International Journal of Biological Macromolecules 150 (2020) 253–260

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Sulfated polysaccharide from the green marine algae Caulerpa racemosa

reduces experimental pain in the rat temporomandibular joint
Natássia Albuquerque Ribeiro a, Hellíada Vasconcelos Chaves b,⁎, Renata Line da Conceição Rivanor a,
Danielle Rocha do Val d, Ellen Lima de Assis b, Felipe Dantas Silveira c, Francisco Isaac Fernandes Gomes b,
Hermany Capistrano Freitas e, Lorena Vasconcelos Vieira b, Deiziane Viana da Silva Costa f,
Gerly Anne de Castro Brito f, Mirna Marques Bezerra d, Norma Maria Barros Benevides a
a
Department of Biochemistry and Molecular Biology, Federal University of Ceará, Avenida Humberto Monte, s/n, Campus do Pici, 60455-760 Fortaleza, Ceará, Brazil
b
Faculty of Dentistry, Federal University of Ceará, Rua Coronel Estanislau Frota, Sobral, Ceará, Brazil
c
Graduate Program in Health Sciences, Federal University of Ceará, Avenida Comandante Maurocélio Rocha Pontes, 100 Derby, 62.042-280 Sobral, Ceará, Brazil
d
Graduate Program in Biotechnology, Northeast Biotechnology Network, Federal University of Pernambuco, Avenida Professor Moraes Rego, 1235 Cidade Universitária, 50670-901 Recife, Pernam-
buco, Brazil
e
Faculty of Medicine, Federal University of Ceará, Avenida Comandante Maurocélio Rocha Pontes, 100 Derby, 62.042-280 Sobral, Ceará, Brazil
f
Faculty of Medicine, Department of Morphology, Federal University of Ceará, Rua Delmiro de Farias, s/n - Rodolfo Teófilo, 60.430-170 Fortaleza, Ceará, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Temporomandibular disorder is a clinical painful condition in the temporomandibular joint (TMJ) region. The pu-
Received 2 December 2019 rified sulfated polysaccharide from the green marine algae Caulerpa racemosa (Cr) has provided anti-
Received in revised form 20 January 2020 inflammatory and antinociceptive activity. This study evaluated these effects on a TMJ hypernociception
Accepted 27 January 2020
model. Wistar rats (180 - 250 g) were pre-treated (i.v.) with Cr at 0.01, 0.1, or 1 mg/kg or vehicle 30 min before
Available online 28 January 2020
formalin (1.5%/50 μL, i.art.), capsaicin (1.5%/20 μL, i.art.), or serotonin (225 μg/50 μL, i.art.) in the TMJ, and noci-
Keywords:
ceptive behaviors were measured for 45 or 30 min upon inflammatory stimuli. Inflammatory parameters vascu-
Caulerpa racemosa lar permeability assay, TNF-α, and IL-1β by ELISA, protein expression of adhesion molecules ICAM-1 and CD55 by
Temporomandibular joint Western blot were assessed. The involvement of heme oxygenase-1 (HO-1) and nitric oxide (NO) pathways were
Heme oxygenase-1 assessed by pharmacological inhibition. Cr (1 mg/kg) reduced nociceptive behavior, plasmatic extravasation,
Nociception TNF-α, and IL-1β levels, as well as ICAM-1 and CD55 expression in periarticular tissues. Cr antinociceptive effect
Inflammation was not prevented by aminoguanidine, but ZnPP-IX did reduce its antinociceptive effect. Therefore, Cr
antinociceptive and anti-inflammatory effects in this experimental model of hypernociception depended on
the HO-1 pathway integrity, as well as reducing peripheral inflammatory events, e.g., TNF-α and IL-1β cytokines
levels, ICAM-1 and CD55 expression.
© 2020 Published by Elsevier B.V.

© 2 0 2 0 p u b l i s h e d b y e l s e v i e r b . v .

1. Introduction
Temporomandibular disorders (TMD) are one type of pathological
conditions that affect the orofacial region and encompass a heteroge-
neous group of musculoskeletal disorders involving the temporoman-
dibular joint, masticatory muscles, and adjacent tissues [1]. TMD
treatment should be preferentially conservative, especially through
⁎ Corresponding author at: Faculdade de Odontologia, Universidade Federal do Ceará,
Avenida Comandante Maurocélio Rocha Pontes, 100, Derby, 62.042-280 Sobral, Ceará,
Brazil.
