Original article

Chin Med J 2008;121(9):832-839

832

Original article

Curcumin improves learning and memory ability and its
neuroprotective mechanism in mice

PAN Rui, QIU Sheng, LU Da-xiang and DONG Jun

Keywords: Alzheimer’s disease; curcumin; apoptosis

Background Increasing evidence suggests that many neurons may die through apoptosis in Alzheimer’s disease (AD).
Mitochondrial dysfunction has been implicated in this process of neuronal cell death. One promising approach for
preventing AD is based upon anti-apoptosis to decrease death of nerve cells. In this study, we observed the memory
improving properties of curcumin in mice and investigated the neuroprotective effect of curcumin in vitro and in vivo.
Methods The mice were given AlCl

orally and injections of D-galactose intraperitoneally for 90 days to establish the

AD animal model. From day 45, the curcumin group was treated with curcumin for 45 days. Subsequently, the
step-through test, neuropathological changes in the hippocampus and the expression of Bax and Bcl-2 were carried out
to evaluate the effect of curcumin on the AD model mice. In cultured PC12 cells, AlCl

exposure induced apoptosis. The

MTT assay was used to measure cell viabilities; flow cytometric analysis to survey the rate of cell apoptosis; DNA-binding
fluorochrome Hoechst 33258 to observe nuclei changes in apoptotic cells and Western blot analysis of Bax, Bcl-2 to
investigate the mechanisms by which curcumin protects cells from toxicity.
Results Curcumin significantly improved the memory ability of AD mice in the step-through test, as indicated by the
reduced number of step-through errors (P <0.05) and prolonged step-through latency (P <0.05). Curcumin also
attenuated the neuropathological changes in the hippocampus and inhibited apoptosis accompanied by an increase in
Bcl-2 level (P <0.05), but the activity of Bax did not change (P >0.05). AlCl

significantly reduced the viability of PC12

cells (P <0.01). Curcumin increased cell viability in the presence of AlCl

(P <0.01). The rate of apoptosis decreased

significantly in the curcumin group (P <0.05) when measured by flow cytometric analysis. Curcumin protected cells by
increasing Bcl-2 level (P <0.05), but the level of Bax did not change (P >0.05).
Conclusions This study demonstrates that curcumin improves the memory ability of AD mice and inhibits apoptosis in
cultured PC12 cells induced by AlCl

. Its mechanism may involve enhancing the level of Bcl-2.

Chin Med J 2008;121(9):832-839

lzheimer’s disease (AD) is a neurodegenerative
disorder that currently affects nearly 5% of people

65-year old and over 30% of those 85-year old. It is now
estimated that there are 18−24 million people suffering
from AD worldwide, two-thirds of whom are living in
developed or developing countries, and this number is
expected to reach 34 million by 2025.

1,2

AD is

characterized by the progressive accumulation of amyloid
beta peptide (Aβ), neurofibrillary tangles (NFTs) and
hyperphosphorylated microtubule-associated tau protein.
Many regions involved in memory and learning processes,
such as the hippocampus and frontal cortex, show neuron
apoptosis several years before clinical signs appear.
Today there is no cure for this devastating disease and
therefore it is of great interest for researchers to find new
drugs that can hinder the disease process. Drugs currently
available on the market include different inhibitors of
acetylcholine esterase and N-methyl D-aspartate
(NMDA)-receptor antagonist. These drugs improve the
function of still intact neurons, but do not inhibit the ongoing
degenerative process leading to neuronal cell death.

Curcumin, a biologically active component of turmeric
(Curcuma longa) is used as a curry spice and herbal

medicine for the treatment of inflammatory conditions,
cancer, AIDS and other diseases.

3-5

Epidemical studies in

India, where turmeric is used routinely, show that the
incidence of AD between the ages of 70 and 79 years is
4.4-fold less than in the USA.

