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antioxidants
Article
Standardized Bacopa monnieri Extract Ameliorates
Learning and Memory Impairments through
Synaptic Protein, Neurogranin, Pro-and Mature
BDNF Signaling, and HPA Axis in Prenatally
Stressed Rat Offspring
Karunanithi Sivasangari and Koilmani Emmanuvel Rajan *
Department of Animal Science, Behavioural Neuroscience Laboratory, Bharathidasan University,
Tiruchirappalli 620024, India; sangarik@bdu.ac.in
*Correspondence: emmanuvel1972@yahoo.com; Tel.: +91-9791-736-009
Received: 4 November 2020; Accepted: 24 November 2020; Published: 4 December 2020
Abstract:
Prenatal stress (PNS) influences offspring neurodevelopment, inducing anxiety-like behavior
and memory deficits. We investigated whether pretreatment of Bacopa monnieri extract (CDRI-08/BME)
ameliorates PNS-induced changes in signaling molecules, and changes in the behavior of Wistar rat
offspring. Pregnant rats were randomly assigned into control (CON)/prenatal stress (PNS)/PNS and
exposed to BME treatment (PNS +BME). Dams were exposed to stress by placing them in a social
defeat cage, where they observed social defeat from gestational day (GD)-16–18. Pregnant rats in the
PNS +BME group were given BME treatment from GD-10 to their offspring’s postnatal day (PND)-23,
and to their offspring from PND-15 to -30. PNS led to anxiety-like behavior; impaired memory;
increased the level of corticosterone (CORT), adrenocorticotropic hormone, glucocorticoid receptor,
pro-apoptotic Casepase-3, and 5-HT
2C
receptor; decreased anti-apoptotic Bcl-2, synaptic proteins
(synaptophysin, synaptotagmin-1), 5-HT
1A,
receptor, phosphorylation of calmodulin-dependent
protein kinase II/neurogranin, N-methyl-D-aspartate receptors (2A,2B), postsynaptic density protein 95;
and conversion of pro and mature brain derived neurotropic factor in their offspring. The antioxidant
property of BME possibly inhibiting the PNS-induced changes in observed molecules, anxiety-like
behavior, and memory deficits. The observed results suggest that pretreatment of BME could be an
effective coping strategy to prevent PNS-induced behavioral impairments in their offspring.
Keywords:
prenatal stress; Bacopa monnieri; caspase-3; synaptophysin; N-methyl-D-aspartate
receptor (NMDAR); brain-derived neurotropic factor (BDNF)
1. Introduction
Animal model and clinical studies have demonstrated the effect of prenatal stress (PNS) and
exposure to antidepressants from the perspective of the offspring [
1
–
4
]. Parallel studies have
documented that PNS-induced behavioral changes were associated with changes in adrenocorticotropic
hormone (ACTH), corticosterone (CORT) and its receptor (glucocorticoid receptor; GR), and the
monoaminergic system [
5
–
7
]. The hyperactivity of the hypothalamic–pituitary–adrenal (HPA) axis
led to the accumulation of corticosterone, which triggered the release of excitatory neurotransmitter
(glutamate, Glu) [
8
]. The level of Glu increased in the synaptic cleft by stress, which caused learning
and memory impairment through neuronal toxicity and degeneration [
9
] and could be linked with the
regulation of pro-apoptotic caspase-3 and anti-apoptotic Bcl-2 [
10
,
11
]. Interestingly, earlier studies have
reported that the interaction of corticotrophin-releasing hormone (CRH) with serotonergic neurons
Antioxidants 2020,9, 1229; doi:10.3390/antiox9121229 www.mdpi.com/journal/antioxidants
Antioxidants 2020,9, 1229 2 of 24
possibly interacted and regulated the release of serotonin (5-hydroxytryptamine, 5-HT) and in turn,
5-HT signals regulated the HPA axis function by regulating the level of CRH [12,13].
Dysregulation of the HPA axis has been known to alter the serotonergic system, thus, synaptic proteins
(synaptophysin (SYP), synaptotagmin-1 (SYT-1)) and 5-HT receptors were also altered [
14
,
15
]. Furthermore,
the synaptic transmission, finely tuned byinteraction between the presynaptic and postsynaptic proteins and
among the presynaptic proteins [
16
], regulated behavior [
17
]. In addition, the 5-HT
1A
/5-HT
2C
receptor has
been known to criticallyregulate anxiety/anxiolytic behavior [
18
–
20
]. Activation/inhibition of 5-HT receptor
critically regulate the signaling pathway mediated by phosphorylation of calmodulin-dependent
protein-kinase II (CaMKII) and N-methyl-D-asparate (NMDA) receptors [
21
,
22
]. Earlier studies have
shown that activation of CaMKII led to formation of a complex with NMDA receptors (NR2A, NR2B),
which was critical for stable long-term potentiation (LTP) [
23
,
24
]. Furthermore, this complex regulates
the localization of postsynaptic density protein 95 (PSD-95) in the spine, and its interactions with
brain-derived neurotrophic factor (BDNF) [
24
,
25
]. Precursor and mature BDNF, and its downstream
signaling molecules are known to influence synaptic plasticity and have been linked with behavioral
disorders [26,27].
In addition, in the Indian traditional system of Ayurvedic medicine, Bacopa monnieri (Linn.)
has been used as a nootropic agent in Ayurvedic herbal formulations and has been classified as
a medhyarasayana, a drug used to cure mental disorders. The characterization and structural
elucidation of B. monnieri extract reveals the presence of bacosides [bacoside A (bacogenins A1, A2, A3,
and A4), bacoside B, bacopacoside II, bacopasaponin X, and bacopasaponin C], which possibly hold the
property to improve cognitive function [
28
–
30
]. Studies in animal models of depression and anxiety
have shown that B. monnieri extract treatment reduced anxiety-like behaviors [
31
,
32
]. In addition,
B. monnieri extract (CDRI-08) supplementation reduced anxiety and depression in elderly people [
33
,
34
].
Homeostatic adaptation of hormones and neurotransmitters mediate stress adaptation, in which a
balancing act of oxidants and antioxidants can play a crucial role [
35
]. Dietary supplement with
antioxidants has been shown to be as effective for protecting against stress-induced abnormality [36].
In this study, we hypothesized that, in our PNS model, exposure to Bacopa monnieri extract
(CDRI-08) may have a resilience effect on PNS outcome in offspring. To test this, we examined
behaviors in an experimental group of adolescent rats and further estimated the level of CORT,
ACTH, expression of GR, caspase-3, synaptic proteins (SYP, SYT-1), 5-HT receptors (5-HT
1A/2C
),
CaMKII/p-CaMKII, NR2A/B receptors, PSD-95, as well as pro-and mature BDNF, in their offspring.
2. Materials and Methods
2.1. Bacopa monnieri Extract
The extract was prepared from the whole plant of Bacopa monnieri (CDRI-08, referred to as BME in
this articles) using ethanol (70%), and then the solvent was removed. Then, the soluble was concentrated
with 95% ethanol, dried in vacuo, and macerated with acetone, obtaining the free flowing powder.
The bacoside (bacopaside A3, bacopacoside II, bacopasaponin X, and bacopasaponin C) enriched
extract was obtained from Dr. Hemant Singh as a generous gift, Lumen marketing company, Chennai,
India (batch# C15030294). The high performance liquid chromatography (HPLC) chromatogram of the
extract is provided as Supplementary Data-1 [37].
2.2. Animal Design
The experimental timeline is depicted in Supplementary Figure S1 in Supplementary Materials.
Female Wistar rats (Rattus norvegicus, 180–250 g) were selected by continuously monitored their
estrous cycle and the distribution of different stages during the estrous cycle [
38
]. Selected Dams
were individually housed with one sexually experienced male for mating. Gestational day (GD)-0
was designated by the presence of sperms in vaginal lavage sample and pregnant rats were housed
individually in standard laboratory cage under a controlled environment (24
±
2
◦
C, 12 h light-dark
Antioxidants 2020,9, 1229 3 of 24
cycle) with ad libitum water and food. Pregnant rats were randomly assigned into the following three
groups: (i) control (CON); (ii) prenatal stress (PNS, received 0.5% gum acacia treatment (per-orally,
p.o.)); and (iii) prenatal stress +exposed to BME (PNS +BME 80 mg/kg +0.5% gum acacia (p.o.))
treatment. In order to select the optimum dose of BME, different concentrations (40, 60, 80, 100 mg/kg)
of BME were provided to the individual dams, and then they were tested using a behavioral task [
36
].
