1. Introduction
Alzheimer’s disease (AD) is a common neurodegenerative disease that is
age-related and features progressive memory decline [1]. Its pathologic
characteristic is the deposition of extracellular amyloid- (A)
peptides and the formation of intracellular neurofibrillary tangles (NFTs).
Although the exact mechanism of AD is not fully clarified, accumulating evidence
supports a crucial role of A in the process of neurodegeneration and
cognitive changes of AD [2, 3, 4]. The overproduction and accumulation of A
in the brain are tightly related to neuronal cell death in AD. It is also
reported that the abnormal deposition of A peptides in the brain can
induce oxidative stress and neuroinflammation and initiate a cascade of
pathologic events, such as tau-hyper-phosphorylation, neurite degeneration and
neuronal loss [5, 6, 7].
A is the product of sequential proteolytic processing of A
precursor protein (APP) by and secretases.
A and A are the primary forms of A
peptide. Compared with the former, A is considered more
amyloidogenic and toxic in the brain [8]. A can be removed from the
brain through enzymatic degradation, cell uptake and blood-to-brain (BBB)
transport [9, 10]. Low-density lipoprotein receptor-related protein-1 (LRP1) and
the receptor for advanced glycation end products (RAGE) are the primary
transporters in BBB A transport. LRP1 modulates the outflow of
A from the brain tissues to peripheral blood, while RAGE is the primary
carrier of soluble A from peripheral blood circulation to the brain [11, 12]. There is growing evidence that the clearance of A is significantly
impaired in most patients with AD [13, 14]. There is reported down-regulation of
LRP1 and a decrease in the amount of A efflux in AD patients. Studies
also showed that the level of RAGE increases in many AD brain regions, and the
blockade of RAGE could alleviate the amyloidosis [13]. Therefore, regulating LRP1
and RAGE expression, responsible for A transportation, may be an
effective strategy to reduce the abnormal A deposition in AD.
To date, many therapies targeting A production have failed in phase III clinical trial, including -secretase and -secretase inhibitors and
A monoclonal antibody [2]. Traditional Chinese medicine research recently
attracted attention in AD treatment [15, 16, 17]. Danggui-Shaoyao-San (DSS) is a
well-known Chinese herbal medicine consisting of Radix angelicae sinensis, Radix
paeoniae alba, and Rhizoma Ligustici, Poria, Rhizoma atractylodis
macrocephalae and Rhizoma Alismatis.
Studies suggest that DSS may be an effective treatment for AD [18, 19, 20]. Our
earlier research demonstrated that DSS effectively alleviates cognitive
impairment in APP/PS1 mice [21]. We also found that DSS can enhance the
expression of LRP1 in rats with vascular cognitive dysfunction [22]. Although
previous studies have proved DSS is effective to reduce the neurotoxicity induced
by A, it remains unknown whether DSS is effective to alleviate the
A deposition of AD, and if so, what is the molecular mechanism. We aim
to examine the effects of DSS on neuronal damage and amyloidosis and explore the
possible molecular mechanism of DSS treatment on AD in an APP/PS1 transgenic mice
model.
2. Materials and methods
2.1 Antibodies and reagents
The primary antibodies: Rabbit monoclonal A1-42 (#39377), Rabbit
monoclonal anti-APP (#32136), Rabbit monoclonal anti-PS1 (#76083), Rabbit
monoclonal anti-BACE1 (#183612), Rabbit monoclonal anti-PSD95 (#18258), Rabbit
polyclonal anti-NGF (#6199), Rabbit monoclonal anti-BDNF (#108319), Rabbit
polyclonal anti-NEP (#227195), Rabbit monoclonal anti-IDE (#133561), Rabbit
polyclonal anti-RAGE (#3611) and Rabbit monoclonal anti-LRP1 (#92544) were
purchased from Abcam, Inc. Mouse monoclonal anti--actin (#A1978) was
purchased from Sigma-Aldrich. Rabbit monoclonal anti-SYN (#4329s) and all
secondary antibodies: Horse anti-mouse (#7076), Goat anti-rabbit IgG HRP-linked
antibody (#7074) and Alexa Fluor 594 Goat anti-Rabbit IgG (#8889) were
purchased from Cell Signaling Technology, Inc. Thioflavin S (ThS) was purchased
from Sigma-Aldrich.
