Research Article

Neuropsychobiology Received: July 13, 2020

Accepted: March 15, 2021
DOI: 10.1159/000515929 Published online: April 29, 2021

Neuroprotective and Antioxidant Effects of

Riparin I in a Model of Depression Induced by
Corticosterone in Female Mice
Iris Cristina Maia Oliveira a Auriana Serra Vasconcelos Mallmann a
   

Francisco Adelvane de Paula Rodrigues a Laura Maria Teodorio Vidal a

   

Iardja Stéfane Lopes Sales a Gabriel Carvalho Rodrigues a

   

Natalia Ferreira de Oliveira a Raquell de Castro Chaves a

   

Victor Celso Cavalcanti Capibaribe a Alyne Mara Rodrigues de Carvalho a

   

Marta Maria de França Fonteles b Stanley Juan Chavez Gutierrez c

   

José Maria Barbosa-Filho d Francisca Cléa Florenço de Sousa a

   

aDepartment of Physiology and Pharmacology, Neuropharmacology Laboratory, Drug Research and Development

Center, School of Medicine, Federal University of Ceará, Fortaleza, Brazil; bDepartment of Pharmacy, Federal
University of Ceará, Fortaleza, Brazil; cDepartment of Pharmacy, Laboratory of Chemical Technology, Federal
University of Piauí, Teresina, Brazil; dDepartment of Pharmacy, Laboratory of Pharmaceutical Technology, Federal
University of Paraiba, João Pessoa, Brazil

Keywords RIP I in the last 7 days. Behavioral and neurochemical analy-

Depression · Cognition · Corticosterone · Brain-derived
neurotrophic factor · Oxidative stress
Abstract
Background: Depression is a common, chronic, and often
recurrent serious mood disorder. Conventional antidepres-
sants present limitations that stimulate the search for new
drugs. Antioxidant and neuroprotective substances are po-
tential antidepressant agents. In this context, riparin I (RIP I)
has presented promising results, emerging as a potential
source of a new therapeutic drug. In this study, the antide-
pressant effect of RIP I was evaluated in an animal model of
depression induced by corticosterone (CORT). The involve-
ment of neuroprotective and antioxidant mechanisms in the
generation of this effect was also assessed. Methods: Female
mice were submitted to CORT for 21 days and treated with
ses were performed. Results: The administration of RIP I re-
versed the depressive and psychotic-like behavior, as well as
the cognitive impairment caused by CORT, in addition to
regulating oxidative stress parameters and BDNF levels in
depression-related brain areas. Conclusion: These findings
suggest that RIP I can be a strong candidate for drugs in the
treatment of depression. © 2021 S. Karger AG, Basel
Introduction
Depression is a common, chronic, and recurrent seri-
ous mood disorder that has been reported as a leading
cause of disability, contributing significantly to the global
burden of disease. Currently, the disorder affects around
300 million people worldwide and is more frequent in
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karger@karger.com © 2021 S. Karger AG, Basel Correspondence to:

www.karger.com/nps Francisca Cléa Florenço De Sousa, cleaflorenco @ yahoo.com.br
Glasgow Univ.Lib.
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women than men. The main symptoms are depressed
mood, fatigue or loss of energy, and decreased pleasure/
interest. In severe cases, psychosis and cognitive deficits
may occur [1, 2].
The etiology of depressive disorders involves genetic
and environmental alterations, with stress being one of
the main factors related to this disease. Hyperactivity of
the hypothalamic-pituitary-adrenal axis was observed
due to high cortisol levels, as well as impairment of its
negative feedback regulatory mechanisms [3–5].
The antidepressant drugs are the basis for the treat-
ment of depressive disorders. However, many patients do
not respond to initial therapy or have recurrences. Pa-
tients with major depression with psychotic features, a
clinical subtype of depression, have a worse disease course
and higher rates of resistance to treatment. Studies have
already reported antipsychotic activity of antidepres-
sants, which seems to be related to their affinity for the
sigma-1 receptor. Thus, a drug with antipsychotic activ-
ity is desirable for the management of depression resis-
tant to treatment or with psychotic characteristics [6, 7].
Currently, natural products have been the target of
several studies because they have great potential as a
source of new drugs [8]. In previous studies from our
group using acute animal models, riparin I (RIP I), an al-
kamide firstly isolated from Aniba riparia, displayed cen-
tral effects, showing antidepressant and anxiolytic activi-
ties. The putative antidepressant mechanism of RIP I
could be related to receptors involving noradrenergic (α1
and α2 receptors), serotonergic (5-HT2A/2C receptor), and
dopaminergic (D1 and D2 receptors) systems [9, 10].
Based on the potential effect of RIP I in the treatment of
depression, we decided to investigate its effects on behav-
ioral, neuroplastic, and oxidative stress parameters in
mice submitted to a corticosterone (CORT)-induced de-
pression model in comparison with fluvoxamine (FLU).
Materials and Methods
Animals
Swiss female mice (19–23 g; age: 8–10 weeks; n = 110) were kept
at a controlled temperature (23 ± 1°C) with a 12-h light/dark cycle
(lights on at 7:00 a.m.) and with free access to food and water. The
animals were ethically treated according to the Guide for the Care
and Use of Laboratory Animals published by the National Insti-
tutes of Health [11].
Drugs
RIP I (Fig. 1), provided by Pharmaceutical Technology Labora-
tory (Federal University of Paraíba, Brazil), was emulsified with
2% polysorbate (Tween® 80; Sigma®, St Louis, MO, USA) and di-
2 Neuropsychobiology
DOI: 10.1159/000515929
NH
O
O
CH3
Fig. 1. Chemical structure of RIP I [13]. RIP I, riparin I.
luted in distilled water (50 mg/kg). CORT (20 mg/kg; Sigma®, St
Louis, MO, USA) was dissolved in a 0.9% saline solution contain-
ing 0.1% polysorbate (Tween® 80; Sigma®, St Louis, MO, USA)
and 0.1% dimethyl sulfoxide (Sigma®, St Louis, MO, USA). FLU
(50 mg/kg; Abbott®, New Jersey, USA) was diluted in distilled wa-
ter. CORT was administered by the subcutaneous route, while the
other drugs were administered by oral gavage. Previous studies
were used to select the dosage and route of administration for all
drugs [10, 12].
Experimental Procedure
Mice were divided into 4 groups: (1) control; (2) corticoste-
rone; (3) corticosterone + riparin I; and (4) corticosterone + flu-
voxamine. Groups 2, 3, and 4 were administrated subcutaneously
with 20 mg/kg CORT for 21 days, while the control group received
only saline solution. After the 14th day of treatment, each group
was treated with RIP I (50 mg/kg) (group 3), FLU (50 mg/kg)
(group 4), or vehicle (distilled water +2% Tween® 80) (groups 1
and 2), per oral gavage, 1 h after CORT treatment.
On the 21st day, the behavioral tests (open field test – OFT,
forced swimming test – FST, sucrose preference test, Y-maze
test – YMT, step-down avoidance – SDA, and prepulse inhibi-
tion – PPI) were performed, 60 min after the last treatment. The
animals were euthanized (24 h later), and brain areas (prefrontal
cortex [PFC], hippocampus [HC], and striatum [ST]) were re-
moved for biochemical analysis (oxidative stress parameters and
BDNF dosage) (Fig. 2).
We tried to minimize the bias and excessive stress to the ani-
mals, so the experimental protocol was performed in 3 timelines
of 36 or 37 animals. The total of 110 animals were divided between
different behavioral tests: first timeline: OFT + FST + SPT (n = 37),