Freshly prepared BME aqueous suspension (BME +gum acacia in double distilled water) or gum
acacia aqueous suspension (gum acacia in double distilled water) were treated orally to the dams
each day (10.00 to 11.00 h) from GD-10 to their pup’s postnatal day (PND)-23 except on the day of
parturition (
≈
8 h after parturition), and to the pups from PND-15 to -30. The day of parturition was
noted as PND-0 and the male pups were separated on PND-24 from their dam and housed (2–3/cage)
in different cages [
39
]. Two or three male pups were randomly selected from each dam (three dam for
each group) for different experimental groups (i.e., control (n=7), PNS (n=6), and PNS +BME (n=6)),
and selected pups were subjected to the behavioral test on PND-31/-32. Throughout the study, care was
taken to minimize the handling stress by partially replacing the bedding material periodically to ensure
the home cage odor, and minimally disrupt the nests. All the experimental group rats were handled
minimally to avoid handling effects. The project was recommended by the Institutional Animal Ethics
Committees of Bharathidasan University, Tiruchirappalli, India (approval no. BDU/IAEC/P22/2018
dated 7 August 2018) and approved by the Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA) (reg. no. 418/GO/Re/S/01/CPCSEA dated 27 April 2018). All the
experiments were conducted in compliance with the guidelines laid down by CPCSEA, Government
of India, India.
2.3. Chemicals
All chemicals used in this study were of analytical grade.
2.4. Prenatal Stress
To induce prenatal stress (PNS), the pregnant rats were allowed to observe social defeat (SDO) from
GD-16 to GD-18, in a specially designed social defeat cage (Supplementary Figure S2 in Supplementary
Materials) for 10 min/day, following the procedure modified from Lee et al. [
40
]. The social defeat cage
consisted of three chambers (observer chamber (OC), intruder chamber (IC), resident chamber (RC))
with equal size (30
×
30
×
30 cm). The RC was connected with a standard laboratory cage through a
transparent plastic pipe (60 cm in length and 10.5 cm in diameter), the OC was permanently partitioned
with wire mesh, and the RC and IC were partitioned with sliding wire mesh doors, which facilitated
the aggressive interaction of the resident male and intruder male. Ten days before the experiment,
the aggressor (senescence male rat) was housed in the standard laboratory cage which was connected
to the RC and allowed the aggressor free access to the RC. First, the intruder (six-month-old male
rat) was introduced into the IC, and after five minutes, the sliding wire mesh door was opened and
the intruder was allowed to interact (10 min/day) with the aggressor in the RC. After the aggressive
interaction the intruder was transferred to the home cage. The control group pregnant rats were
allowed to explore the OC in the absence of aggressor and intruder from GD-16 to GD-18. The PNS
and PNS +BME group were allowed to observe the social defeat (GD-16 to GD-18) from the OC.
2.5. Behavioral Test
2.5.1. Elevated Plus Maze (EPM)
Experimental group pups were subjected to the elevated plus maze (EPM) test from PND-31 to
PND-32. The EPM test was used to identify the anxiety-like behavior in rats; the two closed arm
(30
×
5
×
15 cm) and two open arms (30
×
5
×
0.25 cm) were connected to the central platform and were
50 cm above the floor level [
41
]. Three hours before the behavioral test, animals were shifted to the
behavioral testing room and kept undisturbed. Individually, test animals were placed in the central
Antioxidants 2020,9, 1229 4 of 24
square of the EPM, facing towards an open arm and they were allowed to explore for five minutes.
Individuals’ behavior was video recorded for analysis. The entire behavior test was conducted under
bright illumination (30 W). The apparatus was cleaned with 75% ethanol after every trial to remove
olfactory cues.
2.5.2. Y-Maze Test
Individual rat’s spontaneous alternation behavior was tested using a Y-maze apparatus.
The Y-shaped apparatus was constructed with three horizontal arms to connect symmetrically at
a 120
◦
angle (40
×
5
×
15 cm) [
42
]. The behavioral test was conducted in a behavioral room which was
illuminated with a 100 W bulb. On PND-30, the experimental animal was placed in the start arm end
and allowed to explore the maze for 15 min. The next day, the B arm was closed, and the reward was
placed in the C arm to ensure that the C arm was their first choice for 10 min. On PND-32, the testing
was conducted for 10 min by allowing the rat to investigate all three arms, in which the closed arm
was presented as a novel arm and the reward was placed in the C arm. An entry was calculated when
a rat placed all four paws in an arm. Pure chocolate was provided as the reward without nuts or any
other compounds. Training and testing were conducted as 2 trials/day with four-hour interval. On the
day of testing, individuals’ behavioral profiles, such as total number of arm entries and spontaneous
alternation (number of traits), were recorded and analyzed. As triplet sets (i.e., ABC/BCAo/CAB but
not ABA/BCB/CAC) the entries were designated to calculate spontaneous alternation. The alternations
(% alternations) were calculated as spontaneous alternations/(total number of arm entries −2) ×100.
2.6. Hormone Assay
To estimate the level of corticosterone (CORT) and adrenocorticotropic hormone (ACTH), blood
samples were collected from rats representing each group (control, PNS, PNS +BME, n=6) into
a tube with anticoagulant (sodium citrate, 0.25 mL of 3.8% sodium per 2.0 mL of blood). Plasma was
separated by centrifuging at 1800
×
gfor 10 min [
43
] and stored at
−
20
◦
C. The CORT and ACTH levels
in plasma were estimated by using an ELISA kit (ALPCO Diagnostics, Salem, NH, USA).
2.7. Total RNA Isolation
Animals (n=6) representing each group were sacrificed after the behavioral test. The whole brain
was dissected out and placed on an ice-cold petri dish. The amygdala tissue was carefully dissected
from the brain slice, as reported earlier [
44
]. One portion of the dissected amygdale was used to
isolate RNA and the other side for isolation of protein. Total RNA was isolated by using TRI Reagent
(cat. # FATRR 001; Favogen Biotech Corp, Pingtung, Taiwan) and stored at
−
80
◦
C. Total RNA (2
µ
g)
was used to synthesis cDNA (cat.# 170-8891; Iscript
TM
cDNA synthesis kit, Bio-Rad laboratories Inc.,
Hercules, CA, USA).
2.8. Protein Isolation
Amygdala tissue obtained from the experimental groups were homogenized in ice-cold lysis buffer
(150 mM NaCl, 50 mM Tris-Hcl pH 7.5, 5 mM EDTA, 0.1% v/vNP-40, 1.0 mM DTT, 0.2 mM sodium
orthovanadate, 0.23 mM PMSF) containing protease inhibitor (4
µ
L/mL) (Sigma-Aldrich, Saint Louis,
MO, USA). The homogenate was incubated in ice for 30 min, followed by centrifuged at 4
◦
C (10,000
×
g)
for 30 min. The supernatants were collected in anew tube and again centrifuged at 4
◦
C (12,000
×
g)
for 15 min, after being extracted, the samples as aliquots were stored at
−
80
◦
C [
36
,
45
–
47
]. Protein
was quantified at 595 nm by Bradford method [
48
] (cat. #5000006; Bio-Rad Protein Assay kit, Bio-Rad
laboratories Inc., Hercules, CA, USA) using a Biophotometer Plus (Eppendorf Inc., Hamburg, Germany).
Antioxidants 2020,9, 1229 5 of 24
2.9. Quantitative Real-Time PCR (qRT-PCR)
Total reaction volume (10
µ
L) contains an aliquot of real-time mixture (SYBR green super
mix, Bio-Rad laboratories Inc.) with cDNA (0.2
µ
g) and specific primers (100 pmoles) [
36
].