2.2 Preparation of DSS
The six raw herbs of DSS, including Radix angelicae sinensis,
Radix paeoniae alba, Rhizoma Ligustici, Poria, Rhizoma atractylodis macrocephalae
and Rhizoma Alismatis were obtained from Kangmei pharmaceutical co.,
ltd (Guangdong, P.R. China). They were mixed at a ratio of 3 : 16 : 8 : 4 : 4 : 8.
The DSS extraction process was completed based on our previous study [21]. The
mixed dried herbs were briefly immersed with 8 times (v/w) distilled water for
1 hour and were then decocted for 1 hour. The filtrate was collected, and the
residue was again decocted with six times (v/w) distilled water for 1 h. The
filtrates were mixed, then concentrated. Finally, it was determined that 1 mL of
DSS contained 0.64 g of crude herbs. The mixture was stored at -20 C
and then recomposed with distilled water for use.
2.3 Animals and drug treatment
Male APPswe/PSEN1dE9 (APP/PS1) double-transgenic mice and wild-type mice (Non-Tg
mice, WT) were 3-months old. All mice were purchased from Nanjing University
(Nanjing, P. R. China). All mice were reared in a standard SPF environment (22 C-25 C ,
40% - 60% relative humidity).
After three months of feeding, mice in APP/PS1 group were divided into three
groups randomly: APP/PS1 group (model group), APP/PS1+DSS group (DSS group), and
an APP/PS1 + Donepezil group (Donepezil group). The Donepezil group (3 mg/kg/day)
was used as a positive drug group, and the wild-type mice were set as the control
group (n = 10~12 per group). The dose of DSS 6.4 g/kg/d) used in
the present study was based on our previous study [21]. Mice in the drug
treatment groups received DSS or Donepezil treatment by oral gavage once a day
for 8 weeks. Mice in control and model groups were gavage with distilled water.
2.4 Behavioral measurement
2.4.1 Morris water maze (MWM) test
The Morris water maze test protocols were carried out with our previous studies
with few modifications [23]. The Morris water maze consists of a circular pool
with a circular platform. The circular pool was 120 cm in diameter and 50 cm
deep. The swimming pool was divided into four virtual quadrants. A platform,
which is 10 cm in diameter and 35 cm in height, was placed
in the middle of one of the quadrants. The platform is 1 cm below the water
surface. The water in the pool was controlled at 22 1 C . After drug
treatment, mice in all groups were given an acquisition trail for 4 consecutive
days. For each trial, the mouse was sent into the water at starting points and
allowed to swim freely until they found the hidden platform within 60 s. The time
to reach the platform is defined as escape latency. If the mouse failed to find
the platform with 60 s, the escape latency was recorded as 60 s. The escape
latency and cumulative path were recorded with the corresponding software
(Guangzhou Feidi Biology Technology Co., P.R. China). On the fifth day, the platform
was removed, and the mouse was released at the opposite point of the platform
positioned. The mouse was also allowed to swim 60 s, the time spent in the target
quadrant and the numbers of each mouse to cross the platform were recorded.
2.4.2 Y-maze test
The Y-maze test was conducted as described in our previous study [21]. The
Y-maze was made of three identical arms. Each arm in the Y maze was 35 cm in
length, 5 cm in width and 15 cm in height. The three arms were placed at
120 from each other. The mice were gently placed at the end part
of one arm. The times and sequences of each mouse entering the three arms in a
5-min period were recorded. Alternation was defined as the continuous entries
into the three arms, with no overlapping triplets, such as ABC, CAB or BCA. The
following formula calculated the percentage of alternation:
Alternation% = [(times of alternations)/(total times of arm entries - 2)]
100.
2.4.3 Open field test
The open field apparatus was composed of a black open field box
(50 50 50 cm chamber). The box was divided into twenty-five
squares of 10 10 cm. The central areas were defined as the nine square
areas in the middle of the open field. Mice were placed at the open field and
allowed to explore for 30 min. The time traveled in the central area was recorded
with a video-tracking system (Shanghai XinRuan Information Technology. Co. Ltd,
P.R. China).
2.5 Tissue preparations
After behavioral examination, all mice were anesthetized with a phenobarbitone
(0.5 mg/kg, i.p). All mice were transcardially perfused with 50 mL ice-cold
phosphate-buffered saline (PBS) and sacrificed by decapitation. After that, the
brains were taken out immediately, then the hippocampus and the cortex were
separated on an ice clod board, and they were gathered and stored at -80 C
subsequently for western blotting assays. Mice used for morphological examination
also received an additional 4% paraformaldehyde (PFA) perfusion. Four brain
samples were gathered per group and then fixed in 4% PFA overnight; following
that, they were subsequently transferred to 15% and 30% sucrose solutions until
they sank to the bottom. Coronal sections (25 m) were cut by a freezing
microtome (Leica, Germany) and stored in an antifreeze solution.
