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
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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
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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
.
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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
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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.
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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)
Figure 2 - Illustration of the treatment period on animal models for each group. OVX, ovariectomy group; PG, induction
P. gingivalis
; OPG,
ovariectomy and induction
P. gingivalis
.
Table I - Characteristic of Animal Models Before Treatment
Variables Control (n=7) OVX (n=7) PG (n=7) OPG (n=7) P value
Body Weight (gram) 170.21±4.36 179.67±20.4 201.66±13.27 212.13±14.44 0.179*b
P valuea0.199* 0.251* 0.085* 0.088*
Serum Estrogen (pg/mL) 460.00±58.31 450.28±86.49 462.64±70.64 452.86±50.57 0.195*b
P valuea0.231* 0.157* 0.273* 0.116*
Blood Glucose (mg/dL) 52.15±17.24 62.68±9.94 60.71±9.32 62.14±8.09 0.137*b
P valuea0.292* 0.876* 0.028 0.233*
Total Cholesterol (mg/dL) 108.67±20.56 121.2±17.02 118.57±240.16 119.29±10.5 0.545*b
P valuea0.534* 0.278* 0.718* 0.079*
Blood Uric Acid (mg/dL) 3.1±1.63 3.71±0.76 1.5±1.18 1.98±2.35 0.543*b
P valuea0.573* 0.086* 0.163* 0.018
Data was expressed as a mean (SD, standard deviation) for all variables. P value described statistic analysis using a) Shapiro-Wilktest (p>0.05);
b) Levene analysis (p>0.05). n, number of study subjects; OVX, ovariectomy group; OPG, ovariectomy and induced Pg group; PG, induced
P. gingivalis
group; *significant difference for normality and homogeneity analysis (p>0.05).
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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)
groups and controls, but it was not signicantly
different (p> 0.05). In measuring estrogen levels,
which is one of the steroid sex hormones that
control ovarian function, the results of the study
showed that there was no signicant difference
in estrogen levels in the control and treatment
groups (p>0.05). However, the estrogen levels in
the PG group were the highest among the other
groups (p>0.05). 462.64±70.64). Likewise, for
glucose measurements, even though the control
group had the lowest glucose levels (52.15 ±
17.24), the glucose levels in the groups were
considered the same because there were no
signicant differences (p> 0.05). Even though the
OVX had the highest cholesterol (121.2 ±17.02)
and uric acid level (3.71±0.76), the results
showed that there were no signicant differences
in total cholesterol and uric acid between any of
the groups (p>0.05).
Figure 3 illustrated multiple comparisons
regarding mean differences in body weight and
blood biochemical parameters between the control,
the treatment groups and the observation periods.
The lined chart related to body weight revealed
similar patterns among the treatment groups
following the counter-time. The body weight of
animal models inclined following the observation
Figure 3 - Body Weight, Blood Biochemical Parameters, and Estradiol Level Based on Counter-time of Ovarian Dysfunction. a) Body Weight, b)
Blood Glucose Level, c) Total cholesterol, d) Blood Uric Acid Level, and e) Estradiol Level. Data presented were included mean, standard errors.
Data were analyzed with multiple comparison test. *, significant different between the groups and counter time (p<0.05).
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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)
time, except for the OPG group, which presented
more uctuation than the other treatment groups.
In treatment groups, there were significant
differences between the control group, several
observertion periods and several treatment groups
(p<0.05). In blood glucose levels, the alteration of
blood glucose levels among the treatment group
uctuated more. However, the enhancement peak
was presented on the third day, except the peak of
the OPG group was on the seventh day. The blood
glucose in the OVX and PG groups inclined on the
28th day, while the OPG group declined on the 21st
and continued on the 28th.
Based on multiple comparison analyses,
treatment groups’ mean blood glucose levels
comprised signicant differences following the
observation periods (p<0.05). However, there
were no significant differences between the
control and treatment groups at the beginning
of observation (p>0.05). On the concomitant
of observation time, the treatment groups
signicantly presented differences with control
groups several times (p<0.05), but there were
no signicant distinguishes between treatment
groups (p>0.05). The mean of Total cholesterol
enhanced following the time of observation.
The pattern of Total cholesterol alteration in
the OVX group exhibited the same as the OPG
group; the more extensive observation, the
higher the blood glucose level. However, the
peaks of treatment groups were on the 28th day.
