The Effect of Quercetin towards Adipocytes Count in Toxoplasma gondii Profilin - exposed Adipocytes In Vitro

Abstract

Toxoplasma gondii is one of the protozoan causes of chronic infection that allegedly causes obese (infectobesity). Some previous studies have showed that profilin Toxoplasma gondii has a role in inflammation by promoting interleukin-12 (IL - 12) which induce adipocyte dysfunction through the hyperplasia and hyperproliferation of adipocyte cells. Those processes lead to metabolic syndrome which increase adipocytes count through reducing insulin receptor’s sensitivity. On the other hand, Toxoplasma gondii , as an obligate intracellular parasite, can also damage the pancreatic beta cells. In response to inflammation, adipocytes produce Reactive Oxygen Species (ROS). To scavenge ROS antioxidants are required. Quercetin, an exogenous antioxidant, can be widely found in natural products that might be a promising candidate for development of antioxidant treatment interventions to prevent adipocytopathy. This research aims to explore the effects of quercetin towards Adipocytes Count stimulated from T. gondii profilin-exposed adipocytes. This research using visceral adipocyte rat that was cultured in Dulbecco’s Modified Eagle Medium (DMEM). After 70% confluency, adipocytes were exposed to 20 μΜ T. gondii profilin and treated with four doses of quercetin; 31.25, 62.5, 125, and 250 μΜ that incubated 48 hours. After incubation period, adipocytes were observed using inverted microscope and were captured in high power field magnification using camera. Adipocytes were counted from each captured photo and all groups were analyzed using Analysis of variance (ANOVA) test. The results showed that quercetin significantly reduced adipocyte cell count T. gondii profilin-exposed adipocytes compared to untreated cells (ANOVA p = 0,00). The effective dose to lower adipocyte cell count was 31,25 μΜ. This study implies that quercetin has a potent antioxidant that can prevent toxoplasmosis-mediated adipocytopathy.

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The Effect of Quercetin towards Adipocytes Count in Toxoplasma gondii
Profilin - exposed Adipocytes In Vitro
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The 2nd International Conference on Computer Science and Engineering Technology
Journal of Physics: Conference Series 1430 (2020) 012028
IOP Publishing
doi:10.1088/1742-6596/1430/1/012028
1
The Effect of Quercetin towards Adipocytes Count in
Toxoplasma gondii Profilin - exposed Adipocytes In Vitro
Y D Setia
1*
, S Poeranto
1
, T E Hernowati
2
, A Iskandar
1
, Sudjari
1
1
Parasitology Department, Faculty of Medicine, Universitas Brawijaya, Veteran Street number
1 Malang, Indonesia
2
Clinical Pathology Department, Faculty of Medicine, Universitas Brawijaya, Veteran Street
number 1 Malang, Indonesia
*Corresponding author : dr.yulia.fk@ub.ac.id
Abstract. Toxoplasma gondii is one of the protozoan causes of chronic infection that allegedly
causes obese (infectobesity). Some previous studies have showed that profilin Toxoplasma
gondii has a role in inflammation by promoting interleukin-12 (IL – 12) which induce adipocyte
dysfunction through the hyperplasia and hyperproliferation of adipocyte cells. Those processes
lead to metabolic syndrome which increase adipocytes count through reducing insulin receptor’s
sensitivity. On the other hand, Toxoplasma gondii, as an obligate intracellullar parasite, can also
damage the pancreatic beta cells. In response to inflammation, adipocytes produce Reactive
Oxygen Species (ROS). To scavenge ROS antioxidants are required. Quercetin, an exogenous
antioxidant, can be widely found in natural products that might be a promising candidate for
development of antioxidant treatment interventions to prevent adipocytopathy. This research
aims to explore the effects of quercetin towards Adipocytes Count stimulated from T.
gondii profilin-exposed adipocytes. This research using visceral adipocyte rat that was cultured
in Dulbecco's Modified Eagle Medium (DMEM). After 70% confluency, adipocytes were
exposed to 20 µM T. gondii profilin and treated with four doses of quercetin; 31.25, 62.5, 125,
and 250 µM that incubated 48 hours. After incubation period, adipocytes were observed using
inverted microscope and were captured in high power field magnification using camera.
Adipocytes were counted from each captured photo and all groups were analyzed using Analysis
of variance (ANOVA) test. The results showed that quercetin significantly reduced adipocyte
cell count T. gondii profilin-exposed adipocytes compared to untreated cells (ANOVA p = 0,00).
The effective dose to lower adipocyte cell count was 31,25 µM. This study implies that quercetin
has a potent antioxidant that can prevent toxoplasmosis-mediated adipocytopathy.
Key word : quercetin, adipocyte count, Toxoplasma gondii profilin
1. INTRODUCTION
Due to its high prevalence, obesity is a major global health issue, including in developing countries and
children (Ng M, et al. 2014). The increasing number of obese patients demands more research about
pathogenesis of obesity to identify more effective novel intervention targets. Life style interventions and
body weight reduction alone have shown unsatisfying results, especially because of the problems of
discontinuity (Reever GM, et al. 2013). Several studies described infection through inflammatory
pathways as a potential cause of obesity, further defined as infectobesity (Vasilakopoulou A and Roux
C W 2007; Hedge V and Dhurandar NV 2013). The revelation of infectious agents playing roles in
obesity leads to the idea of specific treatment modalities for obese people due to infection (Hedge V and
Dhurandar NV 2013).

