Phytobacterium plantarum WH021 and application thereof in preventing and / or relieving neuroinflammation

Abstract

The invention discloses plant lactobacillus WH021 and application thereof in preventing and / or relieving neuroinflammation, and belongs to the technical field of microorganisms. The plant lactobacillus WH021 is separated and purified from excrement of healthy babies, is a probiotic with the function of relieving neuroinflammation, and has good application potential in development of food, health care products and drugs for preventing brain inflammation related diseases caused by chronic inflammation of bodies of middle-aged and elderly people.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to Lactobacillus plantarum WH021 and application thereof in preventing and / or alleviating neuroinflammation. Background Art

[0002] Aging is characterized by a chronic, low-grade, and systemic inflammatory state, which is closely related to the decline in physiological function and metabolic dysfunction caused by the decline in oxidative stress adaptability. More importantly, this process promotes the inflammatory response in the central nervous system (CNS), leading to the occurrence and development of neuroinflammation, which has been widely reported to be one of the core mechanisms driving the pathological process of various neurodegenerative diseases (NDs). In clinical practice, drug therapy is the main way to control the disease, but long-term use of drugs may lead to side effects such as constipation, metabolic dysfunction, and even neurological damage. Therefore, the development of safer and more effective new therapies for neuroinflammation, a core pathological link, has become a difficult problem to be overcome, which is of great significance to improving the quality of life of the elderly. With the rapid development of high-throughput sequencing technology and omics analysis methods, the correlation between the nervous system and intestinal flora has gradually entered the public eye. In addition to directly affecting the balance of intestinal microecology, probiotics also play an indirect regulatory role in the pathological process of neuroinflammation through the "microorganism-gut-brain" axis, and have the potential to prevent and alleviate NDs. Although the intestine and the brain are anatomically independent of each other, a variety of mechanisms have been proposed to explain the communication between intestinal microorganisms and the CNS. The process begins by building a healthy intestinal environment by restoring the ecological balance of intestinal microorganisms, maintaining the integrity of the intestinal barrier, and promoting the normal response of the intestinal mucosal immune system. Subsequently, this healthy intestinal environment, with the help of the effects of probiotics and the neuroactive substances, metabolites, and hormones produced by the microbiota, further affects the vagus nerve, enteric nervous system (ENS), neuroendocrine system, immune system, and circulatory system, thereby regulating the central nervous system. Therefore, providing a probiotic that can relieve neuroinflammation has great application potential in the development of foods, health products, and medicines to prevent brain inflammation-related diseases caused by chronic inflammation in the body in middle-aged and elderly people. 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 having the effect of preventing neuroinflammation.

[0004] The second object of the present invention is to provide the application of the above-mentioned Lactobacillus plantarum WH021.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A strain of Lactobacillus plantarum with the effect of preventing neuroinflammation was named Lactobacillus plantarum ( Lactiplantibacillus plantarum ) WH021, and was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province on April 23, 2023, with the deposit number: GDMCCNo: 63388.

[0006] A microbial preparation containing the above-mentioned Lactobacillus plantarum or its culture.

[0007] Furthermore, the microbial preparation is bacterial powder or probiotic capsule.

[0008] Furthermore, the culture medium used for the culture includes MRS solid culture medium and MRS liquid culture medium.

[0009] The use of the above-mentioned Lactobacillus plantarum or biological preparation in the preparation of a product having at least one of the following functions: (1) Prevent and / or alleviate comorbid anxiety and depression, depression or anxiety disorders; (2) Assist in improving memory function; (3) Prevent and / or alleviate neurodegenerative diseases; (4) Prevent and / or alleviate neuroinflammation; (5) Assist in improving tissue structure damage caused by neuroinflammation.

[0010] Furthermore, the neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease and multiple sclerosis.

[0011] Furthermore, the prevention and / or alleviation of neuroinflammation includes at least one of lowering the level of pro-inflammatory factors and increasing the level of anti-inflammatory factors.

[0012] Furthermore, the tissue structure damage includes at least one of oxidative stress damage to brain tissue, dysfunction of the hypothalamus-pituitary-adrenal (HPA) axis, brain pathological damage, blood-brain barrier damage, intestinal pathological damage, intestinal barrier damage, abnormal flora structure in the colon inflammatory state, and abnormal serum metabolism.

[0013] Furthermore, the product exerts its effects by regulating the arachidonic acid metabolic pathway, the 5-hydroxytryptamine synaptic pathway, the GABAergic synaptic pathway, the ferroptosis pathway and the FoxO signaling pathway.

[0014] Furthermore, the products include but are not limited to food, health products, medicines and preparations that can be added to the above products in the form of raw materials.

[0015] Furthermore, the dosage form of the product is an oral preparation or an injection preparation.

[0016] Compared with the prior art, the present invention has the following advantages and effects: The present invention separates and purifies Lactobacillus plantarum WH021 from healthy infant feces. The strain has strong gastrointestinal tolerance. Antibiotic resistance and hemolytic tests, combined with whole genome sequencing analysis, confirm that the strain WH021 is a safe probiotic.

[0017] Three different doses of live bacteria were used for preventive intervention in C57BL / 6J mice, and then lipopolysaccharide (LPS) was injected intraperitoneally to model the mice. The behavioral, peripheral and brain inflammation levels, brain oxidative stress and pathological damage of the mice were analyzed to evaluate the effect of improving brain inflammation. The results showed that the high-dose group (10 9CFU / day) for 28 days, the bacteria can significantly improve the depressive state caused by neuroinflammation in mice, reduce the levels of proinflammatory factors IL-1β and CORT hormone in peripheral blood, and enhance the secretion of anti-inflammatory factor IL-10. In addition, the transcription levels of brain-derived nerve growth factor (BDNF) and postsynaptic density protein-95 (PSD-95) genes were significantly enhanced, and the integrity of the blood-brain barrier was improved, thereby reducing the level of malondialdehyde (MDA), a marker of brain oxidative damage, and increasing the activity of antioxidant enzymes such as glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Pathological tissue staining results showed that the intervention of this strain can significantly reduce the degree of neuronal damage and downregulate the activation state of microglia, thereby effectively alleviating brain inflammation in model mice. By reducing the mRNA transcription levels of colonic inflammatory factors IL-1β and TNF-α, restoring the mRNA transcription levels and histopathological damage of intestinal tight junction proteins Occludin, ZO-1 and Claudin-1, the intestinal barrier of LPS-induced neuroinflammation model mice was effectively protected. Using 16S rRNA amplicon sequencing and serum non-targeted metabolome analysis, it was found that preventive intervention with this bacterium could restore the imbalance of the microbiota structure in neuroinflammation model mice. And by changing the serum metabolic profile, it significantly increased the synthesis of neuromodulators such as phosphatidylserine PS (14:0 / 22:0) and glutamate L-Glutamate, as well as anti-inflammatory substances such as tuftsin, pinolenic acid and 5,6-dehydroarachidonic acid, and promoted the metabolic levels of neurotransmitter metabolites such as 5-hydroxyindole-3-acetic acid in serum. The results of KEGG pathway enrichment analysis showed that the bacteria mainly alleviated neuroinflammation through the serotonergic synapse pathway, GABAergic synapse, arachidonic acid metabolism, ferroptosis pathway and FoxO signaling pathway.

[0018] The Lactobacillus plantarum WH021 provided by the present invention is a probiotic with the effect of alleviating neuroinflammation, and has good application potential in the development of foods, health products and medicines for preventing brain inflammation-related diseases caused by chronic inflammation in the body in middle-aged and elderly people. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the animal experiment process.

[0020] Figure 2 This is a colony image of strain WH021 growing in a blood agar plate.

[0021] Figure 3 The colony (A) and bacterial body (B) morphology of strain WH021.

[0022] Figure 4 Figure 1 shows the 16S rDNA electrophoresis pattern (A) and phylogenetic tree (B) of strain WH021.

[0023] Figure 5 Figure 2 shows the weight change curve (A) and difference (B) of mice.

