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.2023.e3620
1
Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-treated
hamster pouch
Efeito da Timoquinona na regeneração muscular esquelética através da avaliação da expressão das proteínas Pax-7 e Myo-D
em bolsa jugal de hamsters tratados com DMBA
Sara Ali Swidan
1
, Samia Elazab
2
, Magda Mohamed Aly HASSAN
1
1 - Departement of Oral Pathology, Suez Canal University, Faculty of Dentistry, Ismailia, Egypt
2 - Departement of Oral Pathology, Galala University, Suez, Egypt.
How to cite: Swidan SA, El Azabb SA, Hassan MMA. Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7
and Myo-D expression in the DMBA-treated hamster pouch. Braz Dent Sci. 2023;26(2):e3620. https://doi.org/10.4322/bds.2023.e3620
ABSTRACT
Objective: Pax-7 and Myo-D regulate satellite cells’ activation and differentiation, thus muscle regeneration
following damage. This research aimed to investigate the effect of Thymoquinone (TQ) on skeletal muscle
regeneration following 7,12-dimethylbenz-(a)-anthracene (DMBA)-induced injury in the hamster buccal pouch
via immunohistochemical assessment of Pax-7 and Myo-D expression. Material and Methods: 65 male golden
Syrian hamsters were divided into 3 groups: Group 1: (n=5) received no treatment. Group 2: (n=20) served
as a positive control. The left buccal pouches were painted with the carcinogen 3/week/ 6weeks. Group 3:
(n=40) were subdivided into two equal sub-groups as follows: Group 3a: (n=20) were given one i.p. TQ
injection. Group 3b: (n=20) were given two i.p. TQ injections. Five animals from each group (2 and 3) were
euthanized at 24, 48 hrs, one, and two weeks after the last injection. A blood sample (2 ml) was withdrawn for
assessment of TNF-α levels in serum. Serial sections of the pouches were examined histologically (H&E), and
immunohistochemically (IHC) for the detection of Pax-7 and Myo-D proteins. Results: double i.p injections of
TQ resulted in a signicant elevation in the level of TNF-α from the second-day post-injection with a progressive
formation of the muscle bers (MFs) and mononuclear cells (MNCs) around the deeper blood vessels. At 14
days, no statistically signicant difference was found between this group and group ‘2’, while the difference
remained signicant compared to groups ‘1’ and ‘3a’. The muscle bers were more mature and compact. IHC
results showed positive expression of the perivascular mononuclear cells (MNCs) to both Pax-7 and Myo-D
with positive reactivity of the peripheral nuclei of muscle bers to Pax-7 compared to the negative reaction in
the positive control group. Conclusion: early and two TQ injections had a promising effect on the induction of
striated muscle regeneration, mainly by non-myogenic stem cells.
KEYWORDS
Thymoquinone; Pax-7; Myo-D; Buccal pouch; Muscle regeneration.
RESUMO
Objetivo: Pax-7 e Myo-D regulam a ativação e diferenciação de células satélites durante a regeneração muscular
pós-trauma. Assim, objetivamos investigar o efeito da timoquinona (TQ) na regeneração muscular esquelética
após injúria causada por 7,12-dimetilbenzantraceno (DMBA) em bolsa jugal de hamsters, através da análise
imuno-histoquímica de Pax-7 e Myo-D. Material e Métodos: 65 hamsters-sírios machos foram divididos em 3
grupos: Grupo 1: (n=5) controle negativo, sem tratamento. Grupo 2: (n=20) controle positivo. A bolsa jugal
do lado esquerdo recebeu aplicação do DMBA por 3 e 6 semanas. Grupo 3: (n=40) receberam aplicação de
2
Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
INTRODUCTION
The damaged skeletal muscle cells
can regenerate through myogenesis [1].
The muscle-resident stem cells (i.e., satellite
cells) are the key chain in the process of
classic myogenesis. In brief, a comprehensive
review by Musarò (2014) [2], had pointed
out to the essential five inter-related and
time-dependent phases for that classic way of
muscle regeneration [2]. Starting with muscle
bers’ necrosis (a transient inammatory step),
followed by regeneration through activation of
stem cell populations (either satellite or other
non myogenic stem cells). Then remodeling of
the extracellular matrix and angiogenesis. Last
phase is reinnervation of regenerated bers [2].