Specific primers were used to estimate the expression of 5-HT
1A
(accession number NM_012585.1;
forward 5
0
-GACTACGTGAACAAGAGGAC-3
0
and reverse 5
0
-TATAGAAAGCGCCGAAAGTG-3
0
),
5-HT
2C
and (accession number NM_012765.3; forward 5
0
-AAACTGCACAATGCTACCAA-3
0
and
reverse 5
0
-TGATGGACGCAGTTGAAAAT-3
0
) normalized with internal control and glyceroldehydes
-3-phosphate dehydragenase (GAPDH) (accession number NM_017008.4; forward 5
0
-AACATCAT
CCCTGCATCCAC-3
0
and reverse 5
0
-AGGAACACGGAAGGCCATGC-3
0
). The reaction starts with
92
◦
C initial denaturation (3 min), and then 40 cycles of denaturation at 92
◦
C (5 s), annealing (5 s)
(5-HT
1A
(52.4
◦
C), 5-HT
2C
(59.7
◦
C), GAPDH (56
◦
C)), extension at 72
◦
C (5 s), and final extension at 72
◦
C
(10 min). Single specific PCR product amplification was confirmed by observing the dissociation curve
followed by melting curve analysis (CFX-96 Touch Real-Time PCR detection system; CFX manager
version 2 software; Bio-Rad laboratories Inc., Hercules, CA, USA) [
49
,
50
]. The data obtained from
triplicate were normalized with internal control and presented as the mean fold change relative to the
control group.
2.10. Western Blot Analysis
An equal concentration of total protein (80
µ
g) separated on polyacrylamide gel (PAGE 10%) was
mixed with a buffer (2% mercaptoethanol, 100% glycerol, 4% SDS, 125 mM Tris-HCL pH 6.8, 0.006%
bromophenol blue) and boiled (2 min). Semi-dry Western blot apparatus (SD-20; Cleaver Scientific Ltd.,
Rugby, UK) was used to transfer the separated protein to the polyvinylidine difluride membrane
(PVDF) (cat. #IPVH00010; Millipore, Burlington, MA, USA). The membranes were preblocked in
Tris-buffered saline (TBS, 10 mM Tris-base pH 7.5 and 150 mM NaCl) containing Tween-20 (0.1%)
and non-fat milk (5% for 2 h) at room temperature. Membranes were incubated with any one of the
following primary antibodies for 12–15 h at 4
◦
C: rabbit-anti-GR (SC-1004, 1:1000); rabbit-anti-caspase-3
(SC-7148, 1:1000); rabbit-anti-Bcl2 (ab59348, 1:1000); rabbit-anti-SYP (M-04-1019, 1:10,000) (Millipore,
Burlington, MA, USA); mouse anti-SYT1 (BD-610433, 1:4000)(BD Biosciences, San Jose, CA, USA); mouse
anti-total-
α
CaMKII a (t-
α
CaMKII, SC-32288, 1:200); rabbit anti-phosphorylated
α
CaMKII (p-
α
CaMKII,
Thr
286
SC-12886-R, 1:200); rabbit anti-Ng (ab23570, 1:1000); rabbit anti-pNgantibody (ABN 426, 1:1000);
rabbit polyclonal NR2A antibody (BT-AP02388, 1:2000); rabbit NR2B (BT-AP02389, 1:2000); rabbit
anti-PSD-95 (SC-28941, 1:200); rabbit anti-pro-BDNF (SC-20981 (H-117), 1:500); rabbit anti-BDNF
(SC-546 (N-20), 1:500).For the internal control rabbit polyclonal anti-rabbit-
β
-actin (SC-130656) was
used. The membrane was washed with TBS-T (TBS containing 1% Tween-20), and then incubated
(for 4 h) in either alkaline phosphatase (ALP) conjugated goat anti-rabbit (MERK, cat.# 62110080011730,
1:2000) or anti-mouse (MERK cat.# 621100480011730, 1:2000) antibody to detect the membrane bounded
antibodies. Subsequently, ALP activity was detected with the substrates 5 bromo-4-chloro-3-indolyl
phosphate di-sodium salt (BCIP) and nitro blue tetrazolium chloride (NBT) (cat.# S 3771; Promega
Biotech Ltd., Madison, WI, USA), following the manufacturer’s instruction. Individuals’ blot images
were obtained using Molecular Imager and each band trace quantity was measured (Chemi Doc XRS
system, Image Lab 2 software (2.0) Bio-Rad laboratories Inc., Hercules, CA, USA). Differences from
the control group were presented in relative fold [
36
,
45
–
47
]. All uncropped Western blot images are
provided in Supplementary Materials.
2.11. Statistical Analysis
KyPlot (ver 1.0) was used to plot the values (mean
±
standard error of the mean (SEM)) as a graphical
representation. The significant difference among the experimental groups (CON, PNS, PNS +BME)
were tested with one-way analysis of variance (ANOVA) followed by post hoc (Bonferroni test) analysis
Antioxidants 2020,9, 1229 6 of 24
(Sigma Stat version 3.1). Significant difference among groups (* p<0.05, ** p<0.01, *** p<0.001)
and NS, not significantly different were indicated wherever required.
3. Results
3.1. Behavioral Results
3.1.1. Elevated Plus Maze
Behavioral profile in EPM showed that exposure to the BME treatment reduced the PNS-induced
anxiety-like behavior in their offspring. The observed behavioral data showed that there was a
significant difference in the time spent in the open arm across the experimental groups (F
(2,18)
=6.79,
p<0.01). Bonferroni post hoc comparative analysis suggests that, in the open arm, the PNS group
spent less time than the control (p<0.05) and the PNS +BME group (p<0.05), but there was no
significant difference between the control and the PNS +BME group (p=1.00) (Figure 1a). In addition,
for individual time spent in the closed arm, there was a significant difference across the study groups
(F
(2,18)
=7.300, p<0.01). The post hoc analysis demonstrates that the PNS group spent more time
than the control group (p<0.05) and the PNS +BME group (p<0.05). Whereas no difference was
observed between the control group and the PNS +BME group (p=0.984) (Figure 1b). Similarly,
the PNS group resulted in a significant difference in total number of entries (F
(2,37)
=3.76, p<0.05).
The post hoc analysis demonstrated that, unlike the control animals, the PNS group (p<0.05) exhibited
fewer entries and there was no significant difference between the PNS group and the PNS +BME
group (p=0.212) and the control group and the PNS +BME group (p=1.00) (Figure 2). When the
percentage of open entries was calculated, a significant difference was noted across the experimental
group (F
(2,18)
=9.53, p<0.01). The post hoc analysis revealed that the PNS group was significantly
different from the control group (p<0.05) and the PNS +BME group (p<0.05), but the control group
was not significantly different from the PNS +BME group (p=0.759). Similarly, a significant difference
was observed across the groups regarding closed arm entries (F
(2,18)
=9.22; p<0.01). Subsequently,
the post hoc comparison showed a significant difference between the control group and the PNS group
(p<0.01) and the PNS group and the PNS+BME group (p<0.05), where a significant difference was
not observed between the control group and the PNS +BME group (p=1.00).
3.1.2. Spatial Memory
In addition, the Y-maze analysis showed a significant effect of the variable group (F
(2,37)
=14.28,
p<0.001), and the post hoc comparison analysis indicated that, with reference to the control group,
the PNS group spent significantly less arm entries (p<0.001), as well as the PNS +BME group
(p<0.01). However, the PNS group made significantly less entries than the PNS +BME group (p<0.05)
(Figure 3a). When we estimated the percentage of alternation during training, we found a significant
difference across the groups (F
(2,37)
=17.42, p<0.001). The post hoc analysis revealed that the PNS
group exhibited a lower percentage of alternation than the control group (p<0.001) and the PNS +BME
group (p<0.001), but a significant difference was not observed between the control group and the
PNS +BME group (p=0.421). Similarly, during testing, the recorded alternation between groups
was significantly different (F
(2,37)
=21.468, p<0.001). The post hoc analysis showed that the PNS
individuals displayed a lower percentage of alternation than the control group (p<0.001) and the
PNS +BME group (p<0.001). In fact, we did not find a significant difference between the control and
the PNS +BME groups (p =0.11) (Figure 3b). The observed behavioral data in the EPM and the Y-maze
tests demonstrated that the exposure to the BME treatment protected against prenatal stress-induced
anxiety-like behavior and memory impairment in their offspring.
