Differences of Caspase-3 Expression in Liver and Spleen of Rattus

norvegicus Infected with Streptococcus pyogenes and Acinetobacter

baumannii

Nurul Amalia

, Agung Dwi Wahyu Widodo

, and Willy Sandhika

Departement of Immunology Postgraduate School Airlangga University Surabaya, East Java, Indonesia

Departement of Microbiology Clinic, Dr. Soetomo Hospital Surabaya, East Java Indonesia

Departement of Anatomy Pathology, Faculty of Medicine, Universitas Airlangga, Surabaya, Jawa Timur, Indonesia

Keywords: Apoptosis, Caspase-3, Streptococcus pyogenes, Acinetobacter baumannii.

Abstract: Streptococcus pyogenes as exotoxin-producing bacteria are the most common cause of pharyngitis infection.

Acinetobacter baumannii as endotoxin-producing bacteria are found in nosocomial infections. The

infections that has already spread to all organs with decreased immune system undergo apoptosis in liver

and spleen. Caspase-3 is a death protease most commonly activated by apoptotic mediator. The higher the

caspase-3 expression, the higher the severity of the disease, which can cause the organ to experience

dysfunction or failure. This study was aimed to observe the expression of caspase-3 in the liver and spleen

of Rattus norvegicus, infected by Streptococcus pyogenes and Acinetobacter baumannii. This is a true

experimental with a post-test only control-group design. The healthy rats were randomly selected and

injected with 1ml (PZ, suspension of bacteri A. baumannii and S. pyogenes) in the peritoneum in quadrant 3

for each group of experimental animals and observed for 24 hours. After 24 hours, surgery took place for

the removal of the liver and spleen. Organ tissues where fixed into a formalin buffer and tissue was prepared

for IHC caspase-3.The results showed that the mortality rate of Rattus norvegicus infected by A. baumannii

was higher than those infected by S. pyogenes. Caspase-3 expression in the liver of A. baumannii group was

48, S. pyogenes was 22.5, and the control group was 9.5. The mean value of the caspase-3 index in the

spleen of the A. baumannii group was 28.5, S. pyogenes was 17, and the control group was 4.

1 INTRODUCTION

The infection is caused by bacteria that produces

toxins and endotoxins. In general, infectious

diseases are caused by bacteria, fungi, viruses, and

parasites. Streptococcus pyogenes as toxin-

producing bacteria is the most common cause of

infectious pharyngitis. S. pyogenes bacteria is

included in Group A streptococci of serology (Group

A Streptococcus, GAS) (Fidler et al., 2015). S.

pyogenes bacteria can infect the host body's defenses

when dropped or when the organism can penetrate

past the host's defense (Ramachandran, 2017).

The bacteria Acinetobacter baumannii is an

endotoxin- producing bacteria found in nosocomial

infections and harmful gram-negative bacteria

(Bigot and Suzana, 2017). In recent years the

bacteria A. baumannii has increased over the

nosocomial infections in humans. The bacteria A.

baumannii is found as a nosocomial infection-

causing bacteria in the urinary tract, an infection of a

wound, and in vascular surgery, particularly in

patients with low immune systems located in the

ICU. Research in Indonesia has determined the

bacteria Acinetobacter as one gram-negative that

most often infected are 25.8% (Norhamdani, 2004).

Apoptosis has an important role in bacterial

infections of S. pyogens and A. baumannii.

Apoptosis affects the immune cells,which are very

important in the course of an infection. Apoptosis

can be determined not only on a certain type of

bacteria, but in bacterial infections in a variety of

species. The regulation of apoptosis is an important

aspect of the host cell response against stress,

infection, and must be controlled (Ulett and

Elisabeth, 2006).

A variety of stimuli can trigger apoptosis from

within or outside the cell, for example, infection

with microorganisms, cell cycle, or signaling the

258

Amalia, N., Dwi Wahyu Widodo, A. and Sandhika, W.

Differences of Caspase-3 Expression in Liver and Spleen of Rattus norvegicus Infected with Streptococcus pyogenes and Acinetobacter baumannii.

