1. No category

J Korean Med. 2014;35(4):10-23
http://dx.doi.org/10.13048/jkm.14040
pISSN 1010-0695 • eISSN 2288-3339
Original Article
Effect of Guibi-tang on Neuronal Apoptosis and
Cognitive Impairment Induced by Beta Amyloid in Mice
Lee Ju-Won, Cho Dong-Guk, Cho Woo-Sung, Ahn Hyung-Gyu, Lee Hyun-Joon,
Shin Jung-Won, Sohn Nak-Won
Department of Oriental Medical Science, Graduate School of East-West Medical Science
Kyung Hee University, Yongin, 449-701, Korea
Objectives: This study evaluated the effects of Guibi-tang (GBT) on neuronal apoptosis and cognitive impairment
induced by beta amyloid (Aβ), (1-42) injection in the hippocampus of ICR mice.
Methods: Aβ (1-42) was injected unilaterally into the lateral ventricle using a Hamilton syringe and micropump (2
㎍/3 ㎕, 0.6 ㎕/min). Water extract of GBT was administered orally once a day (500 mg/kg) for 3 weeks after the
Aβ (1-42) injection. Acquisition of learning and retention of memory were tested using the Morris water maze.
Neuronal damage and Aβ accumulation in the hippocampus was observed using cresyl violet and Congo red staining.
The anti-apoptotic effect of GBT was evaluated using TUNEL labeling in the hippocampus.
Results: GBT significantly shortened the escape latencies during acquisition training trials. GBT significantly
increased the number of target headings to the platform site, the swimming time spent in the target quadrant, and
significantly shortened the time for the 1st target heading during the retention test trial. GBT significantly attenuated
the reduction in thickness and number of CA1 neurons, and Aβ accumulation in the hippocampus produced by Aβ
(1-42) injection. GBT significantly reduced the number of TUNEL-labeled neurons in the hippocampus.
Conclusion: These results suggest that GBT improved cognitive impairment by reducing neuronal apoptosis and Aβ
accumulation in the hippocampus. GBT may be a beneficial herbal formulation in treating cognitive impairment
including Alzheimer’s disease.
Key Words : Guibi-tang , beta-amyloid, memory impairment, neuronal apoptosis, Alzheimer’s disease
Introduction
Alzheimer's disease is a neurodegenerative disease
and the most common form of senile dementia. It is
characterized by brain atrophy with histology,
showing senile plaques, neurofibrillary tangles, and
1,2)
granulovacuolar degeneration . Beta-amyloid (Aβ)
is united to senile plaque in the extracellular
environment, which causes neuronal apoptosis and
2)
activates neuroglia cells . In the intracellular
environment, tau protein produces neurofibrillary
tangles forming filamentous shapes. It makes empty
space surrounded by basophilic granules and
3,4)
granulovacuolar degeneration .
So far, several possible causes of Alzheimer's
disease have been identified, including the reduction
5)
of acetylcholine, increase of acetylcholinesterase ,
6)
amyloid cascade hypothesis , inflammatory response
7)
by neuroglia cell , gene mutation of Presenilin-1 and
8)
9)
Presenilin-2 , and reactive oxygen species . But the
⋅Received：9 October 2014
⋅Revised：12 December 2014
⋅Accepted：12 December 2014
⋅Correspondence to：Sohn Nak-Won, O.M.D., Ph.D.
Department of Oriental Medical Science, Graduate School of East-West Medical Science,
Kyung Hee University, Yongin, 449-701, Korea
Tel：+82-31-201-2747, E-mail：[email protected]
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Effect of Guibi-tang on Neuronal Apoptosis and Cognitive Impairment Induced by Beta Amyloid in Mice (427)
exact cause and pathogenesis is still unknown and
many efforts have been carried out to clear it.
Currently, drugs for Alzheimer's disease are
directed at elevating the acetylcholine concentration
of neurons by suppressing acetylcholinesterase.
Acetylcholinesterase inhibitors like donepezil,
rivastigmine, and galantamine are used for this
10,11)
. As understanding of the disease’s
purpose
pathogenesis progresses, new treatments which relate
with amyloid cascade hypothesis, are being
12)
developed . The amyloid cascade hypothesis is a
concept that focuses on the accumulation of beta
amyloid as taking central role in Alzheimer’s
disease, wherein its accumulation induces cell
13)
damage and cognitive impairment .