E-mail addresses: helliadachaves@yahoo.com.br (H.V. Chaves), nmbb@ufc.br
(N.M.B. Benevides).
drug therapy, because this therapeutic modality has been more effective
at reducing pain, improving functionality and, as a consequence, the
quality of life [2,3].
In this context, the natural products research field is very promising
as it leads to discoveries that can be used to develop new drugs with an-
algesic potential. This has awakened the scientific community's atten-
tion towards the evaluation of the efficacy and safety of such products
in the last decades [4]. Thus, designing and synthesizing novel therapeu-
tics based on naturally-occurring products have called much attention
[5,6].
Around 30% of all currently available drugs are directly or indirectly
derived from vegetal sources. Marine algae species have been studied
through sulfated polysaccharide obtention to characterize chemical

https://doi.org/10.1016/j.ijbiomac.2020.01.272
0141-8130/© 2020 Published by Elsevier B.V.
254 N.A. Ribeiro et al. / International Journal of Biological Macromolecules 150 (2020) 253–260
and biological properties. Besides, these biomolecules have shown
antinociceptive, anti-inflammatory, antiviral, anticancer, antioxidant,
gastroprotective, and among other biological activities [7–11].
In previous reports, we investigated the biological activities of a
polysaccharide from the green marine algae Caulerpa racemosa (Cr) in
classical models of pain and inflammation such as hot plate test and ab-
dominal writhes in carrageenan-induced paw edema and peritonitis,
respectively. Further, among the Cr-mediated effects, antinociception
and anti-inflammatory activities were reported [9]. However, the litera-
ture lacks information on the Cr effects on the TMJ inflammatory
hypernociception.
Thus, our study aimed to evaluate the antinociceptive and anti-
inflammatory effects of Cr in an experimental model of
hypernociception induced by formalin, capsaicin, and serotonin in the
rat TMJ. Additionally, we investigated the role of inflammatory cyto-
kines, adhesion molecules, and the heme oxygenase-1 (HO-1) and nitric
oxide (NO) pathways in the possible mechanism of action mediated by
Cr in the inflammatory pain in the TMJ.
2. Material and methods
2.1. Animals
Male Wistar rats (Rattus norvegicus) weighing from 180 to 250 g
were housed in a light and dark cycles and temperature-controlled
room (23 ± 2 °C). These animals were randomly allotted in cages of
five animals with food and water ad libitum (n = 5). All the experimen-
tal procedures are in accordance with the “Guidelines on care and use of
laboratory animals” proposed by the Brazilian Society of Science for Lab-
oratory Animals and the experimental protocol was approved by the
Local Ethics Committee of the Federal University of Ceará under the reg-
istration number CEUA 59/13. All the necessary efforts to minimize an-
imal suffering were made.
2.2. Intra-articular injection of formalin, serotonin, and capsaicin
The animals underwent brief inhalation anesthesia with isoflurane
3% for 3 s and received an intra-articular injection of one of the inflam-
matory agents: formalin (1.5%/50 μL), serotonin (225 μg/50 μL), or cap-
saicin (1.5%/20 μL) [12–14]. Drug administration into the
temporomandibular joint was performed by a 30G needle connected
to a micro syringe (Hamilton, 50 μL) through a polyethylene tube P50.
The postero-inferior border of the zygomatic arch was palpated, and
the needle was inserted inferior to this point and advanced in a medial
and anterior direction until the needle made contact with the condyle.
This contact was verified by the moving of the mandible, and the punc-
ture of the needle into the joint space was confirmed by the loss of
resistance.
2.3. Behavioral approaches to evaluate nociceptive responses
Behavioral analyses were carried out during the light phase of the
light and dark cycle (7 am-16 pm) in a temperature-controlled quiet
room at 23 ± 2 °C [15]. To minimize stress during the experimental ses-
sions, animals were previously handled during a seven-day period. No-
ciceptive behavior analyses were done in a mirrored box (30x30x30
cm), in which each animal was individually placed for 10 min for accli-
mation and stress reduction. Immediately after intra-articular injec-
tions, the animals recovered from anesthesia and were replaced into
the box to nociceptive behavior analyses. Scratching the injected region
with forelimbs or hind limbs and head lifting was analyzed for 45 min
after formalin and capsaicin injections or during 30 min after serotonin
injection. The time the animals spent on scratching the orofacial region
was quantified in seconds using a timer, and the number of head lifts
was manually counted. This behavior is considered to follow a uniform
pattern of 1 s of duration. Thus, for this specific behavior, the total sum
of head lifts is added to the time of orofacial scratching as previously
characterized [12–14]. The nociceptive responses were measured by ex-
aminers who were blinded either to the treatments the animals
underwent and to the injection into the temporomandibular joint.
