IMMUNE EFFECTS OF TITANIUM DIOXIDE NANOPARTICLES * Dr. Eman Lecturer of Histology and Cytology, ARTICLE INFO ABSTRACT Introduction and aim of the… [614573]

s

AMELIORATIVE EFFECTS OF

MORINGA OLEIFERA
IMMUNE EFFECTS OF TITANIUM DIOXIDE NANOPARTICLES
*
Dr. Eman
Lecturer of Histology and
Cytology,

ARTICLE INFO

ABSTRACT

Introduction and aim of the work: The protective effects of
against orally administered titanium dioxide nanoparticles (TiO2 NPs) for 3 months on male albino
rats were examined. Methods: The
administrated with saline as a negative control group, • Group 2: Rats were orally administrated with
TiO2 (1200 mg kg
BW), Group 4: co
treatment of TiO2 by
for alterations in cytokines expression as CD3 and TNF
estimation of total proteins and polysaccharides. Results: TiO2 NPs up
expression in spleen where TNF
expression was significantly (P<0.05)
therapeutic normalized cytokines expression.
and therapeutic effects of
rats.

Copyright
©
201
6
,

Eman

Mohammed Mohammed

Abd
permits unrestricted

use, distribution, and reproduction in any medium, provided the original work is properly cited.

INTRODUCTION

The spleen is an organ found in virtually all vertebrate animals.
Similar in structure to a large lymph node, it acts primarily as a
blood filter. The spleen plays important roles in regard to red
blood cells (also referred to as erythrocytes) and the immu
system. It removes old red blood cells and holds a reserve of
blood, which can be valuable in case of hemorrhagic shock,
and also recycles iron. As a part of the mononuclear phagocyte
system, it metabolizes hemoglobin removed from senescent
erythrocytes
. The globin portion of hemoglobin is degraded to
its constitutive amino acids, and the heme portion is
metabolized to bilirubin, which is removed in the liver
and Kraal,
2005
).

The spleen synthesizes antibodies in its
white pulp and removes antib
ody
–
coated bacteria and
antibody
–
coated blood cells by way of blood and lymph node
circulation. A study published in 2009 using mice found that
the spleen contains, in its reserve, half of the body's monocytes
within the red pulp
(
Swirski

et al
., 2009
). Monica premi,
that these monocytes, upon moving to injured tissue (such as
the heart), turn into dendritic cells and macrophages while
promoting tissue healing
(
Monica premi

et al
mentioned that the spleen is a center of activity

*Corresponding author:
Dr. Eman

Mohammed Mohammed
Lecturer of Histology and
Cytology,

Zoology Department, Faculty of Science,
Fayoum University, Egypt.

ISSN
: 0975
–
833X

Article History:

Received xxxxx, 2016

Received in revised form

xxxxxxxx, 2016

Accepted xxxxxxx, 2016

Published
online xxxxxx,

2016

Key words:

TiO
2

NPs,

Moringa,

Spleen,

Immune Responses,

CD3,

TNF
–
α.

Citation:
Eman

Mohammed Mohammed

Abd
–
Ella
titanium dioxide nanoparticles
in male albino rats
”
,
International Journal of Current Research,

RESEARCH ARTICLE

MORINGA OLEIFERA

LAM. LEAF EXTRACT AGAINST
IMMUNE EFFECTS OF TITANIUM DIOXIDE NANOPARTICLES
IN MALE ALBINO RATS

Dr. Eman

Mohammed Mohammed

Abd
–
Ella

Cytology,

Zoology Department
, Faculty of Science, Fayoum

ABSTRACT

Introduction and aim of the work: The protective effects of
Moringa oleifera
against orally administered titanium dioxide nanoparticles (TiO2 NPs) for 3 months on male albino
rats were examined. Methods: The

rats were divided into five groups • Group 1: rats were orally
administrated with saline as a negative control group, • Group 2: Rats were orally administrated with
TiO2 (1200 mg kg
−
1
) (1
\
10 LD50), Group 3: Rats were administrated orally by
BW), Group 4: co
–
treatment of
MOLE

and TiO2 as a protective group for 3 months and Group 5: post
treatment of TiO2 by
MOLE

for 3 successive month as therapeutic treatment. Spleen was examined
for alterations in cytokines expression as CD3 and TNF
–
α, his
topathology and histochemically by
estimation of total proteins and polysaccharides. Results: TiO2 NPs up
expression in spleen where TNF
–
α expression was significantly increased (p<0.05) and CD3
expression was significantly (P<0.05)

decreased but
MOLE

therapeutic normalized cytokines expression.
Conclusion
: present findings confirmed the protective
and therapeutic effects of
MOLE

on TiO2 NPs induced alteration in immune responses in male albino
Abd
–
Ella
.

This
is an open access article distributed under the Creative Commons Att
use, distribution, and reproduction in any medium, provided the original work is properly cited.