Antioxidants 2020,9, 1229 7 of 24
1
Figure 1.
Exposure to BME treatment and resilience effect on the prenatal stress (PNS)-induced
anxiety-like behaviors in the offspring. Behavioral profile in the elevated plus maze (EPM). (
a
) Total
time spent in the closed arm; (
b
) Total time spent in the open arm. The anxiety induced by the
gestational stress was significantly reduced by the maternal exposure to the BME treatment. Data are
expressed as mean
±
SEM (control n=7, PNS n=6, PNS +BME n=6). One-way ANOVA followed by
Bonferroni post hoc test (for all pairwise multiple comparisons, statistical significance is indicated by
*p<0.05; NS—Not significant).
Antioxidants 2020, 9, x FOR PEER REVIEW 7 of 26
Figure 1. Exposure to BME treatment and resilience effect on the prenatal stress (PNS)-induced
anxiety-like behaviors in the offspring. Behavioral profile in the elevated plus maze (EPM). (a) Total
time spent in the closed arm; (b) Total time spent in the open arm. The anxiety induced by the
gestational stress was significantly reduced by the maternal exposure to the BME treatment. Data are
expressed as mean ± SEM (control n = 7, PNS n = 6, PNS + BME n = 6). One-way ANOVA followed by
Bonferroni post hoc test (for all pairwise multiple comparisons, statistical significance is indicated by
* p < 0.05; NS—Not significant).
Figure 2.
Effect of exposure to BME treatment on PNS-induced changes in offspring. Behavioral profile
of EPM showed as the percentage of entries in the open arm and closed arm, maternal exposure to
BME treatment and resilience effect on the anxiety-like behavior in the offspring. Data are expressed
in percentage (mean
±
SEM) calculated against the total number of entries (control n=7, PNS n=6,
PNS +BME n=6). One-way ANOVA followed by Bonferroni post hoc test (for all pairwise multiple
comparisons, statistical significance is indicated by * p<0.05 and ** p<0.01; NS—Not significant).
Antioxidants 2020,9, 1229 8 of 24
Antioxidants 2020, 9, x FOR PEER REVIEW 9 of 26
Figure 3. Exposure to BME treatment protects against PNS-induced memory impairment in offspring.
The behavioral profile in the Y-maze. (a) The BME exposed group (a) made more arm entries than the
PNS group (b) showed a higher percentage of alternations. Data are expressed as mean ± SEM (control
n = 7, PNS n = 6, PNS + BME n = 6). One-way ANOVA followed by Bonferroni post hoc test (for all
pairwise multiple comparisons, statistical significance is indicated by * p < 0.05, ** p < 0.01 and *** p <
0.001; NS—Not significant)
3.2. Hormonal Results
3.2.1. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes in Corticosterone
and Adrenocorticotropic Hormone
Subsequently, we estimated the level of corticosterone and adrenocorticotropic hormone in the
plasma of the experimental groups. As shown in Figure 4a, a significant effect was demonstrated
across the groups (F(2,17) = 104.78; p < 0.001).The post hoc test revealed significant changes in the level
of corticosterone of the PNS group as compared with the control (p < 0.01) and PNS+ BME groups (p
< 0.01). However, exposure to BME treatment significantly reduced the PNS-induced elevation in
corticosterone of the PNS group, and therefore no significant difference between the control and PNS
+ BME groups (p = 0.078). Similarly, we found a significant increase in the level of adrenocorticotropic
hormone of the PNS group as compared with the level of the control group (F(2,37) = 64.16; p < 0.001).
Figure 3.
Exposure to BME treatment protects against PNS-induced memory impairment in offspring.
The behavioral profile in the Y-maze. (
a
) The BME exposed group (
a
) made more arm entries than
the PNS group (
b
) showed a higher percentage of alternations. Data are expressed as mean
±
SEM
(control n=7, PNS n=6, PNS +BME n=6). One-way ANOVA followed by Bonferroni post hoc test
(for all pairwise multiple comparisons, statistical significance is indicated by * p<0.05, ** p<0.01 and
*** p<0.001; NS—Not significant).
3.2. Hormonal Results
3.2.1. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes in Corticosterone and
Adrenocorticotropic Hormone
Subsequently, we estimated the level of corticosterone and adrenocorticotropic hormone in the
plasma of the experimental groups. As shown in Figure 4a, a significant effect was demonstrated across
the groups (F
(2,17)
=104.78; p<0.001).The post hoc test revealed significant changes in the level of
corticosterone of the PNS group as compared with the control (p<0.01) and PNS +BME groups (p<0.01).
However, exposure to BME treatment significantly reduced the PNS-induced elevation in corticosterone
of the PNS group, and therefore no significant difference between the control and PNS +BME groups
(p=0.078). Similarly, we found a significant increase in the level of adrenocorticotropic hormone of
the PNS group as compared with the level of the control group (F
(2,37)
=64.16; p<0.001). The post
hoc analysis indicated a significant increase in the adrenocorticotropic hormone level of the PNS
group as compared with the control group (p<0.01) and the PNS +BME group (p<0.01). However,
there was no significant difference between the PNS +BME and control groups (p=0.844) (Figure 4b).
Antioxidants 2020,9, 1229 9 of 24
The above results indicate that PNS increases the level of corticosterone and adrenocorticotropic
hormone but exposure to BME treatment had resilience effect on the PNS-induced CORT and ACTH
level, which could correlate with the observed anxiolytic behavior in the BME supplemented group.
Antioxidants 2020, 9, x FOR PEER REVIEW 10 of 26
The post hoc analysis indicated a significant increase in the adrenocorticotropic hormone level of the
PNS group as compared with the control group (p < 0.01) and the PNS + BME group (p < 0.01).
However, there was no significant difference between the PNS + BME and control groups (p = 0.844)
(Figure 4b). The above results indicate that PNS increases the level of corticosterone and
adrenocorticotropic hormone but exposure to BME treatment had resilience effect on the PNS-
induced CORT and ACTH level, which could correlate with the observed anxiolytic behavior in the
BME supplemented group.
Figure 4. Maternal exposure to BME treatment and the resilience effect on the PNS-induced changes
in offspring plasma. (a) Corticosterone (CORT); (b) Adrenocorticotropic hormone (ACTH) level.
Exposure to BME treatment significantly reduced the PNS-induced CORT and ACTH level. Data are
expressed as mean ± SEM (n = 6 for each group). One-way ANOVA followed by Bonferroni post hoc
test (for all pairwise multiple comparisons, statistical significance is indicated by ** p < 0.01; NS—Not
significant).
3.2.2. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes on Glucocorticoid
Receptor
As shown in Figure 5 (Supplementary Figure S3), the level of glucocorticoid receptor expression
was significantly different across the groups (F
(2,17)
= 278.96, p < 0.001). The post hoc comparison
suggested that level of glucocorticoid receptor was significantly higher in the PNS group as compared
with the control group (p < 0.001) and the PNS + BME group (p < 0.001). However, the level of
glucocorticoid receptor was significantly lower in the PNS group exposed to BME treatment as
compared with the control group (p < 0.001). The observed results suggest that PNS alters
glucocorticoid receptor expression but exposure to BME treatment has a possible resilience effect on
the PNS-induced changes in glucocorticoid receptor.
Figure 4.
Maternal exposure to BME treatment and the resilience effect on the PNS-induced changes in
offspring plasma. (
a
) Corticosterone (CORT); (
b
) Adrenocorticotropic hormone (ACTH) level. Exposure
to BME treatment significantly reduced the PNS-induced CORT and ACTH level. Data are expressed
as mean
±
SEM (n=6 for each group). One-way ANOVA followed by Bonferroni post hoc test (for all
pairwise multiple comparisons, statistical significance is indicated by ** p<0.01; NS—Not significant).