After the behavioral analyses, the animals were anesthetized and culled
through decapitation for tissue harvesting and further molecular
analyses.
2.4. Total sulfated polysaccharide extraction from the green marine algae
Caulerpa racemosa
To perform total sulfated polysaccharides (TSP) extraction, 5 g of the
dry algae were hydrated in 250 mL of sodium acetate buffer (0.05 M,
pH 6.0) with 1020 mg of papain, 5 mM of cysteine, and 5 mg of EDTA.
This mixture was maintained at 60 °C for 6 h and was filtered. The res-
idue of this filtration was washed with distilled water, filtered once
again, and added to the previous filtrate product. TSP was precipitated
with 16 mL of cetylpyridinium chloride (CPC) for 2 h at room tempera-
ture. The solution was then centrifuged at 2560G, for 20 min, at 5 °C. The
supernatant was discarded and the precipitate obtained was washed
with 610 mL of a 0.05% CPC solution, centrifuged (2560G, 20 min,
5 °C), and dissolved in 172 mL of a solution of NaCl 2 M: ethanol
(100:15, v/v). After, the remaining material was precipitated again
with 305 mL of absolute ethanol for 24 h at 4 °C. The produced precipi-
tate was washed twice through centrifugation (2560G, 20 min, 5 °C)
with 305 mL of 80% ethanol e once with absolute ethanol. The product
was then dialyzed against distilled water, lyophilized, and then
denominated Cr-TSP [16].
2.5. Total sulfated polysaccharides fractioning
A total of 5 mg of Cr-TSP was dissolved in 10 mL of sodium acetate
buffer (50 mM, pH 6.0), centrifuged twice (2560G, 20 min, 5 °C), and
the precipitate was discarded. The supernatant went through a chroma-
tography in the DEAE-cellulose column and the same buffer was used as
equilibration buffer. The column was washed with the equilibration
buffer and the sulfated polysaccharide fractions were eluted in a step-
wise manner with NaCl at the following concentration 0.25, 0.5, 0.75,
1.0, 1.25, and 1.5 M added to the equilibration buffer. The fractions
were monitored through metachromatic properties using 1,9-
dimethylene blue in spectrophotometer (Amersham Biosciences
Ultrospec 1100 PRO) at 525 nm [17]. The major sulfated fraction was di-
alyzed with distilled water, stored for further use, and named Cr.
2.6. Cr effects on formalin-, serotonin-, or capsaicin-induced TMJ
hypernociception
The animals were pre-treated with Cr (0.01, 0.1, or 1.0 mg/kg; i.v.).
Thirty minutes after Cr pre-treatment, the intra-articular injection of
formalin (1.5%/50 μL), serotonin (225 μL/50 μL), or capsaicin (1.5%/
20 μL) was performed and the behavioral nociceptive responses were
quantified as described. As positive controls, the rats received morphine
(5 mg/kg; s.c.) or indomethacin (5 mg/kg; s.c.) 30 min before the intra-
articular injections. After the behavioral analyses, the animals were
anesthetized, culled by decapitation, and the periarticular tissues were
harvested and processed for further molecular assays.
2.7. TNF-α and IL-1β quantification in the rat TMJ periarticular tissues dur-
ing formalin-induced hypernociception
Periarticular tissue samples which correspond to the surrounding
musculature of the temporomandibular joint were obtained and ho-
mogenized in RIPA solution (Santa Cruz, USA). After this step, the sam-
ples were centrifuged (10,000G, 10 min, 4 °C) and the supernatant was
used to quantify TNF-α and IL-1β. Ninety-six-well plates were incu-
bated overnight at 4 °C with anti-rat anti-TNF-α and anti-IL-1β
 N.A. Ribeiro et al. / International Journal of Biological Macromolecules 150 (2020) 253–260
antibodies (10 μg/mL). On the following day, the plates were washed
and incubated for 2 h with a bovine serum albumin solution at 1% to
protect from unspecific binding. After such blockade and washing
steps, standard curves and the samples (triplicate) were added and in-
cubated at 4 °C for 24 h. The plates were then washed three times
with washing buffer and 100 μL per well (1:2000) of the biotinylated
polyclonal antibodies against the TNF-α and IL-1β added to the wells.