Skeletal muscle injury can result from
induction of epithelial dysplasia in hamster buccal
pouch/7,12-dimethylbenz-(a)-anthracene(HBP/
DMBA) model. It is one of the most well-
characterized tumor-induction models that
simulate that occurs in humans [3]. The early
few DMBA paintings result in necrosis of the
distal pouch, with a reduction of its length from
about 5-6 cm to about 2 cm. The pouch does not
regain its full length even after suspending DMBA
painting [4].
Thymoquinone (TQ) [2-isopropyl-5-
methyl-1, 4- benzoquinone (C10H12O2)], is a
major active constituent of
Nigella sativa
(NS)
which is a dicotyledon of the
Ranunculaceae
family [5]. TQ has been extensively studied in
vivo and in vitro and was proven to have many
therapeutic properties including an anticancer
activity [5,6].
TNF-α is expressed by many cells including
monocytes, macrophages, natural killer cells,
and myoblasts [7]. In the scope of muscle
regeneration, TNF-α was found to be expressed
in damaged muscle fibers and perform dual
roles depending on its concentration [8]. At high
concentration, it suppresses myogenesis via
proteolysis of Myo-D, while inducing it at lower
concentrations [9].
Myo-D is a member of the myogenic regulatory
factors (MRFs) that include Myo-D, MRF4,
myogenin, and Myf5 as well [10]. Early after
muscle injury, Myo-D and Myf5 are upregulated
in activated muscle stem cells (satellite cells) as a
primary response to muscle injury; alternatively,
they are not expressed in quiescent satellite
cells [11,12].
The successive expression of key transcription
factors drives satellite cell development through
the myogenic pathway [2]. Pax7 is a paired box
transcription factor that is found in all mature
muscle satellite cells and is required for their
function. Pax7 downregulation is required for
terminal differentiation, whereas Pax7 elevation
after proliferation favors a return to the rest
state [13].
Quiescent satellite cells express the
transcription factor Pax7, which co-expressed
with Myo-D when the cell is activated. Then,
most cells proliferate, downregulate Pax7, and
differentiate. On the other hand, others keep
Pax7 but lose Myo-D, returning to a condition that
resembles quiescence [14]. Other non-myogenic
cells were found to contribute to differentiating
injured muscle cells. These nonmyogenic cells are
DMBA e foram então subdivididos em: Grupo 3a: (n=20) que recebeu 1 injeção intraperitoneal (ip) de TQ e
Grupo 3b: (n=20) que recebeu duas injeções ip de TQ. Cinco animais dos grupos 2 e 3 foram eutanasiados em
24 horas, 48 horas, 7 dias e 14 dias após a administração de DMBA e da última injeção de TQ. Amostras de
sangue (2 ml) foram coletadas para avaliação dos níveis séricos de TNF-α. Cortes seriados da bolsa jugal dos
animais foram analisados histologicamente (H&E), e através de imunohistoquimica (IHC) para avaliação das
proteínas Pax-7 e Myo-D. Resultados: duas injeções ip de TQ aumentaram os níveis séricos TNF-α à partir do
segundo dia pós-administração com formação progressiva de bras musculares (MFs) e células mononucleares
(MNCs) ao redor dos vasos sanguíneos. No dia 14, não houve diferença estatística entre o grupo 3b e o grupo
2, enquanto a diferença permaneceu entre o grupo 1 e 3a. As MFs apresentavam-se mais maduras e compactas.
A IHC mostrou expressão de Pax-7 e Myo-D nas MNCs ao redor dos vasos, e houve expressão nuclear de Pax-7
nas MFs no grupo 2. Conclusão: ambos regimes de administração do TQ, 1 ou 2 aplicações ip, apresentaram
efeito promissor na indução da regeneração muscular esquelética, principalmente nas células não-miogênicas.
PALAVRAS-CHAVE
Timoquinona; Pax-7; Myo-D; Bolsa jugal; Regeneração muscular.