3.2.2. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes on
Glucocorticoid Receptor
As shown in Figure 5(Supplementary Figure S3), the level of glucocorticoid receptor expression
was significantly different across the groups (F
(2,17)
=278.96, p<0.001). The post hoc comparison
suggested that level of glucocorticoid receptor was significantly higher in the PNS group as compared
with the control group (p<0.001) and the PNS +BME group (p<0.001). However, the level
of glucocorticoid receptor was significantly lower in the PNS group exposed to BME treatment as
compared with the control group (p<0.001). The observed results suggest that PNS alters glucocorticoid
receptor expression but exposure to BME treatment has a possible resilience effect on the PNS-induced
changes in glucocorticoid receptor.
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Figure 5. Effect of exposure to BME treatment on PNS-induced changes in offspring’s glucocorticoid
receptor (GR) level in amygdale. (a) Representative Western blot showing that the level of GR varied
across the experimental group; (b) Estimated level of GR suggests that exposure to BME treatment
has a resilience effect on the PNS-induced changes. Data are expressed as mean ± SEM (n = 6/each
group). One-way ANOVA followed by Bonferroni post hoc test (for all pairwise multiple
comparisons, statistical significance is indicated by *** p < 0.001).
3.3. Western Blot Results
3.3.1. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Neuronal Apoptosis
Furthermore, we examined the level of caspase-3 and Bcl-2 to measure the PNS-induced
neuronal damage and the effect of exposure to BME treatment. The Western blot analysis showed
that PNS induced the expression of caspase-3 (Figure 6a, Supplementary Figure S4). The analysis
revealed that the level of caspase-3 significantly varied across the groups (F
(2,17)
= 1714.3, p < 0.001).
The post hoc analysis demonstrated that level of caspase-3 was significantly higher in the PNS group
as compared with the control (p < 0.001) and the PNS + BME groups (p < 0.001). Exposure to the BME
treatment reduced the caspase-3 level, however, the level was significantly higher than in the control
group (p < 0.001) (Figure 6b). Furthermore, the level of Bcl-2 varied significantly across the
experimental groups (F
(2,17)
= 74.2, p < 0.001). The post hoc analysis indicated that the level of Bcl-2
was significantly lower in the PNS group than in the control (p < 0.01) and PNS + BME groups (p <
0.01). This showed that exposure to BME treatment elevated the Bcl-2 level, however, a significant
difference was not detected between the control and the PNS+ BME groups (p = 0.092) (Figure 6c).
Taken together, the observed results suggest that exposure to BME treatment protects against PNS-
induced neuronal damage by balancing the expression of caspase-3 and Bcl-2.
Figure 5.
Effect of exposure to BME treatment on PNS-induced changes in offspring’s glucocorticoid
receptor (GR) level in amygdale. (
a
) Representative Western blot showing that the level of GR varied
across the experimental group; (
b
) Estimated level of GR suggests that exposure to BME treatment has
a resilience effect on the PNS-induced changes. Data are expressed as mean
±
SEM (n=6/each group).
One-way ANOVA followed by Bonferroni post hoc test (for all pairwise multiple comparisons, statistical
significance is indicated by *** p<0.001).
3.3. Western Blot Results
3.3.1. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Neuronal Apoptosis
Furthermore, we examined the level of caspase-3 and Bcl-2 to measure the PNS-induced neuronal
damage and the effect of exposure to BME treatment. The Western blot analysis showed that PNS
induced the expression of caspase-3 (Figure 6a, Supplementary Figure S4). The analysis revealed that
the level of caspase-3 significantly varied across the groups (F
(2,17)
=1714.3, p<0.001). The post hoc
analysis demonstrated that level of caspase-3 was significantly higher in the PNS group as compared
with the control (p<0.001) and the PNS +BME groups (p<0.001). Exposure to the BME treatment
reduced the caspase-3 level, however, the level was significantly higher than in the control group
(p<0.001) (Figure 6b). Furthermore, the level of Bcl-2 varied significantly across the experimental
groups (F
(2,17)
=74.2, p<0.001). The post hoc analysis indicated that the level of Bcl-2 was significantly
lower in the PNS group than in the control (p<0.01) and PNS +BME groups (p<0.01). This showed
that exposure to BME treatment elevated the Bcl-2 level, however, a significant difference was not
detected between the control and the PNS+BME groups (p=0.092) (Figure 6c). Taken together,
the observed results suggest that exposure to BME treatment protects against PNS-induced neuronal
damage by balancing the expression of caspase-3 and Bcl-2.
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Figure 6. Effect of exposure to BME treatment on PNS-induced changes on caspase-3 and Bcl-2 level
in amygdale. (a) Representative Western blot showing that the levels of caspase-3 and Bcl-2 varied
across the experimental groups. Quantitative estimation of caspase-3 (b) and Bcl-2 (c) showing that
exposure to BME treatment induces an opposite pattern of expression. Data are expressed as mean ±
SEM (n = 6/group). One-way ANOVA followed by Bonferroni post hoc test (for all pairwise multiple
comparisons, statistical significance is indicated by ** p < 0.01 and ***p < 0.001; NS—Not significant).
3.3.2. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes in Synaptophysin
and Synaptotagmin-1
Furthermore, we found that exposure to BME treatment had a resilience effect on the PNS-
induced changes in synaptic proteins (Figure 7a, Supplementary Figure S5). In addition, the level of
synaptophysin was also altered by PNS. The observed changes in the level of synaptophysin was
significantly different across the groups (F
(2,17)
= 2097.45, p < 0.001). The post hoc comparison showed
that synaptophysin was significantly higher in the PNS group than that of the control group (p <
0.001) but significantly lower than that of the PNS + BME group (p < 0.001), whereas the level of
synaptophysin was significantly less than the control group (p < 0.001) (Figure 7b). One-way ANOVA
showed a significant difference across the experimental groups (F
(2,17)
= 528.20, p < 0.001), and the post
hoc comparison showed that the level of synaptotagmin-1 was significantly lower in the PNS group
than the control (p < 0.01) and PNS+ BME group (p < 0.001). However, the detected synaptotagmin-1
level in the control group was not significantly different from individuals exposed to BME treatment
(p = 0.08) (Figure 7c).
Figure 6.
Effect of exposure to BME treatment on PNS-induced changes on caspase-3 and Bcl-2 level in
amygdale. (
a
) Representative Western blot showing that the levels of caspase-3 and Bcl-2 varied across
the experimental groups. Quantitative estimation of caspase-3 (
b
) and Bcl-2 (
c
) showing that exposure
to BME treatment induces an opposite pattern of expression. Data are expressed as mean
±
SEM
(n=6/group). One-way ANOVA followed by Bonferroni post hoc test (for all pairwise multiple
comparisons, statistical significance is indicated by ** p<0.01 and *** p<0.001; NS—Not significant).
3.3.2. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes in Synaptophysin and
Synaptotagmin-1
Furthermore, we found that exposure to BME treatment had a resilience effect on the PNS-induced
changes in synaptic proteins (Figure 7a, Supplementary Figure S5). In addition, the level of
synaptophysin was also altered by PNS. The observed changes in the level of synaptophysin was
significantly different across the groups (F
(2,17)
=2097.45, p<0.001). The post hoc comparison showed
that synaptophysin was significantly higher in the PNS group than that of the control group (p<0.001)
but significantly lower than that of the PNS +BME group (p<0.001), whereas the level of synaptophysin
was significantly less than the control group (p<0.001) (Figure 7b). One-way ANOVA showed a
significant difference across the experimental groups (F
(2,17)
=528.20, p<0.001), and the post hoc
comparison showed that the level of synaptotagmin-1 was significantly lower in the PNS group than
the control (p<0.01) and PNS+BME group (p<0.001). However, the detected synaptotagmin-1
level in the control group was not significantly different from individuals exposed to BME treatment
(p=0.08) (Figure 7c).
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Figure 7. Exposure to BME treatment protects against PNS-induced changes in synaptic proteins. (a)
Representative Western blots showing that the level synaptophysin (SYP) and synaptotagmin-1 (SYT-
1) in the experimental groups; (b) SYP and (c) SYT-1 were significantly increased in the BME
supplemented group. Data are expressed as mean ± SEM (n = 6/each group). One-way ANOVA
followed by Bonferroni post hoc test (for all pairwise multiple comparisons, statistical significance is
indicated by ** p < 0.01 and *** p < 0.001; NS—Not significant).
