Introduction
Brains of patients with Alzheimer disease (AD) are characterized
among others by the presence of amyloid deposits in selected
brain regions. Amyloid beta peptides (Abeta) are proteolytically
derived from a larger transmembrane amyloid precursor protein.
The Abeta 1-40 is the predominant form of cerebrovascular
soluble amyloid produced by different cell types and normally
present in the circulation. It is capable of forming stable
amyloid fibres but the rate of aggregation is a very slow
process in the absence of nucleating agents. On the contrary,
the rate of aggregation is increased for the longer Abeta
1-42 that is the major constituent of insoluble amyloid
fibres of senile plaques (1).
Recent research suggests important physiological functions
for Abeta under normal conditions (2). However, depending on
the degree of aggregation, higher concentrations can induce
apoptotic or necrotic neuronal degeneration (1, 2). It seems
that one of possible mechanism of neurotoxicity could be mediated
by reactive free radicals. Concentrations of Abeta 25-35 as
low as 10 nM intercalate into the membrane and inhibit the
lipid peroxidation (LPO) in a dose- and time-dependent manner
as a result of physicochemical interactions of amphiphilic
peptide with the membrane bilayer (3). On the contrary, higher
concentrations of different fragments significantly increase
membrane fluidity and induce the LPO (1, 2, 4).
At the present time, great attention concentrates on the therapy
of AD and a possible administration of some plant or animal
proteases is suggested. There are several reasons for their
therapeutic testing: i) Abnormal metabolisms of endogenous
proteases and their inhibitors play a role in the ethiology
of senile plaques, ii) Abnormally phosphorylated tau proteins
are major components of neurofibrillary tangles. An important
role for proteolytic enzyme calpain in tau metabolism is suggested,
iii) An increased vulnerability of basal forebrain cholinergic
neurons to Abeta during AD has been observed. A potential role
of acetylcholinesterase in the stimulation of the Abeta aggregation
is assumed. In all the three above-mentioned cases, some proteolytic
mechanisms could be influenced by exogenous proteases. iv)
Inflammatory mechanisms play a very important role in AD pathogenesis.
Exogenously administered proteolytic enzymes act as anti-inflammatory,
anti-edematous and immunostimulating agents through the regulation
of cytokines (5). And finally, v) proteases can increase the
permeability of the blood-brain barrier to some exogenous drugs.
Therefore, the application of proteases as supportive agents
together with other medicaments is tested now in neurology
(e.g., cerebrospinal sclerosis).
The aim of this study is to evaluate in vitro experiments with
Abeta and plant proteolytic enzymes (bromelain - BRO, papain
- PAP) using the thiobarbituric acid-test for the determination
of the efficiency of the proteases to influence the effects
of Abeta on LPO in the hippocampal rat and human tissue.
Materials and methods
i) Brain tissue: hippocampal animal tissue (male and female
1-, 3- and 23-month old Wistar rats of the Konarovice breed)
and hippocampal human tissue (cornu Ammoni et gyrus parahippocampalis
of men and women, demented patients with AD or multi-infarct
dementia (MID) as controls)
ii) Chemicals: Abeta (amyloid beta-protein fragment 1-40, Sigma),
BRO (Ananas Comosus, Mucos Pharma), PAP (Carica papaya , Mucos
Pharma) and 10% DMSO (dimethyl sulfoxide, Sigma) as a solvent
iii) Methods: thiobarbituric acid test by Ohkawa et al. (6)
iv) Data analysis: ANOVA and Student's t-test (separate variance)
* p < 0.05, ** p < 0.01, *** p < 0.001
data presented as the means ± S.D.
Results
i) The effect of Abeta on LPO in the hippocampal tissue of
young 3-month animals was significantly time- and dose-dependent.
