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International Journal of
Molecular Sciences
Article
Modified Low-Temperature Extraction Method for Isolation of
Bletilla striata Polysaccharide as Antioxidant for the Prevention
of Alzheimer’s Disease
Yi-Wen Lin 1, Chih-Hsiang Fang 2, Ya-Jyun Liang 1, Hong-Hsiang Liao 1and Feng-Huei Lin 1, 3, *
Citation: Lin, Y.-W.; Fang, C.-H.;
Liang, Y.-J.; Liao, H.-H.; Lin, F.-H.
Modified Low-Temperature
Extraction Method for Isolation of
Bletilla striata Polysaccharide as
Antioxidant for the Prevention of
Alzheimer’s Disease. Int. J. Mol. Sci.
2021,22, 12760. https://doi.org/
10.3390/ijms222312760
Academic Editor: Cristoforo Comi
Received: 19 October 2021
Accepted: 19 November 2021
Published: 25 November 2021
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1Institute of Biomedical Engineering, National Taiwan University, Taipei 10051, Taiwan;
zhew520@gmail.com (Y.-W.L.); kittenor210@gmail.com (Y.-J.L.); hung.ming.is.the.best@gmail.com (H.-H.L.)
2Trauma and Emergency Center, China Medical University Hospital, Taichung City 404332, Taiwan;
danny07291991@hotmail.com
3
Institute of Biomedical Engineering and Nanomedicine Research, National Health Research Institutes, No. 35,
Keyan Road, Miaoli County 35053, Taiwan
*Correspondence: double@ntu.edu.tw; Tel.: +886-2-27327474
Abstract:
Amyloid-
β
(A
β
) peptides play a key role in Alzheimer’s disease (AD), the most common
type of dementia. In this study, a polysaccharide from Bletilla striata (BSP), with strong antioxi-
dant and anti-inflammatory properties, was extracted using a low-temperature method and tested
for its efficacy against AD,
in vitro
using N2a and BV-2 cells, and
in vivo
using an AD rat model.
The characterization of the extracted BSP for its molecular structure and functional groups demon-
strated the effectiveness of the modified method for retaining its bioactivity.
In vitro
, BSP reduced
by 20% reactive oxygen species (ROS) levels in N2a cells (p= 0.0082) and the expression levels of
inflammation-related genes by 3-fold TNF-
α
(p= 0.0048), 4-fold IL-6 (p= 0.0019), and 2.5-fold IL-10
(p= 0.0212) in BV-2 cells treated with A
β
fibrils.
In vivo
, BSP recovered learning memory, ameliorated
morphological damage in the hippocampus and cortex, and reduced the expression of the
β
-secretase
protein in AlCl
3
-induced AD rats. Collectively, these findings demonstrated the efficacy of BSP for
preventing and alleviating the effects of AD.
Keywords:
Alzheimer’s disease; Bletilla striata; amyloid-
β
; oxidative stress; anti-inflammation;
aluminum chloride
1. Introduction
Alzheimer’s disease (AD) is the most common type of dementia, characterized by
progressive cognitive and memory loss accompanied by personality changes [
1
,
2
]. Given
the expected increase in the aged population globally, the prevalence of dementia has been
projected to increase to 131 million by 2050 [
2
]. This impending rise in societal aging could
impose a costly social impact in the coming years.
Several studies have demonstrated that the initial changes in brain physiology as-
sociated with AD begin years before perceptible symptoms of dementia are observed.
In the initial stages, the brain compensates for the changes enabling affected individuals
to function normally. However, with the progress of neuronal damage, the brain fails to
compensatefurther, and individuals show subtle cognitive decline [
3
,
4
]. AD is generally
diagnosed by positron emission tomography (PET) and cerebrospinal fluid (CSF) tests [
5
]
that reveal the elevated amyloid-
β
(A
β
) depositions, which are believed to be related to
pathological changes in preclinical AD.
Several biomarkers detecting specific proteins in blood or body fluids, which could
be correlated to A
β
deposition, pathologic tau, and neurodegeneration, are also used to
uncover the pathological process of AD [
6
]. These include p53-misfolding conformation,
also called Unfolded p53 (U-p532D3A8+), recognized by the antibody 2D3A8. According
to Abate, G. et al., p53 misfolded variants may represent the characteristics of early AD
Int. J. Mol. Sci. 2021,22, 12760. https://doi.org/10.3390/ijms222312760 https://www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2021,22, 12760 2 of 19
pathological events, including Aß accumulation, and redox imbalance, and immune acti-
vation, which ultimately lead to oxidative stress and chronic inflammation, respectively.
Thus, U-p532D3A8+ plasma levels are a promising blood-based candidate biomarker for
AD [7].
In addition, there may be a link between the gut microbiota and neurodegenerative dis-
eases, such as Parkinson’s disease and amyotrophic lateral sclerosis [
8
,
9
]. The microbiota–
gut–brain axis has become the focus of biomedical research and a potential therapeu-
tic target for central nervous system diseases [
10
,
11
]. In particular, the dysfunction of
the microbiota-gut-brain axis is related to the pathogenesis of AD [
12
]. The gut microbiota
is considered a forgotten regulator of host physiology and metabolism [
13
]. The influence
of the microbiota extends to brain development and cognitive functions [14].
A
β
is derived from amyloid precursor protein (APP) by a two-step proteolysis re-
action of two enzyme complexes,
β
-secretase and
γ
-secretase [
15
]. It has been reported
that the accumulation and aggregation of A
β
leading to the formation of senile plaques
in the central nervous system causes progressive cognitive memory loss. Furthermore, A
β
can also increase the levels of reactive oxidative species (ROS) that induces oxidative stress
to the neurons and cause cytotoxicity. In addition, the senile plaques containing the A
β
peptide have also been shown to induce oxidative stress and chronic neuroinflammation
leading to neurotoxicity, which further advances disease progression.
Studies have shown that microglia that secrete neuroprotective growth factors and
anti-inflammatory cytokines phagocytize A
β
and release enzymes responsible for A
β
degradation; they delay the progression of AD by contributing to the clearance of A
β
[
16
,
17
].