second timeline: YMT + SDA (n = 36), and third timeline: PPI
(n = 37) and neurochemical tests.
Behavioral Tests
Open Field Test
This test assesses the influence of drugs on the animal’s loco-
motor activity and was performed in an acrylic device (transparent
walls and black background, dimensions 30 × 30 × 15 cm), divided
into 9 equal quadrants. The animal was placed in the center of the
device, and for 5 min, the number of crossings was observed. The
device was cleaned with 5% alcohol after each animal [14].
Forced Swimming Test
The animals were placed, individually, in an open cylindrical
container (22 × 40 cm) with 20 cm of water (25 ± 1°C). The im-
mobility, recorded during 5 min, is considered when the animal
makes only minimal movements necessary to remain floating. The
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 Saline solution (1) or CORT, s.c., (2) (3) (4)
Distilled water vehicle (1) (2),
RIP I (3) or FLU (4), p.o.
1 15
Groups:
(1) Control
(2) Corticosterone
(3) Corticosterone + riparin I
(4) Corticosterone + fluvoxamine
Fig. 2. Schematic diagram of the experimental protocol. RIP I, riparin I; CORT, corticosterone; FLU, fluvoxamine.
indicative of depressive-like behavior is related to increase in the
duration of immobility time [15].
Sucrose Preference Test
The test was performed as described by Mao et al. [16] and is
based on animals’ clear preference for sweet water in normal con-
ditions, while depressed animals show less interest. In this model,
the mouse can freely choose between 2 solutions for drinking: wa-
ter or sucrose solution. Before the test, on the 18th day of the ex-
periment, the mice were accustomed to a 2% (w/v) sucrose solu-
tion, placing 2 bottles of 2% sucrose solution in each cage for 18 h.
Then, on the 19th day, sucrose solution in 2 bottles was replaced
with tap water for 18 h. After adaptation, on the 20th day, for the
test, the mice were housed in individual cages for 18 h and had free
access to 2 identical bottles, one filled with 2% sucrose solution and
the other with tap water. The test started with the beginning of the
dark (active) phase of the animals’ cycle, and no previous food de-
privation was applied. After this 18 h, on the 21st day of the ex-
periment, the consumption of sucrose and water was recorded,
and the percentage of sucrose preference was calculated.
SDA Test
This test was used to assess the animal’s memory associated
with an aversive event. The apparatus consisted of an acrylic box
(48 × 22 × 22 cm), with an electrified grid floor, having a shock-free
raised zone. The SDA test was performed as described by Souza et
al. [17]. Decrease in the step-down latency indicates impairment
in aversive/affective memory.
Y-Maze Test
The YMT evaluates the spatial working memory by recording
spontaneous alternation behavior [18]. Each mouse was placed at
the end of one of the 3 arms (40 × 25 × 6 cm, in 120° angle) and
allowed to freely move through the maze during 8 min. The total
number of arm entries was recorded, and the percentage of alter-
nations was calculated as described by Chaves et al. [19].
Effect of Riparin I in a Model of
Depression
21 22 Time, days
Behavioral Biochemical
tests analysis
Open field Oxidative stress
Forced swimming BDNF
Step-down avoidance
Sucrose preference
Y-maze
Prepulse inhibition
PPI Test
PPI is the suppression of a startle reflex response to a startle
stimulus that occurs when a weak prepulse stimulus precedes the
startle stimulus. This test has been used to investigate antipsychot-
ic activity of antidepressant drugs [19]. In this test, individual mice
were placed in small metal cages equipped with a sensor recording
vertical movements (Insight, São Paulo, Brazil). The protocol assay
(including pulse and prepulse intensities, frequency, and back-
ground noise) was performed as previously described by Chaves et
al. [19].
Neurochemical Tests
Tissue Preparation
The PFC, HC, and ST homogenates 10% (w/v) were prepared
with ice-cold 0.1 M phosphate buffer (pH 7.4), centrifuged (10,000
g for 15 min), and aliquots of supernatants were separated and used
for oxidative stress determinations. For BDNF measurement, the
samples were homogenized 20% (w/v) with cold PBS buffer (pH
7.4), with a protease inhibitor cocktail (Sigma).
Determination of GSH, TBARS, and Nitrite/Nitrate Levels
Reduced glutathione (GSH) is a thiol bound to proteins, play-
ing an important role as an antioxidant. The detection of GSH
amount, useful indication of cell’s ability to prevent oxidative
stress, was evaluated according to Sedlak and Lindsay [20], based
