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Abstract
Amyloid protein (A) may be neurotoxic during the progression of Alzheimers disease by eliciting oxidative stress. This study was designed
to determine the effect of Polygonum multiorum Thunb water extract (PWE) on A25-35-induced cognitive deficits and oxidative stress in mice.
Mice were fed experimental diets comprising either 0.5 or 1% PWE for 4 weeks, and then received a single intracerebroventricular (i.c.v.) injection
of A25-35 (10 g/mouse). Behavioral changes in the mice were evaluated using passive avoidance and water-maze tests. The consumption of PWE
significantly ameliorated the cognitive deficits caused by i.c.v. injection of A25-35. The A25-35 treatment accelerated the lipid peroxidation, and
PWE attenuated the A-induced increase in brain levels of thiobarbituric acid reactive substances. There was an increase in glutathione peroxidase
activity in PWE-treated groups. The acetylcholinesterase activity in the brain and serum was lower in PWE supplemented groups than in the only
A-injected group. These findings suggest that PWE exerts a preventive effect against cognitive deficits induced by A25-35 accumulation in
Alzheimers disease, and that this effect is mediated by the antioxidant properties of PWE.
2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Amyloid protein; Antioxidative capacity; Cognitive deficits; Polygonum multiorum Thunb
1. Introduction
Alzheimers disease (AD) is characterized by progressive
cognitive function deficits due to the presence of numerous
senile plagues and neurofibrillary tangles in brain regions
(Yankner, 1996). Amyloid (A) peptide is a major component
of these plagues (Golde et al., 1992; Yan et al., 2004). The accumulating evidences suggest that oxidative stress is involved in
the mechanism of A-induced neurotoxicity (Behl et al., 1992;
Yamada et al., 1999; Zhu et al., 2004). The levels of lipid peroxidation, protein carbonyl, and 8-hydroxyl-2-deoxyguanosine are
higher in the brains of AD patients than in aged-matched control
brains (Sayre et al., 1997; Smith et al., 1997; Morishima et al.,
2001). Antioxidants, such as Vitamin E, Ginkgo biloba, and fer-
145
illuminated and one dark, equipped with a grid floor and shock
generator. The day after A25-35 injection, mice were trained
in the passive avoidance task. During the training trial, each
mouse was placed in the lighted compartment, and as soon as
it entered the dark compartment the door was closed, and it
received an inescapable shock (0.3 mA, 3 s). The next day, the
mouse was again placed in the lighted compartment, and the
time until it returned to the dark compartment was measured as
the step-through latency (with a maximum of 300 s).
2.4. Water-maze test
The water maze was slightly modified from the Morris water
task. The experimental apparatus consisted of a circular water
tank (diameter 100 cm; height 35 cm) containing water at 23 C
to a depth of 15 cm and rendered opaque by the addition of powdered milk. A platform was positioned inside the tank with its
top submerged 2 cm below the water surface in the target quadrant of the maze. After several trials, the test was conducted on
the day of injection of A peptide. In each training trial, the time
required to escape onto the hidden platform was recorded. The
number of times the platform was not found was also recorded.
2.5. Measurement of AChE activity
AChE activity in the brain was measured using the method
of Ellman et al. (1961). Acetylthiocholine iodide was used as a
substrate. The hydrolysis of acetylthiocholine was determined
by monitoring the formation of the yellow 5-thio-2-nitrobenzoic
acid at a wavelength of 412 nm. Protein concentration was determined by the method of Lowry et al. (1951). Serum AChE
activity was assayed with cholinesterase kit from Sigma Chemical (St. Louis, MO, USA) following a modified version of the
method of Rappaport et al. (1959).
2.6. Measurement of lipid peroxide levels and antioxidant
enzyme activity
Lipid peroxidation in the brain was determined by measuring the formation of thiobarbituric acid reactive substances
(TBARS) according to the method of Ohkawa et al. (1979).
A standard curve was obtained using 1,1,3,3-tetramethoxypropane. Catalase activity was measured using the method of
Aebi (1974). Brain homogenate was reacted with hydrogen peroxide, and the decomposition of hydrogen peroxide was determined spectrophotometrically at 240 nm. Superoxide dismutase
(SOD) activities were assayed according to Marklund and Marklund (1974). Glutathione peroxidase (GPx) activity was measured according to Lawrence and Burk (1976). The decrease in
NADPH was recorded at 340 nm and is expressed here using a
molar extinction coefficient of NADPH of 6.22 mM1 cm1 .