No changes were observed for 10 nM concentration in the case
of the short (15-30 min) as well as long incubation (60-120
min) (Fig. 1). However, the decrease of LPO after the application
of a small Abeta concentration could be hidden behind the DMSO
effect that acts as a OH radical scavenger (7). 100-500 nM
concentrations increased the LPO in the case of the longer
60 min incubation (Fig. 2). The effect of 1mM Abeta was comparable
for 30 and 60 min incubations (Tables I-IV).
ii) The effect of Abeta on the LPO was signficantly age-dependent
(Tables II and III). The higher effect of 1 mM Abeta was found
in the brain tissue of old compared to young animals.
iii) The effects of both plant proteases on the LPO in hippocampal
tissue of 3-month old animals were more rapid than those of
Abeta. No significant differences between 30 and 60 min incubation
were found (Tables I-III). Concentrations lower than 100 mg/ml
did not influence significantly the LPO (Fig. 3). 100 mg /ml
concentration increased the LPO (Tables I-IV).
iv) The effects of both proteases and especially of BRO were
significantly age-dependent. The higher LPO after application
of proteases was found in the brain tissue of young animals
(Table III).
v) Both proteases eliminated the effect of Abeta previously
applied to hippocampal homogenates of young animals (Table
IV, experiment A). Abeta added to samples previously incubated
with both proteases did not significantly increase the LPO
(Table IV, experiment B).
vi) The lower basal levels of lipid peroxides and the higher
effects of Abeta and of both proteases were found in the AD
compared to the MID group (Table V).
Table I: The effects ofAbeta and of plant proteases on LPO
in rat brain during 30 min incubation
age of animals:
1-month
(n)
3-month
(n)
23-month
(n)
buffer
22.1 ± 1.0
(18)
22.4 ± 1.4
(7)
23.1±1.6
(8)
1 mM Abeta
23.1±1.6
(8)
23.8 ± 1.0
(7)
22.8 ± 1.4
(8)
100 mg/ml BRO
26.4 ± 1.9***
(10)
25.6 ± 0.5***
(7)
24.3 ± 0.7
(8)
100 mg/ml PAP
24.6 ± 1.9**
(10)
23.3 ± 1.2
(7)
24.0 ± 2.1
(8)
ANOVA:
p< 0.001
p< 0.001
p= 0.1771
The experiments were performed on mixed hippocampal homogenates
of eleven male 1-month, six female 3-month and six male 23-month
old animals. The values are expressed as nmoles of thiobarbituric
acid-reactive products per g of tissue. All samples contained
0.9% DMSO.
Table II: The effects of Abeta and of plant proteases on LPO
in rat brain during 60 min incubation
age of animals:
1-month
(n)
3-month
(n)
23-month
(n)
buffer
22.4 ± 1.3
(8)
23.3 ± 1.4
(8)
22.5 ± 0.6
(8)
1 mM Abeta
21.1±1.5
(8)
23.3 ± 1.5
(8)
23.8 ± 0.8**
(8)
100 mg/ml BRO
27.4 ± 1.7***
(8)
27.8 ± 1.6***
(8)
25.4 ± 1.2***
(8)
100 mg/ml PAP
23.1±1.1
(8)
25.3 ± 0.9**
(8)
23.6 ± 1.2*
(8)
ANOVA:
p< 0.001
p< 0.001
p< 0.001
The experiments were performed on mixed hippocampal homogenates
of thirteen female 1-month, six female 3-month and seven male
23-month old animals. The values are expressed as nmoles of
thiobarbituric acid-reactive products per g of tissue. All
samples contained 0.9% DMSO.
Table III: The effects of age on LPO in rat brain influenced
by Abeta and plant proteases
age of animals
Abeta
BRO
PAP
i) 30 min incubation
1-month
104.5 ± 7.3
119.7 ± 8.4
111.4±8.6
3-month
105.9 ± 4.2
114.1 ± 2.4
103.7 ± 5.5*
23-month
98.6 ± 5.9
105.1 ± 3.1***
103.8 ± 8.8
ANOVA:
p= 0.0589
p< 0.001
p-0.0838
ii) 60 min incubation:
1-month
94.2 ± 6.8
122.6 ± 7.4
103.4 ± 4.7
3-month
100.0 ± 6.3
119.3 ± 6.9
108.4 ± 4.0*
23-month
105.7 ± 3.6**
112.9 ± 5.4*
105.0 ± 5.0
ANOVA:
p= 0.0026
p = 0.0246
p= 0.1100
The levels of thiobarbituric acid-reactive products from Tables
I and II were related to the levels with buffer and expressed
in %.
