muscle after forced walking (FW). A: Representative immunoblots probed with antibodies against p-SIRT1, t-SIRT1, and b-actin, as indicated. Quantiﬁcation of the normalized values of p-SIRT1 (C) and t-SIRT1 (D) levels withb-actin and p-SIRT1 (B) with t-SIRT1, respectively. Numbers in square brackets indicate the number of animals in each group. Bars represent means ± standard error mean. #:p < 0.05 vs. control with the forced walking group.

Fig. 3. Effect of EMP administration on the levels of PGC1a (B), SOD1 (C), SOD2 (D), GPx1 (E), and catalase (F) in the soleus muscle after forced walking (FW). A: Representative
immunoblots probed with antibodies against PGC1a, SOD1, SOD2, GPx1, catalase, and b-actin, as indicated. Quantiﬁcation of values for PGC1a, SOD1, SOD2, GPx1, and catalasenormalized with b-actin levels in the soleus muscle. Numbers in square brackets indicate the number of animals in each group. Bars represent means ± standard error mean. *:p < 0.05 vs. control without forced walking group. #: p < 0.05 vs. control with the forced walking group.

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Journal of Pharmacological Sciences 152 (2023) 61e67

at one or another timepoint, improved the forced walkinginduced decrease in locomotor activity (Fig. 1D), suggesting thatthe effect of EMP on forced walking-induced fatigue behavior isrestorative rather than preventive in mice. EMP contains selenoneine, which has strong antioxidative activity. Thus, we hypothesized that the anti-fatigue effect of EMP may be associated with
its antioxidative activity.
Activation of SIRT1 enhances PGC1a activity and improvesmuscle fatigue.6,7 PGC1a is required for the induction of many ROSclearing enzymes, including SOD1, SOD2, GPx1, and catalase.8 In thepresent study, the level of t-SIRT1 expression increased signiﬁcantly when EMP was administered to the forced walking group(Fig. 2D), and no signiﬁcant change was observed when p-SIRT1
was corrected for t-SIRT1 (Fig. 2B). Therefore, the increase in pSIRT1 corrected for b-actin when EMP was administered to theforced walking group (Fig. 2C), was likely due to the increase in tSIRT1. Moreover, we found that in the forced walking groups,
compared with the vehicle, EMP signiﬁcantly increased PGC1a, andcatalase in the soleus muscle expression levels, slightly elevated
SOD1 expression levels, and did not interfere with SOD2 and PGx1
expression levels (Fig. 3). However, in the groups not subjected toforced walking, compared with vehicle, EMP enhanced SOD1 andGPx1 levels in the soleus muscle, whereas it did not inﬂuence
PGC1a level [Fig. 3 (C) and (E)]. In the groups not subjected toforced walking, compared with vehicle, EMP enhanced the activation of AMPK in the soleus muscle, while in those subjected toforced walking, no differences were observed in terms of AMPK
activation (Supplementary Figure S1B). Activation of AMPK enhances SOD1 and GPx1 levels by increasing Forkhead box O (FOXO)
3a activity.30 Thus, we consider that in mice not subjected to forced
walking, EMP treatment increased antioxidant defense by upregulating the AMPK/SOD1-GPx1 pathway. Moreover, we found thatin mice, EX-527 abolished the EMP-induced anti-fatigue effect(Fig. 4B) and enhanced the levels of t-SIRT1, PGC1a, SOD1, andcatalase expression in the soleus muscle (Fig. 5). These results
indicate that in mice subjected to forced walking, EMP enhanced
antioxidant defense via activation of the SIRT1/PGC1a/SOD1catalase pathway in the soleus muscle (Fig. 6).
The anti-fatigue effect of EMP was only observed when it wasadministered before and after forced walking. We have previously
reported that liver hydrolysate (LH) exerts an anti-fatigue effectwhen administered twice before and after forced walking,17 aschedule similar to that used in this study. As LH also has antioxidant properties,31 anti-fatigue substances with anti-oxidant effects may be more effective when administered before and after
forced walking.

