The neuroprotective role of Panax notoginseng saponins in APP/PS1 transgenic mice through the modulation of cerebrovascular

ARTICLE
TMR | September 2020 | vol. 5 | no. 5 | 355
doi: 10.12032/TMR20200227168
Submit a manuscript: https://www.tmrjournals.com/tmr
Traditional Chinese Medicine
The neuroprotective role of Panax notoginseng saponins in APP/PS1
transgenic mice through the modulation of cerebrovascular
Yan Tan1#
, Chen-Chen Song1#
, Zi-Hui Xu2
, Fang He1
, Ya-Li Zhang1
, Ya-Lei Wang1
, Xue Wang1
, Liang-Qin Wan1
, Xu Wang1
,
Ling-Ling Qin1
, Tong-Hua Liu1*
, Qian Hua1*
#
These authors are co-first authors on this work.
1
Beijing University of Chinese Medicine, Beijing 100029, China. 2
Tianjin College, University of Science and Technology
Beijing, Tianjin 301830, China.
*Corresponding to: Tong-Hua Liu. Beijing University of Chinese Medicine, Beijing 100029, China. E-mail:
thliu@vip.163.com; Qian Hua. Beijing University of Chinese Medicine, Beijing 100029, China. E-mail: hqianz@aliyun.com.
Highlights
By modulating cerebrovascular function, PNS can reduce the deposition of amyloid plaques and exhibit the
role of neuroprotection in a preventive strategy, possibly via targeting the vascular related proteins such as
platelet activating factor receptor and vasopressin V1a receptor.
Traditionality
Panax notoginseng is known as Sanqi in Chinese and it is described to removing blood stasis (poor blood
circulation) for promoting tissue regeneration in traditional Chinese medicine. Physician Li Shizhen stated
in Bencao Gangmu (Compendium of Materia Medica, composed during the year of 1552 to 1578) that
"Sanqi is a herb referred to the blood phase of the yang-ming meridian and jue-yin meridians (two of the
twelve meridians in traditional Chinese medicine that are mainly used to run energy and blood, connect
internal and external organs, and communicate with each other), it can be used in all diseases related with
vascular". Panax notoginseng saponins is the main active compound extracted from the root of Panax
notoginseng, which can promote blood circulation and angiogenesis. A bench of clinical trials has been
administrated for the treatment of panax notoginseng saponins in hypertensive intracerebral hemorrhage and
ischemic stroke.

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doi: 10.12032/TMR20200227168
Abstract
Background: Panax notoginseng saponins (PNS) is extracted from Sanqi (Panax notoginseng), which is a valuable
herb and has been widely used in traditional Chinese medicine for the treatment of cerebrovascular diseases and
pain. PNS has been proved to promote blood circulation and angiogenesis by inhibiting platelet aggregation. In our
previous study, PNS accompanied with geniposide can prevent Alzheimer’s disease (AD). However, the efficacy of
PNS and its potential mechanism in AD remain unclear. Methods: Amyloid precursor protein/presenilin-1
(APP/PS1) transgenic (Tg) mice were used as AD-like animal models. Wild-type mice and APP/PS1 transgenic
were administrated with saline solution while mice in PNS treatment group were administrated with PNS at a
dosage of 17 mg/kg/day for three months. Morris water maze (MWM) was applied to evaluate the spatial learning
and memory and step-down test was used to evaluate the cognitive function. 1% Thioflavin-S staining was used to
calculate the average number amyloid plaques in cortex and hippocampus. CD31 staining was detected to observe
the density of cerebrovascular in hippocampus areas and CD105 staining was further detected to evaluate
angiogenesis. Laser Doppler PeriFlux 5000 was further measured the change of cerebrovascular blood flow.