[0024] Figure 6 Figure 2 shows the movement trajectory of mice in the open field experiment (A), total distance (B), and the ratio of the central area movement distance to the total distance (C).

[0025] Figure 7 This is a graph of the immobility time of mice in the tail suspension test.

[0026] Figure 8 The graph shows the effect of strain WH021 on the activities of malondialdehyde (A), glutathione peroxidase (B), and superoxide dismutase (C) in the brain tissue of mice with LPS-induced neuroinflammation.

[0027] Fig. 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.

[0028] Fig.10 This is a picture of H&E staining of brain tissue.

[0029] Fig.11 This is a picture of Nissl staining of brain tissue (scale bar = 40 μm).

[0030] Fig.12 Immunofluorescence staining of IBA-1 and GFAP in brain tissue (green IBA-1; red GFAP; blue DAPI; scale bar = 40 μm).

[0031] Fig.13 The figure shows the effect of strain WH021 on the mRNA expression of BDNF (A) and PSD-95 (B) in the brain tissue of mice with LPS-induced neuroinflammation.

[0032] Fig.14 This figure shows the effect of Lactobacillus plantarum WH021 on the mRNA expression of Claudin-5 (A) and ZO-1 (B) in the brain tissue of mice with LPS-induced neuroinflammation.

[0033] Fig.15 H&E and AB-PAS staining of colon tissue (scale bar = 200 μm, * p <0.05, **** p <0.0001)Fig.

[0034] Fig.16 The mRNA expression levels of colon tight junction proteins Occludin (A), ZO-1 (B) and Claudin-1 (C) (ns p> 0.05,* p <0.05)Fig.

[0035] Fig.17 The mRNA expression levels of inflammatory factors IL-1β (A) and TNF-α (B) (ns p> 0.05,* p <0.05,** p <0.01)Fig.

[0036] Fig.18 Figure 2. Effects of Lactobacillus plantarum WH021 on intestinal microbial β-diversity in mice with LPS-induced neuroinflammation (n=6). (A) PCA, (B) PcoA.

[0037] Fig.19 Figure 2 shows the PLS-DA scores of serum metabolites in each group of mice.

[0038] Fig. 20 Volcano plots of differential metabolites in the serum of mice between the LPS group and the NC group (A and C), and between the H+LPS group and the LPS group (B and D).

[0039] Fig.21 is the relative abundance value of some serum differential metabolites (* p <0.05,** p <0.01,*** p <0.001, **** p <0.0001)Fig.

[0040] Fig. 22 KEGG pathway classification diagram (A) and enrichment column diagram (B) of differential metabolites between H+LPS and LPS groups; Note: The different colored entries in A represent the hierarchical classification annotations of KEGG pathways, corresponding to the pathway name, and the column length represents the number of differential metabolites annotated to the pathway; the x-axis in B is the number of differential metabolites annotated to the pathway, and the y-axis is the pathway name.

[0041] Fig.23It is a bubble chart of KEGG enrichment factors of differential metabolites between the H+LPS group and the LPS group; Note: the x-axis is the enrichment factor of the differential metabolites enriched in the pathway, the y-axis is the P-value of the pathway, and the size of the point represents the number of differential metabolites enriched. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention. Rather than all the embodiments, all other embodiments obtained by ordinary technicians in this field without innovative work based on the embodiments of the present invention are within the scope of protection of the present invention. Example

[0043] 1 Experimental methods 1.1 Isolation and purification of strains: Take 1 g of healthy infant fecal sample and suspend it in 9 mL sterile PBS buffer (pH 7.4, containing 0.5 g / L L-cysteine ​​hydrochloride), homogenize and mix, then dilute in sequence, and apply different concentrations of dilutions on the surface of MRS solid culture medium containing 2% CaCO3, and culture anaerobically at 37°C for 36-48 h. Pick a single colony with a clear transparent circle, smooth surface and round shape, number, record and purify by streaking at least 3 times. During the purification period, the strain can be preliminarily identified and foreign bacteria can be eliminated based on its colony morphology, catalase test and Gram staining microscopy. Finally, the purified single colony was inoculated into MRS liquid culture medium, cultured at 37°C for 18 h, and the fresh bacterial solution was mixed with 25% (w / v) glycerol in equal volumes and frozen in a -80°C refrigerator for use.

[0044] 1.2 Tolerance to simulated gastrointestinal fluid: The present invention evaluated the survival rate of the strain in a simulated gastrointestinal environment. Simulated gastric fluid (SGF) was prepared by dissolving 0.33 g of 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 of bile salts to 100 mL of normal saline (0.85%, w / v), and sterilized at 115°C for 30 min. Subsequently, 0.1 g of trypsin was added to the above sterilized 100 mL of sterile bile solution, and the pH was adjusted to 7.4 using 1.0 M NaOH to obtain simulated intestinal fluid (SIF). The cells were resuspended in 10 mL of SGF and SIF, respectively, and incubated in a 37°C constant temperature shaker with slow shaking for 3 h and 4 h. The solutions in SGF and SIF were sampled at the beginning and end, and the number of viable bacteria was detected by the plate colony count method. The survival rate (%) was calculated by the following formula: Survival rate (%) = N t / N0×100%, where N tand N0 represent the number of viable bacteria in the solution after 3 or 4 hours and in the initial solution, respectively, expressed in CFU / mL.

[0045] 1.3 Safety Assessment 1.3.1 Hemolysis: Streak the bacterial suspension onto Columbia blood agar plates, incubate at 37°C for 36-48 h, and observe whether a hemolytic ring appears around the colonies. Use Staphylococcus aureus as a positive control.

[0046] 1.3.2 Antibiotic sensitivity: The sensitivity of the strain to different types of antibiotics was tested by the paper agar diffusion method (also known as the Kirby-Bauer test). Pipette 100 μL of agar with a concentration of 1×10 8 The test bacterial solution with CFU / mL was spread on the MRS solid culture medium. After the spread was dry, the drug sensitivity test strips were placed on the surface of the culture medium with sterile tweezers. Four test strips could be evenly divided in the culture dish. After marking, they were left to stand for 30 minutes to allow them to fully adsorb and adhere. After inverted culture at 37℃ for 48 hours, the diameter of the inhibition zone around the drug sensitivity test strip was measured with a vernier caliper. The results were judged according to the CLSI drug sensitivity test standards.

[0047] 1.4 Strain identification 1.4.1 Morphological identification: The bacteria were diluted and spread on MRS solid medium, inverted and cultured at 37℃ for 36-48 h, and the colony morphology was observed. In addition, the bacteria were collected by centrifugation and fixed with 2.5% glutaraldehyde at 4℃ overnight, and then dehydrated in 30%, 50%, 70%, 90% and 100% ethanol gradient solutions, each stage lasting 5 minutes. Finally, a small amount of dry sample was evenly attached to the sample stage through conductive glue, gold was sprayed under vacuum conditions, and the sample was scanned and observed and photographed at an appropriate magnification at an accelerating voltage of 2 kV.

[0048] 1.4.2 Molecular biological identification: The strain was inoculated into MRS liquid medium at a 2% inoculation rate, and the bacteria were collected after static culture at 37°C for 18 h. The total bacterial DNA was extracted according to the operating instructions of the bacterial genome extraction kit. This was used as a PCR amplification template for the 16SrRNA gene for gene sequence amplification. The PCR amplification program was 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, for a total of 35 cycles, and finally 72°C extension for 10 min. Part of the amplified product was used for agarose gel electrophoresis detection. After confirming that the product bands were correct, the remaining products were entrusted to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were subjected to BLAST comparison analysis in the NCBI database, and a phylogenetic tree was constructed by the neighbor-joining method in MEGA 11.0 software to analyze the species relationship of the target strain.

