Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Brain, Behavior, and Immunity

journal homepage: www.elsevier.com/locate/ybrbi

Acupuncture inhibits neuroinflammation and gut microbial dysbiosis in a

mouse model of Parkinson’s disease
Jae-Hwan Janga,1, Mi-Jung Yeoma,1, Sora Ahna, Ju-Young Oha, Suhwan Jib, Tae-Hong Kima,
⁎
Hi-Joon Parka,b,
a
Integrative Parkinson’s Disease Research Group, Acupuncture & Meridian Science Research Center, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemoon-gu, Seoul
02447, Republic of Korea
b
Department of Korean Medical Science, Graduate School of Korean Medicine, Kyung Hee University, 26 Kyungheedae-ro, Dongdaemoon-gu, Seoul 02447, Republic of
Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Growing evidences show that gut microbiota is associated with the pathogenesis of Parkinson’s disease (PD) and
Acupuncture the gut-brain axis can be promising target for the development of the therapeutic strategies for PD. Acupuncture
Parkinson’s disease has been used to improve brain functions and inflammation in neurological disorders such as PD, and to recover
Gut microbial dysbiosis the gastrointestinal dysfunctions in various gastrointestinal disorders. Thus, we investigated whether acu-
Neuroinflammation
puncture could improve Parkinsonism and gut microbial dysbiosis induced by 1-methyl-4-phenyl-1,2,3,6-tet-
Microbiome-gut-brain axis
rahydropyridine. First, we observed that acupuncture treatment at acupoints GB34 and ST36 could improve
motor functions and comorbid anxiety in PD mice. Next, we found that acupuncture increased the levels of
dopaminergic fibers and neurons in the striatum and the substantia nigra, respectively. Acupuncture also re-
stored the overexpression of microglia and astrocyte as well as conversion of Bax and Bcl-2 expression in both
the striatum and the substantia nigra, indicating that inflammatory responses and apoptosis were blocked by
acupuncture. Additionally, via 16S rRNA sequence analysis, we observed that the relative abundance of 18
genera were changed in acupuncture-treated mice compared to the PD mice. Of them, Butyricimonas,
Holdemania, Frisingicoccus, Gracilibacter, Phocea, and Aestuariispira showed significant correlations with anxiety
as well as motor functions. Furthermore, the predicted functional analyses showed that acupuncture restored the
physiology functions such as glutathione metabolism, methane metabolism, and PD pathway. In conclusion, we
suggest that the effects of acupuncture on the enhanced motor function and the protection of the dopaminergic
neurons may be associated with the regulation of the gut microbial dysbiosis and thus the inhibition of the
neuroinflammation in the PD mice.

1. Introduction
Parkinson's disease (PD) is the second most common neurodegen-
erative disease after Alzheimer's disease. Nearly one percent of the
population over 60 worldwide have PD (Tysnes and Storstein, 2017).
Pathological hallmarks of PD include the aggregation of α-synuclein in
the central nervous system (CNS) and the loss of dopaminergic fibers
and neurons in the striatum (ST) and the substantia nigra (SN) along
with high levels of neuroinflammation (Dickson, 2012; Hartmann,
2004), resulting in motor dysfunctions such as tremor, rigidity, postural
instability, and bradykinesia (Jankovic, 2008). Recent study reported
that prodromal PD with non-motor symptoms was associated with gut-
brain axis and gut microbiome, which may play important role in PD
(Edwards et al., 1991; Felice et al., 2016).
Gut-brain axis indicates bidirectional communications between the
gut and the brain (Ghaisas et al., 2016; Mulak and Bonaz, 2015). Im-
balance of the gut-brain axis by gut microbial dysbiosis can lead to
increased inflammatory signaling and increases epithelial permeability,
which are recognized to play a role in the neurodegenerative disorders.
Gut microbial dysbiosis related to the PD pathology has been increas-
ingly found in both clinical and preclinical PD studies (Keshavarzian
et al., 2015; Lai et al., 2018; Sampson et al., 2016; Scheperjans et al.,
2015). The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP) is widely used in animal studies for PD model, lead to increased

⁎
Corresponding author at: Acupuncture and Meridian Science Research Center (AMSRC), College of Korean Medicine, Kyung Hee University, 26 Kyungheedae-ro,
Dongdaemun-gu, Seoul 02447, Republic of Korea.
E-mail address: acufind@khu.ac.kr (H.-J. Park).
1
First two authors contributed equally to this article.

