Application of lactobacillus rhamnosus in preparation of product for treating hippocampus injury caused by chronic fatigue

Abstract

The invention discloses application of lactobacillus rhamnosus to preparation of a product for treating hippocampus injury caused by chronic fatigue. The invention discloses that the lactobacillus rhamnosus BD4047 has good neuron and synaptic protection capability for the first time, and can improve hippocampus neuron damage caused by chronic fatigue, increase the density of hippocampus neuron dendritic spines, increase the length of the dendritic spines and increase the number of branch intersection points of the dendritic spines; the synaptic injury caused by chronic fatigue is improved, the synaptic gap width is reduced, the thickness of a synaptic compact region is increased, the length of a synaptic active region is prolonged, the increase of the number of synaptic vesicles and the increase of the number of SYP spots are promoted, and a remarkable synaptic repair effect is achieved; in addition, mouse hippocampus microglial cell activation caused by chronic fatigue can be inhibited, hippocampus injury caused by chronic fatigue and diseases related to the hippocampus injury can be comprehensively improved, and therefore the composition has a wide prospect.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the use of Lactobacillus rhamnosus in the preparation of products for treating hippocampal damage caused by chronic fatigue. Background Technology

[0002] Chronic fatigue (especially chronic fatigue syndrome, CFS) is a complex disease state characterized by persistent fatigue that is difficult to relieve with rest, and may be accompanied by cognitive impairment and emotional problems. Hippocampal synaptic damage caused by CFS can trigger or exacerbate various diseases related to cognitive function and mood regulation. As a key area in the brain responsible for memory formation, learning, and mood regulation, damage to the hippocampus's synaptic structure directly affects neural signal transmission, thus linking to the following diseases: cognitively, it can induce mild cognitive impairment, Alzheimer's disease, vascular dementia, etc., manifested as memory loss and cognitive decline; emotionally and mentally, it is easily associated with depression and anxiety disorders, as synaptic damage impairs the ability to regulate emotions and process fear memories; simultaneously, chronic fatigue syndrome itself is also accompanied by neurocognitive complications such as "brain fog," and may also exacerbate post-traumatic stress disorder and sleep disorders. These diseases are often related to pathological mechanisms such as neuroinflammation and HPA axis abnormalities, and there is a vicious cycle of mutual influence.

[0003] Early intervention in chronic fatigue (such as adjusting sleep patterns and anti-inflammatory treatment) may reduce the risk of related diseases by protecting hippocampal synapses. A deeper understanding of its molecular mechanisms and the development of targeted interventions hold promise for providing new targets and approaches for the treatment of neurological diseases.

[0004] However, existing drug treatments for cognitive impairment and mood disorders caused by chronic fatigue-related hippocampal synaptic damage have significant limitations and drawbacks: most drugs only provide symptomatic relief (e.g., antidepressants improve mood, cholinesterase inhibitors delay cognitive decline), but cannot directly repair synaptic damage or block core pathological mechanisms such as neuroinflammation and HPA axis abnormalities, making it difficult to stop disease progression at its root; efficacy is limited and varies greatly from person to person, for example, Alzheimer's drugs can only delay cognitive decline in some patients, antidepressants have a remission rate of less than 50% for patients with comorbid depression, and some patients cannot adhere to treatment due to poor tolerance; long-term use carries potential neurotoxicity and side effects, such as benzodiazepines. Such drugs may exacerbate hippocampal synaptic inhibition, and long-term use of nonsteroidal anti-inflammatory drugs may hinder synaptic repair. At the same time, it is difficult to break the vicious cycle of "chronic fatigue-synaptic damage-cognitive / emotional abnormalities". Furthermore, the interaction of combined drugs is still unclear for patients with multiple diseases, which may increase the burden on the body. Therefore, comprehensive intervention with non-pharmacological means is required.

[0005] Therefore, screening for strains with high safety, clearly defined functional targets, and diverse mechanisms of action to improve hippocampal injury and related diseases has significant application value and broad development prospects. This may provide new treatment options for CFS patients, helping to break the vicious cycle and improve their quality of life. Summary of the Invention

[0006] In view of the shortcomings of the prior art described above, the object of the present invention is to provide the use of Lactobacillus rhamnosus in the preparation of products for treating hippocampal damage caused by chronic fatigue, in order to solve the problems in the prior art.

[0007] To achieve the above and other related objectives, the present invention provides the use of Lacticaseibacillus rhamnosus BD4047 in the preparation of at least one of the following:

[0008] 1) Products for treating synaptic damage caused by chronic fatigue;

[0009] 2) Products for treating neuronal damage caused by chronic fatigue;

[0010] 3) Products that inhibit microglia activation caused by chronic fatigue.

[0011] In some embodiments, the Lactobacillus rhamnosus BD4047 is accessed under the number CGMCC No. 13310.

[0012] In some embodiments, the product for treating synaptic damage caused by chronic fatigue, and / or for treating neuronal damage caused by chronic fatigue, and / or for inhibiting microglia activation caused by chronic fatigue includes one or more of the following: fermentation broth of Lactobacillus rhamnosus BD4047, live bacteria, and lyophilized powder.

[0013] In some embodiments, the product for treating synaptic damage caused by chronic fatigue has one or more of the functions of A1)-A5):

[0014] A1) Reduce the width of the synaptic cleft in the hippocampus;

[0015] A2) Increase the thickness of the synaptic density region in the hippocampus;

[0016] A3) Increase the length of synaptic active regions in the hippocampus;

[0017] A4) Increase the number of synaptic vesicles in the hippocampus;

[0018] A5) Increase the number of SYP spots in the hippocampus.

[0019] In some embodiments, the product for treating neuronal damage caused by chronic fatigue has one or more of the functions described in B1)-B3):

[0020] B1) Increase the number of dendritic spines in the hippocampus;

[0021] B2) Increase the length of dendritic spines in the horse's body;

[0022] B3) Increase the number of dendritic branch intersections in the horse body.

[0023] In some embodiments, the product that inhibits microglial cell activation caused by chronic fatigue has one or more of the functions of C1)-C3):

[0024] C1) regulates the expression of genes in the hippocampus related to the process of microglia phagocytosis synapses;

[0025] C2) Reduces the number of microglia in the hippocampus;

[0026] C3) Decrease the proportion of M1 microglia in the hippocampus and / or increase the proportion of M2 microglia.

[0027] In some specific embodiments, regulating genes in the hippocampus associated with the microglia phagocytic synapse process refers to increasing the expression levels of SYP and PSD-95, and / or decreasing the expression levels of C1qa, CD68, or Iba-1.

[0028] In some embodiments, the product for treating synaptic damage caused by chronic fatigue, and / or the product for treating neuronal damage caused by chronic fatigue, and / or the product for inhibiting microglia activation caused by chronic fatigue, is for treating diseases associated with hippocampal damage, wherein the diseases associated with hippocampal damage are selected from one or more of Alzheimer's disease, vascular dementia, depression, anxiety disorder, chronic fatigue syndrome, cognitive impairment, post-traumatic stress disorder, and sleep disorder.

[0029] In some embodiments, the product for treating synaptic damage caused by chronic fatigue, and / or the product for treating neuronal damage caused by chronic fatigue, and / or the product for inhibiting microglia activation caused by chronic fatigue is selected from one or more of functional foods, microbial agents, pharmaceuticals, health products, and pharmaceutical raw materials.

[0030] In some specific embodiments, the functional food is selected from fermented milk products, lactic acid bacteria beverage products, probiotic powder products, live bacteria aquatic products, milk powder products, or cheese products containing Lactobacillus rhamnosus BD4047.

[0031] In some specific embodiments, the dosage form of the microbial agent is selected from one or more of lyophilized powder, capsules, tablets, and granules.

[0032] In some more specific embodiments, the method for preparing the fermented milk product containing Lactobacillus rhamnosus is as follows: obtaining raw milk by fermenting raw milk with Lactobacillus rhamnosus BD4047.

[0033] In some more specific embodiments, the bacterial agent is a lyophilized powder obtained by drying a culture of Lactobacillus rhamnosus BD4047.

[0034] In some embodiments, the concentration of *Lactobacillus rhamnosus* BD4047 in the product for treating synaptic damage caused by chronic fatigue, and / or the product for treating neuronal damage caused by chronic fatigue, and / or the product for inhibiting microglial activation caused by chronic fatigue is 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g or higher.