After antibody incubation at room temperature for 1 h, the plates
were washed and 100 μL of avidin-HRP (1:5000) were added to the
samples. After 15 min, 100 μL of the substrate reagent (o-
phenylenediamine dihydrochloride; OPD, Sigma-Aldrich, St. Louis,
MO-USA) were added to the wells and the plates were kept in the
dark at 25 °C for 15–20 min. The enzymatic reaction was then stopped
with H2SO4 1 M and the absorbance was determined at 450 nm.
2.8. Plasma extravasation
To assess inflammatory parameters, all the animals were pre-treated
with Cr at 1.0 mg/kg (i.v.). After 30 min, these rats were anesthetized
with tribromoethanol 1% (i.p.; 0.1 mL/100 g) [18] and then received
an intra-articular injection of formalin (1.5%/50 μL) into the TMJ
followed by a single dose of Evans blue solution 1% through the penile
vein [19]. After 45 min, the animals were culled and, as Evans blue
dye binds plasma proteins, the injection point can be visualized [20],
allowing for adequate harvesting of the TMJ periarticular tissues for
plasma extravasation assays through a spectrophotometer. To perform
Evans blue extraction from tissues, the periarticular tissue of each ani-
mal was weighed, immersed individually in tubes containing formam-
ide, and kept at 60 °C for 24 h [21]. After the extraction, the amount of
Evans blue dye obtained from the tissues was determined in a micro-
plate reader that measured the absorbance of the samples at 620 nm.
The results were compared to the standard curve of known concentra-
tions of Evans blue dye in formamide (4 μg, 2 μg, 1 μg, and 0.5 μg of
Evans blue dye per 1 mL of formamide). Finally, the amount of dye
into the tissues was determined in μg in each sample and normalized
by the initial tissue weight (g). Thus, the plasma extravasation was cal-
culated in micrograms of Evans blue dye per gram of dissected TMJ
periarticular tissue.
2.9. ICAM-1 and CD55 adhesion molecules expression by Western blot
Periarticular tissue samples were homogenized in RIPA buffer (Santa
Cruz Biotechnology) and total protein quantification was carried out.
Then, the proteins were separated by electrophoresis in 10% polyacryl-
amide gel SDS-PAGE, transferred onto a nitrocellulose membrane.
Blocking was made with non-fat dry milk and antibody labelling was
carried out overnight with anti-rat anti-ICAM-1 and anti-CD55 antibod-
ies at 4 °C under gently shaking. After washing away primary antibodies,
secondary antibody incubation was made at room temperature under
gentle shaking. Further, ECL detection system (ECL, Amersham
Pharmacia Biotech, Little Chalfont, R.U.) was used to detect chemilumi-
nescent bands, whose optical density was further analyzed.
2.10. Role of heme oxygenase-1 (HO-1) and nitric oxide (NO) pathways
To study the involvement of NO pathway, the animals were pre-
treated with aminoguanidine (30 mg/kg, i.p.), a non-selective inhibitor
of nitric oxide synthase, whilst the HO-1 pathway role was assessed
through pre-treatment with ZnPP-IX (3 mg/kg; s.c.), a selective inhibi-
tor of HO-1. Thirty minutes after these treatments, Cr-administration
(1 mg/kg, i.v.) was done. After 30 min passed, these animals received
a formalin-injection into the TMJ and the behavioral responses were an-
alyzed as described.
255
2.11. Statistical analyses
Normal distribution was assessed by the Shapiro-Wilk test. The
means were compared by a one-way ANOVA test to analyze the associ-
ation between the variables tested. The post-hoc test for ANOVA was
determined by Levene's test. Variance homogeneity was assumed if p-
value was ≥0.05 and Tukey's test was used as post-hoc test. In case no
variance homogeneity existed (p-value ≤ 0.05), Games-Howell test
was employed. The results were expressed as means ± standard error
of the mean (S.E.M.). All the statistical analyses were carried out using
SPSS 20.0 Windows version (SPSS Inc., Chicago, IL. USA). The graphs de-
signed in GraphPad Prism 6 Windows version (GraphPad Prism, La Jolla,
CA, USA). The probability level was assumed as p b 0.05.
