Journal of Histotechnology

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Grape seed proanthocyanidins improves

depression-like behavior by alleviating oxidative
stress and NLRP3 activation in the hippocampus of
prenatally-stressed female offspring rats

Qinru Sun, Ning Jia, Fei Ren & Xin Li

To cite this article: Qinru Sun, Ning Jia, Fei Ren & Xin Li (2021): Grape seed proanthocyanidins
improves depression-like behavior by alleviating oxidative stress and NLRP3 activation in the
hippocampus of prenatally-stressed female offspring rats, Journal of Histotechnology, DOI:
10.1080/01478885.2020.1861907

To link to this article: https://doi.org/10.1080/01478885.2020.1861907

Published online: 11 Jan 2021.

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JOURNAL OF HISTOTECHNOLOGY
https://doi.org/10.1080/01478885.2020.1861907

Grape seed proanthocyanidins improves depression-like behavior by alleviating

oxidative stress and NLRP3 activation in the hippocampus of prenatally-stressed
female offspring rats
Qinru Suna, Ning Jiab, Fei Renb and Xin Lib
a
Institute of Forensic Medicine, Xi’an Jiaotong University Health Science Center, Xi’an Shaanxi, P.R. China; bDepartment of Human Anatomy,
Histology and Embryology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an Shaanxi, P.R. China

ABSTRACT KEYWORDS
Over several decades, there is a growing evidence, which has shown that prenatal stress (PS) Grape Seed
contributes to depression in offspring. Grape seed proanthocyanidins (GSPs), which contain dimers, Proanthocyanidins;
trimers, oligomers of catechin and epicatechin, are known to possess antidepressant effects. The neuroinflammation; prenatal
present study aimed to investigate the mechanism of antidepressant effects of GSPs on female Stress; depression; rat;
reactive Oxygen Species;
juvenile prenatally stressed offspring rats. The results showed that the female juvenile offspring rats antioxidants; cytokines
exposed to PS exhibited depression-like behavior manifested as longer immobility time and lesser
consumption of sucrose solution. Prenatal stress reduced the number of hippocampal neurons and
increased the level of the reactive oxygen species (ROS) in the hippocampus of the female juvenile
offspring rats. Furthermore, the expression of PYD domains-containing protein 3 (NLRP3) and its
downstream cytokines, Caspase-1, and interleukin-1β (IL-1β), were increased in the hippocampus of
the female juvenile offspring rats exposed to PS. Administration of GSPs not only improved depres­
sion-like behavior and enhanced the number of hippocampal neurons, but also abated excessive
ROS generation and inhibited the activation of the NLRP3-Caspase-1 signaling pathway. Taken
together, GSPs counteract PS-induced hippocampal neuron loss and depression-like behavior by
alleviating oxidative stress and NLRP3 activation. The present study provides a new insight for GSPs
as an effective therapeutic agent for adolescent depression.

Introduction Recent studies has shown that depressive patients

usually exhibit high level of ROS, mitochondrial mem­
Increasing studies across a wide range of species have
brane depolarization, oxidized mitochondrial DNA, and
shown that prenatal exposure to various conditions
altered inflammatory markers. Several studies have shown
including infections, nutritional deficiencies, teratogenic
the levels of pro-inflammatory cytokines, such as tumor
substances, and emotional distress, contribute to adverse
necrosis factor alpha (TNF-α), interleukin-1 (IL-1), inter­
pregnancy outcomes [1–3]. Adverse prenatal exposure
leukin-6 (IL-6) and interleukin-18 (IL-18), are increased in
can lead to cognitive impairments, mental disorders,
patients with depression [10–12]. Compared to placebos,
and metabolic diseases in juvenile and adult offspring
anti-inflammatory agents exhibit antidepressant treatment
[4–6]. However, the mechanism of mental disorder
effects in major depression disorder (MDD) patients [13].
induced by prenatal stress (PS) is unclear. In our previous
A recent study has shown that PS increases depression-like
studies, it was found that the offspring rats exposed to
behavior, pro-inflammatory cytokines (IL-1β and TNF-α)
prenatal stress (PS) had lower birth weight [7], and
and decreases the protein expression of brain-derived
exhibited depression-like behavior at the age of
neurotrophic factor (BDNF) in whole brain of rodent off­
1 month with neuron loss in the hippocampus. In addi­
spring [14]. These data indicate that neuroinflammation
tion, PS offspring showed excessive intracellular reactive
contributes to depression-like behavior in prenatally
oxygen species (ROS) [8] and decreased level of the
stressed offspring. However, how PS caused neuroinflam­
brain-derived neurotrophic factor (BDNF) mRNA in
mation activation in offspring is still needs to be clarified.
the prefrontal cortex and hippocampus [9]. These results
Studies have shown that PYD domains-containing protein
suggest that oxidative damage of neurons in several brain
3 (NLRP3), a kind of inflammasome complex, is increased
regions is related to PS-induced depression.
in the prefrontal cortex rats with depression-like behavior