2.7. Statistics
146
3. Result
3.1. PWE improves cognitive decits in A25-35-treated
mice
It was suggested previously that the i.c.v. injection of A2535 causes memory deficits and lowers choline transferase in
hippocampus (Tang et al., 2002; Stepanichev et al., 2004). In the
present study, an i.c.v. injection of 10 g of A25-35-induced
cognitive dysfunction as assessed by passive avoidance and Morris water-maze tests. Changes in the step-through latency in
passive avoidance are shown in Fig. 1. There was no significant difference between the normal and normal PBS groups
(i.c.v. injection of PBS), but significantly reduced step-through
latency in the A25-35-injected control group (25.3% reduction
compared to normal, P < 0.05). This result confirmed that cognitive deficits induced by A i.c.v. were not attributable to the
i.c.v. injection itself. As shown in Fig. 1, step-through latency
was increased up to 2.5-fold by consumption of the 1% PWE diet
(P < 0.05). As shown in Fig. 2, injection of A25-35 increased
the escape latency and error frequency in the Morris water-maze
test. Treatment of mice with PWE for 4 weeks attenuated the
increase in the escape latency almost to that in the normal group,
but the change was not dose dependent. Changes in error frequency in each group showed a similar pattern to the escape
latency.
Group
N
NP
C
0.5% PW
1% PW
17.0 2.2 ab
17.9 0.9 a
18.4 0.4 a
15.0 0.5 b
14.8 0.3 b
179.9
177.2
219.3
177.6
163.6
27.8 b
8.6 b
11.7 a
8.3 b
2.2 b
(1) Values are means S.E.M. (n = 10). (2) One Rappaport unit is that amount
of cholinesterase that will liberate 1 mol of acetic acid from acetylcholine in
30 min at 25 C and pH 7.8 under the conditions of this test. (3) AChE activities are expressed as the amount of 5-thio-2-nitro-benzoic acid produced by
the hydrolysis of substrate. (4) Values with different letters within a row are
significantly different at = 0.05 by Duncans multiple-range test.
Fig. 3. Effect of PWE on TBARS levels in brain tissue of mice treated with
A25-35 (* P < 0.05 compared to controls; # P < 0.05 compared to normals).
Table 2
The effect of PWE on specific activities of catalase, SOD, and GPx in brain
homogenates
Group
Catalase (units/
(min mg protein))
SOD (units/
(min mg protein))
GPx (nmol/
(min mg protein))
N
NP
C
0.5% PW
1% PW
34.81 7.67 ab
35.87 3.43 a
26.97 1.34 ab
24.68 1.10 ab
34.16 5.78 b
(1) Values are mean S.E.M. (n = 10). (2) Values with different letters within a
column are significantly different at = 0.05 by Duncans multiple-range test.
(3) n.s., not significant.
147
A1-42 impairs antioxidant capacity. We found that consumption of a diet containing PWE ameliorated cognitive deficits
in A25-35-injected mice. Especially, the data show that the
step-through latency in passive avoidance increased in a dosedependent manner, but not in the water-maze test. Also, consumption of PWE decreased the escape latency almost to normal
levels. It is possible that neuroprotection plays a role in the
favorable effect of PWE on A25-35-induced cognitive deficits.
Antioxidants, such as -tocopherol and ferulic acid protect
against learning and memory deficits induced by A (Yamada et
al., 1999; Yan et al., 2001). The AChE activity has been shown to
be increased within and around amyloid plaques in Alzheimers
brain (Ulrich et al., 1990). The enhancement of AChE activity
induced by A25-35 is mediated by oxidative stress (Melo et al.,
2003). The AChE activity in the brain and serum was increased
in mice treated with A25-35 when compared with the normal
in our experiment. In addition, the A25-35-induced increase in
AChE was attenuated by PWE consumption.
PM is a medicinal plant that has antioxidant properties in
vitro (Ryu et al., 2002) and delays aging responses in vivo
(Chiu et al., 2002) involving oxidative stress. Also, previous
studies have shown that PM ethanol extract suppressed lipid
peroxidation in the mitochondria of rat heart (Chen et al., 1999).
Chan et al. (2002) demonstrated that PM ethanol extract significantly improved learning and memory deficits in SAMP8
(a murine AD model), and lowered lipofuscin percentages and
MDA concentrations in hippocampus, and increased total thiol
concentrations. Catalase, SOD, and GPx are involved in the
reduction in reactive oxygen species and peroxides produced in
living organisms as well as in the detoxification of certain compounds of exogenous origin, and thus play a primary role in the
maintenance of a balanced redox status (Kweon et al., 2003).
Treatment of mice with PWE for 4 weeks decreased TBARS
level and increased GPx activity in the brain, while having no
significant effect on catalase and SOD activity. These findings
can be attributed to GPx exhibiting a higher sensitivity to lipid
peroxidation than catalase or SOD. Therefore, we suggest that
accumulation of lipid peroxides by A was reduced by PWE via
antioxidative mechanisms.
In conclusion, we suggest that PWE markedly improves
cognitive deficits induced by A25-35, and that this effect is
mediated by the antioxidant properties of PWE. Future studies
should determine the specific components in PWE responsible
for preventing cognitive impairment.
References
Aebi, H., 1974. In: Bergmeyer, H.U. (Ed.), Catalase. Methods of Enzymatic
Analysis. Academic Press, New York and London, pp. 637684.