Therefore, it is predicted that finding a way to reduce the oxidative stress and chronic
neuroinflammation might slow down the progression of AD or even stop the accumula-
tion of A
β
. However, despite the availability of several reports and significant efforts by
pharmaceutical industries to understand the mechanism, progression, and alleviation of
AD, effective therapies to cure AD or inhibit the progression of AD symptoms significantly
are unavailable [
18
]. Moreover, the drugs currently used to treat AD—donepezil, galan-
tamine, and rivastigmine—mainly focus on anticholinesterase activity rather than ROS and
neuroinflammation reduction.
Bletilla striata (Thunb.) Reichb. f. (Orchidaceae), a traditional Chinese medicine, has
been widely used in East Asian countries for alimentary canal damage, ulcers, bleeding,
bruises, and burns [
19
]. B. striata polysaccharide (BSP) is a water-soluble polysaccharide
isolated from B. striata. The monosaccharides of BSP are composed of (1
→
2)-linked
α
-
D-mannopyranose and (1
→
4)-linked
β
-D-glucopyranose residues in a ratio of 3:1 [
20
].
Studies have reported that BSP could scavenge the ROS and inhibit the activation of
proinflammatory cytokines, including interleukin 6 (IL-6) and tumor necrosis factor
α
(TNF-
α
) in a dose–dependent manner [
21
]. Therefore, we hypothesized that BSP with
strong anti-oxidant and anti-inflammatory properties could be effective in preventing AD.
To test this hypothesis, in this study, we investigated whether BSP could prevent
the diffusion of ROS from A
βin vitro
and the possibility of recovery from the disease
by memory improvement in the AlCl
3
-induced AD rat model
in vivo
(Figure 1). Further-
more, here, we employed a new BSP extraction method at low temperatures to ensure
the retention of its bioactive properties.
Int. J. Mol. Sci. 2021,22, 12760 3 of 19
Figure 1.
The scheme of the experimental design. This study used the N2a cell as an in-vitro model to investigate
the decrease of amyloid-
β
plaque by BSP. In addition, the AD rat model was induced by intraperitoneal (IP) injection of
AlCl
3
three times a week, and the Morris water maze was used to assess working and spatial memory retention in the rats.
Finally, the biochemical analysis was used to evaluate the effect of AD prevention by BSP.
2. Results
2.1. Identification of Molecular Structure and Functional Group
In the study, BSP was extracted from B. striata at low temperature combined with
the use of vacuum system to retain the bioactivity of BSP and to reduce protein extraction.
Figure 2a shows the FTIR pattern of the extracted BSP obtained. The absorption bands at
807 cm
−1
and 874 cm
−1
represented
α
-mannose residues, whereas the sharp absorption at
1022 cm
−1
indicated a
β
-glucose residue. The absorption bands at 1372 cm
−1
, 2906 cm
−1
,
and 3342 cm
−1
corresponded to the C-O-C of glycosidic linkage, C-H of the 6-carbon
ring, and the hydroxyl group (O
−
H), respectively. Furthermore, as shown in Figure 2b,
the pattern of
1
H NMR of the extracted BSP revealed a chemical shift at
δ
4.80 ppm and
δ
4.55 ppm, the characteristic peaks for
α
-mannopyranose and
β
-glucopyranose, respectively.
The signals in the other chemical shifts in the range of 3.3–4.2 ppm corresponded to the non-
anomeric ring protons. The 13C NMR pattern of extracted BSP represented the signals
at
δ
101.1 ppm and
δ
103.5 ppm, attributed to
α
-mannopyranose and
β
-glucopyranose,
respectively, whereas the chemical shifts found in the range of 70–80 ppm indicated the non-
anomeric carbon rings (Figure 2c). The results of
1
H NMR and
13
C NMR are in agreement
with the results of a previous study obtained by fingerprinting analysis [
20
], suggesting
that the molecular structure and functional groups of BSP extracted by the new method
proposed in this study were the same as for the BSP extracted by traditional method.
Int. J. Mol. Sci. 2021,22, 12760 4 of 19
Figure 2.
Characterization of extracted BSP. (
a
) FTIR spectrum; (
b
)
1
H NMR spectrum; (
c
)
13
C NMR spectrum. These were
fully matched to the BSP extracted by the traditional method.
2.2. BSP Inhibit AβFibril Formation
It is known that A
β
42 in PBS gradually aggregates into the A
β
fibril, which, upon
binding to Thioflavin T (ThT), emits fluorescence. As shown in Figure 3a, neither the control
nor the PBS alone showed any fluorescence, which increased markedly with time when ThT
was incubated with A
β
42 and A
β
42 mixed with BSP (A
β
-BSP) in PBS at 37
◦
C as compared
to that in PBS alone. However, the intensity of the emitted fluorescence was markedly
lower with A
β
-BSP treatment than that obtained by A
β
42, indicating the inhibitory effects
of BSP on Aβfibril formation or Aβ42 aggregation.
In addition, TEM imaging data showed that in cells treated with A
β
42 (in PBS), A
β
42
did not aggregate on day 0 but formed thick A
β
fibrils on day 7 (Figure 3b,c). In contrast,
in the A
β
-BSP-treated cells, the aggregation of A
β
42 was relatively loose, and the number
of A
β
fibrils was less than that observed in the A
β
42-treated cells on day 7 (Figure 3d).
These findings further support the findings obtained through ThT assays. In summary,
it can be inferred that BSP effectively inhibits A
β
42 aggregation to block the formation of
Aβfibrils.
Int. J. Mol. Sci. 2021,22, 12760 5 of 19
Figure 3.
The aggregation of A
β
42 into A
β
fibrils. (
a
) The fluorescence intensity of ThT was obtained by
incubation of A
β
42 (A
β
; blue line), A
β
42 mixed with BSP (A
β
-BSP; red line) in PBS at 37
◦
C for 14 days.
The green line represents the fluorescence intensity of ThT in PBS only (PBS). (
b
) The TEM image of A
β
42
monomers without aggregation on day 0; (
c
) the A
β
42 aggregated into thick and compact A
β
fibrils on
day 7; (
d
) BSP reduced aggregation of A
β
42 with reduced thickness and loose packing of A
β
fibrils on day
7. The scale bar is 0.2
µ
m. (n= 6, # p< 0.05 compared to control group, * p< 0.05 compared to A
β
group).