CONTACT Ning Jia jianing@mail.xjtu.edu.cn Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Xi’an
Jiaotong University Health Science Center No. 76, West Yanta Road, Xi’an Shaanxi 710061, P.R. China
© 2020 National Society for Histotechnology
2 Q. SUN ET AL.
[15,16], indicating that NLRP3 is associated with depres­
sion. In addition, ROS has been identified as an important
activator of NLRP3 [17,18]. Taken together, we hypothe­
sized that activation of NLRP3 might be associated with
neuroinflammation and depression-like behavior in pre­
natally stressed offspring.
The adverse effects of ROS on the brain include lipid
peroxidation of neuronal membranes, protein damage,
DNA damage, reduced antioxidant defense capacity, apop­
tosis induction, and neuroinflammation. Antioxidants such
as resveratrol (RES), N-acetylcysteine (NAC) and coenzyme
Q have shown promise in some patients and animals with
depression, although no consensus has yet been reached on
their efficacy [19–21]. Grape seed proanthocyanidins
(GSPs) contain dimers, trimers, oligomers of catechin and
epicatechin, which can resist oxidative stress, promote
DNA repair and prevent mitochondrial dysfunction and
apoptotic cell death [22–24]. Another study showed that
proanthocyanidin could prevent lipopolysaccharide (LPS)-
induced depression-like behavior by inhibiting of neuroin­
flammatory response in mice [25]. Hence, we wondered
whether GSPs could improve depression-like behavior via
alleviating NLRP3-mediated neuroinflammation in prena­
tally-stressed female offspring.
In this study, the prenatal restraint stress in rats was
employed as the animal model. The forced swimming
test (FST) and the sucrose preference test (SPT) were
used to test the depression-like behavior. In order to
explore the mechanism of antidepressant effects of
GSPs, the number of the hippocampal neurons, the
level of intracellular ROS, and the activation of the
NLRP3-Caspase-1 signaling were determined.
Materials and Methods
Animals and groups
Sprague–Dawley rats were purchased from Medical
Experimental Animal Center, Xi’an Jiaotong University.
All rats were maintained at constant temperature (25°C)
and humidity (60%) on a 12 h light/dark cycle, with free
Figure 1. The schematic of experiment. Prenatal stress was performed on pregnant mother from embryo day 14 (E14) to embryo day
20 (E20). Proanthoycyanidins were administered between postnatal day 21 (P21) and postnatal day 30 (P30) before behavioral tests.
access to food and water. All experimental procedures
were approved by the Institutional Animals Care and Use
Committee of Xi’an Jiaotong University. Measurements
were taken to minimize pain or discomfort in accordance
with the National Institutes of Health Guide for Care and
Use of Laboratory [26].
Virgin female rats weighing 250–270 g were housed
with a male rat (280–350 g) for mating (2:1) overnight.
On the following morning, a vaginal smear was examined.
The day 0 of gestation was defined if the vaginal smear was
indicating pregnancy and each pregnant rat was then
housed individually. The pregnant rats were randomly
subjected to either restraint stress or not disturbed. Their
offspring were assigned to either the prenatal stress group
(PS) or control (CON) group, respectively.
Processing of prenatal stress
A transparent cylinder (6.8 cm in diameter) was used as
the restraint stress device with air holes in the cylinder
were for breathing. The length of the cylinder could be
adjusted to accommodate the size of the animals. During
gestation day 14–20, the pregnant rats were subjected to
restraint stress 3 times per day (at approximately 8:00 AM;
12:00 PM; and 4:00 PM), for 45 min per session. After
birth of a litter, each mother and her offspring rats were
placed in same cage until weaning on postnatal day 21
(Figure 1). To reduce differences during breastfeeding, the
litters containing 8–14 pups were selected for next experi­
ments. In this study, only the female offspring were used.
Oral grape seed proanthocyanidins (GSPs)
administration
Grape seed proanthocyanidins (GSPs) (lot number:
002–1501003-04) were purchased from Tianjin Jianfeng
Natural Product R&D Co., Ltd in China. GSPs contain
98.20% total polyphenols, including 85.22% oligomeric
proanthocyanidins, 6.99% catechin, 4.17% epicatechin.
From postnatal day 21 to postnatal day 30, the female

