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 DOI: 10.1177/0960327112467040

2013 32: 721Hum Exp ToxicolNN Fadl, HH Ahmed, HF Booles and AH Sayed

modelSerrapeptase and nattokinase intervention for relieving Alzheimer's disease pathophysiology in rat

  

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Article

Serrapeptase and nattokinaseintervention for relieving Alzheimer’sdisease pathophysiology in rat model

NN Fadl1, HH Ahmed2, HF Booles3 and AH Sayed2

AbstractSerrapeptase (SP) and nattokinase (NK) are proteolytic enzymes belonging to serine proteases. In this study,we hypothesized that SP and NK could modulate certain factors that are associated with Alzheimer’s disease(AD) pathophysiology in the experimental model. Oral administration of aluminium chloride (AlCl3) in a doseof 17 mg/kg body weight (bw) daily for 45 days induced AD-like pathology in male rats with a significantincrease in brain acetylcholinesterase (AchE) activity, transforming growth factor b (TGF-b), Fas andinterleukin-6 (IL-6) levels. Meanwhile, AlCl3 supplementation produced significant decrease in brain-derivedneurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) when compared with control values. Also,AlCl3 administration caused significant decline in the expression levels of disintegrin and metalloproteinasedomain 9 (ADAM9) and a disintegrin and metalloproteinase domain 10 (ADAM10) genes in the brain. Histolo-gical investigation of brain tissue of rat model of AD showed neuronal degeneration in the hippocampus andfocal hyalinosis with cellular as well as a cellular amyloid plaques formation. Oral administration of SP or NK ina rat model of AD daily for 45 days resulted in a significant decrease in brain AchE activity, TGF-b, Fas and IL-6levels. Also, the treatment with these enzymes produced significant increase in BDNF and IGF-1 levels whencompared with the untreated AD-induced rats. Moreover, both SP and NK could markedly increase theexpression levels of ADAM9 and ADAM10 genes in the brain tissue of the treated rats. These findings were wellconfirmed by the histological examination of the brain tissue of the treated rats. The present results supportour hypothesis that the oral administration of proteolytitc enzymes, SP and/or NK, would have an effective rolein modulating certain factors characterizing AD. Thus, these enzymes may have a therapeutic application in thetreatment of AD.

KeywordsAlzheimer’s disease, proteolytic enzymes, serrapeptase, nattokinase, rats

Introduction

In humans, Alzheimer’s disease (AD) develops over

many years and it comprises a chain of subtle altera-

tions in brain function, finally leading to the impair-

ment of memory and cognition.1 The cognitive

disability includes the impairment of behaviour,

visual-spatial skills, speech and motor ability,

depression, delusions, hallucinations, aggressive

behaviour and ultimately, increasing dependence

upon others before death.2 Genetic and neuropatho-

logical evidence suggests that the accumulation of

amyloid-b (Ab) peptides in the brain is a key event

in the pathophysiology of AD. Amyloid fibrils

are an ordered protein aggregate with a lamellar

cross-beta structure. Enhancing amyloid clearance

is one of the targets of the therapy of AD.3 Therefore,

the therapeutic potential of several approaches

1 Medical Physiology Department, National Research Centre,Dokki, Cairo, Egypt2 Hormones Department, National Research Centre, Dokki,Cairo, Egypt3 Cell Biology Department, National Research Centre, Dokki,Cairo, Egypt

Corresponding author:Hanaa H Ahmed, Hormones Department, National Researchcentre, El Bohouth Street, Dokki, Cairo 12622, Egypt.Email: hanaaomr@yahoo.com

Human and Experimental Toxicology32(7) 721–735

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aimed at lowering the level of Ab in the brain is

