Figure 9 Western blot detection
of cAMP, CREB, BDNF protein expression in rat hippocampus
Note: *P <0.05 and **P <0.01 compared
with the CON+NS group; #P <0.05 and##P <0.01 compared with the MS+NS
group.&P <0.05 and&&P <0.01 compared with the MS+P
group.
4.Discussion
This study showed that maternal-infant separation stress
induceddepression-like behavior, led to emotional dysfunction, caused
neurobehavioral changes in SD rats, triggered inflammatory responses in
the body, and decreased the expression levels of related proteins in
5-HT and cAMP/CREB signaling pathways. Probiotics had
antidepressant-like effects on the behavior of their mother-infant
separation rats, modulated the inflammatory response of the body, and
were involved in the regulation of 5-HT and cAMP/CREB signaling and
thedevelopment and improvement of hippocampal neurons, providing an
experimental basis for the application of probiotics in the treatment of
depressive-like behavior.
In general, newborns will have early contact with their mothersafter
birth in order to establish a good intimate bond, and the mother’s
touching, feeding and caring behaviors for her offspring are beneficial
to the early neurological development of the newborn[32]. Walters pointed out that infancy and
childhood are prime periods for the development of hippocampal brain
regions, which are at important stages of behavioral development,
cognitive development, emotional regulation, and character
building[33, 34]. Adverse events experienced early
in life may produce persistent emotional, cognitive, and behavioral
deviations in infants and young children as they grow, providing a risk
basis for the onset and development of depressive-like behaviors, and
such changes have been demonstrated in rodent
models[35, 36]. The mother-infant separation model
can lead to emotional dysfunction in the offspring and the development
of psychological disorders such as anxiety, depression or drug and
alcohol addiction. This early stressful stimulus of mother-infant
separation can affect the immune function of the organism and cause an
inflammatory response, leading to the development of
depression[37, 38]. Leon noted that the
mother-infant separation model harms the normal development and
maturation of the brain, has profound effects on its
futureneuroendocrine system, and has been shown to induce depression,
anxiety-like behaviors, and abnormalities in cognitive function[32]. Arborelius showed that if mother-infant
separation stress is experienced early in life, rats have a reduced
ability to recognize novelty anda reduced central activity walk in
behavioral tests, and these results could suggest that the mother-infant
separation model reduces socialinteraction, interaction behavior, and
even depression and anxiety in offspring rats as
adults[39]. Hao also showed that in an
experimental test in the open field, mother-infant separated mice
exhibited a highfrequency of grooming movements, preferring to move
around and spending little time in the central area, and that this model
of earlylife adverse stress may be related to the inhibition of the HPA
axis,affecting the development and function of neurons, which in turn
induces the onset and development of depression-like
behavior[40]. We also observed a decrease in
sugar-water preference, an increase in floating immobility time, and a
decrease in central activity time, central activity distance, and total
distance in MS rats, where a stressful event such as mother-infant
separation leads to a lack of pleasure in the rats and behavior that
shows despair, depression, or anxiety.
Probiotics are a simple and readily available complementary intervention
that is involved in the development, growth and maturation of multiple
systems, and many diseases have been shown to havea strong association
with probiotics, For example, irritable bowel
syndrome[41]、Metabolic
syndrome[42]、Allergic
diseases[43] etc, And probiotics have a positive
effect on neurological development, cognitivefunction, behavior and
emotional regulation[44]. A growing number of
studies have shown that for infants and young children experiencing
early life stressful events, the addition of probiotics from breast milk
to formula mimics to some extent the nutritional supply of breast milk
to infants and young children to achieve healthy
growth[45, 46]. Breast milk flora mainly consists
of Actinobacteria, Thick-walled Bacteria and Aspergillus, among which
Lactobacillus and Bifidobacterium predominate in the intestinal tract of
breastfed infants and children, and they play an important role in the
homeostasis of the intestinal flora of infants and
children[45, 47]. Leszek also noted that
supplementation with Lactobacillus spp. increased bifidobacteria and
Lactobacillus colonization, alleviated cognitive impairment caused
bydepression, inhibited the growth of potential bacterial fungi,
adhesion, contributed to short-chain fatty acid synthesis, modulated
inflammatory factors, and improved depression[62].