Therefore the PG group altered not signicantly
on the 3rd and the seventh days, following the
time the level in this group increased. Concerning
the blood uric acid level, most of the statistical
analysis demonstrated there were no signicant
differences between the control and treatment
groups following counter-time (p>0.05), except
with OVX (p<0.05). Nevertheless, the pattern
of the lined chart of OPG was likewise the PG
group.
Ovarian dysfunction is associated with
hormonal changes, including estrogen, in the
form of decreased estrogen levels in the blood.
Estradiol is the primary circulating estrogen form.
The results showed that estradiol levels tended
to decline over time. The highest serum estradiol
levels occurred before treatment (0 days) in all
groups (OVX=466.2±17.9; OPG=469.1±38.8;
PG=468.4±39.4). After day 3, estradiol levels
decreased gradually until day 28 (p<0.05).
Regarding correlation analysis in Table II
revealed most of the variables presented
correlation between groups, inflammation
and estrogen level to body weight and blood
biochemical indicators, excepting association
between inammation condition and estrogen
level to blood uric acid level (R<0.1, p>0.05).
The groups and inflammation variables
demonstrated signicantly positive correlation,
which blood glucose level presented signicantly
positive moderate correlation to groups variable
likewise inflammation condition to Total
cholesterol. Interestingly, estrogen level obtained
significantly negative strong correlation with
body weight and blood biochemical parameters
(R>0.5, p<0.05), except correlation with uric
acid level. Moreover, the groups also revealed
significantly negative strong correlation with
blood uric acid level (R>0.5, p<0.05).
DISCUSSION
Metabolic syndromes are multiple and
complex symptoms or disorders affecting
metabolism alteration, and they magnify
cardiovascular diseases and type 2 diabetes
mellitus risks. They are frequently related to
Table II - Association between groups, inflammation condition and estrogen level to body weight and blood biochemical parameters
Groups Estrogen Level
R value P value* R value P value*
Body Weight (gram) 0.724‡ 0.001* -0.637‡ 0.026*
Blood Glucose level (mg/dL) 0.469† 0.054* -0.728‡ 0.007*
Total cholesterol (mg/dL) 0.517‡ 0.028* -0.716‡ 0.009*
Blood Uric Acid level (mg/dL) -0.763‡ 0.000* 0.000# 1.000
Data was expressed as association of variables (R square value) and significant difference (P value) for all variables All of variables were
analyzed with Pearson’s correlation. *significant correlation between variables (p<0.05). #, no significantly correlation; †significant and
moderate correlation. ‡significant and strong correlation; (-), negative correlation. Groups were treatment groups, including OVX (ovariectomy
group), OPG (ovariectomy and induced Pg group), and PG (induced P. gingivalis group).
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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)
dysglycemia, dyslipidemia, visceral obesity,
atherogenic and hypertension. These syndromes
are mainly caused by obesity, physical inactivity,
aging, hormonal alteration and genetics [1].
However, these are currently associated with
inammation, like chronic periodontitis primarily
caused by
P. gingivalis
[21,25].
In this recent study, animal models were
manipulated to mimic ovarian dysfunction or
menopause, thereby ovariectomy and infectious
agent induction utilizing
P. gingivalis
. This
study demonstrated that there was metabolic
syndrome risk in animal models. The syndrome
was characterized by alteration of body weight,
blood glucose level, Total cholesterol and uric
acid level. Body weight observation was related
to investigating the obesity status of animal
models. Regarding the body weight, this study
exhibited the body weight of animal models
magnified following the observation time.
Although there were body weight alterations, the
body weight of animal models typically tended
to be underweight regarding the age increase.
Rats strain Sprague Dawley aged eight weeks
present 200-220 grams [26]. However, based
on the control group, the body weight of animal
models of treatment groups was higher than
the control group, and the OPG group was the
heaviest. The body weight peak was on the 28th
day of observation.
Also, this study revealed that significant
changes in body weight were negatively
correlated with estrogen levels. Compared
to the larger animal models, these exhibited
signicant oestrogen levels. Possible causes of
the estrogen deciency that led to the stimulation
of fat accumulation and the down-regulation of
metabolism include ovariectomy and
P. gingivalis
infection. Moreover, estrogen insufciency and
bacterial infection-induced inammation were
identied as contributing factors to the severity
of fat accumulation [27,28]. Ovariectomy
causes estrogen shortage by imitating the onset
of menopause by affecting reducing estrogen
production [15,29]. This disorder affected visceral
fat deposition and interfered with the metabolism
of the liver and fat tissues [28]. Moreover, a
lack of estrogen promotes hyperphagia and
systemic inammation. This ultimately resulted
in obesity. As a sex steroid hormone, estrogen
plays a crucial part in various homeostatic
processes, metabolism operations, and the release
of cytokines in fat tissues. One of the rules is to
control fat deposition and reduce the danger of
obesity [30,31].