Guibi-tang (GBT) is a herbal prescription used in
traditional Korean medicine. It consists of 12
14)
different herbs . Under admission of Korean Food
and Drug Administration (KFDA), it has been used
as a remedy for anemia and insomnia. The major
ingredients of Guibi-tang are nodakenin, decursin,
15)
ginsenoside Rg1, liquiritin, and glycyrrhizin .
In an in vitro experimental study, it has been
reported that Guibi-tang reduces apoptosis in rat
microglia oxidative damage model and astrocyte
16,17)
. Similarly, in PC12
glutamate damage model
cells treated with 6-Hydroxydopamine (6-OHDG),
Guibi-tang dose-dependently reduces neuronal
18)
apoptosis . In a recent animal study, Guibi-tang has
induced cell proliferation and promoted learning and
19)
memory in the hippocampus dentate gyrus (DG) .
Improved cognitive function in Alzheimer’s disease
20)
patients has been shown in a clinical trial . This
study attempted to examine the impact of Guibi-tang
in Alzheimer’s disease.
This study confirmed the pathological state of
Aβ injected mouse by cresyl violet stain and Aβ
accumulation in the hippocampus CA1 and DG
region. Neuronal apoptosis has been confirmed by
terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) stain. This study examined the
effect of Guibi-tang administration on neuronal
apoptosis in mice induced by intraventricular Aβ
injection and the restoration of cognitive impairment
caused by the neuronal damage using the Morris
water maze.
Materials and Methods
1. Preparation of GBT
The twelve crude herbs of GBT were purchased
from Puremind Medicinal Herbs (Youngchen, Korea).
A decoction of GBT was prepared in our laboratory
from a mixture of chopped crude herbs (Table 1).
GBT was extracted in distilled water at 100°C for
3h. The solution was evaporated to dryness and
freeze-dried (yield: 20.0%). The extracted GBT
powder was stored at 4°C.
Table 1. Herbal Constitution of Guibi-tang (GBT)
Herbal name
Galenical term
Dose (g)
當歸
Andelicae gigantis Radix
4.0
龍眼肉
Longanae Arilus
4.0
酸棗仁
Zizyphi Spinosae Semen
4.0
遠志
Polygalae Radix
4.0
人蔘
Ginseng Radix Alba
4.0
黃芪
Astragali Radix
4.0
白朮
Atractylodis Rhizoma Alba
4.0
白茯神
Hoelen cum Radix
4.0
木香
Aucklandiae Radix
4.0
甘草
Glycyrrhizae Radix
2.0
生薑
Zingiberis Rhixoma Crucus
6.0
大棗
Zizyphi jujubae Fructus
4.0
Total amount
48.0
2. Animals
Male C57BL/6 mice (25-28g, Nara Biotechnology,
Korea) were used for this study. All animal protocols
were approved by the Ethics Committee for the Care
and Use of Laboratory Animals at Kyung Hee
University. The animals were housed in plastic cages
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at constant temperature (22±2°C) and humidity (55±
10%) with 12 h-12 h light-dark conditions. The
animals were allowed free access to food and water
before the experiment.
3. Aβ (1-42)-induced AD mouse Model
The Aβ (1-42) (#A9810, Sigma, St. Louis, MO,
USA) was dissolved in sterile saline to a
concentration of 0.66 mg/ml and incubated at 37°C
for 4 days to allow for fibril formation. The mice
were anesthetized with Zoletil (30 mg/kg, i.p.) and
placed on a stereotaxic instrument (USA). The scalp
of each mouse was incised, and the skull was
adjusted to place the bregma and lambda on the
same horizontal plane. A small bone hole was drilled
through the skull. Aβ (1-42) (3ul＝2ug) or the
vehicle (3 μl) was injected into a lateral ventricle
(－0.5 mm anteroposterior, +1.5 mm medial-lateral,
-2.5 mm dorsal-ventral from the dura, in relation to
the bregma) at a rate of 0.5 μl/min using a 10ul
Hamilton syringe fitted with a 26-gauge stainless
steel needle. The hole was blocked with bone wax
and the scalp was then closed with suture. Sham
group mice received the same surgical procedure
with injection of an identical volume vehicle (sterile
saline).The mice were allowed to recover from
surgery for 2 days. Saline and Guibi-tang (500
mg/kg) were administered intragastrically once daily
for 21 days beginning from two days after the Aβ
(1-42) injection.
