disease (AD) is one of the most prevalent neurodegenerative disease characterized
by the abnormal
amyloid-beta (A) peptide and
neurofibrillary tangles, which leads to
progressively marked deficits in memory and other behavior disorders [1, 2].
Synapse loss occurs at the early stage of
AD, deteriorates progressively, and closely correlates with the cognitive decline
memory is stored in the brain synapses,
synaptic plasticity is thought to underlie
learning and memory . As a primary site of
adult neurogenesis, Hippocampus plays a pivotal role in
the declarative and spatial memory .
loss of hippocampal neurons and synapses in AD causes
impairment of hippocampal-dependent behaviors, including
spatial learning and memory [6, 7].
synaptic plasticity is proposed as the
potential therapeutic strategy for rescuing cognitive dysfunction in AD.
pathway is implicated in neuronal network
maintenance, cell survival and longevity .
Disturbance of this signaling pathway is
the vital mechanism of inducing tau hyperphosphorylation to form neurofibrillary
tangles in AD brain . The phosphorylation
of AKT can inhibit the glycogen synthase kinase-3
(GSK-3) through inducing
phosphorylation inactivation of GSK-3. The “GSK-3 hypothesis
of AD”  proposed that the
aberrant GSK-3 activity
contributes to several features of this pathology such as tau phosphorylation,
memory impairment, microglia-mediated inflammation neuronal apoptosis.
Excessive production of the insoluble 42-amino acid-long A
peptides was almost invariably found in the neocortex and hippocampus of
early-onset autosomal dominant AD the
mutations . Accumulating evidence has
shown that the
GSK-3 is directly disturbed by A exposure in AD brains.
Generally, soluble or diffusible oligomeric aggregates of A [12, 13]
have been implicated in tau
hyperphosphorylation , loss of dendritic
spine density , disruption of memory, neuronal death , and neurotoxic
inflammatory response . As an insulin-suppressing agent, A oligomers
have been shown to inhibit insulin signal transduction by antagonizing insulin
receptors, ultimately causing a dysfunction in the downstream
signaling pathway and a series of
characteristic damage changes in AD, including tau hyperphosphorylation, neuronal
apoptosis, and synaptic loss . Therefore, A
accumulation plays a vital role in the
pathological process related to the PI3K/AKT/GSK-3 signaling pathway.
animal and clinical studies have approved that
electroacupuncture is a potential therapy for
AD [19, 20, 21].
Our previous studies report
an electroacupuncture protocol with
GV29 and IL20 named
“olfactory three-needle” to improve
olfactory dysfunction and cognitive deficits in AD patients in clinical
applications, which has been confirmed an improvement of cognitive
deficits in SAMP8 mice through inhibiting
A deposition, tau phosphorylation, and oxidative stress damage in
hippocampal region [22, 23]. Unlike the
acupoint of other electroacupuncture,
GV29 in the nasal root and IL20 in the
bilateral nasal ala were the main three acupoints used in
three-needle” therapy, which closely related to the olfactory
system through stimulating the olfactory
nerve in front of the olfactory bulb.
Furthermore, we have
further confirmed that olfactory stimulation with “olfactory
three-needle” or eugenol enhances spatial learning and memory abilities of SAMP8
mice [22, 23]. The olfactory system has complex neural connections and can
connect to the hippocampus and prefrontal regions of the brain, involved in
learning and memory abilities . In the present study, we investigate the
effects of electroacupuncture with “olfactory three-needle” on synaptic
function and PI3K/AKT/GSK-3 pathway. Our study may provide a novel
finding of electroacupuncture’s olfactory nerve mechanisms, improving AD’s
2.1 Preparation of A-induced
AD rat model
six-month-old Sprague-Dawley (SD) rats (body weight
220 20 g, SPF grade)
were purchased from
the Chengdu Dashuo Experimental Animal Co.
Ltd, Chengdu, People’s Republic of China (certificate
No. SCXK201302). Rats were housed in normal
plastic cages with free access to commercial water and diet under a
12 h light/dark cycle at an invariable room temperature
(25 C). In this study, AD model rats were established,
referring to the previously described method . In brief, rats were first
injected stereotaxically with 10 g
(Sigma-Aldrich, USA) in the lateral ventricle at accurate positions based on the
mediolateral (ML), dorsoventral (DV), and anteroposterior (AP) coordinates
(2.8 mm, 2.2 mm and 3.0 mm,
respectively), to locate the
CA1 region of the hippocampus.
