Lactobacillus plantarum wh021 and its use in preventing and / or alleviating neuroinflammation

Abstract

The application discloses lactobacillus plantarum WH021 and application thereof in prevention and / or alleviation of neuroinflammation, and belongs to the technical field of microorganisms. The lactobacillus plantarum WH021 is obtained by separation and purification from healthy infant feces, is a probiotic with the effect of alleviating neuroinflammation, and has good application potential in development of food, health products and drugs for preventing brain inflammation related diseases of middle-aged and old people caused by chronic inflammation of the body.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Lactobacillus plantarum WH021 and its application in the prevention and / or relief of neuroinflammation. Background Technology

[0002] Aging is characterized by a chronic, low-grade, and systemic inflammatory state, closely related to decreased oxidative stress adaptation due to physiological functional decline and metabolic dysfunction. Crucially, this process promotes inflammatory responses within the central nervous system (CNS), leading to the occurrence and development of neuroinflammation, which has been widely reported as a core mechanism driving the pathological processes of various neurodegenerative diseases (NDs). In clinical practice, drug therapy is the primary method for controlling the disease, but long-term medication use can lead to side effects such as constipation, metabolic dysfunction, and even nerve damage. Therefore, developing safer and more effective new therapies targeting this core pathological link of neuroinflammation has become an urgent challenge, with significant implications for improving the quality of life for the elderly. With the rapid development of high-throughput sequencing technology and omics analysis methods, research on the correlation between the nervous system and gut microbiota has gradually come into the public eye. In addition to directly influencing the balance of the gut microbiota, probiotics also exert their indirect regulatory function on the pathological processes of neuroinflammation through the "microbe-gut-brain" axis, showing potential for preventing and alleviating NDs. Although the gut and brain are anatomically independent, several mechanisms have been proposed to explain the communication between gut microbiota and the CNS. This process begins with building a healthy gut environment by restoring the balance of the gut microbiota, maintaining the integrity of the intestinal barrier, and promoting a normal response of the intestinal mucosal immune system. Subsequently, this healthy gut environment, through the action of probiotics and the neuroactive substances, metabolites, and hormones produced by the microbiota, further influences the vagus nerve, enteric nervous system (ENS), neuroendocrine system, immune system, and circulatory system, thereby regulating the central nervous system. Therefore, providing a probiotic with the ability to alleviate neuroinflammation has significant application potential in the development of foods, health products, and pharmaceuticals for the prevention of brain inflammatory diseases caused by chronic inflammation in middle-aged and elderly individuals. Summary of the Invention

[0003] In order to overcome the problems existing in the prior art, one of the objectives of the present invention is to provide a strain of *Lactobacillus plantarum* WH021 that has the effect of preventing neuroinflammation.

[0004] The second objective of this invention is to provide applications of the aforementioned *Lactobacillus plantarum* WH021.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] A strain of *Lactiplantibacillus plantarum* with anti-neuritis properties, named *Lactiplantibacillus plantarum* WH021, was deposited on April 23, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCCNo: 63388.

[0007] A microbial preparation containing the aforementioned *Lactobacillus plantarum* or its culture.

[0008] Furthermore, the microbial preparation is a bacterial powder or probiotic capsule.

[0009] Furthermore, the culture medium used for the culture includes MRS solid medium and MRS liquid medium.

[0010] The above-mentioned *Lactobacillus plantarum* or biological agents are used in the preparation of products having at least one of the following functions:

[0011] (1) Prevention and / or relief of comorbid anxiety and depression, depression or anxiety disorder;

[0012] (2) It helps improve memory function;

[0013] (3) Prevention and / or relief of neurodegenerative diseases;

[0014] (4) To prevent and / or alleviate neuroinflammation;

[0015] (5) It helps improve tissue structure damage caused by nerve inflammation.

[0016] Furthermore, the neurodegenerative diseases mentioned include at least one of Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease, and multiple sclerosis.

[0017] Furthermore, the prevention and / or relief of neuroinflammation includes at least one of reducing pro-inflammatory factor levels and increasing anti-inflammatory factor levels.

[0018] Furthermore, the aforementioned structural damage includes at least one of the following: brain tissue oxidative stress damage, hypothalamic-pituitary-adrenal (HPA) axis dysfunction, brain pathological damage, blood-brain barrier damage, intestinal pathological damage, intestinal barrier damage, abnormal colonic flora structure in inflammatory states, and serum metabolic abnormalities.

[0019] Furthermore, the product exerts its effects by regulating the arachidonic acid metabolic pathway, the 5-hydroxytryptamine synaptic pathway, the GABAergic synapse, the ferroptosis pathway, and the FoxO signaling pathway.

[0020] Furthermore, the products include, but are not limited to, food, health products, pharmaceuticals, and preparations that can be added to the products in the form of raw materials.

[0021] Furthermore, the dosage form of the product is an oral preparation or an injectable preparation.

[0022] The present invention has the following advantages and effects compared with the prior art:

[0023] This invention isolates and purifies *Lactobacillus plantarum* WH021 from the feces of healthy infants. This strain has strong gastrointestinal tolerance. Antibiotic resistance and hemolytic tests, combined with whole-genome sequencing analysis, have confirmed that strain WH021 is a safe probiotic.

[0024] Three different doses of live bacteria were used to preventively intervene in C57BL / 6J mice. Subsequently, lipopolysaccharide (LPS) was injected intraperitoneally to model the disease. Behavioral, peripheral and cerebral inflammation levels, oxidative stress in the brain, and pathological damage indicators were analyzed to evaluate its effect on improving brain inflammation. Results showed that the high-dose group (10... 9After 28 days of intervention with CFU / day, this bacterium significantly improved the depressive-like state induced by neuroinflammation in mice, reduced the levels of pro-inflammatory cytokine IL-1β and CORT hormone in peripheral blood, and enhanced the secretion of anti-inflammatory cytokine IL-10. Furthermore, the transcriptional levels of brain-derived neurotrophic factor (BDNF) and postsynaptic dense protein-95 (PSD-95) genes were significantly enhanced, improving the integrity of the blood-brain barrier, thereby reducing the level of malondialdehyde (MDA), a marker of oxidative damage in the brain, and increasing the activity of antioxidant enzymes such as glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Pathological staining results showed that intervention with this strain significantly reduced the degree of neuronal damage and downregulated the activation state of microglia, thus effectively alleviating brain inflammation in model mice. By reducing the transcriptional levels of colonic inflammatory factors IL-1β and TNF-α mRNA, and restoring the transcriptional levels and histopathological damage of intestinal tight junction proteins Occludin, ZO-1, and Claudin-1 mRNA, this bacterium effectively protects the intestinal barrier in LPS-induced neuroinflammation model mice. 16S rRNA amplicon sequencing and serum untargeted metabolomics analysis revealed that prophylactic intervention with this bacterium can restore the imbalanced gut microbiota structure in neuroinflammation model mice. Furthermore, by altering the serum metabolic profile, it significantly increases the synthesis of neuromodulators such as phosphatidylserine (PS) (14:0 / 22:0) and L-glutamate, as well as anti-inflammatory substances such as the prophagocytic peptide tuftsin, pinolenic acid, and 5,6-dehydroarachidonic acid, and promotes the metabolic levels of neurotransmitter metabolites such as 5-hydroxyindole-3-acetic acid in serum. KEGG pathway enrichment analysis results showed that this bacterium mainly exerts its effect of alleviating neuroinflammation through the serotonergic synapse, GABAergic synapse, arachidonic acid metabolism, ferroptosis, and FoxO signaling pathway.

[0025] The *Lactobacillus plantarum* WH021 provided by this invention is a probiotic that can alleviate neuroinflammation and has good application potential in the development of foods, health products, and medicines for the prevention of brain inflammation-related diseases caused by chronic inflammation in the body in middle-aged and elderly people. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the animal experiment process.

[0027] Figure 2 This is a colony diagram of strain WH021 growing on a blood agar plate.

[0028] Figure 3 The images show the colony (A) and cell (B) morphology of strain WH021.

[0029] Figure 4 The image shows the 16S rDNA electrophoresis pattern (A) and phylogenetic tree (B) of strain WH021.