Researchers used the

transgenic mouse APPSw to investigate the curcumin
treatment effect. Results show that a low dose of
curcumin significantly suppressed the inflammatory
cytokine IL-1 and the astrocytic marker GFAP, reduced
oxidative damage and plaque burden and decreased the
amount of insoluble amyloid. Compared to other

Department of Orthopedics, the First Affiliated Hospital, Medical
College of Jinan University, Guangzhou, Guangdong 510632,

China (Pan R)
Department of Pathophysiology, the Key Lab of State
Administration of Traditional Chinese Medicine, Medical College

of Jinan University, Guangzhou, Guangdong 510632, China (Qiu
S, Lu DX and Dong J)
Correspondence to: Prof. DONG Jun, Department of

Pathophysiology, Medical College of Jinan University, Guangzhou,
Guangdong 510632, China (Tel: 86-20-85226129. Fax:
86-20-85221343. Email:dongjunbox@163.com)

This work was supported by grants from Guangdong Provincial
Natural Science Foundation (No. 04010443, 06105246),
Guangdong Provincial Medical Science Foundation (No.

A2004327, A2006334) and Guangzhou City Science and
Technology Plan Foundation (No. 2007J1-C0041).

Chinese Medical Journal 2008; 121(9):832-839

833

antioxidant drugs, such as NSAID or ibuprofen, curcumin
had fewer side effects.

Evidence suggests that metals are

concentrated in the AD brain and curcumin is a chelator
which can bind the iron and copper (but not zinc) on beta
amyloid, which may be one mechanism potentially
contributing to amyloid reduction.

In vivo, curcumin

may protect cells from the beta amyloid attack and
subsequent oxidative stress-induced damage in the
antioxidant pathway.

9,10

The findings of our previous

study prove curcumin can induce cognitive improvement
by enhancing the cholinergic system and its antioxidant
activity. The studies on curcumin are incomplete. We may
further investigate its neuroprotective mechanism.

METHODS

Drugs
Curcumin (Fluka Corporation, USA), huperzine A
(Shanghai Fudan Fuhua Pharmacy Ltd, China),
carboxymethyl cellulose (Guangzhou Chemical Reagent
Factory, China), Hoechst33258 (Binyuntian
Bioengineering Institute, China), monoclonal antibody
against bcl-2 (Santa Cruz Biotechnology, CA, USA),
monoclonal antibody against Bax (Cell Signaling
Corporation, USA), monoclonal antibody against reduced
glyceraldehydes-phosphate dehydrogenase (GAPDH)
(Chemicon, CA, USA), annexin v-fluorescein
isothiocyanate (FITC) staining kit (Beijing Biosea
Biotechnology, China), BCA protein assay kit (Shanghai
Shenneng Bocai, China), Dulbecco’s modified Eagle’s
media (DMEM) (Gibco BRL, Grand Island), horse serum
and fetal bovine serum (Hangzhou Sijiqing
Biotechnology, China), nerve growth factor (Promega
Corporation, USA), MTT (imported separated package
bought from Beijing Probe Company, China) were used
in the present study.

In vivo study
AD animal model
Totally 40 female Kunming mice, weighing from 18 to 22
g were obtained from the Guangdong Experimental
Animal Center (certificate No: 0013627). The mice were
kept in cages with the ambient temperature of (24±2)°C,
relative humidity of (60±10)%, and in 12-hour light-dark
cycle. Free access to food in the form of dry pellets and
water was allowed. The animal experiments were
performed according to the internationally accepted
ethical guidelines.

The mice were randomly assigned into four groups:
control group, AD model group, huperzine A treated
model group and curcumin treated model group. All mice
were administrated AlCl

orally (10 mg/kg) and

intraperitoneal injections of D-galactose (120 mg/kg)
were given for 90 days to set up the AD animal model;
except for the control group. From day 45 water insoluble
curcumin (200 mg/kg) was dissolved in 1%
carboxymethyl cellulose (CMC) and administrated orally
for 45 days. In parallel, huperzine A was also dissolved in

1% CMC and administrated orally for 45 days. Similarly
1% CMC of equal dosage was given to both the control
and AD model group. After 90 days of treatment mice
were tested in the step-through avoidance test.

Step-through avoidance test
The step-through avoidance test was similar to the test
described by Zhang et al.

Histopathological analysis
After the step-through avoidance test, the brains of mice
were removed right after the left ventricle perfusion.
Removed brains were then immersed in 4% neutral
buffered paraformaldehyde for 24 hours. Serial coronal
paraffin sections were cut at 30 µm thickness for
hematoxylin and eosin (HE) staining.

Western blot analysis
Other brains were used for Western blot analysis. The
brain was rapidly dissected over ice and frozen. Protein
extraction was done with gentle homogenization in seven
volumes of cold lyses buffer. The homogenates were
centrifuged at 10 000–14 000 × g at 4°C for 3 minutes.
Protein concentrations were determined with the BCA
protein assay reagent and samples were stored at −80°C
for use in Western blot analysis.