on Ellman’s reagent (DTNB) reaction with free thiol groups. The
levels were determined by the absorbance at 412 nm and expressed
as microgram of GSH/gram of wet tissue.
The lipid peroxidation of cell membranes causes release of the
contents of organelles and formation of cytotoxic products such as
malondialdehyde (MDA) and, consequently, cell damage. MDA
content was measured in the form of thiobarbituric acid reactive
substances (TBARS) and determined by the absorbance at 535 nm,
being represented as microgram of MDA/gram of wet tissue [21].
Nitrite/nitrate level determination was based on Griess reac-
tion to evaluate the nitric oxide products, one of the most impor-
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Neuropsychobiology 3
DOI: 10.1159/000515929
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 50
40
30
Crossing, n
20
10
0 0
Control CORT CORT + RIP I CORT + FLU
Fig. 3. Effect of RIP I (50 mg/kg, p.o.) on locomotor activity in OFT
in a chronic CORT-induced depression mice model. Results are
expressed as mean ± SEM of 8–9 animals/group. No significative
values, according to 1-way ANOVA followed by the Newman-
Keuls’ post hoc test. OFT, open field test; RIP I, riparin I; FLU,
fluvoxamine; CORT, corticosterone.
tant reactive nitrogen species (RNS), as previously described by
Green et al. [22]. The absorbance was measured at 540 nm and was
determined from a standard nitrite curve generated by using
NaNO2 (0.75–100 μM) as standard, and the results were expressed
as micromolar of nitrite/nitrate per gram of tissue.
Determination of BDNF Levels
BDNF levels were determined in the HC with an anti-BDNF
sandwich ELISA (Quantikine® BDNF Immunoassay), according
to the manufacturer’s instructions (R&D Systems®, Minneapolis,
MN, USA). The amount of BDNF was determined by absorbance
in 450 nm and expressed as picogram per gram of wet tissue. The
standard curve demonstrates a direct relationship between optical
density and BDNF concentration.
Statistical Analysis
The behavioral results of PPI were analyzed by 2-way ANOVA
followed by a Bonferroni post hoc test, regarding treatment and
prepulse intensity factors. The results of the other behavioral de-
terminations and neurochemical parameters were analyzed using
1-way ANOVA followed by Student-Newman-Keuls’ post hoc test
for multiple comparisons. Previously, the normal distribution of
the data was evaluated. Significance level was set at p < 0.05. Data
analyses were performed using GraphPad Prism software for Win-
dows (version 6.0; GraphPad Software, San Diego, CA, USA).
Results
Effects of RIP I on Locomotor Activity of Mice in the
OFT
The OFT assesses possible alterations in the locomotor
activity of animals. The results presented no significant
4 Neuropsychobiology
DOI: 10.1159/000515929
250
###
200
Immobility time, s
150 ***
50 ***
Control CORT CORT + RIP I CORT + FLU
Fig. 4. Effect of RIP I (50 mg/kg, p.o.) on immobility time in FST
in a chronic CORT-induced depression mice model. Each bar rep-
resents mean ± SEM of 8–9 animals/group. Significative values:
###
p < 0.001 versus control; ***p < 0.001 versus CORT; according
to 1-way ANOVA followed by the Newman-Keuls’ post hoc test.
FST, forced swimming test; RIP I, riparin I; FLU, fluvoxamine;
CORT, corticosterone.
differences in spontaneous locomotion in any experi-
mental groups, as shown in Figure 3 (F[3, 28] = 3.276;
p > 0.05).
Effects of RIP I on Immobility Time of Mice in the FST
The immobility time in the FST was increased in the
CORT group when compared to the control group (p <
0.001). Figure 4 presents that the administration of RIP I
(p < 0.001) induced a reduction of the immobility time in
stressed animals (F[3, 32] = 43.74).
Effects of RIP I on Anhedonic-Like Behavior of Mice in
the Sucrose Preference Test
Mice treated with CORT, when compared to the con-
trol group, showed a lower preference for sucrose solu-
tion (Fig. 5) (F[3, 32] = 5.789; p < 0.05), and the treatment
with RIP I (p < 0.05) reversed the anhedonic-like behavior
induced by CORT.
Effects of RIP I on Memory of Mice in the SDA Test
The CORT-treated animals decreased the latency to
step-down, as compared to the control group (p < 0.05,
Fig. 6). In addition, an increase in this parameter was seen
in the group treated with RIP I (F[3, 26] = 4.214; p < 0.05).
Effects of RIP I on Memory of Mice in the YMT
As shown in Figure 7, the treatment with CORT pro-
duced a significant reduction in the correct alternations
in the YMT compared to the control group (p < 0.05),
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 100 400