[0030] Figure 5 The graph shows the curve of mouse weight change (A) and the difference (B).

[0031] Figure 6 The graph shows the mouse's movement trajectory (A), total distance (B), and the ratio of movement distance in the central area to the total distance (C) in the open field experiment.

[0032] Figure 7 This is a graph showing the immobility time of mice during the tail suspension experiment.

[0033] Figure 8 The figure shows the effect of strain WH021 on the activities of malondialdehyde (A), glutathione peroxidase (B), and superoxide dismutase (C) in the brain tissue of LPS-induced neuroinflammation mice.

[0034] Figure 9 The figure shows the effect of strain WH021 on the expression of IL-1β (A / B), IL-10 (C / D) and CORT (E) in the brain tissue and serum of mice with LPS-induced neuroinflammation.

[0035] Figure 10 Image of brain tissue stained with H&E.

[0036] Figure 11 Image of Nissl staining of brain tissue (scale bar = 40 μm).

[0037] Figure 12 Immunofluorescence staining of IBA-1 and GFAP in brain tissue (green IBA-1; red GFAP; blue DAPI; scale bar = 40 μm)

[0038] Figure 13 The figure shows the effect of strain WH021 on the expression levels of BDNF (A) and PSD-95 (B) mRNA in the brain tissue of mice with LPS-induced neuroinflammation.

[0039] Figure 14 The figure shows the effect of Lactobacillus plantarum WH021 on the expression levels of Claudin-5 (A) and ZO-1 (B) mRNA in the brain tissue of mice with LPS-induced neuroinflammation.

[0040] Figure 15 Image showing H&E and AB-PAS staining of colon tissue (scale bar = 200 μm, *p<0.05, ****p<0.0001).

[0041] Figure 16 Figure showing the mRNA expression levels of colonic tight junction proteins Occludin (A), ZO-1 (B), and Claudin-1 (C) (ns p>0.05, * p<0.05).

[0042] Figure 17 Figure showing the mRNA expression levels of inflammatory factors IL-1β (A) and TNF-α (B) (nsp>0.05, *p<0.05, **p<0.01).

[0043] Figure 18 Figure 1 shows the effect of Lactobacillus plantarum WH021 on the β-diversity of gut microbiota in LPS-induced neuroinflammation mice (n=6). (A) PCA, (B) PcoA.

[0044] Figure 19 PLS-DA scores for serum metabolites in each group of mice.

[0045] Figure 20 Volcano plots of differential metabolites in the serum of mice in the LPS group and NC group (A and C), and H+LPS group and LPS group (B and D).

[0046] Figure 21 Figure showing the relative abundance values ​​of some differentially metabolites in serum (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0047] Figure 22 The diagram shows the KEGG pathway classification of differential metabolites between the H+LPS and LPS groups (A) and the enrichment bar chart (B). Note: In A, different colored entries represent the hierarchical classification annotation of the KEGG pathway, corresponding to the pathway name, and the length of the bar represents the number of differential metabolites annotated under that pathway; in B, the x-axis represents the number of differential metabolites annotated under that pathway, and the y-axis represents the pathway name.

[0048] Figure 23 A bubble chart showing the KEGG enrichment factor of differential metabolites between the H+LPS group and the LPS group; Note: The x-axis represents the enrichment factor of the differential metabolites enriched in this pathway, the y-axis represents the P-value of the pathway, and the size of the point represents the number of differential metabolites enriched. Detailed Implementation

[0049] The technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without innovative effort are within the scope of protection of this invention.

[0050] Example

[0051] 1 Experimental Methods

[0052] 1.1 Isolation and Purification of Strains: 1 g of healthy infant fecal sample was suspended in 9 mL of sterile PBS buffer (pH 7.4, containing 0.5 g / L L-cysteine ​​hydrochloride), homogenized, and serially diluted. Different concentrations of the diluted solution were spread onto the surface of MRS solid medium containing 2% CaCO3 and anaerobically incubated at 37°C for 36–48 h. Single colonies with a clear transparent zone, smooth surface, and round shape were picked, numbered, recorded, and streaked for purification at least 3 times. During purification, the strains could be preliminarily identified and contaminants removed based on colony morphology, catalase test, and Gram staining microscopy. Finally, the purified single colonies were inoculated into MRS liquid medium and incubated statically at 37°C for 18 h. The fresh bacterial culture was mixed with an equal volume of 25% (w / v) glycerol and frozen at -80°C for later use.

[0053] 1.2 Simulated Gastrointestinal Fluid Tolerance: The survival rate of this strain in a simulated gastrointestinal environment was evaluated. Simulated gastric fluid (SGF) was prepared by dissolving 0.33 g pepsin in 100 mL of sterile deionized water, and the pH of the mixture was adjusted to 3.0 using 1.0 M HCl. A bile solution was prepared by adding 0.3 g bile salt to 100 mL of physiological saline (0.85%, w / v) and sterilizing at 115 °C for 30 min. Subsequently, 0.1 g trypsin was added to the sterilized 100 mL sterile bile solution, and the pH was adjusted to 7.4 using 1.0 M NaOH to obtain simulated intestinal fluid (SIF). The bacterial cells were resuspended in 10 mL of SGF and SIF, respectively, and incubated with gentle shaking at 37 °C for 3 h and 4 h. Samples of the SGF and SIF solutions were taken at the beginning and end, and the viable cell count was determined by plate counting. The survival rate (%) was calculated using the following formula: Survival rate (%) = N t / N0×100%, where N t N0 and N0 represent the number of viable bacteria in the initial solution after 3 or 4 hours, respectively, expressed in CFU / mL.

[0054] 1.3 Security Assessment

[0055] 1.3.1 Hemolytic activity: The bacterial suspension was streaked onto Columbia blood agar plates and incubated upside down at 37°C for 36-48 h. The presence of a hemolytic zone around the colonies was then observed, with Staphylococcus aureus as a positive control.

[0056] 1.3.2 Antibiotic Susceptibility: The susceptibility of this strain to different types of antibiotics was determined by the disk agar diffusion method (also known as the Kirby-Bauer test). 100 μL of a 1×10⁻⁶ antibiotic solution was applied. 8 The CFU / mL bacterial suspension to be tested was spread onto MRS solid medium. After drying, sterile forceps were used to place the drug susceptibility test strips onto the surface of the medium. Four test strips could be placed evenly in each petri dish. After labeling, the strips were allowed to stand for 30 minutes to allow for full adsorption and adhesion. After incubation at 37°C upside down for 48 hours, the diameter of the inhibition zone around the test strips was measured using calipers. The results were evaluated according to the CLSI drug susceptibility testing standards.

[0057] 1.4 Strain Identification

[0058] 1.4.1 Morphological identification: The bacteria were diluted and spread onto MRS solid medium, and incubated upside down at 37°C for 36-48 h. Colony morphology was observed. Furthermore, the bacterial cells were collected by centrifugation and fixed overnight with 2.5% glutaraldehyde at 4°C. Then, they were dehydrated sequentially in a gradient of 30%, 50%, 70%, 90%, and 100% ethanol solutions, each lasting 5 min. Finally, a small amount of dried sample was uniformly attached to the sample stage using conductive adhesive, sputtered with gold under vacuum, and scanned and photographed at an accelerating voltage of 2 kV and an appropriate magnification.

[0059] 1.4.2 Molecular biological identification: The strain was inoculated at a 2% inoculum on MRS liquid medium and incubated at 37°C for 18 h. Bacterial cells were then collected, and total bacterial DNA was extracted according to the instructions of the bacterial genome extraction kit. This DNA was used as a template for PCR amplification of the 16S rRNA gene. The PCR amplification program consisted of 35 cycles: 94°C pre-denaturation for 4 min, 94°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 90 s, followed by a final extension at 72°C for 10 min. A portion of the amplified product was analyzed by agarose gel electrophoresis. After confirming the bands were correct, the remaining product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were analyzed using BLAST alignment in the NCBI database, and a phylogenetic tree was constructed using the neighbor-joining method in MEGA 11.0 software to analyze the species relationship of the target strain.

