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.e4023
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Braz Dent Sci 2024 Jan/Mar; 27 (1): e4023
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.
The effect of calcium hydroxide in combination with propolis
extract on decreasing nerve growth factor and substance
P expression in rat dental pulp neuron
O efeito do hidróxido de cálcio em combinação com o extrato de própolis na diminuição da expressão do fator de
crescimento nervoso e da substância P em neurônio de polpas de ratos
Ira WIDJIASTUTI1 , SETYABUDI1 , Grace Angelina SAMUEL1 , Khadijah Fauzi BASALAMAH1
1 - Universitas Airlangga, Faculty of Dental Medicine, Department of Conservative Dentistry. Surabaya, Indonesia.
How to cite: Widjiastuti I, Setyabudi, Samuel GA, Basalamah KF. The effect of calcium hydroxide in combination with propolis
extract on decreasing nerve growth factor and substance P expression in rat dental pulp neuron. Braz Dent Sci. 2024;27(1):e4023.
https://doi.org/10.4322/bds.2024.e4023
ABSTRACT
Objective: Pain sensitivity was reected in the expression of nerve growth factor (NGF) and substance P (SP), which we
used in a rat tooth perforation model to assess the efcacy of Ca(OH)2 combined with propolis extract direct pulp capping
agent. The model was used to investigate the synergistic effects of Ca(OH)2 and propolis extract. Material and Methods: This
experimental study used 36 Wistar rat mandibular incisors randomly divided into three groups. The rats were euthanized after
12 and 24 h. Specimens underwent histological evaluation under a light microscope to identify the presence of neuron cells; an
immunohistochemistry method using anti-NGF and anti-SP1 was used to evaluate the expression of NGF and SP. The results were
then statistically evaluated by one-way analysis of variance and Tukey’s honestly signicant difference tests. Results: Signicant
differences were found in NGF expression between the Ca(OH)2 + propolis and control group (12h group
p
<0.001; 24h group
p
<0.001) and between Ca(OH)2 and the control group (12h group
p
=0.001; 24h group
p
<0.001). Signicance differences in
SP expression were also found between the Ca(OH)2 + propolis and Ca(OH)2 groups and between the Ca(OH)2 + propolis and
control groups (12h group
p
<0.001; 24h group
p
<0.001). Conclusion: Ca(OH)2 combined with propolis extract as a direct
pulp capping agent can inhibit dental pain response because the main active ingredients of propolis are caffeic acid phenethyl
ester and avonoids which can inhibit the release of prostaglandin, thereby inhibiting SP secretion, but not NGF expression.
KEYWORDS
Calcium hydroxide; Dental pulp capping; Nerve growth factor; Propolis; Substance P.
RESUMO
Objetivo: Avaliar a ecácia do Ca(OH)2 associado com extrato de própolis como agente de capeamento pulpar direto e como
a sensibilidade à dor pode reetir na expressão do fator de crescimento nervoso (NGF) e substância P, em ratos que tiveram
dentes inferiores perfurados. O estudo foi usado para investigar os efeitos sinérgicos do Ca(OH)2 e extrato de própolis.
Material e Métodos: Esse estudo experimental usou 36 incisivos inferiores de ratos Wistar divididos aleatoriamente em três
grupos. Os ratos foram sacricados depois de 12 e 24 horas. Os espécimes foram submetidos à avaliação histológica em um
microscópio de luz para identicar a presença de células neuronais; um método de imuno-histoquímica usando anti-NGF
e anti-SP1 foi usado para avaliar a expressão de NGF e SP. Os resultados então foram analisados estatisticamente análise
de variância one-way e teste de Tukey. Resultados: Diferenças signicativas foram encontradas na expressão de NGF
entre Ca(OH)2 + própolis e grupo controle (grupo 12h p<0.001; grupo 24h p<0.001), e entre Ca(OH)2 e grupo controle
(grupo 12h p=0.001; grupo 24h p<0.001). Diferenças signicativas na expressão de SP também foram encontradas entre
Ca(OH)2 + própolis e grupo Ca(OH)2, e entre Ca(OH)2 + própolis e grupo controle (grupo 12h p<0.001; grupo 24h p<0.001).