2.2 Animal grouping and treatments
One week after the operation,
rats treated with A
were randomly divided into three groups
(n = 10 rats per group) and severally treated with
NS (A group),
4 mg/kg (A + Don group) and “Olfactory
three-needle” (A + Ae group)
once per day for 28
consecutive days. While rats in the sham group and control group were without any
“olfactory three-needle” treatment group,
AD rats were
by mouse bags. The disposable sterile electroacupuncture needles (0.3 mm
13 mm) (Huatuo Medical Instrument Company, Suzhou, PR China) were
used to puncture the GV29 with transverse puncturing to the nasal root direction
at a depth of 10 mm and the bilateral
LI20 with shallowly
puncturing towards the interior and superior at a depth of 2 mm (shown in Fig.
1), which was connected to an electric stimulator (Han Shi, Nanjing, PR China) with electrical stimulation (1.5 mA, 15
Hz) for 10 minutes daily within 28 consecutive days, as description as our
previous study . For the donepezil treatment group, A-induced AD rats were treated with
donepezil hydrochloride tablets (H20050978, Eisai, Co. Ltd, PR China), which were
crushed dissolved in distilled water, with the dose at 4 mg/kg by oral
administration once per day for 28 consecutive days.
The location of acupoints for “olfactory three-needle” applied in this study.
The red point indicated the locations of
GV29 on the head of mice; Blue points
indicated the locations of bilateral IL20
above the nostril of mice.
2.3 Morris water maze test
Morris water maze tests assessed the rats’
spatial learning and memory abilities according to a previous study . At 24 h
after “olfactory three-needle” treatment for 24 days, rats in all the group
were trained for four trials per day and
received place navigation trial for 5 consecutive days in a circular pool (Intex
Recreation Corporation, Long Beach, CA, USA; 91 cm diameter and 40 cm height)
containing a 7 cm-diameter hidden platform submerged 1 cm below the water
surface. Every rat was placed in the water facing the pool wall to locate the
hidden platform within 60 s. The rats that
failed to find the platform were guided to stay there for 10
The time and distance to find the platform (escape latency) were recorded to
reflect the spatial learning ability. On
the sixth day, the quadrant-target duration within the 60s and the number of
times the rats crossed the platform area in a spatial probe trial were recorded
to assess the memory ability.
2.4 Immunohistochemical and
At 24 h after the
behavioral test, five rats selected
randomly from each group were sacrificed and
transcardially with 20 mL of ice-cold sterilized saline followed by 40 mL of 4%
paraformaldehyde. The brain was taken out, and the hippocampus tissue was
stripped and cut into 5 m sections using a cryostat (Leica, CM3050S,
Tokyo, Japan). Immunohistochemistry of
A and p-tau was performed on 5 m paraformaldehyde-fixed
brain sections. After incubation with the primary antibody at
4 C overnight
(anti-beta-Amyloid, Abcam, 1 : 1000;
anti-p-tau, Abcam, 1 : 1000), the brain sections were incubated with gota
anti-rabbit IgG secondary antibody (Boster Biological Technology co., Ltd., PR
China) at 37 C for 30 minutes. All procedures were performed according
to the protocols of the immunoassay kit. The brain slices were concurrently
stained with H&E before assessing the histopathology. Images were captured using
a light microscope (Nikon Eclipse 80i, Nikon, Japan).
2.5 TUNEL staining
TUNEL staining was used to label
apoptotic cells by a fluorescent-TdT
DNA Fragmentation Detection Kit (Calbiochem, Darmstadt, Germany).
the frozen coronal
brain sections of the same brain region
from each rat were incubated with proteinase K for 10 min at room temperature and
washed three times with TBS. These sections were then incubated with TdT
equilibration buffer for 30 min and followed with the TUNEL reaction mixture for
90 min at 37 C in a dark cassette. After washed three times by TBS,
these sections were covered by mounting
media to observe apoptotic cells, which
emitted yellow-green fluorescence in the nucleus under an exciting light of
488 nm. The average number of apoptotic
neurons in six random visual fields per section in penumbra was used for
2.6 Nissl staining
Nissl staining, the brain sections were fixed on the polylysine-coated slides,
dried overnight, rehydrated in distilled water, and immersed in 1% cresyl violet
for 20 min. After being rinsed by distilled water and dehydrated by graded
serried ethanol, these sections were
submerged in xylene and then coverslipped.
cells in the pyramidal layer of the CA1 area were observed to assess neuronal
loss under a microscope (Leica, DM6000 B, Tokyo, Japan) by three pathologists
blind to this study. The average number of
Nissl-positive cells in six random visual fields per section in penumbra was used
for statistical analysis.