2.6 Thioflavin S (ThS) staining
ThS staining detected the amyloid plaques. The brain slices were briefly
incubated in a 0.1% ThS (Sigma, USA) solution for 10 min at room
temperature. Then the slide was eluted with 80% and 70% ethanol for 1 min,
respectively. After being rinsed 3 times with PBS, the slide was allowed to dry
in the dark. The positive plaques with green fluorescence were visualized under a
fluorescent microscope (Model DP80, Olympus). Five stained sections of each
animal were used to analyze.
2.7 Nissl staining
After 10 min of staining with purple tar dye solution (Beyotime, P.R. China), the
slices were incubated in PBS three times for 10 min each time. The samples were
then observed under a microscope (Model DP80, Olympus). Four slices per brain
were used for Nissl-positive cell counting. Four fields of Nissl-positive cells
in the cortex and hippocampal CA1, CA3 and DG were chosen randomly at
400 magnification to analyze.
2.8 Immunofluorescence
The immunofluorescence staining protocols were performed as previously described
with few modifications [24]. The brain sections were incubated with citrate
buffer at 90 C for 30 min and then cooled to room temperature. After being washed
3 times in PBS, the sections were blocked in blocking buffer (containing 0.3%
Triton-X-100 and 10% goat serum in PBS) for 1 h. The slides were then labeled
with primary antibody: anti-A (1 : 200) for 48 h at 4
C. After being washed three times in PBS (10 min each), the slides were
incubated with Alexa Fluor 594 Goat anti-Rabbit IgG (1 : 500) for 1 h. The sections
were washed once more before the addition of DIPA. Finally, the sections were
fixed with an anti-fade mounting medium and imaged using a fluorescence
microscope (Model DP80, Olympus).
2.9 Western blot assay
Western blot assay protocols were performed as previously described with few
modifications [25]. The cortex samples were briefly lysed by SDS lysis buffer
(10% SDS, 0.5 M tris-HCl PH6.8 and glycerin) containing protease inhibitor and
phosphatase inhibitors. They were subsequently sonicated for 2 min with a probe
sonicator and allowed to stand on ice for about 30 min to lyse fully. After that,
they were centrifuged at 4 C for 10 min. The supernatant was collected, and BCA
assays determined the protein concentration. Following that, they were adjusted
with uniform concentration.
The total protein (30 g) were separated with 10%-12% SDS-PAGE gel and
subsequently transferred to PVDF membranes at 300 mA for 2 h. Following that, the
membranes were incubated with corresponding primary antibodies: anti-APP
(1 : 3000), anti-PS1 (1 : 2500), anti-BACE1 (1 : 3000), anti-PSD95 (1 : 3000) , anti-SYN
(1 : 3000), anti-NGF (1 : 3000), anti-BDNF (1 : 3000), anti-NEP (1 : 3000), anti-IDE
(1 : 3000), anti-RAGE (1 : 3000) and anti-LRP1 (1 : 4000) overnight before incubation
with 7% skim milk in 1 TBST for 1 h. After three incubations in TBST of
10 min each, the membranes were incubated with horseradish peroxidase
(HRP)-conjugated anti-mouse (1 : 4000) or Goat anti-rabbit IgG (1 : 4000) for 2 h at
room temperature. Then, the blots were visualized by using a chemiluminescence
kit (WBKLS0500, Millipore). The densitometry of the band was analyzed with Image
J software (NIH, Bethesda, Maryland).
2.10 Statistic analysis
Statistic Package for Social Science (SPSS, v.22.0) statistical software was
used for the data analysis. Repeated measures analysis of variance (ANOVA) was
used to analyze escape latency data in the Morris water maze. One-way ANOVA
followed by post hoc Bonferroni or Tamhane’s T2 test for the analysis of other
data. The statistical significance was set at P 0.05.
3. Results
3.1 DSS ameliorates the cognitive disorders in APP/PS1 mice
The cognitive impairment of APP/PS1 mice was determined by behavioral testing
following DSS treatment. As shown in Fig. 1A, there is a substantial increase in
escape latency in mice’s navigation test in the model group (P 0.05).