https://doi.org/10.1016/j.bbi.2020.08.015
Received 30 April 2020; Received in revised form 4 August 2020; Accepted 17 August 2020
0889-1591/ © 2020 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Jae-Hwan Jang, et al., Brain, Behavior, and Immunity, https://doi.org/10.1016/j.bbi.2020.08.015
J.-H. Jang, et al.
α-synuclein, and induces a change in gut microbial composition in-
cluding phylum Proteobacteria (Lai et al., 2018). Particularly, recent
study found that altered microbiome induces microglial activation and
neuroinflammation, which may promote motor dysfunction in the α-
synuclein overexpressing mice (Sampson et al., 2016).
Acupuncture has been used as the major therapeutic modalities in
East Asian medicine, and gains wide attention worldwide recently
(Chon and Lee, 2013). The functional connectivity and communication
among acupoints, the brain (ancient heart) and the gut has been an
important consideration in acupuncture clinic (Hur, 1991). It has been
reported that acupuncture can regulate the immune system possibly
through the vagus nerve, which is important for the gut-microbiome-
brain axis communication (Torres-Rosas et al., 2014). In the recent
studies, acupuncture relieves abdominal obese by modulating meta-
bolic balance and gut microbial composition in diet-induced model
(Wang et al., 2019), and regulates inflammatory cytokine and gut mi-
crobial dysbiosis in a trinitrobenzene sulfonic acid-induced irritable
bowel syndrome model (Song et al., 2020). Another study reported that
acupuncture might be beneficial for irritable bowel syndrome through
modulation of the motility, visceral sensation, and/or gut-brain inter-
actions (Chey et al., 2011). In our previous studies, we showed that
acupuncture has neuroprotective, anti-neuroinflammatory and anti-
apoptotic effects in PD mice models (Jeon et al., 2008; Kim et al.,
2011a,b; Park et al., 2003; Park et al., 2015; Park et al., 2017). How-
ever, the role of gut microbial function on the effects of acupuncture in
PD is still unknown.
Thus, we hypothesized that acupuncture treatment can improve PD
symptoms and regulate gut microbial composition in MPTP-induced PD
mice model. To this end, we first investigated the effect of acupuncture
on motor function, protecting dopaminergic neurons, and inhibiting
neuroinflammation in PD mice. In addition, we examined the alteration
of gut microbial composition to disclose the role of microbiome in the
effects of acupuncture in the PD mice.
2. Materials and methods
2.1. Animals and group allocation
Seven-week-old male C57BL/6 mice (weight, 23–25 g; Central
Laboratories Animal Inc., Seoul, Republic of Korea) were housed in-
dividually at 24 ± 2 °C under a 12/12 h light/dark cycle with free
access to food and water for at least 7 days before the start of the ex-
periments. All experimental procedures were approved by the Kyung
Hee University Institutional Animal Care and Use Committee (KHUASP
(SE)-14–052). The mice received saline (MPTP vehicle) or MPTP
(30 mg/kg, dissolved in saline; Sigma-Aldrich, St. Louis, MO, USA) via
intraperitoneal injection for 5 consecutive days and were randomly
allocated into four groups: the control group (only saline, n = 6), the
MPTP group (n = 10), the MPTP + Acu group (n = 9), and the
MPTP + Non-acu (n = 5). Because MPTP injection can cause death of
mice, we allocated more mice to the MPTP-treated groups than control
group. During our study, one mouse died in the MPTP-Acu group, and
thus the data from 10 and 9 mice were analyzed in MPTP and MPTP-
Acu group, respectively.
2.2. Acupuncture treatment
The mouse was lightly fixed by hand to minimize stress during
acupuncture treatment. Acupuncture needles (Dong Bang Acupuncture
Inc., Seoul, Republic of Korea) on the acupoints GB34 and ST36 were
inserted bilaterally to a depth of 3 mm at each acupoint, turned at a rate
of two spins per second for 30 s and then removed immediately. The
bilateral acupoints (GB34 and ST36) were stimulated alternately. The
locations of each acupoint were shown in Fig. 1A. Acupoint GB34 has
been used to treat movement disorders in traditional East Asian Medi-
cine, and recent neuroimaging studies have confirmed its effects on
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Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
motor function (Avtandilov et al., 1991; Huang et al., 2010; Na et al.,
2009). Moreover, it is effective in a PD animal models (Jeon et al.,
2008; Kim et al., 2011a,b). ST36 is often used to treat gastrointestinal
diseases. Recent research has verified that stimulating these two acu-
points enhances immunity and improves mobility (Dos Santos et al.,
2008; Kung et al., 2006; Rho et al., 2008). Two non-acupoints were
used for the sham acupuncture treatment. One is located on the lateral
part of biceps femoris 1 cm distal from the gluteal fold and the other is
located on the gluteus maximus 5 mm lateral from the base of the tail.
The depth of insertion and the stimulating methods were the same as
the verum acupuncture treatment. Acupuncture treatments were per-
formed at 2 h after MPTP treatment (1st–5th day), and at 10:00 AM
(6th–12th day).
2.3. Behavioral tests
All behavioral tests were performed on days 11 and 12 after first
MPTP exposure. The detailed schedule is shown in Fig. 1B.
2.3.1. Motor function tests
Motor function of mice was measured using rearing test (Ahn et al.,
2019), and this counted the number of forelimb wall contacts. PD
clinical features of no body movement in mouse was measured using
akinesia test (Ahn et al., 2019; Sedelis et al., 2001). Motor mediation
and balance impairment were analyzed using the rotarod test with the
rod instrument (MED Associates, Inc., Burlington, VT, USA) (Park et al.,
2015; Park et al., 2017).
2.3.2. Open field test (OFT)
We observed anxiety as one of the non-motor features in PD using
the OFT. Mice were individually housed in a rectangular white acryl
box (40 × 40 × 27 cm). Then the behaviors of the mouse including
total distance, central zone distance, and numbers of crossing the cen-
tral zone were recorded for 5 min using video camera and monitored by
a computerized video-tracking system running the S-MART (Pan Lab
Co., Barcelona, Spain).
2.4. Immunohistochemistry
On day 12, the mice were perfused 0.2 M phosphate buffer and
followed with cold 10% formalin or 0.2 M phosphate buffer. The brains
were removed, and cryoprotected. The brains were cut into 40 µm
coronal sections and were incubated with rabbit anti-tyrosine hydro-
xylase (TH) (1:1,000, sc-47708, Santa-Cruz Biotechnology, Dallas, TX,
USA) overnight at room temperature. The sections were incubated with
biotinylated anti-rabbit IgG (1:1,000, BA-1000, Vector Laboratories,
Inc., Burlingame, CA, USA) for 1 h, and were mounted on gelatin-
coated slides, dried, dehydrated, and coverslipped. The histological
images were confirmed using a bright-field microscope (BX51; Olympus
Japan Co., Tokyo, Japan).
2.5. Gut microbiome analyses by 16S rRNA gene sequencing