[0035] As described above, the use of Lactobacillus rhamnosus of the present invention has the following beneficial effects:

[0036] 1) The *Lactobacillus rhamnosus* of this invention possesses significant synaptic protective capabilities, improving synaptic damage or loss caused by chronic fatigue. Specifically, it reduces synaptic cleft width, increases synaptic density thickness, increases synaptic active zone length, increases the number of synaptic vesicles, and increases the number of SYP spots. Therefore, *Lactobacillus rhamnosus* exhibits significant synaptic protective and repair effects.

[0037] 2) The *Lactobacillus rhamnosus* of this invention has significant neuroprotective capabilities, improving damage or loss of dendritic spines in hippocampal neurons caused by chronic fatigue. Specifically, it increases the number of dendritic spines, dendrite length, and the number of dendritic branch intersections. Therefore, *Lactobacillus rhamnosus* has significant neuronal damage protection and repair effects.

[0038] 3) The *Lactobacillus rhamnosus* of the present invention can improve microglia activation caused by chronic fatigue and regulate the expression of genes related to the microglia phagocytic synapse process. Specifically, it reduces the transcriptional level and protein expression of Syp and PSD-95, and increases the transcriptional level and protein expression of C1qa and CD68. In addition, it inhibits the increase of microglia and the high proportion of M1 type caused by chronic fatigue, indicating that it can affect neuroinflammation and even neurodegenerative processes by inhibiting the activation state and function of microglia. Attached Figure Description

[0039] Figure 1This invention demonstrates how Lactobacillus rhamnosus B6 intervention can improve neuronal and synaptic damage in the mouse hippocampus caused by chronic fatigue. (a) Representative images of Sholll analysis of neurons in the hippocampus of 5 groups of mice. (b) Representative images of dendrites of neurons in the hippocampus of 5 groups of mice. Scale bar is 10 μm. (c) Statistical analysis of the number of intersections between neuronal dendrites and concentric circles 10–100 μm away from the cell body in the hippocampus of 5 groups of mice (n=6). (d) Statistical analysis of dendritic spine density ( / 10 μm) of neurons in the hippocampus of 5 groups of mice (n=6). (e) Statistical analysis of dendritic length of neurons in the hippocampus of 5 groups of mice (n=6). (f) Representative transmission microscope images of synapses in the hippocampus of 5 groups of mice. (g) Statistical analysis of synaptic cleft width, synaptic density thickness, and synaptic active zone length in the hippocampus of 5 groups of mice (n=3). (h) Statistical analysis of the number of synaptic vesicles in the hippocampus of 5 groups of mice (n=3).

[0040] Figure 2 This invention demonstrates how *Lactobacillus rhamnosus* B6 intervention can improve the increased phagocytic synaptic process (CMESH) induced by chronic fatigue in mouse hippocampal microglia. (a) Transcriptional levels of phagocytic synaptic-related genes Syp, Psd95, C1qa, and Cd68 in the anterior hippocampus of five groups of mice (n=3). (b) Representative immunoblot images of phagocytic synaptic-related proteins SYP, PSD-95, C1qa, CD68, and Iba-1 in the anterior hippocampus of five groups of mice. (c) Statistical analysis of protein levels of phagocytic synaptic-related proteins SYP, PSD-95, C1qa, CD68, and Iba-1 in the prefrontal cortex of five groups of mice (n=3). (d) Statistical analysis of the total number of microglia in the hippocampus of five groups of mice (n=3). (e) Statistical analysis of the proportion of M1 type microglia in the hippocampus of five groups of mice (n=3). (f) Statistical analysis of the proportion of M2 type microglia in the hippocampus of 5 groups of mice (n=3). (g) Representative images of Iba-1, iNOS, and CD206 immunofluorescence triple staining analysis in the hippocampus of 5 groups of mice. Scale bar is 50 μm. Statistical analysis of (h) the number of Iba-1+ / CD68+ cells per square millimeter, (i) the C1q positive area, and (j) the number of SYP spots / microglia in the hippocampus of 5 groups of mice (n=3). (k) Representative images of Iba-1 and CD68, as well as C1qa and PSD-95 immunofluorescence double staining analysis in the hippocampus of 5 groups of mice. Scale bar is 50 μm. Detailed Implementation

[0041] This invention discovers that Lactaseibacillus rhamnosus BD4047 exhibits good therapeutic effects in diseases (such as cognitive impairment, depression, chronic fatigue syndrome, etc.) caused by chronic fatigue-induced hippocampal synaptic damage, neuronal damage, and / or microglia activation and associated hippocampal damage. Based on this, this invention was completed.

[0042] Chronic fatigue syndrome (CFS) is a neuroendocrine-immune disorder characterized by severe fatigue lasting more than 6 months as its core symptom, accompanied by cognitive impairment, sleep disturbances, and multi-system symptoms (such as muscle pain, sore throat, and swollen lymph nodes). The World Health Organization (WHO) has listed it in the International Classification of Diseases (ICD-11) with the code G93.3.

[0043] Chronic incomplete sleep deprivation (CIBSD), also known as chronic fatigue syndrome (CFS), refers to an individual's daily sleep duration consistently less than physiological needs (usually <5 hours) for more than 3 months. Its core characteristic is prolonged sleep deprivation, but not reaching the extreme state of acute complete deprivation (24-48 hours without sleep). Sleep deprivation models simulate long-term sleep deprivation in humans, with relatively mild stress, and the induced fatigue-like symptoms are closer to the clinical characteristics of chronic fatigue (such as persistent fatigue and cognitive decline). It is currently the preferred model for chronic fatigue research. These models employ "segmented sleep deprivation," depriving mice of 4-8 hours of sleep daily (e.g., during nighttime sleep peaks) for 1-4 weeks; or "intermittent vibration therapy," using low-intensity periodic stimulation to reduce the proportion of deep sleep. Therefore, this invention uses a sleep deprivation device to artificially reduce the amount of normal sleep in animals to simulate chronic fatigue.

[0044] Uses of Lacticaseibacillus rhamnosus BD4047 in the preparation of at least one of the following:

[0045] 1) Products for treating synaptic damage caused by chronic fatigue;

[0046] 2) Products for treating neuronal damage caused by chronic fatigue;

[0047] 3) Products that inhibit microglia activation caused by chronic fatigue.

[0048] In some embodiments, the product for treating synaptic damage caused by chronic fatigue has one or more of the functions of A1)-A5):

[0049] A1) Reduce the width of the synaptic cleft in the hippocampus;

[0050] A2) Increase the thickness of the synaptic density region in the hippocampus;

[0051] A3) Increase the length of synaptic active regions in the hippocampus;

[0052] A4) Increase the number of synaptic vesicles in the hippocampus;

[0053] A5 increases the number of SYP spots in the hippocampus.

[0054] This invention constructed an animal model of chronic fatigue (sleep deprivation) and found that chronic fatigue leads to changes in the synaptic structure of the hippocampus, specifically: increased synaptic cleft width, decreased postsynaptic compactum thickness, shortened synaptic active zone length, and reduced number of synaptic vesicles. Simultaneously, the number of SYP immunopositive spots on the presynaptic membrane increased, leading to synaptic damage. Further research revealed that intervention with *Lactobacillus rhamnosus* BD4047 resulted in decreased synaptic cleft width, increased postsynaptic compactum thickness, prolonged synaptic active zone length, increased number of synaptic vesicles, and an increased number of SYP immunopositive spots. This indicates that *Lactobacillus rhamnosus* BD4047 can exert a neuroprotective effect by regulating synaptic ultrastructure and promoting synaptic repair. The dense region is where postsynaptic membrane receptors and signal transduction proteins accumulate, while the active region is the key area for the release of neurotransmitters from the presynaptic membrane. Lactobacillus rhamnosus BD4047 can increase the thickness of the synaptic dense region and increase the number of synaptic vesicles, thereby compensating for presynaptic membrane damage. At the same time, by narrowing the gap, it restores the tight docking between the presynaptic and postsynaptic membranes and repairs hippocampal synaptic damage induced by chronic fatigue.

[0055] In some embodiments, the product for treating neuronal damage caused by chronic fatigue has one or more of the functions described in B1)-B3):

[0056] B1) Increase the number of dendritic spines in the hippocampus;

[0057] B2) Increase the length of dendritic spines in the horse's body;

[0058] B3) Increase the number of dendritic branch intersections in the horse body.