offspring rats from PS were treated orally with GSPs via
their drinking water to achieve a daily intake of 200 mg/kg
body weight and called the high dose group (GSPs-H).
Meanwhile, a lower daily intake dose of 20 mg/kg body
weight was administered via drinking water and called the
low dose group (GSPs-L) (Figure 1).
Sucrose preference test (SPT)
The rats were habituated to a 1% (w/v) sucrose
(10021418, Sinopharm Group Chemical Reagent Co.
LTD, China) aqueous solution in home cages for 24 h
before the experiment. Rats were fasted for 6 h before
test. During the test, 1 bottle containing tap water and 1
bottle filled with a 1% sucrose solution were simulta­
neously provided to rats randomly for 1 h [27]. The
amount of fluid intake was measured to calculate the
percentage. Sucrose preference was calculated as follows:
Sucrose preference (%) = sucrose solution consump­
tion ÷ (water consumption + sucrose solution con­
sumption) × 100%.
Forced Swimming Test (FST)
The FST was conducted according to a previous report [9].
Each rat was individually placed into a Perspex cylinder
(diameter 18 cm, height 40 cm) filled with 25 °C water to
a height of 30 cm for 6 min as the pre-test. After 24 h, the
rats remained immobile for a 5 min observation period
which was recorded with a video tracking system
(SMART3.0, Panlab, USA). The longer immobile time
indicated increased depressive-like behavior.
Nissl staining
After the behavior test, six offspring rats in each group
were anesthetized and perfused transcardially with
approximately 150 ml physiological saline (SY15932,
Sinopharm Group Chemical Reagent Co.), followed by
approximately cold 200 ml (4 °C) 4% paraformaldehyde
in PBS (PI015, Xi’an Hat Biotechnology Co. Ltd, China)
(pH 7.4). The brains were removed according to pre­
vious study [25], cut into slices containing the hippo­
campus, and post-fixed in the 4% PFA for 24 h at 4 °C.
Subsequently, the hippocampus samples were processed
for paraffin embedding and frontal sections 6 μm thick
were cut from the blocks. Sections were deparaffinized
in xylene (10,023,418, Sinopharm Chemical Reagent
Co.), rehydrated through 100%, 95%, 70%, 50% ethanol
(80,176,961, Sinopharm Chemical Reagent Co.), 5 min
each, and placed in cold running tap water (H2O). All
slides were rinsed in 0.01 M PBS (PW012, Xi’an Hat
Biotechnology Co. Ltd, China) for 5 min. For Nissl
JOURNAL OF HISTOTECHNOLOGY 3
staining, the toluidine blue (89,640, Sigma-Aldrich,
USA) (1% w/v) was dissolved in double-distilled water
(ddH2O) and pipetted onto the sections for 30 min
followed by a tap H2O wash, and successively dipped
in 50%, 70%, 95% ethanol for 5 min each cleared in
xylene for 10 min (two changes [28]. Cover slips were
mounted by using a resinous medium and photo­
graphed under an Olympus light microscope (BX53,
Olympus, Japan). The number of stained cells in each
field was counted at high magnification (400x). For
quantitative analysis, the neuronal cells in hippocampal
CA1 and CA3 areas were counted and expressed as
number of cells/100 μm2. The data were analyzed as
the number of cells per high-power field.
Measurement of ROS level
Isolated hippocampus samples (30 mg/ml in PBS) were
homogenized by Dounce tissue homogenizer on the ice
then centrifuged at 10,000 x g for 5 min at 4 °C. According
to the manufacturer’s instruction, the ROS levels were
measured with the OxiselectTM in vitro ROS/RNS assay
kit (STA-347-T, Cell Biolabs, USA). Briefly, 50 µl of homo­
genate samples were incubated with 50 µl of catalyst and
100 µl of 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (DCF)
for 30 min at RT. The fluorescence (Ex480 nm/Em530 nm)
as % of fluorescence increase was measured using a Multi-
Mode Microplate Reader (Synergy HTX, Bio-Tek, USA).
Protein extraction and Western Blot analysis for
NLRP3, Caspase-1 and IL-1β
Six rats in each group were anesthetized with ether and
euthanized by decapitation. The brain was removed
rapidly, hippocampus dissected away on ice. The hippo­
campal sample was rinsed with PBS 3 times then homo­
genized at 1: 5 (w/v) in an ice-cold RIPA lysis buffer
(PP1201, Shaanxi Xianfeng Biotechnology Co., Ltd,
China) with protease inhibitor cocktail and phosphatase
inhibitor cocktail (78,445, Thermo Fisher Scientific, USA).
Samples were resolved by SDS-PAGE (XF1011,
Shaanxi Xianfeng Biotechnology Co.) and transferred to
Polyvinylidene Fluoride (PVDF) membranes (IPFL00005,
Merck, USA). The blots were probed with the following
primary antibodies: mouse anti β-actin (anti-β-actin,
mouse monoclonal, IgG, clone AC-74, A2228, RRID:
AB_476697, Sigma-Aldrich, USA), rabbit anti-NLRP3
(anti-NLRP3, rabbit polyclonal antibody, IgG, A12694,
RRID:AB_2759538, ABcolonal, China), rabbit anti-
Caspase-1 (anti-Caspase-1, rabbit polyclonal anti­
body, IgG, A0964, RRID:AB_2757485, ABcolonal)
and rabbit anti-IL-1β (anti- IL-1β, rabbit polyclonal
antibody, IgG, A1112, RRID:AB_2758416, ABcolonal),
4 Q. SUN ET AL.