On day 12, fecal samples were collected at 2 h after last acupuncture
treatment. Changes in gut bacterial composition were measured by
sequencing of the bacterial 16S rRNA gene. Fresh mouse feces was
collected in individual sterile EP tubes, quickly frozen in liquid ni-
trogen, and then stored at −80 °C until DNA extraction. Microbial DNA
was extracted using a PowerMax Soil DNA Isolation Kit (MO BIO
Laboratories, Carlsbad, CA, USA) following the manufacturer’s in-
structions. The V3-V4 hypervariable region of the 16S rRNA gene was
amplified by PCR using the 341F (5′-CCTACGGGNGGCWGCAG-3′) and
805R (5′-GACTACHVGGGTATCTAATCC-3′) barcoded primers and se-
quenced using the Illumina MiSeq platform (Illumina, San Diego, CA,
USA). Reads were screened for low quality bases and short read lengths,
assembled and assigned to operational taxonomic units (OTUs) with a
J.-H. Jang, et al.
Fig. 1. Effects of acupuncture on motor functions in MPTP-induced PD mice. (A) Acupuncture on GB34 and ST36 of PD mice treated with MPTP for 12 days
(MPTP + Acu). All motor behavioral tests including (B) cylinder, (C) akinesia, and (D) rotarod tests were performed 11 and 12 days after the saline or MPTP (30 mg/
kg) injection. n = 6–10 per group, ***p < 0.001 vs. Control group, ###p < 0.001 vs. MPTP group, all data are mean ± standard error following one-way ANOVA
test.
similarity threshold of 97%. Taxonomic assignments were performed
using BLASTn against the NCBI 16S Microbial database. The α- and β-
diversity analyses were performed using the QIIME tool (v 1.9). Alpha
diversity was analyzed by the Chao 1, observed OTU, and Shannon
indices. Beta diversity was determined using weighted UniFrac phylo-
genetic distance matrices visualized in principal coordinates analysis
(PCoA) plots, and tested for statistical significance by analysis of si-
milarities (ANOSIM) with 999 permutations. DESeq2 in QIIME was
used to determine significant differences (p < 0.05) in OTU bacterial
relative abundance. Significant differences in the relative abundance of
bacteria at different taxonomic levels between the groups were detected
using linear discriminant analysis (LDA) effect size (LEfSe). Only LDA
values > 2.5 at p-value < 0.05 were considered significantly en-
riched. The LEfSe results were visualized using heatmap, taxonomic bar
charts and cladograms. Microbial functions based on the Kyoto
Encyclopedia of Genes and Genome (KEGG) pathways were predicted
using Phylogenetic Investigation of Communities by Reconstruction of
Unobserved States (PICRUSt) analyses from the 16S rRNA gene se-
quences (Langille et al., 2013).
2.6. Western blotting analyses
Western blot was performed to measure the protein levels in the ST
and SN (Jang et al., 2019). The primary antibodies used were GFAP
(1:200; 14–9892–80, Invitrogen, Carlsbad, CA, USA), tyrosine hydro-
xylase (1:10,000, sc-47708, Santa-Cruz Biotechnology), NF-κB (1:500,
#8242, Cell Signaling Technology, Danvers, MA, USA), Iba-1 (1:200,
MABN92, Millipore), TNF-α (1:500, NBP1–19532, Novus Bio, Cen-
tennial, CO, USA), Bax (1:500, ab216494, Abcam, Cambridge, UK), Bcl-
2 (1:500, # 3498S, Cell Signaling Technology), and β-actin (1:10000,
Sigma-Aldrich). The blots were incubated and mixed with HRP-con-
jugated secondary antibodies (rabbit: PA1–30359, mouse: PA1–30355,
Thermo). All band intensity was analyzed by image QI software.
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2.7. Fecal microbiota transplantation (FMT)
Fresh fecal pellets were collected from MPTP mice (M-FMT, n = 8)
or MPTP + Acu mice (A-FMT, n = 8) at day 11 and day 12. The feces
were pooled, soaked in sterile PBS (1 fecal pellet/ml) for about 15 min,
homogenized and then centrifuged at 1000 rpm for 5 min at 4 °C to
pellet the particulate matter. The suspension was centrifuged at
8000 rpm for 5 min at 4 °C to get total bacteria. The final bacterial
suspension was mixed with a final concentration of 20% sterile gly-
cerol, and then stored at −80 °C until transplantation (Sun et al., 2018).
The suspensions were diluted to an OD at 600 nm of 0.5 in sterile PBS,
corresponding to approximately 108 CFU/ml (Li et al., 2017). Recipient
mice were transplanted with 200 µl of bacterial suspension (108 CFU/
mL) by gavage each day using same protocol. Motor function was
measured in the same methods as in previous experiments using rotarod
and rearing tests. The C57BL/6 mice were divided into four groups of
six animals each: control, MPTP, MPTP + M-FMT (gavaged fecal
samples from MPTP mice), and MPTP + A-FMT (gavaged fecal samples
from MPTP + Acu mice) groups.
2.8. Statistical analysis
GraphPad Prism software (ver. 5; GraphPad Software, Inc., San
Diego, CA, USA) was used for statistical analyses of the behavioral and
histological data. Differences were detected using one-way analysis of
variance (ANOVA) followed by the Newman–Keuls post hoc test. All
results are expressed as mean ± standard error. P-values < 0.05 were
considered significant. The Wilcoxon rank-sum test or the
Kruskal–Wallis test followed by Dunn’s multiple comparison test
(α < 0.05) were used to compared three or four groups of specific taxa
in the microbiota. The correlations between the relative abundance of
microbiota at the genus level and PD clinical features were assessed
using Spearman’s correlation test.
J.-H. Jang, et al.
3. Results
3.1. Acupuncture improves various motor dysfunctions caused by MPTP
Experimental schedule of the acupuncture treatment in the MPTP-
induced PD mice was shown in Fig. 1B. A significant difference was
detected among the three groups in the rearing test (F[2, 22] = 14.88,
p < 0.0001), in the akinesia test (F[2, 22] = 50.29, p < 0.0001), and
in the rotarod test (F[2,22] = 26.76, p < 0.0001). In the rearing test,
the number of rears was lower in the MPTP group than in the Control
group (p < 0.001) and was increased in the MPTP + Acu group
(p < 0.001 vs. MPTP group; Fig. 1C). In the akinesia test, the latency
time increased significantly in the MPTP group compared to the Control
group but decreased significantly in the MPTP + Acu group compared
to the MPTP group (p < 0.001, respectively; Fig. 1D). In the rotarod
test, the reduction of latency time induced by MPTP injection
(p < 0.001 vs. Control group) was restored by the acupuncture
treatment (p < 0.001 vs. PSNL group; Fig. 1E).
3.2. Acupuncture ameliorates the anxiety induced by MPTP
The effects of acupuncture treatment on MPTP-induced non-motor
symptom (anxiety) were evaluated by the OFT (Fig. 2). The routes
traveled by the mice in the open field were measured (Fig. 2A). A
significant difference among the three groups in the main effects (the
total distance, F[2, 22] = 0.9586, p = 0.3995; central distance, F[2,
22] = 109.1, p < 0.0001; the ratio of central/total distance, F[2,
22] = 21.67, p < 0.0001; number of crossings in the central zone, F[2,
22] = 24.48, p < 0.0001). There were decreased significantly in the