Conclusão: Ca(OH)2 associado com extrato de própolis como agente de capeamento pulpar direto pode inibir a resposta à
dor dentária porque os principais componentes ativos do própolis são o éster fenetílico do ácido cafeico e os avonoides, que
podem inibir a liberação de prostaglandina, inibindo assim a secreção de SP, mas não a expressão de NGF.
PALAVRAS-CHAVE
Hidróxido de cálcio; Capeamento da polpa dentária; Fator de crescimento nervoso; Própolis; Substância P.
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Braz Dent Sci 2024 Jan/Mar; 27 (1): e4023
Widjiastuti I et al.
The effect of calcium hydroxide in combination with propolis extract on decreasing nerve growth factor and substance P expression in rat dental pulp neuron
Widjiastuti I et al. The effect of calcium hydroxide in combination with propolis
extract on decreasing nerve growth factor and substance
P expression in rat dental pulp neuron
INTRODUCTION
The inflammation of the pulp and the
activation of the dental pulp nerve bers are the
root causes of dental pain [1]. The pain in the
trigeminal nociceptor is characteristic of the pulp
illness known as pulp inammation or pulpitis [2].
The dental pulp is innervated by sensory nerve
fibers containing neuropeptides, which are
released by afferent nerve bers and are associated
with neurogenic inammation and wound healing.
There are only two types of nerve bers, Aδ and C,
which mainly innervate the pulp tissue and act as
polymodal receptors [3]. Approximately 25-50% of
dental nerve bers are Aδ-myelinated nerve bers
containing the neuropeptides calcitonin gene-
related peptide (CGRP) and nerve growth factor
(NGF) [2]. NGF is a neuropeptide neurotrophic
factor, a well-known mediator for persistent
pain. Pain occurs because a reaction originates
from NGF, a neuropeptide produced when tissue
damage occurs and an important component of
inammation [4]. NGF acts directly on peptidergic
C-ber nociceptors, which express the NGF thyroid
kinase receptor, TrkA, as well as the low-afnity
neurotrophic receptor p75 [5]. When stimulated,
the terminal part of the C nerve ber will produce
NGF receptors and release several neuropeptides,
namely substance P (SP), CGRP, and neurokinin
A (NKA) [6]. SP is considered a major mediator
of neurogenic inflammation and hyperalgesia.
This mechanism not only involves nerve bers
in the area of tissue damage but also extends to
the surrounding damaged tissue, where it causes
secondary hyperalgesia [7].
Direct pulp capping is a procedure that is
used for maintaining the vitality of the dentin-pulp
complex when the dental pulp is exposed and is
still vital, caused by caries, trauma or iatrogenic
causes, such as accidental opening of the pulp
due to tooth preparation and removal of carious
tissue [8]. The pulp requires protection against
bacterial invasion, thermoelectric conduction, and
chemical protection of the overlying restorative
materials. Calcium hydroxide Ca(OH)2 is the
gold standard for pulp capping treatment. The
biocompatibility of Ca(OH)2 and other pulp
capping materials can be used to maximally
reduce the pain and inammation response [9].
One of the alternative pulp capping materials
that can be developed is propolis. Some authors
recommend the use of propolis as a carrier in
a mixture of Ca(OH)2 paste is recommended
because propolis contributes to the antimicrobial
effects of Ca(OH)2, which other carriers, such as
saline and propylene glycol, do not [10-12].
The pharmacologically active chemical
components of propolis that are the most widely
known are avonoids, isoavonoids, phenolics,
caffeic acid and aromatic acid [13]. The content of
avonoids and caffeic acid derivatives in propolis
has an anti-neuroinflammatory effect and can
provide a degree of healing of pulp inammation
and reduce pain [14,15]. Because nerve bers that
innervate the pulp produce NGF and SP molecules
and their receptors, this study aimed to investigate
the effects of expression in nerve cells.
MATERIALS AND METHODS
Materials
Raw propolis extract was combined with
Ca(OH)2 paste at a 1:1.25 ratio (125 mg Ca(OH)2
and 0.1875 propolis extract). This combination
was mixed with a cement spatula until it was
homogeneous. The materials used in this study
were propolis extract, Ca(OH)2 powder (Merck,
Darmstadt, Germany), cotton pellets, paper
point, 10% formalin solution, 70% alcohol, 95%
ketamine HCl (Ketamine®, Kepro BV, Deventer,
Netherland), povidone-iodine (Betadine®, PT
Mahakam Beta Farma, Jakarta, Indonesia), xylazine
HCl (Xyla®, De Adelaar BV, Venray, Netherland),
phosphate-buffered saline (PBS), Cention lling
(Ivoclar Vivadent, Schaan, Liechtenstein), parafn
wax, and hematoxylin and eosin stain.