improved this change [Fig. 3 (B)]. Moreover, EMP slightly elevated
SOD1 (p ¼ 0.0663) expression, and did not change the levels of
SOD2 and GPx1 expression in the soleus muscle compared to thevehicle treatment in the forced walking groups. In the groups not
subjected to forced walking, EMP enhanced SOD1 and GPx1 levelsin the soleus muscle compared to the vehicle administration group,
while it did not affect PGC1a expression level [Fig. 3 (C and E)].
3.4. EX-527 inhibited the EMP-induced anti-fatigue effect
To determine whether activation of SIRT1 was involved in the
EMP-induced anti-fatigue effect in mice, we observed the changes
in locomotor activity at the time of co-administration of the SIRT1
inhibitor EX-527 and EMP. The EMP-induced anti-fatigue effect inmice was attenuated by treatment with EX-527 [Two-way ANOVA,
treatment: F (1, 46) ¼ 4.19, p ¼ 0.0464, inhibitor: F (1, 46) ¼ 6.598,
p ¼ 0.0135, treatment  inhibitor: F (1, 46) ¼ 5.883, p ¼ 0.0193,
Fig. 4 (B)].
3.5. EX-527 prevented EMP-induced enhanced levels of SIRT1,
PGC1a, SOD1, and catalase expression in the soleus muscle
We investigated whether administration of EX-527 inhibited
EMP-induced enhancement in the levels of SIRT1, PGC1a, SOD1, andcatalase in the soleus muscle. Treatment with EX-527 attenuatedthe levels of SIRT1, PGC1a, SOD1, and catalase expression in thesoleus muscle compared to the vehicle group after forced walking[Student's t-test: t ¼ 3.513, df ¼ 10, p ¼ 0.0056, Fig. 5 (A); t ¼ 3.664,
df ¼ 10, p ¼ 0.0044, Fig. 5 (B); t ¼ 4.418, df ¼ 10, p ¼ 0.0013, Fig. 5(C); t ¼ 9.547, df ¼ 10, p < 0.0001, Fig. 5 (D)].
4. Discussion
In this study, EMP showed recovery of the forced walkinginduced fatigue-like behavior in mice following EMP treatment
before and after forced walking. EMP signiﬁcantly increased thelevels of SIRT1, PGC1a, SOD1, and catalase expression in the soleus
muscle of mice after forced walking. These effects of EMP were
inhibited by the SIRT1 inhibitor, EX-527. This shows that EMP may
facilitate recovery from physical fatigue and this effect may involvean increase in the antioxidative-related proteins via the activationof the SIRT1-PGC1a pathway.
Oxidative stress causes the overproduction of ROS which
generally increases during physical exercise.29 Antioxidants might
delay fatigue through the removal of ROS.5 The present study
showed that administration of EMP before and after, and not only

Fig. 4. Effect of EX-527 on EMP-induced anti-fatigue effect in mice. A: Time course of each experimental protocol. Numbers in square brackets indicate the number of animals ineach group. Bars represent mean ± standard error mean. ##: p < 0.01 vs. water with the forced walking group. $$: p < 0.01 vs. EMP with the forced walking group.
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Journal of Pharmacological Sciences 152 (2023) 61e67

Fig. 5. Effect of EX-527 on EMP-induced enhanced levels of SIRT1, PGC1a, SOD1, and catalase expression in the soleus muscle. Quantiﬁcation of values for SIRT1 (A), PGC1a (B), SOD1
(C), and catalase (D) normalized with b-actin levels in the soleus muscle. Numbers in square brackets indicate the number of animals in each group. Bars represent mean ± standard
error mean. $$: p < 0.01 vs. EMP with the forced walking group.

Fig. 6. Schematic representation of anti-fatigue effects of EMP.

In conclusion, the present study demonstrated that EMP
improved physical endurance concomitantly reducing oxidative
stress. Thus, it could be used as a pharmacologic tool for recovery
from fatigue.

In the present study, we determined the effects of EMP on forced
walking-induced fatigue-like behavior. However, it remains unclear
what components in EMPs are responsible for their anti-fatigue
effects in mice. There were no signiﬁcant differences in SIRT1 andcatalase levels in the soleus muscle between the control and forced
walking groups. This result indicates that SIRT1 and catalase in thesoleus muscle are not affected by the fatigue caused by forced
walking for 3 h. However, other researchers have reported thatmoderate exercise (for 2e6 weeks on a treadmill) activated SIRT1
and induced increased catalase expression.32 As the organism responds differently to acute exercise and repeated chronic exercise
regimens, this difference can be explained. The increase in SIRT1
and catalase levels in the soleus muscle following EMP administration indicates that they may be involved in the anti-fatigue effects of EMP. These questions need to be addressed in futurestudies.

Credit authorship contribution statement
Osamu Nakagawasai: Conceptualization, Methodology, Writing- original draft, Project administration, Funding acquisition. Kohei
Takahashi: Formal analysis, Writing - original draft, Visualization,
Funding acquisition. Wakana Sakuma: Formal analysis, Investigation, Data Curation. Wataru Nemoto: Investigation, Data Curation.
Ruka Kobayashi: Investigation. Tomohiro Hoshi: Investigation.
Satoshi Matsumoto: Supervision, Resources. Takeshi Tadano: Supervision. Koichi Tan-No: Supervision.
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Journal of Pharmacological Sciences 152 (2023) 61e67

Funding

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This work was funded by grants 21K15351 and 22K06866 fromthe JSPS KAKENHI and S1511001L from the Ministry of Education,
Culture, Sports, Science and Technology of Japan.
Data availability statement
All data generated or analyzed during this study are included inthis published article (and its supplementary information ﬁles).
Declaration of competing interest
Satoshi Matsumoto is employee of LS Corporation Co. Ltd.
Acknowledgments
The authors would like to thank Ms. Sena Hayasaka, Ms. Yuko
Oshima, and Mr. Kazuaki Shoji of Tohoku Medical and Pharmaceutical University for their technical assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online athttps://doi.org/10.1016/j.jphs.2023.03.001.
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