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
bro-adipogenic precursors (FAPs), bone marrow-
derived stem cells (mesenchymal stem cells), and
pericytes [15]. In conclusion, this research aimed
to prove the assumption that Thymoquinone
(TQ) may stimulate skeletal muscle regeneration
following DMBA-induced injury in the hamster
buccal pouch via immunohistochemical assessment
of Pax-7 and Myo-D expression.
MATERIAL AND METHODS
Chemicals and reagents
Dimethylbenz-(a)-anthracene [or
7,12-Dimethylbenz-(a)-anthracene (DMBA)]
dissolved in heavy mineral oil (Sigma-Aldrich
Company, Saint Louis, Mo, USA). DMBA (0.5%)
solution was prepared by dissolving 1 gram in
200 mL of heavy mineral oil. The carcinogen
was topically applied to the left hamster buccal
pouches (HBPs) using number 4 camel hair brush.
Thymoquinone (TQ) dissolved in propylene glycol
(Sigma-Aldrich Company, Saint Louis, Mo, USA).
TQ solution was dissolved in propylene glycol to
get a solution of concentration 10mg/ml. one mg
dose per hamster was injected intraperitoneally.
Sixty-five male Syrian golden hamsters
(Mesocrietus auratus), 14 weeks old, with
body weight 100-120 grams, were kept ve per
cage in a well-ventilated room with controlled
temperature, 50-70% humidity, and 12 hours
day/night cycle, in the animal house and were
given water and recommended diet ad libitum.
Ethical approval had been obtained from the
institutional ethical review committee and the
experiment followed the “ARRIVE” guidelines for
handling and treating the experimental animals.
Experimental design
The hamsters were divided into 3 groups:
Group 1: 5 animals received no treatment
and served as the negative control
group. They were euthanized on
the day of starting treatment of
other groups.
Group 2: 20 animals served as a positive
control group. The left buccal
pouches were painted with the
DMBA (3 / week / 6 weeks).
Group 3: 40 animals were painted with
DMBA as in group 2, then
subdivided into two equal sub-
groups as follows:
Group 3a: 20 animals were given one intra-
peritoneal (i.p.) TQ injection.
Group 3b: 20 animals were given two i.p.
TQ injections every other day.
Five animals from each group were
euthanized at 24, 48 hrs, one, and two weeks
after the last injection (Figure 1).
SBefore the process of euthanization, the
animals were anesthetized by a cotton-soaked
with ether inside a rmly closed glass container.
A blood sample (2 ml) was withdrawn from the
retro-orbital venous plexus of the eye into a sterile
tube with EDTA, for assessment of TNF-α levels.
The data were statistically analyzed using One-
Way Analysis of Variance (ANOVA) via SPSS® 22.
After euthanization, by inhalation of a lethal
dose of ether, both right and left pouches were
surgically excised, xed in 10% neutral formalin,
and embedded in soft paraffin wax. Sections
of 5μm were cut using a rotary microtome
then mounted on glass slides and stained for
hematoxylin and eosin (for light microscopic
study) and diagnosed by two pathologists.
Serial sections of 5 μm were cut from each
parafn block and mounted on a positively charged
glass slide (Super Frost\Plus slides) for IHC
staining to detect Pax-7 and Myo-D expression,
following the manufacturer’s instructions.
Pax-7 polyclonal (Rhabdomyosarcoma Marker),
(AB clonal, Catalog No. A7335), and Myo-D
(Rhabdomyosarcoma Marker) (clone5.8A&MYD
712, catalog No. RA0233-C.5), were used.
Histopathologic analysis (H&E)
Oral epithelial dysplasia (OED) was graded,
with modification, according Bánóczy and
Csiba (1976) [16] classification into: mild:
when fewer than three dysplastic parameters
were present. Moderate: when three to seven
dysplastic parameters were present. Severe: when
more than seven parameters were present, and
carcinoma in situ: when any number of dysplastic
parameters are distributed from top to bottom or
when all the parameters were seen with an intact
basement membrane [16].
Immunohistochemical analysis (IHC)
Sections were dewaxed by xylene, dehydrated
in ethanol, then rinsed with distilled water.