Seventy micrograms of protein extracts were diluted in
2×SDS loading buffer and loaded onto a 12%
SDS-polyacrylamide denaturing gel. After electrophoresis,
30 minutes at 80 V and 1 hour at 120 V, the gel was
transferred to nitrocellulose membrane by electrophoretic
transfer at 200 mA for 2 hours. The membrane was
washed with TBS for 30 minutes and then incubated in
TBST (TBS containing 0.1% Tween 20) supplemented
with 5% non-fat milk for 2 hours to block nonspecific
protein binding. The membrane was cut according to the
marker and then incubated with the antibody (1:200
dilution of the monoclonal Bcl-2 or Bax antibody, or a
1:800 dilution of the monoclonal GAPDH antibody) over
night at 4°C. Following washing the membrane 3 times
with TBST the membranes were then incubated for 1
hour in horseradish peroxidase linked secondary antibody
(1:2000). Washed 3 times with TBST and then developed
with ECL detection reagent.

In vitro study
Cell culture
Cultures were maintained in DMEM with 1000 U/ml of
penicillin G, 100 µg/ml of streptomycin, 10% horse serum
and 10% fetal bovine serum at 37°C in 5% CO

. The

concentration of cells is 5×10

/cm

. PC12 cells can be

induced to differentiate toward sympathetic neurons after
exposure to nerve growth factor (NGF) 50 ng/ml. We
used differentiated PC12 cells as our study object.

Apoptosis cell culture and cytotoxicity assay
In all experiments, AlCl

was employed as a source of

to investigate effects on cell morphology and

Chin Med J 2008;121(9):832-839

834

viability. AlCl

was prepared as 100× stock solution (in

0.9% sodium chloride solution) of different
concentrations and added in equal volumes to the growth
medium at a concentration of 10, 100, 500 and 1000
µmol/L. Control cultures contained an equal volume of
0.9% sodium chloride solution. Changes of pH due to the
treatment with AlCl

in culture medium were adjusted to

7.3±0.2 with NaHCO

. Cytotoxicity of AlCl

at various

concentrations was determined in 96-well collagen-
coated culture plates by the 3 (4, 5-dimethylthiazolyl-2) 2,
5-diphenyl tetrazolium bromide (MTT) assay, which is
widely used for the measurement of cell viability. The
procedure was done according to the instruction manual.

Water insoluble curcumin was dissolved in cell culture
grade dimethylsulfoxide (DMSO) and stored in the dark
at –20°C. All dilutions of curcumin were made in DMEM
medium and the final concentration of DMSO was 0.1%.
In order to confirm the effect of curcumin on
differentiated PC12 cells, curcumin was incubated with
cells at levels of 20, 40, 80, and 160 µmol/L.

Based on previous study results, AlCl

(1000 µmol/L)

was chosen to incubate with differentiated PC12 cells.
After 12 hours of incubation 20, 40 and 80 µmol/L
curcumin were added to the cells. The MTT assay was
used to determine which concentration of curcumin might
protect cells from AlCl

toxicity.

Assessment of nuclear morphology
Cells were washed in cold PBS twice and fixed in 4%
formaldehyde at 4°C for 10 minutes. Fixed cells were
washed and labeled with Hoechst 33258 (5 µg/ml) at
room temperature in the dark for 10 minutes. Then they
were viewed on an Olympus inverted research
microscope using the filter for 360 nm.

Annexin V /PI assay
The procedure was done according to the instruction
manual.

Western blot analysis
Following treatment, cells were washed twice with cold
phosphate buffered solution (PBS) and protein extracts on
ice for 30 minutes. After centrifugation a total of thirty
micrograms of protein extracted from cells after each
treatment was run in 12% SDS-polyacrylamide gels. The
procedure was the same as previously described.
According to the protein content, which was less than
animal tissue protein, we increased the concentration of
antibody. 1:100 dilution of the monoclonal Bcl-2 and Bax
antibody or a 1:400 dilution of the monoclonal GAPDH
antibody was used, accordingly. The concentration of
horseradish peroxidase linked secondary antibody was
1:1000.

