Different Expression of Caspase-3 in the Spleen and Liver of Rattus
norvegicus Infected with Enterobacter cloacae and Proteus mirabilis
as a Septic Model
Rina Purwati
1
, Yoes Prijatna Dachlan
2
and Agung Dwi Wahyu Widodo
3
1
Department of Immunology Postgraduate School, Universitas Airlangga, Surabaya, East Jawa, Indonesia
2
Department of Anatomical Pathology, Faculty of Medicine, Universitas Airlangga, Surabaya, East Jawa, Indonesia
3
Department of Clinical Microbiology, Faculty of Medicine, Universitas Airlangga, Surabaya, East Jawa, Indonesia
Keywords: Apoptosis, Caspase-3, Eanterobacter cloacae, Proteus mirabilis
Abstract: Enterobacter cloacae and Proteus mirabilis are opportunistic pathogenic bacteria that are the main causes of
high morbidity and mortality in humans from nosocomial infections in hospitals. Caspase-3 is a death protease
that acts as an apoptotic execution on an intrinsic pathway. Caspase-3 plays an important role in the process
of cell death that can divide various dead substrates often causing morphological and biochemical changes in
cells. The increase in caspase-3 expression indicates the severity of the disease, DNA fragmentation, and
condensation of apoptotic chromatin in the examined organs. This research type is true experimental using a
post-test in only the control group. The aims of this study are to determine the expression of caspase-3 in the
spleen and liver organs of experimental animals Rattus norvegicus, infected with E. cloacae and Proteus
mirabilis. E. cloacae and P. mirabilis bacteria were injected through the rats’ peritoneum and then observed
for 24 hours. The rats’ spleens and livers were observed for caspase-3 expression with immunohistochemistry.
Caspase-3 expression increases more in the liver than the spleen. The liver serves as a detoxification of
infection by spending and inducing cleansing of bacterial infections. Excessive inflammatory responses to the
liver can cause harmful effects on the host, with the cell cycle developing cellular stress, causing cell death
through caspase.
1 INTRODUCTION
Nosocomial infection is an infection that occurs in
patients who perform treatment in hospitals or other
health facilities. The most common cause (90%) of
nosocomial infection is from bacterial infection of
Proteus mirabilis, Enterobacter cloacae, Escherichia
coli, Staphylococcus aureus and other causes
protozoa, fungi, and viruses account for only 10%
(Khan et al., 2015). Nosocomial infections in
developing countries are a major cause of high
morbidity and mortality in hospitals (Mohammed et
al., 2014). Data from the WHO indicates that the
largest infection site is sepsis (WHO 2018). In the
case of gram-negative bacterial infections, the
incidence of high sepsis can trigger multiple organ
failure and death.
E. cloacae and P. mirabilis are pathogenic gram-
negative bacteria that tend to increase morbidity and
increase mortality in treatment and hospitalization
cases. Both these gram-negative bacteria infect
various organs and may undergo tissue death
(apoptosis). The second cell wall of the bacteria can
produce endotoxins, which can lead to cellular
response and impact on endothel damage that can
trigger multiple organ damage. Sepsis may also
induce apoptosis in innate and adaptive immune cells.
Several studies have shown that apoptosis
occurs in many organs during sepsis. In animal
models with sepsis, apoptosis is found in the thymus
organ, patch peyer, liver, kidney, lung, intestine, and
skeletal muscle. In patients with sepsis, apoptosis
occurs in the spleen, colon and ileum (Aziz et al.,
2014). The spleen plays an important role in the
modulation of the immune system and in the
maintenance of peripheral tolerance through the
clearance of apoptotic cells, differentiation and
activation of T and B cells (Vincenzo and Mikael,
2013).
Lipopolysaccharides (LPS) of intestinal gram-
negative bacteria was taken periodically to the liver
Purwati, R., Prijatna Dachlan, Y. and Dwi Wahyu Widodo, A.
Different Expression of Caspase-3 in the Spleen and Liver of Rattus norvegicus Infected with Enterobacter cloacae and Proteus mirabilis as a Septic Model.