2.3. Effects of BSP on Aβ-Induced Cytotoxicity and Cell Viability
The protective effects of BSP against A
β
-induced cytotoxicity on N2a cells were
assessed by LIVE/DEAD staining. As shown in Figure 4, the survivability of the N2a
cells treated with BSP and A
β
42+BSP were not significantly different from that observed
in untreated controls. However, the numbers of dead cells in BSP-only treated cells were
lower than those treated with A
β
42-BSP, indicating the cytotoxic effects were induced by
Aβfibrils in N2a cells.
Figure 4.
The LIVE/DEAD staining of N2a cells in (
a
) the control: cells cultured in medium only, (
b
)
positive control: cells treated with zinc diethyldithiocarbamate, (
c
) negative control: cells treated with
aluminum oxide, (
d
) BSP: cells treated with an extracted solution of BSP, and (
e
) A
β
-BSP: cells treated with
Aβ42 and BSP. (f) Quantitative results of survival rate in N2a cells. *** p< 0.001 compared with control.
Int. J. Mol. Sci. 2021,22, 12760 6 of 19
Furthermore, the effects of extracted BSP on the viability of N2a cells evaluated by
WST-1 assay revealed that neither the extracted BSP alone nor that mixed with A
β
42
showed any toxicity to N2a cells compared to the control (Figure 5).
Figure 5.
The effects of the extracted BSP on cell viability were estimated by the WST-1 assay. (n= 6,
*** p< 0.001 compared with control). The abbreviations are as defined in Figure 4.
2.4. Antioxidant Activity of BSP
The antioxidant ability of the extracted BSP was evaluated by DCFDA assay. As shown
in Figure 6, the treatment of Aβ42 alone significantly increased the ROS level in N2a cells
cultured for 1 day relative to the control that did not receive any treatment (p< 0.05),
whereas that in A
β
42- and BSP-treated cells did not vary. However, the ROS level in A
β
-
BSP treated cells was significantly reduced compared to those treated with A
β
42 alone,
indicating the potential of BSP to ameliorate the Aβ-induced increase in ROS levels.
Figure 6.
Antioxidant activity of extracted BSP. DCFDA was used to measure the intracellular ROS
in N2a cells without any treatment (control), treated with A
β
42 (A
β
), and A
β
42 and BSP (A
β
42-BSP).
(n= 6, # p< 0.05 compared to control group, * p< 0.05 compared to Aβgroup).
2.5. Anti-Inflammatory Activity of BSP
As shown in Figure 7, expression of all three inflammatory genes—TNF-
α
, IL-6,
and IL-10—was downregulated in the BV-2 cells in the A
β
-BSP group compared to those
in the Aβgroup, suggesting the strong anti-inflammatory properties of BSP (Figure 7).
Int. J. Mol. Sci. 2021,22, 12760 7 of 19
Figure 7.
Anti-inflammatory effect of the extracted BSP on A
β
-induced inflammation in BV-2 cells estimated
by the expression analysis of inflammation-related genes, TNF-
α
, IL-6, and IL-10. (n= 6,
#p< 0.05
compared
to that in the control group, * p< 0.05 compared to Aβgroup, ** p< 0.01 compared to Aβgroup).
2.6. Morris Water Maze Test
As a next step, we evaluated the effects of BSP
in vivo
. First, we evaluated its effects
on the working and spatial memory of rats. As shown in Figure 8a, a normal rat with
a good memory reached the escape platform following a relatively straight track (Figure 8a),
whereas an AlCl
3
-induced AD rat could not reach the escape platform even after following
a complex track. However, the track followed by a BSP-treated AD rat was like that of
a normal rat. Furthermore, the findings revealed that the time required by the BSP group
did not differ significantly compared to the time required by the control group (Figure 8b).
Collectively, these results indicate the potential of BSP to restore memory in AD rats.
Figure 8.
The MWM track of the normal rats (control), AlCl
3
-induced AD rats (AlCl
3
), and BSP-
treated AD rats (AlCl
3
-BSP). (
a
) The starting point (blue) to endpoint (green) of all the tested rats was
the same. (
b
) The time taken by the tested rats from starting point to reach the escape platform (n= 6
per group, # p< 0.05 compared to control group, * p< 0.05 compared to AlCl3group).
Int. J. Mol. Sci. 2021,22, 12760 8 of 19
2.7. BSP Treatment Downregulated the BACE1 Treatment
To investigate the A
β
fibril formations in AlCl
3
-induced rats, the cortex and hip-
pocampus were harvested from the brain tissue, and the expression of the
β
-secretase
(BACE1) protein was analyzed by western blotting (Figure 9a). As shown in Figure 9, both
in the cortex and hippocampus, BACE1 was highly expressed in AlCl
3
-induced rats, which
was downregulated in BSP-treated AD rats. The measured values of the band intensities
are shown in Figure 9b.
Figure 9.
Bio-chemical analysis. (
a
) The western blot analysis of BACE1 in hippocampus CA1 and
cortex.
β
-actin was used as the loading control. (
b
) The quantitative values of the BACE1 expression
data obtained the ratio of BACE1 protein/actin protein band intensities normalized to 1 in the control
group. (n= 6, # p< 0.05 compared to control group, * p< 0.05 compared to AlCl3group).
2.8. BSP Treatment Reduced Morphological Changes in the Neurons of the AD Rat Model
H&E staining was used to assess the effects of BSP on morphological changes of the cells
in the cortex and hippocampus of the AlCl
3
-induced AD rat. As shown in
Figure 10a
, the hip-
pocampal CA1 and cerebral cortex regions in the control group showed normal morphology,
whereas the cells from AlCl
3
-induced AD rats showed clear damage, such as cellular at-
rophy, shrinkage, necrosis, and pyknosis, indicated by heavy stained and dark nuclei
(hyperchromatic cells). In addition, large cells due to neuronal swelling and vacuolation
were also observed sporadically. In contrast, the damage was substantially decreased
in AlCl
3
-induced AD rats treated with the extracted BSP, indicating the ameliorating ef-
fects of BSP against the damages induced by AlCl
3
in AD rats. As shown in
Figure 10b
,
the dark-brown amyloid plaques were observed in the hippocampus CA1 and cortex
of AlCl
3
-induced rats by BACE1 antibody-positive stain (indicated by the red arrows).
However, no plaques were detected in the control rats, whereas they were barely traced or
observed in very light stains in AlCl3-induced AD rats treated with BSP.
Int. J. Mol. Sci. 2021,22, 12760 9 of 19
Figure 10.