[0060] 1.5 Whole genome sequencing

[0061] Take the centrifugally collected bacterial cells above and prepare the genomic DNA of the target strain by SDS extraction combined with a purification column method. After the extraction, use NanoDrop to detect the purity (OD260 / 280 and OD260 / 230), concentration, and nucleic acid absorption peak of the genomic DNA. Combine with Qubit to accurately detect its concentration, and at the same time compare the Qubit concentration with the NanoDrop concentration to judge the sample purity. Finally, detect the integrity of the genomic DNA by agarose gel electrophoresis. Based on the Illumina NovaSeq second-generation sequencing and Nanopore PromethION third-generation sequencing technologies, combine the sequencing data of the two for hybrid assembly and subsequent analysis processes. This sequencing process was commissioned to be completed by Suzhou Huoxun Biotechnology Co., Ltd. Assemble, annotate, and perform personalized analysis on the sequencing results.

[0062] 1.6 Evaluation of the preventive effect of the strain on neuroinflammation in mice

[0063] 1.6.1 Bacterial liquid preparation: Activate the glycerol tube bacterial liquid stored at -80°C, streak it on an MRS solid medium plate, and incubate it upside down at 37°C for 36 - 48 h. Pick a single colony and inoculate it into an MRS liquid medium, incubate it statically at 37°C for 18 h and passage 2 - 3 times. Take the bacterial liquid and centrifuge it at 5000 rpm / min at 4°C for 5 min to collect the bacterial cells, and resuspend them with a 30% (v / v) glycerol solution to adjust the bacterial liquid concentration to 5×10 9 CFU / mL. All the bacterial liquid required for animal experiments was prepared in the same batch and stored at -80°C for later use. Before gavage, take out the cryotube and melt it in a 37°C water bath, centrifuge it and wash it with sterile normal saline, repeat 2 times to fully remove the residual glycerol component, and finally resuspend the bacterial cells with an equal volume of sterile normal saline again, dilute it to the required concentration for gavage treatment, and use it immediately after preparation.

[0064] 1.6.2 Animal grouping and treatment: 48 SPF-grade C57BL / 6J mice were housed in a standardized SPF-grade experimental animal room (Experimental Animal Center of South China Agricultural University, SYXK (Guangdong) 2022 - 0136), at a temperature of 23 ± 3°C, a humidity of 55 ± 10%, a day-night light-dark cycle of 12 h / 12 h, and free access to food and water. Randomly divided into 6 groups, namely the normal control group (NC), the WH021 control group (WH021, 5×10 9 CFU / mL), the model group (LPS), the high-dose treatment group (H+LPS, 5×10 9 CFU / mL), the medium-dose treatment group (M+LPS, 5×10 8 CFU / mL), and the low-dose treatment group (L+LPS, 5×10 7(CFU / mL), 8 animals per group. After two weeks of acclimatization, animals were continuously treated by gavage (ig) for 4 weeks. The treatment methods for each group were as follows: the WH021 and three dose treatment groups were administered 200 μL of the corresponding bacterial suspension by gavage, while the NC and LPS groups were administered 200 μL of sterile saline by gavage. Starting on day 22, the LPS group and the three dose treatment groups were administered LPS intraperitoneally (ip) at a dose of 250 μg / kg / day to induce neuroinflammation, while the NC and WH021 groups were administered an equal volume of sterile saline intraperitoneally for 1 week. The experimental procedure is as follows. Figure 1 As shown.

[0065] 1.6.3 Status observation: During the experiment, the weight of mice was measured every two days to analyze the trend of weight change in each group, and the basic physiological conditions such as fur characteristics and activity status of mice were observed.

[0066] 1.6.4 Behavioral Experiments: Open Field Test: The open field test evaluates the spontaneous activity and exploratory behavior of mice to reflect their anxiety and depression. The experimental setup is an open box (length × width × height: 50 cm × 50 cm × 35 cm) with an open top. The box is made of black hard plastic, and a camera is placed on top to record the movement trajectory of the mice. To avoid stress caused by the new environment, the test environment is completely identical to the breeding environment. The bottom of the box is covered with white paper that is the opposite color of the mouse's fur. The mice are then placed in the center of the open field and allowed to adapt for 1 minute before their movement trajectory is recorded over 5 minutes. After the experimental monitoring time is over, the mice are returned to their original breeding cages. The bottom of the open field box is cleaned of any residual mouse urine or feces, wiped with 75% alcohol, and allowed to dry before replacing it with new white paper to continue the test. Finally, Tracker software is used to track and analyze the mouse trajectories in the recorded videos and process the data. Tail Suspension Test: The tail suspension test evaluates the behavior of mice in a suspended state from which they cannot escape to reflect their depressive state. The mouse's tail was secured to the tail suspension device with adhesive tape, approximately 1 cm from the end of the tail. The mouse was then hung upside down from the top of the tail suspension box, with its head about 15 cm off the ground. A camera recorded the mouse's activity over 6 minutes, and the cumulative immobile time in the last 4 minutes was statistically analyzed.

[0067] 1.6.5 Sample Collection and Processing: Feces: Feces were collected from mice starting two days before the end of the experiment. Mice to be sampled were placed in clean cages lined with sterile filter paper. Feces were collected immediately after defecation and placed in sterile cryovials. The samples were aliquoted into 4-5 tubes / tube, flash-frozen in liquid nitrogen, and stored at -80°C for later testing. New sterile filter paper was used for samples from different mice to avoid cross-contamination. Blood: Mice were fasted overnight but allowed free water. Blood was collected by enucleation after ether anesthesia. The collected blood was allowed to stand at room temperature for 1 hour, followed by centrifugation at 3000 rpm / min for 15 minutes. The supernatant serum was carefully aliquoted into 50 μL tubes and stored at -80°C for later testing, avoiding repeated freeze-thaw cycles. Brain tissue: After blood collection, mice were euthanized by tailbone dislocation, decapitated, and the brain tissue was carefully dissected. The brain was quickly placed on ice and divided into left and right hemispheres along the midline using a surgical incision. One hemisphere was fixed in 4% (w / v) paraformaldehyde at 4°C for at least 24 hours for pathological sectioning and other related tests. The other hemisphere was flash-frozen in liquid nitrogen and stored at -80°C for later analysis. Colon: The abdomen of the mice was dissected, and a 1 cm distal colon (approximately 1 cm from the anus) was harvested and fixed in 4% (w / v) paraformaldehyde. The remaining colon was cut into three portions, flash-frozen in liquid nitrogen, and stored at -80°C for later analysis.

[0068] 1.6.6 Biochemical Indicator Determination: Oxidative Stress Indicators: Oxidative stress levels in mouse brain tissue homogenate supernatant, including malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities, were determined using commercial kits. Tissue samples and extracts were homogenized at a ratio of 1:9 (g / mL) on ice, centrifuged at 12000 rpm and 4℃ for 10 min, and the supernatant was used to determine the total protein content using a BCA kit. Subsequently, oxidative stress indicators were determined strictly according to the kit instructions. Cytokine Level Determination: Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of pro-inflammatory cytokine IL-1β and anti-inflammatory cytokine IL-10 in mouse brain tissue homogenate supernatant and serum, as well as the serum corticosterone (CORT) level. Similarly, the total protein content in brain and colon tissue homogenate supernatant was determined using a BCA kit, and subsequent determinations were made strictly according to the kit instructions.

[0069] 1.6.7 Histopathology: H&E staining: Fixed brain and colon tissues were subjected to gradient dehydration in a dehydrator. The treatment program consisted of 75% ethanol, 85% ethanol, 90% ethanol, 95% ethanol, anhydrous ethanol I, and anhydrous ethanol II, with treatment times of 4 h, 2 h, 2 h, 1 h, 30 min, and 30 min, respectively. Immediately after treatment, the tissues were immersed in xylene I and II for 5-10 min each for clearing. Subsequently, they were immersed in paraffin I, II, and III (melted at 65°C) for 1 h each for paraffin infiltration. The paraffin-infiltrated tissues were embedded and sectioned using an embedding machine and a microtome, and then immersed in dewaxing solutions I and II for 20 min each, anhydrous ethanol I and II for 5 min each, and 75% ethanol for 5 min each, followed by washing with water. The sections were then immersed in hematoxylin staining solution for 3-5 minutes, followed by water clarification, differentiation, washing, bluing, and washing again. Afterward, they were immersed in 95% alcohol for 1 minute to dehydrate, and finally stained in eosin solution for 15 seconds. The stained sections were dehydrated, sewn together, examined under a microscope, and images were acquired for analysis. Nissl staining: The previously embedded brain tissue pathological sections were immersed in the staining solution for 2-5 minutes, washed with water, slightly differentiated with 0.1% glacial acetic acid, and the reaction was terminated by washing with water. The degree of differentiation was controlled under a microscope. After washing, the sections were dried in an oven. The sections were then cleared in xylene for 10 minutes, mounted with neutral resin, examined under a microscope, and images were acquired for analysis.

