Introduction
The global population is aging at an unprecedented
rate, with the number of individuals aged ≥60 years projected to
>2 billion by 2050(1). Aging is
accompanied by progressive deterioration of physiological
homeostasis and functional decline across multiple organ systems,
with the brain being particularly vulnerable. Age-related brain
dysfunction is characterized by metabolic dysregulation, chronic
neuroinflammation and impaired cellular stress-response mechanisms,
all of which contribute to neurodegenerative processes and
cognitive decline (2-4).
Among the various mechanisms implicated in aging, oxidative stress
has been widely recognized as a central pathological contributor
(5-7).
Oxidative stress occurs when the generation of reactive oxygen
species (ROS) exceeds the capacity of endogenous antioxidant
defenses, resulting in oxidative damage to DNA, proteins and lipids
(7,8). These molecular insults are associated
with age-related neurodegeneration and impairments in learning and
memory (9,10).
The D-galactose (D-Gal)-induced model is widely
employed as an accelerated aging-like paradigm to investigate the
mechanisms underlying age-associated cognitive dysfunction in
rodents. Chronic administration of D-Gal leads to excessive ROS
production, oxidative damage, mitochondrial dysfunction and
neuroinflammation, thereby recapitulating key pathological features
of natural aging (11-15).
In addition to oxidative injury, D-Gal has been shown to disrupt
cholinergic neurotransmission, thereby impairing synaptic
plasticity and cognitive performance (14,15).
Given the important role of the cholinergic system in learning and
memory and its vulnerability to oxidative stress, interventions
that attenuate oxidative damage while preserving cholinergic
function are of considerable interest in aging and
neurodegeneration research.
Perilla seed oil (PSO), commonly known as Nga-mon in
Thailand, is derived from Perilla frutescens (L.) Britt., a
plant belonging to the mint family (Lamiaceae). PSO has been
traditionally consumed throughout Asia as both a medicinal herb and
a functional food. PSO is particularly rich in α-linolenic acid
(ALA), an essential omega-3 fatty acid (16). In addition, it contains phenolic and
flavonoid bioactive compounds (17). These constituents have been reported
to possess antioxidant properties, with ALA in particular
exhibiting anti-inflammatory activity and being associated with
improvements in memory and cognitive performance (17-21).
Notably, previous clinical studies have demonstrated that PSO
supplementation attenuates age-related cognitive decline in healthy
older individuals, improving memory, attention and processing speed
while reducing subjective forgetfulness. These cognitive benefits
were accompanied by enhanced systemic antioxidant capacity and
reduced oxidative stress biomarkers, supporting a potential
neuroprotective role for PSO (19,22).
Despite these clinical observations, the efficacy of
PSO in experimental models of accelerated aging remains
insufficiently investigated, particularly in the D-Gal-induced rat
model. Moreover, the mechanisms by which PSO may influence
cognitive decline, including modulation of oxidative stress,
preservation of cholinergic neurotransmission and changes in
hippocampal neuronal integrity, remain incompletely elucidated.
Therefore, the present study aimed to investigate whether PSO
supplementation could attenuate D-Gal-induced cognitive impairment
in rats and to examine the associated mechanisms, with a particular
focus on oxidative stress, cholinergic function and hippocampal
neuronal integrity.
Materials and methods
Preparation of PSO
Seeds of P. frutescens were collected from
cultivated areas in Mae Hong Son Province, Thailand. The seeds were
cleaned, air-dried, mechanically crushed and subjected to
cold-press extraction. The extracted oil was centrifuged at 4,000 x
g for 10 min at 4˚C, filtered, and stored in airtight amber glass
bottles at 4˚C until use. The chemical composition of the PSO used
in the present study was not reanalyzed because the oil was
obtained from the same production lot previously characterized by
Kangwan et al (20), and the
present study was designed to evaluate the biological effects of
PSO rather than to perform additional compositional
characterization. Using gas chromatography-mass spectrometry
analysis, that PSO lot was shown to contain ALA (59.20±0.11%) as
the major fatty acid, together with linoleic, oleic, palmitic and
stearic acids. Accordingly, the administered doses in the present
study should be interpreted as PSO doses rather than as
individually quantified active constituents.