DOI: 10.5220/0007541202690273
In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 269-273
ISBN: 978-989-758-348-3
Copyright
c
2018 by SCITEPRESS Science and Technology Publications, Lda. All rights reserved
269
through the portal vein then stored by Kupffer cells
(KCs) and macrophages that are stunned at the liver.
The function of the liver is detoxification, the first
barrier against pathogen infection. Kupffer cells and
macrophages are the product of the liver, and can take
bacterial endotoxin and phagocytosis brought
through the portal vein; they play a major role in the
clearance of systemic bacterial infections (Mencin et
al., 2009).
Executor caspases are an important part of the cell
death process during apoptosis. Executor caspases are
necessary for apoptosis, and also in the absence of
cell death (Khalil et al., 2012). Caspase-3 plays an
important role in the process of cell death because of
its function that can cleave dead substrates, ultimately
leading to morphological and biochemical changes in
apoptotic cells (Walsh et al., 2008). Apoptotic
regulation is an important aspect in host cell response
to stress and bacterial infections of E.cloacae and
P.mirabilis. The study on the expression of caspase-3
in the spleen and liver organs that occur in sepsis due
to bacterial infection of E. cloacae and P.mirabilis
needs to be conducted to determine the cleavage of
various dead substrates, which can cause changes to
morphology and biochemistry seen in apoptotic cells
and can be prevented before multiple organ
dysfunction syndrome (MODS) occurs.
2 MATERIALS AND METHOD
2.1 Animal model
The experimental animals used in this study were
Rattus novergicus, the wistar strain, with criteria of
male, body weight 150–200 grama, healthy, and aged
three months.
2.2 Bacteria
Enterobacter cloacae and Proteus mirabilis from the
Installation of Clinical Microbiology RSUD Dr.
Soetomo, Surabaya. The concentration wild type
was105 CFU bacteria with Physiologic Zur (Nacl
0.9%).
2.3 Method
The healthy rats were chosen randomly and separated
into three groups. Each group was injected
intraperitonially with 1ml of PZ, suspension E.
cloacae, P. mirabilis, respectively. The rats were
observed for 24 hours. Surgery was performed for the
removal of the spleen and liver organ. The spleen
organ tissue and liver were fixed onto the formalin
buffer and the tissue was prepared for paraffin
embedded and immunohistochemistry using a rabbit
and mouse caspase-3 reagent.
3 RESULT
3.1 Spleen Organ
The results expression of caspase-3 lymphocyte cells
in the spleen of the treatment group with the E.
cloacae infection were higher than the treatment
group with the P. mirabilis infection (Figure 1).
Figure 1: Box-plot caspase-3 expression of lymphocyte
cells in the spleen
The results of the highest median values infected
with E. cloacae (37.5%) infected with P. mirabilis
(28.5%) and control group (4%). The caspase-3
expression of lymphocytes cell was observed with a
x100 objective lens magnification light microscope at
five viewing fields (Figure 1).
Figure 2: Caspase-3 expression of blue lymphocyte cells in
the spleen (Rattus norvegicus) x1000 magnification of the
control group.
ICPS 2018 - 2nd International Conference Postgraduate School
270
Figure 3: Caspase-3 expressions in the spleen of Ratus
norvegicus infected with E. cloacae are shown by brown
stained lymphocyte cells (x1000 magnification
Figure 4: Caspase-3 expressions in the spleen of Ratus
norvegicus infected with P. mirabilis are shown by brown
stained lymphocyte cells (x1000 magnification).
3.2 Liver Organs
The result showed that the expression of caspase--3
hepatocyte cells in liver organ, treatment group
infected by E. Cloacae was higher than the treatment
group P. mirabilis (Figure 5).
Figure 5: Box-plot of caspase-3 expression of lymphocyte
cells in the liver.
The results of the highest median values with
E.cloacae (81%) were infected with P. mirabilis
(66.5%) and the control group (9.5%). The caspase
expression of three hepatocyte cells was observed in
a five field of view with x100 magnification (Figure
5).
Figure 6: Caspase 3 expressions of hepatocyte cell in liver
Rattus norvegicus 1000x magnification control group.
Figure 7: Caspase-3 expressions in the liver of Rattus
norvegicus infected with E. cloacae are shown by brown
stained hepatocyte cells (x1000 magnification).