ChemDraw was used to draw the molecular structures of five main ingredients of PNS. AlzPlatform were used to
estimate the potential targets of PNS. Results: By a bench of behavioral tests, PNS showed a better tendency in
proving cognitive functions. In addition, the amyloid plaques in both cortex and hippocampus were significantly
reduced after PNS intervention (P < 0.05 and P < 0.001 respectively). Furthermore, the density of cerebrovascular
in the hippocampus areas was increased under PNS administration (P < 0.001), which accompanied with
angiogenesis in dentate gyrus areas and cerebrovascular blood flow promotion (P < 0.05). By AlzPlatform docking
serve, we screened five major ingredients of PNS—R1, Rd, Rb1, Re and Rg1. These screening data suggested that
vascular related proteins could be the one of potential targets of PNS, such as platelet activating factor receptor and
vasopressin V1a receptor. Conclusion: By modulating cerebrovascular function, PNS can reduce the deposition of
amyloid plaques and exhibit the role of neuroprotection in a preventive strategy.
Keywords: Alzheimer’s disease, Angiogenesis, Behavioral test, Cerebrovascular blood flow, Panax notoginseng
saponins
Acknowledgments:
This study was supported by grants of National Natural Science Foundation of China Project (Project No.
81904049), Regional Collaborative Innovation Center of Tibetan Medicine (Project No. 2017XTCX012,
2018XTCX014) and Young Elite Scientists Sponsorship Program by CAST (Project No.
CACM-2018-QNRCC2-C06).
Abbreviations:
PNS, Panax notoginseng saponins; AD, Alzheimer’s disease; APP/PS1, amyloid precursor protein/presenilin-1;
Tg, transgenic; MWM, morris water maze; Aβ, amyloid-β; TCM, Traditional Chinese Medicine; GP, geniposide;
WT, wild-type; PU, perfusion unit; HTDocking, high-throughput docking; DG, dentate gyrus; PAFR, platelet
activating factor receptor; V1AR, vasopressin V1a receptor.
Competing interests:
There are no conflicts of interest.
Citation:
Yan Tan, Chen-Chen Song, Zi-Hui Xu, et al. The neuroprotective role of Panax notoginseng saponins in
APP/PS1 transgenic mice through the modulation of cerebrovascular. Traditional Medicine Research 2020, 5 (5):
355–367.
Executive editor: Nuo-Xi Pi.
Submitted: 24 December 2019, Accepted: 24 February 2020, Online: 23 March 2020.

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doi: 10.12032/TMR20200227168
Background
Alzheimer disease (AD) is an age-related
neurodegenerative disorder, the progress of which
usually starts slowly and worsens over time [1]. At the
early stage of AD, patients usually have no evident
clinical syndromes with normal cognition. But
according to epidemic statistics, a series of
pathophysiological changes observed at preclinical
phase of AD, such as the gradual accumulation of
amyloid-β (Aβ), the changes of cerebral
hemodynamics and hippocampal atrophy [2, 3]. Since
the failure of the present clinical trials target on later
stages of AD, the treatment window should be
re-considered to shift to an early stage. Currently, Food
and Drug Administration (FDA) proposed that “in the
clinical development of drugs for the treatment of the
stages of sporadic AD that occur before the onset of
overt dementia (collectively referred to as early
AD …)…” in Early Alzheimer’s Disease: Developing
Drugs for Treatment published in February 2018. This
guideline provides us two important issues: preventive
strategies and early diagnosis.
In China, Traditional Chinese medicine (TCM) has a
long history in preventing and treating cognitive
decline [4–6]. Panax notoginseng is known as Sanqi in
Chinese, the root of which has plenty of medicinal
properties. In TCM, it is described that “removing
blood stasis (poor blood circulation) for promoting
tissue regeneration”, and physician Li Shizhen stated
in Bencao Gangmu (Compendium of Materia Medica,
composed during the year of 1552 to 1578) that "Sanqi
is a herb referred to the blood phase of the yang-ming
meridian and jue-yin meridians (two of the twelve
meridians in traditional Chinese medicine that are
mainly used to run energy and blood, connect internal
and external organs, and communicate with each other),
it can be used in all diseases related with vascular"; he
also called Sanqi was “not to be exchanged even for
gold”, showing its preciousness and importance in
medication. Panax notoginseng saponins (PNS) is the
main active compound extracted from the root of
Panax notoginseng, which can promote blood
circulation and angiogenesis [7, 8].