In contrast, DSS treatment remarkably reduced the navigation test’s escape
latency (P 0.05). However, Donepezil treatment did not affect escape
latency. The swimming path of mice of each group on the fourth day is shown in
Fig. 1B. On the fourth day, mice’s path in the APP/PS1 group is complex, while
mice in the DSS and Donepezil group find the platform easily(Fig. 1B). As shown in
Fig. 1C,D mice in the APP/PS1 group exhibited a shorter time in the target
quadrant and a lower frequency of crossing the probe test platform than the model
group (P 0.01). However, DSS and Donepezil treatment could improve
this condition (P 0.05 or P 0.01). These data collectively
indicated that DSS exerted beneficial effects on learning and memory in APP/PS1
mice.
The Y-maze test was used to assess working memory. We found that mice in the
APP/PS1 group presented a decrease of alternation percentage relative to those in
the control group. In contrast, the alternation percentage increased remarkably
after DSS treatment (P 0.01, Fig. 1E). The open-field test evaluated
Anxiety-related behavior. We found that mice in the APP/PS1 group exhibited an
approximately 50% decrease in movements in the central area in comparison with
mice in the control group (P 0.01), while both DSS and Donepezil
enhanced movements in the central area of the open field (P 0.05 or
P 0.01, Fig. 1F). These data indicated that DSS could ameliorate the
anxiety-related behavior of APP/PS1 mice.
Fig. 1.
DSS alleviates the cognitive disorders in APP/PS1 mice.
(A–D) Morris water maze test: Escape latency (A) and pathway (B)
during platform trials, time spent in the target quadrant (C) and the number of
crossing through the platform (D) in the probe test. Percentage of alternation
(E) in the Y-maze test. The time spent in the central area (F) in the open field
test. The escape latency was shown as mean SEM. Other data were shown as
mean SD. Compared with the control group, P 0.05 or
P 0.01, compared with the model group, *P 0.05 or
**P 0.01. N = 10~12 in each group.
3.2 DSS alleviates the neuronal degeneration of APP/PS1 mouse brain
The effect of DSS on the neuronal degeneration of AD was assessed with
Nissl staining. We observed numerous vacuoles (the Nissl bodies were disappeared,
which was indicated with yellow arrows) in the neurons of APP/PS1 mouse brains
(not only the cortex area, but also hippocampus CA1, CA3 and DG area), and the
number of Nissl-positive neurons in these regions were reduced remarkably
(P 0.01, Fig. 2A). This suggests the occurrence of neuronal loss and
necrosis in APP/PS1 mice. In contrast, DSS and Donepezil treatment decreased the
vacuoles and increased the Nissl-positive neurons in APP/PS1 mice
(P 0.01, Fig. 2A,B).
Also, the protein levels of PSD95 and SYN, which are related to the neuronal
development and synaptic function and the protein levels of nerve growth factor
(NGF) and brain-derived neurotrophic factor (BDNF), which belong to nerve growth
factors, were detected by Western blotting (Fig. 2C). We found that PSD95 and SYN
levels in the cortex of APP/PS1 mice were reduced compared with that in the
control group (P 0.01, Fig. 2D). Similarly, the expression of NGF and
BNDF was also down-regulated in APP/PS1 mice (P 0.01, Fig. 2E).
Interestingly, DSS and Donepezil treatment enhanced these proteins’ levels in
APP/PS1 mice (P 0.01, Fig. 2D,E). These results indicate that DSS
can ameliorate neuronal and synaptic damage in the APP/PS1 mouse brain.
Fig. 2.
DSS ameliorates the neuronal degeneration in the brain of
APP/PS1 mice. Representative images and quantification of Nissl-positive
cells in the cortex and hippocampal CA1, CA3 and DG region by Nissl staining
(A–B). Scale bar: 50 m (n = 4). The protein bands and quantification data
of PSD95, SYN, NGF and BDNF in the brain (C–E) (n = 3 per group). All data were
shown as mean SD. Compared with the control group, P 0.05 or
P 0.01, compared with the model group, *P 0.05 or **P 0.01.
3.3 DSS mitigates the amyloidosis in the brain of APP/PS1 mice
We detected A plaque deposition and A levels in the
brain by Ths staining and immunofluorescence assays (Fig. 3). We observed a large
amount of A plaque deposition in APP/PS1 mice (Fig. 3A).
Correspondingly, the number of A positive plaques in the
brains of APP/PS1 mice was also increased (Fig. 3B). However, following an 8-week
administration of DSS and Donepezil, the deposition of A plaques and the
number of A immunoreactivity plaques was markedly reduced
compared to that in APP/PS1 group (P 0.01, Fig. 3C,D). These data
indicate that DSS treatment can alleviate amyloidosis in APP/PS1 mice.