**
80 * *
Sucrose consumption, %

* 300

Step-down latency, s
60
# 200 #
40

100
20

0 0
Control CORT CORT + RIP I CORT + FLU Control CORT CORT + RIP I CORT + FLU

Fig. 5. Effect of RIP I (50 mg/kg, p.o.) on anhedonic-like behavior Fig. 6. Effect of RIP I (50 mg/kg, p.o.) on memory in the SDA test
in the sucrose preference test in a chronic CORT-induced depres- in a chronic CORT-induced depression mice model. Each bar rep-
sion mice model. Each bar represents mean ± SEM of 9–10 ani- resents mean ± SEM of 8–9 animals/group. Significative values:
mals/group. Significative values: #p < 0.05 versus control; *p < 0.05 #p < 0.05 versus control; *p < 0.05; versus CORT, according to

versus CORT; **p < 0.01 versus CORT, according to 1-way ANO- 1-way ANOVA followed by the Newman-Keuls’ post hoc test.
VA followed by the Newman-Keuls’ post hoc test. RIP I, riparin I; SDA, step-down avoidance; RIP I, riparin I; FLU, fluvoxamine;
FLU, fluvoxamine; CORT, corticosterone. CORT, corticosterone.

while the administration of RIP I (p < 0.05) increased

80
them (F[3, 31] = 3.177).
*
*
Correct alternations, %

Effects of RIP I on PPI Test 60 #

Acoustic startle response following 75-, 80-, and 85-dB
prepulse intensities (Fig. 8) was used to measure PPI. A sig- 40
nificant effect of treatment was shown (F[3, 96] = 29.33; p <
0.001), with no significance in interaction between treatment
20
× prepulse (F[6, 96] = 0.2337; p > 0.05) or prepulse intensity
(F[2, 96] = 0.8353; p > 0.05). CORT-treated mice exhibited a
significant decrease in PPI for the 70 (p < 0.01) and 80 (p < 0
Control CORT CORT + RIP I CORT + FLU
0.05) dB prepulse stimulus, compared to control mice, while
RIP I treatment was able to normalize the startle response.
Fig. 7. Effect of RIP I (50 mg/kg, p.o.) on memory in YMT in a
chronic CORT-induced depression mice model. Each bar repre-
Effects of RIP I on Oxidative Stress Parameters sents mean ± SEM of 8–9 animals/group. Significative values: #p <
Lipid Peroxidation 0.05 versus control; *p < 0.05 versus CORT, according to 1-way
CORT administration significantly increased TBARS ANOVA followed by the Newman-Keuls’ post hoc test. YMT, Y-
content in PFC (F[3, 27] = 3.456; p < 0.05), HC maze test; RIP I, riparin I; FLU, fluvoxamine; CORT, corticoste-
rone.
(F[3, 31] = 13.61; p < 0.001), and ST (F[3, 30] = 11.12; p <
0.01) as compared to the control group. RIP I-treated
mice exhibited a reduction in TBARS content in PFC
(p < 0.05), HC (p < 0.001), and ST (p < 0.001), altered due (Fig. 10). RIP I treatment reversed CORT alterations in
to CORT injections (Fig. 9). PFC (p < 0.01) and ST (p < 0.01).

Nitrite/Nitrate Levels Reduced GSH Levels

Nitrite and nitrate levels were increased in PFC
(F[3, 31] = 5.240; p < 0.01) and ST (F[3, 29] = 6.910; p <
0.05) but not in HC (F[3, 26] = 1.433; p > 0.05), after
CORT treatment as compared to the control group
As shown in Figure 11, in the CORT group, the GSH
content decreased only in HC (F[3, 30] = 4.936; p < 0.01)
and ST (F[3, 33] = 4.852; p < 0.01), but no alteration was
observed in PFC (F[3, 29] = 2.887; p > 0.05). The treat-
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Effect of Riparin I in a Model of Neuropsychobiology 5

Depression DOI: 10.1159/000515929
Glasgow Univ.Lib.
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 80

*** *** ***

***
60
*** ***

Pre-pulse inhibition, % ■ Control

■ CORT
40 ■ CORT + RIP I
# ■ CORT + FLU
##

20

0
PP 70 PP 75 PP 80

Fig. 8. Effect of RIP I (50 mg/kg, p.o.) on the PPI test in a chronic CORT-induced depression mice model. Each
bar represents mean ± SEM of 8–9 animals/group. Significative values: #p < 0.05 versus control; ##p < 0.001 versus
control; ***p < 0.001 versus CORT, according to 2-way ANOVA followed by the Bonferroni’s post hoc test. PPI,
prepulse inhibition; RIP I, riparin I; FLU, fluvoxamine; CORT, corticosterone.