Statistical analysis
Data were expressed as means ± standard error (SE).
One-way analysis of variance (ANOVA) and Newman

Keel’s test were used for the tests between two or more
groups. The Mann-Whitney U test was used for values
which were not suitable for ANOVA. P values less than
0.05 were considered significant. All statistical analysis
was performed using SPSS statistical software package
version 13.0.

RESULTS

Curcumin improves learning and memory ability in
the AD model mice
Performance of the mice in the step-through passive
avoidance training and testing is shown in Table. During
the first day of training the number of times the mouse
entered the dark chamber was significantly increased in
the model group over the control group (P <0.01). The
increased number of times was significantly reversed by
curcumin (P <0.05). On the testing day errors significantly
increased and step-through latency decreased markedly in
the model group compared to the control group (P <0.01).
The shorter step-through latency and increased errors
were significantly reversed by curcumin and huperzine A
(P <0.05). There was no significant difference between
the curcumin group and huperzine A group (P >0.05).
From the statistics we found that administrating AlCl

combined with D-galactose for 90 days induced mice
memory impairment, which mimic AD pathogenetic and
could be used as an AD animal model. Administration of
curcumin can significantly improve learning ability.

Table. Effect of curcumin on error times and latency of

step-through avoidance test in mice

Testing

Treatment
(n=10)

Training error

times

Error times

Escape latency

Control 0.90±0.32

0.20±0.42

269.30±64.73

Model

2.00±0.94

1.60±0.97

153.10±103.41

Model + huperzine A

1.30±0.48

0.60±0.84

247.00±81.38

Model + curcumin

1.20±0.42

0.60±0.70

240.10±70.11

P <0.05, significantly different from control group.

P <0.01, significantly

different from control group.

P <0.05, significantly different from model group.

Huperzine A is a novel lycopodium alkaloid isolated from
Chinese herbs. Studies have shown it is a reversible and
selective acetylcholinesterase inhibitor improving rats’
memory ability.

In this study, we used it as a positive

drug to compare the effect of huperzine A and curcumin.
Results showed that both of the two drugs induced
memory improvement in mice. There was no significant
difference between the two drugs.

HE staining (Figure 1) shows that there were typical
neuropathological changes in the hippocampus of AD
model mice. In the control group the neurons were full
and arranged tightly, the nuclei were light-stained. By
comparison in the model group mice, the cytoplasm of
neurons were shrunken, the nuclei were side-moved and
dark-stained, neurofibrillary degeneration and neuron loss
were observed in hippocampus. Consecutive administration
of curcumin significantly attenuated these neuropathological
changes. The neurons recovered their characteristic shape,
with prolonged neurofibrillary, the nuclei were light-

Chinese Medical Journal 2008; 121(9):832-839

835

Figure 1. Neuropathological changes in hippocampus of AD model mice induced by AlCl

and D-galactose revealed by hematoxylin and

eosin (HE) staining. A: control group. B: model group. C: curcumin group. D: huperzine A group.

stained and arranged tightly compared to the model
group.

We next investigated the mechanism by which curcumin
protects neurons from AlCl

and D-galactose

neurotoxicity in mice. Changes in the levels of Bcl-2 and
Bax protein in mitochondria were further assessed to
determine programmed cell death in this animal system.
The antibody to Bcl-2/Bax identified a protein band with
an apparent molecular weight of 27/21 kD. We found that
administration of AlCl

and D-galactose resulted in Bcl-2

down-regulation (P <0.05) (Figure 2). The neuroprotective
effect of curcumin treatment resulted in an increase in the
anti-apoptosis protein Bcl-2 content (P <0.05). There was
no significant difference of Bax protein between these
groups (P >0.05). It appears from our studies that level of
the apoptosis regulatory protein Bcl-2 increased, and
triggering signaling led to the initiation and expression of
apoptosis. Curcumin may have potent anti-apoptosis
characterized by up-regulating Bcl-2 level.

Figure 2. Analysis of the Bcl-2/Bax obtained from brain tissue
treated with AlCl

and D-galactose, combined with or without

drug by Western blot. Lane 1: control group. Lane 2: model

group. Lane 3: huperzine A treated model group. Lane 4:
curcumin treated model group. The experiment was repeated
three times with similar results.

P <0.05, significantly different

from control group.

△

P <0.05, significantly different from model

group. GAPDH: glyceraldehydes-phosphate dehydrogenase.