4. Experimental groups
The mice were randomly divided into three
groups. The Aβ and GBT groups were injected
intracerebroventricularly with a Aβ (1-42)(3ul＝2ug).
The sham group received the same surgical
procedures and was injected with an identical
volume of vehicle (sterile saline). The GBT group
received Guibi-tang (500 mg/kg, dissolved in normal
saline, orally), once a day for 21 days from two days
after the Aβ (1-42) injection. The Aβ and sham
group received vehicle (normal saline) orally. A total
of 36 mice were used.
5. Tissue Processing
The mice were anesthetized and transcardially
perfusion-fixed with 4％ formaldehyde in 0.1M
sodium phosphate buffer (pH 7.4). Brains were
removed quickly and postfixed with the same
fixation solution overnight at 4°C. Fifty μm thick
coronal sections of brain tissue were made using a
freezing microtome (Leica, 2800N, Germany).
6. Morris water maze test
The Morris water maze test was performed for 4
days. The acquisition training was performed for 3
Fig. 1. Schematic diagram of experiment schedule
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Effect of Guibi-tang on Neuronal Apoptosis and Cognitive Impairment Induced by Beta Amyloid in Mice (429)
days and the retention test on the 4th day. The
apparatus consisted of a circular water pool 100 cm
in diameter and 40 cm in height. It was filled with
23 ± 1°C water with a depth of 28 cm and covered
a black platform (10 cm in diameter). The platform
was submerged approximately 0.5 cm below the
surface of the water. The pool was divided into four
equal quadrants: northeast (NE), northwest (NW),
southeast (SE), and southwest (SW). The platform
was located in the center of the southwest quadrant.
During the first 3 days acquisition test, mice were
given 8 trials per day to find the hidden platform.
Each mouse (12 mice per group) was gently placed
into the water facing the wall in the direction of
north (N), east (E), south (S), and west (W) in two
series of order. The mouse was allowed to swim
until they reached the hidden platform (maximum
swim time was 60 seconds). The escape latency to
reach the platform was recorded and they were
allowed to remain on the platform for 10 seconds
before being removed. Any mice which failed to find
the platform within 60 seconds were guided to the
hidden platform and then placed on the platform for
10 seconds for reinforcement before being removed.
One trial of the retention test without the platform
was performed on the 4th day to assess the memory
of the correct platform location. The mice were
placed into the pool and swam freely for 60 seconds.
The swimming paths were recorded by a video
camera linked to a computer-based image analyzer
(SMART 2.5 video-tracking system, Panlab, Spain).
The number of target headings and the swimming
time in each zone were analyzed with a grid design
of 4 zones (Fig. 2). This grid design, constructed with
a computer-based image analyzer, was superimposed
over the maze and viewed on a monitor. The mice
were sacrificed after the retention test trial.
7. Cresyl violet staining, Congo red staining,
and TUNEL labeling
Neuronal damage and apoptosis in the CA1 of the
hippocampus were observed using cresyl violet
staining, Congo red staining, and terminal transferase
dUTP nick-end labeling (TUNEL) assay. The
hippocampal tissues analyzed came from the mice
which performed the Morris water maze test. Cresyl
violet staining was used; hippocampus tissues were
immersed in 0.5% cresyl violet solution for 3
minutes. Congo red staining was used; hippocampus
tissues were immersed in 0.3% Congo red stock
solution for 20 minutes. The hippocampus TUNEL
labeling was carried out using In Situ Cell Death
Detection kit, POD (#11684817910, Roche, Germany)
following the manufacturer’s protocol.
8. Image analysis
Fig. 2. Schematic diagram of platform position in the
swimming pool and zones for the retention test.
Relative optical densities of Congo red, counting
of the CA1 and DG neurons stained with cresyl
violet and TUNEL-labeled cells were analyzed using
ImageJ software (Ver. 1.44p, NIH, USA). The
relative optical densities were measured in the CA1
and DG by the mean gray value on an inverted
black-white binary image and were normalized to the
value of the normal group. The number of CA1
neurons were counted in the 500 μm length of the
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pyramidal cell layer of CA1. TUNEL-labeled cells
4
were measured from a 10 µm area of the CA1 and
DG region. Data were normalized with the same
area. The mean values from the four sections
analyzed in each mouse were used for statistical
analysis.