80 Prefrontal cortex 30 Hippocampus

###
#
60
*
MDA, µg/g tissue

MDA, µg/g tissue

20

40
* * ***
10
20

0 0
Control CORT CORT + RIP I CORT + FLU Control CORT CORT + RIP I CORT + FLU

25 Striatum
##
20
MDA, µg/g tissue

15

10
***
***
5

0
Control CORT CORT + RIP I CORT + FLU

Fig. 9. Effect of RIP I (50 mg/kg, p.o.) on MDA levels in the PFC, HC, and ST in a chronic CORT-induced de-
pression mice model. Each bar represents mean ± SEM of 7–9 animals/group. Significative values: #p < 0.05 ver-
sus control; ##p < 0.01 versus control; ###p < 0.001 versus control; *p < 0.05 versus CORT; ***p < 0.001 versus
CORT, according to 1-way ANOVA followed by the Newman-Keuls’ post hoc test. RIP I, riparin I; FLU, fluvox-
amine; CORT, corticosterone; MDA, malondialdehyde; PFC, prefrontal cortex; HC, hippocampus; ST, striatum.
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6 Neuropsychobiology Maia Oliveira et al.

DOI: 10.1159/000515929
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 8 Prefrontal cortex 5 Hippocampus

##
Nitrite/nitrate, µM/g tissue

Nitrite/nitrate, µM/g tissue

4
6

3
4 *
2
**
2
1

0 0
Control CORT CORT + RIP I CORT + FLU Control CORT CORT + RIP I CORT + FLU

10 Striatum
#
Nitrite/nitrate, µM/g tissue

4
**
2
**
0
Control CORT CORT + RIP I CORT + FLU

Fig. 10. Effect of RIP I (50 mg/kg, p.o.) on nitrite/nitrate levels in the PFC, HC, and ST in a chronic CORT-induced
depression mice model. Each bar represents mean ± SEM of 7–9 animals/group. Significative values: #p < 0.05
versus control; ##p < 0.01 versus control; *p < 0.05 versus CORT; **p < 0.01 versus CORT according to 1-way
ANOVA followed by the Newman-Keuls’ post hoc test. RIP I, riparin I; FLU, fluvoxamine; CORT, corticosterone;
PFC, prefrontal cortex; HC, hippocampus; ST, striatum.

ment with RIP I was able to reverse CORT-induced de- can reproduce characteristics similar to depression in hu-
crease in GSH levels in the HC (p < 0.05) and ST (p < mans, thus being a useful tool in the study of depressive
0.05), as shown in Figure 11. disorders, including treatment-resistant depression [25].
In addition to this, exogenous administration of high dos-
Effects of RIP I on BDNF Levels in the HC of Mice es of CORT promotes alterations in brain anatomy, neu-
The BDNF levels significantly decreased after CORT rochemistry, and behavior which may be indicative of a
administration (p < 0.05), while RIP I treatment increased depressive-like phenotype [26, 27].
this parameter in relation to the CORT group (F[3, 31] = The female gender is at a higher risk of developing de-
6.635; p < 0.05) as shown in Figure 12. pressive symptoms, as well as resistance to conventional
treatments [1]. The mechanisms involved in these sexual
differences remain unclear, but some researches point to
Discussion gender-specific characteristics, such as female hormonal
fluctuations throughout life and increased sensitivity to
Cortisol, classically known as the stress hormone, has catecholamines [28, 29]. Because of these important char-
been associated with depressive symptoms, given that pa- acteristics, many research studies are now being conduct-
tients with high blood glucocorticoid levels develop psy- ed using female animals [12, 19, 30–32], like this study. It
chiatric and cognitive symptoms compatible with those is an important point that females housed in groups can
seen in depressive conditions [23, 24]. Some animal mod- have a synchronous cycle or repress each other’s cyclicity,
els, such as those based on chronic exposure to CORT, thus masking the effect of the estrous cycle [33].
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Effect of Riparin I in a Model of Neuropsychobiology 7

Depression DOI: 10.1159/000515929
Glasgow Univ.Lib.
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 Prefrontal cortex Hippocampus
1,000 1,500

800 *
*
GSH, µg/g tissue

GSH, µg/g tissue

1,000
600

##
400
500

200

0 0
Control CORT CORT + RIP I CORT + FLU Control CORT CORT + RIP I CORT + FLU

Striatum
1,500

*
*
GSH, µg/g tissue

1,000
##

500

0
Control CORT CORT + RIP I CORT + FLU

Fig. 11. Effect of RIP I (50 mg/kg, p.o.) on GSH levels in the PFC, HC, and ST in a chronic CORT-induced de-
pression mice model. Each bar represents mean ± SEM of 7–9 animals/group. Significative values: ##p < 0.05
versus control; *p < 0.05 versus CORT, according to 1-way ANOVA followed by the Newman-Keuls’ post hoc
test. RIP I, riparin I; FLU, fluvoxamine; CORT, corticosterone; GSH, glutathione; PFC, prefrontal cortex; HC,
hippocampus; ST, striatum.