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
The slides were immersed in 3% H
2
O
2
for 10 min
and rinsed in distilled water for another 15 min.
For antigen retrieval protocol, the slides were
boiled in a microwave oven for 15 min with citrate
buffer to unmask the antigenicity, then cooled
at room temperature for 20 minutes. Primary
antibodies were added to sections and incubated
overnight at 4°C. They were then rinsed in PBS
(phosphate-buffered saline) three times for two
minutes each. Two drops (100ul) of poly HRP
conjugate were added to cover the tissue, and
incubated for 15 min. The sections were rinsed
with wash buffer for ten min. The slides were
then incubated with a DAB chromogen solution
and incubated at room temperature for ve min.
The sections were counter- stained by dipping in
Harris hematoxylin for one minute and rinsed
under running water. After dehydration in ethanol,
they were immersed in xylene, and coverslips were
applied with synthetic adhesive resin (DPX).
Digital image analysis
The slides were photographed by Olympus
E-330 Evolt Digital Photography camera
using Image Analyzing System (Olympus
BX50 Microscope). Computerized image analysis
of IHC staining “Image J, 1.41a, NIH, USA”
software was used for quantitative IHC analysis
of Pax-7 and Myo-D. A sample of the immune-
positive area was chosen and applied to image J,
then the image was adjusted to 8 bits followed
by thresholding of the whole positive area.
Measurement of the thresholded area was nally
done by the program and the area fraction was
obtained. The mean fraction of the positive
cells for each marker per group was calculated
depending on the following key:
Score 0: Negative stain.
Score 1: when less than 15 positive cells
were found.
Score 2: when 15 to 25 positive cells were
found.
Score 3: when 26 to 50 positive cells were
found.
Score 4: when more than 50 positive cells
were found.
Statistical analysis
The data were analyzed using [One-
Way Analysis of Variance (ANOVA)] via SPSS®
22, for determination of relative protein
expression and evaluation of the serum TNF-α
levels. All values were expressed as mean
± standard deviation. The difference was
considered signicant at a p value ≤ 0.05.
RESULTS
Serum TNF-α level assessment
Statistically signicant elevation (P=0.021)
of the serum TNF-α level was detected in the
Figure 1 - Schematic representation of the experimental timeline and design.
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
positive control group compared to the negative
control group. Statistically signicant elevation
in the level of serum TNF-α one day following
a single TQ injection was recorded between
group ‘3a’, compared to groups ‘1’ (p ≤ 0.002),
and ‘2’ (p ≤ 0.013). The elevation remained
statistically signicant compared to groups ‘1’
(p ≤ 0.003) and ‘2’ (p ≤ 0.024) at 14 days post-
injection. Regarding the levels of serum TNF-α
in group ‘3b’ one day post double TQ injections,
statistically signicant elevation was recorded in
its level compared to groups ‘1’ (p ≤ 0.00), and
group ‘2’ (p ≤ 0.031). At 14 days post-second
injection, no statistically signicant difference
was found between this group and group
‘2’(P=0.631), while the difference remained
signicant compared to groups ‘1’ (p ≤ 0.002)
and ‘3a’ (p ≤ 0.00) (Figure 2).
Clinical results
Both left and right pouches of the animals
in group 1 (negative control) appeared healthy
and normal, with a mean length of 5.2 cm
(Figure 3A, left).
In group 2, the left pouches of the DMBA-
reated animals showed exophytic masses and/
or ulcers and severe inammation with necrotic
distal end. The left pouch’s length was reduced
to a mean of 2 cm (Figure 3A, right).
The left pouches of animals in group (3a)
euthanized after one day of the single TQ
injection, showed gross changes comparable to
those observed in group B. The mean length of the
pouches was 2.2 cm (Figure 3B) which increased
to reach about 3.5 cm at 14 days post-TQ injection
(Figure 3D).
The left pouches of euthanized animals in
group (3b) showed a reduced area of a distal
necrosis and decreased number and size of the
exophytic masses in comparison to groups 2 and
3a. The mean left pouches’ length was 3cm, one
day after the second TQ i.p. injection (Figure 3C)
further increased to about 4.3 cm at 14 days
post-second TQ injection (Figure 3E). The right
pouches of animals in groups 2, 3a, and 3b
appeared normal as in group 1.