currently being investigated.4

Proteases are ubiquitous enzymes that are crucial for

cell growth, development, apoptosis, protein turnover

and cell cycle regulation during life process.5 Several

serine proteases have been well characterized in the

brain. Accumulating evidence demonstrates that some

serine proteases play very important roles in the neural

development, plasticity and neuroregeneration in the

brain.6 The basis of these functions is the unique cor-

rect three-dimensional structures of the proteins, and

all the information necessary for proteolysis are

encoded by the amino acid sequence of protease.7 Dis-

eases such as cystic and even some forms of cancer are

believed to result due to defective protein folding.8

Serrapeptase (SP) is a proteolytic enzyme belong-

ing to serine proteases. It is derived from the non-

pathogenic enterobacteria Serratia E15. It is produced

in the intestines of silkworms to break down cocoon

walls. It has an anti-inflammatory effect.9 This

enzyme is believed to induce the degradation of

insoluble protein products like fibrin, biofilm and

inflammatory mediators.10 Therefore, SP has also

been used for the treatment of a number of inflamma-

tory conditions.11,12

Nattokinase (NK), a bacterial serine protease

derived from Bacillus subtilis, has been reported to

have potent fibrinolytic activity.13 It is considered to

be one of the most active functional ingredients found

in natto, a traditional Japanese food prepared from the

fermented soybeans.14 This enzyme is reported to

directly digest fibrin especially in its cross-linked

form.15 It has a fourfold greater thrombus-dissolving

activity than plasmin and is shown to potentiate endo-

genous fibrinolysis by cleavage and to inactivate plas-

minogen activator inhibitor 1 (PAI-1),16 leading to

efficient lysis of the detrimental coagulation of blood

in the body.15 NK not only dissolves blood clots17 but

also degrades amyloid fibrils.3

In this study, we hypothesized that SP and NK

could correct certain factors including proteins,

growth factors, apoptotic factor, inflammatory factor

and genes that are associated with AD pathophysiol-

ogy in the experimental model. To test this hypoth-

esis, we investigated the effects of SP and NK on

brain cholinesterase activity, brain-derived neuro-

trophic factor (BDNF), transforming growth factor b(TGF-b), insulin-like growth factor-1 (IGF-1), Fas

and interleukin-6 (IL-6) levels. The study was

extended to examine the effects of these enzymes

on the expression levels of disintegrin and

metalloproteinase domain 9 (ADAM9) and disintegrin

and metalloproteinase domain 10 (ADAM10) genes.

Histological investigation of brain tissue was also car-

ried out to confirm the biochemical and molecular

genetics observations.

Materials and methods

Aluminium chloride (AlCl3) was purchased from

Sigma Co. (California, USA). Its molecular weight

is 133.34.

The two proteolytic enzymes, SP (Doctors Best

Serrapeptase 40,000 serratio units per veggie cap.)

and NK (Doctors BEST Nattokinase 2000 FUs per

veggie cap) were purchased from a dietary supple-

ment market in USA.

Reagents for real time-polymerase chain reaction

(RT-PCR) were purchased from Invitrogen (Carlsbad,

USA) and Fermentas (Leon-Rot, Germany).

The human doses of the two proteolytic enzymes

used were converted into rat equivalents with the help

of conversion table described by Paget and Barnes.18

A total of 66 adult male albino rats weighing

120–150 g were enrolled in the current study. The rats

were obtained from the Animal House Colony of the

National Research Centre, Cairo, Egypt. The animals

were housed in polypropylene cages in an environ-

mentally controlled clean air room with a temperature

of 24 + 1�C, an alternating 12 h light–dark cycle, a

relative humidity of 60 + 5% and free access to tap

water and food. Rats were allowed to adapt to these

conditions for 2 weeks before beginning the experi-

mental protocol. The animal experimental protocol

has been approved by the Institutional Animal Ethics

Committee of Medical Research, National Research

Centre, Egypt. AD-like pathology model was

achieved in rats by the oral administration of AlCl3in a dose of 17 mg/kg bw19 daily for 45 days. The ani-

mals used in the present study were classified into six

groups; G1: healthy control group (negative control);

G2: AD group (positive control); G3: AD group

treated orally with water suspension of SP in a dose

of 10.8 U/kg bw daily for 45 days; G4: AD group

treated orally with water suspension of SP in a dose

of 21.6 U/kg bw daily for 45 days; G5: AD group

treated orally with the water suspension of NK in a

dose of 360 FU/kg bw daily for 45 days and G6:

AD group treated orally with water suspension of

NK in a dose of 720 FU/kg bw for 45 days daily.

At the end of the experimental period, the animals

were fasted overnight, sacrificed by decapitation

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under diethylether anaesthesia. The whole brain of

each rat was rapidly and carefully dissected, thor-

oughly washed with isotonic saline, dried on a filter

paper and weighed. The whole brain of a certain num-

ber of animals in each group was fixed in formalin

buffer (10%) for 24 h for histological examination and

the whole brain of the rest number of animals in each

group was mid-sagitally divided into two portions; the

first portion was immediately stored in liquid nitrogen

for gene expression analysis, while the second portion

of the brain was weighed and homogenized immedi-

ately to give 10% (w/v) homogenate in ice-cold

medium containing 50 mM Tris-HCl (pH 7.4) and

300 mM sucrose.20 The homogenate was centrifuged

at 1800g for 10 min in a cooling centrifuge at 4�C.

The supernatant (10%) was stored at �80�C till

further analysis to assess cholinesterase activity,

BDNF, TGF-b, IGF-1, Fas as well as IL-6 levels.

Biochemical analyses

Brain cholinesterase activity was determined kineti-

cally using a kit purchased from Biostc Co (Cairo,

Egypt), according to the method of Young.21 While,

brain BDNF level was assayed by enzyme linked

immunosorbent assay (ELISA) technique using kit pur-

chased from RayBiotech, Inc. (Norcross, USA),

according to the method described by Barde.22 Brain

TGF-b and IGF-1 levels were determined by ELISA

technique using a kit purchased from DRG instrument

GmbH (Marburg, Germany), according to the methods

described by Kropf et al.23 and Lewitt et al.24 respec-

tively. Meanwhile, brain Fas level was detected by the

ELISA technique using kit purchased from Diaclone a

Tepnel Co. (France), according to the method

described by Iio et al.25 Finally, brain IL-6 level was

assayed by ELISA technique using kit purchased from

RayBiotech, Inc, according to the method of Bauer and

Herrmann.26 Quantitative estimation of total protein

content in the brain was carried out according to the

method of Lowry et al.27 to express the concentration

of different brain parameter per milligram protein.28

Gene expression analysis

RNA extraction. Brain tissues of rats in each group

were used to extract the total RNA. Total RNA was

isolated from 100 mg of tissue by the standard TRIzol

extraction method (Invitrogen, USA) and recovered in

100 ml molecular biology grade water. In order to

remove any possible genomic DNA contamination,

the total RNA samples were treated by DNA-free™

DNase and removal reagents kit (Ambion, Austin,

Texas, USA) following the manufacturer’s protocol.