Animals
Male Wistar albino rats (6-8 weeks old; n=35;
initial body weight, 150-180 g) were obtained from Nomura Siam. The
animals were acclimatized for 1 week under controlled environmental
conditions (temperature, 25±2˚C; relative humidity, 60±10%; and a
12-h light/dark cycle). Throughout the experimental period, the
rats had ad libitum access to a standard laboratory diet (feed food
no. 082; Charoen Pokphand Foods PCL) and reverse osmosis water.
Body weight was recorded weekly throughout the experimental period.
All experimental procedures were conducted in accordance with the
guidelines for the care and use of laboratory animals and were
approved by the Institutional Animal Care and Use Committee of the
University of Phayao (approval no. 1-004-66).
Experimental groups and
treatments
After a 1-week acclimatization period, the rats were
randomly assigned to five experimental groups (n=7 per group) and
treated for 8 weeks. The control sham group received daily
subcutaneous injections of normal saline at the nape of the neck
and oral administration of corn oil (CO). The D-Gal group received
5% D-Gal (300 mg/kg/day) subcutaneously in combination with oral
CO. For the positive control group, rats received 5% D-Gal (300
mg/kg/day) subcutaneously together with fish oil (FO; 500
mg/kg/day, orally). The PSO treatment groups received 5% D-Gal (300
mg/kg/day) subcutaneously in combination with PSO at doses of 100
or 500 mg/kg/day, orally. A total of two doses of PSO were included
to assess dose dependence and define an effective range in the
D-Gal-induced accelerated aging model, while FO was used as a
positive control omega-3 intervention to validate model
responsiveness and benchmark the effects of PSO. Cognitive function
was evaluated using the Morris water maze (MWM) on days 50-54,
followed by a probe trial on day 55 and the novel object
recognition (NOR) test on day 56, prior to sacrifice. The
experimental timeline is illustrated in Fig. 1.
Cognitive function test. MWM
The MWM was used to assess spatial learning and
memory, following a protocol adapted from Jeefoo et al
(15). The apparatus consisted of a
circular pool (150 cm in diameter and 45 cm in height) painted dark
blue and divided into four quadrants. The pool was filled with a
starch solution to a depth of 25 cm and maintained at 25˚C. A
circular escape platform (12.5 cm in diameter) was submerged 2 cm
below the water surface and positioned in a fixed target quadrant.
Each rat underwent three training trials per day, with a maximum
duration of 90 sec per trial, for 5 consecutive days (days 50-54).
The animals were released from different starting positions facing
the pool wall, while the platform location remained constant
throughout the training period. Escape latency, defined as the time
required to locate the hidden platform, was recorded as an index of
spatial learning. On day 55, a probe trial was conducted by
removing the platform. Each rat was released from the quadrant
opposite the target quadrant and allowed to swim freely for 90 sec.
Spatial memory retention was assessed by measuring the time spent
in the target quadrant using an overhead video tracking system
connected to a computer.
NOR. Recognition memory was assessed using
the NOR test in a gray plastic arena (54x90x50 cm). On day 55, the
rats were allowed to explore the empty arena freely for 5 min to
habituate, then returned to their home cages for 5 min. During the
training phase, each rat was placed in the arena containing two
identical objects (A1 and A2) and allowed to explore for 5 min.
Object exploration was defined as directing the nose toward an
object at a distance of ≤2 cm. A total of 24 h after the training
session, one of the familiar objects was replaced with a novel
object (B), and the test phase was conducted for 5 min. Recognition
memory was quantified using the discrimination ratio (DR),
calculated as DR=(TB-TA)/(TB + TA), where TA and TB are the times
spent exploring the familiar and novel objects, respectively
(23). A higher DR value indicates
improved recognition memory performance.