Figure 8: Caspase-3 expressions in the liver of Rattus
norvegicus infected with P.mirabilis are shown by brown
stained hepatocyte cells (x1000 magnification).
Different Expression of Caspase-3 in the Spleen and Liver of Rattus norvegicus Infected with Enterobacter cloacae and Proteus mirabilis as
a Septic Model
271
4 DISCUSSION
Based on the result, two rats in E.cloacae group died,
while the Proteus mirabilis infection group and the
control group survived. Those that were alive could
maintain homeostasis in the inflammatory process
due to gram-negative bacterial infection.
The endotoxins content in the E. cloacae bacteria
consisted of glycolipids, LPS macromolecules that
make up about 75% of LPS structures comprise the
domain of hydrophobic lipids A, oligosaccharide
nuclei, and O antigen polysaccharides, which are the
outermost and outer membranes of gram-negative
bacteria involved in sepsis and pathogenesis causing
lethal shock (Nephrol Contrib, 2010). The virulent
factor of pathogenic E.colacae bacteria such as
lipopolysaccharides, type III secretion,
exopolysaccharide and enterotoxin, hemolisyn, thiol
activated toxin, and shiga toxin type II were capable
to colonize in the hosts body, which can result in
organ failure (Krzyminska et al., 2009).
P. mirabilis can differentiate into swarming
motility that contribute to the formation of infection
(Morgenstein et al., 2010). P. mirabilis produces
urease enzymes to improve nutrients in urine,
fimbriae or pili serve as adhesin and avoid host
immune response (Norsworthy et al., 2017). The
spleen is an important role in the modulation of the
immune system and in the maintenance of peripheral
tolerance through clearance of apoptotic cells,
differentiation and activation of T and B cells
(Vincenzo and Mikael., 2013).The spleen acts as a
filter against the antigenic response carried through
the blood by the lymph glands. The antigen carried by
the blood is captured and is concentrated by dendritic
cells as well as macrophages in the spleen. The spleen
contains many phagocyte cells that play a role in
eating and destroying microbes in the blood (Abbas
et al., 2015).
Apoptosis in hepatocytes may be the most
important event in the molecular mechanism of liver
failure; apoptosis is the first liver cell response to
bacterial toxins, including LPS, the caspase pathway
that plays a role in apoptosis including the initiator
and caspase execution. Two main caspase initiators,
caspase-8 and caspase-9, signal death. Caspase-8 is
activated by a signaling of death that binds to a death
receptor on the cell surface. Caspase-9 is activated by
the cytochrome released by the mitochondria. The
caspase initiator's proapoptotic pathway activates the
caspases of the executor, caspase-3. Caspase-3
activation is characterized by protein substrate
division of DNA molecule breakdown and apoptosis.
In gram-negative bacterial infections endotoxin
are 80% injected intravenously in animals to detect in
the liver within 20–30 minutes. LPS begins to enter
from the intestine, periodically taken to the liver
through the portal vein and is then stored by the
Kupffer cells and the macrophages are stunned in the
liver. The first function of the liver is detoxification.
In gram-negative bacterial infections endotoxin
is 80% injected intravenously in animals to detect in
the liver within 20–30 minutes. LPS begins to enter
from the intestine periodically taken to the liver
through the portal vein and then stored by the Kupffer
cells and the macrophages are stunned in the liver.
The function of the liver is detoxification, the first
obstacle to pathogen infection (Mencin et al., 2009)
The process of apoptosis plays an important role
for the development of lymphocytes; the immune
response to homeostasis is due to infection and
maintains tolerance to self-antigen and cell death
infected by pathogenic bacteria (Hardiono., 2016).
Activation of caspase-3 during bacterial infection
involves bacterial invasion, which triggers stress on
host cells associated with cell intracellular
replication. Gram-negative bacterial toxins that have
a large molecular weight can cause cell cycle changes
to cellular stress with cell death through apoptotic
caspase (Wall and McCormick, 2014).
5 CONCLUSION
There was an increased difference in caspase-3
expression in the liver and spleen of the R. norvegicus
group infected with E.cloacae, which was higher than
that infected with P.mirabilis. Increased caspase-3
expression in bacterial infections may increase cell
stress in the host and increase organ mortality.
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