The RNA concentration was determined by spectro-

photometric absorption at 260 nm.

First-strand cDNA synthesis. To synthesize the first-

strand complementary DNA (cDNA), 5 mg of the

complete poly(A)þ RNA isolated from rat brain tissue

was reverse transcribed into cDNA in a total volume

of 20 ml using 1 ml oligo (poly(deoxythymidine)18)

primer.29 The composition of the reaction mixture

was 50 mM MgCl2, 10X reverse transcription (RT)

buffer (50 mM KCl; 10 mM Tris-HCl; PH 8.3),

200 U/ml reverse transcriptase (RNase H free),

10 mM of each dNTP and 50 mM of oligo (dT) primer.

The mixture of each sample was centrifuged for 30 s

at 1000g and transferred into the thermocycler (Bio-

metra GmbH, Gottingen, Germany). The RT reaction

was carried out at 25�C for 10 min, followed by 1 h at

42�C, and finished with denaturation step at 99�C for

5 min. Later, the reaction tubes containing RT pre-

parations were flash-cooled in an ice chamber until

being used for DNA amplification through

RT-PCR.30

RT-PCR assay. The first strand cDNA from each

brain sample was used as a template for the semi-

quantitative RT-PCR with a pair of specific primer

in a 25-ml reaction volume. The sequences of specific

primer and product sizes are listed in Table 1. b-Actin

was used as a housekeeping gene for normalizing

messenger RNA levels of the target genes. The reac-

tion mixture for RT-PCR consisted of 10 mM

dNTP’s, 50 mM MgCl2, 10X PCR buffer (50 mM

KCl; 20 mM Tris-HCl; pH 8.3), 1 U/ml Taq polymer-

ase and autoclaved water. The PCR-cycling para-

meters of the studied genes (ADAM9, ADAM10 and

b-actin) were performed as the PCR condition sum-

marized in Table 1. The PCR products were then

loaded onto 2.0% agarose gel, along with the PCR

products derived from b-actin of the different rat sam-

ples. Each reaction of the RT-PCR was repeated with

the samples obtained from eight rats, generating new

cDNA products at least eight times per each group.

Histological investigation. Brain tissues previously

fixed in formalin buffer (10%) for 24 h were washed

under tap water for 20 min. Then, the serial dilutions

of alcohol (methyl, ethyl and absolute ethyl) were

used for dehydration. Specimens were cleared in

xylene and embedded in paraffin at 56�C in hot air

oven for 24 h. Paraffin beeswax tissue blocks were

Fadl NN et al. 723

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prepared for sectioning at 4-mm-thick using slidge

microtome. The obtained tissue sections were col-

lected on glass slides, deparaffinized and stained by

hematoxylin and eosin stains31 for histological exam-

ination through the light microscope.

Statistical analysis

The results were expressed as mean + SE. Data were

analyzed by one-way analysis of variance and were

performed using the Statistical Package for the Social

Science (SPSS) program, version 11 followed by least

significant difference to compare the significance

between groups.32 Difference was considered signifi-

cant at p < 0.05.

Results

Biochemical results

The data in Table 2 showed the effect of treatment

with SP and NK on brain cholinesterase activity in the

model of AD. The untreated AD group rats showed

significant increase in brain cholinesterase activity

in comparison with the negative control group. In

contrast, the groups of AD group treated with low

or high dose of SP (10.800 or 21.600 U/kg bw) or

NK (360 or 720 Fu/kg bw) showed significant

decrease in brain cholinesterase activity in compari-

son with the untreated AD group.

Table 3 showed the effect of treatment with SP or

NK on brain BDNF, TGF-b and IGF-1 levels in AD

group. The untreated AD group showed significant

decrease in brain BDNF and IGF-1 levels in concomi-

tant with significant increase in brain TGF-b level in

comparison with the negative control group. On the

other hand, treatment with low or high dose of SP

or NK caused significant increase in brain BDNF and

IGF-1 levels associated with a significant decrease in

brain TGF-b level in comparison with the untreated

AD group.

Noteworthy, treatment with a high dose of SP

caused significant increase in brain BDNF level in

comparison with the group of rats treated with low

dose of SP. On the other hand, treatment with

low or high dose of NK produced significant decrease

in brain BDNF level in comparison with low or high

dose of SP. Meanwhile, the group of rats treated with

high dose of NK showed significant increase in brain

TGF-b level in comparison with the group of rats

treated with high dose of SP.