3. Results
3.1. Effects of Cr on the formalin-induced hypernociception
According to Fig. 1, all the tested doses of Cr 0.01 mg/kg (144.80 ±
13.10 s), 0.1 mg/kg (105.40 ± 7.20 s), and 1.0 mg/kg (44.00 ±
10.78 s) reduced formalin-induced hypernociception (223.0 ± 8.56 s).
Morphine (5.6 ± 3.09 s) and indomethacin (29.33 ± 2.43 s) also
strongly prevented the formalin-induced nociceptive behavior in rats.
Strikingly, the 1 mg/kg Cr dose abolished nociceptive behavior to levels
similar to saline-injected rats into the TMJ.
3.2. Effects on Cr on the formalin-induced plasma extravasation
Fig. 2 depicts the significant prevention of plasma extravasation
upon Cr-pre-treatment. Cr 1 mg/kg reduced (14.32 ± 3.08) the amount
of Evans blue dye per mg of TMJ peri-articular tissue compared to for-
malin group (57.44 ± 6.78) and did not show statistical difference in re-
lation to saline-injected animals (18.88 ± 4.29).
3.3. Effects of Cr on TNF-α e IL-1β levels in the TMJ periarticular tissues
TNF-α (3.13 ± 0.13 pg/mL) and IL-1β (5.39 ± 0.46 pg/mL) levels in
the periarticular tissues of the TMJ were significantly reduced upon Cr
1 mg/kg pre-treatment when compared to formalin-injected animals
(TNF-α: 6.89 ± 0.42 pg/mL, IL-1β: 16.24 ± 2.30 pg/mL). The Cr pre-
treatment reduced the cytokine concentrations to levels similar to
Fig. 1. Dose-response curve of Cr treatment on the formalin-induced nociception. Cr 0.01,
0.1, and 1 mg/kg (i.v.) reduced nociceptive responses and the highest dose was statistically
similar to the saline-injected group. Symbols (*) and (#) represent statistically significant
differences (p b 0.05) in comparison to saline- and formalin-treated groups, respectively
(ANOVA, followed by Tukey's test, n = 5).
256 N.A. Ribeiro et al. / International Journal of Biological Macromolecules 150 (2020) 253–260

saline-injected animals (TNF-α: 3.11 ± 0.22 pg/mL, IL-1β: 7.13 ±

0.40 pg/mL) (Fig. 3).

3.4. Analyses of ICAM-1 and CD55 expression

The intra-articular injection of formalin (1.5%/50 μl) increased ICAM-

1 (4.67 ± 0.92) and CD55 (3.30 ± 0.70) expression in the TMJ
periarticular tissues when compared to saline-treated animals (ICAM-
1: 0.96 ± 0.21; CD55: 1.90 ± 0.70). The Cr pretreatment (1 mg/kg) sig-
nificantly reduced the expression of these adhesion molecules ICAM-1
(2.47 ± 0.46) and CD55 (1.83 ± 0.41) in the TMJ periarticular tissues
(p b 0.05) (Fig. 4).
Fig. 2. Plasma extravasation upon Cr pre-treatment in the formalin-induced
hypernociception. Pre-treatment with Cr 1 mg/kg (i.v.) prevented plasma extravasation
levels to the TMJ periarticular tissues. Symbols (*) and (#) represent statistically
3.5. Role of heme oxygenase-1 (HO-1) pathway
significant differences (p b 0.05) in comparison to saline- and formalin-treated groups,
respectively (ANOVA, followed by Tukey's test, n = 5).
Fig. 5 shows that pretreatment with ZnPP-IX (3 mg/kg), a specific
HO-1 inhibitor, prevented (131.80 ± 12.97 s) the antinociceptive effects
of Cr (1.0 mg/kg).

