UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2024.e3880
1
Braz Dent Sci 2024 Jan/Mar;27 (1): e3880
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Effect of ovarian dysfunction induced ovariectomy and
Porphyromonas gingivalis induction to risk of metabolic syndrome:
in vivo study
Efeito da disfunção ovariana por meio de ovariectomia e indução de Porphyromonas gingivalis no risco de desenvolvimento
de síndrome metabólica: estudo
in vivo
Agustin Wulan Suci DHARMAYANTI1 , Zahara MEILAWATY1 , Tantin ERMAWATI1 , Agus Murdojohadi PUTRADJAKA1 ,
Zahreni HAMZAH1
1 - Universitas Jember, Biomedical Department, Faculty of Dentistry. Jember, Indonesia.
How to cite: Dharmayanti AWS, Meilawaty Z, Ermawati T, Putradjaka AM, Hamzah Z. Effect of ovarian dysfunction induced
ovariectomy and Porphyromonas gingivalis induction to risk of metabolic syndrome (in vivo study). Braz Dent Sci. 2024;27(1): e3880.
https://doi.org/10.4322/bds.2024.e3880
ABSTRACT
Periodontal diseases and metabolic syndrome are related to complicated multifactorial conditions. However, the
relationship is not yet evident. Estrogen insufciency might correlate to this condition, possibly caused by ovarian
removal and
Porphyromonas gingivalis
(
P. gingivalis
) infection. This study aimed to evaluate the effect of ovarian
dysfunction caused by ovariectomy and
P. gingivalis
infection to metabolic syndrome development. This study
was an experimental laboratory study using female rats Sprague Dawley Strain. Animal models were divided into
four groups: control, ovariectomy (OVX), ovariectomy-periodontitis (OPG), and periodontitis (PG). The purpose
of every treatment in each group was to induce ovarian dysfunction. The OVX group was undertaken ovaries
removal surgery. PG was performed
P. gingivalis
induction. Therefore OPG was a combination of ovariectomy
and
P. gingivalis
induction. Blood was drawn and observed on days 0, 3, 7, 14, 21, and 28. The blood sample
was examined for uric acid, cholesterol, glucose and estrogen. The collected data were all statistically examined.
All treatment groups presented body weight and blood biochemical observation signicantly higher than the
control group, except total cholesterol (p<0.05). Moreover, most variables presented a correlation between
groups to body weight and biochemical blood indicators, except blood uric acid level (R>0.5). The metabolic
syndrome was triggered by ovarian dysfunction brought on by
P. gingivalis
infection after ovariectomy. They
both took the same risk. Even
P. gingivalis
induction made metabolic syndrome in the group of animal models
which underwent ovariectomy worse.
KEYWORDS
Estrogen deciency; Metabolic syndrome; Ovariectomy; Ovarian dysfunction;
P. gingivalis
.
RESUMO
Doenças periodontais e síndrome metabólica estão relacionadas a condições multifatoriais complicadas. No
entanto, a relação ainda não é evidente. A insuciência de estrogênio pode estar correlacionada a essa condição,
possivelmente causada pela remoção dos ovários e infecção por
Porphyromonas gingivalis
(
P. gingivalis
). Este
estudo teve como objetivo avaliar o efeito da disfunção ovariana causada pela ovariectomia e infecção por
P. gingivalis
no desenvolvimento da síndrome metabólica. Este foi um estudo experimental de laboratório
utilizando ratos fêmeas da linhagem Sprague Dawley. Os modelos animais foram divididos em quatro grupos:
controle, ovariectomia (OVX), ovariectomia-periodontite (OPG) e periodontite (PG). O objetivo de cada tratamento
em cada grupo foi obter disfunção ovariana. O grupo OVX foi submetido à cirurgia de remoção dos ovários; no
grupo PG foi realizada a indução de
P. gingivalis
; e no grupo OPG foi feita uma combinação de ovariectomia e
2
Braz Dent Sci 2024 Jan/Mar;27 (1): e3880
Dharmayanti AWS et al.
