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

, Yoes Prijatna Dachlan

and Agung Dwi Wahyu Widodo

Department of Immunology Postgraduate School, Universitas Airlangga, Surabaya, East Jawa, Indonesia

Department of Anatomical Pathology, Faculty of Medicine, Universitas Airlangga, Surabaya, East Jawa, Indonesia

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

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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