The data in Table 4 showed the effect of treatment

with SP and NK on brain Fas and IL-6 levels in the

model of AD. The untreated AD group showed

Table 2. Effect of treatment with SP and NK on braincholinesterase activity in the model of AD.a

Parameter GroupsCholinesterase(U/mg protein)

Negative control 591.6 + 35.4AD group (positive control) 770.8 + 13.7a

ADþ SP (10.800 U/Kg bw) 680.4 + 20.1b

ADþSP (21.600 U/Kg bw) 633.1 + 13.0b

ADþNK (360 Fu/Kg bw) 682.2 + 12.8b

ADþNK (720 Fu/Kg bw) 686.3 + 22.3b

NK: nattokinase; SP: serrapeptase; AD: Alzheimer’s disease.aSignificant change at p < 0.05 in comparison with the negativecontrol group.bSignificant change at p < 0.05 in comparison with the AD group.

Table 1. Primers and PCR-thermocycling parameters.

Gene Primer sequence (50-30) Conditions of the PCR assayPCR amplicons

(bp)

ADAM9 TGA CCA TCC CAA CGT ACA GA 35 cycles: 94�C for 40 s; 55�C for 40 s; 72�Cfor 45 s

316

TTC CAA AAC TGG CAT TCT C Final extension: 72�C for 5 minADAM10 TCT CCG GAA TCC GTA ACA T 40 cycles: 94�C for 30 s; 60�C for 30 s; 70�C

for 1 min600

TCC AGG AAC TTC TCC ACA C Final extension: 68�C for 2 minBeta-Actin TTG CCG ACA GGA TGC AGA A 25 cycles: 94�C for 30 s; 65�C for 30 s; 72�C

for 1 min540

GCC GAT CCA CAC GGA GTA CT Final extension: 75�C for 2 min

PCR: polymerase chain reaction; ADAM9: disintegrin and metalloproteinase domain 9; ADAM10: disintegrin and metalloproteinasedomain 10.

724 Human and Experimental Toxicology 32(7)

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significant increase in brain Fas and IL-6 levels in

comparison with the negative control group. On the

contrary, treatment with low or high dose of SP or

NK caused significant decrease in brain Fas level in

comparison with the untreated AD group. Also, treat-

ment with high dose of SP produced significant

decrease in brain IL-6 level in comparison with the

untreated AD group. Noteworthy, treatment with low

dose of NK showed significant elevation in brain Fas

level when compared with the group of rats treated

with high dose of SP. Meanwhile, treatment with high

dose of NK caused significant increase in brain Fas

level when compared with the groups of AD treated

with low or high dose of SP as well as low dose of NK.

Molecular genetic results

Expression of ADAM9 and ADAM10 genes. The

results of the expression analysis of ADAM9 and

ADAM10 genes in different studied groups are repre-

sented in Figures 1 and 2.

The current results revealed that the expression

level of ADAM9 gene was significantly decreased in

the brain tissues of AD group in comparison with the

negative control group (Figure 1). In comparison with

the expression level of ADAM9 in the brain tissue of

AD group, low doses of SP and NK did not affect the

expression level of ADAM9 gene in brain tissue of the

treated AD group significantly, while the expression

level of ADAM9 gene in the brain tissue of these

groups was still higher than that in the untreated AD

group. Using high doses of SP or NK increased the

expression level of ADAM9 in the brain tissue of the

treated AD group significantly when compared with

that of the untreated AD group. Significant increase

in the expression level of ADAM9 gene in the brain

tissue of AD group treated with high dose of NK when

compared with that in AD group treated with the low

dose of NK as well as low or high dose of SP

(Figure 1).

Regarding the expression level of ADAM10 gene in

the brain tissue, the results of the present study

revealed that the expression level of ADAM10 gene

was significantly decreased in AD group when com-

pared with the negative control group (Figure 2). Low

dose of SP did not affect the expression level of

ADAM10 gene in the brain tissue of the treated AD

group significantly when compared with that in the

untreated AD group, although the expression level

of ADAM10 gene in the brain tissue of this group was

still higher than that in the untreated AD group

Table 3. Effect of treatment with SP and NK on brain BDNF, TGF-b and IGF-1 levels in the model of AD.a

Parameters Groups BDNF (pg/mg protein) TGF-b (pg/mg protein) IGF-1 (ng/mg protein)

Negative control 88.2 + 2.3 626.7 + 3.4 13.2 + 0.6AD group (positive control) 42.3 + 0.6a 1046.7 + 11.8a 9.5 + 0.2a

AD þ SP (10.800 U/Kg bw) 65.7 + 3.3b 860.9 + 35.3b 11.0 + 0.2b

AD þ SP (21.600 U/Kg bw) 72.5 + 0.8b,c 830.0 + 17.8b 11.3 + 0.2b

AD þ NK (360 Fu/Kg bw) 60.1 + 0.8b,c,d 875.0 + 17.3b 10.7 + 0.2b

AD þ NK (720 Fu/Kg bw) 57.9 + 0.9b,c,d 890.0 + 20.5b,d 10.5 + 0.1b

NK: nattokinase; SP: serrapeptase; AD: Alzheimer’s disease.aSignificant change at p < 0.05 in comparison with the negative control group.bSignificant change at p < 0.05 in comparison with AD group.cSignificant change at p < 0.05 in comparison with AD group treated with low dose of SP.dSignificant change at p < 0.05 in comparison with AD group treated with high dose of SP.