Effect of ovarian dysfunction induced ovariectomy and Porphyromonas gingivalis induction to risk of metabolic syndrome (in vivo study)
Dharmayanti AWS et al. Effect of ovarian dysfunction induced ovariectomy and
Porphyromonas gingivalis induction to risk of metabolic
syndrome (in vivo study)
INTRODUCTION
A metabolic syndrome is a cluster group of
metabolic interference, including dyslipidemia,
visceral obesity, atherogenic, hyperglycemia,
and hypertension, frequently associated with
cardiovascular diseases and type 2 diabetes
mellitus risk [1]. The prevalence of this syndrome
is an individual who presents obesity, physical
inactivity, aging, hormonal alteration, and
genetics [2]. This syndrome correlates to
inammation, such as periodontal diseases [3].
Several studies reviewed metabolic syndrome and
periodontal diseases were complex multifactor
disorders and exhibited correlation [4–6].
However, the relationship between periodontal
diseases and metabolic syndrome remains unclear
and needs advanced investigation.
Most women over forty-ve years old risk
getting metabolic syndrome and periodontal
diseases. Several studies correlated those
disorders with aging and hormonal changes
related highly susceptibility to inammation, such
as gingival inammation and periodontitis, [7-9].
This population is entering the menopause
phase, which decreases ovarian, hypothalamus-
pituitary-gonadal axis function, and sex steroid
hormone, particularly estrogen [10].
Ovaries are the main organs producing
estrogen. This hormone affects both reproductive
and other organs, such as the liver, adipose tissues,
and periodontal tissue [11]. Moreover, estrogen
also involves in several metabolisms, such as
insulin regulation [12], immune cell activation
and regulation [13], and anti-inflammation
agents [14]. Estrogen level alteration stimulates
several disorders and inammation severities [15].
However, this statement has been controversial
until now. Several previous studies exhibited
opposite results each other [16-19].
Besides the aging process affecting estrogen
levels in circulation and the activity, bacterial
infection stimulates estrogen production
disturbance, leading to ovarian dysfunction [20].
Porphyromonas gingivalis
(
P. gingivalis
), as the
major periodontal pathogen, presents several
virulence factors [21]. The virulence factors,
especially lipopolysaccharide (LPS) might
down-regulates estrogen, thereby interrupting
ovary steroidogenesis [20].
P. gingivalis
was
suspected of ovarian function through systemic
inflammation and oxidative stress, which
impacted on metabolism process, especially
glucose, lipid, and uric acid regulation.
However, an explanation about it required
further investigation. This study aimed to
evaluate effect of ovarian dysfunction caused
by ovariectomy and
P. gingivalis
infection to
metabolic syndrome development. This study
used ovariectomy and
P. gingivalis
induction
to get ovarian dysfunction or premature
menopause. Apart from that, there are many
methods for modeling periodontitis. However,
this study used a model of periodontitis induced
by
P. gingivalis
because this method better
describes the condition of chronic periodontitis
caused by periodontal pathogens, especially
P. gingivalis
[22]. Ovariectomy mimics
oophorectomy or ovaries removed surgically,
while
P. gingivalis
induction induced systemic
inammation and possibly triggered premature
menopause. In this study, ovariectomy was
used as a comparison to determine whether
induction of
P. gingivalis
also produced the
same effect as menopause models.
indução de
P. gingivalis
. O sangue foi coletado e observado nos dias 0, 3, 7, 14, 21 e 28. A amostra de sangue
foi examinada para ácido úrico, colesterol, glicose e estrogênio. Os dados coletados foram todos examinados
estatisticamente. Todos os grupos de tratamento apresentaram peso corporal e observações bioquímicas
sanguíneas signicativamente maiores do que o grupo controle, exceto o colesterol total (p<0,05). Além disso, a
maioria das variáveis apresentou uma correlação entre os grupos com o peso corporal e indicadores bioquímicos
sanguíneos, exceto o nível de ácido úrico no sangue (R>0,5). A síndrome metabólica foi desencadeada pela
disfunção ovariana causada pela infecção por
P. gingivalis
após a ovariectomia. Ambos apresentaram o mesmo
risco. Mesmo a indução por
P. gingivalis
piorou a síndrome metabólica no grupo de modelos animais que foram
submetidos à ovariectomia.
PALAVRAS-CHAVE
Deciência de estrogênio; Síndrome metabólica; Ovariectomia; Disfunção ovariana;
P. gingivalis
.