Curcumin inhibits apoptosis in cultured PC12 cells
induced by AlCl

PC12 cells were exposed to 10, 100, 500 and 1000
µmol/L AlCl

, in the growth medium for 3 days. By 48

hours cells in AlCl

-treated cultures presented abnormal

aggregation, shrinkage of cell bodies and degeneration of
processes compared to untreated cultures. We also
observed the significant reduction in the viability of cells

as the dose of AlCl

increased and the time passed (P

<0.01, Figure 3A). When the dose of AlCl

was increased

to 1000 µmol/L for 3 days, the viability of cells was
decreased to (62±2)% compared to control cells. So we
chose 1000 µmol/L AlCl

as the model dosage to incubate

with cells.

In order to observe curcumin exposure on cell viability,
water insoluble curcumin was dissolved in DMSO,
incubated with cells at levels of 20, 40, 80, and 160
µmol/L. The MTT assay found that low dose of curcumin
(20, 40, and 80 µmol/L) did not affect cell viability but
the high dose decreased cell viability in a time
–dependent manner (P <0.01) (Figure 3B).

Based on previous results, high dose curcumin was
deleted in the next test. AlCl

and curcumin were then

incubated together and curcumin protection of cells from
AlCl

toxicity was determined. Results showed that 20

and 40 µmol/L curcumin could decrease AlCl

damage,

as characterized by higher levels of cell viability (P <0.01)
(Figure 3C). Eighty µmol/L curcumin did not show any
protection from toxicity.

Occurrence of apoptosis was further confirmed by the
appearance of changes in the nuclear morphology of
treated cells. As shown in Figure 2, following exposure to
AlCl

, cells exhibited altered nuclei as assessed by

staining of cells with the DNA-binding fluorochrome
Hoechst 33258. The nuclei in the AlCl

-treated groups

appeared either shrunken and irregularly shaped or
degraded, with aggregation and fragmentation of
chromatin (Figure 4B). In the control group, most nuclei
had regular contours and were round and large in size
(Figure 4A). Curcumin (20 and 40 µmol/L) and AlCl

co-treated groups showed DNA condensation but no
fragmentation of chromatin (Figures 4C and 4D). The 80
µmol/L curcumin group showed irregular contours and
fragmentation of chromatin (Figure 4E).

Figure 5 shows a display of propidium iodide vs
annexin-V/PI fluorescence. It was possible to distinguish
early apoptotic, late apoptotic and dead cells. The
intensity of staining for classification of the cells into
positive and negative staining classes were determined
from histogram analyses of signals from propidium
iodide only and annexin-V only. The lower left quadrants
of the histograms showed the viable cells which excluded
propidium iodide and were negative for annexin-V-FITC

Chin Med J 2008;121(9):832-839

836

We next investigated the mechanism by which curcumin
protects PC12 cells from AlCl

neurotoxicity. We have

shown that treatment with curcumin cells results in
inhibition of the AlCl

induced apoptosis. Results of

Western blot analysis showed that the level of the
anti-apoptotic protein Bcl-2 decreased remarkably in the
AlCl

treated group compared to the control group (P

<0.05) (Figure 6). Cells incubated with AlCl

and

curcumin, 20 and 40 µmol/L, together showed higher
levels of Bcl-2 protein compared to the model group (P
<0.05). There was no significant difference in the levels
of Bax protein (P >0.05).

DISCUSSION

Apoptosis is a form of programmed cell death that
involves changes in the cytoplasm, ER, mitochondria and
nucleus. It typically includes the production and/or
activation of proteins such as Bcl-2 and Bax that
decrease/increase the permeability of mitochondrial and
ER membranes resulting in the release into the cytoplasm
of cytochrome c from the mitochondria and calcium from
the ER.

Latter events then activate enzymes called

caspases that cleave various protein substrates to sculpt
morphological and biochemical aspects of the cell
apoptosis process. There are several lines of evidence for
apoptosis in AD. Postmortem analysis of AD brain by
TUNEL analysis showed positive neurons and glia in the
hippocampus and cortex indicating DNA fragmentation.

14-16

Increased expression of Bcl-2 family members,

17,18

well as increased caspase activities and cleavage of
caspase substrates have also been detected in the AD
brain.

The triggers of cell death in AD may include Aβ,

aluminum silicate granules which were surrounded by
insoluble plaques, activation of glutamate receptors,
increased oxidative stress, DNA damage and elevation of
intracellular calcium levels.