The spleen is an organ found in virtually all vertebrate animals.
Similar in structure to a large lymph node, it acts primarily as a
blood filter. The spleen plays important roles in regard to red
blood cells (also referred to as erythrocytes) and the immu
ne
system. It removes old red blood cells and holds a reserve of
blood, which can be valuable in case of hemorrhagic shock,
and also recycles iron. As a part of the mononuclear phagocyte
system, it metabolizes hemoglobin removed from senescent
. The globin portion of hemoglobin is degraded to
its constitutive amino acids, and the heme portion is
metabolized to bilirubin, which is removed in the liver
(
Mebius

The spleen synthesizes antibodies in its
coated bacteria and
coated blood cells by way of blood and lymph node
circulation. A study published in 2009 using mice found that
the spleen contains, in its reserve, half of the body's monocytes
). Monica premi,
stated
that these monocytes, upon moving to injured tissue (such as
the heart), turn into dendritic cells and macrophages while
et al
., 2010
).

Brender

mentioned that the spleen is a center of activity

of the

Mohammed Mohammed

Abd
–
Ella,

Zoology Department, Faculty of Science,

mononuclear phagocyte system and can be considered
analogous to a large lymph node, as its absence causes a
predisposition to certain infections
Titanium dioxide nanoparticles (TiO2 NPs) are widely used in
a number of applications:
as an additive, including as a white
pigment in paint, as a food colorant, in sunscreens and in
cosmetic creams as well as in the environmental
decontamination of air, water and soil by the destruction of
pesticides
(
Medina

et al
., 2007
rapid development of nanotechnology, the potential health
hazards and environmental impact of manufactured TiO2 NPs
have gained increasing attention
added that the smaller the particles of TiO2, the more
reactivity, effectively and toxicity
Intraperitoneal injection of 100
(324
–
2592 mg kg
–
1
bw) caused significant accumulation of
particles, mainly in the
spleen, but also in the liver, kidney and
lung
(
Chen
et al
., 2009
).

Moringa oleifera
(local name Sajna) belongs to the Moringaceae family
et al
., 2007
).

It is a multipurpose tree widely distributed in
Bangladesh, India,
Pakistan, Sri Lanka, Myanmar, Malaysia,
Singapore, the Philippines, Thailand, Cuba, Jamaica and
Nigeria
(
Guevara
et al
., 1999
).

the young leaves and tender pods which are esteemed as very
common vegetable in Bangladesh and

Available online at http://www.journal
cra.
com

International Journal of
Current Research

Vol.
8
, Issue,
11, pp.xxxxxxx,
November
,
201
6

INTERNATIONAL

Ella
,
2016.

“
Ameliorative effects of

Moringa oleifera

Lam. leaf extract against
International Journal of Current Research,
8, (11),
xxxxxx

z

LAM. LEAF EXTRACT AGAINST
SYSTEMIC
IN MALE ALBINO RATS

, Faculty of Science, Fayoum
University
, Egypt

Moringa oleifera

leaves extract (
MOLE
)
against orally administered titanium dioxide nanoparticles (TiO2 NPs) for 3 months on male albino
rats were divided into five groups • Group 1: rats were orally
administrated with saline as a negative control group, • Group 2: Rats were orally administrated with
10 LD50), Group 3: Rats were administrated orally by
MOLE

(50 mg kg
−
1

and TiO2 as a protective group for 3 months and Group 5: post
for 3 successive month as therapeutic treatment. Spleen was examined
topathology and histochemically by
estimation of total proteins and polysaccharides. Results: TiO2 NPs up
–
regulated CD3 and TNF
–
α
α expression was significantly increased (p<0.05) and CD3

administration as co
–
treatment or
: present findings confirmed the protective
on TiO2 NPs induced alteration in immune responses in male albino
is an open access article distributed under the Creative Commons Att
ribution License, which

mononuclear phagocyte system and can be considered
analogous to a large lymph node, as its absence causes a
predisposition to certain infections
(
Brender

et al
., 2005
).

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in
as an additive, including as a white
pigment in paint, as a food colorant, in sunscreens and in
cosmetic creams as well as in the environmental
decontamination of air, water and soil by the destruction of
., 2007
). Hoet
mentioned th
at with the
rapid development of nanotechnology, the potential health
hazards and environmental impact of manufactured TiO2 NPs
have gained increasing attention
(
Hoet

et al
., 2004
).

Sang

added that the smaller the particles of TiO2, the more
reactivity, effectively and toxicity
(
Sang

et al
., 2012
).

Intraperitoneal injection of 100
–
nm TiO2 NPs in high doses
bw) caused significant accumulation of
spleen, but also in the liver, kidney and
Moringa oleifera
(
M. oleifera
) Lam.
(local name Sajna) belongs to the Moringaceae family
(
Goyal

It is a multipurpose tree widely distributed in
Pakistan, Sri Lanka, Myanmar, Malaysia,
Singapore, the Philippines, Thailand, Cuba, Jamaica and

M. oleifera
is valued mainly for
the young leaves and tender pods which are esteemed as very
common vegetable in Bangladesh and
India. It is reported that

INTERNATIONAL
J
OURNAL

OF CURRENT RESEARCH

Lam. leaf extract against
systemic immune effects of

Moringa
leaf is a potential

source of natural antioxidants such
as total phenolics and antioxidant vitamin A, C and E, ascorbic
acid oxidase, polyphenol oxidase and catalase
(
Gyton

and
Hall,

2006
;
Harold,

2006
).