Histopathologic results (H&E stain)
Day 1 post last injection
(Group 1): the mucosa of the pouch
appeared normal with regular
epithelial stratification, and
normal underlying connective
tissue. The muscle layer is
formed of scattered mature
muscle bers. The same nding
was seen in the right pouches of
all other groups (Figure 4A).
(Group 2): the left pouches revealed
degenerated distal end, with
different grades of epithelial
Figure 2 - Representative graph of serum TNF-α levels in different
groups at different time intervals: *p ≤ 0.05 compared to group “2”;
#p ≤ 0.05 compared to group “1”.
Figure 3 - Clinical presentation of the pouches’ length of different groups at different time intervals. (A): (left) pouch length is almost 5cm in
Group1, and (right) the left pouch of an animal from group 2 at end of the experiment. (Day 1): (B & C): pouches of animals in groups 3a & 3b,
respectively. (Day 14): (D & E): pouches of animals in groups 3a & 3b, respectively, 14 days post TQ injections.
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
dysplasia from moderate
dysplasia to carcinoma in situ
(CIS). The underlying lamina
propria was highly inflamed,
with a loosely arranged muscular
layer, due to degenerated bers
and widely inltrating fat tissue
(Figure 4B).
(Group 3a): the lamina propria was inamed,
thicker, and more fibrotic
comparable to that of group 2.
(Group 3b): progressive formation of the MFs
and MNCs around the deeper
blood vessels (BVs) (Figure 4C).
Day 2 post last injection
The histopathological picture was
comparable to that in the previous section.
Day 7 post last injection
(Group 2): degeneration of the distal end
with focal supercial invasion
could be seen.
(Group 3a): the pouches showed moderate
dysplastic epithelium with
remnants of a necrotic area at
the distal end (Figure 4D). An
intra-epithelial accumulation
of inflammatory cells was
seen focally along the rest of
epithelial lining (Figure 4E).
Mesial to the necrotic area, the
underlying muscle bers (MFs)
were progressively forming
but loosely separated by fat
tissue. There was an increase
of mononuclear cells (MNCs)
Figure 4 - H&E-stained sections from different groups at different time intervals. (G1): (A) the normal pouch mucosa formed of thin keratinized
epithelium, collagenous lamina propria, and loose muscular layer. (Day 1): (G2) (B): showing superficial invasion. (G3b): (C) thick, collagenous
lamina propria and the MFs were progressively forming but loosely separated. (Day 7): (G3a): (D): the area near the necrotic end with MFs.
MNCs were found between MFs and around the BVs. (E): accumulation of inflammatory cells in the superficial epithelium along the pouch
length. (F): the MFs are mature and progressively forming but loosely separated by fat cells. MNCs are seen around the BVs. (G3b) (G): the
healed necrotic area at the distal end with newly formed MFs and MNCs between them. (H): multiple perivascular MNCs in areas of muscle
fiber’s formation (arrow), and intraepithelial inflammatory cells. (Day 14): (G3a) (I): remains of the distal necrotic end. (J): near the distal end,
the MFs were more compact. MNCs were around the BVs, and between MFs. (G3b) (K): along the pouches’ length, mature MFs were more
compact. (L): normal-appearing epithelium with no necrosis at the distal end. The lamina propria was thin and free of inflammatory cells.
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Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
around the blood vessels (BVs)
under the forming MFs, along
the pouches’ length (Figure 4F).
(Group 3b): healing of the distal necrotic end
with mild to moderate dysplastic
epithelium with a thinner
fibrotic lamina propria could
be seen. Newly formed MFs and
MNCs between them and around
blood vessels were prominent
findings (Figure 4G). Along
the pouch’s length, MFs were
more condensed and mature
(with peripheral nuclei). The
perivascular mononuclear cells
were abundant in the areas of
muscle formation. Aggregates of
inammatory cells could be seen
extruded from the epithelium
as a constant finding in the
TQ-treated group (Figure 4H).