2.7 Western blotting
Isolated hippocampus tissues were removed from the remaining five rats of every
group and rapidly homogenized.
protein extracts were mixed with sample buffer, heated for 10 min and then
centrifuged for 10 min at 12,000 g. After estimating the protein
concentration with a bicinchoninic acid
(BCA) kit (Sigma-Aldrich), 50 g total proteins were transferred to
difluoride (PVDF) membranes (Millipore,
Billerica, MA, USA) by 10% SDS-PAGE in
Tris-glycine transfer buffer. After blocked in PBST within 5% milk for 1 h,
membranes were incubated at 4 C overnight with the following primary
antibodies: rabbit anti-Synaptophysin
(ab14692, 1 : 1000, Abcam), rabbit anti-PSD95 (ab18258, 1 : 1000, Abcam), rabbit
anti-GAP43 (ab134075, 1 : 1000, Abcam),
rabbit anti-PI3K (#4249, 1 : 1000,
Cell Signaling Technology), rabbit
anti-AKT (sc-8312, 1 : 1000, Santa Cruz, CA, USA), rabbit
anti-phospho-AKT (sc-271964, 1 : 1000, Santa Cruz), mouse anti-GSK-3
(#9832, 1 : 1000, Cell Signaling Technology),
anti-phospho-GSK-3 (#5558, 1 :
1000, Cell Signaling Technology), and mouse anti--actin (A1978, 1 :
10000, Sigma), conjugated to horseradish peroxidase were used as secondary
antibodies. After washing three times by
PBST, PVDF membranes were incubated with an anti-mouse or anti-goat IgG antibody
(PerkinElmer Life Sciences, Waltham, MA, USA) for 1h at room temperature.
bands were visualized by enhanced
chemiluminescence (Millipore), and images were captured under a Bio-Image system
(Bio-Rad Laboratories, Inc., Hercules, CA, USA).
2.8 Statistical analysis
Statistical analysis was performed with the SPSS 23.0, and the data were
exhibited as the mean SD. Two-tailed Student’s t-test and
two-way ANOVA with repeated measures
were used to analyze the orientation navigation test data in different groups
simultaneously. Also, data of spatial probe test, WB and other data were analyzed
by One-Way ANOVA.
spatial learning and memory impairment of
investigate the effect of
“olfactory three-needle” on AD rats’
behavior deficits, the Morris water maze
test was employed to measure the ability of spatial learning and memory after the
treatments on day 31 to 35 and day 36, respectively (Fig. 2A). In
an orientation navigation test of five consecutive days (Fig. 2B),
rats showed an apparent deficit in spatial memory with longer
escape latency than control or sham rats.
Still, it could be significantly rescued by “olfactory three-needle” treatment.
Despite apparent improvements in days 1, 2, and 3 in the orientation navigation
test, donepezil could not shorten the escape latency of
A-treated rats on days 4
“Olfactory three-needle” ameliorated the spatial
learning and memory impairment of A-induced
AD rats. (A) Experimental design for the animal study. (B) Escape latency time
during the orientation navigation test of
five consecutive days after “olfactory three-needle” treatment. (C) Time was
spent in the target quadrant during the
spatial probe test. (D) Platform crossing
numbers in the spatial probe test. (E) Swimming trajectories of groups.
Data are expressed as means SD,
*P 0.05, **P 0.01, ***P 0.001 vs.
A treated rats.
In the spatial probe test,
rats performed impaired spatial memory with
fewer cross-platform (Fig. 2C) and more time spent in
crossing platforms than
control or sham rats (Fig. 2D). After 28 days of treatments,
“olfactory three-needle” and
significantly improved the spatial memory of
A-treated rats by
increasing the cross-platform numbers and shortening the time cross-platform
(Fig. 2C, D, E). Overall, the defect of spatial learning and memory in
A-treated rats was ameliorated by “olfactory three-needle”
treatment by improving spatial learning and memory abilities.
3.2 “Olfactory three-needle”
deposition and tau hyperphosphorylation in the hippocampus of
The arrangement of vertebral neuron cells in the
CA1 area of A-treated rats was disordered,
with a small number of vertebral cell necrosis. 28 days after treatments,
vertebral neuron cells’ arrangement in the
hippocampus was improved by “olfactory three-needle” and donepezil
(Fig. 3A). Immunohistochemistry revealed the presence of A and p-tau
immunoreactivity in the hippocampus CA1
area of A-treated rats.