Figure 3. Viability of PC 12 cells after exposure to AlCl

with/without curcumin. A: dose and time-dependency of AlCl

neurotoxicity. After exposure to various concentrations of AlCl

for 3 days cell viability was obtained as the percentage relative
to that of control (no addition) cells. Data show means ± SE

(n=6).

P <0.01, significantly different from control group. B:

dose and time-dependency of curcumin on cell viability. After
exposure to various concentrations of curcumin for 3 days, cell

viability was obtained as the percentage relative to that of
control (no addition) cells. Data show means ± SE (n=6).

P<0.01, significantly different from control group. C: after

exposure to AlCl

and various concentrations of curcumin for 3

days, cell viability was obtained as the percentage relative to
that of control (no addition) cells. Data show means ± SE (n=6).

P <0.01, significantly different from control group.

P <0.01,

significantly different from model group.

There is no animal model available that can mimic all the
cognitive, behavioral, biochemical, and histopathological
abnormalities observed in patients with AD.

However,

partial reproduction of AD neuropathology and cognitive
deficits have been achieved with pharmacological
approaches, such as cholinergic dysfunction-related
animal models, amyloid peptide related animal models,
and transgenic animal models.

21-23

In this study we set up

a different AD animal model, administered AlCl

combined with D-galactose for 90 consecutive days.
Many scientific studies have brought to light the potential
toxicity of aluminum in experimental animal models and
in humans under different clinical conditions.

Candy et

al,

using energy dispersive X-ray microanalysis, found

the presence of minuscule insoluble Al silicate granules in
the brains of patients with AD. A very detailed study by
Savory et al

clearly showed that aged rabbits are more

susceptible to Al toxicity compared to young rabbits.
Al-induced neuropathological events (alteration in Bcl-2:
Bax ratio, oxidation, apoptosis, redo-active iron, etc.) in
aged rabbits mimic AD neuropathology. Some findings

binding. The lower right quadrants represented the early
apoptosis cells, propidium iodide negative and
annexin-V-FITC positive. The upper right quadrants
contained late apoptosis and necrotic cells, positive for
propidium iodide and for annexin-V binding. The upper
left quadrants represented cells damaged during the
procedure.

In the control group, most cells were healthy and the rate
of apoptosis was about (9.20±0.20)% (Figure 5A). PC12
cells exposed to AlCl

had a significantly increased rate of

apoptosis, (34.33±3.30)% compared to control group (P
<0.01) (Figure 5B). Cells incubated with AlCl

and

curcumin (20 and 40 µmol/L) together showed lower
levels of apoptosis compared to the model group
(23.47±1.15)% and (24.43±0.91)% (P <0.05) (Figures 5C
and 5D). The high dose of curcumin, 80 µmol/L, did not
show any protection from AlCl

intoxication (Figure 5E).

Chinese Medical Journal 2008; 121(9):832-839

837

Figure 4. Fluorescence images of Hoechst 33258-stained cells after a 72-hour exposure
to AlCl

in the absence or presence of curcumin. A: control cells. B: AlCl

(1 mmol/L)

treated model cells. C, D, and E: AlCl

and curcumin (20, 40, and 80 µmol/L) co-treated

cells.

Figure 5. Flow cytometric analysis of apoptosis and
necrosis in treated cells. A: control cells. B: AlCl

mmol/L) treated model cells. C, D, and E: curcumin

(20, 40, and 80 µmol/L) and AlCl

co-treated cells.

<0.01, significantly different from control group.

<0.05, significantly different from model group.

Figure 6. Western blot of Bcl-2 and Bax in cultured PC12 cells
extracts. Lane 1: control group. Lane 2: AlCl

treated model

group. Lanes 3–5: curcumin (20, 40, and 80 µmol/L) treated
groups. The experiment was repeated three times with similar
results.

P <0.05, significantly different from control group.

△

<0.05, significantly different from model group.

suggest an almost threefold increased absorption of Al in
AD patients compared to healthy controls.

Chronic administration with a low dose of D-galactose
(D-gal) induces changes that resemble natural aging in
animals, such as a shortened life span, cognitive
dysfunction, neurodegeneration, oxidative stress,
decreased immune responses and gene transcriptional
changes.

28-31

D-gal-induced senescence acceleration has

been widely used as a model for studying aging
mechanisms and for screening drugs.