PNS has the effect of inhibiting platelet aggregation,
increasing the heart and cerebral blood flow and plays
a protective role in cardiovascular and cerebrovascular
diseases [7, 9]. A bench of clinical trials has been
administrated for the treatment of PNS in hypertensive
intracerebral hemorrhage and ischemic stroke (Table 1).
In addition, in our previous study, we found PNS
accompanied with geniposide (GP) prevented
cognitive decline in different AD animal models [10,
11]; in vitro, PNS protected endothelial cells from
oxidative stress, suggesting that the neuroprotection
role of PNS may work on vascular. Here, in order to
evaluate the preventive role of PNS in AD, we made
use of both virtual screening methods and amyloid
precursor protein/presenilin-1 (APP/PS1) transgenic
(Tg) mice to explore its potential targets and efficacy
in neuroprotection.
Materials and methods
Animal
APP/PS1 Tg mice were used as AD-like animal
models. The double Tg mice expressing a human
amyloid precursor protein (HuAPP695swe) and a
mutant human presenilin 1 (PS1-dE9), both associated
with early-onset AD. The typical pathological products,
such as Aβ40 and Aβ42 were gradually accumulated
around 4-month age; the deposition of amyloid plaques
can be detected around 6-month age. Accordingly, the
cognitive impairments can be observed at 7-month age
[12, 13]. This model is useful in studying neurological
disorders of the brain, specifically AD and amyloid
plaque formation. In this study, 4-month age of
APP/PS1 Tg mice and their littermates of wild-type
(WT) mice were purchased from Model Animal
Research Center of Nanjing University (Cat. No.
N000175). These Tg mice were randomly divided into
two groups—APP/PS1 group and PNS treatment group
(n = 12). Littermate WT mice were considered as
control, WT group (n = 14). The animals were
maintained at a constant temperature on a 12-h
light-dark cycle with accessing to food and water
freely. All procedures concerning care, treatment and
dissection are in accordance with the Animal Ethics
Committee of Beijing University of Chinese Medicine
(No. BUCM-2016103101-1008).
Administration
Based on previous studies [11], the dosage of PNS was
converted according to body weight index between
human being and mouse, orally administrating with a
dosage of 17 mg/kg/day dissolved in saline solution.
WT and APP/PS1 groups were administrated with
same volumes of saline solution. A preventive strategy
has been applied that the administration started at
four-month age, and lasting for 3-month. PNS was
purchased from National Institute for the Control of
Pharmaceutical and Biological Products (Cat. No.
84527, UV95%, China).
Morris water maze
To evaluate hippocampal dependent spatial learning
and memory, mice were tested in a standard morris
water maze (MWM) task. Briefly, mice were trained 4
trials per day in 6 consecutive days. During the
training section, mice were placed on the hidden
platform for 10 sec at the end of each trial. 24 hours
after the training section, a testing trial was carried out
with the platform removed. Mice started the test from
the opposite quadrant, and escape latency, percentage
of dwelling time in the target quadrant were measured.

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doi: 10.12032/TMR20200227168
Step-down test
To evaluate the fear memory, mice were tested in the
step-down test. A inhibitory avoidance apparatus was
used. Briefly, during training test, mice were placed
into the apparatus for a 5-minute habituation. Then, an
electric current of 36V was delivered to the copper grid,
and lasted for another 5 min. The escape latency to
step up to the platform with all paws was measured. 48
hours after the training test, a retention test was carried
out without electronic shock. The escape latency of
step down to the floor with all paws was measured.