[0070] 1.6.8 Immunofluorescence: Brain tissue pathological sections that have been embedded were heated in EDTA antigen retrieval buffer (pH = 9.0) for antigen retrieval. After cooling, they were washed with PBS buffer (pH = 7.4) and blocked with BSA solution for 30 min. Primary antibody working solution (1:1000) was then added to the antibody incubation chamber and incubated overnight at 4°C. After incubation, the sections were thoroughly washed and incubated in secondary antibody working solution (1:500) at room temperature in the dark for 1 h. After incubation, the sections were thoroughly washed again and incubated with DAPI staining solution at room temperature in the dark for 10 min. The sections were mounted with anti-fluorescence quenching mounting medium and examined under a fluorescence microscope at the following wavelengths for image acquisition and analysis. DAPI excitation wavelength 330–380 nm, emission wavelength 420 nm; 488 excitation wavelength 465–495 nm, emission wavelength 515–555 nm; CY3 excitation wavelength 510–560 nm, emission wavelength 590 nm; CY5 excitation wavelength 608–648 nm, emission wavelength 672–712 nm.

[0071] 1.6.9 RT-qPCR: Add 1 mL of RNA tissue lysis buffer and 3 zirconia grinding beads to a 2 mL grinding tube and pre-chill on ice. Take approximately 20 mg of tissue sample frozen at -80°C and add it to the grinding tube. Grind thoroughly using a low-temperature tissue homogenizer until no visible tissue fragments remain. Subsequent procedures should be performed according to the FastPure® Cell / Tissue Total RNA Kit instructions.

[0072] 1.6.10 Microbial Community Sequencing Analysis: Genomic DNA was extracted from fecal samples of mice in each group according to the experimental procedures provided by the manufacturer. DNA quality was assessed by 1.8% agarose gel electrophoresis, and its concentration and purity were determined using a NanoDrop 2000 micro-spectrophotometer. After passing the tests, PCR amplification of the V3 and V4 hypervariable regions of the 16S rRNA gene was performed using universal primers (338F: 5′-ACTCCTACGGGAGGCAGCA-3′; 806R: 5′-GGACTACHVGGGTWTCTAAT-3′). The products were purified using an Omega DNA purification kit, quantified and homogenized using Qsep-400 to form sequencing libraries. Finally, the constructed libraries underwent quality control, and those that passed quality control were sequenced using an Illumina NovaSeq 6000. After sequencing, reads were assembled, filtered, clustered, or denoised to obtain valid data. Species annotation and abundance analysis were then performed to reveal the species composition of mouse fecal samples from each group. Further alpha and beta diversity analyses were conducted, and linear discriminant analysis (LEfSe) was used to identify taxa or pathways with rich differences.

[0073] 1.6.11 Non-targeted metabolomics sequencing analysis: Serum sample pretreatment: Take 100 μL of mouse serum sample collected, centrifuged and frozen at -80℃, add 500 μL of extraction buffer containing internal standard (methanol:acetonitrile solution volume ratio = 1:1, internal standard L-2-chlorophenylalanine concentration 20 mg / L), vortex to mix for 30 s. Sonicate in an ice-water bath for 10 min, and incubate at -20℃ for 1 h. Then centrifuge the sample at 12000 rpm for 15 min at 4℃, carefully aspirate 500 μL of supernatant into an EP tube, and dry the extract in a vacuum concentrator. Add 160 μL of extraction buffer (acetonitrile:water volume ratio = 1:1) to the dried extract to reconstitute, vortex for 30 s, and sonicate in an ice-water bath for 10 min. Centrifuge the sample again at 12000 rpm for 15 min at 4℃. Carefully aspirate 120 μL of supernatant into a 2 mL injection bottle. Take 10 μL of each sample and mix them to form a QC sample for instrument testing.

[0074] Chromatography-mass spectrometry (LC-MS) detection: The LC-MS system used for detection and analysis consisted of an Acquity I-Class PLUS ultra-high performance liquid chromatograph (Waters, USA) and a Xevo G2-XS QTOF high-resolution mass spectrometer (Waters, USA), equipped with an AcquityUPLC HSS T3 column (1.8 μm 2.1*100 mm, Waters, USA). Positive ion mode (POS): Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% ethyl acetone formate; Negative ion mode (NEG): Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% ethyl styrene formate. The injection volume was 1 μL, and the specific elution conditions for the chromatographic mobile phases are shown in Table 1.

[0075] Table 1 Chromatographic mobile phase conditions

[0076]

[0077] The Xevo G2-XS QTof high-resolution mass spectrometer can perform primary and secondary mass spectrometry data acquisition in MSe mode under the control of acquisition software (MassLynx V4.2, Waters). In each data acquisition cycle, it can simultaneously acquire data from both low-collision and high-collision energies in dual channels. The low-collision energy is 2V, and the high-collision energy range is 10–40V, with a scan frequency of 0.2 s. The ESI ion source parameters are as follows: capillary voltage: 2500V (positive ion mode) or -2000V (negative ion mode); cone voltage: 30V; ion source temperature: 100℃; desolvation gas temperature: 500℃; backflushing gas flow rate: 50L / h; desolvation gas flow rate: 800L / h; mass-to-nucleus ratio acquisition range: 50–1200 m / z.

[0078] Mass spectrometry data processing: Raw data acquired using MassLynx V4.2 was processed using Progenesis QI software for peak extraction, peak alignment, and other data processing operations. Identification was performed based on the online METLIN database, public databases, and a self-built library by BGI Genomics. Theoretical fragment identification was also performed, with a parent ion mass number deviation within 100 ppm and a fragment ion mass number deviation within 50 ppm. The raw peak area information and total peak area were normalized before subsequent analysis. Identified compounds were retrieved for classification and pathway information in the KEGG, HMDB, and Lipidmaps databases. Based on the grouping information, fold differences were calculated and compared, and a t-test was used to calculate the p-value for the significance of differences between compounds. OPLS-DA modeling was performed using the R package ropls, and 200 permutation tests were conducted to verify the model's reliability. Multiple cross-validation was used to calculate the VIP value of the model. A combination of fold differences and OPLS-DA model p-values ​​and VIP values ​​were used to screen differentially metabolites. The screening criteria were FC>1, p<0.05, and VIP>1. The significance of KEGG pathway enrichment in differentially metabolites was calculated using the hypergeometric distribution test.

[0079] 2 Results and Discussion

[0080] 2.1 Simulated Gastrointestinal Tolerance: To effectively exert their functional activities in the host, probiotics must possess a certain survival ability in the harsh environment of the gastrointestinal tract. Table 2 shows the survival rate and viable cell loss (in logarithmic order) of strain WH021 and reference strain LGG in an in vitro simulated gastrointestinal environment. After incubation in a simulated gastric fluid environment at pH 3.0 for 3 h, the survival rate of this strain was comparable to that of strain LGG (97.8%), while the survival rate after incubation in a simulated intestinal fluid environment for 4 h was higher than that of strain LGG, reaching 97.22%. Whole-genome sequencing analysis revealed the presence of the genes atpA-atpF encoding the FoF1-ATPase system in the genome of this strain. Numerous studies have shown that the FoF1-ATPase (F-type proton pump, also known as ATP synthase) mechanism is a key factor in the adaptation of probiotics to acidic conditions. In an acidic environment, probiotics can use this enzyme to work in reverse, i.e., hydrolyze ATP to drive protons (H+). + This substance is transported from inside the cell to the outside, thereby reducing the acid load within the cell and helping to stabilize the intracellular pH within a suitable range for survival. Furthermore, the genome has been found to encode Na+. + / H + The NhaC gene, a reverse transporter protein, is present in cells to maintain intracellular pH homeostasis and Na+. + It plays an important role in the dynamic equilibrium of ions.