Propolis preparation
Propolis extract was created by treating
dried Apis mellifera honeycomb (100% propolis)
and processed at Balai Penelitian dan Konsultasi
Industri, Surabaya, East Java, Indonesia. One kg
of propolis was macerated and cut to a thickness
of 0.5 to 1 cm. The mixture was then homogenized
for 24 h by shaking 1,000 mL of 96% ethanol into
the closed container at 80 rpm. The mixture of
propolis and ethanol that has been shaken is
then ltered with a vacuum lter. The ltrate
liquid is transferred into an evaporator cup,
then evaporated in a vacuum evaporator until
all the solvent is separated at a temperature of
50-60 °C for 3-5 h. The resulting extract residue
(thick, brownish liquid) is transferred into a
brown glass bottle and stored at a temperature
of 20-25 °C [16].
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Braz Dent Sci 2024 Jan/Mar; 27 (1): e4023
Widjiastuti I et al.
The effect of calcium hydroxide in combination with propolis extract on decreasing nerve growth factor and substance P expression in rat dental pulp neuron
Widjiastuti I et al. The effect of calcium hydroxide in combination with propolis
extract on decreasing nerve growth factor and substance
P expression in rat dental pulp neuron
Tools
The tools used consisted of a cage for
containing the subjects, 3 cc and 1 cc syringes,
a micromotor, a low-speed handpiece (NSK,
Tokyo, Japan), a 0.8 mm round diamond bur,
a 0.46 mm diameter probe tip, a half-moon
explorer, tweezers, glass plates, cement spatulas,
cement stoppers, a plastic filling instrument,
a headlamp, pot urine, surgical masks, surgical
gowns, surgical nurse caps, gloves, cutting tools
and a digital camera.
Subjects
The procedures used in this study were in
accordance with the ethical standards instituted
by the Health Research Ethical Clearance
Commission of the Universitas Airlangga
Faculty of Dental Medicine No: 259/HRECC.
FODM/V/2020. The subjects were male Wistar
rats
(Rattus novergicus)
between 12-18 weeks
old with a body weight of around 200-300 grams
each. The sample number was calculated using
the Federer formula of (t-1) (n-1) ≥15 [17]. Six
groups were created, with six rats for each group
and 36 samples in total were obtained from
that estimation. The groups were divided into
Ca(OH)2 and propolis extract groups, Ca(OH)2
groups and control groups. The tools used in
this research were sterilized in an autoclave at
121 °C for 30 minutes. During the procedure in
the treatment group, the tools used were rst
disinfected with 70% alcohol for aerobic bacterial
decontamination [18]. All rats were prepared
with povidone-iodine antiseptic at the injection
site, then anesthetized by peritoneal injection
with ketamine HCl and xylazine HCl. The incisal
surface of the mandibular central incisor was cut
4 mm, and the cavity was prepared on the incisal
surface using a low-speed handpiece with a round
tapered ring bur (diameter 0.8 mm) until the pulp
chamber perforated. The depth of preparation
was estimated to be 1.5 mm. Perforation of the
pulp chamber was accomplished using a probe
(0.46 mm tip diameter). Then the cavity was
dripped with sterile saline solution and dried
with a cotton pellet and paper point. In groups
3 and 4, Ca(OH)2 mixed with distilled water
was applied until its thickness reached 0.8 mm.
In groups 5 and 6, Ca(OH)2 combined with
propolis extract was applied until its thickness
was 0.8 mm. A carrier was used to apply the
material on the pulp surface, and the material was
compacted with an ultrane micro brush. After
applying the pulp capping material, the cavity
was restored with a Cention filling material.
The rats were observed in the cage after being
labeled. At the end of the experimental period of
12 and 24 h after procedure, the animals were
euthanized by guillotine decapitation. Sections of
the teeth were obtained. The specimens were the
alveolar bone and the teeth; they were taken, and
decalcication continued. The specimens were
xed for four days using a 10% formaldehyde
solution. Decalcication was accomplished using
a 10% EDTA solution 10% for 30 days. The EDTA
solution was changed every day. The specimen
was cut at the pulp site and xed again for 24 h
using 2% sodium sulfate. The specimens were
placed in parafn wax, labeled, then sliced using
a 6µm width microtome and put on a glass plate.