Fig. 3. TNF-α and IL-1β levels in the periarticular tissues after Cr treatment. (A) Cr 1 mg/kg (i.v.) pre-treatment reduced TNF-α levels in the periarticular tissue of the TMJ after formalin
injection (B) Cr 1 mg/kg (i.v.) pre-treatment reduced IL-1β levels in the TMJ periarticular tissue following formalin injection. Symbols (*) and (#) represent statistically significant
differences (p b 0.05) in comparison to saline- and formalin-treated groups, respectively (ANOVA, followed by Tukey's test for A and Games-Howell post-hoc test for B, n = 5).

Fig. 4. Effects of Cr pretreatment on ICAM-1 and CD55 expression in the TMJ periarticular tissues. (A) Cr at 1 mg/kg reduced ICAM-1 expression (2.47 ± 0.46) in the periarticular tissues
after formalin injection. (B) Cr at 1 mg/kg decreased CD55 expression (1.83 ± 0.41) in the periarticular tissues after formalin injection. Symbols (*) and (#) represent statistically
significant differences (p b 0.05) in comparison to saline- and formalin-treated groups, respectively (ANOVA, followed by Tukey's test, n = 5).
 N.A. Ribeiro et al. / International Journal of Biological Macromolecules 150 (2020) 253–260
Fig. 5. Analysis of the HO-1 involvement in the Cr antinociceptive mechanism.
Pretreatment with ZnPP-IX (3 mg/kg), a selective HO-1 inhibitor, prevented the
antinociceptive effects mediated by Cr treatment (1 mg/kg). Symbols (*), (#), and (#)
represent statistically significant differences (p b 0.05) in comparison to saline-,
formalin-, Cr-treated groups, respectively (ANOVA, followed by Tukey's test, n = 5).
3.6. Role of nitric oxide (NO) pathway
Fig. 6 depicts that pre-treatment with aminoguanidine (30 mg/kg, i.
p.), an inducible nitric oxide synthase inhibitor (iNOS), did not prevent
(64.17 ± 6.68 s) the antinociceptive effect mediated by Cr (1 mg/kg)
treatment (44.00 ± 10.78 s).
3.7. Analysis of the Cr-mediated effects on serotonin-induced
hypernociception
According to Fig. 7, Cr 1.0 mg/kg (56.40 ± 5.51 s) reduced serotonin-
induced hypernociception (105.6 ± 3.09 s), and no difference was ob-
served between Cr-treated animals and the saline-injected control
Fig. 6. Analysis of the NO involvement in the Cr antinociceptive mechanism. Pretreatment
with aminoguanidine (30 mg/kg), an iNOS inhibitor, did not prevent the antinociceptive
effects mediated by Cr treatment (1 mg/kg). Symbols (*) and (#) represent statistically
significant differences (p b 0.05) in comparison to saline-, formalin-, Cr-treated groups,
respectively (ANOVA, followed by Tukey's test, n = 5).
257
Fig. 7. Effects of Cr-treatment on serotonin-induced hypernociception. Pretreatment with
indomethacin (5 mg/kg) and Cr (1 mg/kg) abolished the serotonin-induced
hypernociception. Symbols (*) and (#) represent statistically significant differences
(p b 0.05) in comparison to saline-, serotonin-, Cr-treated groups, respectively (ANOVA,
followed by Tukey's test, n = 5).
(54.8 ± 7.13). Indomethacin-treated animals (29.33 ± 2.43 s) behaved
similarly to the negative control, the saline-injected animals.
3.8. Analysis of the Cr-mediated effects on capsaicin-induced
hypernociception
Upon capsaicin injection (Fig. 8), Cr 1.0 mg/kg (80.25 ± 6.92 s) re-
duced the time spent on nociceptive behaviors elicited by capsaicin
alone (182.2 ± 12.82 s) and no difference existed between the negative
control of this experiment, saline-injected animals (54.80 ± 7.13 s).
Fig. 8. Effects of Cr-treatment on capsaicin-induced hypernociception. Pretreatment with
Cr (1 mg/kg) diminished the capsaicin-induced hypernociception. Symbols (*) and (#)
represent statistically significant differences (p b 0.05) in comparison to saline- and
capsaicin-injected groups, respectively (ANOVA, followed by Games-Howell's test, n = 5).