[0059] This invention constructed an animal model of chronic fatigue (sleep deprivation) and found that chronic fatigue leads to changes in the dendrites of hippocampal neurons, specifically a decrease in the number of dendritic spines, shortening of dendritic length, and a decrease in the number of dendritic branching points. Further research revealed that intervention with *Lactobacillus rhamnosus* BD4047 increased the number and length of dendritic spines in the hippocampus and increased the number of dendritic branching points, indicating that *Lactobacillus rhamnosus* BD4047 can exert a neuroprotective effect by regulating dendritic plasticity and repair neuronal damage induced by chronic fatigue.

[0060] In some embodiments, the product that inhibits microglial cell activation caused by chronic fatigue has one or more of the functions of C1)-C3):

[0061] C1) regulates the expression of genes in the hippocampus related to the process of microglia phagocytosis synapses;

[0062] C2) Reduces the number of microglia in the hippocampus;

[0063] C3) Decrease the proportion of M1 microglia in the hippocampus and / or increase the proportion of M2 microglia in the hippocampus.

[0064] In some embodiments, the regulation of genes related to the hippocampal and microglia phagocytic synapse processes refers to increasing the expression levels of SYP and PSD-95, and / or decreasing the expression levels of C1qa, CD68, or Iba-1.

[0065] This invention constructed an animal model of chronic fatigue (sleep deprivation) and found that chronic fatigue leads to microglia activation, specifically manifested as an increase in the number of microglia in the hippocampus and an increased proportion of M1-type microglia, thereby activating microglia. Further research revealed that intervention with *Lactobacillus rhamnosus* BD4047 reduced the number of microglia in the hippocampus and the proportion of M1-type microglia, indicating that *Lactobacillus rhamnosus* BD4047 can alleviate chronic fatigue-induced neuroinflammatory damage by inhibiting microglia activation.

[0066] In some embodiments, the chronic fatigue refers to chronic incomplete sleep deprivation (CIBSD), which is part of chronic fatigue syndrome (CFS), and is caused by insufficient sleep or sleep deprivation.

[0067] In some embodiments, the Lactobacillus rhamnosus BD4047 strain has the accession number CGMCC No. 13310. This strain was deposited on November 15, 2016, at the China General Microbiological Culture Collection Center (CGMCC), Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing. It is also known as Lactobacillus rhamnosus B6, as can be found in references 1 (Han Jin. Screening, identification and optimization of sugar-producing lactic acid bacteria. Journal of Microbiology [J]. 2021, 5: 43-51) and 2 (Li Ruiying et al. Study on antioxidant activity and cell-protective effect of Lactobacillus rhamnosus B6. Food and Fermentation Industries [J]. 2022, 17: 57-63). In this invention, Lactobacillus rhamnosus BD4047 and Lactobacillus rhamnosus B6 can be used interchangeably.

[0068] In some embodiments, the product for treating synaptic damage caused by chronic fatigue, and / or for treating neuronal damage caused by chronic fatigue, and / or for inhibiting microglia activation caused by chronic fatigue includes one or more of the following: fermentation broth of Lactobacillus rhamnosus BD4047, live bacteria, and lyophilized powder.

[0069] In some embodiments, the product for treating synaptic damage caused by chronic fatigue, and / or the product for treating neuronal damage caused by chronic fatigue, and / or the product for inhibiting microglia activation caused by chronic fatigue, is for treating diseases associated with hippocampal damage, wherein the diseases associated with hippocampal damage are selected from one or more of Alzheimer's disease, vascular dementia, depression, anxiety disorder, chronic fatigue syndrome, cognitive impairment, post-traumatic stress disorder, and sleep disorder.

[0070] In some embodiments, the product for treating synaptic damage caused by chronic fatigue, and / or the product for treating neuronal damage caused by chronic fatigue, and / or the product for inhibiting microglia activation caused by chronic fatigue is selected from one or more of functional foods, microbial agents, pharmaceuticals, health products, and pharmaceutical raw materials.

[0071] In some specific embodiments, the functional food is selected from fermented milk products, lactic acid bacteria beverage products, probiotic powder products, live bacteria aquatic products, milk powder products, or cheese products containing Lactobacillus rhamnosus BD4047.

[0072] In some specific embodiments, the dosage form of the microbial agent is selected from one or more of lyophilized powder, capsules, tablets, and granules.

[0073] In some more specific embodiments, the fermented milk product containing *Lactobacillus rhamnosus* BD4047 is prepared by fermenting raw milk with *Lactobacillus rhamnosus* BD4047. The fermented milk is prepared by inoculating the raw milk with the aforementioned BD4047 (i.e., *Lactobacillus rhamnosus* B6) and fermenting. The raw milk can be any type of raw milk suitable for yogurt preparation, for example, it can include one or more combinations of raw milk and reconstituted milk. For example, raw milk can meet relevant standards such as GB 19301 National Food Safety Standard for Raw Milk, and reconstituted milk can meet relevant standards such as GB 19644 National Food Safety Standard for Milk Powder; preferably, the reconstituted milk is skim milk powder. The source of the raw milk is preferably cow and / or sheep. Specifically, the seed culture of *Lactobacillus rhamnosus* is inoculated into 10% skim milk at an inoculation rate of 3% (v / v) and fermented at 37°C.

[0074] In some specific embodiments, the bacterial powder product is a lyophilized powder obtained by drying a culture of *Lactobacillus rhamnosus* BD4047 (i.e., *Lactobacillus rhamnosus* B6). The bacterial agent also contains a protectant and / or auxiliary additives. The protectant is selected from skim milk powder. The auxiliary additives include one or more of inulin, fructooligosaccharides, galactooligosaccharides, mannose oligosaccharides, trehalose, soybean oligosaccharides, resistant dextrin, spirulina, polydextrose, α-lactalbumin, or lactoferrin. *Lactobacillus rhamnosus* BD4047 (i.e., *Lactobacillus rhamnosus* B6) can be used alone in related products or in combination with other strains. The culture of *Lactobacillus rhamnosus* BD4047 is obtained by inoculating *Lactobacillus rhamnosus* BD4047 into a culture medium. The culture medium can be MRS liquid medium, whose formulation, by example, includes: peptone, beef extract, glucose, sodium acetate, yeast extract, diammonium citrate, K₂PO₄·3H₂O, MgSO₄·7H₂O, MnSO₄, L-cysteine, and Tween 80. The culture temperature is 30-40℃, for example, 30℃, 32℃, 35℃, 36℃, 37℃, 38℃, etc.; the culture time is 12-30 hours, for example, 12 hours, 14 hours, 18 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 30 hours, etc. In one specific embodiment, *Lactobacillus rhamnosus* BD4047(B6) is inoculated into MRS medium and cultured at 37℃ for 56 hours.

[0075] In one specific embodiment, the preparation method of the bacterial powder product is as follows: centrifugation and drying of a culture of *Lactobacillus rhamnosus* BD4047 (i.e., *Lactobacillus rhamnosus* B6). Preferably, the drying refers to vacuum freeze drying or other bacterial liquid drying processes.

[0076] In some embodiments, the viable count of *Lactobacillus rhamnosus* BD4047 in the product for treating synaptic damage caused by chronic fatigue, and / or the product for treating neuronal damage caused by chronic fatigue, and / or the product for inhibiting microglial activation caused by chronic fatigue is not less than 1 × 10⁻⁶. 6 CFU / mL or 1×10 6 CFU / g, for example 1×10 6 1×10 7 CFU / g, 1×10 8 CFU / g, 1×10 9 CFU / g, 2×10 9 CFU / g, 5×10 9 CFU / g, 8×10 9 CFU / g, 1×10 10 CFU / g, 5×10 10CFU / g, 1×10 11 CFU / g, etc., those skilled in the art can select the range of values ​​according to actual needs.

[0077] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0078] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0079] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0080] Data in the examples and figures are expressed as mean ± standard deviation. Multiple comparisons of univariate data were performed using one-way ANOVA, with post-hoc LSD correction tests. P < 0.05 was considered statistically significant.

[0081] Under aseptic conditions, a complete culture medium was prepared using high-glucose DMEM medium, fetal bovine serum, and a penicillin-streptomycin antibiotic mixture at a volume ratio of 10:1:0.1 for subsequent in vitro experiments. Specifically, the commonly used working concentration of penicillin was 100 U / mL; the commonly used working concentration of streptomycin was 100 μg / mL. Before use, the mixture was diluted 100-fold to form the antibiotic mixture, i.e., the culture medium used was 445 mL DMEM + 50 mL serum + 5 mL antibiotics. The high-glucose DMEM medium was Hyclone SH30022.01 DMEM high-glucose medium.

[0082] For convenience, in the following embodiments of this application, Lacticaseibacillus rhamnosus BD4047 is uniformly referred to as Lacticaseibacillus rhamnosus B6 (also abbreviated as strain B6).