Application of the combination of calcium
hydroxide and propolis
After pulp chamber perforation in the
mandibular central incisor, the 36 rats were
divided into six groups:
Group 1: Control group. The pulp chamber is
applied with Cention. Decapitation after 12 h;
Group 2: Control group. The pulp chamber is
applied with Cention. Decapitation after 24 h;
Group 3: Treatment group. The pulp chamber is
applied with calcium hydroxide. Decapitation
after 12 h;
Group 4: Treatment group. The pulp chamber is
applied with calcium hydroxide. Decapitation
after 24 h;
Group 5: Treatment group. The pulp chamber
is applied with a combination of calcium
hydroxide and propolis. Decapitation after 12 h;
Group 6: Treatment group. The pulp chamber
is applied with a combination of calcium
hydroxide and propolis. Decapitation after 24 h.
Histopathology and immunohistochemistry
A light microscope (Olympus BX51, Olympus®,
Tokyo, Japan) was used to perform histopathology
and immunohistochemistry. The calculation was
done by counting brown dots as visualized at
the site. The brown color formed because of the
reaction of DAB (diaminobenzidine) and HRP
(enzyme peroxidase). Two different people did the
calculation, and then the average was obtained.
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Widjiastuti I et al.
The effect of calcium hydroxide in combination with propolis extract on decreasing nerve growth factor and substance P expression in rat dental pulp neuron
Widjiastuti I et al. The effect of calcium hydroxide in combination with propolis
extract on decreasing nerve growth factor and substance
P expression in rat dental pulp neuron
Statistical analysis
All data are expressed as means and SD. This
research was experimental. Levene’s test was used
to test homogeneity. Saphiro-Wilk was chosen for
the normality test because the sample was less than
50. One-way ANOVA testing was implemented
to determine whether there was an effect on the
treatment group. Tukey’s honestly significant
difference (HSD) test measured the differences
between each group post hoc. Results were considered
statistically different at p < 0.05. Data were analyzed
using SPSS version 25.0 for Windows.
RESULTS
Histopathology (HPA) was done to make
sure the sliced specimens were exemplary.
Figure 1 shows the histopathology results.
After confirmation with histopathology,
immunohistochemistry (IHC) was done to
determine the differences between NGF and SP
expression (Figure 2 and Figure 3). The mean
and standard deviation results of NGF IHC can
be seen in Table I and SP in Table II.
Normality and homogeneity tests were
carried out prior to data analysis. The results of
the Saphiro-Wilk test for normality and Levene’s
test for homogeneity obtained
p
> 0.05 for all
treatment groups, establishing that the data
were normally distributed and homogeneous.
Then the ANOVA test was conducted to see
if the treatment group affected NGF and SP
expression. In this test,
p
= 0.000 (
p
< 0.05) at
12 h and 24 h indicated a signicant difference
between the treatment groups regarding NGF
and SP expression. The analysis was continued
post hoc using Tukey’s HSD test to determine
the differences in each group. NGF results are
reported in Tables III and IV, and Tables V and VI
show SP results.
Figure 1 - Histopathology morphology pulp nerve cells of rat lower incisor (A) 100x magnification, (B) 400x magnification.
Table I - Sample size, mean, and standard deviation of NGF expression at 12 and 24 h
Group N
12 h 24 h
Mean SD Mean SD
Ca(OH)2 + Propolis Extract 6 3.83 2.137 3.17 1.169
Ca(OH)26 6.00 1.789 4.83 2.229
Control 6 11.67 2.503 12.33 2.582
Table II - Sample size, mean, and standard deviation of NGF expression at 12 and 24 h
Group N
12 h 24 h
Mean SD Mean SD
Ca(OH)2 + Propolis Extract 6 4.00 1.673 2.83 1.472
Ca(OH)26 10.83 2.317 12.17 2.317
Control 6 11.17 2.994 12.67 2.066
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Widjiastuti I et al.