MPTP group (p < 0.001, p < 0.01 vs. Control group), but were in-
creased significantly in the MPTP + Acu group (p < 0.001 vs. MPTP
group; Fig. 2B–E).
3.3. Acupuncture restores the deficit in dopaminergic fibers and neurons
following MPTP
We confirmed the neuroprotective effects of acupuncture on dopa-
minergic fibers and neurons in the ST and SN of MPTP-induced mice. In
the ST, a significant differences were observed among the three groups
in the optical density (F[2, 10] = 519.5, p < 0.0001) and expression
level (F[2, 10] = 519.5, p < 0.0001) of TH. There were decreased
significantly after the MPTP injection (p < 0.001, p < 0.05 vs.
Control group), but were increased significantly after the acupuncture
treatment (p < 0.001, p < 0.01 vs. MPTP group; Fig. 3A and B). In
the SN, a significant differences were observed among the three groups
in the number of TH-positive neurons (F[2, 10] = 28.64, p < 0.0001)
and the expression level (F[2, 6] = 91.10, p < 0.0001) of TH. There
were decreased in the MPTP group (p < 0.01, p < 0.001 vs. Control
group) and were increased in the MPTP + Acu group (p < 0.001 vs.
MPTP group, respectively; Fig. 3C and D).
3.4. Acupuncture alters dysbiosis of the gut microbiome in MPTP-induced
PD mice
Dysbiosis of the gut microbiome was associated with PD. Thus, 16S
rRNA gene sequencing analyses of fecal samples were carried out to
assess the effects of acupuncture on dysbiosis of the gut microbiome in
PD. An average of 108,656 reads was obtained per sample, and 203
discrete bacterial taxa (OTUs) were identified. A significant reduction
in α-diversity was observed in the MPTP-treated mice compared to the
controls based on the Chao1 index, an estimate of microbial community
richness. However, the Chao1 index increased significantly following
acupuncture treatment (Fig. 4A). Interestingly, the acupuncture treat-
ment significantly increased the number of observed OTUs and the
Shannon index. We also found that Chao1 index, observed OTUs, and
Shannon index were not increased significantly in the MPTP + Non-acu
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group compared to the MPTP group (Fig. S1A–C). The extent of the
similarity of gut microbial communities between the Control, MPTP,
and MPTP + Acu groups was measured using PCoA based on the
weighted UniFrac distance at the OTU level. As shown in Fig. 4B and C,
the gut microbiota of the groups were distinctly separated (ANOSIM
R = 0.758, p = 0.001), suggesting that the overall structures of the
bacterial communities in the Control, MPTP, and MPTP + Acu groups
were significantly different. In addition, we also found differences in
structures of the bacterial communities between the MPTP + Acu and
MPTP + Non-acu groups (Fig. S1D and E). Differential abundance
analyses at the phylum level revealed that the relative abundance of
Proteobacteria was significant in the MPTP group. It tended to decrease
following acupuncture treatment, but the change was not significant
(Fig. 4D). Furthermore, LEfSe analyses were performed to identify the
bacterial taxa that significantly differed after acupuncture treatment. A
significant shift in the microbiota based on relative abundance is shown
in the cladogram (Fig. 4F). These LEfSe comparisons identified 25 taxa
(1 class, 1 order, 4 families, 9 genera and 10 species) that were dif-
ferentially abundant among the three groups (Fig. 4E–G). Significant
enrichments in class Betaproteobacteria, order Burkholderiales, families
Sutterellaceae and Tannerellaceae, gerera Faecalibaculum, Harryflintia,
Parabacteroides and Parasutterella, and species Faecalibaculum rodentium,
Harryflintia acetispora, Parasutterella excrementihominis, Ruminococcus
champanellensis were identified in MPTP-treated mice, while the genus
Absiella and species Absiella dolichum were significantly more abundant
in fecal samples from normal control mice. Families Rikenellaceae and
Vallitaleaceae, genera Alistipes, Vallitalea, Lachnoclostridium, and Pseu-
doclostridium, and the species Bacteroides xylanolyticus, Vallitalea pro-
nyensis, Clostridium aerotolerans, Pseudoclostridium thermosuccinogenes
and Roseburia faecis were significantly enriched following acupuncture
treatment (Fig. 4G).
3.5. Acupuncture treatment affects the abundance of certain bacteria in
MPTP-induced PD mice
Genus-level analyses showed a clear distinction between the gut
microbiota of the Control and MPTP groups. A total of 44 genera were
identified and differed significantly among the three groups (Table S1;
p < 0.05, Dunn’s post hoc test following Kruskal–Wallis one-way
ANOVA). Of these genera, 26 were significantly different between the
Control and MPTP groups (Table S1; adjusted p < 0.05); 16 genera in
the MPTP group were enriched, but 10 genera were depleted compared
to the Control group. The acupuncture treatment significantly reversed
the changes in the relative abundance of the 18 genera, which was si-
milar to those in the Control group (Fig. 5A and B). Additionally, genera
Bacteroides and Parabacteroides did not differ between the MPTP and
Control groups, but decreased in the MPTP + Acu group compared to
the MPTP group (Fig. 5C, Table S1).
To examine whether the gut microbiota was correlated with PD
clinical features, and also to determine whether the extensive changes
in the fecal microbiota and clinical features of PD following acu-
puncture treatment are associated, we further analyzed the relationship
between the gut microbiota and motor functions using Spearman’s
correlation, based on 44 genera (Table S1; passed the statistical
threshold (adjusted p < 0.05) by Kruskal-Wallis one-way ANOVA test
with Dunn’s post hoc analysis) and the representative parameters for
behavioral deficits in the MPTP mouse model, rearing activity and time
on the rotarod. The correlation analyses indicated that 17 and 10
genera were significantly correlated with rearing activity and time on
the rotarod, respectively (Table S2). Rearing activity was highly posi-
tively correlated with the genera Gracilibacter, Phocea, Flintibacter, Os-
cillibacter, Alkalibacter, and Desulfitobacterium, but highly negatively
correlated with Holdemania, Phascolarctobacterium, Erysipelatoclos-
tridium, Faecalibaculum, Rhodospirillum, Parasutterella, Frisingicoccus,
Marvinbryantia, Sporobacter, Aestuariispira, and Parabacteroides (Table
S2). Time on the rotarod was highly positively correlated with the
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Fig. 2. Effects of acupuncture on comorbid anxiety in MPTP-induced PD mice. The non-motor behaviors test and the open-field test (OFT) were conducted 11 days
after the saline or MPTP (30 mg/kg) injection. All mice were measured for (A) tracking, (B) total distance, (C) central distance, (D) ratio of central/total distance, and
(E) number of crossings in the central zone in the open field. n = 6–10 per group, **p < 0.01, ***p < 0.001 vs. Control group, ###p < 0.001 vs. MPTP group;
results are mean ± standard error by one-way ANOVA.