[0049] 1.5 Whole genome sequencing Take the above centrifugation collected bacteria, and prepare the genomic DNA of the target strain by SDS extraction combined with purification column method. After extraction, use NanoDrop to detect the purity (OD260 / 280 and OD260 / 230), concentration, and nucleic acid absorption peak of the genomic DNA. And combine Qubit to accurately detect its concentration, and compare the Qubit concentration with the NanoDrop concentration to determine the purity of the sample. Finally, the integrity of the genomic DNA was detected by agarose gel electrophoresis. Based on Illumina NovaSeq second-generation sequencing and Nanopore PromethION third-generation sequencing technology, the two are combined to mix and assemble their sequencing data and perform subsequent analysis processes. The sequencing process was commissioned to Suzhou Hongxun Biotechnology Co., Ltd. The sequencing results were assembled, annotated, and personalized analyzed.

[0050] Evaluation of the effect of 1.6 strains in preventing neuroinflammation in mice 1.6.1 Preparation of bacterial solution: Activate the bacterial solution in the glycerol tube stored at -80℃, streak it on an MRS solid medium plate, and invert and culture it at 37℃ for 36-48 hours. Pick a single colony and inoculate it in MRS liquid medium, incubate it at 37℃ for 18 hours, and pass it 2-3 times. Centrifuge the bacterial solution at 5000 rpm / min and 4℃ for 5 minutes to collect the cells, and resuspend them in 30% glycerol solution to adjust the concentration of the bacterial solution to 5×10 9 CFU / mL. The bacterial solution required for gavage in animal experiments was prepared from the same batch and temporarily stored at -80°C for use. Before gavage, take out the cryopreserved tube and place it in a 37°C water bath to thaw, centrifuge and wash with sterile saline, repeat twice to fully remove the residual glycerol component, and finally use an equal volume of sterile saline to resuspend the bacteria again, dilute to the required concentration for gavage treatment, and use it immediately after preparation.

[0051] 1.6.2 Animal grouping and treatment: 48 SPF C57BL / 6J mice were housed in a standardized SPF experimental animal room (Experimental Animal Center of South China Agricultural University, SYXK (Guangdong) 2022-0136), with a temperature of 23±3℃, a humidity of 55±10%, a light-dark cycle of 12h / 12h, and free access to food and water. They were randomly divided into 6 groups, namely normal control group (NC), WH021 control group (WH021, 5×10 9 CFU / mL), model group (LPS), high-dose treatment group (H+LPS, 5×10 9 CFU / mL), medium-dose treatment group (M+LPS, 5×10 8 CFU / mL and low-dose treatment group (L+LPS, 5×10 7CFU / mL), 8 mice in each group. After two weeks of adaptive feeding, the mice were treated by gavage (ig) for 4 weeks. The treatment methods of each group were as follows: WH021 and the three-dose treatment groups were gavaged with 200 μL of the corresponding bacterial suspension, while the NC group and LPS group were gavaged with 200 μL of sterile saline. Starting from the 22nd day, the LPS group and the three-dose treatment groups were given intraperitoneal injection (ip) of LPS at a dose of 250 μg / kg / day to induce neuroinflammation, while the NC group and WH021 group were given intraperitoneal injection of an equal volume of sterile saline for 1 week. The experimental process is as follows Figure 1 shown.

[0052] 1.6.3 Status observation: During the experiment, the mice were weighed every two days to analyze the changing trend of the weight of each group. At the same time, the basic physiological conditions such as the hair characteristics and activity status of the mice were observed.

[0053] 1.6.4 Behavioral experiment: Open field experiment: The open field experiment was used to evaluate the autonomous activities and exploratory behaviors of mice to reflect their anxiety and depression. The experimental device was an open box with an open top (length × width × height: 50 cm × 50 cm × 35 cm). The box was made of black hard plastic and a camera was installed on the top to record the movement trajectory of the mice. In order to avoid stress caused by the new environment to the animals, the test environment was exactly the same as the breeding environment. The bottom of the box was covered with white paper of the opposite color to the mouse's fur. Then the mouse to be tested was placed in the center of the open field. After it was adapted for 1 min, the movement trajectory video within 5 min was recorded. After the experimental monitoring time was over, the mouse was put back into the original breeding cage, the mouse urine or feces remaining on the bottom of the open field box was cleaned, and it was wiped with 75% alcohol. After it was dried, new white paper was replaced and laid on the bottom to continue the test. Finally, the Tracker software was used to track and analyze the mouse trajectory in the recorded video and process the data; Tail suspension experiment: The tail suspension experiment was used to evaluate the behavior of mice in a suspended state where they could not escape to reflect their depressive state. The mouse's tail was fixed to the tail suspension device with adhesive tape, and the fixed position was about 1 cm away from the end of the mouse's tail, so that it was hung upside down on the top of the tail suspension box, and the mouse's head was about 15 cm above the ground. The camera recorded the mouse's activities within 6 minutes, and the cumulative immobility time of the mouse within the next 4 minutes was counted and analyzed.

[0054] 1.6.5 Sample collection and processing: Feces: Two days before the end of the experiment, mouse feces were collected. The mice to be sampled were placed in clean mouse cages lined with sterile filter paper. Feces were collected immediately after defecation and placed in sterile cryopreservation tubes. After 4-5 pellets / tube, they were quickly frozen in liquid nitrogen and stored at -80℃ for testing. New sterile filter paper must be replaced for sampling from different mice to avoid cross-contamination of samples. Blood: Mice were fasted but not watered overnight. Eyeballs were removed after ether anesthesia to collect blood. The collected blood was left at room temperature for 1 hour, then centrifuged at 3000 rpm / min for 15 minutes at room temperature. The upper serum was carefully aspirated and aliquoted in 50 μL / tubes, and frozen at -80℃ for testing to avoid repeated freezing and thawing. Brain tissue: After blood collection, mice were killed by tail dislocation, decapitation and careful removal of brain tissue, and quickly placed on ice and divided into left and right hemispheres along the midline of the brain using a surgical knife. Half of the brain tissue was fixed in 4% (w / v) paraformaldehyde at 4°C for more than 24 h for pathological sections and other related tests; the other half of the brain tissue was quickly frozen in liquid nitrogen and stored at -80°C for testing. Colon: The abdominal dissection of the mouse was performed, and 1 cm of the distal colon (about 1 cm from the anus) was cut off and fixed in 4% (w / v) paraformaldehyde. The remaining colon was cut and divided into 3 parts, then quickly frozen in liquid nitrogen and stored at -80°C for testing.

[0055] 1.6.6 Determination of biochemical indices: Determination of oxidative stress indices: Oxidative stress levels in the supernatant of mouse brain tissue homogenate were determined using commercial kits, including malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities. The tissue sample and the extract were homogenized in an ice bath at a ratio of 1:9 (g / mL), centrifuged at 12,000 rpm and 4°C for 10 min, and the supernatant was taken to detect the total protein content using the BCA kit, and then the oxidative stress indices were detected in strict accordance with the kit instructions; Cytokine level determination: Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of pro-inflammatory factors IL-1β and anti-inflammatory factors IL-10 in the supernatant of mouse brain tissue homogenate and serum, as well as the level of corticosterone CORT in serum. The total protein content in the supernatant of brain and colon tissue homogenate was also detected using the BCA kit, and then the detection was carried out in strict accordance with the kit instructions.

[0056] 1.6.7 Histopathology: H&E staining: The fixed brain tissue and colon tissue were placed in a dehydrator for gradient dehydration. The processing program was set to 75% alcohol, 85% alcohol, 90% alcohol, 95% alcohol, anhydrous ethanol I, and anhydrous ethanol II. The processing time was 4 h, 2 h, 2 h, 1 h, 30 min, and 30 min. After the end, it was immediately immersed in xylene I and II for 5-10 min each for transparent treatment. Then it was placed in paraffin I, II, and III melted at 65°C for 1 h each and immersed in wax. The tissues that were immersed in wax were embedded and coronally sliced ​​by an embedding machine and a slicer, and placed in dewaxing liquid I and II for 20 min each, anhydrous ethanol I and II, and 75% alcohol for 5 min each, and washed with water. Then, the slices were immersed in hematoxylin solution for 3-5 min, and then dehydrated in 95% alcohol for 1 min after water clarification, differentiation, water washing, blueing, and water washing. Finally, they were placed in eosin solution for staining for 15 s. The stained slices were dehydrated and slit, examined under a microscope, and image analysis was collected; Nissl staining: The above-mentioned embedded brain tissue pathological slices were taken, immersed in the stain solution for 2-5 min, washed with water, slightly differentiated with 0.1% glacial acetic acid, and washed with water to terminate the reaction. The degree of differentiation was controlled under a microscope, and the slices were placed in an oven to dry after washing. The slices were immersed in xylene for transparent treatment for 10 min, sealed with neutral gum, examined under a microscope, and image analysis was collected.