This study was able to demonstrate that exposure to

400 the chronic treatment with CORT induced a depressive-
like behavior in the FST. On the other hand, the results
300 obtained show antidepressant-like activity of RIP I ex-
**
BDNF, pg/g tissue

* pressed by reduced immobility time in this test.

200
The FST is considered a gold standard animal test to as-
## sess the antidepressant-like effect of compounds based on
a behavioral despair measured by the immobility behavior
100
of animals. Because of this, the psychomotor stimulant/sed-
ative effects of a substance can lead to false positive/negative
0 results, thus the influence of the test drug on the locomotor
Control CORT CORT + RIP I CORT + FLU
activity of the animal is a governing concern [34]. Based on
this, the OFT was run as a control paradigm to ensure that
Fig. 12. Effect of RIP I (50 mg/kg, p.o.) on BDNF levels in the HC the behavior of interest does not depend on differential mo-
in a chronic CORT-induced depression mice model. Each bar rep- bility of the control and experimental groups. Since there
resents mean ± SEM of 8–9 animals/group. Significative values: was no significant difference in the locomotor activity be-
##p < 0.01 versus control; *p < 0.05 versus CORT; **p < 0.01 versus

CORT, according to 1-way ANOVA followed by the Newman- tween groups, the tests which evaluate immobility in this
Keuls’ post hoc test. RIP I, riparin I; FLU, fluvoxamine; CORT, study are reliable. Our results reinforce the data previously
corticosterone; HC, hippocampus. observed by Sousa et al. [10] in an acute depression model.
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8 Neuropsychobiology Maia Oliveira et al.