A deposition on the intercellular substance between
neurons and p-tau
accumulation in neurons is the hallmark characteristic in AD and AD-like brain
aging, which is also the leading cause of neurodegeneration. However, the
integrated optical density of A and p-tau immunostaining was
significantly decreased in Ae and donepezil groups (Fig. 3B, C), which suggested
that both “olfactory three-needle” and donepezil could decrease the production
of A and
p-tau in the hippocampus.
“Olfactory three-needle” reduced A deposition and tau hyperphosphorylation in the hippocampus of A-treated rats. (A) Representative HE and
immunohistochemical staining for A and p-tau positive
areas in the hippocampal CA1 region.
The red arrow indicated that cells scattered outside the pyramidal cell layer. The black
arrow indicated the necrosis of pyramidal cells. (B, C)
Quantification of the integrated optical
density of A and p-tau by immunoreactivity. Blue arrow
indicated that A deposition on the intercellular substance between
cells. The green arrow indicated that p-tau accumulation in neuron cells. Data
are expressed as means SD, *P 0.05, **P 0.01,
***P 0.001 vs. A treated rats.
3.3 “Olfactory three-needle”
neuroinflammation in the hippocampus of
A-induced AD rats
A deposition is neurotoxic to neurons and will lead to neuronal
apoptosis and neuro-inflammation. Compared to control or Sham rats, the
apoptotic neurons were significantly increased in
A-treated rats. The
apoptotic neurons’ level was decreased by the treatments of
“olfactory three-needle” and donepezil
(Fig. 4A, B). Moreover, the level of apoptotic neurons in the
“olfactory three-needle”-treated group was lower than the donepezil-treated
group. The mRNA expression of
IL-1, TNF-, and IL-6
in the hippocampus was significantly increased in
A-treated rats. Still, there was no
difference in the expression of those inflammatory cytokines between
A-treated rats and donepezil-treated rats.
Moreover, “olfactory three-needle” treatment decreased the
expression of IL-1 and TNF- but not included IL-6 (Fig. 4C, D, E).
“Olfactory three-needle” inhibited neuro-apoptosis and
neuroinflammation in the hippocampus of A-induced AD rats. (A)
Representative pictures of TUNEL staining
in the hippocampus, the green signal represented TUNEL positive nucleus, and the
blue signal represented a normal nucleus.
The white arrows point to
apoptotic cells. (B) Percent of
apoptotic cells in the hippocampus. (C, D,
E) The mRNA expression of IL-1, TNF-, and IL-6. Data are
expressed as means SD, *P 0.05, **P 0.01,
***P 0.001 vs. A treated rats.
3.4 “Olfactory three-needle” improved neuronal activity
synaptic plasticity in
the hippocampus of
The ratio of viability neurons in the CA1 area of the
hippocampus was decreased in
A-treated rats compared
to control or sham rats, and “olfactory three-needle” and
donepezil treatments improved the ratio of viability neurons
(Fig. 5A, B). Moreover, A-treated rats showed a pronounced
decrease in hippocampal synaptic protein expression, including
SYN, PSD95, and GAP43, compared with control or sham rats.
Remarkably, the levels of SYN, PSD95, and GAP43 were
significantly increased by “olfactory three-needle”
treatment compared with the A-treated
group. In contrast, there were no significant changes between the donepezil
treated group and the A-treated group
(Fig. 5C, D). Taken together, these results
demonstrated that “olfactory three-needle” treatment
substantially increased the expression of SYN, PSD95, and GAP43 in the
hippocampus of A-treated rats, suggesting that “olfactory
three-needle” likely alleviates the decline in MWM performance in these rats by
improving hippocampal synaptic plasticity.
“Olfactory three-needle” improved neuronal activity and
synaptic plasticity in the hippocampus of A-induced AD rats.
(A) Nissl staining for the hippocampal CA1 region of rats.
The red arrows point to viability neurons
with Nissl’s body. (B) The ratio of viability neurons in the CA1 area of rats.