[0057] 1.6.8 Immunofluorescence: Take the above embedded brain tissue pathological sections, place them in EDTA antigen retrieval buffer (pH= 9.0) and heat them for antigen retrieval. After cooling, wash them with PBS buffer (pH= 7.4) and place them in BSA solution for blocking for 30 minutes. Then add the primary antibody working solution (1:1000) in the antibody incubation box and incubate them at 4°C overnight. After incubation, wash the sections thoroughly and place them in the secondary antibody working solution (1:500) for 1 hour at room temperature in the dark. After incubation, wash the sections thoroughly again and incubate them in DAPI staining solution for 10 minutes at room temperature in the dark. Seal the sections with anti-fluorescence quenching sealing agent and place them under a fluorescence microscope for microscopic examination at the following wavelengths, and collect and analyze images. The excitation wavelength of DAPI is 330-380 nm, and the emission wavelength is 420 nm; the excitation wavelength of 488 is 465-495 nm, and the emission wavelength is 515-555 nm; the excitation wavelength of CY3 is 510-560 nm, and the emission wavelength is 590 nm; the excitation wavelength of CY5 is 608-648 nm, and the emission wavelength is 672-712 nm.

[0058] 1.6.9 RT-qPCR: Add 1 mL RNA tissue lysis buffer and 3 zirconium oxide grinding beads to a 2 mL grinding tube and pre-cool on ice. Take about 20 mg of tissue sample frozen in a -80℃ refrigerator, add it to the grinding tube, and grind it thoroughly with a low-temperature tissue grinder until no tissue blocks are visible. Subsequent operations are carried out according to the instructions of the FastPure® Cell / Tissue Total RNA Kit.

[0059] 1.6.10 Bacterial flora sequencing analysis: Genomic DNA from the fecal samples of each group of mice was extracted according to the experimental procedures provided by the manufacturer. The DNA quality was tested by 1.8% agarose gel electrophoresis, and its concentration and purity were determined using a NanoDrop 2000 ultra-micro spectrophotometer. After the test was qualified, the V3 and V4 hypervariable regions of the 16SrRNA gene were PCR amplified with universal primers (338F: 5′-ACTCCTACGGGAGGCAGCA-3′; 806R: 5′-GGACTACHVGGGTWTCTAAT-3′), and the products were purified using the Omega DNA purification kit, quantified and normalized by Qsep-400 to form a sequencing library. Finally, the constructed library was first subjected to library quality inspection, and the qualified library was sequenced using Illumina NovaSeq 6000. After sequencing is completed, reads are spliced, filtered, clustered or denoised to obtain valid data, and species annotation and abundance analysis are performed to reveal the species composition of each group of mouse fecal samples. Alpha diversity analysis (AlphaDiversity) and beta diversity analysis (Beta Diversity) are further performed, and linear discriminant analysis effect size (LEfSe) analysis is used to mine differentially abundant taxa or pathways.

[0060] 1.6.11 Non-targeted metabolomics sequencing analysis: Serum sample pretreatment: Take 100 μL of mouse serum sample collected, centrifuged and frozen at -80°C, add 500 μL of extract containing internal standard (methanol: acetonitrile solution volume ratio = 1:1, internal standard L-2-chlorophenylalanine concentration 20 mg / L), and vortex mix for 30 s. Place in an ice water bath for ultrasonication for 10 min and stand at -20°C for 1 h. Then centrifuge the sample at 12000 rpm for 15 min at 4°C, carefully pipette 500 μL of supernatant into an EP tube, and dry the extract in a vacuum concentrator. Add 160 μL of extract solution (acetonitrile: water volume ratio = 1:1) to the dried extract for re-dissolution, vortex for 30 s, and ultrasonicate in an ice water bath for 10 min. Centrifuge the sample again at 12000 rpm for 15 min at 4°C. Carefully pipette 120 μL of supernatant into a 2 mL injection vial, and take 10 μL of each sample to mix as QC samples for testing.

[0061] Chromatography-mass spectrometry detection: The LC-MS system used for detection and analysis consists of an Acquity I-Class PLUS ultra-high performance liquid chromatography (Waters, USA) and a Xevo G2-XS QTOF high-resolution mass spectrometer (Waters, USA), equipped with an AcquityUPLC HSST3 column (1.8 μm 2.1*100 mm, Waters, USA). Positive ion mode (POS): mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in ethyl acetone; negative ion mode (NEG): mobile phase A: 0.1% formic acid in water; mobile phase B: 0.1% formic acid in ethyl styrene. The injection volume was 1 μL, and the specific elution conditions of the chromatographic mobile phase are shown in Table 1.

[0062] Table 1 Chromatographic mobile phase conditions 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 the acquisition software (MassLynx V4.2, Waters). In each data acquisition cycle, dual-channel data acquisition can be performed simultaneously for low collision energy and high collision energy. The low collision energy is 2V, the high collision energy range is 10-40V, and the scanning frequency is 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℃; backflush gas flow rate: 50L / h; desolvation gas flow rate: 800L / h; mass-to-nuclear ratio acquisition range 50-1200 m / z.

[0063] Mass spectrometry data processing: The raw data collected using MassLynx V4.2 were processed by Progenesis QI software for peak extraction, peak alignment and other data processing operations. Identification was performed based on the Progenesis QI software online METLIN database, public databases and Biomarker's self-built library. Theoretical fragment identification was performed at the same time. The deviation of the parent ion mass number was within 100ppm and the deviation of the fragment ion mass number was within 50ppm. The original peak area information was normalized with the total peak area for subsequent analysis. The classification and pathway information of the identified compounds were retrieved in the KEGG, HMDB and lipidmaps databases. According to the grouping information, the difference multiples were calculated and compared, and the T test was used to calculate the significance of the difference of each compound. p The OPLS-DA model was built using the R package ropls, and 200 permutation tests were performed to verify the reliability of the model. The VIP value of the model was calculated using multiple cross-validation. pThe differential metabolites were screened by using FC>1, p <0.05 and VIP> 1. The hypergeometric distribution test was used to calculate the KEGG pathway enrichment significance of differential metabolites.

[0064] 2 Results and discussion 2.1 Simulated gastrointestinal tolerance: In order to effectively exert their functional activity in the host, probiotics must have a certain ability to survive in the harsh environment of the gastrointestinal tract. Table 2 shows the survival rate and live bacterial loss (expressed in logarithmic order) of strain WH021 and reference strain LGG in a simulated gastrointestinal environment in vitro. The survival rate of this strain after incubation for 3 hours in a simulated gastric fluid environment at pH = 3.0 was equivalent to that of strain LGG (97.8%), while the survival rate after incubation for 4 hours in a simulated intestinal fluid environment was higher than that of strain LGG, reaching 97.22%. Whole genome sequencing analysis revealed that the genome of this strain contains genes encoding the FoF1-ATPase system. atpA - 5f A large number of 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 work in reverse through this enzyme, that is, hydrolyzing ATP to drive protons (H + ) from inside the cell to outside, thereby reducing the acid load in the cell and helping the intracellular pH to stabilize in a suitable survival range. In addition, it was found that the genome contains a gene encoding Na + / H + The NhaC gene of the antiporter protein plays an important role in maintaining intracellular pH homeostasis and Na + It plays an important role in ion homeostasis.