Hippocampal tissue preparation. At the end of
the experimental period, the rats were deeply anesthetized
intraperitoneally with thiopental sodium (100 mg/kg; Jagsonpal
Pharmaceuticals Ltd., India) prior to transcardial perfusion with
normal saline. Adequate depth of anesthesia was confirmed by
absence of tail reflex before perfusion. After perfusion, mortality
was confirmed by cessation of respiration and heartbeat. The brains
were then rapidly removed, and the cerebral hemispheres were
separated along the midline. The left hippocampi were immediately
microdissected on an ice-cold glass plate, homogenized in 10% (w/v)
PBS (1 M, pH 7.4) using a handheld homogenizer (D-160; DLAB
Scientific Co., Ltd) and centrifuged at 8,000 x g for 15 min at
4˚C. The resulting supernatants were collected and stored at -80˚C
for subsequent lipid peroxidation and enzymatic activity
assays.
In parallel, the right cerebral hemispheres were
fixed overnight in 4% buffered formalin at 4˚C, cryoprotected in
30% sucrose for 48 h at 4˚C and sectioned coronally at 30-µm
thickness using a cryostat microtome (CM1950; Leica Microsystems).
The sections were stored in antifreeze solution at 4˚C until
further processing. For histological analysis, sections were
stained with 0.2% cresyl violet (MilliporeSigma) for 10 min at
25˚C, dehydrated through graded ethanol solutions (70, 95 and
100%), cleared in xylene and covered with a coverslip using DPX
mounting medium (Distyrene-Plasticizer-Xylene). All images were
captured using the ECLIPSE Ni-U | Upright Microscopes (Nikon
Corporation).
Biochemical assays. Lipid
peroxidation
Lipid peroxidation was assessed by measuring
malondialdehyde (MDA), a major end product of lipid peroxidation,
as an index of oxidative stress. MDA levels were determined using
the thiobarbituric acid-reactive substances assay, as previously
described (15). Briefly,
hippocampal tissue supernatants were mixed with SDS, acetic acid
and thiobarbituric acid, followed by incubation in a boiling water
bath for 60 min. After cooling, the absorbance of the resulting
chromogen was measured at 540 nm using a microplate reader. MDA
concentrations were expressed as µmol per mg protein.
Reduced glutathione (GSH). GSH levels in
hippocampal tissue homogenates were measured using a commercially
available assay kit (GSH Assay Kit; cat. no. DIGT-250; BioAssay
Systems), according to the manufacturer's instructions. GSH
concentrations were expressed as nmol per mg protein.
Superoxide dismutase (SOD) activity. SOD
activity in hippocampal tissue homogenates was determined using a
commercially available assay kit (SOD Assay Kit-WST; cat. no.
S311-500; Dojindo Laboratories, Inc.), according to the
manufacturer's instructions. Enzyme activity was expressed as the
percentage inhibition of water-soluble tetrazolium salt-1formazan
formation.
Acetylcholinesterase (AChE) activity. AChE
activity was determined according to the method described by
Nakdook et al (24), with
minor modifications. The reaction mixture (225 µl) consisted of
hippocampal tissue supernatant, 0.1 M phosphate buffer (pH 7.4), 1
mM acetylthiocholine iodide (ATCI) and 1 mM
5,5'-dithiobis-(2-nitrobenzoic acid). The change at 405 nm was
monitored every 10 sec for 3 min using a microplate reader. Enzyme
activity was expressed as nmol of ATCI hydrolyzed per min per mg
protein.
Cell count analysis. Coronal hippocampal
sections (30-µm thick) were stained with cresyl violet (Nissl
staining) for 10 min at 25˚C. Images of the pyramidal cell layers
in the cornu ammonis 1 (CA1) and cornu ammonis 3 (CA3) subregions
were captured under bright-field microscopy at x40 magnification
using a Nikon microscope and analyzed using NIS-Elements imaging
software version 5 (Nikon, Corporation) (25). Neuronal cell bodies were manually
counted within a fixed-size region of interest (ROI; 100x100 µm)
positioned over the pyramidal cell layer. Only clearly identifiable
neuronal cell bodies with a visible nucleus and nucleolus within
the pyramidal layer were included in the counts. For each animal,
one anatomically matched coronal section at the mid-hippocampal
level was selected for analysis, based on the Paxinos and Watson
rat brain atlas (26). A total of
two non-overlapping fields were analyzed for each hippocampal
subregion (CA1 and CA3) and the mean value for each region was
calculated per animal for subsequent statistical analysis. Cell
counting was performed by an investigator blinded to the
experimental groups. Neuronal density was expressed as cells/mm²,
calculated from the mean number of neurons counted within
fixed-size ROIs (100x100 µm).