Table IV: Shared effects of Abeta and both plant proteases
on LPO in rat brain
Experiment A
Experiment B
composition
n
nmoles/g of tissue
composition
n
nmoles/g of tissue
Abeta
5
31.0 ± 2.2
Abeta
6
34.0 ± 1.7
BRO
5
27.5 ± 1.0
BRO
6
21.9 ± 2.2
PAP
5
29.0 ± 1.1
PAP
6
31.3±3.2
Abeta + BRO
5
28.3 ± 1.2*
BRO + Abeta
6
23.5 ± 2.5***
Abeta + PAP
5
27.5 ± 1.3*
PAP + Abeta
6
30.2± 1.1**
ANOVA:
p = 0.0053
ANOVA:
p< 0.001
Experiment A: samples were firstly preincubated for 30 min
with 1 mM Abeta or 1.7% DMSO, subsequently proteases (100 mg/ml)
were added and incubated for further 30 min, the experiment
was performed on mixed homogenates of four male 3-month old
rats.
Experiment B: samples were firstly preincubated for 30 min
with 100 mg/ml proteases or 1.7% DMSO, subsequently 1 mM Abeta
was added and incubated for further 30 min, the experiment
was performed on mixed homogenates of five male 3-month old
rats t-test was calculated with respect to the samples with
Abeta.
Table V: The effects of Abeta and plant proteases on LPO in
human brain of patients with Alzheimer disease (AD) and multi-infarct
dementia (MID) during 60 min incubation
composition
AD (n=4)
nmoles/g of tissue
MID (n=4)
nmoles/g of tissue
buffer
32.6 ± 4.5
38.7 ± 0.5
1 mM Abeta
38.2 ± 1.1
37.9 ± 1.8
100 mg/ml BRO
41.0 ± 1.1*
41.0 ± 1.1*
100 mg/ml PAP
37.5 ± 2.0
38.6 ± 3.5
ANOVA:
p = 0.0049
p=0.2311
The experiments were performed on mixed hippocampal homogenates.
All samples obtained 0.9 % DMSO. Statistical significance (t-test)
between AD and MID for samples with buffer: p = 0.0724.
group
n
sex
(M/F)
age
(years)
postmortem interval
(hours)
brain weigh
(g)
AD
3
0/3
86.3 ± 2.1
5.3 ± 0.6
983.3 ± 104.1
MID
3
2/1
83.7 ± 6.4
6.0 ± 1.0
1100.0 ± 0.0
Fig. 1: The effect of time on LPO for Abeta and DMSO
% DMSO: related to DMSO at 15 min ANOVA
for DMSO: p=0.0062
% Abeta: related to corresponding DMSO ANOVA
for Abeta: p=0.2056
Fig. 2: The effect of Abeta on LPO
ANOVA for 30 min: p=0.0085
ANOVA for 60 min: p=0.0002
Fig. 3: The effect of proteases on LPO
ANOVA for BRO: p=0.1045
ANOVA for PAP: p=0.1653
Discussion
Increased levels of lipid peroxides in the individual brain
regions of patients with AD have been observed for many times
(8). A marked role for Abeta in the induction of oxidative
stress is suggested. The effect of normal aging on the susceptibility
of brain tissue to undergo the LPO has been also found (e.g.,
9). It is well known that some nootropic drugs act as free
radical scavengers (10, 11). Therefore, a question of whether
drugs increasing the LPO can be applied to patients with
AD should be raised here.
Our results with BRO and PAP are in accordance with the other
studies where the enhanced release of reactive oxygen species
after application of some plant proteases in vivo and in
vitro have been found (e.g., 12). It is suggested that this
release is one of the mechanism of anticancer activity of
proteolytic enzymes. Regarding the AD therapy, some of our
experiments in vitro suggest certain positive circumstances
for the BRO and PAP human application in vivo in future.
Firstly, the effects of Abeta and of both proteases on LPO
are significantly age-dependent with antagonistic trends.
While the modified crosslinked brain proteins of aging animals
are more resistant to proteolysis (13, Tables I-III), the
effect of Abeta is more pronounced in old animals (Tables
II and III). Secondly, both proteases are able to eliminate
the negative effects of Abeta on LPO (Table IV). However,
more detailed experiments and especially the application
in vivo to young and old rats must be further performed.
Our experiments on demented patients suggest that oxidative
impairment of hippocampal tissue in AD group can be lower
(decreased basal levels of lipid peroxides, higher effects
of proteases) when compared with the MID group. On the contrary,
the higher sensitivity to Abeta have been found in AD patients
(Table V).
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