genera Butyricimonas, Phocea, Acutalibacter and Gracilibacter, but ne- Gracilibacter were correlated with rearing activity and time on the ro-
gatively correlated with Desulfovibrio, Aestuariispira, Frisingicoccus, tarod (Fig. 6A and B). Furthermore, we also analyzed the relationship
Holdemania, Turicibacter, and Bifidobacterium (Table S2). Interestingly, between the comorbid anxiety and gut microbiota using Spearman’s
Butyricimonas, Holdemania, Frisingicoccus, Aestuariispira, Phocea, and correlation, based on anxiety parameters and 6 genera, which has a

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Fig. 3. Neuroprotective effects of acupuncture on dopaminergic fibers and neurons in the ST and SN of MPTP-induced PD mice. Immunohistostaining for tyrosine
hydroxylase (TH) in the (A) ST and (C) SN of brain extracted from Control, MPTP, and MPTP + AP treated mice. n = 3–5 per group. Scale bar: 500 μm (ST) and
200 μm (SN). Representative Western blotting bands and quantification of TH and β-actin in the (B) ST and (D) SN. n = 3 per group, *p < 0.05, **p < 0.01,
***
p < 0.001 vs. Control group, ##p < 0.01, ###p < 0.001 vs. MPTP group, all results are mean ± standard error analyzed by one-way ANOVA.