Histological staining. (
a
) Hematoxylin and eosin (H&E) staining of hippocampal CA1 and cortex regions of
the tested rats. Brain sections from rats treated with AlCl
3
show that pyramidal cells in the hippocampal CA1 region exhibit
more severe morphological changes. (
b
) BACE1 reveals plaque-like staining in the brains of AD rats by immunohistochem-
istry. Brain sections from control rats stained with BACE1 show the normal BACE1 immunoreactivity pattern, whereas
those from AlCl
3
-induced rats display plaque-like BACE1 immunoreactivity, which was reduced in BSP-treated AD rats.
Arrowheads indicate plaque-like BACE1-immunopositive deposits. Scale bars: 50 µm.
3. Discussion
In this study, the BSP was successfully isolated and purified from the tubers of B. striata
following a modified extraction method at low temperature that could effectively preserve
BSP bioactivity and reduce protein loss. We obtained a new polysaccharide with a molecu-
lar weight of 2.35
×
10
5
Da containing mannose, glucose, and galactose in a molar ratio of
9.4:2.6:1.0. The results indicated that the backbone of BSP consisted of (1
→
4)-linked manno-
syl residues and (1
→
4)-linked glucosyl residues in a molar ratio of 2:1. A monosaccharide
composition analysis revealed multiple repetitive sequences. The molecular structure
and functional groups of the extracted BSP characterized by NMR and FTIR are fully
in agreement with that obtained by the traditional method [
20
]. Furthermore, the extracted
BSP demonstrated good survivability and no harm to the N2a cells
in vitro
. It also alle-
viated the damage to the hippocampus and cortex and recovered the neurodegeneration
in AlCl3-induced AD rats in vivo.
Aluminum treatment increased A
β
(1–42) levels in the brain by up-regulating BACE1
expression [
22
]. In addition, aluminum treatment inhibited the expression of LRP1 and
NEP (A
β
clearance related protein) [
23
]. The present study demonstrated the aggregation
of A
β
42 into A
β
fibrils in A
β
42 in BSP-treated cells, which could be because of the A
β
42
binding to the ThT, which restricts the rotation of the benzylamine and benzothiazole
rings of ThT, consequently emitting fluorescence. Furthermore, it has been shown that
the deposition of A
β
fibrils as plaques are enriched with the
β
-sheet structure, which
blocks neurotransmitter transportation, consequently leading to AD [
24
]. The findings
showed that the formation of A
β
fibrils was reduced by treating the cells with BSP, which
could be attributed to the inhibition of the aggregation of A
β
42 due to the stereo-obstacles.
Meanwhile, our results also indicated that BSP could down-regulate BACE1 expression.
Therefore, BSP might be a potential supplement for AD prevention.
It has been reported that microglia surrounding the A
β
fibrils form a protective barrier
around them, limit further recruitment of A
β
, and release enzymes responsible for A
β
fibril
degradation that delay the progression of AD [
25
]. Furthermore, it has been established
that A
β
plays a significant role in inducing and regulating microglial ROS production. Al-
though the breakdown by microglial proteases could enhance the clearance of A
β
deposits,
the clearance rate is relatively slow for diffusing the stress from deposited A
β
fibrils fully,
consequently, over years, this leads to accumulation of A
β
fibrils resulting in induction of
ROS and chronic inflammation [
26
,
27
]. In addition, the production of ROS by microglial
cells and subsequent oxidative stress are strongly implicated in the pathogenesis of AD.
Int. J. Mol. Sci. 2021,22, 12760 10 of 19
The oxidative stress to the surrounding cells subsequently induces chronic inflammation,
which might cause further damage to accelerate AD progression and activate the defense
cells to secrete pro-inflammation factors [
17
]. As is evident from the findings of this study,
BSP effectively inhibited ROS production from the accumulated A
β
fibrils and downreg-
ulated the expression of inflammation-related genes in BV-2 cells, indicating the strong
antioxidant and anti-inflammatory properties of BSP.
Oxidative stress and neuroinflammation are the two main factors involved in the pro-
gression of neurodegenerative diseases. Therefore, compounds with antioxidant and
anti-inflammatory properties should provide significant neuroprotection. More and more
evidence has shown that vitamins protect against central nervous system diseases in recent
years. Like other neurodegenerative diseases, one of the leading causes of progressive
neurodegenerative disorders in AD is oxidative stress [
28
]. There is increasing evidence
that the antioxidant activity of vitamins may be beneficial in the treatment of AD. Therefore,
vitamins have been used as adjuvants in the treatment of AD [
29
]. Vitamins are considered
familiar and effective antioxidants. Studies have shown that a diet rich in antioxidant
vitamins can slow the progression of Alzheimer’s disease. A meta-analysis indicated that
vitamin A, B, C, D, and E deficiencies might increase the risk of AD [
30
]. Multiple pieces
of evidence show that, compared with healthy individuals with complete neurocognitive
functions, AD patients have reduced serum levels of VitA, VitB [
31
,
32
], VitC, VitD, VitE,
and VitKin [
33
,
34
]. Interestingly, a cross-sectional and prospective study reported that
the combination of VitC and VitE supplements reduced the prevalence and incidence of
AD; however, they did not report VitA and found that VitB intake was not associated with
AD [35].
Low levels of VitD in the serum are associated with increased risk of AD [
36
,
37
].
Calcium levels, parathyroid hormone, and specific cytokines regulate the concentration of
the active form of VitD, calcitriol. The inactive form of VitD crosses the BBB, where it is
converted into the active form by the enzyme CYP27B1 in glial and neuronal cells [
38
,
39
].
Microglia are also responsible for converting provitamin D into the active form of VitD [
40
].
Calcitriol controls the synthesis of nerve growth factors (NGF), and ultimately controls
the differentiation and maturation of neuronal cells. In addition, calcitriol regulates the syn-
thesis of glial cell line-derived neurotrophic factor (GDNF) [
41
,
42
]. Both NGF and GDNF
regulate learning and memory through the hippocampal pathway. The concentration
of amyloid precursor protein (APP) is effectively regulated by NGF [
43
]. Interruption
of NGF signaling leads to an up-regulation of APP levels and an increase in aggregates
in Abeta cells [
44
]. It is interesting that the active form of VitD and its analogs induce
NGF expression [
45
], which suggests a possible mechanism by which VitD can indirectly
improve the pathology of AD.