The effect of calcium hydroxide in combination with propolis extract on decreasing nerve growth factor and substance P expression in rat dental pulp neuron
Widjiastuti I et al. The effect of calcium hydroxide in combination with propolis
extract on decreasing nerve growth factor and substance
P expression in rat dental pulp neuron
Figure 2 - Immunohistochemistry pulp nerve cells of rat lower incisor that express NGF at 1000x magnification. Black arrows indicate NGF
expression at 12 h and 24 h: (A) NGF expression after perforation at 12 h; (B) NGF expression after Ca(OH)2 application at 12 h; (C) NGF
expression after application of the combination of Ca(OH)2 and propolis at 12 h; (D) NGF expression after perforation at 24 h; (E) NGF
expression after Ca(OH)2 application at 24 h; (F) NGF expression after application of the combination of Ca(OH)2 and propolis at 24 h.
Figure 3 - Immunohistochemistry pulp nerve cells of rat lower incisor that express SP at 1000x magnification. Black arrows indicate NGF
expression at 12 h and 24 h: (A) SP expression after perforation at 12 h; (B) SP expression after Ca(OH)2 application at 12 h; (C) SP expression
after application of the combination of Ca(OH)2 and propolis at 12 h; (D) SP expression after perforation at 24 h; (E) SP expression after
Ca(OH)2 application at 24 h; (F) SP expression after application of the combination of Ca(OH)2 and propolis at 24 h.
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Braz Dent Sci 2024 Jan/Mar; 27 (1): e4023
Widjiastuti I et al.
The effect of calcium hydroxide in combination with propolis extract on decreasing nerve growth factor and substance P expression in rat dental pulp neuron
Widjiastuti I et al. The effect of calcium hydroxide in combination with propolis
extract on decreasing nerve growth factor and substance
P expression in rat dental pulp neuron
DISCUSSION
This study used Wistar rats as animal models
because apart from being easy to handle and
relatively economical compared to primates,
the rat’s dental pulp reaction to a substance is,
in principle, similar to the reaction in human
dental pulp [19]. The selection of lower incisors
in rats was based on the consideration that the
structure of the rat incisors was the same as the
rat molar teeth. The molars’ innervation differs
from the incisors’ innervation only in that they
project toward the roof of the pulp chamber and
the pulp horn, where the radicular and coronal
structures are formed. In addition, the nerve
innervation of the rat incisor teeth is straighter
and does not form a woven nerve plexus in the
sub-odontoblastic area. In contrast, the rat molar
tooth’s nerve innervation does form a woven
nerve plexus in the sub-odontoblast area [20].
It is considered easier to read and make histology
with the incisors’ simpler structure and larger
morphology than the molars.
In this study, observations were made about
the expression of NGF and SP because NGF
is a neurotrophic factor neuropeptide, a well-
known mediator for persistent pain, and has a
role in inammatory hyperalgesia [21,22]. SP is
considered a signicant mediator of neurogenic
inammation and associated hyperalgesia and is a
promising target for therapy aimed at controlling
pain and minimizing the adverse consequences
of tissue injury [7].
The control group showed significant
differences compared with the Ca(OH)2 group
and the Ca(OH)2-propolis extract group. This
means that the release of the neuropeptides
NGF and SP was mainly observed in the control
group. Cention restorative materials are alkasite
restorative materials, a new category of lling
material such as compomer, a subgroup of the
composite material class. This new category of
materials uses alkaline llers that can release
acid-neutralizing ions. Cention N is a self-
cure, tooth-colored direct restorative filling
material with an additional light-curing option.
Table III -
Multiple Comparison
Tukey HSD between groups on NGF expression at 12 h
Group Ca(OH)2 + Propolis Extract Ca(OH)2Control
Ca(OH)2 + Propolis Extract
Ca(OH)20.225
Control < 0.001 0.001
Table IV -
Multiple Comparison
Tukey HSD between groups on NGF expression at 24 h
Group Ca(OH)2 + Propolis Extract Ca(OH)2Control
Ca(OH)2 + Propolis Extract
Ca(OH)20.372
Control < 0.001 < 0.001
Table V -
Multiple Comparison
Tukey’s HSD between groups on SP expression at 12 h
Group Ca(OH)2 + Propolis Extract Ca(OH)2Control
Ca(OH)2 + Propolis Extract
Ca(OH)2< 0.001
Control < 0.001 0.968
Table VI -
Multiple Comparison
Tukey’s HSD between groups on SP expression at 24 h
Group Ca(OH)2 + Propolis Extract Ca(OH)2Control
Ca(OH)2 + Propolis Extract
Ca(OH)2< 0.001
Control < 0.001 0.901
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Widjiastuti I et al.