Day 14 post last injection
(Group 2): severe dysplastic epithelium
with superficial invasion. The
underlying layer shows inamed
lamina propria and degenerated
MFs.
(Group 3a): the dysplastic criteria were mild
to moderate with a decrease in
the size of the distal necrotic
end. (Figure 4I-4J).
(Group 3b): normal-appearing epithelium
with focal hyperplasia, and
thinner inflammation-free
lamina propria could be seen
(Figure 4K). The necrotic tissue
at the distal end had completely
disappeared and the MFs were
more mature and compact
(Figure 4L).
Immunohistochemical results
Pax-7
Day 1 post last injection
(Group 1): IHC results of Pax-7 revealed
mild positively stained nuclei of
epithelial cells, broblast of the
lamina propria, and endothelial
cells (score +1). Whereas the
nuclei of MFs were negative
(score 0) (Figure 5A).
(Group 2): negatively stained nuclei of
epithelial cells and MFs (score
0) till the end of the experiment
(Figure 5B).
(Group 3a): negatively stained nuclei of
epithelial cells and MFs (score
0).
(Group 3b): negatively stained nuclei of
epithelial cells and MFs. Mild
positive reaction of MNCs (sore
+1) (Figure 5C).
Day 2 post last injection
(Group 3a): the positive reaction of MNCs’ and
MFs’ nuclei to Pax-7(score +1)
was rst recorded (Figure 5D).
(Group 3b): positive stain of the lamina
propria cells, the perivascular
MNCs, and some MFs peripheral
nuclei (score +2) was recorded.
(Figure 5E).
Day 7 post last injection
(Group 3a): the positive reaction to Pax-7
was more intense (score +2)
(Figure 5F).
(Group 3b): The positive stain intensity
of MNCs & MFs to Pax-7 was
sustained. (Figure 5G)
Day 14 post last injection
(Group 3a): milder immunoreactivity of the
peripheral nuclei of MFs and
MNCs to Pax-7 was recorded
(score +1) (Figure 5H).
(Group 3b): The positive reactivity to Pax-7
declined to score +1 (Figure 5I).
Myo-D
Day 1 post last injection
(Group 1&2): Negative expression of mature
muscle bers was recorded in
every time interval till the end
of the experiment (score 0)
(Figure 6A-6B).
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
Figure 5 - Pax-7 IHC stained sections from different groups. (G1): (A): mild positively stained nuclei of epithelial and connective tissue cells.
Negatively stained nuclei of MFs (arrow). (Day 1): (G2): (B): negatively stained nuclei of MFs (arrow) and nuclei of epithelial cells. (G3b): (C):
negatively stained nuclei of epithelial cells, and MFs (broad arrows). The nuclei of perivascular MNCs were positively stained (thin arrows).
(Day 2): (G3a): (D): positive stain of MNCs (thin arrows) and the peripheral nuclei of some MFs (wide arrows). (G3b): (E): negatively stained
nuclei of upper epithelial cells, positively stained nuclei of perivascular MNCs, and some MFs peripheral plaque (arrows). (Day 7): (G3a): (F):
positive stain of MNCs (narrow arrows) and peripheral nuclei of some MFs (wide arrows). (G3b): (G): positive reaction of MNC & MFs to pax-7
(arrows). (Day 14): (G3a): (H): positive stain of MNCs (narrow arrows), and peripheral nuclei of some MFs (wide arrows). (G3b): (I): positive
staining of the peripheral nuclei of some MFs (arrows).
Figure 6 - Myo-D IHC stained sections from different groups. (G1): (A): negative Myo-D reaction. (G2): (B): negative expression of the
multinucleated muscle fibers (arrows). (Day 7): (G3a) (C): positive reaction of mononuclear perivascular cells near the area of necrosis
(arrows). (G3b) (D): positive reaction of perivascular cells (arrow). (Day 14): (G3a) (E): positive immunoreaction of MNCs (arrow). (G3b) (f):
positive immunoreaction of MNCs and muscle fibers (arrows).
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Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
(Group 3a&3b): All the newly formed MF &
MNCs were negative to Myo-D
(score 0).