[0083] Example 1: Preparation of Lactobacillus rhamnosus B6 lyophilized powder and fermented milk lyophilized powder

[0084] 1.1 Preparation of Lyophilized Powder of Lactobacillus rhamnosus B6 Cells

[0085] 1) Single colonies of *Lactobacillus rhamnosus* B6 were selected using an inoculation loop and placed in a 10 mL sterile tube containing 5 mL of MRS liquid medium (purchased from Merck Co., Germany), and incubated at 37°C for 18 h. Then, a 3% (w / v, i.e., g / mL) inoculation was added to 150 mL of MRS liquid medium, and incubated at 37°C for another 18 h. Finally, a 3% (w / v) inoculation was added to 1 L of MRS liquid medium, and incubated at 37°C for 20 h to obtain the MRS fermentation broth of *Lactobacillus rhamnosus* B6.

[0086] 2) Centrifuge the MRS fermentation broth of *Lactobacillus rhamnosus* B6 obtained in step 1) at 8000 rpm for 20 min, discard the supernatant, and collect the precipitated bacterial cells. Wash the precipitated bacterial cells with ultrapure water 3-5 times until the precipitated bacterial cells change from brown to transparent. Place the washed precipitated bacterial cells on a tray and freeze-dry them in a vacuum freeze dryer at -50℃ for 48 h to obtain *Lactobacillus rhamnosus* B6 lyophilized bacterial powder.

[0087] The viable cell count of the B6 lyophilized powder was 2.6 × 10⁻⁶. 10 CFU / g.

[0088] 1.2 Preparation of Lyophilized Milk Fermented with Lactobacillus rhamnosus B6

[0089] 1) Select 2-3 single colonies of *Lactobacillus rhamnosus* B6 using an inoculation loop and incubate them in 10 mL of skim milk liquid medium at 37°C for 18 h. Then, inoculate 10% (w / v) of this culture into 100 mL of skim milk liquid medium and continue incubation at 37°C for another 18 h. Finally, inoculate 10% (w / v) of this culture into 1 L of skim milk liquid medium and incubate at 37°C for 20 h to obtain fermented milk from *Lactobacillus rhamnosus* B6. The skim milk liquid medium consists of 10 g of skim milk added to 90 mL of ddH2O to obtain 10% skim milk, i.e., skim milk liquid medium.

[0090] 2) Place the fermented milk of Lactobacillus rhamnosus B6 obtained in step 1) on a tray and freeze-dry it at -50℃ for 48 hours in a vacuum freeze dryer to obtain freeze-dried powder of Lactobacillus rhamnosus B6 fermented milk.

[0091] The viable count of B6 fermented milk freeze-dried powder was 2.6 × 10⁻⁶. 10 C number FU / g.

[0092] When intervening in animals, use 1g of 2.6×10 10 CFU / g of B6 bacterial powder was dissolved in 26 mL of sterile water to obtain 1×10 9 CFU / mL B6 bacterial cell solution; 1 g 2.6 × 10 10 CFU / g of B6 fermented milk lyophilized powder was dissolved in 26 mL of sterile water to obtain 1×10 9 B6 fermented milk solution with CFU / mL.

[0093] When intervening in animals, the above-mentioned B6 lyophilized bacterial powder was suspended in sterile water, i.e., 1g = 2.6 × 10⁻⁶. 10 CFU / g bacterial powder dissolved in 26mL of sterile water yields a concentration of 1×10⁻⁶. 9 A CFU / mL solution of *Lactobacillus rhamnosus* B6 cells (i.e., B6 powder solution) was administered orally at a dose of 200 μL per animal; the above B6 fermented milk lyophilized powder was suspended in 26 mL of sterile water to obtain a concentration of 1 × 10⁻⁶. 9 CFU / mL Lactobacillus rhamnosus B6 fermented milk solution.

[0094] Example 2: Experimental Animals and Grouping

[0095] Sixty SPF (Specified Pathogen Free) male C57BL / 6J mice, 8 weeks old and weighing 22-28g, were purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd. All mice were housed in an SPF-grade animal facility with a 12 / 12h light / dark cycle, a temperature maintained at 22±2℃, and free access to food and water. The mice were allowed one week to acclimatize to the SPF-grade animal facility in preparation for subsequent experiments.

[0096] Sixty mice were randomly divided into five groups, and the experimental schedule for each group was as follows:

[0097] ① Control group (CON, n=12): Mice were placed in small chambers of sleep deprivation devices ZT8-ZT2 with the devices turned off. They were allowed to eat and drink freely and were given 200 μL of physiological saline by gavage daily for 25 days.

[0098] ② Chronic fatigue group (SD, n=12): Mice were placed in a rat sleep deprivation device. The sleep deprivation device was kept on from ZT8 to ZT2 (Zeitgeber times, timing factor, ZT0 is the time when artificial light was turned on). A metal rod was rotated during the period from ZT2 to ZT8 (6 hours of mouse sleep time). When the mouse was moved by the metal rod, it needed to be turned over (i.e., the dynamic state was maintained for 18 hours). The mice could eat and drink freely. They were given 200 μL of physiological saline by gavage every day for 25 days.

[0099] ③ Lactobacillus rhamnosus B6 bacterial powder group (B6P, n=12): The sleep deprivation device was kept on from ZT8 to ZT2 (Zeitgebertimes, timing factor, ZT0 is the time when artificial light was turned on). A metal rod was rotated during the period excluding ZT2-ZT8 (6 hours of mouse sleep). Mice needed to be turned over when propelled by the metal rod (i.e., maintaining a dynamic state for 18 hours). Mice could eat and drink freely. 200 μL (5 × 10⁻⁶) of Lactobacillus rhamnosus B6 bacterial solution was administered by gavage daily. 8 / day), lasting for 25 days.

[0100] ④ Lactobacillus rhamnosus B6 fermented milk group (B6FM, n=12): The sleep deprivation device was kept on from ZT8 to ZT2 (Zeitgebertimes, timing factor, ZT0 is the time when artificial light was turned on). A metal rod was rotated during the period excluding ZT2-ZT8 (6 hours of mouse sleep). Mice needed to be turned over when propelled by the metal rod (i.e., maintaining a dynamic state for 18 hours). Mice could eat and drink freely. 200 μL (5 × 10⁻⁶) of Lactobacillus rhamnosus B6 fermented milk solution was administered by gavage daily. 8 / day, to ensure consistent live bacteria count with group ③), for 25 consecutive days.

[0101] ⑤ Lactobacillus rhamnosus B6 heat-inactivated fermented milk group (HB6FM group, n=12): 200 μL (5×10⁻⁶) of heat-inactivated Lactobacillus rhamnosus B6 fermented milk solution was administered by gavage daily. 8 / day)(When administering the heat-inactivated fermented milk group via gavage, the corresponding live bacteria dose is 5×10 8 / day), for 25 days. The preparation of heat-inactivated Lactobacillus rhamnosus B6 fermented milk solution is as follows: 50 minutes before gavage, the B6 fermented milk solution is placed in a 100℃ water bath for 1-2 minutes to heat-inactivate Lactobacillus rhamnosus B6.

[0102] Example 3: Lactobacillus rhamnosus B6 can repair hippocampal neuronal damage and synaptic damage.

[0103] Mice in each group of Example 2 were euthanized by excessive anesthesia with isoflurane. The mouse brain was divided into two hemispheres along the central axis. For RT-qPCR, the hippocampus (approximately 8-16 mg) and prefrontal cortex tissue (approximately 20-80 mg) were extracted from each hemisphere, treated with liquid nitrogen, and then quickly stored in a -80°C freezer.

[0104] For samples used in immunofluorescence, the hemispheres were fixed in 4% paraformaldehyde solution and stored at 4°C in the dark.

[0105] For samples used for Golgi staining, the hemisphere is removed and directly transferred to a storage bottle containing Golgi staining fixative.

[0106] For samples used in tissue transmission electron microscopy (TEM), the hippocampus is extracted and cut into 1mm pieces. 3 After measuring approximately the same size, transfer the sample to an EP tube containing electron microscopy fixative and fix it. Store at 4°C in the dark.

[0107] 3.1. Transmission electron microscopy was used to observe hippocampal samples from the hemisphere.

[0108] ① Post-fixation: The cut hippocampal sample was removed from the electron microscopy fixative and rinsed three times with 0.1M phosphate buffer PB (pH 7.4), 15 min each time. Then, it was fixed in 1% osmium tetroxide prepared with 0.1M phosphate buffer PB (pH 7.4) at room temperature in the dark for 2 h. Afterwards, it was rinsed three times with 0.1M phosphate buffer PB (pH 7.4), 15 min each time.