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doi:10.1088/1742-6596/1430/1/012028
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One of the potential pathogens involved in obesity pathogenesis is Toxoplasma gondii (T. gondii). This
apicomplexan protozoa infected 30% of population around the globe (Reever GM, et al. 2013). As an
intracellular parasite, T. gondii can infect all nucleated cells, including adipocytes (Toulah, Fawzia H,
et al. 2011). As other members of the apicomplexan phylum, the actin cytoskeleton change during
gliding movement is essential for T. gondii during host cell invasion. Profilin is an important component
for actin polymerisation during actin-dependent gliding movement of T. Gondii (Plattner, Fabienne et
al. 2008). Profilin-like protein of T. gondii is also an immunogenic element that stimulates inflammatory
pathway through its recognition by the endosomal pattern recognition receptor (PRR), Toll-like receptor
11 (TLR 11) (Andrade WA. et al. 2013; Susanto, et al. 2014; Yarovinsky F 2014; Iskandar A et al.
2016).
As a response to inflammation, cells will generate of reactive oxygen species (ROS). ROS production
leads to random and unregulated intracellular oxidation, which in turn triggers oxidation of iron,
intracellular lipids, proteins, and DNA, resulting in vast intracellular molecular damage. Many diseases
potentially arise as the consequence, such as neurodegenerative diseases, atherosclerosis, aging process,
and metabolic syndrome (Holmström, Kira M and Toren F. 2014).
To scavenge ROS and intracellular damage, antioxidants are needed. Body cells are equipped with
endogenous enzimatic antioxidant. Glutathione (GSH), a cystein protein contain tripeptide, has an
important role in cellular redox (Holmström, Kira M and Toren F. 2014).
Synthetic antioxidants are commercially available, but the safety and toxicity risks of synthetic
antioxidants are higher than natural antioxidant (Ebrahimzadeh, et. al. 2008). Flavonoid is one of the
most well recognized exogenous natural antioxidants, which is produced outside the body. Quercetin, a
type of flavonoid, can be widely found in natural products, such as onion, cherry, tomato, broccoli,
apple, green tea, black tea, grape, or blueberry (Fazel S et al. 2015) ; Ratnawati R and Hernowati TE.
2015). Previous studies showed that quercetin inhibits proliferation and differentiation of pre-adipocyte
culture by decreasing the expression of adipocytokines, such as CCAAT/enhancer binding protein alpha
(C/EBPα) and sterol regulatory element binding protein 1c (SBREP-1c) (Ratnawati R and Hernowati
TE. 2015).
This study aims to explore the potency of quercetin to scavenge ROS free radicals and to stimulate GSH
endogenous antioxidant by exposure of T. gondii profilin-exposed adipocytes. T. gondii profilin can be
recognised by TLR 11 and stimulate inflammatory pathway that has causal relationship with
adipocytopathy. The results of this study could be implied in development of potential antioxidant
treatment interventions to prevent toxoplasmosis-mediated adipocytopathy.
2. MATERIAL AND METHOD
2.1. Experimental design
This research used true experimental study using adipocyte culture that exposed to profilin T.
gondii and treated by quercetin. Samples were divided into 6 groups. Each group contains four
replication samples each, namely : Negative control (maturated only); Positive control (maturated and
20 µM T. gondii profilin exposed); Q 31.25 (maturated, 20 µM T. gondii profilin and Quercetin 31.25
µM exposed); Q 62.5 (maturated, 20 µM T. gondii profilin and Quercetin 62.5 µM exposed); Q 125
(maturated, 20 µM T. gondii profilin and Quercetin 125 µM exposed), and Q 250 (maturated, 20 µM T.
gondii profilin and Quercetin 250 µM exposed).
Adipocyte culture
Adipocyte culture was developed from adipose tissue of 1 month old wistar rats. Adipose tissue was
collected from peritoneal and retroperitoneal regions. The tissue was munched mechanically by scalpel
or scissors and digested enzimatically by type 1 collagenase (Worthington).
The obtained cells were maintained in a culture flask and nourished using Dulbecco’s Modified Eagle
Medium (DMEM) (Gibco©) containing sodium bicarbonate, L-glutamine, antibiotics (100U/ml