DOI: 10.5220/0007541002580262

In Proceedings of the 2nd International Conference Postgraduate School (ICPS 2018), pages 258-262

ISBN: 978-989-758-348-3

 2018 by SCITEPRESS – Science and Technology Publications, Lda. All r ights reserved

death of cell-surface receptors, development, DNA

damage and occurrence of inflammation.

Inflammatory Cytokines

(TNF) can constantly

induce activation of caspase-8,

caspase-3, and the

fragmentation of DNA through membrane

receptors. This is apoptosis pathway activation

directly from the extrinsic pathway, called caspase.

Metabolic disorders of intracellular reactive oxygen

species

or overload can cause damage to t h e

mitochondria, which produce cytochrome c release

and the activation of caspase-9. Caspase-9

reactivates further trigger activation of caspase-3

and apoptosis. High apoptosis hemeostasis systems

cannot keep the organ systems, causing multiple

organ dysfunction syndrome (MODS). Organ system

failure is

very harmful to humans and can even

cause death (Caspian, 2016). Expressions of

Caspase-3 were examined in the liver and spleen of

the rat (Rattus norvegicus) where the apoptosis

occurred.

2 METHODS

The research was done in a purely experimental

laboratory using a true experimental research

design. Post-tests only Control Group Design (data

retrieval is performed after the given treatment) and

compared with the control group.

The research was performed in, the Animal

Models Department of Biochemistry, Microbiology

Clinic Laboratory, Dr. Sutomo Hospital as a place of

clinical isolation for the culture of S. pyogenes and

A. baumannii. The Anatomic Pathology Laboratory

Faculty of Medicine Airlangga University processed

checks and the observation of the expression of

caspase-3 in organ livers and lien on the Rattus

Norvegicus. The research was carried out over three

months. (October 2017–December 2017).

The object of the research is the white R.

norvegicus male wistar strain, aged 3 months with a

weight of 200–250 grams. Four R. norvegicus were

used in this study

Pure liquid bacteria was isolated from S.

pyogenes at

A. baumannii laboratory clinical microbiology of

the Dr. Sutomo Hospital. Expressions of caspase-3 in

rats’ livers and spleen who had infection with

bacteria S.pyogenes and A.baumannii are detected

by IHC method (Immunohistochemistry) using the

caspase-3 p12 subunits of antibodies.

Treatment for animal models:

R. norvegicus were given injections in the

peritoniumnya with bacteria S. pyogenes and A.

baumanni. Each anesthetic had 2.52mg and 0.25mg

of ketamine dexamethasone, followed by an

injection of bacteria S. pyogenes and A. baumannii

of which the doses were 1x109 per CFU/Rat. After

24 hours, if the rat was not dead, it was dissected

and the liver and spleen were taken.

Tissue Preparation:

The liver and spleen were removed from the

treated rats and fixed with 10% formalin. Further

cutting of the organs at 1x1x2cm continued using

paraffin blocks that are used in the pathology

laboratory.

Observation of Caspase-3 expression

Observation on the expression of caspase-3 in

rats liver and spleen was conducted with primary

antibody caspase-3 p12 subunits. Expressions of

caspase-3 were observed using the IHC method by

using caspase-3 p12 subunit primary antibodies. The

cells expressing caspase-3 were counted within five

fields of view x1000 magnification and the numbers

were compared between two bacteria-treated rats.

Cell expression of caspase-3 were calculated on an

examination under microscope light with

magnification 100x objective with five field of view

and magnification of 10x to 40x and photographed

for comparison of each liver and spleen.

3 RESULTS

3.1 The results of the expression of

Caspase-3 in the liver

The number of cells expressing results obtained

demonstrated that the average number of

expressions of caspase-3 in the group injected with

A. baumannii is higher than the group injected with

S. pyogenes and the control group.

Table 1: The Distribution of liver cells expressing caspase-

3 Note SD is Standard Deviation.

Percentage

of cell

C(n=4) S. pyogenes

.baumanni

X± SD 9.5 ± 2.38 20.75 ± 7.136 48 ± 21.74

Median 9.5 22.5 48

Min - Max 7

–

12 11

–

27 26

–

The number of cells expressing caspase-3 in the

livers of rats infected with A. baumannii was higher

than those infected with S. pyogenes

Differences of Caspase-3 Expression in Liver and Spleen of Rattus norvegicus Infected with Streptococcus pyogenes and Acinetobacter

baumannii

259

Figure 1: Box plot of the number of cells express caspase-

3 in the liver infected with A. baumannii and S. pyogenes.