[0081] Table 2 Survival rates of potential strains under simulated gastrointestinal conditions

[0082]

[0083] 2.2 Safety Assessment: Antibiotic Sensitivity Analysis: Antibiotic resistance is a significant threat to global public health. Current research indicates that resistance genes can be transferred through probiotics as transmission vectors. Therefore, evaluating the sensitivity of probiotics to different types of antibiotics is also an important safety indicator in probiotic screening standards. The sensitivity of strains was determined by the diameter of the inhibition zone to different types of antibiotics. The results are shown in Table 3. This strain was sensitive to seven antibiotics: penicillin, amoxicillin, ceftriaxone, cefadroxil, gentamicin, erythromycin, and rifampin, but resistant to kanamycin and streptomycin. This aligns with the findings of Liu et al., who suggested that some lactic acid bacteria are resistant to aminoglycoside antibiotics such as kanamycin and streptomycin. However, this strain does not exhibit gene transfer characteristics, and the reference strain LGG has long been widely used in food as a safe edible strain. Annotation analysis of the whole genome sequencing data in this invention did not reveal any genes related to antibiotic resistance, which is consistent with the results of antibiotic sensitivity testing. Combining the epigenetic results of the antibiotic inhibition zone with the background analysis of whole-genome genetics, it was found that strain WH021 has good safety as a potential probiotic.

[0084] Table 3. Susceptibility of potential strains to antibiotics

[0085]

[0086] Note: S represents sensitive; I represents moderately sensitive; R represents resistant.

[0087] Hemolytic activity analysis: During their growth and reproduction, some bacteria produce hemolysin to dissolve red blood cells, leading to a series of adverse reactions in the host, such as hemolytic anemia. Therefore, hemolytic activity is a crucial safety indicator that must be considered during probiotic screening. Results are as follows: Figure 2 As shown, Staphylococcus aureus, which exhibits β-hemolytic activity, served as a positive control, and a distinct white hemolytic area was observed around the colony. In contrast, the standard strain Lactobacillus rhamnosus LGG, used as a safety reference strain, showed a normal morphology with milky-white colonies and no hemolytic area. Comparatively, strain WH021 showed a colony morphology consistent with LGG, with no hemolytic area, indicating that neither strain exhibited hemolytic activity.

[0088] 2.3 Strain identification: Morphological identification: such as Figure 3 As shown, the colonies of the strain on the culture medium are milky white, opaque, raised, flattened round bodies with smooth surfaces and edges, and a diameter of approximately 1-3 mm, which is typical of lactic acid bacteria colonies. Scanning electron microscopy revealed that the bacterial cells are approximately 1-1.5 μm in length and have a cross-sectional diameter ranging from 0.7-1 μm, exhibiting a short rod-like shape.

[0089] Molecular biological identification: PCR amplification of the 16S rRNA gene was performed using the strain's DNA as a template. The electrophoresis results of the products are as follows: Figure 4 As shown in Figure A, the band is located at approximately 1500 bp and is clearly defined. Sequencing results indicate that the 16S rRNA gene sequence of this strain is 1455 bp, and its specific sequence is provided in the appendix. Alignment analysis using the BLAST program on the NCBI website with the 16S rRNA gene sequences of known strains in the GeneBank database revealed that this strain has the highest homology with *Lactobacillus plantarum*. Figure 4 As shown in B, a phylogenetic tree was constructed using the neighbor-joining method to identify other closely related genera. The results showed that strain WH021 and *Lactiplantibacillus plantarum* 4320 (accession number: MT544862.1) belong to the same branch, with a similarity of 99%. Finally, combining morphological characteristics and molecular biological identification results, the strain was determined to be *Lactiplantibacillus plantarum*. This strain was named *Lactiplantibacillus plantarum* WH021 and deposited on April 23, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), Building 59, No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No: 63388.

[0090] 2.4 Observation of Conditions: To investigate the effects of oral gavage with *Lactobacillus plantarum* WH021 and intraperitoneal injection of LPS on the basic vital signs of normal mice, the weight of the mice was monitored regularly during the experiment. For example... Figure 5 As shown in Figure A, the body weight of mice in each group showed a slow increase and then stabilized from day 0 to day 20, indicating that gavage with *Lactobacillus plantarum* WH021 before intraperitoneal injection of LPS had no significant effect on mouse body weight (p > 0.05). However, after intraperitoneal injection of LPS on day 21, the body weight of mice in the model group and the three dose experimental groups rapidly decreased, and intervention with different doses of the strain could not prevent the weight loss. However, with the extension of LPS induction time and continuous strain intervention, the weight loss trend of mice in the experimental group was somewhat mitigated, indicating that ingestion of *Lactobacillus plantarum* WH021 had a certain alleviating effect on the weight loss caused by abnormal inflammatory response. Furthermore, starting from day 24, the body weight of mice in each group gradually recovered. This phenomenon may be attributed to the inflammatory stress response caused by LPS in vivo, which caused discomfort to the mice. As the induction time was extended, the body gradually adapted and could repair itself through its own immune system. The weight difference throughout the experimental period was as follows: Figure 5 As shown in B in the diagram.

[0091] Observations on fur color and activity status in mice revealed that mice in the LPS-induced model group exhibited abnormal reactions such as huddling together, reduced activity, and decreased responsiveness. Their fur became brittle and dull, they tended to arch their backs and curl up, and their reactions were sluggish; some even had discharge from the corners of their eyes. Based on the above monitoring results of mouse weight and activity status, this preliminarily demonstrates the successful establishment of the LPS-induced neuroinflammation model, and that high and medium doses of *Lactobacillus plantarum* WH021 administered via gavage alleviated the abnormal physiological states produced by the neuroinflammation model.

[0092] 2.5 Behavioral Experiments: Open Field Test: This behavioral experiment investigated the preventative protective effect of *Lactobacillus plantarum* WH021 against LPS-induced neuroinflammatory abnormalities in mice. The open field test was recognized as a comprehensive assessment method for spontaneous activity and exploratory behavior. The open field test was conducted 4 hours after the last intraperitoneal injection of LPS. Figure 6 Figure A shows the movement trajectories of mice in each group, and statistical analysis was performed on the total movement distance and the ratio of the movement distance in the central region to the total distance. The results are as follows: Figure 6 As shown in Figure B, compared with the NC group which received intraperitoneal injection of saline, the LPS group mice exhibited less spontaneous activity, with the total movement distance decreasing from 2199 cm to 750 cm (p < 0.01). Furthermore, the ratio of the distance traveled into the central area to the total distance was significantly lower in the LPS group than in the NC group (p < 0.01), and the trajectory plots showed that the mice in this group moved entirely around the edges and corners of the enclosure. These results indicate that intraperitoneal injection of LPS for 7 days significantly reduced spontaneous activity and exploration desire in mice, inducing an anxiety- and depression-like state, demonstrating the successful establishment of a neuroinflammation model based on behavioral state. In contrast, supplementation with *Lactobacillus plantarum* WH021 in healthy mice had no effect on their mental state, indicating that this strain has no significant side effects on the host's mental health. When three different doses of *Lactobacillus plantarum* WH021 were administered to the host in advance, the high-dose group (H+LPS) significantly reversed the total movement distance and the proportion of movement distance in the central region of mice (p < 0.01), while the medium-dose group (M+LPS) and the low-dose group (L+LPS) showed no significant difference from the model group (LPS). Therefore, the high-dose (10) 9 Lactobacillus plantarum WH021 (CFU / day / mouse) significantly improved spontaneous activity and exploratory behavior in mice under neuroinflammatory conditions.