3
Braz Dent Sci 2024 Jan/Mar;27 (1): e3880
Dharmayanti AWS et al.
Effect of ovarian dysfunction induced ovariectomy and Porphyromonas gingivalis induction to risk of metabolic syndrome (in vivo study)
Dharmayanti AWS et al. Effect of ovarian dysfunction induced ovariectomy and
Porphyromonas gingivalis induction to risk of metabolic
syndrome (in vivo study)
MATERIAL AND METHODS
Animals
This study was approved and conducted
by the Health and Research Ethics Committee
of the Dental Faculty, Gadjah Mada University.
This study followed national and international
guidelines for the care and welfare of laboratory
animals.
This experimental laboratory study used
rats (Rattus norvegicus) Sprague Dawley Strain,
aged 6 to 8 weeks, female, and 150-200 grams.
The rats adapted to constant room temperature
and relative humidity on a 12-h day and night
cycle, with direct access to food and water
(diet and water ad libitum). Animal models
were divided into four groups: control (without
any treatments), ovariectomy group (OVX),
periodontitis group (PG), and ovariectomy-
periodontitis group (OPG). All of the treatment
groups were performed to get ovarian dysfunction.
The control group in this study was used to
determine the baseline value (standard value) of
the animal models. This OVX group manipulated
animal models to mimic menopause or ovarian
dysfunction. Animal models of OVX groups were
given 3 days before blood sampling was carried
out for the recovery period after ovariectomy
surgery and to avoid the impact of surgery
on blood test results. The PG group showed
mimicking periodontitis due to the induction of
major periodontal pathogens. Meanwhile, the
OPG group is mimicking menopause accompanied
by periodontitis due to the induction of major
periodontal pathogens. In the OPG group,
induction of p. gingivalis is given after 3 days
post-surgery for the recovery period.
The grouping of animal models was carried
out randomly. However, in this study, there
were difculties due to the non-uniformity of the
body weight of the animal models, so one or two
animal models that had body weights outside the
average affected the results of the body weight
of the model animals.
Preparation of
P. gingivalis
Suspension
P. gingivalis
was obtained from
P. gingivalis
stock (Porphyromonas gingivalis ATCC 33277,
Thermo Fisher Scientific, USA).
P. gingivalis
stock was inoculated on solid Brain Heart
Infusion Agar (BHI-A) (Oxoid, Thermo Fisher
Scientific, USA). After that, it was put in a
desiccator for 2x24 hours with CO2 gas pack
(Oxoid, Thermo Fisher Scientic, USA ) to make
anaerobic condition. Subsequently, one ose of
P. gingivalis
on BHI was taken and put in 2 mL
of Brain Heart Infusion Broth (BHI-B) (Oxoid,
Thermo Fisher Scientific, USA). Afterwards,
the suspension was homogenized using vortex
(Thermo Fisher Scientic, USA) for 30 seconds
and then incubated in a desiccator with CO2 gas
pack at 37° C for 24 hours. The growth marked
the turbidity of BHI media, then it was diluted
with sterile aquadest, shaken till homogenous
and measured the concentration manually with
1.5 of Mc. Farland standard 2.109 cells/ ml of
concentration [23].
Surgical procedure (Ovariectomy)
The OPG and PG groups were prepared for
ovariectomy procedures. They were anaesthetized
with ketamine/xylazine (80/10 mg/kgBW)
intraperitoneally (Sigma Aldrich, Singapore).
After anesthetization, the dorsal area of animal
models was disinfected with povidone-iodine.
On the right side of the dorsal, approximately
1-1.5 cm of the spine was performed with a small
transverse incision (0.4–0.6 cm) using surgical
scalpel blade no. 11 on the right side. After the
peritoneal cavity was accessed, the adipose tissue
was pulled away until the right uterine tube and
the ovary, surrounded by a variable amount of
fat, were identied. The ovary and associated
fat were located and exteriorized by gentle
retraction. The uterine horn was bound, and then
the ovaries were cut. After that uterine horn was
reimplanted in the peritoneal cavity. The wound
was closed by sterile sutures. The procedure is
repeated for the left ovary through the same
incision. Povidone iodine was applied to the area
to disinfect the skin after suturing. A signicant
degree of aseptic procedure was maintained
throughout the operation. After surgery, the rats
were housed individually in cages for a week
(7 days) to allow recovery and then re-grouped
in their home cages [24].