9. Statistical analysis
All data in this study are presented as means ±
standard errors and were evaluated using Student’s
t-test. A probability value of less than 0.05 was used
to indicate a significant difference. Differences between
groups in the acquisition test were evaluated using
one-way ANOVA.
Results
Guibi-tang improved the spatial learning and
memory deficits in Aβ (1-42)-injected mice.
1. Acquisition trials analysis.
Fig. 3 demonstrates escape latencies in the training
period for three days. The Aβ group had
significantly longer acquisition time than the sham
group at the 5th, 6th, 7th, and 8th trials (p<0.05,
p<0.01, p<0.05, p<0.01, respectively) on the 1st day,
6th and 8th trials (p<0.01, p<0.05, respectively) on
the 2nd day and 4th, 5th, 6th, 7th and 8th trials
(p<0.01, p<0.05, p<0.01, p<0.01, p<0.01, respectively)
on the 3rd day. The GBT group showed significantly
less acquisition time at the 6th and 8th trials
(p<0.05, p<0.01) on the 2nd day and 4th, 7th, and
8th trials (p<0.05, respectively) on the 3rd day.
There were many trials that had shorter escape
latency on the 3rd day compared to the 1st and 2nd
days. On days 1, 2, and 3, the shortened escape
latencies occurred towards the latter half of the 8
trials.
Fig. 3. Effect of Guibi-tang (GBT) on escape latencies in the acquisition training trials. Aβ(1-42) (2 ㎍/3 ㎕) or sterile
saline (3 ㎕) was injected into the lateral ventricle 19 days before the first training day. A solution of Guibi-tang
or normal saline was given orally once daily for 21 days after surgery. The training trial was performed eight times
a day for three days. Data are represented by mean ± SEM (n=12 in each group). Statistical significances are
compared between Sham and Aβ groups (*, p<0.05; **, p<0.01) or between Aβ and Aβ+GBT groups (#,
p<0.05; ##, p<0.01).
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Effect of Guibi-tang on Neuronal Apoptosis and Cognitive Impairment Induced by Beta Amyloid in Mice (431)
(A)
(B)
Fig. 4 (A, B) Effects of Guibi-tang (GBT) on escape latency and number of target headings in the retention test trial.
The retention trial was conducted the day after the training trial. (A) Escape latency. (B) Number of target
headings. Data are represented by mean ± SEM (n=12 in each group). Statistical significances are
compared between Sham and Aβ groups (*, p<0.05; **, p<0.01) or between Aβ and Aβ+GBT groups (#,
p<0.05; ##, p<0.01).
Fig. 5. Effect of Guibi-tang (GBT) on the swimming time spent in discrete quadrants in the retention trial (Fig. 2). The
retention trial was conducted the day after the training trial. Data are represented by mean ± SEM (n=12 in each
group). Statistical significances are compared between Sham and Aβ groups (*, p<0.05; **, p<0.01) or between
Aβ and Aβ+GBT groups (#, p<0.05; ##, p<0.01).
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(A)
(B)
(C)
Fig. 6. (A, B, C) Effects of Guibi-tang (GBT) on the thickness and number of CA1 neurons in the hippocampus. (A)
Representative photographs showing cresyl violet stained neuron in the hippocampus. (B) Neuron count in CA1
region of the hippocampus. (C) Depth of CA1 region of the hippocampus. Original magnification: HIPPOCAMPUS
(40 ×), CA1 (200 ×). Scale bar: HIPPOCAMPUS (1 mm), CA1 (200 μm). Data are represented by mean ± SEM
(n=6 in each group). Statistical significances are compared between Sham and Aβ groups (*, p<0.05; **, p<0.01)
or between Aβ and Aβ+GBT groups (#, p<0.05; ##, p<0.01).
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Effect of Guibi-tang on Neuronal Apoptosis and Cognitive Impairment Induced by Beta Amyloid in Mice (433)
(A)
(B)
Fig. 7. (A, B) Effect of Guibi-tang (GBT) on Aβ accumulation in the hippocampus. (A) Representative photographs
showing Congo-red stained beta amyloid in the hippocampus. (B) The mean of optical density of beta amyloid
in CA1 and DG region of the hippocampus. Original magnification: CA1 (400 ×), DG (200 ×). Scale bar: CA1
(400 μm), DG (200 μm). Data are represented by mean ± SEM (n=6 in each group). Statistical significances
are compared between Sham and Aβ groups (*, p<0.05; **, p<0.01) or between Aβ and Aβ+GBT groups (#,
p<0.05; ##, p<0.01).