3.3.3. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes on 5-HT1A and 5-
HT2C Receptors
Subsequently, we estimated the expression level of 5-HT1A and 5-HT2C receptors in the
experimental groups (Supplementary Figure S6). The one-way ANOVA analysis showed a significant
difference in the estimated level of 5-HT1A between the groups (F(2,17) = 44.94,p< 0.001). The post hoc
analysis indicated that 5-HT1A receptor expression was significantly lower in the PNS group than in
the control (p < 0.05) and PNS + BME groups (p < 0.01). In fact, the level of 5-HT1A was significantly
higher in the PNS + BME group than in the control group (p <0.01) (Figure 8a). When we examined
the 5-HT2C receptor, the level of expression between groups varied significantly (F(2,17) = 390.92,p<
0.001). The post hoc comparison revealed that the level of 5-HT2C receptor expression was elevated in
the PNS group as compared with the control (p < 0.01) and PNS + BME groups (p < 0.01). Exposure to
the BME treatment significantly reduced the PNS-induced changes in 5-HT2C receptor, and therefore
a significant difference was observed between the control and PNS + BME groups (p < 0.001) (Figure
8b). The obtained data demonstrates that exposure to BME treatment differentially regulates 5-HT1A
and 5-HT2C receptors, with a possible resilience effect on the PNS-induced impact on the serotonergic
system.
Figure 7.
Exposure to BME treatment protects against PNS-induced changes in synaptic proteins.
(
a
) Representative Western blots showing that the level synaptophysin (SYP) and synaptotagmin-1
(SYT-1) in the experimental groups; (
b
) SYP and (
c
) SYT-1 were significantly increased in the BME
supplemented group. Data are expressed as mean
±
SEM (n=6/each group). One-way ANOVA
followed by Bonferroni post hoc test (for all pairwise multiple comparisons, statistical significance is
indicated by ** p<0.01 and *** p<0.001; NS—Not significant).
3.3.3. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes on 5-HT1A and
5-HT2C Receptors
Subsequently, we estimated the expression level of 5-HT
1A
and 5-HT
2C
receptors in the
experimental groups (Supplementary Figure S6). The one-way ANOVA analysis showed a significant
difference in the estimated level of 5-HT
1A
between the groups (F
(2,17)
=44.94, p<0.001). The post hoc
analysis indicated that 5-HT
1A
receptor expression was significantly lower in the PNS group than in the
control (p<0.05) and PNS +BME groups (p<0.01). In fact, the level of 5-HT
1A
was significantly higher
in the PNS +BME group than in the control group (p<0.01) (Figure 8a). When we examined the 5-HT
2C
receptor, the level of expression between groups varied significantly (F
(2,17)
=390.92, p<0.001). The post
hoc comparison revealed that the level of 5-HT
2C
receptor expression was elevated in the PNS group as
compared with the control (p<0.01) and PNS +BME groups (p<0.01). Exposure to the BME treatment
significantly reduced the PNS-induced changes in 5-HT
2C
receptor, and therefore a significant difference
was observed between the control and PNS +BME groups (p<0.001) (Figure 8b). The obtained data
demonstrates that exposure to BME treatment differentially regulates 5-HT1A and 5-HT
2C
receptors,
with a possible resilience effect on the PNS-induced impact on the serotonergic system.
Antioxidants 2020,9, 1229 13 of 24
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Figure 8. Exposure to BME treatment protects the PNS-induced alternations in the serotonergic
system. Quantitative real-time PCR analysis showing that exposure to BME treatment significantly
ameliorates the PNS-induced changes in (a) 5-HT
1A
and (b) 5-HT
2C
receptor expression. Data are
expressed as mean ± SEM (n = 6/group). One-way ANOVA followed by Bonferroni post hoc test (for
all pairwise multiple comparisons, statistical significance is indicated by * p < 0.05 and ** p < 0.01).
3.3.4. Effect of Exposure to BME Treatment on Gestational Stress-Induced Changes on Neurogranin
(Ng) and CaMKII Signaling
Subsequently, we examined the effect of PNS on activation of CaMKII. The western blot analysis
demonstrated that the level of CaMKII was altered (Figure 9a, Supplementary Figure S7). The one-
way ANOVA reported a significant difference across the variable groups (F
(2,17)
= 132.10, p < 0.001).
The post hoc comparison analysis showed that the level of p-CaMKII/CaMKII was significantly
reduced in the PNS group (p < 0.001) as compared with the control and PNS + BME groups (p < 0.001).
Furthermore, the level of p-CaMKII/CaMKII was significantly higher in the PNS + BME group than
in the control group (p < 0.001) (Figure 9b).
Figure 8.
Exposure to BME treatment protects the PNS-induced alternations in the serotonergic system.
Quantitative real-time PCR analysis showing that exposure to BME treatment significantly ameliorates
the PNS-induced changes in (
a
) 5-HT
1A
and (
b
) 5-HT
2C
receptor expression. Data are expressed as
mean
±
SEM (n=6/group). One-way ANOVA followed by Bonferroni post hoc test (for all pairwise
multiple comparisons, statistical significance is indicated by * p<0.05 and ** p<0.01).
3.3.4. Effect of Exposure to BME Treatment on Gestational Stress-Induced Changes on Neurogranin
(Ng) and CaMKII Signaling
Subsequently, we examined the effect of PNS on activation of CaMKII. The western blot analysis
demonstrated that the level of CaMKII was altered (Figure 9a, Supplementary Figure S7). The one-way
ANOVA reported a significant difference across the variable groups (F
(2,17)
=132.10, p<0.001). The post
hoc comparison analysis showed that the level of p-CaMKII/CaMKII was significantly reduced in the
PNS group (p<0.001) as compared with the control and PNS +BME groups (p<0.001). Furthermore,
the level of p-CaMKII/CaMKII was significantly higher in the PNS +BME group than in the control
group (p<0.001) (Figure 9b).
Antioxidants 2020,9, 1229 14 of 24
1
Figure 9.
Exposure to BME treatment protects against PNS-induced changes in the activation of CaMKII.
(
a
) Representative Western blot showing variations in the level of CaMKII/p-CaMKII and Ng/p-Ng.
Estimated ratio of phosphorylation of CaMKII (
b
) and Ng/p-Ng (
c
) in experimental groups. Data are
expressed as mean
±
SEM (n=6/each group). One-way ANOVA followed by Bonferroni post hoc test
(for all pairwise multiple comparisons, statistical significance is indicated by * p<0.05, ** p<0.01 and
*** p<0.001).
Next, we examined whether the neurogranin expression was associated with the CaMKII
expression. Our analysis revealed that the level of neurogranin expression among the groups was
significantly different (F
(2,17)
=94.08, p<0.001). In addition, the post hoc analysis showed that the level
of p-Ng/Ng in the PNS group was significantly lower than in the control (p<0.01) and PNS +BME
groups (p<0.05). The comparison analysis showed that the level of p-Ng/Ng was significantly lower
in the PNS +BME group than in the control group (p<0.05) (Figure 9c). These analyses suggest that
PNS reduced the level of phosphorylation of Ng and CaMKII but the exposure to BME treatment
reduced the effect.
3.3.5. Effect of Exposure to BME Treatment on Gestational Stress-Induced Changes on
N-Methyl-D-Aspartate Receptors (2A,2B)
Exposure to BME treatment protects against the PNS-induced alteration in N-methyl-D-aspartate
receptors (Figure 10a, Supplementary Figure S8). The Western blot analysis exhibited the differential
expression pattern in the N-methyl-D-aspartate receptors among the experimental groups. One-way
ANOVA showed that the level of NR2A varied significantly across the experimental groups
(F
(2,17)
=719.59, p<0.001), and the post hoc analysis suggested that the level of NR2A was significantly
low in PNS group than in the control (p<0.001) and PNS +BME groups (p<0.001). BME treatment
protects against the PNS-induced changes in NR2A expression, however, the level of NR2A was
significantly low in the PNS +BME group (p<0.001) (Figure 10b). Similarly, the NR2B level was
Antioxidants 2020,9, 1229 15 of 24
also altered and the variations among the groups were significantly different (F
(2,17)
=72.00, p<0.01).