Atack, J.R., Perry, E.K., Bonham, J.R., Perry, R.H., Tomlinson, B.E., Blessed,
G., Fairbairn, A., 1983. Molecular forms of acetylcholinesterase in senile
dementia of Alzheimer type: selective loss of the intermediate (10S) form.
Neuroscience Letters 40, 199204.
Behl, C., Davis, J., Cole, G.M., Schubert, D., 1992. Vitamin E protects nerve
cells from amyloid beta protein toxicity. Biochemical and Biophysical
Research Communications 186, 944950.
Behl, C., Sagara, Y., 1997. Mechanism of amyloid beta protein induced
neuronal cell death: current concepts and future perspectives. Journal of
Neural Transmission. 49, 125134.
148
Chan, Y.C., Cheng, F.C., Wang, M.F., 2002. Beneficial effects of different Polygonum multiorum Thunb. extracts on memory and hippocampus morphology. Journal of Nutritional Science and Vitaminology 48,
491497.
Chen, Y., Wang, M., Rosen, R.T., Ho, C.T., 1999. 2.2-Diphenyl-1picrylhydrazyl radical-scavenging active components from Polygonum
multiorum Thunb. Journal of Agricultural and Food Chemistry 47,
22262228.
Chiu, P.Y., Mak, D.H., Poon, M.K., Ko, K.M., 2002. In vivo antioxidant action of a lignan-enriched extract of Schisandra fruit and an
anthraquinone-containing extract of Polygonum root in comparison with
schisandrin B and emodin. Planta Medica 68, 951956.
Christen, Y., 2000. Oxidative stress and Alzheimer disease. American Journal
of Clinical Nutrition 71, 621S629S.
Ellman, G.L., Courtney, K.D., Andres, V.J., Featherstone, R.M., 1961. A
new and rapid colormetric determination of acetylcholinesterase activity.
Biochemical Pharmacology 7, 8895.
Golde, T.E., Estus, S., Younkin, L.H., Selkoe, D.J., Younkin, S.G., 1992.
Processing of the amyloid protein precursor to potentially amyloidogenic
derivatives. Science 255, 728730.
Jhoo, W.K., Kim, H.C., Yamada, K., Im, D.H., Shin, E.J., Park, S.J., 2000.
Prolonged exposure to -amyloid protein enhances 4-hydroxy-2-nonenal
modifies proteins in the rat brain. AbstractsSociety for Neuroscience
26, 1020.
Kim, H.C., Yamada, K., Nitta, A., Olariu, A., Tran, M.H., Mizuno, M., Nakajima, A., Nagai, T., Kamei, H., Jhoo, W.K., Im, D.H., Shin, E.J., Hjelle,
O.P., Ottersen, O.P., Park, S.C., Kato, K., Mirault, M.E., Nabeshima, T.,
2003. Immunocytochemical evidence that amyloid beta (1-42) impairs
endogenous antioxidant systems in vivo. Neuroscience 119, 399419.
Kweon, S.H., Kim, Y., Choi, H.M., 2003. Grape extracts suppress the formation of preneoplastic foci and activity of fatty acid synthase in rat liver.
Experimental and Molecular Medicine 35, 371378.
Laursen, S.E., Belknap, J.K., 1986. Intracerebroventricular injections in mice.
Some methodological refinements. Journal of Pharmacological Methods
16, 355357.
Lawrence, R.A., Burk, R.F., 1976. Glutathione peroxidase activity in
selenium-deficient rat liver. Biochemical and Biophysical Research Communications 71, 952958.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry
193, 265275.
Marklund, S., Marklund, G., 1974. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide
dismutase. European Journal of Biochemistry 47, 469474.
Melo, J.B., Agostinho, P., Oliveira, C.R., 2003. Involvement of oxidative
stress in the enhancement of acetylcholinesterase activity induced by amyloid beta-peptide. Neuroscience Research 45, 117127.
Morishima, Y., Gotoh, Y., Zieg, J., Barrett, T., Takano, H., Flavell, R., Davis,
R.J., Shirasaki, Y., Greenberg, M.E., 2001. Beta-amyloid induces neuronal
apoptosis via a mechanism that involves the c-Jun N-terminal kinase
pathway and the induction of Fas ligand. Journal of Neuroscience 21,
75517560.
Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95,
351358.
Rappaport, F., Fischl, J., Pinto, N., 1959. An improved method for the estimation of cholinesterase activity in serum. Clinica Chimica Acta 4, 227230.
Ryu, G., Ju, J.H., Park, Y.J., Ryu, S.Y., Choi, B.W., Lee, B.H., 2002. The
radical scavenging effects of stilbene glucosides from Polygonum multiorum. Archives of Pharmacol Research 25, 636639.
Sano, M., Ernesto, C., Thomas, R.G., Klauber, M.R., Schafer, K., Grundman,
M., Woodbury, P., Growdon, J., Cotman, C.W., Pfeiffer, E., Schneider,