Brain tissue preparation
Mice were deeply anesthetized with a cocktail of
1.25% avertin (0.6 mL/20 g) via intraperitoneal
injection, and transcardially perfused with ice-cold
phosphate-buffered saline, followed by ice-cold 4%
paraformaldehyde (PFA) in PBS. Brains were carefully
eviscerated and placed in 4% PFA at 4°C for overnight,
following by 15% sucrose and 30% sucrose at 4℃ for
overnight. Brains were embedded with OCT
compound (Tissue Tek, Japan) in cryostat, and coronal
cut at 10 μm thickness and mounted on positively
charged microscope slides. The slides were kept at
-80°C until used.
Amyloid plaques deposition
Cryostat slices prepared above were collected and
stained by 1% Thioflavin-S (Sigma, Cat. No. T1867,
USA). Briefly, three consecutive sections at three
positions (15 slices interval) were picked, and
calculated the average number amyloid plaques (in
cortex and hippocampus, respectively) of each position,
as average (Ave) 1, Ave 2 and Ave 3. Then, a sum total
from Ave 1 to Ave 3 was calculated as one piece of
data for one hemisphere. There were three animals in
each group. All images were obtained by inverted
fluorescence microscope (Olympus, CKX41, Japan).
10× or 20× objective lens have been used for each field.
The strategy for data collection and calculation per
animal as Figure 1.
Figure1 A brief strategy for data collection and calculation per animal. Upper panel: sagittal plates were
collected for amyloid plaque deposition (left-up corner). On the right hemisphere, we targeted on the
parietal-temporal lobes and the hippocampus beneath (red box). Every 15 slices interval, 3 positions were collected.
Each position was picked serial three slices. Lower panel: average number of each position was calculated, and Ave
1 for position 1, Ave 2 for position 2, and Ave 3 for position 3. Then, a sum total from Ave 1 to Ave 3 was
calculated as one piece of data as one hemisphere. A, anterior; P, posterior; Ave, average.

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doi: 10.12032/TMR20200227168
Table 1 Clinical trials list of PNS mainly in cerebrovascular diseases
Clinical trials.
government identifier
Condition or
disease
Intervention/treatment Phase Study completion date
NCT02999048
Hematoma
absorption and
neurological
function recovery
Drug: PNS Phase 4 May 2016
NCT01636154 Ischemic stroke
Drug: Ixeris of
sonchifolia Hance
Drug: PNS
Drug: Ixeris of
sonchifolia Hance
combined with PNS
Not
applicable
August 2016
NCT02544087 Ischemic stroke
Drug: Ixeris of
sonchifolia Hance
Drug: PNS
Drug: Ixeris of
sonchifolia Hance
combined with PNS
Not
applicable
April 2018
PNS, Panax notoginseng saponins.
Immunofluroscence staining
Cryostat slides were prepared and incubated with
CD31 or CD105 primary antibody (Abcam, Cat. No.
ab24590 and ab107595, host in rabbit, USA) at a
dilution of 1:100 at 4℃ overnight, and followed by
Alexa Fluor488 labeled anti-rabbit secondary antibody
(Thermo Fisher, Cat. No. A11008, USA) at room
temperature for 1 h. The sections were mounted with
DAPI (Solarbio, Cat. No. 28718-90-3, China). All
images were obtained by inverted fluorescence
microscope (Olympus, CKX41, Japan). 20× or 40×
objective lens have been used for each field.
Cerebral blood flow
Mice were deeply anesthetized with isoflurane (1.0 L
/min). Brains were fixed by stereotaxic apparatus, and
local dissected to expose the skull of brains. The
detector of Laser Doppler flowmetry (Perimed AB,
PeriFlux System5000, German) was placed in the
midline of the brain, then right-handed adjust to 2 mm
and pointed on the right branch of superior saggital
sinus on the right hemisphere. A continuous
measurement for 120 seconds was collected to analysis
cerebrovascular perfusion, and analyzed as perfusion
unit (PU). The fluctuation of cerebral blood flow was
further calculated. Ten points in every 12-second
interval were picked. Each point contained 3-second
long, and the difference values of maximum and
minimum PU (∆PU) in these 3-second were calculated
to evaluate the fluctuation of blood flow.