Statistical analysis
All statistical analyses were performed using
GraphPad Prism version 9.0 (GraphPad Software; Dotmatics). Data are
presented as mean ± SEM. Escape latency during the acquisition
phase of the MWM was analyzed using a two-way repeated measures
ANOVA (Group x Day). Probe trial data, DR, biochemical parameters
and histological data were analyzed using one-way ANOVA followed by
Dunnett's post hoc test. Paired Student's t-tests were used only
for within-group comparisons of object exploration times during the
NOR test, specifically between A1 and A2 during the training phase
and between the familiar object (A) and the novel object (B) during
the test phase, within the same animals in each group. These tests
were not used for between-group comparisons or as post hoc tests
following ANOVA. P<0.05 was considered to indicate a
statistically significant difference.
Results
No significant differences in body weight were
observed among experimental groups throughout the study period
(data not shown).
Effect of PSO on cognitive function.
MWM test
During the acquisition phase of the MWM test, escape
latency progressively decreased across the training days in all
experimental groups (Fig. 2A).
Two-way repeated measures ANOVA revealed a significant effect of
day [F(2.548, 76.45)=88.15; P<0.001], but no significant effect
of group [F(4, 30)=0.3644; P>0.05] or group x day interaction
[F(10.19, 76.45)=0.6452, P>0.05] (Table SI). Thus, all groups showed
improvement during the acquisition phase, with no significant
differences among groups. The day-by-day escape latency data for
the acquisition phase are provided in Table SI. By contrast, significant
between-group differences were observed in the probe trial
(Fig. 2B). In the probe trial, rats
in the D-Gal group spent significantly less time in the target
quadrant (26.60±3.09 sec) compared with the control sham group
(44.44±4.29 sec; P<0.001), indicating impaired spatial memory
(Fig. 2B). Treatment with PSO at
doses of 100 or 500 mg/kg significantly increased the time spent in
the target quadrant (42.92±2.59 sec and 41.95±2.77 sec,
respectively) compared with the D-Gal group (P<0.01 for both).
Similarly, administration of FO at a dose of 500 mg/kg
significantly increased target quadrant time (45.83±1.86 sec)
compared with the D-Gal group (P<0.001; Fig. 2B).
NOR test. Recognition memory was evaluated
using the NOR test (Fig. 3). During
the training phase, no significant within-group differences were
observed between exploration of the two identical objects (A1 and
A2) in any group (Fig. 3A). During
the test phase, control sham rats and D-Gal-treated rats receiving
PSO (100 or 500 mg/kg) or FO (500 mg/kg) showed significantly
greater exploration of the novel object than the familiar object
within the same group (Fig. 3B). By
contrast, the D-Gal group showed no significant preference for the
novel object. Comparisons of DR among groups are shown in Fig. 3C. The DR of the D-Gal group
(0.07±0.06) was significantly lower compared with that of the
control sham group (0.45±0.04; P<0.0001), indicating impaired
recognition memory. Treatment with PSO at doses of 100 or 500 mg/kg
significantly increased the DR values (0.30±0.04 and 0.33±0.05,
respectively) compared with the D-Gal group (P<0.05 and
P<0.01, respectively). Similarly, FO treatment (500 mg/kg)
significantly increased the DR value (0.32±0.04) compared with the
D-Gal group (P<0.01).