[0065] Table 2 Survival rates of potential strains under simulated gastrointestinal conditions 2.2 Safety Assessment: Antibiotic Sensitivity Analysis: Antibiotic resistance is one of the important issues threatening global public health. Current studies have shown that resistance genes can be transferred through probiotics as a transmission carrier. Therefore, evaluating the sensitivity of probiotics to different types of antibiotics is also an important indicator of safety in the screening of probiotic standards. The sensitivity of the strain to different types of antibiotics is determined by the diameter of the inhibition zone. The results are shown in Table 3. The strain is sensitive to seven antibiotics, including penicillin, amoxicillin, ceftriaxone, cefradine, gentamicin, erythromycin and rifampicin, and is resistant to kanamycin and streptomycin. This is consistent with the research results of Liu et al. that some lactic acid bacteria are resistant to aminoglycoside antibiotics such as kanamycin and streptomycin, but do not have the characteristics of transferring genes, and the reference strain LGG has been widely used in food as a safe edible strain for a long time. The present invention annotates and analyzes the whole gene sequencing data, and no genes related to antibiotic resistance are found, which is consistent with the results of the antibiotic sensitivity test. Combining the apparent experimental results of antibiotic inhibition zone with the background analysis of whole genome genetics, it was shown that strain WH021 has good safety as a potential probiotic.

[0066] Table 3 Sensitivity of potential strains to antibiotics Note: S stands for sensitive; I stands for moderately sensitive; R stands for resistant Hemolytic analysis: During the growth and reproduction process, some bacteria will produce hemolysin to dissolve red blood cells, which will then cause a series of adverse reactions such as hemolytic anemia in the host. Therefore, hemolytic activity is a safety indicator that must be examined in the screening process of probiotics. Figure 2 As shown, the β-hemolytic Staphylococcus aureus was used as a positive control, and an obvious white hemolytic area was observed around the colony. The standard strain Lactobacillus rhamnosus LGG was used as a safety reference strain, and its colony showed a normal morphology of milky white and no hemolytic area. In comparison, the colony morphology of strain WH021 was consistent with that of LGG, and no hemolytic area appeared, indicating that both were non-hemolytic.

[0067] 2.3 Strain identification: Morphological identification: Figure 3 As shown in the figure, the colony morphology of the strain on the culture medium is milky white, opaque, convex and flat, with a smooth surface and edges and a diameter of about 1-3 mm, which is a typical lactic acid bacteria colony morphology. Scanning electron microscopy showed that the length of the bacteria was about 1-1.5 μm, the cross-sectional diameter ranged from 0.7-1 μm, and it was short rod-shaped.

[0068] Molecular biological identification: PCR amplification of the 16srRNA gene was performed using the strain DNA as a template. The electrophoresis results of the product were as follows: Figure 4As shown in A in the figure, it is located at about 1500 bp and the band is clear. The sequencing results show that the 16srRNA gene sequence of this strain is 1455 bp in total. The specific sequence is shown in the appendix. By comparing the 16srRNA gene sequence of known strains in the GeneBank database with the Blast program on the NCBI website, it was found that the strain has the highest homology with the genus Lactobacillus plantarum. Figure 4 As shown in B, the phylogenetic tree of other closely related bacterial genera was constructed by the neighbor-joining method, showing that strain WH021 and Lactiplantibacillus plantarum 4320 (accession number: MT544862.1) is in the same branch with a similarity of 99%. Finally, combined with the morphological characteristics and molecular biological identification results, it was determined that the strain was Lactobacillus plantarum. Lactiplantibacillus plantarum The strain was named Lactobacillus plantarum ( Lactiplantibacillus plantarum ) WH021, and was deposited in the Guangdong Microbiological Culture Collection Center (GDMCC) at Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province on April 23, 2023, with the deposit number: GDMCC No: 63388.

[0069] 2.4 Status Observation: In order to explore the effects of oral administration of Lactobacillus plantarum WH021 and intraperitoneal injection of LPS on the basic vital signs of normal mice, the weight of mice was regularly monitored during the experiment. Figure 5 As shown in A, the body weight of mice in each group increased slowly from day 0 to day 20 and tended to be stable, indicating that intragastric administration of Lactobacillus plantarum WH021 before intraperitoneal injection of LPS had no significant effect on the body weight of mice ( p >0.05). However, after intraperitoneal injection of LPS on the 21st day, the body weight of mice in the model group and the three doses of experimental groups rapidly showed a sharp decline, and intervention with different doses of strains could not prevent the weight loss of mice. However, with the extension of LPS induction time and continuous strain intervention, the weight loss trend of mice in the experimental group was alleviated, indicating that the intake of Lactobacillus plantarum WH021 has a certain degree of alleviating effect on the weight loss caused by abnormal inflammatory response of the body. Furthermore, it was observed that the weight of mice in each group gradually recovered from the 24th day. This phenomenon may be attributed to the inflammatory stress response caused by LPS in the body, which caused discomfort to the mouse body. As the induction time prolonged, the body gradually adapted and could be repaired by its own immune system. The weight difference during the entire experimental period was as follows: Figure 5 As shown in B.

[0070] From the fur color and activity status of mice, it can be observed that after intraperitoneal injection of LPS, the mice in the model group began to show abnormal reactions such as huddling, reduced activity and decreased response. Their hair stood up and became dull, they tended to arch their backs and curl up, and their reactions were slow, and even secretions appeared in the corners of their eyes. Based on the above monitoring results of the weight and activity status of mice, it was preliminarily proved that the LPS-induced neuroinflammation model was successfully established, and that high and medium doses of Lactobacillus plantarum WH021 administered intragastricly had an alleviating effect on the abnormal physiological state produced by the neuroinflammation model.

[0071] 2.5 Behavioral experiment: Open field test: The preventive protective effect of Lactobacillus plantarum WH021 on the abnormal mental state of LPS-induced neuroinflammatory mice was investigated through behavioral experiments. The open field test was identified as a comprehensive evaluation method for autonomous activity and exploratory behavior. The open field test was started 4 hours after the last intraperitoneal injection of LPS. Figure 6 A in the figure shows the movement trajectory 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 area to the total distance. Figure 6 As shown in Figure B, compared with the NC group injected with saline, the mice in the LPS group showed less spontaneous activity, and the total movement distance decreased from 2199 cm to 750 cm ( p <0.01). In addition, the ratio of the distance of entering the central area to the total distance was significantly lower than that of the NC group ( p <0.01), and it can be observed from the trajectory diagram that this group of mice moved completely on the edges and corners of the box. The above results show that intraperitoneal injection of LPS for 7 days can significantly reduce the autonomous activity and exploration desire of mice, and induce anxiety and depression-like states in mice, that is, the behavioral state shows that the neuroinflammatory model has been successfully constructed. However, supplementation of healthy mice with Lactobacillus plantarum WH021 had no effect on their mental state, indicating that this strain has no obvious side effects on the host's mental health. When three different doses of Lactobacillus plantarum WH021 were used to intervene in 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 area of ​​the mice ( p <0.01), while the medium dose group (M+LPS) and low dose group (L+LPS) had no significant difference with the model group (LPS). 9 CFU / day / mouse) of Lactobacillus plantarum WH021 has a significant effect on improving the autonomous activity and exploratory behavior of mice in the neuroinflammatory state.

[0072] Tail suspension test: By observing the struggling behavior of mice in a suspended state where they cannot escape within 6 minutes, their state of despair is assessed, thereby reflecting their level of depression. Figure 7As shown in the figure, compared with the NC group treated with normal saline, the supplementation of Lactobacillus plantarum WH021 in healthy mice had no effect on their struggling time in a desperate state, which again showed that the intake of this strain had no obvious side effects on the host's mental health. However, the non-struggling time of mice in the LPS-induced model group increased significantly ( p <0.05), and this despair injury 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 CFU / day / mouse) of Lactobacillus plantarum WH021 has a significant improvement effect on the despair behavior of mice in the neuroinflammatory state.