followed by incubation with the appropriate horseradish
peroxidase-conjugated secondary antibodies (SA00001-1,
SA00001-2, Proteintech Group WUHAN SANYING,
China). The blots were incubated with a chemilumines­
cence substrate solution (SuperSignal™ West Pico PLUS
Chemiluminescent Substrate, 34580, Thermo Fisher
Scientific) and veiwed with a chemiluminescent detection
system (SYSTEM GelDoc XR+ IMAGELA, Bio-Rad,
USA). The optical density of immunoreactive bands was
quantified using the Quantity One 1-D analysis software
(Bio-Rad).
Statistics analysis
All values were represented as mean ± SEM, and data
analyses were performed using the software of SPSS V21.0
(IBM, USA). One-way ANOVA followed Dunnett post-hoc
test was used to examine differences among the groups.
A difference was considered significant at p < 0.05 level.
Results
GSPs improved depression-like behavior induced by
PS
To understand the role of GSPs in depression-like beha­
vior in PS offspring rats, sucrose preference test (SPT)
and forced swimming test (FST) was performed. For the
sucrose preference (%) in offspring rats, the 1% sucrose
preference (%) was significantly lower in the PS group
than that in the CON group (F3, 37 = 93.120, p < 0.01,
Figure 2a). As compared with the PS group, the sucrose
preference (%) was significantly increased in the GSPs-
H group (F3, 37 = 93.120, p < 0.01) but not in the GSPs-L
group (Figure 2a).
Forced swimming test (FST) results showed that the
immobility time in the PS group was significantly longer
than that in the CON group (F3, 37 = 603.58, p < 0.01,
Figure 2b). With comparison to the PS group, the
immobility time was significantly shortened in the
GSPs-H group (F3, 37 = 603.58, p < 0.01, Figure 1c) but
not in the GSPs-L group.
The body weight of offspring rats were recorded on
the postnatal day 30. The results showed that the body
weight of PS offspring rats were significantly lower than
offspring rats in CON group. Administration with GSP-
L or GSP-H did not show significant effect on the body
weight in PS offspring (Table 1).
The data revealed that GSPs improved the sucrose
preference and decreased immobility time of PS off­
spring, indicating GSPs could improve depression-like
behavior of PS offspring rats.
GSPs attenuated PS-induced hippocampal neuron
loss
To determine whether GSPs can protect the neurons
from PS-induced damage, hippocampal cellular mor­
phology was evaluated by using the Nissl staining. As
shown in Figure 3a, the hippocampus of the PS group
displayed injured neurons. Most damaged neurons

Figure 2. GSPs improved depression-like behavior induced by Prenatal Stress. (a) Sucrose preference test (SPT) shows the 1% sucrose
preference in PS group was lower than that in CON group. Administration of high dosage of GSPs counteracted the decrease of
increased sucrose preference induced by PS. (b) Forced swimming test (FST) shows PS group displayed longer immobility time in
seconds (sec) as compared with CON group. Administration of high dosage of GSPs decreases immobility time of PS offspring rats in
FST Control (CON), Prenatal Stress (PS), Grape Seed Proanthocyanidins (GSPs), PS offspring rats administrated with GSPs Low Dose
daily 20 mg/kg b.w. (GSPs-L), PS offspring rats administrated with GSPs High Dose daily 200 mg/kg b.w. (GSPs-H). *p < 0.01 versus
CON; #p < 0.01 vs PS (n = 10 rats in CON group. n = 10 in PS group. n = 8 in GSPs-L group and n = 10 in GSPs-H group.).
 JOURNAL OF HISTOTECHNOLOGY 5