Fig. 3.
DSS reduces A amyloidosis in the brain of
APP/PS1 mice. Representative images of A plaques (A) and
A immunoreactivity plaques (B) were examined by Ths staining
and immunohistochemistry assays. Scale bar: 200 m (n = 4, four sections per
mice). The statistical data of A and A immunoreactivity plaques (C–D). All data were shown as mean SD.
Compared with the control group, P 0.05 or P 0.01, compared
with the model group, *P 0.05 or **P 0.01.
3.4 The effect of DSS treatment on production, degeneration and
transport of A of APP/PS1 mice
Proteins including APP, PS1, BACE1 for A production, NEP and IDE for
A degeneration, RAGE and LRP1 for A transportation were
detected by Western blot.
We observed the level of RAGE, which is responsible for A influx into
the brain, was increased, whereas the level of LRP1, which is responsible for
A transferring out of the brain, was reduced in APP/PS1 mice
(P 0.01, Fig. 4A,B). It is noteworthy that DSS and Donepezil
intervention down-regulated the level of RAGE and up-regulated the level of LRP1
in APP/PS1 mice (P 0.01, Fig. 4A,B). We also observed increased
protein expression of APP, PS1 and BACE1 (P 0.01, Fig. 5A,B) and
decreased protein levels of NEP and IDE in APP/PS1 mice (P 0.01,
Fig. 6A,B). Unfortunately, the protein levels of APP, PS1, BACE1 in the
A production pathway, NEP and IDE in the A degeneration pathway
were not alerted following DSS treatment (Fig. 5 and Fig. 6). These findings
indicate that DSS might have no effect on A production and degeneration,
yet can regulate the transport of A.
Fig. 4.
DSS increases the level of LRP1 and decreases RAGE level
in APP/PS1 mice’s brain. The protein bands and quantification data of
LRP1 and RAGE (A–B) in the cortex (n = 3 per group). All data were shown as mean
SD. Compared with the control group, P 0.05 or
P 0.01, compared with the model group, *P 0.05 or **P 0.01.
Fig. 5.
Effects of DSS on the main protein of A
production in APP/PS1 mice’s brain. The protein bands and
quantification data of APP, PS1 and BACE1 (A–B) in the cortex (n = 3 per group).
All data were shown as mean SD. Compared with the control group,
P 0.05 or P 0.01.
Fig. 6.
Effects of DSS on the main protein of A
degeneration in APP/PS1 mice’s brain. The protein bands and
quantification data of NEP and IDE (A–B) in the cortex (n = 3 per group). All
data were shown as mean SD. Compared with the control group, P 0.05 or P 0.01.
4. Discussion
Several studies have demonstrated that DSS is effective against AD. However, the
mechanisms of DSS in AD are still largely unclear. This study explored the
possible mechanisms of DSS on amyloidosis and neuronal degeneration in APP/PS1
transgenic mice. We found DSS alleviated the cognitive deficits and neuronal
degeneration of APP/PS1 mice. Moreover, DSS reduced the amyloidosis in the
APP/PS1 mouse brain and down-regulated the level of RAGE and up-regulated the
level of LRP1. However, DSS did not alter the primary protein levels of
A production and degeneration. These findings collectively demonstrate
DSS ameliorates the amyloidosis, and neuronal degeneration of AD is associated
mainly with its up-regulation of LRP1 and down-regulation of RAGE.
The APP/PS1 transgenic mouse is a classic model to mimic the pathology of
A deposition and behavioral changes of AD [26]. This study evaluated the
effects of DSS on cognitive deficits and neuronal degeneration in APP/PS1 mice.
As a result, we found DSS could ameliorate the cognitive deficits, including
spatial memory, working memory and anxiety-like behavior of APP/PS1 mice, which
is consistent with previous reports in AD animal models [21, 27, 28, 29]. Meanwhile, we
also observed that the improvement effect of DSS on neuronal survival and
neuronal function in APP/PS1 mice. DSS promoted neuronal survival by decreasing
vacuoles in neurons and increased the overall number of Nissl-positive neurons in
APP/PS1 mouse brains. Also, DSS enhanced the protein levels of PSD95 and SYN,
which are near related to neuronal development and the protein expressions of NGF
and BDNF, which are belong to nerve growth factors of APP/PS1 mice. These results
collectively indicate the potential of DSS to treat AD.