32,33

Because

D-gal-induced senescence is accompanied by
neurodegeneration, when combined with neurotoxin Al
treatment, it would be an ideal model for studying the
molecular mechanisms involved with age-associated
neurodegeneration disease— Alzheimer’s disease. Based
on previous studies, in our experiment, the mice were
administrated AlCl

10 mg/kg orally and given

intraperitoneal injections of D-galactose 120 mg/kg for

Chin Med J 2008;121(9):832-839

838

90 days to set up our AD animal model.

In the in vivo study, the mice treated with AlCl

combined

with D-galactose for 90 days have induced morphology
and biochemistry changes. This kind of animal model is
based upon the proven theory of AlCl

neurotoxin and

D-gal induced senescence. This chronic animal model
developed a simulated slow evolution of the
neurodegenerative disease; it is used for assessing the
validity of therapeutic interventions with drugs. Results
showed that behavioral deficits in step-through tests are
similar to the cognitive impairments in AD. HE staining
in the hippocampus also suggests that the condensed,
dark stained neurons, neurofibrillary degeneration and
neuron loss were similar to the pathology in AD.

Apoptosis, or programmed cell death, plays a critical role
in the normal development and maintenance of tissue
homeostasis and is also a process by which brain cells die
in AD. Mitochondrial changes following cytotoxic stimuli
represent a primary event in apoptotic cell death. The
apoptotic generated factor, cytochrome c, release from
mitochondria into the cytoplasm has been shown to
involve three distinct pathways. One implicates the
opening of the mitochondrial permeability transition pore
(MTP), the second is triggered by the translocation to
mitochondria of the pro-apoptogenic Bax which can form
a channel by itself, and the third may result from the
interaction of Bax with the voltage-dependent anion
channel (VDAC) to form a larger channel which is
permeable to cytochrome c. In contrast to Bax, the
anti-apoptotic Bcl-2 has the ability to block the release of
cytochrome c from mitochondria by mechanisms such as
a direct blockade of the MTP opening, or by functioning
as a docking protein.

33,34

We have shown that

administration with AlCl

combined with D-galactose

results in Bcl-2 down-regulation. These results indicate
that AlCl

and D-galactose target the signaling pathway

of mitochondria. Furthermore, the neuroprotective effect
of curcumin treatment resulted in inhibition of the AlCl

and D-galactose induced apoptosis with enhanced levels
of the anti-apoptotic proteins Bcl-2.

The PC12 cell is a cloned rat pheochromocytoma cell line
that retains a number of chromaffin cell characteristics,
such as the presence of nicotinic cholinergic receptors,
the synthesis and secretion of catecholamine and the
expression of a number of neuropeptide genes. The PC12
cell line is a useful model for the study of neuron
development, since PC12 cells can be induced to
differentiate toward sympathetic neurons after exposure
to nerve growth factor (NGF).

Although the viability of

cells decreased significantly as the dose of AlCl

increased, we used this exposure time and dose to
establish a marked model of apoptosis and then to study
the mechanism by which curcumin protects cells through
anti-apoptosis pathways.

The MTT assay found that curcumin (20 and 40 µmol/L)

could protect cells from AlCl

toxin and increase cell

viability. DNA-binding fluorochrome Hoechst 33258 and
flow sytometric analysis revealed the decreased apoptosis
in curcumin treated cells. The mechanism may inhibit the
activation of Bax and promote the activation of Bcl-2.
Other studies have shown that administration of curcumin
to mice at a dose of 2000 mg/kg, which is 83 times
greater than the dose utilized by Lim et al,

36,37

was

non-toxic. In this study curcumin at a dose of 200 mg/kg
in mice and 20–40 µmol/L in cells was effective in
blocking apoptosis, indicating that a low dose of
curcumin may be effective in preventing Alzheimer’s
disease. But our results also showed that high doses of
curcumin had no effect on anti-apoptosis. This is not in
concordance with other results.

In summary, our study provides one mechanism, among
several multifactor effects, by which curcumin hampers
the process of apoptosis by decreasing Bax activity. Our
in vitro studies also provide support for the hypothesis of
the anti-apoptosis property of curcumin. These studies
provide a rationale for the therapeutic use of curcumin.
Curcumin may be a potent anti-apoptosis drug for the
prevention of Alzheimer’s disease.

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(Received January 3, 2008)

Edited by LIU Dong-yun