[0109] ② Room temperature dehydration and permeation embedding: Hippocampal bodies were sequentially dehydrated in 30%-50%-70%-80%-95%-100%-100% ethanol for 20 min each time. Then, they were treated with 100% acetone for 15 min each time. Next, acetone embedding medium 1 (acetone: 812 embedding medium = 1:1) was prepared and infiltrated at 37°C for 4 h. Then, acetone embedding medium 2 (acetone: 812 embedding medium = 1:2) was prepared and infiltrated at 37°C overnight. Afterwards, pure 812 embedding medium was used and infiltrated at 37°C for 5 h. The pure 812 embedding medium was poured into an embedding plate, the sample was inserted into the plate, and embedded overnight at 37°C.

[0110] ③ Polymerization, Positioning, and Ultrathin Sectioning: The embedding plate was placed in a 60℃ oven for polymerization for 48 hours, and the resin block was removed for later use. The resin block was sectioned into 1.5μm sections using a microtome, stained with toluidine blue, and positioned under a light microscope. The resin block was then ultrathinly sectioned into 60nm sections using an ultratome, and then slides were retrieved using a 150-mesh copper screen.

[0111] ④ Staining: Stain copper mesh in 2% uranium acetate saturated alcohol solution for 8 min in the dark; wash 3 times with 70% alcohol; wash 3 times with ultrapure water; stain in 2.6% lead citrate solution for 8 min in the dark; wash 3 times with ultrapure water, and blot dry with filter paper. Place the copper mesh sections in a copper mesh box and dry at room temperature overnight.

[0112] ⑤ Photography: Observe under a transmission electron microscope and acquire images for subsequent analysis. For hippocampal samples, acquire images at 30,000x magnification.

[0113] ⑥ Analysis: The analysis was performed by transmission electron microscopy (TEM) specialists. For hippocampal samples, CaseViewer 2.4 scanning software was used to select the target region of the tissue for imaging. After imaging, Image-Pro Plus 6.0 analysis software was used to calculate the synaptic cleft width, synaptic compaction thickness, synaptic active zone length, and synaptic vesicle number for each image. Results are shown below. Figure 1 f-1h.

[0114] 3.2 Golgi staining of hippocampal samples from the hemisphere.

[0115] ① Staining: For bottles containing Golgi staining solution for the hemisphere, pour out the staining fixative, replace with fresh Golgi staining solution, completely immerse the tissue block, and place in a cool, ventilated place at 26°C, protected from light for 14 days (replace with fresh staining solution after 48 hours of soaking, and then every 3 days thereafter). The Golgi staining solution consists of: 5% potassium dichromate : 5% mercuric chloride = 1:1. The mixture is as follows: Solution A: 10ml DW of 5% potassium dichromate + 0.5g potassium dichromate (dissolved with a magnetic stirrer); Solution B: 10ml DW of 5% mercuric chloride + 0.5g mercuric chloride (dissolved in a glass or with a magnetic stirrer). Solution B is stirred with a magnetic stirrer, and Solution A is added slowly.

[0116] ② Tissue washing treatment: After staining, change the tissue treatment solution and treat for 1 hour, then change the tissue treatment solution again, and treat at 4℃ in the dark for 3 days. The tissue treatment solution consists of a gradient dehydration solution of 50%, 75%, 95%, 100%, 100%, 100%, and 100% ethanol for 4 minutes each.

[0117] ③ Vibratory sectioning: The brain tissue was adhered to the tray of the vibratory microtome using 502 super glue and immersed in tissue processing solution. The sections were 60μm in diameter. After slicing, the sections were attached to the glass slides with the help of a brush. After retrieving the slides, tissue processing solution was dropped onto the surface of the brain tissue sections. After slightly drying, they were ready for development.

[0118] ④ Development: Wash the sections with ultrapure water, add Golgi developer for 30 minutes, and then wash with water. The developer composition is: alkalizing solution: mercuric sulfide: water = 1:1:2.

[0119] ⑤ Mounting: Wipe off excess moisture from the slide, mount with glycerin gelatin, and store in a light-proof slide box at room temperature.

[0120] ⑥ Scanning: Turn on the slide scanner, fluorescence light source, and Pannoramic Scanner software. Place the prepared slide on the scanner slide tray. Click "Start Scanning," and the scan will end after 5 minutes.

[0121] ⑦ Analysis: CaseViewer 2.4 scanning software was used to select the target region of the tissue for 400x and 800x imaging. After imaging, Image-Pro Plus 6.0 analysis software was used to measure the length of all dendrites in each 400x image and calculate the average value; the total number of dendritic spines was measured in each 800x image, and the density was calculated as the number of dendritic spines per 10μm (Spines / 10μm) = number of dendritic spines / dendritic length * 10. The NeuronJ plugin in ImageJ 1.51K analysis software was used to plot the central neuron cell body structure of the 400x image. Using the Sholl analysis plugin, 10 concentric circles with a spacing of 10μm were drawn around the cell body, and the number of intersections between the dendrites and each concentric circle was counted. See... Figure 1 a-1e diagram.

[0122] 3.3 Results

[0123] The hippocampus, located in the medial temporal lobe of the brain, is found in one of each cerebral hemisphere and is closely related to emotion regulation, learning, and memory formation. Neurons in the hippocampus receive and integrate information through dendrites, participating in memory formation; the complexity and dynamic changes of dendritic branches directly affect the functional efficiency of the hippocampus. Together, they complete crucial steps in information processing and memory storage within one cerebral hemisphere. Dendrites receive input signals from multiple neurons through their branches and dendritic spines.

[0124] Dendritic spines are branches of neurons, and their number can reflect the complexity and connectivity density of neurons. A higher number of dendritic spines per 10 μm suggests that neurons in the experimental group may have more branches, which may be associated with better neural plasticity, stronger neural network connectivity, or higher neural activity. Dendritic length is the extension length of neuronal branches, reflecting the projection range of the neuron and the area covered by potential neural connections. An increase in dendritic length may indicate that neurons in the experimental group have longer branches, which may be associated with enhanced neurotransmission capacity and wider neural network connectivity.

[0125] from Figure 1As shown in a-1c, with the increase of distance from the cell body, the number of dendritic branch intersections generally showed a trend of first increasing and then decreasing. However, the number of intersections in the B6 bacterial powder group (B6P) and the B6 fermented milk group (B6FM) was higher than that in the chronic fatigue group (SD). The overall trend of the B6 bacterial powder group (B6P) and the B6 fermented milk group (B6FM) was similar to that of the control group (Con) and was significantly higher than that in the chronic fatigue group (SD).

[0126] from Figure 1 The results from f-1h showed that the hippocampus of the chronic fatigue group (SD) mice had increased synaptic clet width, thinned synaptic density (PSD) thickness, shortened active zone length, and decreased number of synaptic vesicles, all of which were statistically significant compared to the CON group (P<0.05). Compared with the chronic fatigue group (SD), the B6 bacterial powder group (B6P) and the B6 fermented milk group (B6FM) showed decreased synaptic cleft width (B6P; B6FM: P<0.05), increased synaptic density thickness (B6P; B6FM: P<0.05), prolonged synaptic active area length (B6P; B6FM: P<0.05), and increased number of synaptic vesicles (B6P; B6FM: P<0.05) in the hippocampus of mice, and all of these differences were statistically significant compared with the chronic fatigue group (SD).

[0127] from Figure 1 As shown in Figure d-1e, in the Golgi staining experiment, the number of dendritic spines per 10 μm and the dendritic length of the Golgi apparatus in the chronic fatigue group (SD) mice were reduced, and both were statistically significant compared to the CON group (P<0.05). The number of dendritic spines per 10 μm in the B6 bacterial powder group (B6P) was significantly higher than that in the chronic fatigue group (SD) (p<0.01), and the dendritic length was also significantly higher in the B6 fermented milk group (B6FM) than in the chronic fatigue group (SD) (p<0.01). Figure 1 There were no significant differences between the B6 bacterial powder group (B6P) and the B6 fermented milk group (B6FM). However, the B6 heat-inactivated fermented milk group (HB6FM) showed no significant differences from the chronic fatigue group (SD) in the number of dendritic spines per 10 μm and dendritic length (p > 0.05).