(C) Representative western blot bands of SYN, PSD95, and
GAP43 in the hippocampus. (D) Relative protein expression of SYN, PSD95, and
GAP43. Data are expressed as means SD, *P 0.05, **P 0.01, ***P 0.001 vs. A
signaling in the hippocampus of
Western blot analysis showed a significant decrease in PI3K expression
and the activity levels of p-AKT/AKT and
A-treated rats compared with those in
control or sham rats, which suggested that the
PI3K/AKT/GSK-3 signaling in the
hippocampus was inhibited by A (Fig. 6). However, the protein
expression of PI3K (Fig. 6A, B), as well as the activity levels of p-AKT/AKT and
p-GSK-3/GSK-3 (Fig. 6A, C, D), were both enhanced by
“olfactory three-needle” treatment. In contrast, the
donepezil-treated rats showed no significant changes compared with
A-treated rats. Those data indicated that
the “olfactory three-needle” greatly enhanced
PI3K/AKT/GSK-3 signaling in the hippocampus of
A-treated rats, a potential mechanism the “olfactory
three-needle” to improve synaptic plasticity.
“Olfactory three-needle” enhanced PI3K/AKT/GSK-3
signaling in the hippocampus of A-induced AD rats. (A)
Representative western blot bands of PI3K, p-AKT, AKT,
p-GSK-3 and GSK-3 in
the hippocampus. (B) Relative protein expression of PI3K. (C)
The relative expression of p-AKT/AKT. (D) The relative expression of
p-GSK-3/GSK-3. Data are expressed as means SD,
*P 0.05, **P 0.01, ***P 0.001 vs.
A treated rats.
Synaptic dysfunction and neuronal loss are
the leading causes of the early development of AD ;
recovering synaptic dysfunction has been
proposed as a promising therapeutic approach for AD. The present study
three-needle” rescued the spatial learning and memory dysfunction through
improving neuro-apoptosis and neuro-inflammation, and synaptic plasticity in the
hippocampus of A-induced AD rats. Also, we show that
the “olfactory three-needle” treatment
enhanced the PI3K/AKT/GSK-3 signaling pathway, which
is involved in AD-related synaptic pathophysiology .
In general, these results suggest that
“olfactory three-needle” could improve
synaptic function probably via activating the
PI3K/AKT/GSK-3 signaling pathway.
been suggested as an effective therapeutic
intervention for AD in many clinical and animal studies [20, 27, 28]. Our
previous studies have reported that “olfactory three-needle” improved cognitive
deficits of SAMP8 mice through inhibiting A deposition, tau
phosphorylation, and oxidative stress damage in the hippocampal region .
acupoint of other electroacupuncture, GV29 in the nasal root and IL20 in the
bilateral nasal ala were the main three acupoints used in “olfactory
three-needle” therapy, which is implicated
in the olfactory system. In the multiple
steps of adult neurogenesis, the survival and integration of newborn neurons are
strongly affected by the olfactory system [29, 30].
generation of newborn neurons from the olfactory bulb after adulthood is a
remodeling of structural and functional
plasticity. Olfactory learning has been
shown to selectively promote remodeling of
both inhibitory and excitatory inputs
the deep dendritic domain of adult-born neurons
Cognitive deficits of
AD are attributable to the disruptions of
synaptic functions and neuronal loss,
correlated to the memory deficit’s severity
in AD .
we believe that the olfactory system’s stimulation through electroacupuncture may
be a valid alternative to improve synaptic plasticity and
neuronal survival of neurodegenerative diseases.
A peptide deposition and the
subsequent formation of amyloid plaques in the brain have been recognized as an
important event in the neuro-pathogenesis
of AD . The neurotoxicity of A
has been proven to involve intracellular hyperphosphorylated tau neurofibrillary
tangles. The activation of microglia-mediated neuroinflammation response resulted
in neuronal damage, apoptosis, and,
eventually, spatial memory impairment [34, 35]. In this study, we found that
“olfactory three-needle” not only
decreased A deposition in the hippocampus of
A-induced AD rat but
also inhibit the expression of tau
hyperphosphorylation and pro-inflammatory cytokines IL-1 and
TNF-, as well as the damage and apoptosis of hippocampal neurons.
Therefore, this clearance of A may
be one possible mechanism of “olfactory
three-needle”, stimulating the olfactory system in ameliorating cognition
degeneration in rats.