[0093] Tail suspension test: This test assesses a mouse's despair state and reflects its level of depression by observing its struggles while suspended in a position from which it cannot escape for 6 minutes. Figure 7As shown, compared with the saline-treated NC group, supplementation with *Lactobacillus plantarum* WH021 in healthy mice had no effect on their struggle time in a state of despair, further indicating that the strain has no significant side effects on the host's mental health after ingestion. However, the LPS-induced model group mice showed a significant increase in non-struggle time (p < 0.05), and this despair impairment could be prevented by pretreatment with high (H+LPS group) and medium (M+LPS group) doses of *Lactobacillus plantarum* WH021 (p < 0.01). Therefore, high and medium doses (10... 9 and 10 8 Lactobacillus plantarum WH021 (CFU / day / mouse) significantly improved despair behavior in mice under neuroinflammatory conditions.

[0094] 2.6 Effects of WH021 on Oxidative Stress in Brain Tissue: The brain requires a large amount of oxygen and contains abundant highly reactive metals (such as iron and copper) and easily oxidized polyunsaturated fatty acids. LPS-induced inflammatory responses generate more free radicals, leading to further oxidative stress. This cycle repeatedly exacerbates neuroinflammation, resulting in neuronal degeneration and cognitive impairment in the mouse brain. Therefore, this invention investigated the effects of *Lactobacillus plantarum* WH021 on LPS-induced oxidative stress damage in the mouse brain by measuring the levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) in mouse brain tissue. MDA, a product of membrane lipid peroxidation, can be used as a biomarker to assess the degree of oxidative damage. GSH-Px and SOD, as typical antioxidant enzymes, not only scavenge free radicals and their derivatives but also reduce the formation of lipid peroxides, enhancing the body's ability to resist oxidative damage. Results are as follows... Figure 8 As shown, the MDA level in the brain tissue of mice induced by LPS was significantly increased (p < 0.01), while the activities of antioxidant enzymes GSH-Px and SOD were significantly decreased (p < 0.05). Normal mice supplemented with only *Lactobacillus plantarum* WH021 showed no significant difference compared to the NC group, indicating that consuming this strain did not affect the level of oxidative stress in the brains of healthy mice (p > 0.05). However, in the experimental groups supplemented with different concentrations of *Lactobacillus plantarum* WH021, only the high-dose group (H+LPS) significantly improved LPS-induced oxidative stress damage in brain tissue (p < 0.05).

[0095] 2.7 Effects of WH021 on Brain Inflammatory Factors and Serum CORT Levels: A series of imbalanced inflammatory responses induced by LPS lead to abnormal levels of inflammatory factor secretion. IL-1β, a typical pro-inflammatory cytokine, is mainly secreted by monocytes, macrophages, and dendritic cells, and is crucial for the host's defense against infection and injury. IL-10, an anti-inflammatory cytokine, can also be produced by various immune cells such as monocytes and B cells to jointly maintain immune homeostasis and suppress inflammatory responses. This invention uses enzyme-linked immunosorbent assay (ELISA) to detect the expression levels of these two pro-inflammatory and anti-inflammatory cytokines, evaluating the effects of *Lactobacillus plantarum* WH021 on the secretion levels of inflammatory cytokines in the brains of mice with LPS-induced neuroinflammation. Figure 9 As shown, compared with the NC group, LPS significantly increased the expression of the pro-inflammatory cytokine IL-1β in the brain tissue and serum of mice and significantly decreased the expression of the anti-inflammatory cytokine IL-10 in the brain tissue and serum of mice (p < 0.05), indicating that LPS successfully induced brain and peripheral inflammation in mice. The intervention with *Lactobacillus plantarum* WH021 inhibited the changes in the above indicators to varying degrees, with the H+LPS group significantly reducing the levels of pro-inflammatory factors and increasing the levels of anti-inflammatory factors (p < 0.05).

[0096] Furthermore, the hypothalamic-pituitary-adrenal (HPA) axis, as a bidirectional signaling system, plays a crucial role in the brain-gut axis interaction. It can be activated by gut microbiota imbalance or an increase in pro-inflammatory cytokines, thereby stimulating the pituitary gland to secrete adrenocorticotropic hormone (ACTH), which in turn increases cortisol (CORT) secretion. This allows the HPA axis to exert a negative feedback mechanism to regulate brain function. Therefore, CORT is one of the major stress hormones affecting various parts of the body, including the brain and gut. The study assessed the effect of *Lactobacillus plantarum* WH021 intervention on the HPA axis in neuroinflammatory mice by measuring its levels in host serum. Figure 9 As shown in Figure E, the CORT level in the serum of mice induced by LPS was significantly increased (p < 0.05). Such results indicate that chronic inflammation characterized by increased CORT can cause dysregulation and dysfunction of the HPA axis, which is considered to play an important role in the pathogenesis of neurological diseases such as depression. In this invention, compared with the LPS model group, pretreatment with high (H+LPS group) and medium (M+LPS group) doses of *Lactobacillus plantarum* WH021 significantly reversed the abnormality of this hormone level (p < 0.05). Based on the above analysis results, it is preliminarily shown that high doses (10... 9 The CFU / day / mouse of Lactobacillus plantarum WH021 significantly improved the secretion level of inflammatory factors and abnormal HPA axis function in mice under neuroinflammatory conditions.

[0097] 2.8 Effects of WH021 on Neuronal Cells: Neuronal cell damage is one of the main pathological phenomena in the brain caused by neuroinflammation. This invention first uses hematoxylin and eosin (H&E) staining to detect the structural morphology and distribution of neurons in the hippocampus and cortex of coronal sections of mouse brain tissue from each group. For example... Figure 10 As shown, in the NC group, the morphological structure of neurons in the hippocampal DG region, CA1 region, and cortex was clear, intact, and densely arranged, with normal neuronal morphology and no obvious pathological changes. In contrast, the morphological structure of neurons in the LPS model group was irregular, loosely arranged with widened gaps, and some cells showed signs of lysis and nuclear shrinkage, indicating significant damage to neurons in LPS-induced neuroinflammation mice. Different doses of *Lactobacillus plantarum* WH021 intervention alleviated the damage to varying degrees. In the high-dose H+LPS group, the neuronal layers were clearer, the arrangement of neurons in the CA1 region was significantly more orderly and dense, the number of neurons increased, they appeared fuller, and nuclear shrinkage was significantly reduced. These results indicate that high-dose (10) LPS intervention... 9 Lactobacillus plantarum WH021 (CFU / day / mouse) significantly improved pathological brain damage in mice under neuroinflammatory conditions.

[0098] Nissl bodies are widely distributed within neuronal cell bodies and are a key characteristic structure of neurons, serving as the primary site of protein synthesis. A large number of Nissl bodies indicates a stronger protein synthesis function in the neuron; conversely, the number of Nissl bodies decreases or even disappears when the neuron is damaged. Therefore, Nissl staining is used to observe Nissl bodies in neuronal cells to assess neuronal damage. Figure 11 As shown, the hippocampus in the NC group was morphologically healthy, with densely packed neurons in the dentate gyrus and a large number of Nissl bodies. Conversely, the neurons in the LPS group were loosely arranged, with obvious gaps between them, and the number of Nissl bodies was reduced and their dispersion was unclear. Pretreatment with different doses of *Lactobacillus plantarum* WH021 showed a reduction in damage, resulting in denser cell arrangement and higher density. The high-dose H+LPS group (10... 9 Lactobacillus plantarum WH021 (CFU / day / mouse) significantly improved pathological brain damage in mice under neuroinflammatory conditions.

[0099] 2.9 Effect of WH021 on Glial Cell Activation: Astrocytes and microglia play important immunomodulatory roles in neuroinflammatory responses. Peripheral LPS induction exacerbates the activation of these two cell types, thereby increasing the expression of pro-inflammatory cytokines in the brain. In this invention, ionized calcium-binding adaptor molecule 1 (Iba-1) and glial fibrillary acidic protein (GFAP) were used to perform immunofluorescence staining on microglia and astrocytes associated with neuroinflammatory responses in the hippocampus, and the results are as follows: Figure 12 As shown in the figure, compared with the NC group, the immunofluorescence intensities of IBA-1 and GFAP in the LPS-induced neuroinflammation model group were significantly increased. Meanwhile, the ingestion of *Lactobacillus plantarum* WH021 alone by healthy mice did not cause changes in the immunofluorescence intensities of IBA-1 and GFAP, indicating that astrocytes and microglia were extensively activated in the hippocampus of the LPS-treated mice, while the WH021 group, like the NC group, remained at normal levels. This strain does not induce a neuroimmune stress response in the host. In the experimental groups, it was observed that the H+LPS group significantly inhibited the activation of astrocytes and microglia in the mouse hippocampus, maintaining a relatively stable neuroimmune state.