[0073] 2.6 Effect of WH021 on oxidative stress state of brain tissue: The brain consumes a lot of oxygen to operate, and is rich in metals with high redox activity (such as iron and copper) and polyunsaturated fatty acids that are susceptible to oxidative damage. The inflammatory response induced by LPS will produce more free radicals, leading to further oxidative stress, which will repeatedly aggravate neuroinflammation and cause neuronal degeneration and cognitive dysfunction in the mouse brain. Therefore, the present invention explores the effect 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. Among them, MDA is a product of membrane lipid peroxidation and can be used as a biomarker to judge the degree of oxidative damage. GSH-Px and SOD, as typical antioxidant enzymes, not only have the function of scavenging free radicals and derivatives, but also can reduce the formation of lipid peroxides and enhance the body's ability to resist oxidative damage. The results are as follows Figure 8 As shown in Figure 2, the MDA level in the brain tissue of mice induced by LPS was significantly increased ( p <0.01), the activities of antioxidant enzymes GSH-Px and SOD were significantly decreased ( p <0.05). There was no significant difference between normal mice supplemented with Lactobacillus plantarum WH021 and the NC group, indicating that the consumption of this strain by healthy mice would not affect the oxidative stress level in their brains ( p >0.05). In the experimental groups supplemented with different doses and concentrations of Lactobacillus plantarum WH021, only the high-dose group (H+LPS) could significantly improve LPS-induced oxidative stress damage in brain tissue ( p <0.05).

[0074] 2.7 Effect of WH021 on brain inflammatory factors and serum CORT levels: A series of unbalanced inflammatory responses induced by LPS lead to abnormal secretion levels of inflammatory factors, among which IL-1β is a typical proinflammatory cytokine, which is mainly secreted by monocytes, macrophages and dendritic cells, and is crucial for the host's defense response to infection and injury. IL-10, as an anti-inflammatory cytokine, can also be produced by a variety of immune cells such as monocytes and B cells to jointly maintain immune homeostasis and inhibit inflammatory responses. The present invention detected the expression levels of the above two proinflammatory and anti-inflammatory cytokines by enzyme-linked immunosorbent assay (ELISA) to evaluate the effect of Lactobacillus plantarum WH021 on the secretion levels of inflammatory cytokines in the brain of mice with LPS-induced neuroinflammation. Fig. 9 As shown in the figure, compared with the NC group, LPS significantly increased the expression of pro-inflammatory cytokine IL-1β in the brain tissue and serum of mice, and significantly decreased the expression of 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. After intervention with Lactobacillus plantarum WH021, the changes in the above indicators were inhibited to varying degrees, among which the H+LPS group could significantly reduce the levels of pro-inflammatory factors and increase the levels of anti-inflammatory factors ( p <0.05).

[0075] In addition, the hypothalamus-pituitary-adrenal axis (HPA) plays a key role in the interaction of the "brain-gut axis" as a bidirectional signaling system. It can be activated by an imbalance in the intestinal flora or an increase in proinflammatory cytokines, thereby stimulating the pituitary gland to secrete adrenocorticotropic hormone (ACTH), which increases the secretion of cortisol (CORT), thereby allowing the HPA axis to exert a negative feedback mechanism to regulate brain function. Therefore, CORT is one of the main stress hormones that affects various parts of the human body, including the brain and intestines. The determination of its content in the host serum was used to evaluate the effect of Lactobacillus plantarum WH021 intervention on the HPA axis of mice with neuroinflammation. Fig. 9 As shown in E, the CORT content in the serum of mice was significantly increased after LPS induction ( p <0.05). Such results indicate that chronic inflammation characterized by increased CORT can cause dysregulation and dysfunction of the HPA axis, which is believed to play an important role in the pathogenesis of neurological diseases such as depression. In the present invention, high (H+LPS group) and medium (M+LPS group) doses of Lactobacillus plantarum WH021 pre-intervention treatment can significantly reverse the abnormal hormone levels compared with the LPS model group ( p <0.05). Based on the above analysis results, it is preliminarily shown that high dose (10 9CFU / day / mouse) has a significant effect on improving the secretion level of inflammatory factors and the abnormal function of the HPA axis in mice under neuroinflammatory state.

[0076] 2.8 Effect of WH021 on neuronal cells: Neuronal cell damage is one of the main brain pathological phenomena caused by neuroinflammation. The present invention firstly detects the structural morphology and distribution of neuronal cells in the hippocampus and cortex of the coronal sections of the brain tissue of each group of mice by hematoxylin-eosin staining (H&E). Fig.10 As shown in the figure, the morphological structure of neurons in the DG and CA1 regions of the hippocampus and the cortex of the NC group was clear and complete, and the arrangement was neat and dense. The neuronal morphology was normal without obvious pathological changes. However, the morphological structure of neurons in the LPS model group was irregular, the arrangement was loose, the gaps were widened, and some cells were dissolved and the nuclei were shrunken. These pathological phenomena showed that the neurons of mice with neuroinflammation induced by LPS were significantly damaged. Different doses of plant lactobacillus WH021 intervention can alleviate the degree of damage to different degrees. In the H+LPS high-dose group, the neuronal layers were clearer, the arrangement of neurons in the CA1 region was obviously neat and dense, the number increased, and the neurons were fuller and the nuclear shrinkage was significantly reduced. The above results show that high doses (10 9 CFU / day / mouse) has a significant improvement effect on brain pathological damage in mice under neuroinflammatory state.

[0077] Nissl bodies are widely present in the cell bodies of neurons. As one of the characteristic structures of neurons, they are the main site for protein synthesis. A large number of them indicates that the nerve cells have a strong ability to synthesize proteins. On the contrary, when nerve cells are damaged, the number of Nissl bodies will decrease or even disappear. Therefore, Nissl staining is used to observe Nissl bodies in nerve cells to evaluate the damage of neurons. Fig.11 As shown in the figure, the hippocampus of the NC group was healthy, the neurons in the dentate gyrus were densely arranged, and the number of Nissl bodies was large. In contrast, the neurons in the LPS group were loosely arranged, with obvious gaps between them, and the number of Nissl bodies was reduced and unclear. Pretreatment with different doses of Lactobacillus plantarum WH021 showed relief from the damage, making the cells show a tighter arrangement and higher density. Among them, the high-dose H+LPS group (10 9 CFU / day / mouse) has a significant improvement effect on brain pathological damage in mice under neuroinflammatory state.

[0078] 2.9 Effect of WH021 on the activation state of glial cells: Astrocytes and microglia play an important role in immunoregulation in neuroinflammatory response. When peripheral LPS is induced, the activation of these two types of cells is aggravated, thereby increasing the expression of proinflammatory cytokines in the brain. The present invention uses ionized calcium binding adapter molecule 1 (Iba-1) and glial fibrillary acid protein (GFAP) to perform immunofluorescence staining on neuroinflammation-related microglia and astrocytes in the hippocampus, and the results are as follows: Fig.12 As shown. Compared with the NC group, the immunofluorescence intensity of IBA-1 and GFAP in the LPS-induced neuroinflammation model group increased significantly. At the same time, the intake of Lactobacillus plantarum WH021 alone by healthy mice did not cause changes in the immunofluorescence intensity of IBA-1 and GFAP, indicating that astrocytes and microglia in the hippocampus of the brain tissue of mice in the LPS group were activated in large quantities, while the WH021 group was at a normal level like the NC group, and the strain did not cause the host's neuroimmune stress response. In the experimental group, it can be observed that the H+LPS group can significantly inhibit the activation of astrocytes and microglia in the hippocampus of mice, keeping the body's neuroimmune state at a relatively steady level.

[0079] 2.10 Effect of WH021 on neurotrophic factors: Changes in neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and 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 and thus damage synaptic function, directly affecting memory, emotion, cognitive function and other disorders. The present invention quantitatively analyzed the expression levels of BDNF and PSD-95 genes in the brain tissues of each group of mice. The results are as follows: Fig.13 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 decreased ( p <0.05), indicating that LPS-induced treatment caused a decrease in brain-derived neurotrophic factor and synaptic marker postsynaptic density protein 95 in mice, leading to abnormal synaptic connections of neurons and thus affecting the function of neural networks. Compared with the LPS model group, different doses of Lactobacillus plantarum WH021 can significantly increase the mRNA expression level of BDNF in brain tissue ( p <0.01), the high-dose H+LPS group could significantly enhance the expression of PSD-95 mRNA ( p <0.05).