high correlation with motor functions. Numbers of crossing in the
central zone was positively correlated with genera Butyricimonas,
Phocea, and Gracilibacter, but negatively correlated with Holdemania,
Frisingicoccus, and Aestuariispira (Fig. 6C).
3.6. Acupuncture changes the functionality of the gut microbiota in MPTP-
induced PD mice
To gain insight into the functional categories and pathways asso-
ciated with acupuncture treatment in MPTP-induced PD mice, we
compared the PICRUSt-predicted functional KEGG pathway enrichment
between the Control, MPTP, and MPTP + Acu groups using PIC

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(caption on next page)

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J.-H. Jang, et al.
Fig. 4. Effects of acupuncture on the imbalance in the gut microbiota of MPTP-induced PD mice. (A) α-diversity (Chao1) comparison of the gut microbiota. (B and C)
2D (B) and 3D (C) principal component analyses (PCoA) plots of the weighted UniFrac distances using the relative abundance of OTUs. Each point represents a
sample from the Control, MPTP, or MPTP + Acu groups. Ellipses in the 2D PCoA plot indicate 95% confidence intervals. (D) The average relative abundance of the
microbial community for each group at the phylum level. (E–G) Comparison of gut microbiota using LES among the Control, MPTP, and MPTP + Acu groups at OTU
level. Heatmap (E), cladogram (F), and histogram (G) of bacterial taxa that significantly differed among the three groups (LDA > 3.0 and p < 0.05). The red, green,
or blue shading in the cladogram depicts bacterial taxa that were significantly higher in either the Control, MPTP, or MPTP + Acu groups, as indicated. c, class; o,
order; f, family; g, genus; s, species. n = 5 per group, *p < 0.05, **p < 0.01 vs. Control; #p < 0.05 vs. MPTP group; results are mean ± standard deviation by one-
way ANOVA.
analyses designed to predict the functional patterns from the 16S rRNA-
based taxonomic profile. A total of 41 KEGG pathways at level 3 were
significantly altered in the MPTP group (exact p < 0.05), and many of
the predicted functional differences between the microbiomes of the
MPTP-treated and control mice were in metabolic pathways (Table S1).
Among them, 16 KEGG pathways were selected and showed at least a
0.03% difference in the mean proportion between the MPTP and Con-
trol (Fig. 7A). Photosynthetic proteins, photosynthesis, propanoate
metabolism, valine, leucine, and isoleucine degradation, glutathione
metabolism, the sulfur relay system, tetracycline biosynthesis, lysine
degradation, and fatty acid metabolism were significantly enriched in
the MPTP-induced PD mice, whereas methane metabolism, strepto-
mycin biosynthesis, arginine and proline metabolism, drug metabolism-
other enzymes, glycosphingolipid biosynthesis-globo series, and poly-
ketide sugar unit biosynthesis were depleted. Among them, seven KEGG
pathways were affected by acupuncture treatment.
After the acupuncture treatment, metabolic pathways, such as
photosynthesis, glutathione metabolism, tetracycline biosynthesis, and
photosynthetic proteins, were significantly enriched, but other meta-
bolic pathways, such as methane metabolism and drug metabolism
were depleted. Interestingly, the PD pathway enriched in MPTP-in-
duced PD mice was silent after the acupuncture treatment (Fig. 7B).
3.7. Acupuncture inhibits activation of neuroinflammation and apoptosis
induced by MPTP
To investigate the effects of acupuncture on activation of the in-
flammatory pathway, we evaluated the expression levels of TH, GFAP,
Iba-1, NF-κB, and TNF-α in the ST and the SN using Western blotting
analyses. In the ST, a significant differences were observed among the
three groups in the expression levels of GFAP (F[2, 6] = 125.4,
p < 0.0001), Iba-1 (F[2, 6] = 6.055, p = 0.0364), NF-κB (F[2,
6] = 69.72, p < 0.0001), and TNF-α (F[2, 6] = 33.80, p = 0.0005)/
β-actin. The GFAP, NF-κB, and TNF-α were increased significantly in
the MPTP group (p < 0.05, p < 0.001 vs. Control group, respectively)
but were decreased significantly in the MPTP + Acu group (p < 0.05,
p < 0.001 vs. MPTP group, respectively; Fig. 8A, C, D). The Iba-1
tended to increase in the MPTP group compared to the Control group,
but was decreased in the MPTP + Acu group (p < 0.05 vs. MPTP
group; Fig. 8B). In the SN, a significant differences were shown among
the three groups in the expression levels of GFAP (F[2, 6] = 9.486,
p = 0.0139), Iba-1 (F[2, 6] = 13.86, p = 0.0056), NF-κB (F[2,
6] = 8.840, p = 0.0163), TNF-α (F[2, 6] = 9.625, p = 0.0134)/β-
actin. There were increased significantly in the MPTP group (p < 0.05,
p < 0.01, p < 0.001 vs. Control group, respectively) but decreased
significantly in the MPTP + Acu group (p < 0.05, p < 0.001 vs.
MPTP group, respectively; Fig. 8G–J).
To investigate the effects of acupuncture on apoptosis, we measured
the expression levels of Bax and Bcal-2 in the ST and the SN. A sig-
nificant differences were observed among the three groups in the ex-
pression levels of Bax (F[2, 6] = 10.83, p = 0.0102) and Bcl-2 (F[4,
105] = 12.00, p = 0.0080)/β-actin in the ST. The Bax was increased in
the MPTP group (p < 0.01 vs. Control group) and was decreased in the
MPTP + Acu group (p < 0.05 vs. MPTP group; Fig. 8E). In contrast,
the Bcl-2 was decreased in the MPTP group compared to the Control
group (p < 0.05), and was increased in the MPTP + Acu group
8
Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
compared to the MPTP group (p < 0.01; Fig. 8F). A significant dif-
ferences were observed among the three groups in the expression levels
of Bax (F[2, 6] = 15.70, p = 0.0041) and Bcl-2 (F[2, 6] = 324.2,
p < 0.0001)/β-actin in the SN. The Bax was increased in the MPTP
(p < 0.01 vs. Control group) and was decreased in the MPTP + Acu
groups (p < 0.05 vs. MPTP group Fig. 8K). The Bcl-2 was decreased
significantly in the MPTP group (p < 0.001 vs. Control group), and
was higher in the MPTP + Acu group than the MPTP group
(p < 0.001; Fig. 8L).
3.8. Acupuncture-fecal microbiota transplantation improves motor
dysfunction induced by MPTP
Finally, to demonstrate the causality between the improvement of
PD symptoms and the change of gut microbiota composition by acu-
puncture treatment, we measured the motor function using rotarod and
rearing test after FMT treatment in the MPTP-induced PD mice. A sig-
nificant difference was shown among the four groups in the numbers of
rears (F[3, 16] = 85.45, p < 0.0001) in the latency time (F[3,
17] = 21.87, p < 0.0001), and there were decreased in the MPTP
group (each p < 0.001 vs. Control group) and was increased in the
MPTP + A-FMT group compared to the MPTP (p < 0.001, p < 0.05)
or MPTP + M-FMT group (each p < 0.001), respectively.
Interestingly, we found that the numbers of rears and latency time were
lower in the MPTP + M-FMT group than in the MPTP group (p < 0.01,
p < 0.05; Fig. 9A and B).
4. Discussion
Imbalance of the gut-microbiome-brain axis by gut microbial dys-
biosis plays an important role in pathogenesis and development of PD,
and induces production of neuroinflammation and deficiency of dopa-
minergic neurons (Baizabal-Carvallo and Alonso-Juarez, 2020;
Sampson et al., 2016). Previous PD studies have shown that acu-
puncture has neuroprotective, anti-inflammatory and anti-apoptotic
effects (Jeon et al., 2008; Kim et al., 2011a,b; Park et al., 2003, Park
et al., 2015, Park et al., 2017), but still unknown on the association
with gut microbial dysbiosis in PD. In the present study, we found that
acupuncture can induce normalization of gut microbial dysbiosis as
well as inhibition of neuroinflammation in the SN and the ST, which are
associated with PD phenotypes.
Increasing evidence indicates that different levels of the gut-brain
axis may be involved in PD (Keshavarzian et al., 2015; Lai et al., 2018;
Scheperjans et al., 2015). The gut microbiota composition of germ-free
mice receiving fecal microbiota transplantation from PD patients were
similar to that of host, and α-synuclein overexpressing mice trans-
planted with stool of PD patient showed motor dysfunctions (Sampson
et al., 2016). The α-synuclein-containing inclusions termed Lewy
bodies are found in the enteric nervous system (ENS) as well as CNS.
They spread from gut to brain in a prion-like fashion via vagus nerve,
which may reduce dopaminergic neuron in the SN (Kim et al., 2019).
Recent studies have demonstrated an active role for the gut microbiota
in inflammatory capabilities of microglia (Erny et al., 2015). Bacterial
product, lipopolysaccharide (LPS) induces increases of blood–brain
barrier (BBB) permeability and release of pro-inflammatory cytokine
including TNF-α, which induces loss of dopaminergic neuron in the SN
J.-H. Jang, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx

Fig. 5. Effects of acupuncture on changes in the relative abundance of the gut microbial community at the genus level in MPTP-induced PD mice. (A) The relative
abundance of 18 selected bacterial genera. *p < 0.05, **p < 0.01 vs. Control; #p < 0.05, ##p < 0.01 vs. MPTP group; results are mean ± standard deviation by
one-way ANOVA.

(Villaran et al., 2010). An inflammatory environment can enhance α-
synuclein aggregation, which further activates microglia upon contact
and promote a feed-forward cascade that leads to additional α-synu-
clein aggregation and propagation, and progression of disease (Gao
et al., 2011). In the MPTP-induced PD mice model, glial-mediated
neuroinflammation, dopaminergic neuronal loss, and gut microbial
dysbiosis are shown (Lai et al., 2018; Sun et al., 2018). Healthy mice
receiving fecal microbiota transplantation from MPTP mice display
decreases of dopamine and dopamine metabolites, resulting in im-
pairment of motor functions (Sun et al., 2018). In this study, we found

9
J.-H. Jang, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx

Fig. 6. Correlation between the bacteria of genus and the behavior functions. Spearman’s correlation analyses between the relative abundance of gut microbiota at
the genus level and the value in (A) rotarod, (B) rearing test, or (C) open field test, respectively. Significant correlations were determined based on < –0.7 Spearman
r < 0.7 and p < 0.01.