Blood glucose level observation was aimed at
investigating glucose dysregulation. This recent
study exhibited that the level uctuated following
the time and tended to increase on the 28th day,
except for the OPG group. Notwithstanding,
the blood glucose level of the treatment group
presented higher than the control group; blood
glucose level enhancement could not be indicated
as a diabetic condition. The blood glucose level
in treatment groups was slightly higher than
the physiologic value in rats. The standard
value is 93.2±10.3 mg/dL [32,33]. It might
be caused by the treatments (ovariectomy,
P. gingivalis
induction and combination) only
induced hyperglycemia and influenced the
glucose intolerance and regulation in the
bloodstream. Nevertheless, it might not affect
glucose metabolism and insulin regulation
permanently. The mechanism might be related
to the type of treatments, inammation severity
and estrogen deficiency. This suggestion
was substantiated by correlation analysis.
The analysis revealed a significantly positive
correlation between blood glucose alteration
and inammation and a signicantly negative
correlation with serum estrogen level.
P. gingivalis
induction related to bacterial endotoxemia
stimulated early hyperglycemia, representing a
general sign of metabolic syndromes [34,35].
Moreover,
P. gingivalis
induction affected
inflammation, in which the host released
proinflammatory cytokine in response
to
P. gingivalis
[21]
.
This response caused insulin
resistance. The effect of insulin resistance on
blood glucose level among treatment groups were
suspected to be the same and depended on the
sensitivity of the host response [36].
Total cholesterol level indicates lipid
prole as a screening parameter for the risk of
atherosclerosis and cardiovascular diseases. This
study demonstrated that total cholesterol among
treatment groups signicantly enhanced following
the period of observation, presenting a negatively
signicant correlation with serum estrogen level.
The treatment might stimulate inammation and
sex steroid hormone alteration. Inflammation
and hormone alteration possibly generated lipid
metabolism disturbance.
P. gingivalis
stimulates
the released proinflammatory cytokines as a
host response, which the cytokines stimulate
lipid discharge and hyperlipidemia [37].
9
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)
In comparison, ovariectomy might influence
estrogen production, and ovariectomy impaired
lipid metabolism. Estrogen regulates cholesterol
metabolism, accelerates cholesterol catabolism,
enhances fatty acids oxidation and plays
anti-inflammation [15,38,39]. Furthermore,
estrogen deficiency stimulated ovariectomy
generated triglyceride accumulation liver and
impacted the enhancement of total cholesterol [40].
Uric acid acts as a potent natural antioxidant
and free radical scavenger. It serves cellular
defence against oxidative stress [41]. However,
excessive uric acid leads to ischemia and perfusion
of tissues [42]. A recent study showed that only
the OVX group presented elevated uric acid
levels. There was no correlation between uric
acid alteration to serum estrogen level. Estrogen
alteration might not affect uric acid fraction
alteration [43]. The previous study associated
signicant uric acid alteration with obesity [44],
while in this study, the animal model’s body weight
normally increased. Furthermore, the advanced age
of animal models in this study did not inuence uric
acid levels. Although previous studies exhibited
middle age, estrogen deciency and periodontal
disease increased uric acid levels [45].
This study revealed changes in estrogen
levels in the treatment group.
P. gingivalis
infection might affect hormone production in
the ovaries, such as during an ovariectomy.
P. gingivalis
encloses potent virulence factors that
may inuence the activity of estrogen synthesis
and production in the ovaries, such as LPS. Several
studies described that LPS reduced estradiol
synthesis in the ovaries by decreasing the activity
of the aromatase cytochrome P450 enzyme
(CYP19A1). This enzyme is needed to convert
androsterone into estradiol. LPS will modulate
inflammation in the ovaries and increase the
activity of CCAAT enhancer binding protein
beta (CEBPβ), which plays a role in regulating
cytokine expression and possibly the expression
of aromatase, the pro-inflammatory cytokine
IL-6 or TNFα which increases after LPS exposure
can also regulate estradiol synthesis [46-48].