It has been shown that Lactobacillus and Bifidobacterium can convert the
amino acid glutamate to gamma-aminobutyric acid (GABA), and that
disorders of the GABA receptor signaling pathway are associated with
anxiety and depression[50]. Lebovitz showed that
Lactobacillus rhamnosus GG modulates gastrointestinal metabolism,
motility, and reduction of stress-induced visceral sensitization in
mother and infant separation[48]. It has been
shown that the mother-infant separation model can lead to increased
colonic mucosal permeability and elevated inflammatory factors, while
Bifidobacterium bifidum G9-1 can improve mucosal permeability, protect
intestinal mucosal integrity and restore intestinal flora
homeostasis[49]. Consistent with the results of
previous studies, our findings suggest that the probiotic intervention
resulted in an improvement in the behavioral expression of despair,
depression or anxiety in mother-infant separated rats.
5-HT is a monoamine neurotransmitter that plays an important role in
mood regulation and is closely related to the HPA axis, while CORT is a
product secreted by the HPA axis, both of which areimportant substances
that protect the nervous system[51]. Nisin
vesicles in the hippocampus are unique to neurons and are distributed in
the cytoplasm and dendrites, and their role is closely related to
synthetic proteins and neurotransmitters, while 5-HT as a monoamine
neurotransmitter has a positive effect on synaptic transmission and
plasticity, neuronal repair and regeneration, and regulates mood,
cognition, and autonomic neuron function[52]. Wu[53, 54] showed that oral administration of
Lactobacillus casei reversed the elevation of 5-HT levels in the
hippocampus of mice induced by chronic adverse stress, causing an
increase in the number of neurons, improvement in neuronal morphology,
clarity and integrity of cell structure, and inhibition of apoptosis or
necrosis. Tian showed that oral administration of a subgenus of
Bifidobacterium longum upregulated 5-HT concentration levels and was
confirmed in behavioral assays, improving depressive, anxiety-like
behavior in mice[55]. LI showed that probiotics
have antidepressant effects and have improved efficacy in the regulation
of 5-HT and CORT metabolism and have protective effects on neuronal
regeneration and repair[56]. In this experiment,
after the probiotic intervention, there was an increase in the number of
Nisus vesicles, an increase in the number of neurons and an improvement
in the morphology of neurons in the hippocampal CA1 region of MS rats;
the serum 5-HT and CORT concentrations were detected by Elisa and it was
found that the serum 5-HT concentration increased and the CORT
concentration decreased in MS rats after the probiotic intervention; the
results of 5-HT detection in the hippocampus of rats using
immunohistochemistry found that After the probiotic intervention, the
cell arrangement of 5-HT in the hippocampus of MS rats was neat, the
cell structure was clear, the phenomenon of lysis destruction was
alleviated, and the expression of 5-HT was more obvious. It can be seen
that probiotics can improve the expression of 5-HT in the hippocampus of
MS rats, and have a regulatory effect on the concentration of 5-HT and
CORT in the serum, which in turn promotes the repair and regeneration of
neurons in the hippocampus of MS rats, and has a certain protective
effect on neurons.
Cellular inflammatory factors are often accompanied by alterations in
the immune system, and once pro-inflammatory and anti-inflammatory
factors are dysregulated[57], It accelerates
oxidative stress, which affects neuronal repair or regeneration and
plays a dangerous role in the pathogenesis of mental
disorders[58]. It has been shown that immune
inflammation is a crucial factor in the pathogenesis of depression, and
that these inflammatory factors such as IL-1β, IL-6 and TNF-α are the
most widely studied pro-inflammatory factors, are important indicators
of the neuro-endocrine system and play a crucial role in the
pathophysiology of depression[52, 59]. The
beneficial effects of probiotics on anxiety and depression may be
through competitive elimination of harmful intestinal pathogens,
reduction of pro-inflammatory cytokines and communication with the
central nervoussystem through vagal sensory fibers, leading to changes
in neurotransmitter levels or function.Russo noted that Bifidobacterium
longum was effective in alleviating gastrointestinal disorders in their
infants and children, and the possible mechanism is related to the
regulation of intestinal flora by probiotics, which produces resistance
through mediated immune inflammatory signaling pathways and thus
improves gastrointestinal discomfort[60]. Westfall
showed that oral administration of probiotics or prebiotics, by
alleviating the body’s inflammatory response and improving the body’s
immunity, can reduce depression-like behavior in mice due to chronic
stressful events[61]. Studies have shown that oral