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(A)
(B)
Fig. 8. (A, B) Effect of Guibi-tang (GBT) on TUNEL-labeled neurons in the hippocampus. (A) Representative
photographs showing TUNEL stained neurons in the hippocampus. (B) Number of positive TUNEL stained neurons
in CA1 and DG region of the hippocampus. Original magnification: CA1, DG (200 ×). Scale bar: CA1, DG (200
μm). Data are represented by mean ± SEM (n=6 in each group). Statistical significances are compared between
Sham and Aβ groups (*, p<0.05; **, p<0.01) or between Aβ and Aβ+GBT groups (#, p<0.05; ##, p<0.01).
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2. Retention trials analysis
Upon completion of the acquisition trials, the
escape latency and number of target headings were
examined without the platform for 60 seconds on the
4th day. The escape latency of the Aβ group was
44.0±6.2 s and was significantly longer than in the
sham group, which was 18.4±5.3 s (p<0.01). The
escape latency of the GBT group was 20.4±4.4, a
significantly shorter time compared to that of the
Aβ group (p<0.01). The number of target headings
for the Aβ group was 1.1±0.5. This was significantly
smaller compared to the sham group’s 2.3±0.4
(p<0.05). The number of target headings for the
GBT group was 2.4±0.4. This was significantly
larger compared to that of the Aβ group (p<0.05).
3. Swimming time spent in discrete
quadrants
After completion of the acquisition trials, the
tracking path without the platform was examined for
60 seconds on the 4th day. Swimming time in
discrete quadrants for the sham group were: 21.7±
3.3 in Quadrant A (where the platform was placed
previously), 12.0±1.8 in Quadrant B, 15.4±2.0 in
Quadrant C, and 11.0±1.2 in Quadrant D. Swimming
time in discrete quadrants for the Aβ group were:
14.5±0.7 in Quadrant A (where the platform was
placed previously), 18.0±1.5 in Quadrant B, 13.8±1.5
in Quadrant C, and 13.7±0.9 in Quadrant D. The
Aβ group swam for a significantly shorter time in
Quadrant A compared to the sham group (p<0.05).
The Aβ group swam in Quadrants B and D for a
significantly longer time compared to the sham
group (p<0.01, p<0.05, respectively). Swimming time
in discrete quadrants for the GBT group were:
21.3±1.5 in Quadrant A (where the platform was
placed previously), 12.4±1.7 in Quadrant B, 14.5±1.6
in Quadrant C, and 11.7±1.4 in Quadrant D. The
GBT group showed significantly more time in
Quadrant A (p<0.01) and significantly less time in
Quadrant B compared to the Aβ group (p<0.05).
Guibi-tang attenuated neuronal cell loss in the
hippocampus of Aβ (1-42)-injected mice.
Neuronal cell loss was assessed by counting the
number and observing the morphology of neuron
cells in the hippocampus using cresyl violet staining.
The Aβ group showed short and thin neuron cell
layers in the hippocampus CA1 region compared to
the sham group. This change meant that the number
of neurons decreased. In contrast, the GBT group
exhibited thick and deep neuron cell layers.
The number and depth of neuron cell layers was
inspected in certain hippocampus CA1 regions. The
number of CA1 neurons was 123.2±7.1 in the sham
group and 94.8±6.0 in the Aβ group. The Aβ
group showed significantly fewer CA1 neurons
compared to the sham group (p<0.01) while the GBT
group showed significantly more CA1 neurons
(114.4±4.2) compared to the Aβ group (p<0.01).
The depth of the CA1 region in the sham group was
54.6±0.9 and was 50.3 ± 1.3 in the Aβ group. The
Aβ group showed significantly shallower CA1
compared to the sham group. The GBT group
showed significantly deeper CA1 (54.1±1.1) compared
to the Aβ group.
Guibi-tang attenuated Aβ accumulation in the
hippocampus of Aβ (1-42)-injected mice.