Table 1. The body weight of offspring rats on postnatal day 30 of the PS group were dramatically decreased, which was
before behavior experiments. counteracted by administrated of GSPs-H but not GSPs-
Treatment Group Number of rats/Group Body weight (g) L (CA1 F3, 23 = 21.02, CA3 F3, 23 = 35.89, p < 0.01)
CON 12 77.6 ± 1.1
PS 12 70.3 ± 0.6*
(Figure 3b,c).
GSPs-L 10 71.4 ± 0.9*
GSPs-H 12 70.8 ± 0.8*
Control (CON), Prenatal Stress (PS), Grape Seed Proanthocyanidins (GSPs), PS
offspring rats administrated with GSPs Low Dose daily 20 mg/kg b.w. GSPs reversed the increase of intracellular ROS
(GSPs-L), PS offspring rats administrated with GSPs High Dose daily generation in the hippocampus induced by PS
200 mg/kg b.w. (GSPs-H). *p < 0.01 vs CON.
To understand the antioxidant effects of GSPs, the ROS
showed shrinkage with condensed nuclei and sparse level in the hippocampus were measured. As shown in
Nissl bodies. However, in the GSPs-H group, neurons Figure 4, the level of ROS in the hippocampus increased
had larger cell bodies compared to the PS group, and significantly in the PS group as compared to the CON
most of the neurons contained visible Nissl bodies. group (F3, 23 = 554.89, p < 0.01). GSPs-H treatment
However, treatment with GSPs-L did not show signifi­ significantly attenuated ROS generation in the hippo­
cantly changes. Quantitative data showed that the num­ campus (F3, 23 = 554.89, p < 0.01). However, adminis­
ber of Nissl staining cells in hippocampal cornu tration of low dosage of GSPs (GSPs-L) did not show
ammonis 1 (CA1) and cornu ammonis 3 (CA3) regions significant changes.

Figure 3. GSPs attenuated hippocampal neuron loss induced by Prenatal Stress. (a) Representative images of Nissl staining of
hippocampus. (b) Comparison of neuron number in the hippocampal CA1 region among different groups. PS increased neuron loss,
which was attenuated by administration of high dosage of GSPs. (c) Comparison of neuron number in the hippocampal CA3 region
among different groups. Administration of high dosage of GSPs attenuated neuron loss induced by PS. Neurons stained with Toluidine
Blue (arrows). Scale bar = 400 µm for the images of hippocampus. Scale bar = 40 µm for images of CA1 or CA3. *p < 0.01 vs CON;
#
p < 0.01 vs PS (n = 6 in CON, PS, GSPs-L and GSPs-H group, respectively).
6 Q. SUN ET AL.

Figure 4. GSPs decreased intracellular ROS generation in the hippocampus induced by Prenatal Stress. The level of ROS was
increased in hippocampus of the PS group compared with that in CON group. Administration with high dosage of GSPs
decreased PS-induced ROS increase. Control (CON), Prenatal Stress (PS), Grape seed proanthocyanidins-Low dose (GSPs-L) and
Grape seed proanthocyanidins-High dose (GSPs-H). *p < 0.01 vs CON; #p < 0.01 vs PS (n = 6 rats in CON, PS, GSPs-L and GSPs-
H groups, respectively).