The leaves are a

rich source of essential
amino acids such as methionine, cysteine, tryptophan, lysine,
vitamins and minerals
(
Jahn,

1988
).

Moringa
leaves have been
reported to act as a hypocholesterolemic agent, thyroid
hormone regulator, antidiabetic agent, antitumor agent and
hypotensive agent
(
Moore

et al
., 2010
).

Despite the above
mentioned beneficial effect of
Moringa
leaves, its efficacy in
red
ucing particular toxicity in general and Nano
–
titanium
toxicity in particular, has not yet been studied enough.
Therefore, we aimed to investigate the efficacy of
M. oleifera
leaves on nano
–
titanium toxicity in rats' model.

MATERIALS AND METHODS

Chemicals: Titanium Dioxide nano
–
particles (TiO2

NPs
):
Anatase form, particle size (25
–
100 nm) was purchased from
Sigma Aldrich chemical Co., USA.

Animals:

Tow
–
month
old (160
–

200 g body weight)
male
albino rats (
Rattus rattus
) were selected from animal

house of
National Research Center, Giza, Egypt. The animals were
housed under controlled environment conditions (12 h
light/dark cycle) at a temperature of 25
˚C + 10˚C and humidity
of 60% + 5% and fed standard diet and water Ad libitum for
the experiment
al period.

Plant material:

Moringa oleifera

leaves powdered

obtained
as capsules (natural product drug) from local pharmacy of
Texas, USA.

Extract preparation of
Moringa oleifera

leaves (
MOLE
)

The

ethanol extracts was prepared by soaking 100
gm

of
Moringa oleifera

leaves powdered in 300 ml ethanol (95%)
shaking (24 h) then covered by a piece of aluminum foil and
kept in the
refrigerator
. The infusion was filtered by a piece of
double gauze and the filtrate was centrifuged at 3000 rpm for
10 minutes,

then the supernatant (ethanol) was evaporated
using a rotatory evaporator apparatus attached
to a

vacuum

pump. The 100 gm of dried grape seeds powder yield 26.7 gm
ethanol
(
Balu

et al
., 2016
).

Experimental protocol:

The rats were randomly divided into
5
groups of 12 animals each as follows:

Group 1:

served as negative control (rats were orally
administrated with ml of normal saline for 3 month.

Group 2:

(positive control group) TiO2 NPs
–
intoxicated rats:
received 1200 mg kg
−
1

body weight TiO2 NPs by
gavage (1/10 LD 50) in 1 mL saline solution as a
solvent once daily for 3
months (
Wang

et al
., 2007
).

Group 3:

(MOLE control group): Rats were administrated
orally MOLE (50 mg/kg b·w/day) diluted with
saline solution; once daily for three months.

Group 4:

(MOLE +TiO2); co
–
treatment group: received 50
mg kg
−
1

body weight
MOLE

one hour before TiO2
by gavage once daily for 3 months.

Group 5:

(therapeutic group), rats received 1200 mg kg
−
1

body weight TiO2 by gavage (1/10 LD 50) in 1 mL
saline soluti
on as a solvent once daily for 3 months,
then treated daily with a
single

dose of MOLE (50
mg/kg b·w/day) diluted with saline solution; for
successive 3 months.

Examinations

Rats of each group were
sacrificed

by cervical dislocation at
the end of the experimental periods and decapitation. Spleen of
each animal was obtained and divided and fixed in buffered
neutral formalin 10% solution for 24 hrs, dehydrated through
alcohols, cleared in xylene and embedded in
paraffin wax.
Five
–
micrometer

thickness paraffin sections were prepared and
mount on clean slides.
For histopathological studies
, such as
sections were stained with Ehrlich
.s
hematoxylin

and
counterstained with eosin
(
Drury

and Wallington, 1967
).

For
histo
chemical investigations,

the periodic acid Schiff’s (PAS)
technique of
Hotchkiss (1948); (
Hotchkiss,

1948
)

was used for
the detection of

total carbohydrates;
1, 2 glycol group.

Bromophenol blue method to demonstrate total proteins
(Maize, 1953); (
Maize

et
al
., 1953
).

For immunohistochemical
studies;
other sections were de
–
paraffinized, placed on charged
slides, and used for localization of CD3;
(
cluster of

differentiation

3
),

T
–
cell co
–
receptor helps to activate the
cytotoxic T
–
Cell;

and TNF
–
α; (
tumor necrosis factor alpha),

It
is produced chiefly by activated

macrophages
,

where it's
primary role is in the regulation of

immune cells
;

on cells.
Anti
–
CD3 and TNF
–
α antibodies respectively were employed to
stain the cells in an avidin
–
biotin
–
complex (ABC
)
immunoperoxidase technique. Specifically, the sections were
incubated in 5% H
2
O
2

(in methanol) solution for 10 min to
block endogenous peroxidase activity and then incubated with
primary anti
–
CD3 and TNF
–
α rabbit monoclonal antibody
respectively (1:50 di
lution in 1% bovine serum albumin
solution; Pan
–
T Clone SP7, Thermo Scientific, Lab Vision,
Fremont, CA) for 60 min at room temperature. After rinsing
with phosphate
–
buffered saline (PBS, pH

7.4) to remove
unbound primary antibody, the samples were incubat
ed with
diaminobenzidine (DAB) chromogenic solution for 5 min at
25°C. The sections were then counterstained with
haematoxylin for 15 sec.
(
Kiernan
, 2007
).