10
J.-H. Jang, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx

Fig. 7. Effects of acupuncture on the PICRUSt-predicted functional KEGG pathways in MPTP-induced PD mice. (A) Changes in the KEGG pathways of MPTP-treated
mice compared to normal controls. Only the significantly different (exact p < 0.05) pathways for MPTP vs. Control groups were included, based on a two-sided
Wilcoxon rank-sum test. The bar plots on the left display the mean proportion of OTUs belonging to each order. The dot plots display the difference in mean
proportions between the MPTP and Control groups with the exact p-value. Error bars on the dot plots represent the 95% confidence intervals (CIs). (B) The relative
abundance of the predicted KEGG pathways among the Control, MPTP, and MPTP + Acu groups. n = 5 per group, *p < 0.05 vs. Control; #p < 0.05 vs. MPTP
group; results are mean ± standard deviation by one-way ANOVA.

that fecal microbiota was significantly altered in the MPTP-induced PD
mice, as demonstrated by a pronounced decrease in the Chao1 (rich-
ness) of the microbial community, a high degree of overall dissimilarity
in microbiota composition compared to the Control group. In contrast,
acupuncture treatment to PD mice distinctly increased not only Chao1
but also the number of observed OTUs (richness) and Shannon (richness
and evenness) index comparable to those of the controls. The higher the
richness and more even the distribution within a sample, the higher the
intra-sample diversity.
MPTP-induced PD mice possessed a significantly higher abundance
of Proteobacteria, a phylum known to comprise several pathogens and to
have pro-inflammatory properties, which is consistent with previous
reports describing an increase in the relative abundance of
Proteobacteria in the gut microbiome in patients and mice with PD
(Gorecki et al., 2019; Keshavarzian et al., 2015; Sampson et al., 2016;
Sun et al., 2018). Acupuncture resulted in robust reduction in Proteo-
bacteria in MPTP-induced PD mice; however, the difference was not
significant.
Thus, genus-level comparisons were made to identify specific taxa
that might contribute to the global microbiota changes following acu-
puncture treatment. Contrary to our expectations, we observed a sig-
nificant difference in genera belonging to mainly phylum Firmicutes, not
Proteobacteria. MPTP led to fundamental alterations in a total of 26
genera, including decreases in Butyricimonas and Alistipes as well as

11
J.-H. Jang, et al. Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx

Fig. 8. Anti-inflammatory and anti-apoptotic effects of acupuncture in the ST and SN of MPTP-treated PD mice. Western blotting analyses of the (A–F) ST and (G–L)
SN from the Control, MPTP, and MPTP + AP treated mice. Representative Western blotting bands and quantification of (A and G) GFAP, (B and H) Iba-1, (C and I)
NF-kB, (D and J) TNF-α, (E and K) Bax, and (F and L) Bcl-2. n = 3 per group, *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group, #p < 0.05, ##p < 0.01,
###
p < 0.001 vs. MPTP group; all data are mean ± standard error following one-way ANOVA.