258 N.A. Ribeiro et al. / International Journal of Biological Macromolecules 150 (2020) 253–260
4. Discussion
This study demonstrated that the sulfated polysaccharide from the
green marine algae Caulerpa racemosa possesses antinociceptive and
anti-inflammatory activities in an experimental model of temporoman-
dibular joint hypernociception induced by formalin, serotonin, and cap-
saicin. Further, Cr also reduced plasma extravasation and ICAM-1, CD55,
and cytokines TNF-α and IL1-β levels in the periarticular tissues. The
mechanism of action through which Cr exerted antinociception seems
to be dependent upon HO-1 pathway integrity, while the nitric oxide
pathway might not be involved in Cr effects.
Natural products play such an important role in drug discovery [22].
Other studies report that biomolecules isolated from algae present bio-
logical activities, demonstrating their capacity to produce primary me-
tabolites with high complexity and diversity of biological potential
different from the ones encountered in terrestrial plants [8–11,23–26].
In the last decades, much emphasis on controlled harvesting of ma-
rine algae has led to large scale production of algae-derived products
[27]. Among the bioactive molecules extracted from marine algae, the
polysaccharides have emerged in the past few years as a rich source of
natural products. For this reason, the applications of these polysaccha-
rides as therapeutic agents have fostered intense research on this poten-
tial [28]. As these compounds are not derived from animal sources, they
present a low risk of contamination by viral particles [29], as well as a
relatively low toxicity profile [30].
In previous reports, the sulfated polysaccharide from the green ma-
rine algae Caulerpa racemosa presented antinociceptive and anti-
inflammatory effects on experimental models of acetic acid-induced
writhes and carrageenan-induced paw edema in rodents [9]. Another
study using a Caulerpa racemosa-derived bioproduct proved its
antinociceptive and anti-inflammatory efficacy in capsaicin-induced
ear edema and carrageenan-induced peritonitis [31]. To evaluate the
Cr-mediated effects on the TMJ hypernociception, we used the formalin
test [12]. Cr prevented nociceptive responses in rats elicited by formalin
injection into the TMJ. Similarly, sulfated polysaccharide fractions from
the species Gracilaria cornea, Solieria filiformis, and Caulerpa cupressoides
also exerted antinociceptive effects [7,8,32].
Regarding inflammatory parameters, Cr diminished plasma extrava-
sation of Evans blue dye to periarticular exudates. It is already well
established that plasma extravasation promotes protein and leukocyte
migration to inflamed tissues (Chicre-Alcântara et al., 2012). A polysac-
charide fraction from Solieria filiformis could also prevent such events to
regions of high ongoing inflammatory processes (De Araujo et al.,
2011).
As a component of leukocyte migration, ICAM-1, intracellular adhe-
sion molecule 1, is the protein that plays such a crucial role in adhesion
and migration of neutrophils through endothelium, a phenomenon
known as diapedesis, and its expression is upregulated in epithelium
of inflamed tissues [33,34]. Further, CD55 is usually present as a soluble
molecule in body fluids and functions as an anti-adhesive factor for leu-
kocytes [34–36]. Upon Cr pre-treatment, ICAM-1 was reduced in the
periarticular tissues after formalin injection into the TMJ. Additionally,
CD55 levels also reduced upon Cr treatment, suggesting that Cr exerted
anti-inflammatory effects through inhibition of leukocyte migration via
adhesion molecules.
TNF-α has a fundamental role in cell adhesion to endothelial walls as
it stimulates NF-κβ and, consequently, induces the expression of a cas-
cade of genes related to inflammation, including adhesion molecules
such as VCAM-1, ICAM-1, and E-selectin. As a result, activated endothe-
lial cells exhibit an increase in surface adhesion molecules, facilitating
leukocytes transmigration through blood vessels towards inflamed tis-
sues [37]. Besides, a recent report demonstrated that a polysaccharide
from the red algae Porphyridium sp. presented anti-inflammatory and
vasorelaxation activities through inhibition of ICAM-1 expression in an
experimental model of TNF-α-induced inflammation in endothelial
cells of human coronary arteries [38].
Corroborating with these findings, our data showed a significant re-
duction in the TNF-α and IL-1β levels in the TMJ periarticular tissues in
Cr-treated animals, suggesting that the reduction of cytokines produc-
tion could also reduce adhesion molecules expression, ultimately
diminishing plasma extravasation. Recent studies also using polysac-
charides from marine algae reported a reduction in nociceptive behav-
iors and expression of TNF-α and IL-1β [7,11,26,39]. In fact, during the
induction of inflammatory hypernociception, a cascade of cytokines is
released. These cytokines further mediate the release of prostanoids,
which in turn sensitizes primary sensory neurons and lowers the
threshold of nociceptive fibers [40,41].