For statistical
analysis, each section was counted manually at high power
(X400) after identifying
at low power (x100). The
representative areas with the highest concentration of stained
cells were detected according to the

recommendation of Cohen
and Hogan (1994); (
Cohen

and Hogan, 1994
)
.

About 1000
cells/slide were counted in each of five microscopic
fields from
well
–
labeled areas to determine the average of TNF
–
α Labelling
index. TNF
–
α was expressed as number of labeled cells
(positive for TNF
–
α) as a percentage of the total number of
cells counted in each specimen. All identifiable staining was
regarded as positive. Then this method applied with CD3
expression.

Statistical analysis

The obtained results of each of, TNF
–
α and CD3 were
expressed as mean + Standard Error (SE). They were also
statistically analyzed by using the SPSS11 computer software
program (ANOVA) analyses.

RESULTS

Histopathological results

The spleen tissues of

the negative control group are illustrated
in Figures 1(
A
). This spleen tissue is normal in histological

presentation and appeared with normal architecture. The spleen
tissue sections consist of the white and red pulps. Where the
white pulp contain the sp
lenic follicles and the marginal zone,
while red pulp consists of many splenic sinusoids, small
macrophages and large
–
sized megakaryocytes. In Figure 1 (
B
);
the photomicrograph illustrate the spleen tissue of the MOLE
control group. The pulps are also well

defined and the entire
tissues structural integrity is normal. The white pulps appear
relatively quiet prominent. The specimens of TiO2 NPs treated
group (positive control group) show the white pulp contains
lymphocytic proliferation around the central ve
in, peri
–
arterial
lymphocytic sheath (PALS) with congestion in the blood
vessels and degenerated areas
with apoptotic cells; Figure 1 (
C,
D & E
). The spleen of the MOLE and TiO2 NPs co
–
administered rats (protective group) showed decrease in the
lymphocytic

proliferation especially around the PALS, while
the congestion still persist; Figure 1 (
F
).

Most of the tissue
sections of spleen of
group 5

(therapeutic group),
show
apparent normal structure for white and red pulps as it was
seen in the normal control group
Figure 1 (
G
).

Table 1.

Means of

TNF
–
α

antibody values recorded at different
treatments

a significant difference with the group I

b significant difference
with the group II

Results are expressed as mean ± SD

LSD
0.05
–

0.01

(
1.04
–

1.39
)

Table 2.

Means of CD3 antibody values recorded at different
treatments

a significant difference with the group I

b significant difference with the group II

Results are
expressed as mean ± SD

LSD
0.05
–

0.01

(
1.24
–

1.69
)

Histochemical results

Negative control and MOLE control spleen sections stained
with PAS method are shown in Figure 2 (
A & B
). This spleen
tissue is normal in polysaccharides contents and the pulps are
also well defined and the entire tissues structural integrity is
normal. The white pulps appear relatively quiet prominent.
Spleens of rats administered TiO2 NPs had an alteration in
carbohydrate content. Glycogen appeared around the cell

membranes Figure

2 (
C & D
).
Most of the tissue sections of
spleen of
group 4 & 5

(protective and therapeutic group),
show
apparent normal structure for white and red pulps as it was
seen in the normal control group and positive reaction of PAS
at the wall of blood vessels
Figure 2 (
E & F
).

Examination of
spleen sections from the negative control and MOLE control
groups,
spleen sections stained with by bromophenol blue
method, showed normal protein content and the pulps are also
well defined and the entire tissues structural integrity is
normal. The white pulps appear relatively quiet prominent,
Figure 2 (
G & H
). Protein c
ontent was moderately decreased in
the splenic cells of rats administered with TiO2 NPs, Figure 2
(
I
).
Most of the tissue sections of spleen of
group 4 & 5

(protective and therapeutic group),
show apparent normal
structure for white and red pulps as it was

seen in the normal
control group
Figure 2 (
J & K
).

Immunohistochemical results

TNF
–
α labeled cells in spleen sections were increased after
administration of TiO2

NPs;

Figure 3 (
C
);
when compared
with

Spleen sections from the negative control rats of gro
up 1

as well as the
sections of spleen of

MOLE control, group 3;

Figure 3 (
A & B
).
Sections of spleen from
co
–
treatment
(groups 4
) revealed shortage of the numbers of
TNF
–
α labeled
cells when compared with TiO2

NPs
–
intoxicated, group 2,
but
more pronounced increasing
at sections of group 5 than spleen
sections of

TiO2

NPs

group 2

Figure 3 (
D & E
) respectively.

The mean optical density of
TNF
–
α
expression for all groups
presented in Table 1. There was a significant increase (P<0.05)
in
TNF
–
α
expression in
TiO2

NPs
–
intoxicated, group 2 with
mean value
49.70
+

2.25

compared with
negative control,
group 1 with mean value 12.00
+

0.97

as well as the

sections
of spleen of

MOLE control, group 3 with mean value 14.60
+
1.05
. There was a significant decrease (P<0.05) in
TNF
–
α
expression in
co
–
treatment (groups 4
) with

mean value

23.20
+

2.00

when compared with TiO2
–
intoxicated, group 2. But
TNF
–
α
expression
s more pronounced increased;

significant
increase (P<0.05);

at group 5

in mean value
70.10
+

6.11

when
compared with TiO2
–
intoxicated.