Day 2 post last injection
(Group 3a): The deeper MNCs around blood
vessels are positive for Myo-D
(score +1).
(Group 3b): Positive Myo-D expression of
MNCs was recorded (score +1).
Day 7 post last injection
(Group 3a): The deeper MNCs around blood
vessels were positive for Myo-D
(score +1) (Figure 6C).
(Group 3b): A positive reaction of the MFs to
Myo-D was rst recorded (score
+1) (Figure 6D).
Day 14 post last injection
(Group 3a): The deeper MNCs around blood
vessels were still positive to
Myo-D (score +1) (Figure 6E).
(Group 3b): Both MNCs and MFs were
positively stained to Myo-D
(score +2) (Figure 6F).
DISCUSSION
The present work aimed to evaluate the
inuence of TQ on skeletal muscle regeneration
following DMBA-induced muscle injury.
Shortening of the buccal pouch was induced
when DMBA was painted for consecutive 6 weeks.
The muscle regeneration potentiality of TQ
was assumed based on its documented anti-
inflammatory effect [17]. The animals were
euthanized on the 1st, 2nd, 7th, and 14th days
post single and double TQ injections. This
sequence was determined based on the classic
order of muscle satellite cells’ differentiation
as early as 2 days post-injury till maturation
after two weeks, with a focus on Myo-D and
Pax-7 expression sequence [1,2].
TNF-α is normally produced from activated
macrophages, CD4+ lymphocytes, NK cells,
neutrophils, mast cells, eosinophils, and other
stromal cells where it performs diverse cellular
events resulting in acute phase reactions [9].
This can explain the significant elevation of
TNF-α in group ‘2’ following DMBA painting
for 6 weeks compared to the negative control
group. This elevated level of TNF-α following
DMBA application was reported in comparable
research [18]. DMBA, despite being applied
topically has a systemic effect and is known
to have toxic effects on multiple organs [19].
This toxicity and severe inammatory effect are
presented in the current study by severe necrosis
of the painted animals’ pouches.
Levels of TNF-α were further elevated
following TQ injections in comparison to the
DMBA-only group. This can be possibly explained
by the effective systemic anti-inflammatory
activity of TQ, which resulted in the expulsion of
the formed TNF-α from the affected tissue cells to
the circulation. This explanation is demonstrated
in the present research via histological sections
of pouches from the TQ-treated groups, showing
absence of inflammatory cells (TQ-treated
groups), and necrosis in the DMBA-only painted
group. This nding was further supported by a
study in which the IHC staining technique was
used to detect the presence of the TNF-α and
NF-ĸB in the surface epithelium and inammatory
cells in the pouches of TQ-treated animals.
The researchers reported negative expression
of both markers in the surface epithelium and
inammatory cells as well [20].
Histologically, pouches of the negative control
group and right pouches of other groups showed
normal-appearing mucosa and loosely arranged
mature muscle bers (peripherally located nuclei)
were Myo-D negative. The left pouches of the
DMBA-painted pouches (positive control) showed
a necrotic distal end, that resulted in a signicant
reduction in the length of the pouch (average
2cm). This observation was reported in comparable
studies [4,20]. On the other hand, animals that
received one and two i.p. TQ injections, after
cessation of DMBA application, showed gradual
elongation in pouches’ length with no ulceration
or inflammation. They showed grossly and
histologically, normal-appearing epithelium with
inammatory-free collagenized lamina propria.
The muscle layer showed progressively increased
bulk till fourteen days after two TQ injections. This
result was reported in other studies [4,20,21].
Myo-D was negatively expressed in group
“2”, one possible explanation may be based on the
histopathological picture of the painted pouches
that revealed degenerated muscle fibers, with
hyperplastic and hyper-keratinized epithelium
10
Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
close to the necrotic end, with highly inamed
lamina propria. At the mesial end of the pouches,
the newly formed MFs were multinucleated,
so were Myo-D negative. Myo-D may have
been expressed during the carcinogen painting
(i.e. during the 6 weeks of DMBA painting).
The negative expression may be regarded to
destabilization of Myo-D caused by increased
TNF-α levels in NF-kappa B-dependent manner, as
reported by Langen et al. [22]. The authors reported
that TNF-α / NF-κB signaling pathways play an
essential role in the process of myogenesis [22].