The optical density of Congo red staining of Aβ
accumulation was observed in certain hippocampus
CA1 and DG regions. The optical density of Congo
red staining in the CA1 region for the sham and Aβ
group were 87.8±0.5 and 89.1±0.7, respectively. The
Aβ group’s value increased compared to the sham
group’s but not significantly. The optical density of
Congo red staining in the CA1 region for the GBT
group was 85.7±0.7, which was significantly lower
compared to the Aβ group (p<0.01). The optical
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density of Congo red staining in the DG region for
the Aβ group was higher compared to the sham
group but not significantly. There was also no
significant difference in the optical density of Congo
red staining in the DG region between the GBT and
Aβ groups.
Guibi-tang attenuated neuronal apoptosis in the
hippocampus of Aβ (1-42)-injected mice.
Neuronal apoptosis was examined using TUNEL
assay that stains DNA fragments as a result of cell
death. TUNEL-labeled cells were rarely seen in the
CA1 and DG regions of the sham group. Many
TUNEL labeled cells were seen in the Aβ and GBT
groups. The number of TUNEL-labeled cells were
4
measured from a 10 µm area of the CA1 and DG
region. The Aβ group showed significantly
increased TUNEL-labeled cells in the CA1 region
compared to the sham group (29.8 ± 5.0 vs. 2.7 ±
4
1.0 cells/10 μm, p<0.01). The GBT group showed
significantly fewer TUNEL-labeled cells in the CA1
region compared to the Aβ group (20.6 ± 2.4 vs
4
29.8 ± 5.0 cells/10 μm, p<0.05). The Aβ group
showed significantly more TUNEL-labeled cells in
the DG region compared to the sham group (26.8 ±
4
5.5 vs. 1.2 ± 0.6 cells/10 μm, p<0.01). The number
of TUNEL-labeled cells in the DG region was
similar between the GBT and Aβ groups (22.5 ±
4
3.0 vs 26.8 ± 5.5 cells/10 μm).
Discussion
Available evidence indicates that Guibi-tang is
effective in the management of learning, memory,
19)
and cognitive impairment . We analyzed the effects
of Guibi-tang administration as a possible Alzheimer’s
disease treatment. Using an Alzheimer’s disease
animal model, we tested Guibi-tang’s effect on the
amyloid cascade hypothesis, neuronal apoptosis, and
cognitive impairments as well as Guibi-tang’s
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potential neuron protective effect.
The Morris water maze test, which confirms space
reference memory and space working memory, was
conducted to determine improvement of cognition.
The GBT group showed improvement in advanced
learning memory and cognition. Significant results
during the training trials were mostly concentrated
towards the tail end of the trials (14th, 16th, 20th,
23th, and 24th of 24 innings). The favorable effect
of Guibi-tang was significant during the 2nd and 3rd
day out of the three-day training period. Another
Guibi-tang study showed similar favorable results in
the rear of the training trials (11th, 12th and 13th of
21)
16 inning) . Concentration of significant results
towards the rear of trial series suggests that
Guibi-tang had excellent effect in repeated learning
memory. Learning and memory are major problems
in Alzheimer’s disease, which impedes the patients
from having a normal daily life. An improvement in
learning memory among patients with Alzheimer’s
disease as a result of Guibi-tang can be valuable.
Retention tests showed that the GBT group had 90.2
% recovery compared to the sham group on escape
latency. In particular, the 98.2% recovery on time
spent in the target quadrant showed that Guibi-tang
had a greater effect than ferulic acid, which effected
an 86% recovery in another study that employed a
22)
similar experimental design . A study that induced
dementia by scopolamine showed that GBT treatment
shortened the escape latency more than tacrin
23)
treatment , wherein tacrin is already a known
remedy for dementia. From these experimental
results, it appears that Guibi-tang can effect an
improvement of memory deficit and cognitive
impairment in diverse conditions.
The Aβ (1-42) induced brain tissue damage,
confirmed by using cresyl violet staining, was
attenuated by Guibi-tang. Abnormal accumulation of
Aβ is a major factor in Alzheimer’s disease. It is
closely related with memory and cognition
impairment, wherein Aβ forms senile plaques
Effect of Guibi-tang on Neuronal Apoptosis and Cognitive Impairment Induced by Beta Amyloid in Mice (437)
outside the neurons. Alzheimer’s disease is also
associated with a reduction in neurons that are
related with memory and cognition in the cerebral
24, 25)
. A study that utilized
cortex and hippocampus
Aβ injection confirmed that neurons were reduced
in the hippocampus including CA1, CA2, and DG
after 6 weeks26). According to the cresyl violet
staining we conducted, there was significant decrease
in the number of neurons and layer depth in the
CA1 region of the hippocampus 3 weeks after Aβ
(1-42) was injected into the lateral ventricle of the
brain. The GBT-treated group recovered 92.8% of
the neuron cell numbers and 99.1% of the neuron
cell layer depth compared to the sham group. This
showed that Aβ-induced brain tissue damage can be
restored by Guibi-tang. We hypothesize that
Guibi-tang may have a neuron protective effect by
disrupting neuronal apoptosis.