Target prediction
ChemDraw was used to draw the molecular structures
of five main ingredients of PNS—notoginsenoside R1,
ginsenoside Rb1, ginsenoside Rg1, ginsenoside Rd and
ginsenoside Re. Both Corey-Padi-Koltun molecular
models and 2D molecular structures were provided at a
situation of their minimal energy statements.
For virtual screening, AlzPlatform has been used to
estimate the potential targets of PNS in the treatment
of AD. AlzPlatform is an AD domain-specific database
for target identification [14]. It is a free web-based
computing tool to explore interactions between
compounds and proteins. In the program of
high-throughput docking (HTDocking)
(http://www.cbligand.org/AD/docking_search. php), it
automates docking procedure to search for targets
between compound and protein. Briefly, water
molecules and ligands were removed, hydrogen atoms
were added. By using AutoDock utility scripts, the
active sites of each protein were defined. AutoDock
provides predicted binding affinity values (ΔG values)
from different docking poses for each compound in a
binding pocket of a protein. The overall score of a
queried compound from each protein structure is used
to assess and rank the potential protein partners or
targets.
Statistical analysis
All data are reported as mean ± SEM. Paired or
unpaired Student’s t-test (for two group means) or one
or two-way analysis of variance (ANOVA) with
post-hoc Tukey test, Scheffe’s test, or Bonferroni test,
as appropriate, were conducted using the SPSS 17.0.
The significance level for the two-sided analyses was
set at P ≤ 0.05. Prism and Photoshop Illustrator were
used for photographing.
Results
PNS improves cognitive functions of 7-month age of
APP/PS1 Tg mice in both MWM and step-down
test
MWM was applied to evaluate the spatial learning and

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doi: 10.12032/TMR20200227168
memory. In the training trials, all the groups showed a
progress in spatial learning at the first three days, but
the deficits occurred at day 4 in APP/PS1 Tg mice.
Compared with Tg mice, both WT and PNS groups
significantly shortened the latency to platform (P <
0.001, P < 0.05, Figure 2a). In the testing trial, the time
percentage in target quadrant has been measured. WT
group showed a significant longer occupancy
compared with Tg mice (P < 0.05), while there is a
longer tendency in PNS groups but no significant
change (Figure 2b, P = 0.068).
Step-down test also has been used to evaluate the
cognitive functions. 48h after the training trial, only
WT mice maintained a significant longer latency than
Tg mice (P < 0.05, Figure 2c). But, when measuring
the percentage of animals in the safe zone up to 5 min,
there is only 64.3% of Tg mice maintained the shock
memory; both WT and PNS groups showed better
performance—more than 85% of them maintained the
shock memory (Figure 2d). Taken together, at 7-month
age, the cognition is impaired in Tg mice; PNS can
prevent the deficit in both spatial learning and fear
memory.
Figure 2 PNS improves cognitive functions of 7-month age of APP/PS1 Tg mice. a. During the 6-day training of
MWM, all groups showed a reduced latency to platform at the first three days, but PNS and WT groups maintained
the progress in the following days of training. Compared with APP/PS1 Tg in the training test, PNS and WT
showed significant reduced latency to platform. b. In the probe test of MWM, the percentage in target quadrant was
significantly higher in WT group compared with Tg mice. Although there was no significant change in PNS group,
it enjoyed a better tendency in spatial memory (P = 0.068). c. In the step-down test after training, WT mice showed
significant longer latency compared with Tg mice. d. 48h after step-down test training, the number of animals
remained fear memory up to 5 min was measured. Like WT mice, PNS could maintain the number of animals to
86.7%, while Tg group only enjoyed a percentage of 64.3. Data are represented as mean ± SEM. *P < 0.05, ***P <
0.001.