[0128] The above results indicate that both B6 bacterial powder and B6 fermented milk can reduce the width of the synaptic cleft in hippocampal neurons, increase the thickness of the synaptic compact area, increase the length of the synaptic active area, and increase the number of synaptic vesicles, thereby significantly improving synaptic damage caused by chronic fatigue. In addition, they can also increase the number of dendritic spines, dendrite length, and number of intersections, thereby promoting synaptic plasticity and significantly improving nerve damage caused by chronic fatigue, as well as neuroinflammation caused by subsequent nerve damage.

[0129] Example 4: Lactobacillus rhamnosus B6 inhibits microglia activation

[0130] 4.1 Immunofluorescence

[0131] 4.1.1 Paraffin Sectioning and Sealing

[0132] Hemibrain sample paraffin embedding: The hemibrain sample was removed from the paraformaldehyde solution and placed in a dehydration box. The dehydration box was then placed in a dehydrator for sequential dehydration with alcohols in the following order: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, anhydrous ethanol for 30 minutes, benzene for 10 minutes, and xylene for 10 minutes. The paraffin was melted at 65°C. The melted wax was first placed into the embedding frame. Before the wax solidified, the hemibrain was removed from the dehydration box and placed into the embedding frame according to the required embedding surface, and the corresponding label was affixed. The sample was cooled on a -20°C freezing stage. After the wax solidified, the wax block was removed from the embedding frame and trimmed.

[0133] Paraffin sectioning: Place the trimmed paraffin block on a paraffin microtome and section it. Set the section thickness to 4μm (for conventional scanning) or 10μm (for laser confocal scanning). Float the section on 40℃ warm water in a slide spreader to flatten the tissue. Remove the tissue from the slide and bake it in a 60℃ oven. After the paraffin has melted in the water, remove the slide and store it at room temperature for later use.

[0134] Circle-based serum blocking: Draw a circle around the tissue with a histochemical pen, and add BSA (bull serum albumin; if the primary antibody is from goat, use 10% donkey serum for blocking; if the primary antibody is from rabbit, mouse, or rat, use 3% BSA for blocking) inside the circle, and block for 30 minutes.

[0135] 4.1.2 Immunofluorescence double labeling of the hippocampus

[0136] Iba-1 and CD68, C1q and PSD-95, and Iba-1 and SYP were co-localized in the hippocampus.

[0137] The specific steps are as follows:

[0138] ① Add the first primary antibody: Add the prepared primary antibody (dilution ratio: Iba-1 antibody, 1:1000; PSD-95 antibody, 1:500) to the slide, place the slide flat in a light-proof humidified box, and incubate overnight at 4°C.

[0139] ② Add the corresponding species-labeled secondary antibody: Place the slide in PBS solution and wash it three times on a pendulum shaker for 5 minutes each time. After slightly drying the slide, place it flat in a light-proof humidified box and add HRP-labeled secondary antibody corresponding to the species of the primary antibody to cover the tissue. Incubate at room temperature for 50 minutes.

[0140] ③ Add the corresponding tyramide derivative: Place the slide in PBS solution and wash three times on a pendulum shaker for 5 minutes each time. After slightly drying the slide, place it flat in a light-protected humidified chamber and add the corresponding tyramide derivative drop into the circle. Incubate at room temperature for 10 minutes in the dark. After incubation, place the slide in TBST (Tris buffered saline tween) and wash three times on a pendulum shaker for 5 minutes each time.

[0141] ④ Microwave treatment: Place the tissue sections in a repair box containing citric acid repair solution and heat in a microwave oven, maintaining boiling for about 10 minutes to remove the primary and secondary antibodies that have been bound to the tissue.

[0142] ⑤ Serum blocking: Place the slide flat in a light-proof, humidified chamber and add serum for blocking at room temperature for 30 minutes. If the primary antibody is of goat origin, use 10% rabbit serum for blocking; if the primary antibody is of other origin, use 3% BSA for blocking.

[0143] ⑥ Add the second primary antibody: Add the second primary antibody prepared in a certain ratio (dilution ratio: CD68 antibody, 1:1000; C1q antibody, 1:2000; PSD-95 antibody, 1:1000) to the slide, place the slide flat in a light-proof humidified box, and incubate overnight at 4°C.

[0144] ⑦ Add the corresponding species secondary antibody: Place the slide in PBS solution and wash it three times on a pendulum shaker for 5 minutes each time. After slightly drying the slide, add the secondary antibody corresponding to the species of the second and primary antibodies in the circle and incubate at room temperature for 50 minutes.

[0145] ⑧ Counterstaining cell nuclei with DAPI: Place the slide in PBS solution and wash three times on a pendulum shaker for 5 minutes each time. After slightly drying the slide, place it flat in a light-protected humidified box, add DAPI staining solution to the circle, and incubate at room temperature for 10 minutes in the dark.

[0146] ⑨ Quenching tissue autofluorescence: Place the slide in PBS (pH 7.4) and wash three times on a decolorizing shaker, 5 min each time. Add autofluorescence quencher solution B for 5 min, then rinse with running water for 10 min.

[0147] ⑩ Mounting: Mount with anti-fluorescence quenching mounting medium.

[0148] Scanning: Open the slide scanner, fluorescence light source, and Pannoramic Scanner software. Place the prepared immunofluorescence slides on the scanner slide tray. Click "Start Scan," and the scan will end after 5 minutes.

[0149] For laser confocal imaging: Turn on the computer, then the laser stage power switch, turn the laser stage key 90° to the right, then the microscope, stage, mercury lamp, and C2 imaging system. Start the "NIS-Elements" software and fix the prepared immunofluorescence slides in the slide clips. The scanned images were then quantitatively analyzed using ImageJ. Results are shown below. Figure 2 g and 2k.

[0150] 4.1.3 Immunofluorescence labeling of the hippocampus

[0151] Three-way immunofluorescence labeling: labeling the co-localization expression of Iba-1, iNOS, and CD206 in the hippocampus. The method was the same as step 4.1.2, and the results are shown below. Figure 2 g.

[0152] 4.1.4 Immunoblotting in the hippocampus

[0153] Expression of CD68, SYP, PSD-95, Iba-1, C1qa and GAPDH in the hippocampus.

[0154] ①Total protein extraction from tissues: Wash hippocampal or prefrontal cortex tissue blocks 2-3 times with pre-cooled PBS to remove blood contamination, cut into small pieces and place in homogenization tubes, add 2 4mm... 2 Homogenized beads were mixed with RIPA lysis buffer containing 10% protease inhibitor (hippocampus: 80 μL; prefrontal cortex: 300 μL), and homogenized using a homogenization program (60 Hz, 30 sec, 2 cycles, with a 15 sec pause between cycles). The mixture was then placed on ice for 20 min of lysis, with vortexing repeated every 5 min. Finally, the mixture was centrifuged at 12000 rpm at 4 °C for 10 min, and the supernatant was collected to obtain the total protein solution.

[0155] ② Nucleoprotein extraction: Nucleoprotein extraction was performed according to the instructions. The specific steps are as follows: The hippocampal or prefrontal cortex tissue block was washed 2-3 times with pre-cooled PBS to remove blood contamination, cut into small pieces, placed in a homogenization tube, and two 4mm particles were added. 2Add reagent A (hippocampus: 100 μL; prefrontal cortex: 400 μL) containing 10% protease inhibitor to the homogenized beads and set the homogenization program (60 Hz, homogenize for 30 sec, cycle twice, pause for 15 sec in between) for homogenization and lysis. Then place on ice and continue lysis for 15 min, vortexing every 5 min to ensure complete tissue lysis. Add reagent B (hippocampus: 5 μL; prefrontal cortex: 20 μL), vortex at high speed for 5 sec, and incubate on ice for 1 min. Then centrifuge at 12000 rpm, 4°C for 10 min, discard the supernatant, retain the precipitate, add reagent A (hippocampus: 100 μL; prefrontal cortex: 400 μL) to the precipitate again, vortex at high speed for 5 sec. Then centrifuge at 13000 rpm, 4°C for 5 min, discard the supernatant, and retain the precipitate. Add reagent C containing 10% protease inhibitor (hippocampus: 50 μL; prefrontal cortex: 200 μL) to the precipitate, vortex at high speed for 15 sec, place on ice for 40 min, and repeat vortexing once every 10 min. Then centrifuge at 13000 rpm, 4 °C for 10 min, collect the supernatant to obtain the nuclear protein solution.