GSPs blocked the activation of the NLRP3-Caspase-1
signaling pathway in the hippocampus induced by PS
To further determine whether GSPs inhibited ROS-
induced hippocampal inflammation in PS offspring,
the levels of NLRP3, Caspase-1 and IL-1β in hippocam­
pus were detected by Western blotting. Compared to the
CON offspring, PS offspring displayed increased levels
of NLRP3, Caspase-1 and IL-1β in the hippocampus
(Figure 5a). After administration of GSPs-H, PS-
induced levels of NLRP3, caspase-1 and IL-1β were
significantly reduced in the hippocampus (NLRP3
F3, 23 = 540.36; caspase-1 F3, 23 = 412.09; IL-1β, F3, 23
= 522.473, p < 0.01) (Figure 5b–d). Not surprisingly,
administration of low dosage of GSPs (GSPs-L) did not
show significantly changes (Figure 5b–d).
Discussion
Although the monoamine hypothesis has been widely
accepted as a molecular mechanism to explain the etiology
of depression, the causes of depression are multifactorial
and complex, including genetic and environmental fac­
tors. Up to now, the efficacy of antidepressants against
monoamine system is limited. In addition, the safety of
long-term use of antidepressants still needs to be further
evaluation, especially in children and adolescents. Over
the past two decades, extracts and prescriptions of
Traditional Chinese Medicine have been considered to
be the pursuit of antidepressants [29]. Proanthocyanidins
are widely available in many plants, such as fruits, vege­
tables, nuts and seeds, especially in grape seeds [30].
According to an epidemiological investigation report,
higher dose of proanthocyanidins intakes may be benefi­
cial for reducing depression risk, particularly in elderly
women [31]. However, the antidepressant mechanism of
proanthocyanidins remains unclear.
Prenatal stress (PS) could predispose of the offspring
toward vulnerability to neurobehavioral disorders [32,33].
In the present study, we found that high dosage of GSPs
(daily intake of 200 mg/kg body weight b.w.) from P21 to
P30 significantly reversed the shrinkage of immobility
time and the decrease of consumption of sucrose solution
induced by PS. These results demonstrated that adminis­
tration of GSPs counteracted depression-like behaviors of
juvenile offspring rats induced by PS. The Jiang et al study
indicated that GSPs possessed effective therapeutic effects
against lipopolysaccharide-induced depressive-like beha­
vior [25]. To explore the mechanism of antidepressant
effect of GSPs, the number of neurons in hippocampus,
the level of the intracellular ROS and the activation of
NLRP3-Caspase-1 signaling pathway were determined.
The results showed that administration of high dosage
of GSPs significantly reversed the decline of neuron num­
ber and the increase of intracellular ROS generation in PS
offspring hippocampus. These results suggest that admin­
istration of GSPs may inhibit accumulation of intracellu­
lar ROS and protect neurons from PS-induced oxidative
stress. Our results are consistent with the finding by
 JOURNAL OF HISTOTECHNOLOGY 7

Figure 5. GSPs blocked the activation of the NLRP3-Caspase-1 signaling pathway in the hippocampus induced by Prenatal Stress. (a)
The representative immunoblot bands for NLRP3, Caspase-1 and IL-1β. Protein expression levels were normalized to β-actin. The
quantification analysis of (b) NLRP3, (c) Caspase-1and (d) IL-1β among different groups. Control (CON), Prenatal Stress (PS), Grape seed
proanthocyanidins-Low dose (GSPs-L) and Grape seed proanthocyanidins-High dose (GSPs-H). *p < 0.01 vs CON; #p < 0.01 vs PS (n = 6
in CON, PS, GSPs-L and GSPs-H group, respectively).