VitE plays an essential role in improving cognitive function and memory deficits [
46
].
Furthermore, VitE can be combined with other antioxidants to enhance the efficacy of
treatment strategies effectively [
47
]. Although there is some evidence for the antioxidant
activity of VitE, the role of VitE is controversial; clinical trials on VitE activity in AD have
shown conflicting findings [
48
]. Research reported on the effect of VitE combined with
donepezil supplementation on patients with mild cognitive impairment (MCI) (early AD)
found that supplementation with VitE had no additional benefit [
49
]. MCI patients were
treated with 2000 IU VitE daily and 10 mg donepezil or placebo daily for three years
in this double-blind study. VitE does not affect MCI; however, donepezil reduced the rate
of progression from MCI to AD in the first 12 months and reduced the longer duration
of the apolipoprotein E4 carrier [
50
]. In short, since it is not clear whether long-term
co-treatment with VitE is beneficial to the management of AD, a more comprehensive
population study is needed.
Finally, since there are no international guidelines on dietary recommendations for
AD, data on supplementation of vitamins A, B, C, D, and E are unclear [
51
]. Nevertheless,
some clinical trials still support the potential role of vitamins in treating neurodegenerative
diseases in the future. Stimulants are an essential factor and should be considered when
Int. J. Mol. Sci. 2021,22, 12760 11 of 19
designing vitamin dosages. Stimulants differentiate between beneficial and toxic activities
of vitamins at specific doses. Compared with a single vitamin-based option, multivitamin
supplements show therapeutic potential for neurodegenerative diseases. Multiple signaling
pathways involve multiple vitamin methods that can enhance the antioxidant response [
52
].
Several studies have shown that aluminum exposure induces AD with strikingly
similar clinical and pathological symptoms [
53
,
54
]. For instance, a study has shown that
aluminum has been detected in both senile plaques and neurofibrillary tangle-bearing
neurons in the brains of AD patients. In addition, neuropathological and neurobehavioral
changes resulting in impaired learning ability induced by aluminum exposure are also
evident from animal studies. Here, we demonstrated that AD rats lost cognition, with in-
creased escape latency and reduction in the percentage of time spent in the target quadrant,
which was recovered by treating the AD rats with the extracted BSP, suggesting the po-
tential efficacy of BSP for ameliorating the working and learning memory impairment
induced by AlCl
3
in AD rats. The hippocampus is related to learning and memory; which
is the initial site of Aβaccumulation [55].
BACE1 plays a key role in the production of A
β
; the digestion of APP by BACE1
is the first step in the chain reaction of A
β
aggregation, and its gene deletion has been
shown to produce mild phenotypes. Several transcription-factor-binding sites are lo-
cated within the BACE1 promoter region, including NF-
κ
B, Sp1, and PPAR
γ
, and the reg-
ulatory effects at the transcriptional level are probably involved in the expression of
BACE1 [
55
]. Moreover, several groups have reported that BACE1 level and activity is
increased in the AD brain [
32
,
33
], indicating that it presents an attractive therapeutic target
for AD. Therefore, studies have attempted to identify factors that can suppress BACE1
expression [
15
]. In this study using an anti-BACE1 monoclonal antibody, we showed
that BACE1 levels become elevated in the brains of AlCl
3
-induced AD rats, which was
subsequently reduced in the BSP-treated rats, suggesting that BSP could be a potential
source for the development of BACE1 inhibitor drugs.
Furthermore, western blot analysis of BACE1 and BACE1-staining supports the effi-
cacy of BSP for alleviating the damages to the hippocampus and cerebral cortex in AlCl
3
-
induced AD rats. The protective effects of BSP against AD could be attributed to its antiox-
idative and anti-inflammatory effects. The antioxidative effects of BSP are shown to be medi-
ated through the nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4)/p22phox
signaling pathway [
21
]. Therefore, it is speculated that BSP might be involved in the inhibi-
tion of the expression of NOX4 and p22phox, consequently blocking angiotensin II-induced
ROS generation. However, further studies are required to delineate the exact underlying
mechanism of action of BSP.
4. Materials and Methods
4.1. Materials
B. striata was obtained from Seng Chang Pharmaceutical Co., Ltd., (Taoyuan, Taiwan).
A
β
42 peptides were synthesized by Gendanio Biotech Inc. (New Taipei City, Taiwan). 2,7-
dichlorodihydrofluorescein diacetate (DCFDA) and aluminum chloride were purchased
from Sigma Aldrich (St Louis, MO, USA). The RT-PCR primers were synthesized by
MDBio Inc. (Taipei, Taiwan). The anti-BACE1 antibody and the A
β
(C-Terminal) antibody
were purchased from Merck (Darmstadt, Germany) and Proteintech (Rosemont, IL, USA),
respectively.
4.2. BSP Extraction and Purification
In the study, a new method was developed to extract BSP at low temperature by com-
bining a cold extraction method with a vacuum system at 0.5 atm to retain the bioactivity of
BSP and reduce protein contamination. Briefly, the as-received B. striata was chopped into
pieces and ground to dry powder. Then 100 g B. striata dry powder was homogenized and
dispersed by double-distilled water (1000 mL) in a low-pressure container at room temper-
ature for 4 h [56]. The solution was then centrifuged at 5000 g for 10 min; the supernatant
Int. J. Mol. Sci. 2021,22, 12760 12 of 19
was collected and precipitated by adding 3000 mL 95% (v/v) ethanol and allowed to stand
for overnight. Afterward, the solution was filtered using filter paper, and the precipitate
was lyophilized and kept in a desiccator for later use. Subsequently, 100 g extracted dried
powder was resuspended in 1800 mL distilled water and then mixed with 600 mL Sevage
reagent (n-butanol: chloroform = 1:4); the solution was magnetically stirred overnight.
On the next day, the mixture was centrifuged at 6000 rpm for 10 min, and the supernatant
was collected and dialyzed using an MWCO-3500 dialyzer (60035515, Orange Scientific,
Braine-I’ Alleud, Belgium) against double-distilled water to remove n-butanol. The final
extract was lyophilized to obtain BSP.