[0100] 2.10 Effects of WH021 on Neurotrophic Factors: Changes in neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and the synaptic marker postsynaptic density protein 95 (PSD-95) are closely related to neuronal survival and synaptic plasticity. Neuroinflammation can lead to a decrease in their expression levels, thereby causing synaptic dysfunction and directly affecting memory, emotion, and cognitive function. This invention quantitatively analyzed the expression levels of BDNF and PSD-95 genes in the brain tissue of mice in each group. The results are as follows: Figure 13 As shown in the figure, compared with the NC group, the expression levels of BDNF and PSD-95 mRNA in the brain tissue of mice in the LPS group were significantly reduced (p<0.05), indicating that LPS-induced treatment reduced brain-derived neurotrophic factor and the synaptic marker postsynaptic density protein 95, leading to abnormal synaptic connections in neurons and thus affecting the function of neural networks. Compared with the LPS model group, different doses of Lactobacillus plantarum WH021 significantly increased the mRNA expression level of BDNF in brain tissue (p<0.01), and the high-dose H+LPS group significantly enhanced the expression level of PSD-95 mRNA (p<0.05).

[0101] 2.11 Effects of WH021 on the Blood-Brain Barrier: The blood-brain barrier (BBB), as a crucial structure for maintaining brain homeostasis, dynamically isolates the central nervous system (CNS) from the peripheral circulatory system. On one hand, it restricts the entry of toxic substances and inflammatory factors from the blood into the brain and selectively transports certain nutrients for brain tissue use; on the other hand, it removes metabolic waste products from the brain, thus maintaining the stability of the CNS internal environment. Endothelial cells form a tight junctional state through tight junctions such as claudins, occludins, junction adhesion molecules, and zonula occludins, thus ensuring the low permeability of the BBB. In this invention, quantitative fluorescence analysis of the expression levels of Claudin-5 and ZO-1 genes in the brain tissue of LPS-induced neuroinflammation model mice indirectly reflects changes in the permeability of the BBB. Figure 14 As shown, the expression levels of tight junction proteins Claudin-5 and ZO-1 genes in the brain tissue of model mice under neuroinflammatory conditions were significantly reduced (p < 0.05), indicating that during LPS-induced systemic inflammation, a large amount of soluble inflammatory mediators circulating in the peripheral blood affect the permeability of the blood-brain barrier, thereby triggering inflammation within the nervous system. Early intervention with high-dose *Lactobacillus plantarum* WH021 significantly alleviated the expression levels of these two tight junction protein genes in brain tissue. This result demonstrates that disruption of the blood-brain barrier is a marker of neuroinflammatory diseases in mice, with the high-dose H+LPS group (10... 9 Lactobacillus plantarum WH021 (CFU / day / mouse) significantly improved blood-brain barrier damage in mice under neuroinflammatory conditions.

[0102] 2.12 Effects of WH021 on Colonic Tissue Pathological Damage: The intestine, as the most important digestive and immune organ in the body, contains 70% of the body's immune tissues and cells. Due to its special location and function, it plays a crucial role in the body's immune response, acting as the "front line" and "main battlefield" of the immune system. In this invention, the degree of damage to the colonic tissue and the distribution of goblet cells in each group of mice were detected by hematoxylin-eosin staining (H&E) and alicin blue staining (AB-PAS), respectively, to evaluate the degree of relief of intestinal inflammation in a neuroinflammatory model mouse by prophylactic intervention with different doses of *Lactobacillus plantarum* WH021. Figure 15As shown, the H&E results indicate that the colonic glands of healthy mice in the NC group were straight, long, densely packed, and numerous, with regular and intact crypt structures. No inflammatory cell infiltration or related lesions were observed. The AB-PAS results showed that the mucosal epithelium contained a large number of goblet cells (blue area). In contrast, the intestinal gland structure of the LPS model group was significantly shorter and looser, with distorted crypt structures and inflammatory cell infiltration (red arrows). The number of goblet cells was significantly reduced, leading to a decrease in mucin secretion levels and ultimately damage to the colonic mucus layer. This type of colonic pathological damage has also been observed in other studies of neuroinflammatory mouse models, such as those observed by Wu et al. In this invention, by scoring the colonic tissue and assessing the number of goblet cells in each intestinal gland (black arrows), it was found that high (H+LPS group) and medium (M+LPS group) doses showed inhibitory effects on the aforementioned inflammatory lesions. Specifically, prophylactic treatment with *Lactobacillus plantarum* WH021 significantly reduced colonic tissue scores in mice (p < 0.0001), particularly showing a direct improvement in intestinal gland morphology damage and inflammatory cell infiltration. Furthermore, it significantly increased the number of goblet cells in the colonic glands (p < 0.05), promoted mucus production, and improved crypt structure lesions. In summary, intake of high and medium doses (10... 9 CFU / Tianhe 10 8 Lactobacillus plantarum WH021 (CFU / day) has a repairing effect on intestinal pathological damage in mice with neuroinflammation.

[0103] 2.13 Effects of WH021 on Colonic Barrier Integrity: Tight junctions (TJs) consist of a group of protein complexes located at the cell apex and play a crucial role in maintaining cell morphology and forming a barrier against the invasion of pathogens and other harmful substances. This is significant, as demonstrated in the previous chapter regarding its impact on blood-brain barrier function and in this chapter regarding its role in protecting the intestinal epithelial barrier. To evaluate the effect of *Lactobacillus plantarum* WH021 on colonic barrier integrity in neuroinflammated mice, this invention analyzed the expression levels of ocludin, claudins, and zonaOccludens 1 (ZO-1) mRNA. The results are as follows: Figure 16 As shown, compared with the NC group, the expression levels of Occludin, ZO-1, and Claudin-1 mRNA were significantly reduced after LPS induction treatment (p<0.05), while high-dose (H+LPS group) bacterial culture treatment significantly reversed this phenomenon. This result indicates that 10 9 Prophylactic intake of CFU / day of Lactobacillus plantarum WH021 can repair damage to the intestinal barrier under LPS-induced neuroinflammatory conditions.

[0104] 2.14 Effects of WH021 on Colonic Inflammatory Factors: It has been reported that LPS can be recognized by Toll-like receptor 4 (TLR4) to activate the innate immune system, leading to NF-κB activation and the production of pro-inflammatory cytokines through a signaling cascade, thus triggering an intestinal inflammatory response. Therefore, this invention evaluated the effects of *Lactobacillus plantarum* WH021 on intestinal inflammation in neuroinflammatory mice by measuring the transcriptional levels of IL-1β and TNF-α genes in colonic tissue. The results are as follows... Figure 17 As shown, there was no significant difference in the transcription levels of pro-inflammatory cytokines IL-1β and TNF-α genes in the colon of mice in the NC group and the WH021 group (p > 0.05), indicating that the ingestion of *Lactobacillus plantarum* WH021 in healthy mice does not induce an abnormal response in their intestinal inflammation levels. However, the mRNA expression levels of IL-1β and TNF-α in the LPS model group mice were significantly increased (p < 0.05), and pre-treatment with different doses of *Lactobacillus plantarum* WH021 significantly downregulated the mRNA expression of these two pro-inflammatory factors in the colonic tissue of LPS-induced neuroinflammatory mice. Therefore, these results indicate that *Lactobacillus plantarum* WH021 at all three dose concentrations can alleviate the colonic inflammation state in neuroinflammatory mice.

