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].