Effect of PSO on oxidative stress
markers in the hippocampus
As shown in Fig. 4,
hippocampal MDA levels were significantly higher in the D-Gal group
(0.47±0.09 µmol/mg protein) compared with the control sham group
(0.14±0.01 µmol/mg protein; P<0.05; Fig. 4A). In addition, the antioxidant
parameters GSH and SOD activity were significantly decreased in the
D-Gal group (4.49±0.67 nmol/mg protein and 88.71±1.92%,
respectively) compared with the control sham group (6.99±0.65
nmol/mg protein and 95.61±0.38%; P<0.05 and P<0.001,
respectively; Fig. 4B and C). Treatment with PSO at 500 mg/kg
significantly increased SOD activity (93.76±0.94%) compared with
the D-Gal group (P<0.05). Similarly, FO (500 mg/kg) treatment
significantly increased SOD activity (94.40±1.05%) relative to the
D-Gal group (P<0.01; Fig. 4C).
Although PSO or FO treatment did not result in statistically
significant changes in MDA levels or GSH activity compared with the
D-Gal group (P>0.05), both parameters exhibited an improving
trend and did not differ significantly from control sham values
(Fig. 4A and B).
Effect of PSO on hippocampal AChE
activity
Hippocampal AChE activity was significantly higher
in the D-Gal group (0.90±0.11 nmol of ATCI hydrolyzed/min/mg
protein) compared with the control sham group (0.49±0.04 nmol of
ATCI hydrolyzed/min/mg protein; P<0.001; Fig. 4D). Treatment with PSO at doses of
100 or 500 mg/kg significantly reduced AChE activity (0.54±0.06 and
0.54±0.06 nmol of ATCI hydrolyzed/min/mg protein, respectively)
compared with the D-Gal group (P<0.01). Similarly, FO (500
mg/kg) treatment significantly decreased AChE activity (0.50±0.03
nmol of ATCI hydrolyzed/min/mg protein) relative to the D-Gal group
(P<0.001; Fig. 4D).
Effect of PSO on hippocampal neuronal
density
Representative Nissl-stained images of the pyramidal
cell layers in the CA1 and CA3 regions are shown in Fig. 5A. Neuronal density was quantified
and expressed as cells/mm². In the CA1 region (Fig. 5B), neuronal density did not differ
significantly among the experimental groups (P>0.05). The
control group showed a neuronal density of 2,229±207.80 cells/mm²,
whereas the D-Gal group exhibited 1,657±148.20 cells/mm². Treatment
with PSO at 100 and 500 mg/kg resulted in neuronal densities of
1,693±221.00 and 1,721±290.70 cells/mm², respectively, while the
FO-treated group showed 1,929±277.3 cells/mm².
By contrast, in the CA3 region (Fig. 5C), neuronal density was
significantly reduced in the D-Gal group compared with the control
group (1,786±70.47 vs. 1,136±105.10 cells/mm²; P<0.001).
Treatment with PSO (100 mg/kg), PSO (500 mg/kg) and FO (500 mg/kg)
resulted in neuronal densities of 1,364±81.31, 1,271±116.90 and
1,386±116.40 cells/mm², respectively. However, none of these
treatment groups differed significantly from the D-Gal group
(P>0.05).
Discussion
The present study demonstrated that PSO
supplementation attenuated cognitive impairment induced by chronic
D-Gal administration in rats. PSO treatment improved spatial
learning and memory performance in the MWM probe trial and enhanced
recognition memory in the NOR test. Notably, no significant group
differences were observed during the acquisition phase of the MWM,
whereas PSO- and FO-treated rats showed improved performance in the
probe trial. This pattern may suggest that PSO exerted a greater
influence on memory retention or consolidation than on the initial
acquisition of spatial learning. At the biochemical level, PSO (500
mg/kg) significantly increased SOD activity, and both doses reduced
hippocampal AChE activity. Although MDA and GSH levels did not
differ significantly from those in the D-Gal group, both parameters
showed a tendency toward normalization, suggesting a partial
improvement in hippocampal redox balance. Histological analysis of
the hippocampus revealed region-specific changes in neuronal
density: no significant differences were observed in the CA1
region, whereas neuronal density in the CA3 region was
significantly reduced in the D-Gal group compared with the control
group. Consistent with this observation, PSO treatment did not
markedly alter neuronal cell counts in these regions. Collectively,
the results indicate that PSO mitigates D-Gal-induced cognitive
dysfunction, potentially by enhancing antioxidant enzyme activity
and modulating cholinergic signaling, but does not significantly
restore hippocampal neuronal density. Importantly, these findings
suggest that cognitive and neurochemical dysfunctions may co-occur
with region-specific structural vulnerability during early-stage
brain aging, underscoring the relevance of functional, biochemical
and histological markers as complementary indicators of brain
aging.