[0105] 2.15 The Impact of WH021 on Gut Microbiota Structure: β-diversity was used to further compare the degree of difference in the composition of microbial communities among different samples. Its representation can be presented by Principal Component Analysis (PCA) and Principal Coordinates Analysis (PCoA), respectively. As commonly used dimensionality reduction methods, PCoA can reflect the differences and distances in the microbiota structure by analyzing the composition of characteristic OTUs (Optical Units) of different samples. PCA uses variance decomposition to reflect the differences of multiple sets of data on a two-dimensional coordinate graph, with the coordinate axes taking the two eigenvalues ​​that best reflect the variance. PCoA, based on PCA analysis, can perform dimensionality reduction based on the distance or similarity matrix between samples, more effectively preserving the relative positional relationships between samples, and is particularly suitable for exploring sample classification or clustering patterns in complex datasets. Therefore, the combination of the two methods can be applied to the analysis and presentation of β-diversity in the composition of gut microbiota structure in this invention. The results are presented by… Figure 18 Figures A and B visually demonstrate a significant separation of the gut microbiota between the NC and LPS groups, indicating a substantial difference in gut microbiota structure between LPS-induced neuroinflammation mice and healthy NC mice. When pre-treated with live *Lactobacillus plantarum* WH021, although the gut microbiota structure overlapped to some extent with the LPS group, it showed a trend towards a normal gut microbiota structure, suggesting that this bacterium has a restorative effect on the abnormal gut microbiota structure caused by LPS.

[0106] 2.16 Effects of WH021 on Serum Metabolites: Metabolites are the basis and direct manifestation of an organism's phenotype. Gut microbiota metabolites can cross the intestinal barrier into the peripheral blood circulation and act on the brain. To explore the potential mechanism by which *Lactobacillus plantarum* WH021 alleviates neuroinflammation, serum non-targeted metabolomics was used to investigate differential metabolites among different groups of mice. After processing the raw data, 2,670 and 3,254 peaks were detected in positive and negative ion modes, respectively, of all samples, with 1,081 and 1,181 metabolites annotated, respectively. This invention integrates and analyzes the metabolites detected and annotated in both positive and negative ion modes, and uses partial least squares discriminant analysis (PLS-DA) to show the overall differences between groups and the magnitude of variation within groups. Figure 19 Observation of sample distribution revealed distinct clusters among the NC normal control group, LPS model group, and H+LPS high-dose group, indicating significant differences in serum metabolites between LPS-induced neuroinflammation model mice and normal mice. Furthermore, the H+LPS high-dose group showed a clear distinction from the LPS model group and tended to converge towards the NC normal control group, suggesting that *Lactobacillus plantarum* WH021 has a positive restorative regulatory effect on serum metabolites in neuroinflammation mice.

[0107] Based on the OPLS-DA model analysis results, differentially expressed metabolites between different groups were screened using parameters such as Variable Importance in Projection (VIP) > 1, p-value < 0.05, and fold change > 1 (upregulation) or < 1 (downregulation). Overall, 395 differentially expressed metabolites were identified between the normal control (NC) group and the LPS model group, and 403 were identified between the LPS model group and the high-dose H+LPS experimental group. Figure 20 As shown, the differential distribution results from the metabolite volcano plot can be seen as follows ( Figure 20 (A, C) Compared to the NC group, the LPS group showed significant upregulation of 137 differentially expressed metabolites and significant downregulation of 258 differentially expressed metabolites; the H+LPS group showed significant upregulation of 270 differentially expressed metabolites and significant downregulation of 133 differentially expressed metabolites compared to the LPS group. Further cluster analysis of these metabolites was performed to obtain cluster heatmaps, such as... Figure 20As shown in B and D, significant clustering differences in serum metabolites in mice treated with LPS and *Lactobacillus plantarum* WH021 are clearly visible. The focus was on the reversal of differentially expressed metabolites in the serum of mice with a neuroinflammation model after *Lactobacillus plantarum* WH021 intervention. In the serum of mice in the H+LPS group, the expression levels of the same metabolites showed varying degrees of reversal. These metabolites mainly included glycerophosphates, glycosides, organic acids and their derivatives, amino acids and peptide analogs, steroids and their derivatives, etc.

[0108] Specifically, among their differentially metabolites, phosphatidylserine (PS(14:0 / 22:0)), L-glutamate, tuftsin, pinolenic acid, and 5,6-dehydroarachidonic acid are all neuroactive and inflammatory substances that can participate in the metabolism of the nervous system and the inflammatory regulation of the immune system. Their specific changes in different groups are as follows: Figure 21 As shown, after prophylactic administration of *Lactobacillus plantarum* WH021, the above metabolites completely returned to normal levels. It has been reported that abnormal lipid metabolism can lead to nerve cell damage and even death, thus playing an important role in the central nervous system. Phosphatidylserine (PS) is a key phospholipid in the brain, playing a crucial role in intercellular communication, neurotransmitter release, and signal transduction by regulating cell membrane fluidity and permeability. Glutamate is the main excitatory neurotransmitter in the mammalian brain, and it can interconvert with γ-aminobutyric acid (GABA) and maintain a relative balance. Studies have shown that glutamate-mediated neurotransmitter disorders are involved in the development of various neuropsychiatric diseases, including schizophrenia, Alzheimer's disease, and mood disorders. Tuftsin, a tetrapeptide (Thr-Lys-Pro-Arg), is an immune-enhancing molecule that can bind to and activate many immune cells, including macrophages or monocytes, neutrophils, and dendritic cells, and has a relieving effect on systemic chronic inflammation in mice. In addition, pinolenic acid and arachidonic acid and their derivatives have been shown by numerous studies to have significant anti-inflammatory effects. For example, in a neuroinflammatory model of mouse microglia BV-2 cells stimulated with LPS, Chen et al. found that pinolenic acid could significantly reduce the secretion level of pro-inflammatory mediators.

[0109] Furthermore, comparisons of serum differential metabolites between the H+LPS group and the LPS group, and between the LPS group and the NC group, revealed the presence of new differential metabolites. Specifically, these new metabolites appeared after LPS-induced neuroinflammation model treatment, whereas no differential expression was observed initially. Among them, 5-hydroxyindole-3-acetic acid (5-HIAA) is one of the major metabolites of serotonin (5-HT), an important neurotransmitter. As an indole derivative, 5-HIAA is considered a ligand for the aryl hydrocarbon receptor (AhR), which is a crucial hub for regulating intestinal immunity and stimulating downstream cascades, playing a vital role in maintaining the balance of the immune-inflammatory system.

[0110] 2.17 Differential Metabolic Pathway Analysis: Complex metabolic responses and their regulation in organisms do not occur in isolation; they are often formed by complex pathways and networks of different genes and proteins. Their interactions and regulation ultimately lead to systemic changes in the metabolome. Analyzing these metabolic and regulatory pathways can provide a more comprehensive and systematic understanding of biological processes altered by changes in experimental conditions, the mechanisms of trait or disease development, and drug action mechanisms. This invention uses the KEGG (Kyoto Encyclopedia of Genes and Genomes) database to study the content of the aforementioned differential metabolites as a whole network, primarily focusing on the classification and enrichment of KEGG metabolic pathways for differential metabolites between the H+LPS group and the LPS group. Results are as follows... Figure 22 As shown in Figure A, the metabolic pathways annotated with a large number of differentially metabolites are mainly lipid metabolism (arachidonic acid metabolism pathway and steroid hormone biosynthesis pathway) and the nervous system (5-hydroxytryptaminergic synaptic pathway), etc. Further enrichment of differentially metabolites using KEGG in the bar chart (…) Figure 22 (B) and enrichment factor bubble chart ( Figure 23 It is known that the main mechanism by which *Lactobacillus plantarum* WH021 exerts its effect in alleviating neuroinflammation is through the regulation of the arachidonic acid metabolism pathway, the serotonergic synapse pathway, the GABAergic synapse pathway, the ferroptosis pathway, and the FoxO signaling pathway.

[0111] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus plantarum* with preventive effects against neuroinflammation, characterized in that: The plant lactobacillus is named *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum The accession number is WH021, and it was deposited on April 23, 2023, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number: GDMCC No: 63388.

2. A microbial preparation, characterized in that: Contains *Lactobacillus plantarum* or a culture thereof as described in claim 1.

3. The microbial preparation according to claim 2, characterized in that: The microbial preparation is a bacterial powder or probiotic capsule; The culture medium used for the culture is MRS solid medium or MRS liquid medium.

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