12
J.-H. Jang, et al.
Fig. 9. Effects of fecal microbiota transplantation on motor functions in the MPTP-induced PD mice. The saline, M-FMT, or A-FMT was treated in the MPTP-induced
mice. Motor behavioral tests including (A) cylinder and (B) rotarod tests were performed. n = 4–6 per group, ***p < 0.001 vs. Control group, #p < 0.05,
##
p < 0.01, ###p < 0.001 vs. MPTP group, all data are mean ± standard error following one-way ANOVA test.
increases in Holdemania, Aestuariispira, Erysipelatoclostridium,
Parabacteroides, and Paraprevotella. In contrast, after acupuncture
treatments, seventy percent of microbiome composition (18 out of 26)
were restored compared to that of the MPTP mice. Moreover, we
showed that the abundances of the genera Butyricimonas, Holdemania,
Aestuariispira, Frisingicoccus, Gracilibacter and Phocea were highly cor-
related with behavioral metrics of comorbid anxiety (OFT) as well as
motor deficits (both rearing and rotarod tests).
We found that the genus Butyricimonas was positively correlated
with the motor function and the anxiolytic behaviors, indicating that
the enhanced motor and anxiolytic effects of acupuncture are asso-
ciated with the increase of Butyricimonas in the gut microbiome of PD
mice. Previously, the decrease of Butyricimonas was identified in the
germ free mice colonized with microbiota derived from PD donors,
compared to healthy controls (Sampson et al., 2016). Gut bacteria
metabolites, short chain fatty acids (SCFAs) such as acetate, butyrate
and propionate are transmitted throughout the whole body through the
blood and can affect the brain by passing through the BBB, which may
modulate microglial activation (Sampson et al., 2016). Considering that
Butyricimonas is butyrate producers and butyrate has anti-inflammatory
properties among SCFAs (Shirai et al., 1988), it is speculated that the
increase of Butyricimonas might play pivotal roles in the anti-in-
flammation by acupuncture treatments in PD mice.
Recently, it was reported that Holdemania is enriched in the feces of
Chinese PD patients and closely related to PD clinical characteristics
(Qian et al., 2018). Our data support this finding as the relative
abundance of Holdemania was positively correlated with motor deficits,
as indicated by a negative correlation of Holdemania with time on ro-
tarod as well as rearing. Another recent report showed that Holdemania
exhibited moderate correlation with anxiety in patients with major
depressive disorder (Chung et al., 2019). Here, we showed that mice
with high levels of fecal Holdemania exhibited decreased entries to
center during the OFT, indicative of increased anxiety-like behavior.
These findings suggest that the decrease of Holdemania might have
association with the anxiolytic effects as well as motor improvements
induced by acupuncture treatments in PD mice. However, it was not
well established how Holdemania deteriorates PD symptoms, and thus
further studies are needed to elucidate the role of Holdemania in the
pathogenesis of PD.
Of note, the MPTP + Acu group exhibited remarkably reduced level
of genus Bacteroides compared to the Control and MPTP groups. The
Bacteroidetes constitutes the largest phylum of Gram-negative bacteria
in the species of Bacteroides, such as Bacteroides fragilis, has the potential
13
Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
to secrete a remarkably complex array of pro-inflammatory neurotoxins
including surface LPS and toxic proteolytic peptides (Choi et al., 2016).
In addition, Bacteroides species have been shown to stimulate macro-
phages and monocytes to secrete TNF-α by LPS-mediated pathways
(Delahooke et al., 1995). Thus, the decrease in Bacteroides following
acupuncture might contribute, at least in part, to the beneficial effects
of acupuncture in MPTP-induced PD mice.
Then, to understand whether the changes of microbiome was closely
correlated with the effects of acupuncture against PD-like phenotypes,
we performed FMT using fecal samples from MPTP- or MPTP + Acu
mice, respectively. Interestingly, the MPTP + M-FMT group resulted in
more aggravated motor deficits than MPTP group, while MPTP + A-
FMT group dramatically recovered the motor functions compared to
MPTP or MPTP + M-FMT groups. This might imply that the changes of
microbiota induced by acupuncture can be one of the potential me-
chanisms related to the antiparkinsonian effects of acupuncture.
PICRUSt-predicted functional KEGG pathway analysis revealed the
aberrant functional pathways in MPTP-treated mice, especially those
related to methane metabolism. There is emerging evidence that me-
thane delays intestinal transit, possibly acting as a neuromuscular
transmitter, and has been epidemiologically and clinically associated
with constipation related diseases (Triantafyllou et al., 2014). It is
conceivable that abnormal methane metabolism might be related to
constipation shown in the subjects with PD. We also observed MPTP-
induced significant increase of ‘Parkinson disease pathway’ in the KEGG
pathway, suggesting that alterations in the gut microbiota represent a
risk factor for PD. The functionally identified pathways in PD group
might be related with the following pathological process of PD: the
bacterium major metabolites, SCFA and/or LPS may cross the BBB and
impact the physiology of cells in CNS, or they may have peripheral
effects, which indirectly activate and mature microglia (Erny et al.,
2015; Mitchell et al., 2011); the increase of apoptosis as well as pro-
duction of pro-inflammatory cytokines by glial cells activation can lead
to neuronal dysfunction and cell death, and that motor dysfunctions in
models of PD (Liu and Bing, 2011; Sampson et al., 2016). In contrast,
the functionally analyzed pathways, such as metabolism-, biosynthesis-,
and Parkinson’s disease pathways associated with PD, were restored by
acupuncture treatment.
We also investigate whether acupuncture can ameliorate the neu-
roinflammation against the MPTP challenges together with the micro-
bial alterations, we observed the changes of inflammatory and apop-
totic markers in both the SN and the ST. As expected, acupuncture
treatment reduced the activation of microglia and astrocyte, which
J.-H. Jang, et al.
were observed by the expression levels of Iba-1 and GFAP induced by
MPTP. Acupuncture treatments also inhibited the increase of Bax ex-
pression as well as the level of NF-κB and TNF-α. It was also observed
that acupuncture restored anti-apoptosis regulator such as Bcl-2. Taken
together, acupuncture has neuroprotective effects on the dopaminergic
neurons through inhibiting the activation of neuroinflammation and
apoptosis, which might contribute to the improvements of motor dys-
function and comorbid anxiety in PD mice. Given the role of gut mi-
crobiota in the immune system (Erny et al., 2015), it is likely that anti-
inflammatory action of acupuncture can play crucial role in the neu-
roprotective effects possibly through the immunomodulatory role of the
gut microbiota. Nevertheless, further studies are needed to elucidate
how the change of the gut-microbiome is associated with the anti-in-
flammatory and neuroprotective action of acupuncture in the brain.
Though further studies are awaited to reveal how acupuncture
works in gut-brain axis of PD, we suggest the vagus nerve can play the
crucial role in the connection between the gut microbiota and the brain
induced by acupuncture. The communication among the microbiota,
the gut, and the brain through the microbiota-gut-brain axis is a bi-
directional manner, where the autonomic nervous system plays crucial
role (Bonaz et al., 2018; Borovikova et al., 2000). The vagus nerve, the
major component of the parasympathetic nervous system, is a mixed
nerve with both afferent and efferent fibers. It can sense the microbiota
metabolites, and transfer the gut information to the CNS. This inter-
oceptive information is integrated in the central autonomic network,
and then produces the response via autonomic pathways (Bonaz et al.,
2018). Acupuncture effects have been well known to be associated with
ANS (Park and Namgung, 2018). These changes are initiated from the
local response of acupuncture. Acupuncture stimulation produces the
release of adenosine (Goldman et al., 2010) and histamine (Huang
et al., 2018) as well as the expressions of pERK (Park et al., 2014) and
TRPV1 (Abraham et al., 2011) in the skin and muscle layers, which
activate the neurotransmission to the CNS. The signal transmitted to the
brain affects various parts such as prefrontal cortex, hypothalamus,
limbic areas, basal ganglia, and nucleus solitary tract (Cai et al., 2018;
Chae et al., 2009; Park et al., 2017; Wang et al., 2005; Yeo et al., 2014),
whereby central autonomic network is activated to generate the sti-
mulation of efferent vagus nerve (Borovikova et al., 2000). It has been
shown that vagus nerve mediates the anti-inflammatory action of acu-
puncture via cholinergic anti-inflammatory pathway (Du et al., 2013;
Hu et al., 2013; Wang et al., 2015). It is also suggested that activated
efferent vagus nerve can mediates peripheral anti-inflammatory re-
sponse, resulting in alleviating the intestinal permeability, and thus
regulating the changes of microbial composition (Borovikova et al.,
2000; Zhou et al., 2013). The altered changes of gut microbiome by
acupuncture can contribute to the antiparkinsonian effects in the brain,
possibly via neural (i.e. vagal) and/or humoral pathways. It is necessary
to identify more detailed mechanism regarding the gut-brain connec-
tion induced by the acupuncture in PD model.
In conclusion, this study suggests that the improvement of motor
function as well as the anxiolytic effects of acupuncture in PD model
may be partly mediated by the restoration of the gut microbiota, which
might result in the decrease of neuroinflammation. Furthermore, the
specific effect of acupuncture on the genus levels of gut microbiota may
explain its robust antiparkinsonian action on PD model. It is suggestive
that gut-microbiome-brain axis may be a potential mechanism of acu-
puncture for the PD mice.
5. Data availability statement
The data that support the findings of this study are available from
the corresponding authors upon reasonable request.
Conflict of interest
All authors declare no competing financial interests.
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Brain, Behavior, and Immunity xxx (xxxx) xxx–xxx
Acknowledgments
The present research was supported by grants from the National
Research Foundation of Korea funded by the Korean government (NRF-
2017R1A2B4009963 and 2020R1A4A1018598) and from the Korea
Institute of Oriental Medicine (grant K18182).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bbi.2020.08.015.
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