Although systemic senescence-associated biomarkers
were not directly measured in the present study, the D-Gal-induced
accelerated aging model has been extensively characterized in
previous investigations. Chronic D-Gal administration has been
reported to induce oxidative stress, mitochondrial dysfunction,
neuroinflammation and increased expression of senescence-related
markers (27). A previous
systematic review and meta-analysis further supported this model by
demonstrating consistent effects on cognitive impairment and
oxidative imbalance across multiple experimental conditions
(27). In the present study, the
observed cognitive deficits, increased MDA levels, reduced
antioxidant capacity and elevated AChE activity collectively
support the presence of aging-associated alterations under the
experimental conditions used.
The detrimental effects of D-Gal administration on
cognitive performance and oxidative balance observed in the present
study are consistent with previous reports demonstrating that
chronic D-Gal exposure induces oxidative stress and memory
impairment (14,15). Protective effects on cognitive
function associated with antioxidant-related mechanisms have also
been reported for various nutraceutical interventions, including
resveratrol (28), curcumin
(29) and omega-3 fatty acids
(30,31). In addition, ALA, the predominant
fatty acid component of PSO, has been associated with enhanced
cognitive performance and resistance to oxidative stress-related
neuronal dysfunction (32,33). In particular, Lee et al
(32) demonstrated that ALA derived
from PSO attenuates amyloid-β-induced memory impairment. Although
the individual contributions of ALA and minor bioactive
constituents such as tocopherols and polyphenols were not directly
assessed in the present study, their combined presence may
contribute to the overall biological effects of PSO.
Chronic D-Gal exposure has been shown to disrupt
endogenous antioxidant defenses, promote oxidative damage and
neuroinflammation and thereby contribute to age-related cognitive
decline (14,15,34).
In the present study, PSO supplementation at the higher dose
significantly increased hippocampal SOD activity, suggesting
enhancement of enzymatic antioxidant defenses, particularly SOD
activity. By contrast, alterations in MDA and GSH levels were less
pronounced, with PSO- and FO-treated groups exhibiting only a
tendency toward normalization compared with the D-Gal group. These
findings indicate that the observed biochemical effects of PSO may
be more closely associated with enhancement of enzymatic
antioxidant activity, particularly SOD at the higher dose, rather
than changes in non-enzymatic antioxidant components such as GSH
under the present experimental conditions. Similar findings in the
FO-treated group are consistent with previous reports demonstrating
that omega-3 fatty acids can enhance antioxidant enzyme activity in
D-Gal-induced aging models (35-37).
Because SOD reflects enzymatic antioxidant defense, whereas MDA
represents a downstream product of lipid peroxidation, the
significant increase in SOD activity without a corresponding
reduction in MDA may suggest that PSO enhanced antioxidant capacity
before producing a measurable reduction in accumulated oxidative
damage. The lack of statistical significance in MDA and GSH levels
may be partially attributable to the relatively small sample size
(n=7 per group), which may limit the statistical power to detect
modest biochemical changes. In addition, the 8-week experimental
duration may have been insufficient to induce more pronounced lipid
peroxidation or depletion of non-enzymatic antioxidant
reserves.
In addition, the antioxidant-related effects
observed in the present study are in agreement with previous
evidence indicating that ALA, the major fatty acid constituent of
PSO, suppresses lipid peroxidation and enhances antioxidant enzyme
activities in experimental models of amyloid-β-induced
neurotoxicity (32,33). Although inflammatory markers were
not directly evaluated in the present study, prior investigations
have suggested that PSO and ALA can modulate inflammatory signaling
pathways, including the NF-κB pathway, and reduce pro-inflammatory
cytokine expression in cellular and animal models (38,39).