[0156] ③ BCA method for determining protein concentration and denaturation: A standard curve was prepared, and the samples were diluted 5 times before concentration measurement. After calculation, the concentration of each group of protein samples was adjusted to be the same using phosphate buffer. Then, the protein solution was added to 5× reducing protein loading buffer at a ratio of 4 μL: 1 μL, and denatured in a boiling water bath for 15 min, ready for electrophoresis.

[0157] ④ Gel preparation, sample loading, and electrophoresis: Follow the instructions for gel preparation. The specific steps are as follows: Take 4 mL each of the lower gel solution and lower gel buffer, and add 20 μL of modified coagulant, then mix well. Pour the mixed solution into the gel casting plate, and gently tap the gel casting apparatus to level the liquid surface with the table. Next, take 0.5 mL each of the upper gel solution and upper gel buffer, and add 5 μL of modified coagulant, then mix well. Pour the mixed solution into the gel casting plate until slightly overflowing, insert the comb, and after 15 minutes, obtain the gel ready for electrophoresis. Add 4 μL of the modified protein to each well, and electrophores at 150V constant current mode for 45 minutes.

[0158] ⑤ Transfer and blocking: The proteins from electrophoresis were then transferred to a PVDF membrane and electrophoresed at 300mA in constant voltage mode for 50 min. The PVDF membrane was then removed, placed in an incubation chamber, and 5 mL of 3% BSA blocking buffer was added for 1 h. Then, 5 mL of TBST was added and incubated for 5 min. This process was repeated three times.

[0159] ⑥ Primary antibody incubation: Incubate with primary antibodies (dilution ratios: SYP: 1:5000; PSD-95: 1:3000; Iba-1: 1:2000, CD68: 1:2000, C1qa:GAPDH: 1:5000) overnight at 4°C on a shaker. Afterward, remove the membrane, add 5 mL of TBST, and incubate for 5 min. Repeat 3 times.

[0160] ⑦ Secondary antibody incubation: Incubate with the secondary antibody at room temperature for 1 hour. Then remove the membrane, add 5 mL of TBST, and incubate for 5 minutes. Repeat 3 times.

[0161] ⑧ Development and Analysis: Chemiluminescence solution was added, and the images were exposed and imaged in a chemiluminescence analyzer. The results were saved. The results were analyzed using Image-Pro Plus 6.0 software, and the target protein was compared using GAPDH expression data. See [link to results]. Figure 2 b and 2c.

[0162] 4.2 Real-time quantitative PCR

[0163] (1) Extraction of total RNA

[0164] The total RNA extraction process was performed in accordance with the instruction manual. The specific experimental steps are as follows:

[0165] ① Tissue homogenization: Take out a 1.5 mL centrifuge tube containing hippocampal tissue from a -80℃ freezer, add one zirconia grinding bead to each tube, then add 0.5 mL Transzol Up and 0.1 mL RNA Extraction Agent to each tube, place the centrifuge tube into an automated grinder, grind thoroughly, and then vortex at room temperature for 5 min to fully lyse the tissue;

[0166] ② Centrifuge at 4℃, 10000xg, for 15 min. Transfer approximately 400 μL of the upper aqueous phase solution to a new RNA-free tube, add 400 μL of ethanol, and invert 5 times to mix.

[0167] ③ Transfer the solution into a centrifuge column twice; centrifuge at 4°C, 12000 x g, for 30 seconds. Discard the eluent.

[0168] ④ Add 500 μL of CB9, centrifuge at 4°C, 12000 x g for 30 seconds. Discard the eluent. Repeat this step twice.

[0169] ⑤ Add 500 μL of WB9, centrifuge at 4°C, 12000 x g for 30 seconds. Discard the eluent. Repeat this step twice.

[0170] ⑥ Centrifuge at 4℃, 12000xg, for 2 minutes. Discard the effluent.

[0171] ⑦ Discard the effluent tube at the bottom of the centrifuge column and replace it with a new RNA-free tube. Add 50 μL of RNA-free water to the center of the centrifuge column and let it stand at room temperature for 2 minutes.

[0172] ⑧ Centrifuge at room temperature, 12000xg, for 2 min. Repeat this step twice. Obtain the RNA solution for subsequent experiments.

[0173] ⑨ Measure the total RNA concentration: Use a photometer to measure the concentration of RNA solution in each tube. Dilute samples with high concentrations with RNA-free water to control the total RNA concentration in each tube between 250 and 300 ng / μL.

[0174] (2) First-strand cDNA synthesis

[0175] The reverse transcription process for cDNA synthesis should be performed according to the manufacturer's instructions. The specific reaction system and conditions are as follows:

[0176] ① Reaction system: Prepare the reaction system using 100μL sterile tubes. Add 2μL of the total RNA solution obtained in the above steps to each tube, and 4μL of... All-in-One Super Mix for qPCR: Add 1 μL of gDNA remover and bring the total volume to 20 μL with RNA-free water. After preparation, briefly centrifuge to ensure no liquid adheres to the sidewalls.

[0177] ②Reaction conditions: Incubation, 45℃, 15min; 85℃, 5sec.

[0178] (3) Primer design

[0179] Table 1. Primer Sequence List for RT-qPCR

[0180]

[0181] (4) Amplification reaction

[0182] For each sample, the RT-qPCR experiment was performed in triplicate. The reaction procedure was followed according to the manufacturer's instructions. The specific reaction system and conditions are as follows:

[0183] ① Reaction system: Prepare the reaction system using 100μL sterile eight-tube sets. Add 1μL of the cDNA solution obtained in the above steps, 0.4μL of forward primer (0.1μM), 0.4μL of reverse primer (0.1μM), and 10μL of... to each tube. Add Green qPCR SuperMix and bring the total volume to 20 μL with RNA-free water. After preparing the mixture, briefly centrifuge to ensure no liquid adheres to the sidewalls.

[0184] ②Reaction conditions:

[0185]

[0186] 4.3 Results

[0187] 4.3.1 Effects on the transcriptional levels of genes related to phagocytic synapses in the hippocampus and prefrontal cortex

[0188] from Figure 2 As shown in a-2c, in the chronic fatigue group (SD), the transcriptional levels of SYP and PSD-95, genes related to microglia phagocytic synapses, were decreased in the hippocampus, while the transcriptional levels of C1qa and CD68 were increased (3a). Furthermore, in the prefrontal cortex, the transcriptional levels of SYP and PSD-95, genes related to microglia phagocytic synapses, were decreased, while the transcriptional levels of C1qa, CD68, and Iba-1 were increased (3a). Figure 2 b and 2c) indicate that the phagocytic activity of microglia in the malachite body and prefrontal cortex is weakened. Gavage administration of B6 can improve this situation, increasing the transcriptional levels of SYP and PSD-95 while decreasing the transcriptional levels of C1qa, CD68, and Iba-1. Compared with the chronic fatigue group (SD), the B6 bacterial powder group (B6P) significantly increased the transcriptional levels of Syp and PSD-95 (p < 0.01), the B6 fermented milk group (B6FM) significantly increased the transcriptional levels of Syp and PSD-95 (p < 0.05), and the B6 heat-inactivated fermented milk group (HB6FM) showed no significant difference.

[0189] 4.3.2 Effects on total microglia in the hippocampus

[0190] Microglia are immune cells in the central nervous system. They regulate the density and function of neural connections by phagocytizing synapses, a process that is crucial for neural development and synaptic pruning.

[0191] from Figure 2As shown in d-2f, in the chronic fatigue group (SD), the total number of microglia in the hippocampus increased (2d) and the proportion of total microglia in the M1 microglia station increased (2e), while the proportion of total microglia in the M2 microglia station decreased (2f), indicating that chronic fatigue leads to damage to the hippocampal structure. Oral administration of B6 improved this situation, reducing the total number of microglia and the proportion of total microglia in the M1 microglia station, while increasing the proportion of total microglia in the M2 microglia station. Compared with the chronic fatigue group (SD), the B6 microbial powder group (B6P) significantly reduced the total number of microglia and the proportion of total microglia in the M1 microglia station, and significantly increased the proportion of total microglia in the M2 microglia station; the B6 fermented milk group (B6FM) significantly reduced the total number of microglia and the proportion of total microglia in the M1 microglia station; while the B6 heat-inactivated fermented milk group (HB6FM) showed no significant difference.

[0192] 4.3.2 Effects on the three-color and two-color fluorescent labels in the hippocampus

[0193] from Figure 2 g indicates that strain B6 and the fermented milk group can reduce the expression of Iba-1 and iNOS, and increase the expression of the M2 polarization marker CD86.