PNS, Panax notoginseng saponins; APP/PS1, amyloid precursor protein/presenilin-1; Tg, transgenic; MWM, morris
water maze; WT: wild-type mice.

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doi: 10.12032/TMR20200227168
PNS reduces the number of amyloid plaques in both
cortex and hippocampus
In previous studies, we have proved that the
combination of PNS and GP can significantly reduce
the number of amyloid plaques in brains of several AD
animal models [11, 15, 16]. In order to illustrate if PNS
contributes to this efficacy, amyloid plaques deposits
has been detected. By 1% Thioflavin-S staining, we
chose three-consisting slices of every 10–15 slice
interval, to estimate the deposition of amyloid plaques
in both cortex and hippocampus areas. In WT mice,
there was no amyloid plaque observed, while there
were amount of plaque deposition in both cortex and
hippocampus areas. Compared with Tg mice, PNS
significantly reduced the number of plaques deposition
(P < 0.05, P < 0.01, Figure 3). This result indicates that
PNS can contribute the clearance of amyloid plaques
in AD brains.
Figure 3 PNS reduces the number of amyloid plaques in both cortex and hippocampus. By Thioflavin-S
staining, there was no deposition of amyloid plaques detected in WT mice. Compared with APP/PS1 Tg mice, the
number of amyloid deposits was significantly reduced under the treatment of PNS in both cortex and hippocampus
areas. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar: 100µm.
PNS, Panax notoginseng saponins; WT, wild-type mice; APP/PS1, amyloid precursor protein/presenilin-1; Tg,
transgenic.

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doi: 10.12032/TMR20200227168
PNS promotes angiogenesis and cerebral blood flow
in APP/PS1 Tg mice
Since PNS is used in cerebrovascular diseases, we next
want to detect if PNS can work on cerebrovascular
function in APP/PS1 Tg mice. CD31 refers to a typical
marker of the vascular. The structure of cerebral
vascular in the hippocampus was detected. Compared
with WT mice, the density of vessels was reduced in
Tg mice; while PNS reversed the phenomena (Figure
4a). In addition, since CD105 is widely considered as a
crucial role in angiogenesis, we next want to detect if
the increased density of vessels is contributed by
angiogenesis. By CD105 immunofluorescence staining,
the number of CD105 positive cells was measured. In
the dentate gyrus (DG) area of hippocampus,
compared with Tg mice, the number of CD105 positive
cells was significantly increased under the treatment of
PNS (P < 0.001), which is identical to WT groups
(Figure 4b).
Figure 4 PNS increases the density of cerebrovascular and the number of CD105 positive cells in the
hippocampus areas. a. CD31 staining was detected to observe the density of cerebrovascular in hippocampus
areas. From the overview, the density of vessels was evidently reduced in APP/PS1 mice compared with WT mice,
while PNS reversed the phenomena. Scale bar: 500 µm. b. CD105 staining was further detected to evaluate
angiogenesis. In APP/PS1 Tg mice, few CD105 positive cells was detected (arrow indicator). Compared with Tg
group, the number of CD105 positive cells was significantly increased in both WT and PNS mice.Scale bar: 50µm.
c. The number of CD105 positive cells was counted. Data are represented as mean ± SEM. ***P < 0.001.
PNS, Panax notoginseng saponins; APP/PS1, amyloid precursor protein/presenilin-1; WT, wild-type mice; Tg,
transgenic.

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doi: 10.12032/TMR20200227168
By Laser Doppler PeriFlux 5000, we further
measured the change of cerebrovascular blood flow in
the right branch of superior saggital sinus, sketched
according to Dorr’s work [17]. Within 120-second of
whole view recording, the blood fluctuation was much
smoother in WT mice, which indicated a normal
vasoconstriction; however, Tg mice showed a severe
variation. In addition, the blood fluctuation was
calculated within 3 seconds of every 12 seconds
interval. Again, compared with Tg mice, PNS
significantly smoothed the vasoconstriction (Figure 5a).