The post hoc analysis revealed that the NR2B level was significantly lower in the PNS group than
in the control (p<0.001) and PNS +BME groups (p<0.05). Exposure to BME treatment protected
against the PNS-induced effect, however, the NR2B levels in the control and PNS +BME groups were
significantly difference (p<0.001) (Figure 10c). The observed results suggest that exposure to BME
treatment had a resilience effect on the PNS-induced changes in N-methyl-D-aspartate receptors.
1
Figure 10.
Exposure to BME treatment protects against the PNS-induced changes in NR2A and NR2B.
(
a
) Representative Western blots showing that the level of NR2A and NR2B; (
b
) NR2A and (
c
) NR2B
levels were significantly altered by exposure to BME treatment. Data are expressed as mean
±
SEM
(n=6/each group). One-way ANOVA followed by Bonferroni post hoc test (for all pairwise multiple
comparisons, statistical significance is indicated by * p<0.05 and *** p<0.001).
3.3.6. Effect of Exposure to BME Treatment on Postnatal Stress-Induced Changes in Postsynaptic
Density Protein-95
Furthermore, our analysis showed PNS-induced changes in expression of postsynaptic density
protein-95 (Figure 11; Supplementary Figure S9), which varied significantly across the experimental
groups (F
(2,17)
=88.62, p<0.001). The post hoc analysis indicated that the level of postsynaptic density
protein-95 in the PNS group was significantly lower than in the control (p<0.01), and PNS +BME
groups (p<0.05). Furthermore, exposure to BME treatment elevated the postsynaptic density protein-95
level, which was not significantly different from the control group (p=0.14) (Figure 11a). Taken together,
these results demonstrated that exposure to BME treatment elevated the expression of postsynaptic
density protein 95.
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together, these results demonstrated that exposure to BME treatment elevated the expression of
postsynaptic density protein 95.
Figure 11. Effect of exposure to BME treatment on postsynaptic density protein 95 (PSD-95) in PNS
offspring. (a) Representative Western blot depicting the level of PSD-95 in the experimental groups;
(b) Exposure to BME treatment significantly increased the expression of PSD-95. Data are expressed
as mean ± SEM (n = 6/each group). One-way ANOVA followed by Bonferroni post hoc test (for all
pairwise multiple comparisons statistical significance is indicated by * p < 0.05 and ** p < 0.01; NS—
Not significant).
3.3.7. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes in Expression of
Pro and Mature Brain-Derived Neurotrophic Factor
The level of pro and mature brain-derived neurotrophic factor was altered by PNS (Figure 12a;
Supplementary Figure S10). One-way ANOVA found that ratio of pro and mature brain-derived
neurotrophic factor varied significantly across the experimental groups (F(2,17) = 132.10, p < 0.001). The
post hoc analysis demonstrated that the estimated ratio of pro and mature BDNF in the PNS group
was significantly lower as compared with the control (p < 0.05) and PNS + BME groups (p < 0.05).
Interestingly, a significant difference was not detected between the control and PNS + BME groups
(p = 0.495) (Figure 12b). the observed results suggest that PNS alters the conversion of mature brain-
derived neurotrophic factor but the exposure to BME treatment has a resilience effect on the PNS-
induced effect on brain-derived neurotrophic factor maturation.
Figure 11.
Effect of exposure to BME treatment on postsynaptic density protein 95 (PSD-95) in
PNS offspring. (
a
) Representative Western blot depicting the level of PSD-95 in the experimental
groups; (
b
) Exposure to BME treatment significantly increased the expression of PSD-95. Data are
expressed as mean
±
SEM (n=6/each group). One-way ANOVA followed by Bonferroni post hoc test
(for all pairwise multiple comparisons statistical significance is indicated by * p<0.05 and ** p<0.01;
NS—Not significant).
3.3.7. Effect of Exposure to BME Treatment on Prenatal Stress-Induced Changes in Expression of Pro
and Mature Brain-Derived Neurotrophic Factor
The level of pro and mature brain-derived neurotrophic factor was altered by PNS (Figure 12a;
Supplementary Figure S10). One-way ANOVA found that ratio of pro and mature brain-derived
neurotrophic factor varied significantly across the experimental groups (F
(2,17)
=132.10, p<0.001).
The post hoc analysis demonstrated that the estimated ratio of pro and mature BDNF in the PNS
group was significantly lower as compared with the control (p<0.05) and PNS +BME groups
(p<0.05). Interestingly, a significant difference was not detected between the control and PNS +BME
groups (p=0.495) (Figure 12b). the observed results suggest that PNS alters the conversion of mature
brain-derived neurotrophic factor but the exposure to BME treatment has a resilience effect on the
PNS-induced effect on brain-derived neurotrophic factor maturation.
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Figure 12. Effect of exposure to BME treatment on pro and mature brain-derived neurotrophic factor
(BDNF) in PNS offspring.(a) Representative Western blots depicting the level of pro BDNF and
mature BDNF; (b) Exposure to BME treatment silences the PNS-induced changes in the ratio of
proand mature BDNF. Data are expressed as mean ± SEM (n = 6/each group). One-way ANOVA
followed by Bonferroni post hoc test (for all pairwise multiple comparisons, statistical significance is
indicated by * p < 0.05; NS—Not significant).
4. Discussion
Prenatal stress can alter the architecture of the developing brain program [51], more specifically
the amygdala region [52] by a mother’s physiological and emotional environment [53]. The outcomes
of animal and human studies have linked maternal stress and epigenetic changes; PNS has been
known to alter the methylation pattern of GR, and thereby influence the HPA axis regulation in
offspring and induce depression and anxiety-like behavior [54]. In this study, we observed that PNS
induced anxiety-like behavior in offspring, which was similar to earlier reports in other models
[55,56] and we validated our social defeat observation model. However, the observed anxiolytic
behavior in the PNS + BME group showed that exposure to BME treatment had a resilience effect on
prenatal stress and reduced the anxiety-like behavior in their offspring, as reported in other anxiety
animal models [31,32]. Exposure to stress during gestation has been known to alter the level of stress
hormone in the dam and also in the amniotic fluid [57], which can induce alterations in the HPA axis
in offspring [58]. When the level of CORT and ACTH was estimated, we found that the level of CORT
and ACTH was elevated in individuals experiencing PNS, as reported earlier [7]. However, exposure
Figure 12.
Effect of exposure to BME treatment on pro and mature brain-derived neurotrophic factor
(BDNF) in PNS offspring. (
a
) Representative Western blots depicting the level of pro BDNF and
mature BDNF; (
b
) Exposure to BME treatment silences the PNS-induced changes in the ratio of proand
mature BDNF. Data are expressed as mean
±
SEM (n=6/each group). One-way ANOVA followed by
Bonferroni post hoc test (for all pairwise multiple comparisons, statistical significance is indicated by
*p<0.05; NS—Not significant).
4. Discussion
Prenatal stress can alter the architecture of the developing brain program [
51
], more specifically
the amygdala region [
52
] by a mother’s physiological and emotional environment [
53
]. The outcomes
of animal and human studies have linked maternal stress and epigenetic changes; PNS has been known
to alter the methylation pattern of GR, and thereby influence the HPA axis regulation in offspring
and induce depression and anxiety-like behavior [
54
]. In this study, we observed that PNS induced
anxiety-like behavior in offspring, which was similar to earlier reports in other models [
55
,
56
] and
we validated our social defeat observation model. However, the observed anxiolytic behavior in the
PNS +BME group showed that exposure to BME treatment had a resilience effect on prenatal stress and
reduced the anxiety-like behavior in their offspring, as reported in other anxiety animal models [
31
,
32
].