This study proposed the mechanism by which
ovariectomy and
P. gingivalis
contribute to the
development of metabolic syndrome. The rst,
metabolic syndrome, was related to the treatments’
estrogen-altering effects. The treatments had
a significant effect on serum estrogen levels,
causing them to be substantially lower than in
the control group. Reductions in estrogen levels
induced ovarian dysfunction and metabolic
disturbances in the treatment groups. In adipose,
muscle, and liver tissues, estrogen controls lipid
and glucose metabolism. When estrogen levels are
insufcient, calorie intake and energy expenditure
are impaired. It generates inefficient glucose
utilisation, resulting in hyperglycemia and glucose
dysregulation [12,30]. In addition, estrogen
deciency causes insulin sensitivity and resistance,
which worsens hyperglycemia and disrupts
immune cell activity. This condition stimulates
macrophages and adipose tissue to produce pro-
inammatory cytokines. This condition performed
oral microenvironment and periodontal disease
severity tests conclusively [49-51].
Subsequently, disruption of glucose
homeostasis also inuences lipid metabolism in
adipose and liver tissue. Both estrogen deciency
and disturbance of glucose metabolism will
stimulate lipid deposition and accumulation
of vascular and tissue. This accumulation will
increase triglyceride, total cholesterol and
low-density lipoprotein (LDL), enhancing
cardiovascular disease risk. This hyperlipidemia
will trigger leukocytes to be hyperactive to
produce ROS. Imbalance ROS and endogen
antioxidant lead to oxidative stress, insulin
resistance and inflammation. Briefly, glucose
and lipid metabolism inferences influence
bidirectional. Moreover, these interferences
stimulate systemic inammation [52,53].
The second proposed mechanism is via the
inammation process.
P. gingivalis
, an infectious
agent, caused systemic inammation. This local
induction spreads via the bloodstream and causes
endotoxemia. The endotoxemia of the virulence
factor of
P. gingivalis
, LPS, involves ovarian
inflammation and dysfunction. LPS disturbs
steroidogenesis and estrogen production. This
interruption will lead to immune cell impairment
to produce cytokines and ROS. Excessive cytokine
and ROS also influence steroidogenesis and
estrogen excretion. Finally, it causes estrogen
deciency and leads to the serial of metabolic
interruptions [24,54,55].
The limitation of our study was using
periapical x-ray to detect alveolar bone resorption,
as the animal models got periodontitis, so it did
not detect detailed bone destruction. Although,
the radiographic showed that the induction was
performed in distobuccal and distolingual of rst
10
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)
molar mandible caused expansion of damage to
the mesial and distal second molar.
CONCLUSION
Briefly, ovarian dysfunction caused by
ovariectomy and
P. gingivalis
infection might
increase the metabolic syndrome development,
especially cardiovascular disease and glucose
intolerance, in which signed by increase glucose
and cholesterol blood level.
P. gingivalis
induction
not only leaded metabolic syndrome risk but
also worsens metabolic syndrome in ovarian
dysfunction-induced ovariectomy due to estrogen
deciency. However, this study required further
studies to investigate the molecular and cellular
mechanism of periodontal pathogen involvement in
ovarian dysfunction-induced metabolic syndrome.
Acknowledgements
The authors are grateful to Bangun Febrianto
and Indria Cahyani for support during the animal
models treatment and collecting data
Author’s Contributions
AWSD: Conceptualization, Methodology,
Resources, Investigation, Writing – Original
Draft Preparation, Writing – Review & Editing.
ZM: Data Curation, Formal Analysis, Software,
Validation, Visualization. TE: Methodology,
Validation, Visualization, Data Curation. AMP:
Investigation, Project administration. ZH:
Funding Acquisition, Supervision.
Conict of Interest
The authors have no proprietary, nancial,
or other personal interest of any nature or kind
in any product, service, and/or company that is
presented in this article
Funding
This study was totally supported by Research
and Community Service Institution of Universitas
Jember.
Regulatory Statement
This study was conducted in accordance
with all the provisions of the medical research
oversight committee guidelines and policies of:
Faculty of Dentistry, Gadjah Mada University,
Yogyakarta, Indonesia
The approval code for this study is: 0029/
KKEP/FKG-UGM/EC/2022
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v20i3.1443.
Agustin Wulan Suci Dharmayanti
(Corresponding address)
Biomedical Department, Faculty of Dentistry, University of Jember,Jember, East
Java, Indonesia.
Email: agustinwulan.fkg@unej.ac.id
Date submitted: 2023 May 05
Accept submission: 2024 Apr 10