administration of probiotics or prebiotics, by alleviating the body’s
inflammatory response and improving the body’s immunity, can reduce the
depression-like behavior in mice due to chronic stressful events. It has
been shown that Bifidobacterium has an ameliorative effect on the
pathological changes in the hippocampus of chronically stressed mice,
and the results by Tunel staining showed that the number of positive
cells was elevated and apoptosis was alleviated, and biochemically, the
intervention of Bifidobacterium decreased the expression of inflammatory
factors such as IL-1β and TNF-α levels in serum and down-regulated the
content of pro-apoptotic proteins[55]. Maria
showed that depression leads to systemic inflammation in the body and
that this inflammation disrupts the blood-brain barrier in different
pathways to reach the central nervous system, activating the central
immune system and inducing an inflammatory response in the nervous
system[63, 64]. Satya showedthat acetate-3 and
acetate-5 inhibited monoamine oxidase-A enzymes,increased norepinephrine
(NA) and 5-HT levels in rat brain, decreased plasma CORT levels, reduced
TNF-α and IL-6 inflammatory factor levels, and improved depression-like
symptoms[65]. It has been shown that in clinical
practice, depressed patients receiving probiotic-assisted intervention
therapy suppress inflammatory responses, reduce the release of
inflammatory mediators, alleviate neurological damage,and reduce
depression-like symptom[66]. It has been shown
that in the acute phase, depressed patients have elevated serum levels
of TNF-α and IL-1 inflammation, which can inhibit synaptic function and
lead to anxiety and depression-like behaviors [52,
67]. Yoo showed that oral administration of Lactobacillus plantarum,
which inhibited the expression of inflammatory factors such as IL-6 and
TNF-α, attenuated the level of neuroinflammation in the brain of mice
and led to the alleviation of their depression-like
behavior[68]. In this experiment, we found that
the levels of IL-1β, IL-6 and TNF-α inflammatory factors in the serum of
MS rats were reduced after probiotic intervention by Elisa assay, and MS
rats showed improvement in depression-like behavior, suggesting that
probiotic intervention can inhibit the production of pro-inflammatory
factor cells and reduce the neuroinflammatory response, and then improve
the depression-like behavior of offspring rats due to maternal-infant
separation, which is consistent with the findings of park[69].
The cAMP signaling pathway is one of the most classic and earliest
studied, and plays an important role in neural cell and functional
repair[70]. cAMP/CREB is involved in cell
signaling and is closely related to neuronal growth and development,
cell repair, and synaptic plasticity[71].BDNF is
one of the most abundant and important neurotrophic factors in the
body’s brain, and is responsible for protecting neurons, participating
in axonal growth, dendrite number, morphological
regulation[72]. It is assumed that BDNF deficiency
may lead to impaired neurological growth and development in the brain,
which in turn makes it a risk factor for the pathogenesis of
depression[73]. It has been shown that 5-HT can be
regulated through the G protein-coupled cAMP/CREB signaling pathway, and
that activation of the cAMP/CREB signaling pathway can in turn affect
5-HT production, thus inducing depression or anxiety-like disorders if
the pathway is abnormal[74, 75]. Wu showed that
the pathogenesis of depression is related to the fact that some drugs
with antidepressant effects activate the PKA/CREB signaling pathway and
modulate the expression of BDNF activity[28]. Li
Zhiyong showed that Chai Shao An Shen Jie Yu granules could reduce the
secretion of inflammatory factors in the body, upregulate hippocampal
PKA/CREB-related proteins, activate anti-inflammatory factors, and help
the effect of hippocampal neuron repair,thus improving post-stroke
depression[76]. In thepresent study we evaluated
the effect of probiotics on the cAMP/CREB signaling pathway in the
hippocampal region of MS rats. We found that the cell structure of cAMP,
CREB, and BDNF in the hippocampal region of MS rats was unclear,
misaligned and lax, withsigns of lysis and disruption, and reduced
protein expression, indicating a correlation between depression-like
behavior and abnormal protein expression in hippocampal tissue. After
probiotic intervention, the cell edges of hippocampal area in MS rats
were relatively intact,relatively neatly arranged, with clearer
structure, and the expression of cAMP, CREB and BDNF proteins were
increased, indicating thatprobiotics would regulate the 5-HT content in
hippocampus and serum, inhibit the inflammatory factors of the body, and
then bind to the receptors, activate the cAMP/CREB signaling pathway,
and improve the hippocampal related tissue protein levels and improve
depression-like behavior in mother-infant separated offspring rats,
which is consistent with the study of Mato[77].