[0080] 2.11 Effect of WH021 on blood-brain barrier: The blood-brain barrier (BBB), as an important structure for maintaining the homeostasis of the brain environment, can form a dynamic isolation between the central nervous system (CNS) and the peripheral blood circulation system. On the one hand, it limits the entry of toxic substances and inflammatory factors in the blood into the brain, and can selectively transport certain types of nutrients for use by brain tissue; on the other hand, it discharges waste products from brain tissue metabolism out of the brain to maintain the stability of the internal environment of the central nervous system. Among them, endothelial cells form a tight connection state through tight junctions such as claudins, occludins, junction adhesion molecules and zonula occludins, thereby ensuring the low permeability of the blood-brain barrier. In the present invention, the fluorescence quantitative analysis of the Claudin-5 and ZO-1 gene expression levels in the brain tissue of LPS-induced neuroinflammation model mice can indirectly reflect the changes in the permeability of the blood-brain barrier. Fig.14 As shown in Figure 2, 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 number of soluble inflammatory mediators in the peripheral blood circulation affect the permeability of the blood-brain barrier, thereby inducing inflammation within the nervous system. When high-dose plantarum Lactobacillus WH021 was used in advance, the expression levels of the genes corresponding to these two tight junction proteins in brain tissue were significantly alleviated. This result proves that the destruction of the blood-brain barrier is a sign of neuroinflammatory diseases in mice, among which the high-dose H+LPS group (10 9 CFU / day / mouse) has a significant improvement effect on blood-brain barrier damage in mice under neuroinflammatory state.

[0081] 2.12 Effect of WH021 on pathological damage of colon tissue: The intestine is the most important digestive and immune organ of the body, containing 70% of the body's immune tissues and immune cells. Due to its special position and function, it plays a key role in the body's immune response and plays the role of "frontline" and "main battlefield" in the immune system. In the present invention, the degree of damage to the colon tissue and the distribution of goblet cells in each group of mice were detected by hematoxylin-eosin staining (H&E) and Alcian blue staining (AB-PAS) to evaluate the degree of relief of intestinal inflammation in neuroinflammatory model mice by preventive intervention with different doses of Lactobacillus plantarum WH021. Fig.15As shown, the H&E results show that the colon glands of healthy mice in the NC group are straight and long, closely arranged, numerous, with regular and complete crypt structures, no inflammatory cell infiltration and related pathological damage. The AB-PAS results show that the mucosal epithelium contains a large number of goblet cells (blue area); while the intestinal gland structure of the LPS model group is significantly shortened and loose, the crypt structure is distorted, and there is inflammatory cell infiltration (red arrows). The number of goblet cells is significantly reduced, resulting in a decrease in the level of mucin secretion, and ultimately leading to damage to the colon mucus layer. This type of colon pathological damage has also been shown in other studies of neuroinflammatory mouse models, such as Wu et al., who also observed similar pathological manifestations. In the present invention, by scoring colon tissue and evaluating the number of goblet cells in each intestinal gland (black arrow), it was found that high (H+LPS group) and medium (M+LPS group) doses can show an inhibitory effect on the above-mentioned inflammatory damage. Specifically, the preventive treatment of Lactobacillus plantarum WH021 significantly reduced the score of mouse colon tissue ( p <0.0001), especially the intestinal gland morphological damage and inflammatory cell infiltration were improved visually. In addition, the number of goblet cells in the colon glands was significantly increased ( p <0.05), promoted the production of mucus and improved the pathological damage of crypt structure. 9 CFU / day and 10 8 CFU / day) of Lactobacillus plantarum WH021 has a repairing effect on intestinal pathological damage in neuroinflammatory mice.

[0082] 2.13 Effect of WH021 on colon barrier integrity: Tight junction (TJ) is composed of a group of protein complexes located at the top of the cell. It plays an important role in maintaining cell morphology and forming a barrier to prevent the invasion of harmful substances such as pathogens. For example, it has an important impact on the blood-brain barrier function in the previous chapter and in this chapter on the protection of the intestinal epithelial barrier function.

[211] In order to evaluate the effect of Lactobacillus plantarum WH021 on the colon barrier integrity of mice with neuroinflammation, the present invention detected and analyzed the expression levels of occludin, claudins and zona occludens 1 (ZO-1) mRNA. Fig.16 As shown in Figure 2, compared with the NC group, the expression levels of Occludin, ZO-1, and Claudin-1 mRNA were significantly decreased after LPS induction ( p <0.05), while the high-dose (H+LPS group) bacterial solution treatment significantly reversed this phenomenon. The results showed that 10 9CFU / day preventive intake of Lactobacillus plantarum WH021 can repair the damage of the intestinal barrier under LPS-induced neuroinflammatory state.

[0083] 2.14 Effect of WH021 on colon inflammatory factors: It is reported that LPS can be recognized by Toll-like receptor 4 (TLR4) to activate the innate immune system, leading to the activation of NF-κB and the production of proinflammatory cytokines and inducing intestinal inflammatory response through signal cascade reaction. Therefore, the present invention evaluated the effect of Lactobacillus plantarum WH021 on intestinal inflammation in neuroinflammatory mice by measuring the transcription levels of IL-1β and TNF-α genes in colon tissue. The results are as follows Fig.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 between the NC group and the WH021 group ( p >0.05), firstly indicating that the intake of Lactobacillus plantarum WH021 by healthy mice did not cause abnormal intestinal inflammation. However, the mRNA expression levels of IL-1β and TNF-α in mice in the LPS model group were significantly increased ( p <0.05), and different doses of Lactobacillus plantarum WH021 in advance intervention can significantly downregulate the mRNA expression of the above two pro-inflammatory factors in the colon tissue of mice with LPS-induced neuroinflammation. Therefore, the results show that Lactobacillus plantarum WH021 at three dose concentrations can alleviate the colonic inflammatory state of mice with neuroinflammation.

[0084] 2.15 Effect of WH021 on the structure of intestinal flora: β-diversity was used to further compare the degree of difference in the composition of microbial communities of different samples. Its representation method can be presented by principal component analysis (PCA) and principal coordinates analysis (PCoA), respectively. As a commonly used dimensionality reduction analysis method, the differences and distances in the flora structure can be reflected by analyzing the characteristic OTUs composition of different samples. Among them, PCA principal component analysis uses variance decomposition to reflect the differences between multiple groups of data on a two-dimensional coordinate graph, and the coordinate axes take the two eigenvalues ​​that can maximize the variance. PCoA can reduce the dimension based on the distance or similarity matrix between samples on the basis of PCA analysis, and more effectively maintain the relative position relationship between samples, which is particularly suitable for exploring sample classification or clustering patterns in complex data sets. Therefore, the combination of the two can be applied to the analysis and display of β-diversity in the structure and composition of intestinal flora in the present invention, and the results are shown in the figure. Fig.18A and B in the figure can intuitively show that there is a clear separation of the intestinal flora of mice between the NC group and the LPS group, indicating that the intestinal flora structure of mice with LPS-induced neuroinflammation is significantly different from that of healthy mice in the NC group. After pre-intervention with live Lactobacillus plantarum WH021 bacteria, although the distribution of its intestinal flora structure overlapped to a certain extent with the LPS group, it showed a trend of transition to a normal intestinal flora structure, indicating that the bacteria have a restorative effect on the abnormal flora structure caused by LPS.