The 2nd International Conference on Computer Science and Engineering Technology
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doi:10.1088/1742-6596/1430/1/012028
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Pennicillin and 100 mg/ml Streptomycin (MP Biomedicals, LCC)), and supplemented with 10% of heat-
inactivated fetal bovine serum (Gibco©).
Adipocyte culture were kept at 37° C, 5% CO
2
environment. Culture media were changed every 48 hours
until confluency was achieved (Ratnawati R and Hernowati TE. 2015; Zhu S et al 2010). After the cells
in the culture flask were confluent, the researchers then subcultured the cells into 12-well culture plates.
The cells in the culture plates achieve the same treatment as in the culture flask.
2.2. Profilin and quercetin exposure
All of the confluent groups of pre-adipocyte culture were maturated by 0,1 µM dexamethasone, 0,5 mM
isobutylmethylxanthine, and 0,1 µM insulin (Ratnawati R and Hernowati TE. 2015). Simultaneously
with maturation process, some of the cell cultures were exposed to 20 µM T. gondii profilin
(MyBioSource) (positive control and four quercetin treatment groups) and quercetin (Sigma) (four
quercetin treatment groups) dissolved in DMEM then incubated for 48 hours (Ratnawati R and
Hernowati TE. 2015; Mochamad R, et al. 2013).
2.3. Adipocyte count measurement
After 48 hours of incubation, adipocytes were observed using inverted microscope and were captured
in high power field magnification using camera.
2.4. Statistical analysis
The number of adipocytes in each groups were presented as mean and standard error of the mean (SEM)
of four independent replications. Homogen and normal data (p>0,05) were analysed by ANOVA test.
2.5. Ethical statement
All procedures involving animals were in accordance with the ethical standards of Faculty of
Medicine, Brawijaya University (No 99/EC/KEPK-PSPD/03/2017).
3. Result
The results showed that the mean of adipocyte number in positive control group (95) was higher
compared to negative control (81.33). The decline in ROS levels were observed in all quercetin
treatment groups (Q 31.25= 84.67, Q 62.5 = 71.67, Q 125 = 73.67 and Q 250 = 57.33) compared to
negative or positive control groups (Figure 1).

The 2nd International Conference on Computer Science and Engineering Technology
Journal of Physics: Conference Series 1430 (2020) 012028
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Figure 1.
Mean number of adipocyte in each
groups
From statistical analysis using ANOVA, the results showed that quercetin
significantly reduced adipocyte cell count T. gondii profilin-exposed adipocytes compared to untreated
cells (ANOVA p = 0,00).
4. DISCUSSION
During T. gondii infection, profilin acts as a pathogen associated molecular pattern (PAMP)
recognised by the endosomal Toll-like receptor, TLR-11 (Yarovinsky F 2014). This recognition further
stimulates the inflammatory pathway, eventually leading to the production of ROS (Furukawa S, et al.
2004).
The results showed there was significant difference between negative and positive control, which
corresponds with the theoretical basis that profilin of T. gondiigenerates intracellular oxidative stress.
The excessive ROS production stimulated by T. gondii profilin was unable to be neutralized by
endogenous antioxidants alone (Marı M, et al. 2009), thus it stimulates intracellular molecular damage
of lipids, proteins, and DNA. These damages in turn becomes the basic mechanisms of diseases,
including hiperplasia of adipocyte that can lead to obesity and metabolic syndrome (Furukawa S, et al.
2004); Holmström, Kira M and Toren F. 2014).
The results here showed significant differences between control and all doses of quercetin groups.
These results confirmed the theorical basis that quercetin acts as an antioxidant in T. gondii profilin-
exposed by significantly decreasing ROS levels (Fazel S, et al. 2015; Mochamad R, et al. 2013).
Consistent with this results, a previous study by Lee et al. in 2013 observed quercetin effectivity as a
ROS scavenger using H2DCFDA staining in fibrosarcoma culture in vitro. The study revealed that
quercetin treatment was able to scavenge ROS level at doses of 5, 10, or 50 50 µg/ml (Lee DE, et al.
2013). In this study, the researchers found no significant difference among three different doses (31,25,
62,5 and 125 µM) of quercetin groups. This indicated that all doses employed in this study have similar
effectivity as antioxidant, where the minimum dose of 31.25µM showed similar effectivity to those of
higher doses (62.5 and 125 µM).
81.33
95.00
84.67
71.67 73.67
57.33
0.00
20.00
40.00
60.00
80.00
100.00
120.00
negative
control
positive
control
Q 31,25 Q 62,5 Q 125 Q 250
Adipocyte count

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5. Conclusion
This study implies that quercetin has a potent antioxidant that can prevent toxoplasmosis-mediated
adipocytopathy.
Acknowledgements
This research was supported by BPPM of Faculty of Medicine, Universitas Brawijaya, and Indonesia’s
Ministry of Research and Technology and High Education.
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