Compared with Tg mice, cerebrovascular blood
perfusion was significantly higher in both WT and
PNS groups (P < 0.001, P < 0.05, Figure 5b, 5c).
Taken together, under a three-month treatment of PNS,
the angiogenesis and cerebral blood flow are promoted
at 7-month age of APP/PS1 mice.
By virtual screening servers, vascular related
proteins are found to be the potential targets of
PNS
Since PNS can work on cerebrovascular function, we
next want to further explore if vascular related protein
is its potential target. Firstly, by ChemDraw software,
the structure of R1, Rd, Rb1, Re and Rg1, the five
major ingredients of PNS has been drawn. At their
minimal energy statements, both CPK molecular
models and 2D molecular structures are provided
(Figure 6a).
Secondly, potential targets was predicted by a free
serve for AD drugs. AlzPlatform
(www.cbligand.org/AD/) provides a user-friendly
interface with a HTDocking program on AD
chemicals and targets. By docking analysis, , the five
ingredients were input into the interface separately.
Overall score refers to the degree of feasibility. We
found that thyroid stimulating hormone receptor
(TSHR), muscarinic acetylcholine receptor M1-5,
platelet activating factor receptor (PAFR) and
vasopressin V1a receptor (V1AR) were always listed
on the top 10 of target identification (Figure 6b),
suggesting that vascular related proteins could be the
potential targets of PNS.
Figure 5 PNS promotes cerebrovascular functions in 7-month age of APP/PS1 Tg mice. a. A graph was showed
the perfusion and fluctuation of right branch of superior saggital sinus. Compared with APP/PS1 Tg and PNS
groups, the fluctuation was more smoother in WT mice within 120 sec recording. b. To measure the cerebral blood
flow, the signal of PU was qualified. Compared with Tg mice, the signal was significantly increased in both WT
and PNS group. c. Since the changes of vasoconstriction was observed in recording. The fluctuation was measured
within 3sec every 12sec interval. Again, both WT and PNS significantly smoothed the vasoconstriction compared
with Tg mice. Data are represented as mean ± SEM. (*P < 0.05, ***P < 0.001).
PNS, Panax notoginseng saponins; APP/PS1, amyloid precursor protein/presenilin-1; Tg, transgenic; WT, wild-type
mice; PU, perfusion unit; ∆PU, the difference values of maximum and minimum PU.

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doi: 10.12032/TMR20200227168
Figure 6 The structures of five major ingredients of PNS and the potential targets of PNS. A. There are five
main ingredients of PNS—R1, Rd, Rb1, Re and Rg1, by ChemDraw, the CPK molecular models and 2D molecular
structures were provided as their minimal energy statement. B. By THDocking, vascular-related proteins were
found to be the potential targets of PNS. PAFR and VIAR were in the top ten identifications. Overall score
indicated the degree of feasibility based on THDocking database.
PNS, Panax notoginseng saponins; TSHR, thyroid stimulating hormone receptor; PAFR, platelet activating factor
receptor; VIAR, vasopressin V1a receptor.
Discussions
Currently, cerebrovascular diseases are getting
increased attention. In clinic, insufficient cerebral
perfusion is closely related with cognition impairment
[18]. According to long-term clinical observation,
Torre addressed that neurodegeneration could be
caused by cerebrovascular diseases, and he pointed
that insufficient cerebral perfusion could be considered
as a diagnostic indicator of AD in clinic [19]. Further,
Zlokovic has proposed “neurovascular hypothesis” —
the dysfunction of neurovascular units could lead to
neurological dysfunctions, which would result in
neuron damage or even cognition impairment [20].
These suggest that cerebrovascular dysfunction is
occurred on the early stage of AD and closely related
with AD processing; it can be a new drug target of AD.