Abhijit et al, who believe that combine of GSPs and Conclusion

aerobic exercise can effectively reduce ROS generation
In conclusion, administration of GSPs can counteract
and increasing neuron number in hippocampus [34].
neuron loss and depression-like behavior in juvenile pre­
Kun Fu et al reported that GSPs could protect Neuro-2a
natally stressed offspring rats. In addition, GSPs can sup­
cells (N2a) cells from OGD/R insult by decreasing ROS
press excessive ROS generation and the activation of
level [35]. The present study provides evidence for the
NLRP3-Caspase-1 in hippocampus, thereby blocking sub­
neuronal protection and potential antidepressant effects
sequent hippocampal neuroinflammation. The present
of GSPs by decreasing ROS level.
study provides a new theoretical foundation for GSPs as
Furthermore, administration of high dosage of GSPs
an effective therapeutic agent for adolescent depression.
abated the activation of the NLRP3-Caspase-1 signaling
pathway. These results illustrate that NLRP3-mediated
neuroinflammation is involved in PS-induced neuron
Disclosure statement
loss in hippocampus, which are ameliorated by GSPs via
inhibiting NLRP3-Caspase-1 signaling pathway. Previous The authors declare no conflict of interest.
study has shown that NLRP3-Caspase-1 signaling pathway
mediates neuroinflammation in the prefrontal cortex of
depressive rats [15]. Administration of proanthocyanidins Funding
reversed LPS-induced depression-like behavior in male This work was supported by National Natural Science Foundation
mice by inhibiting neuroinflammation [25]. In the present of China [81500927]; Key Research and Development Program of
study, we found that the NLRP3-Caspase-1 signaling path­ Shaanxi [2020SF-133]; National Science Foundation for Post-doc­
way contributes to neuroinflammation in the hippocam­ toral Scientists of China [2018T111074]; National Science
Foundation for Post-doctoral Scientists of China [2017M623190];
pus of juvenile prenatally stressed offspring rats.
Foundation for Post-doctoral Scientists of Shaanxi Province
Administration of GSPs could reverse the activation of [2018BSHERZZ97]; Natural Science Basic Research Plan in
NLRP3-Caspase-1, thereby further blocking PS-induced Shaanxi Province of China [2017JM8017]; and Natural Science
hippocampal neuroinflammation. Basic Research Plan in Shaanxi Province of China [2016JM8041].
8 Q. SUN ET AL.
References
[1] Christian LM. Psychoneuroimmunology in pregnancy:
immune pathways linking stress with maternal health,
adverse birth outcomes, and fetal development. Neurosci
Biobehav Rev. 2012;36:350–361.
[2] Fekadu Dadi A, Miller ER, Woodman RJ, et al. Effect of
antenatal depression on adverse birth outcomes in
Gondar town, Ethiopia: A community-based cohort
study. PLoS One. 2020;15:e0234728.
[3] Martines F, Salvago P, Ferrara S, et al. Factors influen­
cing the development of otitis media among Sicilian
children affected by upper respiratory tract infections.
Braz J Otorhinolaryngol. 2016;82:215–222.
[4] Moukarzel S, Ozias M, Kerling E, et al. Maternal
Vitamin D status and infant infection. Nutrients.
2018;10:111.
[5] Cannizzaro E, Martire M, Gagliano M, et al. Reversal of
prenatal diazepam-induced deficit in a spatial-object
learning task by brief, periodic maternal separation in
adult rats. Behav Brain Res. 2005;161:320–330.
[6] Hellemans KG, Verma P, Yoon E, et al. Prenatal alcohol
exposure and chronic mild stress differentially alter
depressive- and anxiety-like behaviors in male and female
offspring. Alcohol Clin Exp Res. 2010;34:633–645.
[7] Jia N, Sun Q, Su Q, et al. Taurine promotes cognitive
function in prenatally stressed juvenile rats via activat­
ing the Akt-CREB-PGC1alpha pathway. Redox Biol.
2016;10:179–190.
[8] Zhu Z, Li X, Chen W, et al. Prenatal stress causes
gender-dependent neuronal loss and oxidative stress
in rat hippocampus. J Neurosci Res. 2004;78:837–844.
[9] Jia N, Li Q, Sun H, et al. Alterations of Group I mGluRs
and BDNF associated with behavioral abnormity in
prenatally stressed offspring rats. Neurochem Res.
2015;40:1074–1082.
[10] Hiles SA, Baker AL, de Malmanche T, et al. A
meta-analysis of differences in IL-6 and IL-10 between
people with and without depression: exploring the
causes of heterogeneity. Brain Behav Immun. 2012;26:
1180–1188.
[11] Kohler CA, Freitas TH, Maes M, et al. Peripheral cyto­
kine and chemokine alterations in depression: a
meta-analysis of 82 studies. Acta Psychiatr Scand.
2017;135:373–387.
[12] Ma L, Demin KA, Kolesnikova TO, et al. Animal
inflammation-based models of depression and their
application to drug discovery. Expert Opin Drug
Discov. 2017;12:995–1009.