Therefore, it is plausible that the beneficial effects of PSO
observed in the present study may also involve interactions between
oxidative stress regulation and inflammatory processes. Moreover,
minor bioactive constituents of PSO, such as polyphenols and
tocopherols, possess antioxidant properties (16-19)
and may collectively contribute to the overall biological effects
of PSO. From a translational perspective, modulation of oxidative
and inflammatory balance at this functional stage may be
particularly relevant for delaying or mitigating subsequent
neurodegenerative processes.
In addition to oxidative and inflammatory
mechanisms, disruption of cholinergic neurotransmission is a
well-recognized contributor to age-related cognitive decline.
Increased AChE activity accelerates acetylcholine degradation and
has been associated with memory deficits in D-Gal-induced aging
models (14,15,40).
In the present study, supplementation with PSO and FO significantly
reduced hippocampal AChE activity compared with the D-Gal group,
suggesting a preservation of cholinergic function. The inclusion of
two PSO doses allowed assessment of dose dependency, while the FO
group served as a benchmark omega-3 intervention to contextualize
the magnitude of PSO effects. This reduction was accompanied by
improvements in spatial learning and recognition memory, indicating
that modulation of cholinergic activity may contribute to the
observed cognitive benefits. Similar cholinergic-related cognitive
benefits have been reported for other omega-3-rich interventions.
For example, krill-derived phosphatidylserine improved learning and
memory performance while modulating hippocampal choline
acetyltransferase and AChE activity (41), and omega-3 fatty acids were shown to
prevent ketamine-induced elevations in AChE activity (42). Together, these findings suggest that
omega-3 fatty acids, including ALA-rich PSO, may support cognitive
function through combined antioxidant-related and
cholinergic-modulatory effects. Supporting this concept, a previous
in vitro study have demonstrated that docosahexaenoic acid
(DHA)-containing phospholipids, such as
1,2-di-DHA-phosphatidylcholine, directly inhibit AChE activity and
exhibit antioxidant properties, underscoring the close interplay
between redox regulation and cholinergic signaling (43).
Histological analysis revealed region-specific
alterations in hippocampal neuronal density. While no statistically
significant differences were observed in the CA1 region among the
experimental groups, neuronal density in the CA3 region was
significantly reduced in the D-Gal group compared with the control
group. These findings indicate that chronic D-Gal exposure under
the present experimental conditions induced selective structural
vulnerability within the CA3 subregion of the hippocampus. This
region-specific pattern may be biologically plausible, as recent
evidence indicates that hippocampal vulnerability can vary across
experimental models and that CA3 damage may, under some conditions,
be more pronounced than CA1 depending on factors such as species,
age, experimental protocol and observation period (44). In addition, CA3 neurons may exhibit
increased susceptibility to metabolic and oxidative stress due to
differences in intrinsic cellular properties, including higher
metabolic demand and lower calcium-buffering capacity (45). Although PSO and FO treatment did not
significantly restore neuronal density in the CA3 region,
improvements in cognitive performance and biochemical parameters
were observed in the treated groups. This discrepancy suggests that
the beneficial effects of PSO on cognitive function may be more
closely associated with modulation of oxidative stress and
cholinergic signaling than with robust structural recovery of
neuronal populations in the hippocampus.
Several limitations of the present study should be
acknowledged. First, although the D-Gal paradigm is widely used as
an accelerated aging-like model, systemic senescence-associated
biomarkers were not assessed in the present study. Future studies
should include direct assessment of established
aging/senescence-associated markers, such as p16, p21,
SA-β-galactosidase activity and inflammatory cytokines, to
strengthen validation of the accelerated aging-like phenotype.
Second, although oxidative stress and cholinergic activity were
assessed, inflammatory markers and synaptic plasticity-related
markers, such as synaptophysin and PSD-95, were not directly
examined, limiting mechanistic insight into the molecular pathways
underlying the observed effects of PSO. Third, although fatty acid
composition data for the PSO production lot used in the present
study were available from prior characterization, total polyphenol
content and tocopherol levels were not determined for this lot.