Spleen sections from the
negative control rats of group 1 showed normal distribution of
CD3
+
T
–
lymphocytes through the cells of white pulp;

Figure 3
(
F
);

while the sections of spleen of

MOLE
control group 3 as
well as co
–
treatment group 4 showed high distribution of
CD3
+
T
–
lymphocyte numbers
;
Figure 3 (
G & I
);

when
compared with

TiO2

NPs
–
intoxicated, group 2 that showed
high decrease in CD3
+
T
–
lymphocyte distribution through the
PALS of
spleen sections that the majority of cells were weakly
stained, and only a few had a strongly positive reaction;

Figure 3 (
H
)
. Sections of spleen from
therapeutic groups
(groups 5
);
Figure 3 (
J
)

revealed slight increasing in the
numbers of CD3
+
T
–
lymphoc
ytes when compared with

TiO2

NPs
–
intoxicated, group 2

but
more pronounced
at sections of
group 4.

The mean optical density of CD
–
3 expression for all
groups presented in Table 2. There was a significant decrease
(P<0.05) in CD
–
3 expression in
TiO2

NPs
–
i
ntoxicated, group
2 with mean value 19.10 + 2.20
compared with
negative
control, group 1 with mean value 39.00
+

0.93 and

MOLE
control, group 3

with mean value
50.20 + 1.61
. There was a
significant increase (P<0.05) in CD
–
3 expression in both
co
–
treatment
and

therapeutic groups (groups 4 & 5

respectively)
with
TiO2

NPs
–
intoxicated, group 2 with mean value 19.10 +
2.20, but
CD
–
3 expression
s was more pronounced at group 4 in
mean value
52.40 + 2.86

than sections of group 5 in mean
value
32.90 + 1.31
.

Figure
1
.
(A)

Normal histological features of splenic tissue of the negative control group 1 stained with hematoxylin and eosin, it consist
s of the capsule, white
pulps (wp) with the central arteries, trabeculea) and red pulps (rp), vascular organization and cellular c
omposition (400X).
(B)

Normal histological features of
splenic tissue of the MOLE control group III stained with hematoxylin and eosin, it consists of the capsule, white pulps (wp)

and red pulps (rp), vascular
organization and cellular composition (400X).

(C, D, & E)

Spleen sections of rats administered TiO2 NPs group II show enlarged white pulps, thickened capsule,
increased proliferation of lymphocytes with some degenerated areas (arrow) and apoptotic cells, hematoxylin
–
eosin (400, 800, 400 x respectivel
y).
(F)

Spleen
section of co
–
treatment group IV, slightly there is no histopathological alterations, hematoxylin
–
eosin (400 x).
(G)

Spleen section of therapeutic group V, there is
no histopathological alterations but there is slightly increasing proliferat
ion of lymphocytes, hematoxylin
–
eosin (400 x)

Figure 2.

Total carbohydrates in spleen sections. (A): Normal features of splenic tissue of the negative control group I stained with P
AS (400x).
(B)

Normal
features of splenic tissue of the MOLE control group III stained with PAS (400x).
(C, D
) Spleen sections of rats administered TiO2 NPs group II show a slight
decrease in carbohydrate content, stained with PAS (400x).

(E)

Spleen section of co
–
treatm
ent group IV, mild alterations in carbohydrate content, stained with
PAS (400x).
(F)

Spleen section of therapeutic group V, slight normal distribution of carbohydrate contents, stained with PAS (400x).
–

Protein content in spleen
sections.
(G):

splenic tis
sue of the negative control group I with a considerable amount of protein elements, stained with bromophenol blue (400x).
(H)

Normal
features of splenic tissue of the MOLE control group III stained with bromophenol blue (400x).

(I
) Spleen section of rat ad
ministered TiO2 NPs group II show a
slight decrease in protein content with a weak response towards bromophenol blue reaction, stained with bromophenol blue (400
x).

(J)

Spleen section of co
–
treatment group IV, mild alterations in protien content, stained w
ith bromophenol blue (400x).
(K)

Spleen section of therapeutic group V, slight normal
distribution of protien contents, stained with bromophenol blue (400x)

DISCUSSION

Increased use of artificial nanoparticles in a wide range has
introduced a potential inhaled pollutant. As a result of
expanding usage of TiNPs in various products as well as in
biological and medical applications, it is important to study
their environmen
tal safety, transport and possible toxicological
effects on humans.
Recordati
explained that the toxicity of
TiNPs is size
–

and dose
–
dependent as well as coating
–

and cell
type
–
dependent
(
Recordati
et al
., 2016
).