4.3. The Characterization of Extracted BSP by Fourier-Transform Infrared (FTIR) Spectroscopy
and Nuclear Magnetic Resonance (NMR)
The FTIR analysis was carried out by mixing 10 mg BSP with KBr in a ratio of 1:9
and then pressing it into a disc in an aluminum ring at 10 MPa. After that, the ring was
mounted on an FTIR spectrophotometer (Spectrum 100 FTIR Spectrometer, PerkinElmer,
Boston, MA, USA) at the wavenumber range of 450 to 4000 cm
−1
and 400 nm/min scanning
rate. The molecular structure of BSP was analyzed by
1
H NMR and
13
C NMR spectra mea-
surements. BSP was dissolved by chloroform-d (CDCl
3
) at a concentration of 50 mg/mL,
and then the spectra were recorded on a Bruker ARX-600 instrument (600 MHz, Bruker
Co., Ltd., Fällanden, Switzerland) [56].
4.4. Preparation of AβFibrils
A
β
42 peptides were purchased from Peptide Institute, Inc. (ASIA BIOSCIENCE CO.,
LTD., Taipei, Taiwan). The A
β
42 peptides were dissolved in hexafluoro-2-propanol (HFIP,
Oakwood Products, Estill, SC, USA) for monomerization to obtain a final concentration
of 1 mM. Then the A
β
42-HFIP solution was transferred to microfuge tubes in aliquots
and kept at room temperature to evaporate HFIP, and then stored at
−
80
◦
C for later
use. Immediately before use, the monomerized A
β
42 peptides in microfuge tubes were
completely resuspended in 5 mM in anhydrous dimethyl sulfoxide (DMSO, catalog number
D-2650, Sigma) by pipette mixing, and diluted to 100
µ
M with DMEM medium addition
(Dulbecco’s modified Eagle’s medium, Sigma). The mixture was homogenized in a shaker
at 37
◦
C for 7 days to aggregate into A
β
fibrils, the final concentration of A
β
fibrils was
100 µM [57,58].
4.5. Measurements of Thioflavin T Fluorescence
Thioflavin T (ThT), a commonly used probe to monitor A
β
fibril formation, emits
fluorescence upon binding to A
β
fibrils and the intensity of fluorescence is used to measure
the concentration of A
β
fibrils. To monitor the aggregation of A
β
42 into A
β
fibrils, 10
µ
M
A
β
42 peptides were added to phosphate-buffered saline (PBS, pH 7.4) at 37
◦
C and trans-
ferred onto 96-well plates (Bio-One, Greiner, Kremsmünster, Austria) containing 10
µ
M
ThT. The fluorescence intensity at 485 nm was measured by an ELISA reader (Infinite
200Pro, Tecan, Männedorf, Switzerland) under an excitation wavelength of 440 nm, after 1,
4, 7, 11, and 14 days [59].
4.6. Transmission Electron Microscopy (TEM)
400-mesh copper grid covered with Formvar and carbon (01754-F) was obtained from
Ted Pella, INC, Taiwan. Ten microliters of A
β
42 (10
µ
M) were dropped onto the copper
grids and dried at room temperature. It was then stained with 10
µ
L 1% (w/v) uranyl
acetate (UA, 2%) for 20 s. The grid was washed with 10
µ
L distilled water twice and dried
at room temperature. The images of aggregated A
β
fibrils at different time were examined
using a transmission electron microscope (TEM, Hitachi H7650, Tokyo, Japan) operated at
an acceleration voltage of 70 kV [60].
Int. J. Mol. Sci. 2021,22, 12760 13 of 19
4.7. Evaluation of Cell Viability
The effects of BSP on cell viability were evaluated by the water-soluble tetrazolium
(WST-1) assay (TaKaRa, Kusatsu, Shiga, Japan) using the N2a cell lines obtained from
Bioresource Collection and Research Center (Hsinchu, Taiwan). The N2a cells were cul-
tured in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) containing 1% antibiotic-
antimycotic (Gibco) and 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) and
incubated at 37
◦
C and 5% CO
2
in a 95% humidified incubator. The overall process was
based on the regulation of ISO 10993-5 [
61
]. Prior to the assessment, first, the extraction
solution was obtained by soaking 1 mg/mL BSP in 10 mL culture medium for a day. In ad-
dition, the pre-cultured N2a cells were seeded onto 96-well plates with a density of 1
×
10
4
cells/well and cultured for one day. Subsequently, the extraction solution was added to
the culture wells and further cultured for one day. Following this, 200
µ
L WST-1 reagent
was added to each well and incubated at 37
◦
C for 2 h. The OD value of each well was
determined at 450 nm by using an ELISA plate reader (SpectraMax iD3, Molecular Devices,
Inc, San Jose, CA, USA). The cell viability was calculated using the following formula:
Cell viability (%)=(OD experiment −OD background)×100
(OD control −OD background)(1)
Zinc diethyldithiocarbamate (ZDEC, Sigma-Aldrich) and aluminum oxide (Sigma-
Aldrich) were used as positive and negative controls, respectively. The cells cultured in only
the fresh medium were used as the control group. The observations were repeated six
times (n= 6) in each group.
4.8. Evaluation of Cytotoxicity
Cytotoxicity was evaluated using the LIVE/DEAD staining kit (L3224, Invitrogen,
Waltham, MA, USA) according to the manufacturer’s instructions [
62
]. Briefly, 0.2 g
specimen was immersed in 1 mL culture medium at 37
◦
C for 24 h, and the medium
was used as the extracted medium for subsequent experiments. As described earlier,
the pre-cultured N2a cells were seeded on a 24-well plate at a density of 2
×
10
4
cells/well
and incubated for 24 h. Afterwards, the extracted medium was added to each well,
followed by the simultaneous addition of 10
µ
M A
β
fibrils and 1 mg/mL BSP, and cultured
for 1 day. Subsequently, 2
µ
M calcein AM and 4
µ
M ethidium homodimer-1 (EthD-1)
were added to each well and incubated at 37
◦
C for 30 min to stain the live and dead
cells, respectively. Finally, the LIVE/DEAD staining was observed using a fluorescence
microscope (IX81, Olympus, Tokyo, Japan) at excitation and emission wavelengths of
488 nm and 515 nm, respectively. N2a cells cultured in the extracted medium only were
used as the control group. ZDEC and aluminum oxide were used as positive and negative
controls, respectively.