Figure 2: No cell expression caspase-3 in the liver control

group, Figure 3: Expression of caspase-3 in rats’ liver

Hepatocyte Cell Group A.baumannii x1000 zoom. Figure

4: Expression of caspase-3 in rats liver Hepatocyte Cell

Group S.pyogenes x1000 zoom.

3.2 The results of the expression of

caspase-3 in the spleen

From the results obtained, the average number of

expressions of caspase-3 in the group infected with

A. baumannii is higher than the group infected with

S. pyogenes and the control group.

Table 2 : Note SD is Standard Deviation

Percentage

of cell

K(n=4) S. pyogenes

.baumanni

X± SD 4.25 ± 0.5 17.75 ± 7.136 27.25 ± 7.58

Median 4 17 28.5

Min - Max 4

–

5 15

–

22 17

–

Based on the data in the table above, it can be noted

that the expression of caspase-3 in rats in the group

infected with A.baumannii is the highest, i.e. SD

27.25 ± 7.58, while the expression of caspase-3 in

the group of rats’ spleens infected with S. pyogenes

is 17.75 ± 3,403.

Figure 5: Box-plot the number of expression of Caspase-3 in

rats’ spleens.

The highest median value, i.e. the A.baumannii

group was 28.5, the second was the S.pyogenes

group at 17, and the control group was 4.

Description of caspase-3 expression was observed

under the light microscope with a magnification of

x100 and x1000 and the x% fields of view are as

follows:

Figure 6: Expression of caspase-3 in the spleen control

group, Figure 7: Expression of caspase-3 on rat

lymphocyte cells spleen Group A.baumannii x1000 zoom,

and Figure 8: Expression of caspase-3 in the spleen of rats

infected with S.pyogenes magnification x1000.

3.3 Expression of Caspase-3 in the liver

and spleens of rats infected with

A.baumannii and S.pyogenes

Based on the averages of cells, those that expressed

caspase-3 in the livers and spleens of rats infected

with A.baumanii and S.pyogenes showed that the

group infected with A.baumannii was higher (48)

than that of rats infected with S.pyogenes (22.5) and

the control group (9.5). The average number of cells

expressing caspase-3 in the spleens of rats infected

with A.baumanii was higher (28.5), compared with

that of rat infected with S.pyogenes (17) and the

control group, which was only 4 as shown in Figure

ICPS 2018 - 2nd International Conference Postgraduate School

260

Figure 9: Box-plot Average number expression of Caspase-

3 in rats’ spleens and livers.

4 DISCUSSION

Apoptosis as a type of cell death is highly organized

and genetically controlled. It is characterized by a

number of different morphological changes, such as

cell condensation and marginalization, the shrinking

of cells, and plasma membrane blebbing. This is

accompanied by biochemical features, such as DNA

fragmentation, changes in membrane (e.g. exposure

to fosfatidilerin on the outside of the plasma

membrane), and specific cell protein degradation, as

a result of the activation of a massive amount of

intracellular protease and endonuclease (Guicciardi

and Scratch, 2005). The cleavage of caspase was

mediated by caspase-3 and caspase-7, while the last

two caspase activations are generally a function of

initiator caspase. Initiator caspase’s apoptosis signal

pathway determines, after activation of caspase

executor, that caspase-3 and caspase-7 can process

at least 100 proteins. The cleavage of a caspase-3

substrate can lead to profit or loss of the function of

proteins, ultimately causing cellular changes

associated with apoptosis (Rogers et al., 2015).

1. The death of rats due to A.baumannii and S.

pyogenes.