Comparable studies had demonstrated the effect
of elevated TNF-α on the expression of Myo-D and
the process of myogenesis. When TNF-α was added
to proliferating myoblast, it inhibited myoblasts’
differentiation and downregulated Myo-D and
myogenin expression [22,23]. Coletti et al. [24]
reported muscle wasting and impaired muscle
regeneration following TNF-α gene transfer [24].
However, in the present study, Myo-D was
positive in MNCs, connective tissue cells near the
necrotic area, and lamina propria, suggesting that
these cells had the main role in myogenesis and
elongation of the pouches due to TQ injection(s).
In groups, 3a/3b, Myo-D negative expression
of the peripheral nuclei of newly formed muscle
bers, in early timing following TQ injection. This
possibly could be regarded to the non-myogenic
origin of these fibers that were positive for
Myo-D in early timings. However, the positive
reactivity of the nuclei of fat cells, interstitial
cells, and broblasts suggests stimulation of FAPs
cells’ differentiation into a myogenic lineage.
A comparable study suggested a possible transition
of brotic to myogenic linage as evidenced by
Masson’s trichrome stain [20]. The transition of
thick bulky brotic lamina propria into myogenic
cells, as they were positive to the proposed
markers, may explain the reduction of the thick
lamina propria after DMBA treatment to thinner
propria in the TQ- treated groups. This nding
indicates that the lamina propria was replaced by
the increased muscle bulk. Other studies reported
positive reactivity of broblasts to Myo-D during
differentiation into myogenic lineage [25,26].
The third proposed non-myogenic lineage
is the perivascular mononuclear cells (MNCs)
that were positive to Pax-7 and Myo-D at 24-
and 48-hours post-TQ injection(s), respectively.
The origin of these MNCs is questionable. Based
on their location int proximity to the local vessels
of the newly formed muscle bers, it is suggested
that their origin is either bone marrow-derived
mesenchymal stem cells or pericytes [27,28].
Positive expression of Pax-7 in the TQ-treated
groups was recorded in the nuclei of MNCs and
muscle bers starting from the 2nd day post-
injection. This observation could indicate the
activation of satellite cells from the second day after
injection and their proliferation at about seven
days post-injection. It was found that following
activation of satellite cells, Pax7 is activated
and then progressively became transcriptionally
inactive in most satellite cell progeny as they are
committed to differentiation [1,29]. This can
explain the decline of the staining intensity by
end of the experiment (at 14 days).
CONCLUSION
The present protocol introduces a novel
mechanism for skeletal muscle regeneration,
depending on non-myogenic cells, as revealed by
early maturation of new bers (peripheral nuclei),
as well as different sequences and localization of
Myo-D and Pax-7 expression (MNCs, and FAPs)
following muscle degeneration. This mechanism
was mainly due to the absence of TNF-α and
inammatory cells, following TQ injection.
Author’s Contributions
SAS: Conceptualization. SAS: Methodology.
SAS: Software. SAEA: Investigation. SAEA,
MMAH: Data Curation. SAS, MMAH: Writing –
Original Draft Preparation. SAEA, MMAH: Writing
– Review & Editing. SAEA: Visualization.
Conict of Interest
No conicts of interest declared concerning
the publication of this article.
Funding
The authors declare that no nancial support
was received.
Regulatory Statement
This study was conducted in accordance with
all the provisions of the local human subjects
oversight committee guidelines and policies of:
265/2020
11
Braz Dent Sci 2023 Apr/Jun;26 (2): e3620
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via assessment of Pax-7 and Myo-D expression in the DMBA-treated hamster pouch
Swidan SA et al.
Effect of Thymoquinone on skeletal muscle regeneration via
assessment of Pax-7 and Myo-D expression in the DMBA-
treated hamster pouch
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Sara Ali Swidan
(Corresponding address)
Suez Canal University, Faculty of Dentistry, Ismailia, Egypt.
Email: sara_sowidan@dent.suez.edu.eg
Date submitted: 2022 Aug 18
Accept submission: 2023 Apr 15