4.9. Determination of Cellular ROS Generation
The ability of BSP to diffuse ROS induced by A
β
fibrils was measured by a DCFDA-
cellular ROS detection assay kit (ab113851, Abcam, Cambridge, UK) [
63
]. In brief, N2a
cells were seeded on 96-well plates at a density of 2.5
×
10
4
cells/well and cultured
for 1 day. Afterward, 1 mg/mL BSP and 10
µ
M A
β
fibrils were added to each well to
induce ROS generation. On the next day, the medium was removed and washed with PBS.
Subsequently, a culture medium containing 20
µ
M 2,7-dichlorodihydrofluorescein diacetate
regent (DCFDA; Thermo Fisher, Waltham, MA, USA) was added to each well and further
cultured for 45 min. The fluorescence intensity representing the ROS level induced by A
β
fibrils was detected using a multimode microplate reader (Molecular Devices, SpectraMax
i3x, San Jose, CA, USA). The excitation and emission wavelengths were 495 and 535 nm,
respectively.
Int. J. Mol. Sci. 2021,22, 12760 14 of 19
4.10. Gene Expression Analysis
To assess the anti-inflammatory effects of BSP, we analyzed the expression levels of
three inflammation-related genes— tumor necrosis factor-
α
(TNF-
α
), interleukin-6 (IL-6),
and interleukin-10 (IL-10)—using BV-2 as target cells. The relative expressions of the genes
were estimated from three experimental groups: (1) control group: BV-2 cells seeded on
96-well plate with a density of 1
×
10
5
cells/mL were cultured without any treatment,
(2) A
β
group: BV-2 cells at 1
×
10
5
cells/mL were cultured and treated with 10
µ
M A
β
fibrils, and (3) A
β
-BSP group: BV-2 cells at 1
×
10
5
cells/mL were cultured and treated
with 1 mg/mL BSP and 10
µ
M A
β
fibrils. Total RNA was extracted using Qiazol reagent
(Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. Random hexamers
(Vivantis Inc., California, CA, USA) and reverse transcriptase (Vivantis Cat No: RTPL12)
were used for the first-strand cDNA synthesis with the following PCR parameters: 95
◦
C
for 3 min (denaturation), 40 cycles of 95
◦
C for 20 s, 60
◦
C for 30 s (annealing), and 72
◦
C for
30 s (elongation). The real-time RT-PCR was performed using the TOOLS 2X SYBR qPCR
Mix (Biotools Co., Ltd., Taipei, Taiwan) on a CFX Connect Real-Time PCR Detection System
(Bio-Rad, Hercules, CA, USA). The specific primers (Biotools Co., Ltd., Taipei, Taiwan)
used for real-time RT-PCR are shown in Table 1. The changes in expression of the target
genes were calculated using GAPDH as an endogenous control [64].
Table 1. Primers used for gene expression analysis.
Name Sequence
TNF-α-Forward 50-CATCTTCTCAAAATTCGAGTACAA-30
TNF-α-Reverse 50-TGGGAGTAGACAAGGTACAACCC-30
IL-6-Forward 50-GGAGCCCACCAAGAACGATAGTCA-30
IL-6-Reverse 50-GAAGTAGGGAAGGCCGTGGTT-30
IL-10-Forward 50-TAAGGCTGGCCACACTTGAG-30
IL-10-Reverse 50-GTTTTCAGGGATGAAGCGGC-30
GAPDH-Forward 50-TGCTGAGTATGTCGTGGAGTCT-30
GAPDH-Reverse 50-AATGGGAGTTGCTGTTGAAGTC-30
4.11. AlCl3-Induced AD Rat Model to Develop Alzheimer’s Disease (AD)
Eight-week-old Sprague Dawley (SD) male rats were purchased from BioLASCO
Taiwan Co., Ltd. (Taipei City, Taiwan) and adapted to the new environment for two weeks.
All experiments were carried out in compliance with the National Taiwan University Col-
lege of Medicine’s Institutional Animal Care and Use Committee (IACUC no. 20130429).
We maintained the animals according to the Guide for the Care and Use of Laboratory
Animals. The behavioral tests performed were approved by the Animal Ethics Committee
of the National Taiwan University Hospital, Taiwan. AD was induced by intraperitoneal
(IP) injection of AlCl
3
(100 mg/mL AlCl
3
dissolved in normal saline) three times a week as
described in a previous study [65]. Animal models play a central role in AD research [66].
Long-term exposure to aluminum (Al
3+
) is considered a pathogenic factor in the pathogen-
esis of AD [
67
]. Previous animal studies have shown that Al
3+
induces neuropathological,
neurochemical, and neurobehavioral changes similar to AD [
22
]. In addition, it has been
reported that Al
3+
is found in SP and neurons with neurofibrillary tangles in the brains
of AD patients [
67
]. Therefore, Al
3+
administration can be used to induce AD in animal
models. The SD rats (n= 18) were randomly categorized into 3 groups with 6 rats per group:
(1) Control group: rats injected with normal saline, (2) AlCl
3
group: rats injected with
AlCl
3
(100 mg/kg body weight) three times every week and consecutively for eight weeks,
(3) AlCl
3
-BSP group: rats were injected with AlCl
3
(100 mg/kg body weight), and then
treated with BSP (10 mg/kg/day) by oral administration during the AD induction period.
At the end of the experiment (18-week-old SD rats after purchasing), the rats were subjected
to the Morris Water Maze test to assess their memory and cognitive functions. The blood
was collected to assess the blood elements, and serological analysis to check the safety.
Int. J. Mol. Sci. 2021,22, 12760 15 of 19
The cortex and hippocampus were harvested after sacrificing the rats by euthanasia for
western blotting and histological examination.
4.12. Morris Water Maze Test
The Morris Water Maze was used to assess working and spatial memory retention in the rats,
following a previous study [
68
]. Briefly, the circular pool (160 cm in diameter and 35 cm in height)
was divided into four quadrants. It contains a
10 cm (diameter) ×25 cm (height)
round escape
platform, the same color as the rest of the basin (to eliminate any false-positive results
due to vision), placed in the constant quadrant of the basin during the entire test kept
2 cm below the water surface. Animals had 4 trials per day separated by 10 min for
4 successive days. For each training session, the rat was gently placed in water at a different
drop location and allowed to find the submerged platform; the rat was guided toward
the platform if it could not find the platform within 2 min. Once the platform was reached,
the rat was allowed to stay on it for 30 s. The training was continued in each quadrant
for four consecutive days. After four training sessions, the time taken by the rat to reach
the escape platform was recorded using the EthoVision software (The Observer XT, Noldus
Information Technology, Wageningen, Netherlands). The retrieval tests of working and
spatial memories were performed as described in a previous study [
69
]. To assess spatial
memory, we recorded the time taken (s) by each rat to reach the escape platform from their
initial position, whereas working memory was assessed from the time spent (s) by the rat
in the same quadrant (maximum 120 s) without the escape platform [70].