The effect of calcium hydroxide in combination with propolis extract on decreasing nerve growth factor and substance P expression in rat dental pulp neuron
Widjiastuti I et al. The effect of calcium hydroxide in combination with propolis
extract on decreasing nerve growth factor and substance
P expression in rat dental pulp neuron
Cention can release uoride ions, calcium ions,
and hydroxide ions. The reactive parts of resin-
based restorative materials are monomers, along
with initiators, catalysts, and other additives.
The monomers contained in Cention N are
urethane dimethacrylate (UDMA), tricyclodecan-
dimethanol dimethacrylate, etramethyl-xylylen-
diurethane dimethacrylate and polyethylene glycol
400 dimethacrylate. Dimethacrylate contains
an irritant that can cause inammation [23].
UDMA exposure has the potential to induce
inammation and toxicity in pulp cells, where it
can cause cell cycle dysregulation and trigger the
accumulation of reactive oxygen species (ROS).
An imbalance between ROS and antioxidants
causes oxidative stress, which in turn will
trigger tissue inflammation [24]. This tissue
inammation is thought to be related to the high
expression of NGF and SP in the control group.
The study’s results on NGF expression at
12 h showed a signicant difference between the
control group with Ca(OH)2 and the control group
with the combination of Ca(OH)2 and propolis.
This was also the case with NGF expression at
24 h. The results of this study indicate that the
application of Ca(OH)2 can reduce NGF expression
because Ca(OH)2 can dissolve NGF per the opinion
of Tomson et al. [25], who state that Ca(OH)2 can
dissolve several growth factors including stem
cell factor (SCF), macrophage colony-stimulating
factor (M-CSF), granulocyte-macrophage colony-
stimulating factor (GM-CSF), insulin-like growth
factor-binding proteins-1 (IGFBP-1), NGF and
glial cell line-derived neurotrophic factor (GDNF).
Rosa et al. [26], share this opinion and note that
pulp capping materials such as mineral trioxide
aggregate and Ca(OH)2 can dissolve dentin
bioactive molecules such as TGF-β1, NGF, and
GDNF, stimulating tertiary dentinogenesis.
The avonoid content in propolis, in addition
to inhibiting the COX2 enzyme so that it suppresses
the synthesis of PGE2 and reduces NGF expression,
also has a Caffeic acid effect that can induce
NGF [27,28]. Consequently, there was no
signicant difference between the 12- and 24-h
measures of NGF expression in the Ca(OH)2 group
compared with the Ca(OH)2-propolis combination
group. In addition, the ingredients in propolis,
such as quercetin and artepillin C, also have
a neuroprotective effect and can induce NGF.
These results are consistent with Ni et al. [29],
which indicated that the content of artepillin C
in propolis could increase the production of NGF,
and also supported by Xu et al.’s [30], study which
noted that 33 identied kinds of avonoids could
induce the synthesis and secretion of neurotrophic
factors including NGF, GDNF and brain-derived
neurotrophic factor (BDNF).
The expression of SP at 12- and 24-h
results showed there were signicant differences
between the control group and the Ca(OH)2-
propolis combination group, as well as with the
comparison between the Ca(OH)2 group and
the Ca(OH)2-propolis combination group. These
results establish that the administration of propolis
creates the most signicant results in reducing
pain, as evidenced by the drastic decrease in SP
expression. This decrease in SP occurs because
the acidic environment (acidosis) produced by the
inammatory reaction is inhibited, so sensitization
to TRPV1, which triggers the release of SP, is also
inhibited. TRPV1 is a cation permeable channel
expressed in nociceptive bers and is responsible
for detecting noxious stimuli from the periphery
such as low pH, temperature rise (> 42 °C),
changes in osmolality, AA metabolites, second
inammatory messengers and capsaicin (an irritant
in chili peppers) [31]. Acidosis conditions up to pH
6 make TRPV1 sensitive to agonists, including heat
and capsaicin, whereas acidosis, when pH < 6, can
directly open the receptor gate [32]. However, the
presence of OH- ions, which have a high pH, can
neutralize the acidic environment produced by the
inammatory reaction so that TRPV1 activation
is inhibited [33,34]. With inhibition of TRPV1
activation, the release of SP will also decrease.