Table 4. Effect of treatment with SP and NK on brain Fasand IL-6 levels in the model of AD.a

Parameters GroupsFas (pg/mgprotein)

IL-6 (pg/mgprotein)

Negative control 28.8 + 0.4 26.6 + 1.4AD group (positive

control)65.9 + 2.9a 35.9 + 0.3a

AD þ SP (10.800 U/Kg bw) 45.6 + 1.6b 33.9 + 3.3AD þ SP (21.600 U/Kg bw) 40.7 + 3.6b 30.0 + 2.0b

AD þ NK (360 Fu/Kg bw) 48.1 + 1.4b,d 32.7 + 0.9AD þ NK (720 Fu/Kg bw) 58.8 + 2.4b,c,d,e 34.5 + 0.4

NK: nattokinase; SP: serrapeptase; AD: Alzheimer’s disease.aSignificant change at p < 0.05 in comparison with the negativecontrol group.bSignificant change at p < 0.05 in comparison with the AD group.cSignificant change at p < 0.05 in comparison with AD grouptreated with low dose of SP.dSignificant change at p < 0.05 in comparison with AD grouptreated with high dose of SP.eSignificant change at p < 0.05 in comparison with AD grouptreated with low dose of NK.

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(Figure 2). However, the treatment of AD group with

the low dose of NK was able to increase the expres-

sion level of ADAM10 gene in the brain tissue signif-

icantly when compared with that in the untreated AD

group. On the other hand, using a high dose of SP or

NK increased the expression level of ADAM10 in the

brain tissue of the treated AD group significantly

when compared with that in the untreated AD group.

Similarly, treatment of AD group with low dose of SP

produced significant decrease in the expression level

of ADAM10 gene in the brain tissue when compared

with that in AD group treated with high dose of NK,

low dose of NK as well as high dose of NK (Figure 2).

Histological results. Our histological study showed

that there is no histopathological alteration observed

in the meninges, cerebral cortex and cerebrum

(Figure 3(a)), hippocampus (Figure3(b)), cerebellum

(Figure 3(c)) and medulla oblongata (Figure 3(d)) of the

brain of healthy control rats (negative control group).

(a)

(b)

a

bcb

bcc

a

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Control

Treatments

Rel

ativ

e ex

pres

sion

of

AD

AM

9 (A

DA

M9/

Bet

a ac

tin)

Control AD AD+SP1 AD+SP2 AD+ NK1 AD+NK2

ADAM9 316 bp-

β-actin 540 bp-

AD AD + SP1 AD + SP2 AD + NK1 AD + NK2

Figure 1. Semiquantitative RT-PCR of ADAM9 gene in brain tissue of AD group treated with SP and NK at low (a) andhigh (b) doses for 45 days. Superscripts with different letters within each column means significantly different (p < 0.05).RT-PCR: real time-polymerase chain reaction; ADAM9: disintegrin and metalloproteinase domain 9; SP: serrapeptase;NK: nattokinase; ADAM10: disintegrin and metalloproteinase domain 10; AD: Alzheimer’s disease.

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Oral administration of AlCl3 in a dose of 17 mg/

kg bw for 45 days induced AD-like pathology model

(AD group). This event is confirmed by our histologi-

cal investigation of brain tissues of rats in this group

(Figures 4(a) to (g)). These figures showed the

appearance of focal gliosis in the cerebral cortex

(Figure 4(a)) associated with neuronal degeneration

in the hippocampus (Figure 4(b)). The hippocampus

also showed focal hyalinosis with cellular

(Figure 4(c)) as well as a cellular amyloid plaques

formation (Figure 4(d)). Encephalomlacia was

detected in the cerebral matrix with the degeneration

of the surrounding neurons (Figure 4(e)), while the

blood vessels showed perivascular cuffing (Figure

4(f)). Also, there was degeneration in the Purkenje

cells of the cerebellum (Figure 4(g)).

Treatment of AD group rats with the low dose of

SP (10.8 U/kg bw) for 45 days resulted in an appreci-

able improvement in the histological feature of the

different brain areas in concomitant with the disap-

pearance of amyloid plaques in the hippocampus.

There was only focal gliosis in the cerebrum

(Figure 5(a)). Treatment of AD group with the high

dose of SP (21.6 U/kg bw) led to a complete dissolu-

tion of amyloid plaques in the hippocampus. Only, the

presence of focal gliosis (Figure 5(b)) and perivascu-

lar cuffing (Figure 5(c)) was observed in the

cerebrum.