Aβ (1-42) accumulation in brain tissue was
confirmed by using Congo red staining. Congo red
staining is most commonly used to observe
27)
formation and cohesion of Aβ fibril . Congo red
stains a cross β-pleated sheet present on the Aβ
fibril. Congo red does not stain the monomer and
dimer states of Aβ, allowing Congo red to confirm
28)
pathological condition of Aβ accumulation . Aβ
can accumulate in various brain regions after it is
injected into the lateral ventricle of the brain. A
study was able to immediately confirm Aβ
accumulation by injecting fluorescent labeled Aβ
(25-35) into the lateral ventricle, the 3rd and 4th
ventricles, but accumulation of Aβ in the
hippocampus was detected three days after
29)
injection . At least three days is needed for Aβ to
accumulate in the hippocampus after it is injected
into the lateral ventricle. Our experiment was done
over a 3-week period after the Aβ injection.
The Congo red staining showed the Aβ group
had more Aβ accumulation in the hippocampus
CA1 and DG region than the sham group, but their
optical densities did not differ significantly. Methods
that can better quantitate Aβ accumulation like
western blotting or ELISA are needed for further
study. The GBT group had reduced Aβ
accumulation compared to the Aβ group with the
accumulation in the hippocampus CA1 region, being
suppressed significantly. CA1 has more neuronal
apoptosis than other hippocampus regions in
30)
Alzheimer's disease patients . It can be relevant to
Alzheimer’s disease that Guibi-tang reduced Aβ
accumulation in CA1.
We confirmed neuronal apoptosis by TUNEL
labeled neuron. Neurons have normal morphology
during apoptosis. Cohering a chromatin in the
31)
nucleus, the neuron starts to atrophy and fragment .
DNA fragments are formed by the cutting of the
nucleosome.
TUNEL
staining
detects
this
32)
fragmentation . The Aβ group had significantly
more TUNEL-labeled cells in the hippocampus CA1
and DG regions compared to the sham group. The
GBT group had a reduced number of
TUNEL-labeled cells with significant reduction in
the CA1 region.
Guibi-tang reduced Aβ accumulation in the CA1
region and reduced neuronal apoptosis induced by
Aβ. Guibi-tang’s cognition improvement effect
appears to be due to the reduction of neuronal
apoptosis. Considering the irreversible nature of
Alzheimer’s disease, the above result is meaningful.
Our study is the first to confirm that Guibi-tang
reduced tissue damage through suppression of
neuronal apoptosis in an Alzheimer’s animal model,
in vivo study. We did not compare Guibi-tang and
other chemical drugs because Guibi-tang is a crude
water extract and it is difficult to compare dosage
with chemical drugs. Further study about comparative
efficacy with chemical drugs is needed.
Guibi-tang improved Aβ-induced memory
impairment in mice, and attenuated Aβ accumulation
and neuronal apoptosis in the hippocampus CA1
region. As a result, this can explain improvement of
cognitive impairment.
http://dx.doi.org/10.13048/jkm.14040
21
(438) Journal of Korean Medicine 2014;35(4)
Conclusions
9. Mhatre M, Floyd RA, Hensley K. Oxidative stress
and neuroinflammation in Alzheimer’s disease and
The results suggest that GBT improved cognitive
impairment by reducing neuronal apoptosis and Aβ
accumulation in the hippocampus. GBT may be a
beneficial herb formulation that can be used to treat
cognitive impairment, including Alzheimer’s disease.
amyotrophic lateral sclerosis: Common links and
potential therapeutic targets. J Alzheimers Dis.
2004;6(2):147-57.
10. Roberson MR, Kolasa K, Parsons DS, Harrell LE.
Cholinergic denervation and sympathetic ingrowth
result in persistent changes in hippocampal
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