The decrease in SP expression was also due to
the content of propolis ingredients. Propolis has
anti-inammatory components, such as avonoids
and caffeic acid phenethyl ester (CAPE), which
can inhibit eicosanoid synthesis from arachidonic
acid and suppress the activity of COX1 and COX2
enzymes. This occurs because it inhibits the
release of inammatory mediators such as PGE2,
leukotrienes, and thromboxane, causing an increase
in IL-10 expression with an increasing propolis
dose. The decrease in prostaglandins is caused
by the inhibition of prostaglandin synthesis from
arachidonic acid, catalyzed by the transformation
of the cyclooxygenase enzyme produced by
galangin compounds from avonoids and CAPE.
Furthermore, galangin from avonoid materials
can also inhibit leukotrienes from arachidonic
acid lipoxygenase (LOX) enzymes [35], and the
quercetin component in propolis can inhibit LOX
enzymes [27,36].
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Braz Dent Sci 2024 Jan/Mar; 27 (1): e4023
Widjiastuti I et al.
The effect of calcium hydroxide in combination with propolis extract on decreasing nerve growth factor and substance P expression in rat dental pulp neuron
Widjiastuti I et al. The effect of calcium hydroxide in combination with propolis
extract on decreasing nerve growth factor and substance
P expression in rat dental pulp neuron
There was no significant difference in SP
expression in the Ca(OH)2 control group at 12 and
24 h. From this analysis, it can be concluded that
the Ca(OH)2 administration cannot signicantly
suppress pain. This is because when pure Ca(OH)2
is applied to the pulp, it will destroy some of the
pulp tissue causing continuous inflammation,
according to Dwiandhono et al. [37], Inammation
that occurs can trigger the release of SP so that SP
expression in the Ca(OH)2 group was not different
from the control group. The SP expression that
did not experience a signicant decrease due to
the administration of Ca(OH)2 may have occurred
because the continuous Ca(OH)2 solubility process
made the Ca2+ concentration high. Calcium
receptors on cells can thus act as sensors for
increased concentration and activate chemotaxis
from deeper tissues to the affected site. Increasing
the concentration of Ca2+ ions may increase
calcium inux through the VGCC gate and increase
intracellular Ca2+, which can increase SP release
from nerves [38]. It is expected that this research
can be used as the basis for the development of a
combination of calcium hydroxide and propolis
extract as an alternative material for pulp capping
processing in humans. This research is limited by
the the lack of investigation into the antimicrobial
properties of propolis and the inadequacy of
considering different propolis extraction techniques.
CONCLUSION
SP expression in the combination of Ca(OH)2
with propolis extract exhibit a signicant decrease
compared to the application of Ca(OH)2. NGF
expression in the combination of Ca(OH)2 with
propolis extract did not show a signicant decrease
compared to the application of Ca(OH)2.
Acknowledgements
All co-authors have seen and agree with the
contents of the article and there is no nancial
interest to report.
Author’s Contribution
IW: Conceptualization, Methodology, Writing –
Original Draft Preparation, Supervision, Validation. S:
Writing – Review & Editing, Supervision, Validation.
GAS: Writing – Review & Editing, Software, Formal
Analysis, Resources, Data Curation. KFB: Writing –
Review & Editing, Visualization, Investigation, Data
Curation, Project Administration, Funding Acquisition.
Conict of Interest
No conicts of interest were declared concerning
the publication of this article.
Funding
The authors declare that no nancial support
was received.
Regulatory Statement
Before the commencement of research, the
ethical approval for the current study was obtained
from the Health Research Ethical Clearance
Commission of the Universitas Airlangga Faculty of
Dental Medicine. The approval code for this study
is No. 259/HRECC.FODM/V/2020.
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Ira Widjiastuti
(Corresponding address)
Universitas Airlangga, Faculty of Dental Medicine, Department of
Conservative Dentistry, Surabaya, Indonesia.
Email: ira-w@fkg.unair.ac.id Date submitted: 2023 Aug 17
Accept submission: 2024 Jan 11