Treatment of AD group with a low dose of NK

(360 Fu/ kg bw) for 45 days revealed an obvious

improvement in the histology of different brain areas

associated with the lysis of amyloid plaques in the

(a)

(b)

ab

bb

cc

a

0.000

0.200

0.400

0.600

0.800

1.000

1.200

1.400

1.600

Rel

ativ

e ex

pres

sion

of

AD

AM

10 (

AD

AM

10/B

eta

acti

n)600 bp- ADAM10

β-actin 540 bp-

Control AD AD+SP1 AD+SP2 AD+ NK1 AD+NK2

Control

Treatments

AD AD + SP1 AD + SP2 AD + NK1 AD + NK2

Figure 2. Semi-quantitative RT-PCR of ADAM10 gene in brain tissue of AD group treated with SP and NK at low (a) andhigh (b) doses for 45 days. Superscripts with different letters within each column means significantly different (p < 0.05).RT-PCR: real time-polymerase chain reaction; SP: serrapeptase; NK: nattokinase; ADAM10: disintegrin and metallopro-teinase domain 10; AD: Alzheimer’s disease.

Fadl NN et al. 727

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hippocampus. Focal gliosis was observed only in the

striatum of the cerebrum (Figure 6(a)). Treatment of

AD group with a high dose of NK (720 Fu/kg bw)

resulted in the disappearance of amyloid plaques in

the hippocampus. The cerebrum showed focal gliosis

(Figure 6(b)) as well as diffuse gliosis in the striatum

of the cerebrum (Figure 6(c)).

Discussion

Several proteins can form amyloid fibrils in vivo,

which are related to various diseases, such as AD.

Enhancing amyloid clearance is one of the targets of

the therapy of these amyloid-related diseases.3 In this

study, we investigate whether the proteolytic

enzymes, namely SP and NK, could ameliorate cer-

tain factors involved in AD pathophysiology in an

experimental rat model in an attempt to explore new

modalities for the management of AD.

Protoelytic enzymes are a group of enzymes that

break the long chain-like molecules of proteins into

shorter fragments (peptides) and eventually into their

components called amino acids.33

The current study demonstrated that the AlCl3administration (AD-like pathology model) revealed a

significant increase in brain acetylcholinesterase

(AchE) activity. This result coincides with that of

Zhang et al.34 The two suggested mechanisms for

aluminium-induced stimulation of AchE activity in the

brain are: (1) aluminium can induce caspase-1 activa-

tion and IL-1b secretion.35 IL-1b has been found to

promote the activity and expression of AchE both in

cell cultures and in vivo.36 (2) aluminium can augment

the accumulation of insoluble Ab protein.37 Abinduced elevation in the AchE activity through the

induction of lipid peroxidation in neuronal membranes

due to the production of hydrogen peroxide (H2O2).38

H2O2 may have a direct action on the AchE enzyme

as it acts as a modulator (may be allosteric) in the activ-

ity of functionally important proteins, receptors and

enzymes.39 Based on these findings, aluminium’s

action on AchE activity may be related to the involve-

ment of aluminium in the aetiology of AD pathological

process,40 as the impairment of cholinergic neurotrans-

mission became a well established fact in AD.41

In the present study, the induction of AD-like

pathology in rats via AlCl3 supplementation resulted

in a significant decrease in brain BDNF level. Inflam-

matory stimulation strongly decreases the generation

and release of the neurotrophic factors, which further

contributes to neurodegeneration.42 Aluminium has

been found to induce the inflammatory genes leading

to the production of a number of proinflammatory

cytokines.43 Parallel to the increasing generation of

these cytokines, a dramatic decrease in the nerve

growth factor and BDNF was demonstrated after

aluminium intoxication.44 The second possible

mechanism by which aluminium could decrease brain

BDNF in the present study is related to aluminium-

induced Ab formation in the brain. Ab has been

shown to reduce BDNF content and function45 via

imparing BDNF retrograde transport. Thus, Ab could

reduce BDNF signalling and this may underlie the

synaptic dysfunction observed in AD patients.46 In

Figure 3. (a) Photomicrograph of brain section of rat in the healthy control (negative control) group showing the normalhistological structure of the meninges (m) cerebral cortex (CC) and cerebrum (C). (H&E, �40). (b) Photomicrograph ofbrain section of rat in the healthy control (negative control) group showing the normal histological structure of thehippocampus (hp). (H&E,�40). (c) Photomicrograph of brain section of rat in the healthy control (negative control) groupshowing the normal histological structure of the cerebellum (Cr) (H&E,�40). (d) Photomicrograph of brain section of ratin the healthy control (negative control) group showing the normal histological structure of the medulla oblongata (mo).(H&E, �40). H&E: hematoxylin and eosin stain.

728 Human and Experimental Toxicology 32(7)

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view of neuroprotective effects of BDNF, the demon-

strated decrease in BDNF in AD patients may contrib-

ute to the development of this neurodegenerative

disease due to a lack of neurotrophic support.47 These

observations assess the contribution of aluminium in

the pathophysiology of AD.