[0194] 4.3.3 Number of Iba-1+ / CD68+ cells per square millimeter in the hippocampus

[0195] Iba-1 is a marker associated with microglial activation, and CD68 is typically associated with anti-inflammatory responses. (See results below.) Figure 2 h and 2k.

[0196] from Figure 2 h and 2k showed that in the chronic fatigue group (SD), the number of Iba-1+ / CD68+ cells per square millimeter of the hippocampus was significantly increased, indicating that chronic fatigue leads to neuroinflammation. Gavage administration of B6 improved the increase in the number of Iba-1+ / CD68+ cells per square millimeter; compared with the chronic fatigue group (SD), the B6 bacterial powder group (B6P) and the B6 fermented milk group (B6FM) showed significant decreases, while the B6 heat-inactivated fermented milk group (HB6FM) showed no significant difference.

[0197] 4.3.4. Area of ​​C1q positivity in the hippocampus

[0198] The detection method for increased C1q positive area and number of SYP spots in mouse hippocampus: Immunofluorescence assay, as above.

[0199] The core identity of C1q is that it is the initiating component of the classical complement pathway, possessing dual attributes of "immune regulation" and "neurological function regulation." Its function is not simply "protection" or "damage," but rather exhibits a bidirectional nature depending on the pathological stage and changes in the microenvironment. The role of C1q in Alzheimer's disease (AD) in the mouse hippocampus is closely related to Aβ pathology. In the early stages of nerve damage, local neuronal stress and glial cell (microglia and astrocytes) activation occur in the hippocampus, at which time C1q expression is significantly upregulated (mainly secreted by activated microglia and astrocytes).

[0200] Figure 2 As shown in Figure i, the C1q-positive area in the hippocampus of mice in the chronic fatigue group (SD) was significantly increased compared to the control group (CON) (P < 0.01), indicating that chronic fatigue leads to increased microglial cell activation, prompting microglia to release inflammatory factors. Gavage administration of B6 improved the increase in C1q-positive area. Compared to the chronic fatigue group (SD), the C1q-positive area in the hippocampus of mice in the B6 bacterial powder group (B6P) was significantly decreased (P < 0.05), and the C1q-positive area in the B6 fermented milk group (B6FM) was significantly decreased (P < 0.01), while there was no significant difference in the B6 heat-inactivated fermented milk group (HB6FM). The difference between the B6 fermented milk group (B6FM) and the chronic fatigue group (SD) was statistically significant (P < 0.05).

[0201] 4.3.5, Number of SYP points in the hippocampus / number of microglia

[0202] Method for detecting the number of SYP spots in the mouse hippocampus: Immunofluorescence assay, as above.

[0203] from Figure 2 As can be seen, in the chronic fatigue group (SD), the number of SYP puncta / microglia was significantly increased, indicating that chronic fatigue leads to increased microglia activation, prompting microglia to release inflammatory factors. Oral administration of B6 improved the increase in the number of SYP puncta / microglia. Compared with the SD group, the B6 probiotic powder group (B6P) showed a significant decrease, the B6 fermented milk group (B6FM) showed a highly significant decrease, and the B6 heat-inactivated fermented milk group (HB6FM) showed no significant difference.

[0204] 4.3.6 Effects on SYP spots in the hippocampus

[0205] Synaptophysin (SYP) plays a crucial role in nerve damage and Alzheimer's disease (AD). It is a marker protein highly enriched on synaptic vesicle membranes, its core function being the formation, transport, and neurotransmitter release of synaptic vesicles. It is considered one of the "gold standards" for reflecting synaptic number, density, and functional integrity. Unlike the "active regulation" of C1q, SYP's role is more that of an "indicator" of synaptic functional state, and changes in its expression can conversely affect synaptic function, thus participating in the pathological process of the disease. See [link to relevant documentation]. Figure 2 l, where 2D is a two-dimensional graph and 3D is a three-dimensional graph.

[0206] from Figure 2 It was found that the hippocampal microglia of mice in the chronic fatigue group (SD) had more SYP spots, significantly higher than those in the control group (CON) (P < 0.05). Compared with the chronic fatigue group (SD), the number of SYP spots in the hippocampus of mice in the B6 bacterial powder group (B6P) (P < 0.05) and the B6 fermented milk group (B6FM) (P < 0.05) was significantly reduced. SYP spots in microglia represent the number of synapses phagocytosed by microglia. Microglia in the chronic fatigue group (SD) were activated to phagocytose and digest SYP. After intervention in the B6 bacterial powder group (B6P) and the B6 fermented milk group (B6FM), the phagocytosis of SYP by microglia was reduced, combined with... Figure 2 The results showed that the total SYP in the chronic fatigue group (SD) decreased, and the B6 group recovered.

[0207] In summary, this embodiment shows that chronic fatigue leads to abnormal activation of microglia, such as an increase in the number of Iba-1+ / CD68+ cells and the release of inflammatory mediators (such as increased transcriptional levels of C1qa, CD68, and Iba-1), which further damages synaptic structure and function (such as decreased transcriptional levels of SYP and PSD-95; and an increase in the number of SYP spots). In contrast, strain B6 can significantly inhibit the activation of microglia and regulate the expression of synapse-related genes, thereby treating neuroinflammation.

[0208] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. Use of Lacticaseibacillus rhamnosus BD4047 in the preparation of at least one of the following: 1) a product for treating synaptic impairment caused by chronic fatigue; 2) a product for treating neuronal impairment caused by chronic fatigue; 3) a product for inhibiting microglial activation caused by chronic fatigue.

2. Use according to claim 1, characterized in that, The Lacticaseibacillus rhamnosus BD4047 has a preservation number of CGMCC No. 13310.

3. Use according to claim 1, characterized in that, The product for treating synaptic impairment caused by chronic fatigue has any one or more of A1) to A5) functions: A1) reducing synaptic cleft width in the hippocampus; A2) increasing synaptic density in the hippocampus; A3) increasing synaptic active zone length in the hippocampus; A4) increasing synaptic vesicle number in the hippocampus; A5) increasing SYP puncta number in the hippocampus.

4. The use according to claim 1, characterized in that, The product for treating neuronal impairment caused by chronic fatigue has any one or more of B1) to B3) functions: B1) increasing dendritic spine number in the hippocampus; B2) increasing dendritic spine length in the hippocampus; B3) increasing dendritic branch point number in the hippocampus.

5. The use according to claim 1, characterized in that, The product for inhibiting microglial activation caused by chronic fatigue has any one or more of C1) to C3) functions: C1) regulating expression of genes related to microglial phagocytosis of synapses in the hippocampus; C2) reducing the number of microglia in the hippocampus; C3) reducing the proportion of M1 microglia among total microglia and / or increasing the proportion of M2 microglia among total microglia in the hippocampus.

6. Use according to claim 5, characterized in that, The regulating expression of genes related to microglial phagocytosis of synapses in the hippocampus refers to increasing the expression level of SYP and PSD-95, and / or, reducing the expression level of C1qa, CD68 or Iba-1.

7. The use according to claim 2, characterized in that, The product for treating synaptic impairment caused by chronic fatigue, and / or the product for treating neuronal impairment caused by chronic fatigue, and / or the product for inhibiting microglial activation caused by chronic fatigue is for treating a disease related to hippocampal impairment, which is selected from one or more of chronic fatigue syndrome, cognitive impairment, sleep disorder, Alzheimer's disease, vascular dementia, depression, anxiety, post-traumatic stress disorder and sleep disorder.

8. Use according to claim 7, characterized in that, The product for treating synaptic impairment caused by chronic fatigue, and / or the product for treating neuronal impairment caused by chronic fatigue, and / or the product for inhibiting microglial activation caused by chronic fatigue is one or more of a fermented milk product containing Lacticaseibacillus rhamnosus BD4047, a lactic acid bacteria beverage product, a bacterial powder product, a live bacteria water product, a milk powder product and a cheese product.

9. Use according to claim 8, characterized in that, The preparation method of the fermented milk product containing Lacticaseibacillus rhamnosus BD4047 is: obtaining by fermentation of Lacticaseibacillus rhamnosus BD4047 raw milk; And / or, the bacterial powder product is a freeze-dried powder obtained by drying the culture of Lacticaseibacillus rhamnosus BD4047.

10. The use according to claim 1, characterized in that, The number of viable cells of Lactobacillus rhamnosus BD4047 in the product for treating synapse damage caused by chronic fatigue, and / or the product for treating neuron damage caused by chronic fatigue, and / or the product for inhibiting microglial cell activation caused by chronic fatigue is not less than 1 x 10 6 CFU / mL or 1 x 10 6 CFU / g.

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