[0085] 2.16 Effect of WH021 on serum metabolites: Metabolites are the basis and direct manifestation of the phenotype of an organism. Gut flora metabolites can enter the peripheral circulation of the blood through the intestinal barrier and act on the brain. In order to explore the potential mechanism by which Lactobacillus plantarum WH021 alleviates neuroinflammation, serum non-targeted metabolomics was further used to explore the differential metabolites between mice in different groups. After processing the raw data, 2,670 and 3,254 peaks were detected in the positive ion mode and negative ion mode of all samples, respectively, of which 1,081 and 1,181 metabolites were annotated, respectively. The present invention integrates and analyzes the metabolites detected and annotated in the positive and negative ion modes, and uses partial least squares discriminant analysis (PLS-DA) to display the overall differences between samples in each group and the variation range between samples within the group. Fig.19 Observation of the sample distribution showed that there were obvious separate clusters among the NC normal control group, LPS model group, and H+LPS high-dose group, indicating that the serum metabolites of LPS-induced neuroinflammatory model mice were significantly different from those of normal mice. The H+LPS high-dose group was also clearly distinguished from the LPS model group and tended to approach the NC normal control group, indicating that Lactobacillus plantarum WH021 had a positive restorative regulatory effect on the serum metabolites of neuroinflammatory mice.

[0086] Based on the analysis results of the OPLS-DA model, the importance projection (Variable Importance in Projection, VIP)> 1, p -value<0.05 and fold change>1 (upregulation) or <1 (downregulation) were used as parameter conditions to screen the differential metabolites between different groups. Overall, 395 differential 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. Fig. 20 As shown, it can be seen from the difference distribution results of the metabolite volcano map ( Fig. 20In A and C, there were 137 significantly upregulated metabolites and 258 significantly downregulated metabolites in the LPS group compared with the NC group; there were 270 significantly upregulated metabolites and 133 significantly downregulated metabolites in the H+LPS group compared with the LPS group. Cluster analysis of metabolites was performed to obtain cluster heat maps, as shown in Figure 1. Fig. 20 As shown in B and D, it can be seen intuitively that there are obvious cluster differences in the metabolites of mouse serum after LPS and Lactobacillus plantarum WH021 treatment. The focus was on the reversal of differential metabolites in the serum of neuroinflammatory model mice after Lactobacillus plantarum WH021 intervention. In the serum of mice in the H+LPS group, the expression levels of the same metabolites showed different degrees of reversal, and the metabolite categories mainly covered glycerophosphates, glycosides, organic acids and their derivatives, amino acids and peptide analogs, steroids and their derivatives, etc.

[0087] Specifically, among its differential metabolites, phosphatidylserine (PS (14:0 / 22:0)), glutamic acid (L-Glutamate), tuftsin, pinolenic acid and 5,6-dehydro arachidonic acid (5,6-dehydro Arachidonic Acid) are all active substances in nerves and inflammation, which can participate in the metabolism of the nervous system and the inflammation regulation process in the immune system. The specific changes in different groups are as follows: Fig.21 As shown in the figure, after preventive administration of Lactobacillus plantarum WH021, the above metabolites completely returned to normal levels. It is reported that abnormal lipid metabolism may lead to damage or even death of nerve cells, so it plays an important role in the central nervous system. Among them, phosphatidylserine (PS) is a key phospholipid in the brain. By regulating the fluidity and permeability of cell membranes, it plays a very important role in cell membrane communication, neurotransmitter release, and signal transmission; while glutamate is the main excitatory neurotransmitter in the mammalian brain, which can be mutually converted with γ-aminobutyric acid and maintain a relative balance. Studies have shown that glutamate-mediated neurotransmitter disorders are involved in the occurrence and development of a variety of neuropsychiatric diseases, including schizophrenia, Alzheimer's disease, affective disorders, etc. Tuftsin is a tetrapeptide (Thr-Lys-Pro-Arg). As an immune-enhancing molecule, it 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, a large number of studies have confirmed that arachidonic acid, arachidonic acid and their derivatives have significant anti-inflammatory effects. Among them, Chen et al. found that arachidonic acid can significantly reduce the secretion level of pro-inflammatory mediators in a neuroinflammation model of mouse microglial BV-2 cells stimulated with LPS.

[0088] In addition, by comparing the serum differential metabolites between the H+LPS group and the LPS group and between the LPS group and the NC group, new differential metabolites were found. That is, no differential expression was observed during the treatment of the LPS-induced neuroinflammation model, while intervention with Lactobacillus plantarum WH021 led to the appearance of these new metabolites. Among them, 5-hydroxyindole-3-acetic acid (5-HIAA) is one of the main metabolites of serotonin (5-HT), an important neurotransmitter in the body. As an indole derivative, 5-HIAA is considered to be a ligand of the aryl hydrocarbon receptor (AhR), which is an important hub for regulating intestinal immunity and stimulating downstream cascades, and is of great significance for maintaining the balance of immune-inflammatory related systems.

[0089] 2.17 Analysis of differential metabolic pathways: Complex metabolic reactions and their regulation in organisms do not proceed independently, but are often formed by complex pathways and networks of different genes and proteins. Their mutual influence and mutual regulation ultimately lead to systematic changes in the metabolome. The analysis of these metabolic and regulatory pathways can provide a more comprehensive and systematic understanding of biological issues such as changes in biological processes caused by changes in experimental conditions, the mechanisms of occurrence of traits or diseases, and drug action mechanisms. The present invention uses the KEGG (Kyoto Encyclopedia of Genes and Genomes) database to study the above-mentioned differential metabolite contents as a whole network, focusing mainly on the classification and enrichment of the KEGG metabolic pathways of differential metabolites between the H+LPS group and the LPS group. The results are as follows: Fig. 22 As shown in A in the figure, it can be observed that the metabolic pathways with a large number of differential metabolites are mainly lipid metabolism (arachidonic acid metabolic pathway and steroid hormone biosynthesis pathway) and nervous system (5-hydroxytryptamine synaptic pathway). Further enrichment of KEGG of differential metabolites ( Fig. 22 B) and enrichment factor bubble chart ( Fig.23 ) It can be seen that the main mechanism by which Lactobacillus plantarum WH021 alleviates neuroinflammation is through regulating the arachidonic acid metabolism pathway, serotonergic synapse pathway, GABAergic synapse, ferroptosis pathway and FoxO signaling pathway.

[0090] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum having the effect of preventing neuroinflammation, characterized in that: The strain was named Lactobacillus plantarum ( Lactiplantibacillus plantarum ) WH021, and was deposited in the Guangdong Provincial Microbiological Culture Collection Center, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province on April 23, 2023, with the deposit number: GDMCC No: 63388.

2. A microbial preparation, characterized in that: The invention comprises the plant lactobacillus or its culture as claimed in claim 1.

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

4. Use of the plant lactobacillus described in claim 1 or the biological preparation described in any one of claims 2 to 3 in the preparation of a product having at least one of the following functions: (1) Prevent and / or alleviate anxiety-depression comorbidity, depression or anxiety disorders; (2) Assist in improving memory function; (3) Prevent and / or alleviate neurodegenerative diseases; (4) Prevent and / or alleviate neuroinflammation; (5) Assist in improving tissue structure damage caused by neuroinflammation.

5. The use according to claim 4, characterized in that: The neurodegenerative disease includes at least one of Alzheimer's disease, Parkinson's disease, Lewy body dementia, Huntington's disease and multiple sclerosis.

6. The use according to claim 4, characterized in that: The prevention, alleviation and / or treatment of neuroinflammation includes at least one of lowering the level of pro-inflammatory factors and increasing the level of anti-inflammatory factors.

7. The use according to claim 4, characterized in that: The tissue structure damage includes at least one of oxidative stress damage to brain tissue, dysfunction of the hypothalamus-pituitary-adrenal axis, brain pathological damage, blood-brain barrier damage, intestinal pathological damage, intestinal barrier damage, abnormal flora structure in the colon inflammatory state, and abnormal serum metabolism.

8. The use according to claim 4, characterized in that: The products described herein work by regulating the arachidonic acid metabolic pathway, the serotonergic synaptic pathway, the GABAergic synaptic pathway, the ferroptosis pathway, and the FoxO signaling pathway.

9. The use according to any one of claims 4 to 8, characterized in that: The products include but are not limited to food, health products, medicines and preparations that can be added to the above products in the form of raw materials.

10. The use according to any one of claims 4 to 8, characterized in that: The dosage form of the product is an oral preparation or an injection preparation.

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