A. baumannii produces endotoxin as OmpA capsule

(outer membrane protein A) induces apoptosis in

human laryngeal epithelial cells. OmpA is purified

and localized and the mitochondria and apoptosis

are induced through the release of the proapoptotic

cytochrome c molecules and the driving factors of

apoptosis, suggesting that this is the path where the

A.baumannii induces damage to the cells of the

respiratory tract during infection (Peng et al., 2016).

Gram-negative can cause the onset of sepsis and

sepsis shock (Girardt et al., 2016).

Streptolisin O is the basic nature of the toxin beta-

hemolysis with toxins from Gram-positive bacteria

S.pyogenes. Streptolisin O is potentially cell poison

affecting many cell types including neutrophil,

platelets, and organella subsel. This toxin is capable

of producing a large cellular immune response that

can lead to fatal toxic shock (Regnier et al., 2016).

S. pyogenes is a species of gram-positive bacteria

that contain peptidoglycan cell walls and

lipoteichoic acid (LTA) discovered by the immune

system as the PAMPs, is in line for the bacteria

S.pyogenes as a TLR-Peptidoglycan and LTA

interaction with TLR-2 produces a signalling

pathway via the adapter MyD88 and TRIF

activation that can trigger the formation of NF-kB

and cytokines expression of MAPKs so it can be

(Pyrshev et al., 2017).

Expression of caspase-3 in livers of rats

infected with A. baumannii and S. pyogenes

The increased of caspase-3 expression in the livers

of rats infected with A.baumanni and S. pyogenes

indicate the death of cells due to apoptosis but no

damage to the organ. Hepatocyte death is common

in the aftermath of inflammatory disease in the liver.

An increase of apoptosis in the liver can be caused

by a high inflammatory process, which triggers the

apoptotic hepatocyte cell to cause most of the

damage to the hepatocytes, mediated by the reactive

oxygen species (ROS) that initiates inflammatory

reactions and the occurrence of apoptosis in

hepatocytes (Rinaldi, 2014). In hepatocyte damage,

the liver will induce the onset of signals to stimulate

the release of monocyte chemoattractant Chemokin

protein-1 (MCP-1), which will enhance kupffer

cells/macrophages, as well as the release of pro-

inflammatory cytokines, such as interleukin (IL )-1

β, IL-1 and Tumor Necrosis Factor (TNF)-α, which

can enable the Nuclear Factor κB activation (NF-κB

activation) and mitogen-activated protein kinase

(MAPK) (Guicciardi et al., 2013).

Caspase-3 expressions in the spleens of rats

infected with A. baumannii and S. pyogenes.

Potential complications from splenic swelling are

bacterial infections; this is because the spleen

swells, which reduces the number of healthy red

blood cells, platelets, and white blood cells in the

bloodstream, exposing it to the infection (Bronte

and Mikael, 2013).

High bacterial infections activate the immune

system to attack the bacteria present in the blood

(Tan et al., 2017). The spleen will induce

proinflamasi cytokines, such as interleukin (IL)-1 β,

Differences of Caspase-3 Expression in Liver and Spleen of Rattus norvegicus Infected with Streptococcus pyogenes and Acinetobacter

baumannii

261

IL6, the tumor necrosis factor (TNF)-α, γ interferon

(IFN) and the synthesis of nitric oxide (NO).

Patients infected with bacteria undergo

overproduction of proinflammatory cytokines, such

as, TNF-α, IFN-γ, IL-2, ROS and NO. Excessive

TNF-α will increase the production of NO acting as

free radicals. In addition, TNF-α can also increase

the Intercellular Adhesion Molecule-1 (ICAM-1)

and cause obstruction within the brain (Bronte and

Mikael, 2017).

5 CONCLUSION

1. Based on the average of cells expressing

caspase-3 of rats infected with S.pyogenes in

hepar was higher (20.75 ± 7.136) than that in

the spleen (17.75 ± 3.403).

2. Based on the average of cells expressing

caspase-3 of rats infected with A.baumannii in

hepar was higher (48± 21.74) than that in the

spleen (27.25 ±7.58).

3. The highest number of cells expressing

caspase-3 was observed in the group of rats

infected with A. baumannii, compared with

that of the group of rats infected with S.

pyogenes. The liver demonstrated a higher

expression of caspase-3 than the spleen.

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