The induction of AD-like pathology in rats by AlCl3administration in the present study produced a signifi-

cant elevation in brain TGF-b level. This finding could

be explained by the stimulatory effect of aluminium

on Ab deposition in the brain, as it has been suggested

that Ab could induce a remarkable inflammatory cas-

cade leading to the production of inflammatory cyto-

kines.48 Also, the experimental studies on microglial

cells stimulated by 1–42 peptide of Ab showed high

transcription level of many proinflammatory cytokine

genes such as Tumor necrosis factor-alpha (TNF-a),

monocyte chemoattarctant protein-1 (MCP-1), IL-8

and TGF-b.49,50 These observations are in consistent

with other reports that showed elevations of TGF-bexpression and immunoreactivity in AD patients.51 Our

findings are greatly supported by the study of Salins

et al.52 who stated that TGF-b level was markedly

upregulated in cortical brain regions of mouse model

of familial AD.

Our results showed a significant decrease in brain

IGF-1 level in AD-like pathology model due to alumi-

nium administration. On the basis of decreased IGF-1

concentrations in AD, some authors have suggested

that disrupted IGF-1 input into the brain may be

involved in the pathogenesis of amyloidosis and that

changed the IGF-1 signalling may potentially lead

to amyloidosis.53 IGF-1 resistance at the blood–brain

barrier is a pathogenic event in AD, and the local

alterations in insulin like growth factor binding pro-

teins (IGFBPs) may lead to a loss of IGF-1 input at the

Figure 4. (a) Photomicrograph of brain section of rat (AD group) showing focal gliosis in cerebral cortex (g) (H&E,�64). (b)Photomicrograph of brain section of rat (AD group) showing neuronal degeneration in the hippocampus (arrow) (H&E,�80). (c) Photomicrograph of brain section of rat (AD group) showing focal hyalenosis with cellular Plaque formation (h) inhippocampus (H&E,�80). (d) Photomicrograph of brain section of rat (AD group) showing hyalenosis with a cellular plaqueformation and encephalomalacia (h) (H&E, �64). (e) Photomicrograph of brain section of rat (AD group) showing ence-phalomalacia and neuronal degeneration (C) (H&E x 160). (f) Photomicrograph of brain section of rat (AD group) showingpesivascular caffing (C) (H&E �160). (g) Photomicrograph of brain section of rat (AD group) showing degeneration inpurkenji cells (arrow) in cerebellum (H&E, �160). AD: Alzheimer’s disease; H&E: hematoxylin and eosin stain.

Fadl NN et al. 729

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blood–brain barriers in AD.54 Moreover, as the

increased levels of oxidative stress are associated with

the aging process,55 and also impair neuronal IGF-1

signalling,56 oxidative stress may constitute a

mechanism for IGF-1 resistance in the aging brain.

This mechanism could also involve in our AD-like

pathology model as aluminium is a well known

pro-oxidant that induces oxidative stress in the brain

tissue57 leading to IGF-1 resistance in AD brain.

Significant increase in brain Fas level has been

detected after 45 days of AlCl3 administration to

induce AD-like pathology model in the current study.

A number of studies suggested a possible connection

between oxidative stress and increases in Fas/FasL

expression in brain astrocytes, microglia and neu-

rons.58–60 Moreover, Fas and FasL have been reported

to participate in Ab-induced neuronal death.61 Mor-

ishima et al.62 provided evidence that Ab induces

FasL expression in a Jun N-terminal kinase (JNK3)-

dependent manner. In agreement with our results Choi

et al.63 reported that Fas and FasL are elevated in the

compromised AD brains.

The present findings indicated that our AD-like

pathology model showed a significant increase in

Figure 5. (a) Photomicrograph of brain section of rat in AD group treated with low dose of SP (10.8 U/kg bw) daily for45 days showing focal gliosis in cerebrum (g) (H&E,�40). (b) Photomicrograph of brain section of rat in AD group treatedwith high dose of SP (21.6 U/kg bw) daily for 45 days showing focal gliosis in cerebrum (g) (H&E, �40). (c) Photo-micrograph of brain section of rat in AD group treated with high dose of SP (21.6 U/kg bw) daily for 45 days showingpesivascular caffing (arrow) (H&E, �40). AD: Alzheimer’s disease; H&E: hematoxylin and eosin stain; SP: serrapeptase.

Figure 6. (a) Photomicrograph of brain section of rat in AD group treated with low dose of NK (360 Fu/kg bw) daily for45 days showing focal gliosis in striatum of cerebrum (g) (H&E, �40). (b) Photomicrograph of brain section of rat in ADgroup treated with high dose of NK (720 Fu/kg bw) daily for 45 days showing focal gliosis in cerebrum (g) (H&E, �40). (c)Photomicrograph of brain section of rat in AD group treated with high dose of NK (720 Fu/kg bw) daily for 45 daysshowing diffuse gliosis in striatum of cerebrum (arrow) (H&E,�64). AD: Alzheimer’s disease; H&E: hematoxylin and eosinstain; NK: nattokinase.