4.13. Blood Analysis
The safety
in vivo
was evaluated by blood element analysis and serological analysis.
Blood from the rats was collected by cardiac punctures at the end of the experiment. Serum
was obtained by centrifuging the collected blood samples at 1500 rpm at 4
◦
C for 15 min.
The collected serum samples were stored at
−
80
◦
C for subsequent analyses. For biochemical
tests, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen
(BUN), and lactate dehydrogenase (LDH) in serum were measured following a previously
described method [
36
]. For blood element analysis, red blood cells (RBC), hemoglobin
(HGB), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin (MCH),
mean corpuscular hemoglobin concentration (MCHC), reticulocytes (RET), platelets (PLT),
white blood cells (WBC), neutrophil (NEUT), lymphocytes (LYMPH), monocytes (MONO),
eosinophils (EO), and basophils (BASO) were measured. Blood element and serum were
measured by the National Taiwan University Veterinary Hospital, Taiwan. Reference: Charles
River Laboratories, CD®IGS Rat Model Information Sheet [71,72].
4.14. Western Blotting
The total protein was extracted from the hippocampus and cortex tissues of the exper-
imental animals using RIPA lysis buffer (Thermo Fisher, Waltham, MA, USA) containing
a protease inhibitor cocktail. The protein concentration was measured by the Bradford
protein assay kit (Z5030028, BioChain Institute Inc., San Francisco CA USA). The samples
were resolved with equal amounts of protein (25
µ
g) using 12% SDS-PAGE. Three samples
from each group were washed, lysed, and equal amounts of protein were separated and
transferred onto a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA),
blocked, and incubated with primary antibodies against BACE1 and
β
-actin (Anti-BACE1,
cat. no. ab183612; dilution, 1:500, and anti-
β
-actin, T5168, Sigma-Aldrich; dilution, 1:5000).
BACE1 protein was visualized by enhanced chemiluminescence [73].
4.15. Histological Analysis and Immunohistochemical (IHC) Staining
The cortex and hippocampus harvested from the experimental rats were treated with
a series of alcohol and fixed by 4% glutaraldehyde. The specimens were then bisected
and embedded in paraffin. Subsequently, the paraffin-embedded tissue blocks were cut
into 5-
µ
m-thick sections, placed on the glass slides, and stained with BACE1 immuno-
Int. J. Mol. Sci. 2021,22, 12760 16 of 19
histochemical anti-body. Hematoxylin and eosin (H&E) stain was used for contrast imaging.
The slides were then deparaffinized and rehydrated with 0.1% hydrogen peroxide (Sigma-
Aldrich, St. Louis, MO, USA) in PBS solution for 10 min to block endogenous peroxidases.
For retrieval, nonspecific background staining was blocked by 20
µ
g/mL proteinase K
(Sigma-Aldrich, USA) solution [
74
]. The solution containing the slides was incubated
in a humidified chamber at 37
º
C for 20 min. The primary antibodies against BACE1 (Anti-
BACE1, cat. no. ab183612), were diluted with 1% BSA in a ratio of 1:500 (v/v). The diluted
primary antibody was then applied to the slides and incubated at 4
◦
C overnight. After
incubation, the tissue sections were washed by TBS containing 0.025% Triton-X 100 with
gentle agitation. The sections were further incubated in 1% BSA containing goat anti-rabbit
HRP IgG secondary antibody at 1:5000 (v/v) dilution. Finally, the sections were further
treated with 3, 3
0
-diaminobenzidine (DAB, Sigma-Aldrich, USA) substrate solution as
an enhancer to more clearly reveal colors under the optical microscope [75].
4.16. Statistical Analyses
The data were expressed as mean
±
standard deviation (SD). All statistical analyses
were done using Student’s t-test or one-way analysis of variance (ANOVA) post hoc
tests and by Tukey’s test using GraphPad Prism software (Prism 9, GraphPad Software,
Inc., La Jolla, CA, USA), where p-values of less than 0.05 were considered statistically
significant [76].
5. Conclusions
In this study, BSP was successfully isolated and purified from dry B. striata by a mod-
ified extraction method. The molecular structure and characterized functional groups,
identified and confirmed by NMR and FTIR, respectively, are consistent with those re-
ported in previous studies. Though previous studies have shown its antioxidant and
anti-inflammatory effects, this study, for the first time, investigated the role of BSP in AD
therapy. The findings demonstrated that the extracted BSP could effectively diffuse ROS
from the A
β
fibrils and had good biocompatibility with N2a cell lines, whereas its anti-
inflammatory ability was evident in BV-2 cell lines. It also suppressed the expression of
BACE1, a prime therapeutic target for the development of drugs to treat AD. Moreover, BSP
treatment in AD rats reduced the damage to the hippocampus and cortex and recovered
the neurodegeneration induced by AlCl
3
. Collectively, the findings of this study indicate
that BSP could be a potential therapeutic agent in AD treatment.
Author Contributions:
Y.-W.L. collected the data and drafted the manuscript. H.-H.L. executed most
of the experiments. C.-H.F. and Y.-J.L. contributed to the active discussion of experimental design and
performing of the animal study. F.-H.L. supervised the study, assisted with study conceptualization,
and made a substantial contribution to the revision of the manuscript. All authors have read and
agreed to the published version of the manuscript.
Funding:
This work was supported by the Institute of Biomedical Engineering, National Taiwan
University, Taipei, Taiwan, ROC, Ministry of Science and Technology, Taipei, Taiwan, ROC and
National Health Research Institutes (109-1901-01-19-07), Taiwan, Republic of China.
Institutional Review Board Statement:
All animal procedures and experiments were performed
following a protocol approved by the National Taiwan University College of Medicine and Public
Health Institutional Animal Care and Use Committee (IACUC no. 20180104).
Data Availability Statement: The datasets used and/or analyzed in the current study are available
from the corresponding author on reasonable request.
Conflicts of Interest: The authors declare no conflict of interest.
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