This limits the interpretation of the possible contributions of
other bioactive constituents in PSO in addition to its fatty acid
composition. Fourth, histological evaluation was restricted to
neuronal cell density in the CA1 and CA3 regions, and additional
analyses of dendritic architecture or synaptic markers may provide
a more comprehensive understanding of hippocampal integrity. In
addition, neuronal quantification was performed using a
standardized sampling approach based on anatomically matched
sections and fixed regions of interest rather than unbiased
stereological methods. Although this approach is commonly used for
comparative analyses, a more comprehensive stereological assessment
across multiple serial sections and larger sample sizes would
provide greater sensitivity for detecting subtle neuronal
alterations in future studies. Finally, the present study focused
on a single time point of D-Gal exposure; longer treatment periods
may be required to determine whether prolonged oxidative stress
ultimately leads to structural neuronal loss. Future studies
incorporating extended aging paradigms and additional molecular
endpoints will be valuable for further elucidating the
neurobiological effects of PSO in brain aging. In addition, food
intake was not quantitatively monitored, as it was not included in
the original experimental design, therefore no food intake records
were available. This should be considered a limitation when
interpreting potential confounding effects related to nutritional
intake. Moreover, only male rats were included in the present
study. Considering that sex hormones influence oxidative stress
(46), cholinergic signaling
(47) and cognitive aging (48), future studies incorporating female
models, particularly those mimicking perimenopausal or
postmenopausal conditions, would provide valuable insight into
potential sex-specific effects of PSO.
In conclusion, the present study demonstrates that
PSO supplementation attenuates D-Gal-induced cognitive impairment
in rats and is associated with partial attenuation of oxidative
imbalance, reflected by increased hippocampal SOD activity,
together with modulation of cholinergic function. Histological
analysis revealed region-specific alterations in hippocampal
neuronal density: Significant neuronal loss in the CA3 region
following D-Gal administration, with no significant differences in
the CA1 region. Although PSO treatment did not significantly
restore neuronal density, improvements in cognitive performance and
biochemical parameters suggest that its beneficial effects may be
associated, at least in part, with modulation of antioxidant enzyme
activity and cholinergic signaling. These findings highlight the
potential of PSO as a nutritional intervention to support cognitive
function amid aging-related neurobiological changes. Given the
known influence of sex hormones on cognitive aging (48), the potential relevance of PSO for
the cognitive health of women warrants further investigation,
particularly in female models reflecting perimenopausal or
postmenopausal conditions. Further studies are warranted to
elucidate the long-term effects and molecular mechanisms underlying
the beneficial effects of PSO in aging-related cognitive
decline.
Supplementary Material
Escape latency during the acquisition
phase of the Morris water maze across training days.
Acknowledgements
Not applicable.
Funding
Funding: The present study was supported by the Thailand Science
Research and Innovation Fund and the University of Phayao (grant
no. FF66-RIM058).
Availability of data and materials
The data generated in the present study may be
requested from the corresponding author.
Authors' contributions
WPJ conceived and designed the study, developed the
methodology and provided project supervision. WPJ, RS and SP
performed the animal experiments and behavioral assessments. WPJ,
AK, NK and JK conducted the biochemical assays. ST performed
histological staining and guided image interpretation. SP and JK
conducted neuronal cell counting and WPJ acquired and compiled
histological images and data. WPJ, AK, ST and NK analyzed and
interpreted the data. WPJ was responsible for statistical analysis,
project administration, funding acquisition and overall
supervision. WPJ drafted the original manuscript. WPJ, ST, AK and
NK reviewed and edited the manuscript. WPJ and ST confirm the
authenticity of all the raw data. All authors have read and
approved the final version of the manuscript.
Ethical approval and consent to
participate
All animal experimental procedures were reviewed and
approved by the Institutional Animal Care and Use Committee of the
University of Phayao, Thailand (approval no. 1-004-66). All methods
were carried out in accordance with the relevant guidelines and
regulations for the care and use of laboratory animals.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing
interests.
Use of artificial intelligence tools
During the preparation of this work, ChatGPT
(OpenAI) was used solely to improve the readability and language of
the manuscript. The authors subsequently reviewed and edited the
content as necessary and take full responsibility for the final
content of the manuscript. No artificial intelligence tools were
used in data analysis, interpretation, or generation of
figures.
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