730 Human and Experimental Toxicology 32(7)

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brain IL-6 level. It has been assumed that Ab induced

by aluminium administration activates microglia and

astrocytes upregulating cytokines production includ-

ing IL-6.48 Also, Ab could induce IL-6 expression

in several types of brain cells.64,65 Furthermore, IL-

6 enhances neuronal damage induced by Ab.66 The

destructive processes including neurodegneration

gliosis and progressive neurological diseases are

mediated by the overexpression of many cytokines

including IL-6.67 Apelt and Schliebs68 reported that

in patients with AD, IL-6 is increased locally around

amyloid plaques.

AD is characterized by the excessive deposition of

amyloid b-peptides (Ab peptides) in the brain. In the

non-amyloidogenic pathway, the amyloid precursor

protein (APP) is cleaved by the a-secretase within the

Ab peptide sequence. Proteinases of the ADAM fam-

ily (a disintegrin and metalloproteinase) are the main

candidates as physiologically relevant a-secretases.69

Three members of the ADAM family, ADAM9,

ADAM10 and ADAM17, can act as a-secretases in

various cell lines.70,71 ADAM10 can cleave APP-

derived peptides at the main a-secretase cleavage site

between the positions 16 and 17 of the Ab region and

it is expressed in mouse and human brain.72,73

In the present study, a significant decrease in

expression levels of ADAM9 and ADAM10 genes was

demonstrated in AlCl3-administered rats, which fur-

ther confirmed the implication of aluminium in the

pathogenesis of AD. This finding indicates that alumi-

nium could promote the amyloidogenic pathway in

the brain that results in Ab deposition as shown in our

histological study (Figures 4(a) to (g)).

The data in the present work revealed that the treat-

ment of AD group with either SP or NK resulted in

significant modulation in the different studied bio-

chemical parameters. The detected positive effects

of these enzymes could be attributed, at least in part,

to the ability of SP74 and NK75 to cross the blood–

brain barrier.

The proposed mechanisms for the beneficial

effects of SP and/or NK may be related to the ability

of SP to induce the degradation of insoluble pro-

teins.10 Similarly, NK possesses high amyloid-

degrading ability, which suggests the usefulness of

this enzyme in the treatment of amyloid-related

diseases.3 Thus, these enzymes might dissolve Abprotein that accumulates in the brain due to alumi-

nium neurotoxicity. This explanation was well docu-

mented by our histological findings (Figures 5(a)

and 6(c)). As mentioned above, Ab plays an important

role in the activation of AchE activity in the brain

besides its reducing effect on brain BDNF content.

Therefore, the degradation of Ab by these enzymes

leads to the inhibition of brain AchE activity and

restoration of BDNF brain level. Moreover, the disso-

lution of Ab by SP and/or NK enzymes results in the

blocking of the inflammatory cascade and preventing

the production of the proinflammatory cytokines such

as TGF-b and IL-6 in the brain. Also, the elimination

of Ab deposits helps in correcting the disruption of

brain IGF-1 level and managing the process of apop-

tosis and neuronal death due to the suppressing of Fas

expression and in turn its level in the brain.

SP has been found to have anti-inflammatory

effect9,76 and the efficacy of SP in the treatment of

a number of inflammatory conditions has been

reported.11,12 This property of SP represents an addi-

tional mechanism by which SP could correct the

activity of brain AchE and the levels of BDNF,

TGF-b and IL-6 in the brain.

SP and NK are belonging to proteolytic enzymes

family. These enzymes have been reported to have

free radical-scavenging property,77 which enables

these enzymes to reduce reactive oxygen species

accumulation in the brain. By this way, both SP and

NK could elevate IGF-1 and suppress Fas levels in the

brain.

Regarding the effect of SP and/or NK on the

expression levels of ADAM9 and ADAM10 genes in

the brain tissue, our results revealed that the high

doses of these enzymes could increase the expression

levels of ADAM9 and ADAM10 genes. Also, the low

dose of NK could elevate the expression level of

ADAM10 gene in the brain tissue of the treated ani-

mals. These results indicate that these enzymes may

have the ability to shift the amyloidogenic pathway

to the non-amyloidogenic one via increasing the

expression level of ADAM9 and ADAM10 genes in

the brain. To the best of the author’s knowledge, this

is the first published study that highlighted the effi-

cacy of SP and NK in alleviating the generation

of Ab in the brain via promoting the activity of

a-secretase-like action.

Several studies investigating the systemic use of

SP10,12,78 and NK79 have demonstrated no adverse

effects. In the present study, the lack of a dose depen-

dence may confirm the safety of these enzymes as

they are recommended as dietary supplements.

In conclusion, the results of the present work sup-

port our hypothesis that the oral administration of

SP or NK would have beneficial effects against AD.

Fadl NN et al. 731

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Thus, they may represent good remedies for the man-

agement of AD via their proteolytic activity, antioxi-

dant capacity and anti-amyloidogenic effect.

Funding

This research received no specific grant from any funding

agency in the public, commercial, or not-for-profit sectors.

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