Since there is no effect drugs anti-AD, when we
come back to TCM, which has a long history
prescribed in oriental countries, in China, a widely
used herb, PNS has been administrated in the diseases
of vascular dysfunctions, such as hemorrhage, stroke,
pain [21, 22]. In our previous studies [11],
accompanied with GP, the combination has been
administrated for acute strokes and improved the
cognition of stroke patients with memory deficits. In
addition, we found the combination could improve
impaired cognition in several AD animal models [11,
15, 16]. In vitro, the combination could protect
vascular endothelial cells from oxidative stress [23].
These results indicate that PNS and its combination
have a neuroprotective role in AD. Therefore, in order
to further explore the efficacy and its potential targets
of PNS, especially in preventive strategy, we mainly
focused on the changes of vascular functions in both
animal model and computer simulation technology.
In vivo, we focused on cerebrovascular function of
PNS in AD-like animal model. In behavioral tests,
PNS could improve cognitive functions of spatial
learning and fear memory; in addition, the number of
amyloid plaques deposition was significantly reduced
in both cortex and hippocampus in PNS group.
Furthermore, under the three-month long
administration, angiogenesis especially in
hippocampus DG areas was observed. Adult
neurogenesis contributes to hippocampal plasticity [24].
Accumulating evidence suggests that adult-born
neurons in this area play distinct physiological roles in
memory encoding and mood regulation [25].
Angiogenesis may control function of neuronal stem
cells via VEGF factors [26]. VEGF shows a role in

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doi: 10.12032/TMR20200227168
learning and memory improvement by VEGF-induced
modulations of neuronal activity [27]. What’s more,
cerebral blood perfusion was significantly higher
compared with APP/PS1 mice, and the fluctuation of
blood flow was significantly smoother. These data
suggest that PNS recovers the cerebrovascular
function—angiogenesis and cerebrovascular blood
flow in APP/PS1 Tg mice.
There are five main ingredients of PNS—R1, Rd,
Rb1, Re and Rg1. By HTDocking tools [14], we
further found that its main gradients might work on
two vascular-related proteins, PAFR and V1AR. In
addition, according to PHAROS server
(www.pharos.nih.gov/idg/targets), these two proteins
might work on APP, which is the precursor protein of
Aβ. In addition, V1AR is one of major receptors for
vasopressin, highly present throughout brain, such as
cortex, hippocampus, hypothalamus and adrenal gland
[28]. Besides working on APP, V1AR also had a
potential effect on one of subunits of
γ-secretase—Aph1, screening by PHAROS server.
Further, 20-year ago, it was found that there was a
significant correlation between PAF binding and
degree of cognitive impairment in AD patients [29]; in
depressed coronary artery disease patients,
platelet-activating factors were associated with
cognitive deficits [30]; in addition, V1AR was
indispensable for normal social recognition and
cognitive empathy [31, 32]. By other virtual screening
serves, such as ChamMapper and PharmMapper,
VEGF receptors are also the top-level of potential
targets of PNS, as well as other neuron-related proteins,
such as dopamine receptors, estrogen receptor.
However, computer simulation can provide an assistant
analysis in target identification, but a validation
required [33].
Conclusions
PNS has been used for the treatment of intracerebral
hemorrhage and ischemic stroke, and improves
cognition deficits of stroke patients as well as in a
bench of AD animal models. In this study, we find that
PNS can partially contribute to the improvement of
cognitive functions in 7-month age of APP/PS1 mice.
In addition, PNS can promote angiogenesis in DG
areas of hippocampus and cerebrovascular blood flow.
These results indicate that the contribution of PNS in
neuroprotection is via the modulation of
cerebrovascular functions (Figure 7).
Figure 7 The hypothesis of neuroprotective role of PNS in cerebrovascular targets. Based on experimental
studies and virtual screening, the neuroprotective role of PNS may work on cerebrovascular to promote vascular
function, the latter of which could lead to the reduction of amyloid plaques and eventually result in AD prevention.
PNS, Panax notoginseng saponins; AD, Alzheimer’s disease.

ARTICLE
doi: 10.12032/TMR20200227168
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