[13] Kohler-Forsberg O, Lydholm CN, Hjorthøj C, et al.
Efficacy of anti-inflammatory treatment on major depres­
sive disorder or depressive symptoms: meta-analysis of
clinical trials. Acta Psychiatr Scand. 2019;139:404–419.
[14] Enayati M, Mosaferi B, Homberg JR, et al. Prenatal
maternal stress alters depression-related symptoms in
a strain - and sex-dependent manner in rodent
offspring. Life Sci. 2020;251:117597.
[15] Pan Y, Chen XY, Zhang QY, et al. Microglial NLRP3
inflammasome activation mediates IL-1beta-related
inflammation in prefrontal cortex of depressive rats.
Brain Behav Immun. 2014;41:90–100.
[16] Wang M, Yan S, Zhou Y, et al. Trans-cinnamaldehyde
reverses depressive-like behaviors in chronic unpredict­
able Mild stress rats by inhibiting NF-kappaB/NLRP3
inflammasome pathway. Evid Based Complement
Alternat Med. 2020;(2020):4572185.
[17] Abais JM, Xia M, Zhang Y, et al. Redox regulation of
NLRP3 inflammasomes: ROS as trigger or effector?
Antioxid Redox Signal. 2015;22:1111–1129.
[18] Du RH, Wu FF, Lu M, et al. Uncoupling protein 2 mod­
ulation of the NLRP3 inflammasome in astrocytes and its
implications in depression. Redox Biol. 2016;9:178–187.
[19] Moore A, Beidler J, Hong MY. Resveratrol and depres­
sion in animal models: a systematic review of the bio­
logical mechanisms. Molecules. 2018;23:2197.
[20] Chakraborty S, Tripathi SJ, Srikumar BN, et al. N-acetyl
cysteine ameliorates depression-induced cognitive def­
icits by restoring the volumes of hippocampal subfields
and associated neurochemical changes. Neurochem Int.
2020;132:104605.
[21] Abuelezz SA, Hendawy N, Magdy Y. Targeting oxida­
tive stress, cytokines and serotonin interactions via
indoleamine 2, 3 dioxygenase by coenzyme Q10: role
in suppressing depressive like behavior in rats.
J Neuroimmune Pharmacol. 2017;12(2):277–291.
[22] Chen Y, Liang Z, Blanchard J, et al. A non-transgenic
mouse model (icv-STZ mouse) of Alzheimer’s disease:
similarities to and differences from the transgenic
model (3xTg-AD mouse). Mol Neurobiol. 2013;47
(2):711–725. .
[23] Yang X, Liu T, Chen B, et al. Grape seed proanthocya­
nidins prevent irradiation-induced differentiation of
human lung fibroblasts by ameliorating mitochondrial
dysfunction. Sci Rep. 2017;7(1):62. .
[24] Sun Q, Jia N, Li X, et al. Grape seed proanthocyanidins
ameliorate neuronal oxidative damage by inhibiting
GSK-3beta-dependent mitochondrial permeability tran­
sition pore opening in an experimental model of spora­
dic Alzheimer’s disease. Aging (Albany NY). 2019;11:
4107–4124.
[25] Jiang X, Liu J, Lin Q, et al. Proanthocyanidin prevents
lipopolysaccharide-induced depressive-like behavior in
mice via neuroinflammatory pathway. Brain Res Bull.
2017;135:40–46.
[26] National Research Council, editor. Guide for the care
and use of laboratory animals. 8th ed. Washington
(DC): National Academies Press, 2011.
[27] Lu Y, Jiang J, Si J, et al. PDLIM5 improves depression-like
behavior of prenatal stress offspring rats via methylation
in male, but not female. Psychoneuroendocrinology.
2020;115:104629.
[28] Wang R, Ma WG, Gao GD, et al. Fluoro
jade-C staining in the assessment of brain injury after
deep hypothermia circulatory arrest. Brain Res. 2011;
1372:127–132.
[29] Wang YS, Shen CY, Jiang JG. Antidepressant active
ingredients from herbs and nutraceuticals used in
TCM: pharmacological mechanisms and prospects for
drug discovery. Pharmacol Res. 2019;150:104520.
[30] Li WG, Zhang XY, Wu YJ, et al. Anti-inflammatory
effect and mechanism of proanthocyanidins from grape
seeds. Acta Pharmacol Sin. 2001;22:1117–1120.

[31] Chang SC, Cassidy A, Willett WC, et al. Dietary flavo­
noid intake and risk of incident depression in midlife
and older women. Am J Clin Nutr. 2016;104:704–714.
[32] Smith JW, Seckl JR, Evans AT, et al. Gestational stress
induces post-partum depression-like behaviour and
alters maternal care in rats. Psychoneuroendocrinology.
2004;29:227–244.
[33] Castelli V, Lavanco G, Brancato A, et al. Targeting the
stress system during gestation: is early handling
a protective strategy for the offspring? Front Behav
Neurosci. 2020;14:9.
JOURNAL OF HISTOTECHNOLOGY 9
[34] Abhijit S, Tripathi SJ, Bhagya V, et al. Antioxidant
action of grape seed polyphenols and aerobic exercise
in improving neuronal number in the hippocampus is
associated with decrease in lipid peroxidation and
hydrogen peroxide in adult and middle-aged rats. Exp
Gerontol. 2018;101:101–112.
[35] Fu K, Chen L, Miao L, et al. Grape seed proanthocyani­
dins protect N2a cells against ischemic injury via endo­
plasmic reticulum stress and mitochondrial-associated
pathways. CNS Neurol Disord Drug Targets.
2019;18:334–341.