Although the reports
on toxicity of nanoparticles are now increasing, immunity
–
related responses of nanoparticles have not been well studied.
In this study, we focused on the systemic immune response
induced by repeated exposure of titanium dioxide
nanopar
ticles, involved in immune organ formation,
lymphocyte proliferation, lymphocyte distribution and
cytokine induction.
Chen
demonstrated that acute toxicity
induced by various doses of TiO2 NPs in mice, that
accumulation of TiO2 NPs (80 nm, 100 nm, anatase)

was high
in spleen, liver, kidneys and lung in a decreasing manner
(
Chen
et al
., 2009
).

Some of the particles were excreted from
the kidney and
Linglan

found that the inflammatory cytokines
cascade may cause inflammatory cell chemotaxis and
apoptosis, res
ulting in serious spleen injury
(
Linglan
et al
.,
2009
).

According to
Shi,

the cellular damage and oxidative
stress of nanoparticles in the spleenocytes were related to the
particle size and chemical compositions of nanoparticles
(
Shi
et al
., 2013
).

Most of

nanoparticles tend to accumulate in the

liver
(
Sadauskas
et al
., 2007
)

as well as spleen and kidney
(
Xue
et al
., 2011
).

In the present study spleen of the TiO2 NPs
treated groups showed many drastic changes in the white
pulps. As progressive dilation and congestion of blood sinuses,
increased number of connected white pulps with increased
proliferation of lymphocytes, dila
ted and elongated trabecular.
The dilated and elongated splenic vein may referred to
decreased ribosomes, glycogen granules and cristae of
mitochondria may led to corrugated membranes,
(
Khaki
et al
.,
2006
).

Also
Melamed

mentioned that progressive dilation
and
congestion of blood sinusoids could be considered as a reactive
change that may be related to the inhibitory effect on the
vascular smooth muscles which induced relaxation and
consequent vasodilatation
(
Melamed
et al
., 2003
).

Danz
reported that this va
sodilatation and increased vascular
permeability should lead to loss of fluid from the blood which
would result in degeneration and necrosis in the tissues and
may be considered as a reaction to progressive epithelial cell
death and atrophy of the tissue
(
Danz
et al
., 1998
).
TiO2 NPs
treated group showed degenerative and atrophic changes in rat
spleen with small sized lymphatic follicles and absence of
germinal centers. The differentiation between red and white
pulps was indistinct. This results are in agree
ment with
(
Ciric

et al
., 2005
).

Highly reduced total protein was detected in the
splenic tissue with moderately stained capsule of TiO2 NPs
treated groups.
Yee and Choi

found that mercury treatment
caused biochemical damage to tissues, total protein depletion
and genes through diverse mechanisms, such as disrupting

Figure 3.

(A):

splenic tissue of the negative control group I with a

weak diffuse of
immunostaining TNF
–
α (
Immunoperoxidase, X400
).

(B)

Normal features of
splenic tissue of the MOLE control group III shows week to mild diffuse of
immunostaining TNF
–
α (
Immunoperoxidase, X800
).
(C
) Spleen sections of rats
administered TiO2 NPs group II show a

high diffuse of
immunostaining TNF
–
α (
Immunoperoxidase, X800
).

(D)

Spleen section of co
–
treatment group IV, with a

slight diffuse of
immunostai
ning TNF
–
α (
Immunoperoxidase, X800
).
(E)

Spleen section of therapeutic group V, show a

high diffuse of
immunostaining TNF
–
α
(
Immunoperoxidase, X400
).
(F):

splenic tissue of the negative control group I with a

moderate diffuse of
immunostaining CD3 (
Immunop
eroxidase, X400
).

(G)

Normal features of splenic tissue of the MOLE control group III shows slightly high diffuse of
immunostaining CD3 (
Immunoperoxidase, X400
).
(H
) Spleen
sections of rats administered TiO2 NPs group II show a

weak diffuse of
immunostaini
ng CD3 (
Immunoperoxidase, X400
).

(I)

Spleen section of co
–
treatment
group IV, with high

diffuse of
immunostaining CD3 (
Immunoperoxidase, X400
).
(J)

Spleen section of therapeutic group V, show a

moderate diffuse of
immunostaining CD3 (
Immunoperoxidase, X400
)

membrane potential, altering protein synthesis and
mitochondrial damage
(
Yee and Choi, 1996
).

Anthony and
A
llison

&

Gomez
–
Vargas

found that the protein depletion of
TiO2 NPs treated groups may be due to the ability of TiO2 to
induce lysis of target cells by initiation of mitochondrial
reaction, or tissue reaction as liposomes stimulating complexes
(4 & 26)
.
Har
old

added, it may also result by tissue destruction
as activated vascular permeability and result in systemic
effects that include fever and the production of acute
–
phase
proteins
(
Harold, 2005
).

Also decreased protein content in
tissue may be due to the d
rastic effect on the rough
endoplasmic reticulum (RER), mitochondria and Golgi
apparatus and increased lysosomes in the cells
(
Eid and Al
–
Dossary, 2007
).

Highly decreased total glycogen was detected in the splenic
tissue with moderately stained capsule of

TiO2 NPs treated
groups. Decreased polysaccharides content in the degenerated
epithelial cells of bronchioles and haemolysed RBCs was
detected also by
Abu El Naga;
She stated that this decrease
may be due to decreased T3 and T4 hormones of the thyroid
gla
nds, which lessen entrance of glucose to the cells, this
investigation and the result of the present study were insured
the finding of
Viera
; who reported that the nanoparticles effect
may contributed to the cascade of reactions called "Gulf War
syndrome",

in which antithyroid effects were documented in its
symptoms
(
Abu El Naga, 1989; Viera, 2001
).