Furthermore, counter-regulatory pathways act to ease off the in-
flammatory process such as the heme oxygenase-1 (HO-1) pathway.
HO-1 induction is negative feedback on the production of inflammatory
mediators and takes part in the anti-oxidant system, modulating in-
flammatory pain processing [42]. Besides, in an experimental model of
zymosan-induced inflammation, HO-1 expression and activity in-
creased, enabling endothelial cells to endure oxidative stress and
inhibited adhesion molecules expression. These events, reduced leuko-
cyte influx to inflammatory foci [43]. In our study, ZnPP IX prevented
the Cr antinociceptive effect, which shows that Cr effects depend upon
HO-1 integrity. We can also infer that the reduction of adhesion mole-
cules mediated by Cr treatment could be mediated by HO-1. The litera-
ture shows the interrelationship between HO-1 and nitric oxide
pathways [42,44,45]. However, our study showed that Cr effects are in-
dependent of nitric oxide pathway integrity.
Evaluating the antinociceptive effects of polysaccharides from ma-
rine algae in hypernociception induced by other stimuli such as seroto-
nin [32,39]. A study on the nociception induced by serotonin injected in
the TMJ demonstrated that serotonin exerts an indirect action on pri-
mary nociceptive neurons. This effect occurs through polymorphonu-
clear leukocytes, cyclo-oxygenase, norepinephrine in synaptic
terminals, and local activation of β1 or β2-adrenergic receptors [13].
Cr treatment prevented the serotonin mediated nociception, suggesting
that Cr effects also depend upon inhibition of sympathomimetic amines,
as well as prostaglandin synthesis inhibition. In fact, it is known that a
cascade of cytokines leads to a final production of prostaglandins and
sympathetic amines [40], showing that Cr treatment, by reducing TNF-
α and IL-1β, can also reduce norepinephrine release, thus, reducing
the activation of β1 and β2-adrenergic receptors [41].
Another pronociceptive substance is capsaicin, an active component
of red peppers from the Capsicum genus. It provokes a burning sensa-
tion by activating sensory terminals. The capsaicin receptor is known
as the transient receptor potential cation channel subfamily V member
1 (TRPV1), which detects noxious heat stimuli (b43 °C) [46]. TRPV1
can be permanently activated in inflamed tissues as the low extracellu-
lar pH and temperature increase are frequently found during inflamma-
tion [47]. Capsaicin injection into the TMJ provokes nociceptive
responses in animals [48], and our findings show that Cr pre-
treatment prevented capsaicin-induced nociceptive behaviors in rats.
It is also important to emphasize that our experimental model of
temporomandibular joint pain is an acute model of inflammatory pain
and might not entirely translate the actual chronicity of the TMJ pain
burden. Given the promising results found here, we reinforce the need
to elucidate the Cr-mediated effects on chronic experimental models
of pain to afford further conclusions on its potential analgesic effects.
5. Conclusion
In summary, our findings provide evidence that Cr exerts
antinociceptive and anti-inflammatory effects in an experimental
model of TMJ hypernociception. These effects can be linked to a periph-
eral induction of HO-1, plasma extravasation reduction, as well as TNF-
α, IL-1β, and ICAM-1 reduction. Further, the results also suggest that Cr
antinociception might also depend upon sympathomimetic amines re-
lease and TRPV1 receptors inhibition. Altogether, the sulfated
 N.A. Ribeiro et al. / International Journal of Biological Macromolecules 150 (2020) 253–260
polysaccharide from the green marine algae Caulerpa racemosa might
open avenues as a novel therapeutic to temporomandibular joint
disorders.
Financial support
This study was supported by Brazilian grants from Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq),
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES), Instituto de Biomedicina do Semi-Árido Brasileiro (INCT),
and Fundação Cearense de Apoio ao Desenvolvimento Científico e
Tecnológico (FUNCAP).
Declaration of competing interest
The authors declare that there is no conflict of interests regarding
this study.
Acknowledgments
None to declare.
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