Accumulating studies indicated that the imbalance between A production
and clearance leads to extracellular A accumulation, which is considered
to trigger AD’s cognitive dysfunction [30, 31]. However, clinical trials that
target inhibiting A production have been declared a failure [2].
Nowadays, promoting A clearance to reduce its aggregation in the brain
to alleviate the amyloidosis and cognitive deficits have attracted more and more
attention [14, 32].
We found DSS treatment can alleviate the deposition of A plaques and
A levels in the brain of APP/PS1mice. LRP1 and RAGE are the
primary transport receptors of A in BBB, binding A directly. It
is reported that there is an impairment of A transportation of BBB in
AD. In animal and AD patients, the level of LRP1 is decreased [33, 34]. Studies
in AD animals studies have shown the decreased level of LRP1 could affect
A efflux across the BBB, leading to an increase of plasma A
levels and further aggravating cognitive deficits [35].
In contrast, the level of RAGE is up-regulated in AD. Many studies have
demonstrated that RAGE blockade could alleviate amyloidosis and cognitive
deficits [32, 36, 37]. Our previous studies indicated that DSS increases the
level of LRP1 and decreases the level of RAGE in a vascular cognitive deficit
rat [22]. Therefore, we sought to further investigate whether DSS affects RAGE and
LRP1 in APP/PS1 mice.
As a result, we found that DSS up-regulated the level of LRP1 and down-regulated
RAGE level in APP/PS1 mice. We also examined the A production pathway
(including APP, PS1 and BACE1) and A degeneration pathway (including NEP
and IDE). However, a two-month DSS treatment showed no effects on protein levels
related to A generation and A degeneration in this study. These
findings collectively indicate that DSS can alleviate the neuronal degeneration
and amyloidosis of AD, which may be through up-regulation of LRP1 and
down-regulation of RAGE (Fig. 7).
Fig. 7.
Schematic diagram of the mechanism
for DSS to improve neuronal degeneration and amyloidosis in APP/PS1 mice. DSS alleviates the neuronal degeneration and amyloidosis of AD, which may be through up-regulation of LRP1 and down-regulation of RAGE.
Due to the lack of effective treatment for AD currently, we selected donepezil,
specific central acetylcholinesterase (AChE) inhibitor used in the clinic for AD
therapy, as a positive control drug present study. Recently, studies also showed
that donepezil is also useful in reducing the amyloidosis and A
accumulation of AD [38, 39, 40].
We found that DSS effectively alleviates the amyloidosis and neuronal
degeneration in the APP/PS1 transgenic mouse, which could achieve the same effect
as Donepezil. This indicates that DSS can be used in the clinical treatment of
AD. However, there are some limitations. First, due to the lack of RAGE
overexpression or LPR1 knockout, it is unclear whether DSS ameliorates the
neuronal injury and amyloidosis of AD directly via the upregulation of LRP1 and
downregulation of RAGE. Second, apart from neurons, RAGE and LRP1 are extensively
expressed in microglia and endothelial cells [41]. To further
understand and explore the therapeutic targets of DSS for modulating the
transport of A in AD, future studies should focus on the co-expression
of RAGE and LRP1with A with microglia and endothelial cells.
5. Conclusions
In conclusion, our findings demonstrated that DSS could ameliorate neuronal
injury and amyloidosis related to AD. Also, the effects of DSS on amyloidosis in
AD may be mediated via up-regulation of LRP1 and down-regulation of RAGE. Our
findings indicate that DSS is an effective AD treatment, and increased focus on
regulating A transport signaling may be productive.
Author contributions
QW and QD designed the present research. CY and YSM
wrote the manuscript. CY, YSM, HFC, YHH,
SLL conducted the experiments. XC conducted the data analysis.
HW and SQH revised the grammar of the manuscript.
Ethics approval and consent to participate
The present research was approved by the Ethics Committee of laboratory animals
of Guangzhou University of Chinese Medicine (No.00107331).
Acknowledgment
We thank two anonymous reviewers for excellent criticism of the article.
Funding
This work was supported by Key laboratory project of colleges and universities in Guangdong province (No. 2019KSYS005), Guangzhou Science Technology and Innovation Commission Technology Research Projects (No. 201805010005, No. 201803010047), Guangdong province science and technology plan international cooperation project (No.2020A0505100052) and National Natural Science Foundation of P.R. China (No.81673627, No.81704131, No.81904168).
Conflict of interest
The authors declare that there is no conflict of interest.