Szelényi

stated
that cytokines are cell
–
signaling proteins secreted to mediate
the

immune response of a body and to regulate inflammatory
processes. The family of cytokines includes pro
–
inflammatory
proteins, such as tumor necrosis factor (TNF)
–

α cytokines
allow organisms to respond to infectious agents and induce
inflammation
(
Szelényi
, 2001
);

where
Abou
–
Raya and
Kanterman

found that over
–
production of these agents can
occur as well, leading to chronic inflammation and/or
autoimmunity
(
Abou
–
Raya and Abou
–
Raya, 2006; Kanterman

et al
., 2012
).

While the immune system and its cells have
m
echanisms to inhibit the inflammation induced by these
cytokines
(
Kim
et al
., 2004
),

changes in levels of these proteins
can serve as an indirect index to assess immune function status
(
Liu
et al
., 2014
).

In the present study the values of TNF
–
α produced
by TiO2
NPs group showed significant increased difference. These
results are in agreement with those of
Carter and Swain
, who
stated that T lymphocytes were stimulated with TiO2 NPs
increased TNF
–
α cytokine production after 24h post stimulation
(
Carter and

Swain,
1997
).

Where
Roman
–
Ramos R,

mentioned
that administration of TiO2 NPs to mice increased their mRNA
expression of inflammatory genes including those for IL
–
6 and
TNF α, once again, oxidative stress might also be involved in
this process
(
Roman
–
Ramos
et al
., 2011
).

In support of this
notion, it was found that N
–
acetyl cysteine (anti
–
oxidant)
attenuated lipopolysaccharide induced apoptosis in splenic
lymphocytes (
Martin
et al
., 2000
).

The data presented here
indicated that in fact there was
a strong positive correlation
among the changes in morphometrical results 0f TNF
–
α and the
loss of splenic anti
–
oxidants; this lends credence to the
potential role that oxidative stress might be having in mediating
the immunotoxicity of TiO2 NPs.

As T
–
lymp
hocytes play an
important role in maintaining host immune status
(
Ayuob,
2013
),

the alterations in T
–
lymphocyte levels in response to
TiO2 NPs consumption here may reflect a wider TiO2 NPs
–
induced immunotoxicity. where TiO2 NPs induced a decrease
in CD3+
T
–
lymphocytes in the splenic PALS. These were
confirmed by morphometrical results which showed a
significant decrease (P<0.05) in optical density of CD
–
3
expression in TiO2 NPs
–
treated group (II) compared with
control group (I).
Cemerski S,

stated that ox
idative stress
induces structural modifications in T
–
lymphocytes, leading to
their becoming hypo
–
responsive consequently
(
Cemerski
et al
.,
2003
),

the oxidative stress induced by TiO2 NPs could be
responsible for the reduction in T
–
lymphocytes in the spleen
.
Support for this hypothesis has been bolstered by a recent study
wherein aluminum
–
induced oxidative stress reduced the
number and density of T
–
lymphocytes in the spleen of pregnant
rats
(
Ayuob, 2013
).

Plants have played a significant role in
maintaining
human health and improving the quality of human
life for thousands of years and have served humans well as
valuable components of medicines, seasonings, beverages,
cosmetics and dyes
(
Agaie, 2004
).

Newman

stated that herbal
medicine is based on the premise that plants contain natural
substances that can promote health and alleviate illness
(
Newman
et al
., 2000
).

One such plant, Moringa oleifera, (Family: Moringaceae) is a
multipurpose tree, used as vegeta
ble, spice, a source of
cooking and cosmetic oil and as a medicinal plant (
Fahey,
2005
).

Moringa oleifera is one of the leading names recently in
plants and drug research. Moringa Oleifera leaves contain
flavonoid pigments, such as kaempferol, rhamnetin,
i
soquercitrin and kaempferitrin. Flavonoid compounds have
various biological activities, including antiinflammatory and
anti
–
cancer ones (
Jahn, 1988
)
.

In addition, the Moringa
Oleifera leaves are rich in a group of the glycoside compounds,
glucosinolates an
d isothiocyanates (
Caceres
et al
., 1992
), as
well as in beta
–
sitosterol, glycerol
–
1
–
(9
–
octadecanoate), 3
–
O
–
(6'
–
O
–
oleoyl
–
beta
–
D
–
glucopyranosyl), beta
–
sitosterol

and

beta
–
sitosterol
–
3
–
O
–
beta
–
D
–
glucopyra

noside, all of which have
demonstrated anti
–
cancer properties in
–
vitro
(
Chaurasia’s,
2010
).

In this study we investigated the toxicological impacts
of TiO2 NPs on the spleen as well as the potential role of
Moringa Oleifera leaves extract (MOLE) in the cle
arance of
TiO2 NPs from treated spleen and protection of spleen against
toxic impacts caused by TiO2 NPs.

The mechanism action of
flavonoid pigments of Moringa Oleifere leaves are best known
as antioxidant free radical for scavenging and inhibition of
lipi
d peroxidation
(
Flora
et al
., 1998
).