P. gingivalis
induction (Periodontitis Model)
P. gingivalis
induction was undergone
under light UV protection to prevent bacteria
from being transmitted to a human.
P. gingivalis
induction was performed in OPG and PG groups.
Each animal model was injected with 0.05 ml
P. gingivalis
suspension, suspected of having
4
Braz Dent Sci 2024 Jan/Mar;27 (1): e3880
Dharmayanti AWS et al.
Effect of ovarian dysfunction induced ovariectomy and Porphyromonas gingivalis induction to risk of metabolic syndrome (in vivo study)
Dharmayanti AWS et al. Effect of ovarian dysfunction induced ovariectomy and
Porphyromonas gingivalis induction to risk of metabolic
syndrome (in vivo study)
a concentration of 2.109 cells/ ml dissolved
in saline. The bacteria were injected into the
distolingual and distobuccal gingival sulcus
area of the mandible rst molar. The induction
was repeated every three days for 19 days.
The animal model was x-rayed to nd out that
the mouse model had experienced periodontitis,
and X-ray results showed alveolar bone resorption
(Figure 1). In the OPG group, the
P. gingivalis
induction was taken seven days after the surgical
procedure of ovariectomy [24].
Body weight measurement
Body weight measurements in this study
were only to evaluate the health and safety level
of animal models after receiving treatment. Apart
from that, body weight is also associated with
systemic changes due to metabolic syndrome.
Blood sampling
The blood sampling for each group was
various because the end of the treatment for each
group was different. However, the collection period
remained the same for each group. The blood
collection was carried out a day before and on the
3rd, 7th, 14th, 21st, and 28th day after the treatment.
So, each animal model in each group was taken
blood collection six times, except the control
group, which was taken once (Figure 2). Before
blood sampling, the animal models fasted for
8 hours. Blood sampling was from the infraorbital
plexus, about 5-7.5% of body weight or about
0.8-1.0 cc. Blood collection with this volume was
highly considered because it was the safest and did
not cause stress and weakness in animal models.
So that after taking blood, the animal stayed alive,
and the recovery process was fast.
Blood was directly measured glucose,
cholesterol, and uric acid and stored in tubes
without anticoagulants aimed to get the serum.
Measurement of glucose, cholesterol, and uric
acid utilized GCUmeter (easy touch, Taiwan).
Therefore, the serum for estrogen level analysis.
The estrogen level used estradiol ELISA kit,
which all of the procedures followed the manual
of ELISA kit (BT-Lab, USA). All obtained data
were analyzed with one-way analysis variance
(p<0.05), multiple comparisons (p<0.05), and
Pearson’s correlation (r=1). However, before
analysis, all data was tested for normality using
Saphiro-Wilk analysis (p>0.05) and homogeneity
using Levene test (p>0.05).
Blinding (masking)
Five researchers conducted this study, and
all researchers were involved during the research
process. The blinding process was carried out
from the randomization of the grouping of
animal models. Then, the researcher applied the
experimental animals according to the treatment
given to the experimental animals. To avoid
blinding, when collecting blood and measuring
serum biochemical and estrogen levels, only
researchers in the treatment section knew the
coding on blood tubes. In contrast, researchers
in the data analysis section did not know the
code. This step was done to avoid manipulation
of research results.
RESULT
Table I existed to evaluate the characteristics
of animal models before treatment. Before
treatment, animal models enclosed varying body
weights, estrogen levels and blood biochemistry.
Even though the experimental animals were
selected randomly, when measuring body
weight, each group had the same normality and
homogeneity values regarding body weight,
serum levels of estrogen, glucose, cholesterol
and uric acid (p>0.05). In measuring body
weight, the OPG group (212.13 ± 14.44) had the
heaviest body weight among the other treatment
Figure 1 - Radiographic of the mandible. a) did not indicate
periodontitis; b) periodontitis. Periodontitis was characterized by
bone resorption between the first, second and third molars.