DOI: 10.1159/000515929
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 Anhedonia is an important feature of all forms of de-
pression and can greatly influence general symptoms [2].
This may be related to interactions between brain reward
systems and stress, involving dysfunction of mesolimbic
dopamine pathways [35]. Several authors [12, 16, 30, 31,
36] previously described that high levels of glucocorti-
coids can reduce preference for sweetened solution in an-
imals, an anhedonic-like behavior. The ability of RIP I
treatment to reverse this parameter is an important result,
given that anhedonia is a difficult symptom to be treated
with conventional pharmacotherapies, such as SSRI [37].
Various neuropsychiatric conditions, such as depres-
sive disorders with psychotic features and schizophrenia,
are related to failure of PPI. This inhibition is an attenu-
ation in startle reflex magnitude that occurs when a star-
tling stimulus (a pulse) is closely preceded by a weak sen-
sory event (a prepulse or prestimulus), related to a senso-
rimotor gating which coordinates the input of sensory
stimuli, avoiding sensory overload in the brain [19]. In
this study, the chronic treatment with CORT was able to
cause impairment in the sensorimotor gating, implicat-
ing in a relation between the neurobiology of schizophre-
nia, stress response, and PPI. This effect is similar to that
obtained after ketamine administration, a known psy-
chotic drug previously described [19].
Similarly, a positive correlation between cortisol levels
and progression and severity of the psychotic symptoms
was also observed in a clinical study conducted by Walk-
er et al. [38]. In addition, Duval et al. [39] showed that
patients with unipolar major depression presenting mel-
ancholic and psychotic features and concomitant cortisol
hypersecretion exhibited monoaminergic dysfunction.
Thus, the hypothalamic-pituitary-adrenal axis hyperac-
tivity can alter the neurotransmission of various systems
and circuits related to the pathophysiology of psychosis.
Therefore, this could be related, at least in part, to the
genesis of psychotic symptoms in depressed patients [40].
In addition to the emotional symptoms, some studies
report the onset of cognitive impairment in depressed pa-
tients. This dysfunction can remain even in periods of
remission [41–43]. The lack of satisfactory treatments for
cognitive deficits that usually accompany psychiatric dis-
orders presents a constant challenge for psychopharma-
cological research [41]. The ability of RIP I to reverse the
memory and sensorimotor gating impairment caused by
CORT is an interesting finding, since depressive disor-
ders are commonly associated with several disabling co-
morbidities that affect the patient’s life quality and cause
difficulty in pharmacotherapy.
Effect of Riparin I in a Model of
Depression
The disruption of the redox signaling is important in
the pathophysiology and progression of unipolar and bi-
polar depression. When present in abundance, ROS and
RNS, such as peroxynitrite, superoxides, peroxides, and
nitric oxide, can induce functional and structural chang-
es that result in cell damage [44]. The effects of ROS and
RNS are strongly harmful to the brain tissue since it con-
tains high concentrations of polyunsaturated fatty acids
that are susceptible to lipid peroxidation. Therefore, en-
hanced accumulation of free radicals results in oxidative
stress that culminates in the development of neuropsy-
chobiological deficits such as depressive disorders [45,
46].
Previous studies have shown that repeated administra-
tion of CORT results in increased pro-oxidant markers
and decline in antioxidant mechanisms in the brain [19,
30, 47]. This suggests that stress hormones have a causal
role in oxidative processes inducing local damage that can
be correlated with the cognitive deficit and depressive-
like behavior observed in the mice in this study. Some
research involving depressed patients has shown an in-
creased lipid peroxidation in brain areas, as well as elevat-
ed levels of peroxynitrite (and its precursor, nitric oxide)
and low levels of GSH, pointing to the pathophysiology
of stress-induced depression [48–50].
In addition, Lopes et al. [30], Sato et al. [51], and Mel-
lo et al. [52] demonstrated these same oxidative stress pa-
rameters altered in the brain of animals submitted to
stress induced by CORT [30, 51] and lipopolysaccharide
[52], corroborating our results that demonstrated chron-
ic administration of CORT leading to an oxidative imbal-
ance. The demonstration of RIP I treatment reversing this
alteration is also in line with previous studies, in which
antidepressants were able to decrease oxidative damage
[30, 47]. Consequently, it is suggested that RIP I may be
a promising therapeutic strategy for depression, with the
reduction of oxidative damage being a possible mecha-
nism of its antidepressant effect.
Some studies indicate that the HC is one of the most
susceptible brain areas to CORT-induced oxidative im-
balance. This may be related to the greater density of glu-
cocorticoid receptors in this area and, consequently, to
the greater impact of the deregulation of the glucocorti-
coid system on the redox balance, since these receptors
are considered as regulatory factors for antioxidant en-
zymes [47, 53].
The oxidative stress is related to a reduction of BDNF,
cAMP response element-binding protein, and synapsin I
level, proteins implicated in cellular plasticity. It is known
that BDNF functions as a relevant mechanism of regula-
130.209.6.61 - 8/12/2021 3:49:45 PM
Neuropsychobiology 9
DOI: 10.1159/000515929
Glasgow Univ.Lib.
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tion of synaptic plasticity and synaptogenesis, which are
involved in learning and memory in the mature central
nervous system, as well as in mood regulation. These
functions may be adversely affected by the reduction of
BDNF levels [54]. In conclusion, the present study sug-
gests that the putative antidepressant mechanism of RIP
I can be related to the production of neurotrophic factors
and reestablishment of oxidative stress balance, indicat-
ing a potential neuroprotective activity of the substance,
which makes RIP I a promising candidate in the treat-
ment of depression.
Statement of Ethics
The experimental protocols followed the ethical principles in
animal experimentation adopted by the Brazilian College of Ani-
mal Experimentation (COBEA), as well as the Guide for the Care
and Use of Laboratory Animals published by the National Insti-
tutes of Health. This study was approved by the Animal Research
and Ethics Committee of the Federal University of Ceará (protocol
58/2013).
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
This work was supported by grants from Conselho Nacional
de Desenvolvimento Científico e Tecnológico (CNPq) (306746/
2013-1; 407567/2013-5; 465536/2014-0; 446120/2014-6; and
308141/2017-2).
Author Contributions
Oliveira I. designed the study and wrote the first draft of the
manuscript. Mallmann A., Rodrigues F., and Vidal L. performed
behavioral tests. Rodrigues G., Oliveira N., and Capibaribe V. con-
tributed to neurochemical analyses. Carvalho A. and Chaves R.
performed statistical analyses. Gutierrez S. and Barbosa-Filho J.
worked on riparin synthesis. Sales I. and Fonteles M. revised the
manuscript. Sousa F. was responsible for funding acquisition and
project administration.

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