MOLE has been
reported to support the immune cells through its antioxidant
activity and free
–
radical removing action
(
Basaga
et al
., 1997
).

In this study, the MOLE gradients; flavonoid pigments might
be able to react wi
th the free radicals produced from the
reaction of TiO2 NPs and the spleen proteins and enzymes.
Additionally MOLE could induce the immune systems in the
treated rats with TiO2 NPs. In our finding we reported that
TiO2 NPs decreased total protein and polys
accharides contents
as well as decreased CD3 secretion and increased TNF
–
α
secretion but MOLE administration as co
–
treatment (group 4)
or therapeutic treatment (group 5) slightly normalized it in a
way to initiate chemo
–
attractant mechanism. On the same li
ne
CD3 a cytokine produced primarily by monocytes and to a
lesser extent by lymphocytes, has pleiotropic effects in
immune
–
regulation and inflammation. It down
–
regulates the
expression of Th1 cytokines and acts as anti
–
inflammatory
cytokine

(
Heinrich
et al
., 2003
)
.
Pestka

reported that CD3 acts
as an essential immune
–
regulator. This studies may explain the
role of MOLE that ameliorate the toxic effect of TiO2 NPs by
inducing the immune system and increased the secretion of
CD3
(
Pestka
et al
., 2004
).

TNF
–
α
inhibits IL
–
1 and IL
–
6
production from macrophages
(
Fiorentino
et al
., 1991
).

TNF
–
α

plays a critical role in shaping the development of the immune
response by blocking class II major histocompatibility
complex expression and decreasing pro
–
inflammatory cyt
okine
expression
(
Newman
et al
., 2000
).

Where the primary role of TNF is in the regulation of

immune
cells
. TNF, being an endogenous pyrogen, is able to induce
fever,

apoptotic

cell death,

cachexia
, inflammation and

to inhibit

tumorigenesis

and

viral replication

and respond
to

sepsis

via

IL1

&

IL6

producing cells. Dysregulation of TNF
production has been implicated in a variety of
human

diseases

including

Alzheimer's

disease

(Swardfager

et al
., 2010
),

cancer

(
42
),
major depression

(
Dowlati
et al
.,
2010
),
psoriasis

(
Victor and Gottlieb, 2002
)

and

inflammatory
bowel d
isease

(IBD)

(
Brynskov
et al
., 2002
).

So, the increase in TNF
–
α secretion and expression as shown in
the therapeutic treatment by MOLE may referred to control
degree of toxicity induced by TiO2 and to counteract the
increase in expression and secretion of IL
–
1 and 6. But the
decrease in antibo
dy secretion as shown in the co
–
treatment by
MOLE may referred to the protective ability of MOLE that
dependent on ameliorating the TiO2 NPs toxicity and
protection spleen cells from this toxicity and this is coincided
with the decrease in IL
–
8 and IL
–
10,
because it has been
reported that T cells and B cells besides antibody production
they can secret various interleukins as IL
–
8 and IL
–
10
(
Smolen

And Maini, 2006
).

IL
–
10 has pleiotropic effects in
immunoregulation and inflammation. It enhances B cell
surviv
al, proliferation and antibody production
(
Pestka
et al
.,
2004
).

Xu

stated that in spleen, TiO2 nanoparticles
administration caused an increase in proliferation of local
macrophages
(
Xu
et al
., 2013
).

It has been shown that long
term exposure to low dose
of TiO2 NPs may cause spleen
injury, resulting from alteration of inflammatory and apoptotic
cytokines expression and reduction of immune capacity
(
Shi
et
al
., 2013
).

Fakurazi
, also showed that MOLE accelerated
recovery of hepatic cells after intoxicated w
ith TiO2 NPs. It
was evidenced from the histopathological observation; the
ability of MOLE to reverse the hepatic lesions was comparable
to the treatment with TiO2 NPs
(
Fakurazi
et al
., 2008
).

A study
of

Blakely & McDonald

revealed that animals pretreated with
MOLE were able to prevent further damage by TiO2 NPs
intoxication. The presence of focal infiltration of lymphocytes
were observed within 24h and after 48h the damage was
reduced to only focal hydropic degeneration
(
B
lakely and
McDonald, 1995
).

Where Hydroethanol extract was thought to
contain high phenolic content that may reduce the oxidative
stress in spleen tissue
(
Ferguson, 2001
).

Certain phenolic
compounds may also have induced phase II drug metabolism
enzymes th
at will enhance the excretion of oxidizing species
and inhibit cytochrome P450 activities.

Conclusion

In conclusion,

based on the above findings, we suggest that
ethanolic extract of leaves of Moringa oleifera could suppress
the cellular
and immune

resp
onse in rat induced by TiO2 NPs
through modulation of cytokines
expression and secretion
. It
may be rewarding to run detailed immunopharmacological
studies with this plant to explore its potential as an alternative
herbal medicine that could overcome the t
oxicity

problems of allopathic immunosuppressants. The plant
–
derived
immunomodulators have tremendous potential to be developed
as new pharmaceutical products.

Acknowledgement

The author would like to thank all participants for their
contribution in this study including animal house technicians
and histology technicians.

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