Exposure to stress during gestation has been known to alter the level of stress hormone in the dam and
also in the amniotic fluid [
57
], which can induce alterations in the HPA axis in offspring [
58
]. When the
level of CORT and ACTH was estimated, we found that the level of CORT and ACTH was elevated
in individuals experiencing PNS, as reported earlier [
7
]. However, exposure to BME treatment had
a resilience effect on the PNS-induced changes and the level of CORT and ACTH was close to the basal
level in their offspring. The adoptogenic properties of BME possibly have a resilience effect on the
Antioxidants 2020,9, 1229 18 of 24
PNS-induced effect on CORT and ACTH [
32
,
59
]. We observed that the level of GR was up regulated
in individuals who experienced PNS, and exposure to BME treatment resulted in down regulation
of GR, which might be due to GR-CORT interactions [
60
,
61
]. In addition, it has been documented
that stress induced an elevated level of CORT, overexpression of the GR known to activate oxidative
mediators [
62
,
63
], and then up-regulation of the potential regulatory molecule caspase-3 in respond to
the oxidative stress [
63
,
64
] and neuronal damage [
65
]. Caspase-3 is the key effector molecule in the
caspase family, which is involved in the final execution of apoptosis, and activates the apoptosis process
through the signal conduction pathway mediated by caspase-3 [
66
,
67
]. We observed that the level of
caspase-3 was up regulated by PNS. Furthermore, we noted that exposure to BME treatment lowered
the caspase-3 expression, suggesting that BME possibly reduced the oxidative stress, as reported
earlier [
32
,
68
]. Interestingly, anti-apoptotic Bcl-2 was reduced by PNS, while exposure to BME treatment
increased Bcl-2 expression, which has been known to suppress apoptosis [
69
,
70
]. Our data clearly
demonstrate that the trends of caspase-3 and Bcl-2 expression are opposite. The levels of caspase-3 and
Bcl-2 were dynamically balanced in this model. These results support the neuroprotective effects of
BME in the PNS-exposed offspring and are consistent with those of another models [71,72].
The HPA axis receives serotonergic input, and its feedback mechanism it a critical regulator
of behavior [
73
–
75
]. In line with earlier reports with other stress models [
74
,
76
,
77
], a reduced level
of SYP was observed in the individuals who experienced PNS and displayed anxiety-like behavior.
Whereas, exposure to BME treatment restored the level of SYP and behavior; the changes in level of
SYP possibly associated with modulation of the HPA axis [
75
,
77
]. Notably, the HPA axis mediated
changes in SYP have been known to drive the changes in SYT-1 [
78
,
79
]. Similarly, we found that PNS
induced reduction of SYT-1 expression, however, exposure to BME treatment restored the level of
SYT-1 and their behavior. Similar to other animal models, the observed lower level of SYT-1 was
possibly associated with an imbalance in the synaptic vesicle exocytosis, which has been implicated in
regulating synaptic transmission, synaptic strength, and plasticity [
80
,
81
]. Taken together, these results
demonstrate that expression of SYP and SYT may be associated with synaptic plasticity and decreased
long-term potentiation (LTP). Earlier studies have demonstrated that synaptic transmission could
be mediated by Ca
2+
dependent SYT-1 interaction with synaptic protein and 5-HT receptors [
82
,
83
].
In addition, activation/inhibition of 5-HT receptors regulates the CRH expression [
84
]. Stress-induced
changes in synaptic transmission possibly induce adverse effects on serotonergic systems (level of 5-HT,
5-HT receptors, and SERT), affecting fetal development [
85
,
86
]. We found that the level of 5-HT
1A
receptor expression was significantly decreased, whereas the 5-HT
2C
receptor expression was elevated.
In basolateral amygdale, the 5-HT
1A
receptor was involving in neural circuits and its activation reduced
fear-related anxiety [
87
]. PNS-induced reduction in the 5-HT
1A
receptor could reflect the reactive
anxiety-like behavior [
88
]. Exposure to BME treatment facilitates activation of the 5-HT
1A
receptor
and is possibly related to the observed anxiolytic behavior [
18
,
20
]. Earlier studies on stress model
have demonstrated that up regulation of the 5-HT
2C
receptor contributed to increased response to
anxiety [
21
]. The underlying mechanism in the differential expression of the 5-HT receptor may be
possibly associated with stress-mediated elevated level of corticosterone [
89
,
90
]. It is worthwhile
mentioning that exposure to BME treatment and the resilience effect on the PNS-induced changes in
5-HT1A and 5-HT2C receptors were similar to the other stress models [91].
The CaMKII has been known to be more sensitive to 5-HT transmission, intracellular calcium
level, and its kinase activity mediated by phosphorylation [
20
,
21
]. As reported by Fumagalli et al. [
92
],
we observed that PNS did not alter the total CaMKII, but the level of phosphorylation was decreased.
However, exposure to BME treatment elevated the phosphorylation level of CaMKII [
93
], which
subsequently activated the target genes and may be linked with the observed anxiolytic behavior.
Earlier studies have shown that at low concentrations or in the absence of Ca
2+
, Ng could bind with
calmodulin (CaM), which was linked with the activity-dependent synaptic plasticity and LTP,a substrate
for learning and memory [
94
,
95
]. In this study, we observed the level of Ng was significantly low in
the PNS group, but BME treatment protected against the effect of PNS and the individuals showed less
Antioxidants 2020,9, 1229 19 of 24
anxiety-like behavior and memory. Supporting our observation, earlier studies demonstrated that stress
reduced the level of Ng and led to a reduction in LTP and a functional deficit in spatial learning [
96
,
97
].
The observed behavioral phenotype in this study could be linked with the PNS-induced reduction in
Ng and CaMKII.
As reported earlier, PNS down regulates the NMDA receptor expression (NR2A, 2B) [
98
,
99
],
possibly through the interaction of p-CaMKII [
23
]. It should be noted that exposure to BME treatment
and the resilience effect on the PNS-induced effects on NR2A and NR2B receptor expression may be
correlated with the observed anxiolytic behavior. Subsequently, we found that the PNS mediated
effect on PSD-95 and further exposure to BME treatment increased the level of PSD-95, possibly due to
the differential effects mediated expression [
24
]. Exposure to the stress has been known to alter the
BDNF level either directly by glucocorticoid level or through the signaling molecules [
7
]. We found
that the level of proand mature BDNF was decreased in the amygdala of offspring that experienced
PNS, similar to another prenatal study report on hippocampus [
100
]. Stress reduced the proteolytic
conversion of pro-BDNF to mature BDNF; therefore, the lower level of BDNF estimated in individuals
who experienced PNS [
27
]. However, exposure to BME treatment increased the level of pro-BDNF,
as well as the mature BDNF level. The observed mechanism in the present study could be the effect of
BME treatment mediating BNDF level, as in earlier reports on other models [32,101].
5. Conclusions
In conclusion, behavioral data suggest that PNS induces anxiety-like behavior in offspring which
may be linked to an alteration in the HPA axis (CORT, ACTH, GR) and overexpression of GR, activating
oxidative stress and possibly the neuronal damage. Interestingly, exposure to BME treatment balances
caspase-3 and Bcl-2 expression to protect the neurons. In addition, the feedback mechanism of the
HPA axis and the serotonergic system alter synaptic proteins (synaptophysin, synaptotagmin-1),
5-HT receptors, phosphorylation of Ng and its interaction CaMKII, NMDA receptors (2A, 2B), PSD-95,
and conversion of pro and mature BDNF. Exposure to BME treatment protects against the PNS-induced
neuronal damage and changes in proand mature BDNF through the HPA axis and synaptic protein
mediated signaling, which may be linked with the observed anxiolytic behavior in offspring.
Supplementary Materials:
The following are available online at http://www.mdpi.com/2076-3921/9/12/1229/s1.
Supplementary Data-1: High performance liquid chromatography (HPLC) chromatogram of Bacopa monnieri
extract, Supplementary Information: Supplementary figures showing all the uncropped full Western blot images.
Supplementary Data for Western blot: Representative Western blots each band trace quantity.
Author Contributions:
K.E.R. designed the experiment; K.S. performed the experiment, K.S. and K.E.R. analyzed
the data and prepared the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding:
K.S. is a recipient of a junior research fellowship through DST-Promotion of University Research
and Scientific Excellence (PURSE). DST-FIST is supporting the Instrumentation Facility at the Department of
Animal Science.
Acknowledgments: We thank Hemant Singh for his generous gift of Bacopa monnieri extract (CDRI-08).
Conflicts of Interest: The authors declare that they have no conflict of interest.
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