To explore the potential molecular mechanism of
“olfactory three-needle” to improve
A-induced learning and memory impairment, the expression of
synapse-related molecules forming the structural basis of
synaptic plasticity of learning and memory
were investigated. SYN and PSD95, which are the markers of
post-synaptic terminals, respectively, are
considered to represent the structural bases of plasticity underlying learning
and memory. SYN expression reflects the synaptic density and
distribution, which is implicated in the formation and reconstruction of synapsis
[36, 37]. SYN may directly affect the synaptic structure and influence synaptic
plasticity by regulating neurotransmitter release . PSD95, as the structural
basis of post-synaptic plasticity, is also
vital for information
transmission and memory formation . It is reported that the reduced level of
PSD95 in the hippocampus results in the impairment of learning and memory
function . GAP43 is a growth-associated phosphoprotein with high expression
levels during neuronic development, axonal
regeneration, and neuronic sprouting
Therefore, stimulating the olfactory system to increase
of synapse-related SYN, PSD95, and GAP43 of
hippocampal CA1 was the molecular basis for
“olfactory three-needle” to improve
synaptic plasticity and cognitive deficits in A-induced
As the most commonly used
anticholinesterase inhibitor for AD clinical treatment, Donepezil has been proven
to enhance neurogenesis, improve cognition, learning, and memory and prevent
aging progress by decreasing cholinergic loss neurons . Recent studies have
revealed that donepezil can inhibit the A deposition by inhibiting
acetylcholinesterase and its receptors, which plays an essential role in
promoting A peptides in AD neuro-pathogenesis . Many studies have
shown that donepezil possesses neuroprotective activity.
Still, it could not improve the structure and density of neural synapses [44, 45], consistent with our results. The
synapse-related protein expressions of
hippocampal CA1 were not significantly improved by donepezil in
A-induced AD rats.
However, “olfactory three-needle” could
improve synaptic function by promoting the expression of synapse-related
proteins. As a potential therapy for AD, the combination of electroacupuncture
and donepezil applied in AD is worthy of expectation and attention.
It has been proposed that GSK-3 activity might exert a central role in
A production, tau phosphorylation, and neurodegeneration during the
development of AD . Overexpression of
GSK-3 in the conditional
transgenic mice engendered tau hyperphosphorylation pathology and neuronal death
[46, 47]. Mainly, GSK-3 plays a
vital role in the stress response for neurons through
compromising the transcriptional activity
of the cAMP response element-binding (CREB) protein to
downregulate the expression levels of
brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and other
neurotrophic factors, which are important for the regulation of long-term memory
and the maintenance of synaptic plasticity, thereby contributing to
the neuronal degeneration of AD [48, 49].
The GSK-3 activity also has a critical role in inducing the differentiation and migration
of inflammatory cells. Thus, the secretion of pro-inflammatory cytokines 
could potentially deteriorate microglia-induced inflammatory responses in the
vicinity of A plaques.
Furthermore, GSK-3 is involved in modulating
critical steps of apoptotic signaling pathways . PI3K/AKT
signaling is the upstream regulator of GSK-3, which
phosphorylates and inhibits GSK-3 . Our current
results showed that “olfactory three-needle” treatment enhanced the activation
level of PI3K/AKT signaling and promoted the phosphorylation
inactivation of GSK-3. Thus,
“olfactory three-needle” enhanced
PI3K/AKT signaling to inhibit GSK-3, thereby increasing synapse-related
molecule expression, inhibiting inflammatory cytokines (IL-1 and
TNF-) and neuronal apoptosis, which may be a potential mechanism of the
“olfactory three-needle”-mediated protection of synaptic
we provided evidence that
“olfactory three-needle” could rescue the cognitive deficits of
A-induced AD rats by
improving synaptic plasticity, neuro-apoptosis and neuro-inflammation through
enhancing PI3K/AKT/GSK-3 signaling pathway.
YW and AZ conceived and designed the experiments. YW, HY, QW, BR, TG, TG and HC
performed the experiments. YG, LX and ZL analyzed the data. ZL and HL contributed
to the reagents and materials. YW and AZ wrote the manuscript.
Ethics approval and consent to participate
The experiment was carried out in the Shaanxi Key Laboratory of Acupuncture and
Medicine of the Shaanxi University of Chinese Medicine. All the animal
experiments were in accordance with the guidelines of the Shaanxi Province
Experimental Animal Management Committee with a permit number (AM2019-0161).
This research was supported by the National Natural Sciences Foundation of China
(Grant Nos. 82074552 and 81503667), the Shaanxi Science and Technology Department Project
(Grant No. 2018JM7041), and Program for Meridian-Viscera Correlationship
Innovative Research Team of Shaanxi University of Chinese Medicine (Grant No.
Conflict of interest
The authors declare no competing interests.