Application of a lactobacillus reuteri in preparation of a medicine for preventing, alleviating and / or treating cognitive dysfunction

Abstract

This invention provides the application of a strain of *Lactobacillus reuteri* in the preparation of drugs for the prevention, relief, and / or treatment of cognitive impairment, belonging to the field of neuroprotection and microecological regulation technology. The *Lactobacillus reuteri* strain is designated ATCC 23272; the cognitive impairment is caused by exposure to environmental pollutants. In this invention, colonization of *Lactobacillus reuteri* ATCC 23272 can activate the IAA / AHR / Wnt9a / PLD3 signaling pathway, increase the expression levels of Wnt9a and PLD3 proteins in DEP-exposed mice, effectively restore DEP-induced neuronal damage, and has a certain effect in preventing, alleviating, or treating DEP-induced cognitive impairment.

Description

Technical Field

[0001] This invention belongs to the field of neuroprotection and microecological regulation technology, and in particular relates to the application of a strain of Lactobacillus reuteri in the preparation of drugs for the prevention, relief and / or treatment of cognitive impairment. Background Technology

[0002] In recent years, with the acceleration of global urbanization, neurological diseases caused by air pollution have received increasing attention. Diesel exhaust particulate matter (DEP), as a major component of fine particulate matter (PM2.5), has been proven to be closely related to cognitive impairment, Alzheimer's disease, and other neurodegenerative diseases. DEP can induce neuroinflammation, oxidative stress, and neuronal damage through the blood-brain barrier or the gut-brain axis, but there are currently no targeted interventions available clinically. Summary of the Invention

[0003] The purpose of this invention is to provide the application of a strain of Lactobacillus reuteri in the preparation of a drug for the prevention, relief and / or treatment of cognitive impairment, which can prevent, improve or treat cognitive impairment caused by DEP exposure, and solve the problem of the lack of effective intervention methods for nerve damage caused by environmental pollutants in the prior art.

[0004] This invention provides the use of a strain of Lactobacillus reuteri in the preparation of a medicament or cognitive protectant for the prevention, relief and / or treatment of cognitive impairment, wherein the strain number of Lactobacillus reuteri is ATCC 23272; and the cognitive impairment is cognitive impairment caused by exposure to environmental pollutants.

[0005] Preferably, the environmental pollutants include air pollutants; the environmental pollutants include environmental neurotoxins.

[0006] Preferably, the air pollutants include diesel engine exhaust particulate matter.

[0007] Preferably, the prevention, mitigation and / or treatment of cognitive impairment includes at least one of the following: 1) improving the exploratory ability of patients with cognitive impairment; 2) improving the motor coordination of patients with cognitive impairment; 3) improving the ability of patients with cognitive impairment to recognize new objects.

[0008] Preferably, the cognitive impairment includes one or more of the following: decreased ability to explore new environments, weakened voluntary motor ability, and reduced ability to recognize new objects.

[0009] Preferably, the mode of exposure to the environmental pollutants includes nasal inhalation.

[0010] This invention also provides the application of a strain of Lactobacillus reuteri in the preparation of a drug that activates the IAA / AHR / Wnt9a / PLD3 signaling pathway, wherein the strain number of Lactobacillus reuteri is ATCC 23272; the cognitive impairment is cognitive impairment caused by exposure to environmental pollutants.

[0011] Preferably, the dosage form of the drug includes an oral formulation or a gavage formulation.

[0012] Preferably, the concentration of Lactobacillus reuteri in the drug is 10. 8 CFU / mL.

[0013] Preferably, the drug is a unit-dose formulation; based on mice, the unit-dose formulation is prepared to facilitate administration at a dose of 200 μL / mouse.

[0014] This invention provides the application of a strain of *Lactobacillus reuteri* in the preparation of drugs or cognitive protective agents for the prevention, alleviation, and / or treatment of cognitive impairment. The strain number of *Lactobacillus reuteri* is ATCC 23272. The cognitive impairment is cognitive impairment caused by exposure to environmental pollutants. In this invention, colonization of *Lactobacillus reuteri* ATCC23272 can activate the IAA / AHR / Wnt9a / PLD3 signaling pathway, increase the expression levels of Wnt9a and PLD3 proteins in DEP-exposed mice, effectively restore DEP-induced neuronal damage, and has a certain effect in preventing, alleviating, or treating cognitive impairment caused by DEP exposure. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Figure 1 shows the effect of DEP exposure on the ability of mice to explore novel environments; where a is the movement trajectory of the mouse in the box; b is the total distance the mouse moves in the box (mm); c is the time the mouse spends in the area around the box (s); and d is the time the mouse spends on the central platform of the box (s). Figure 2 The figure shows the effect of DEP exposure on the cognitive function of SPF mice; where a is the movement trajectory of the mouse in the box; and b is the recognition index (%) of the mouse for new objects. Figure 3Figure 1 shows the results of Lr bacterial colonization in alleviating the decreased exploration ability of mice exposed to novel environments after DEP exposure; where a is the movement trajectory of the mouse in the box; b is the total distance the mouse moves in the box (mm); c is the time the mouse spends in the area around the box (s); and d is the time the mouse spends on the central platform of the box (s). Figure 4 Figure 1 shows the results of Lr bacterial colonization in alleviating cognitive impairment in DEP-exposed mice; where a is the movement trajectory of the mouse in the box; and b is the recognition index (%) of the mouse for new objects. Figure 5 Figure showing the results of Lr bacterial colonization alleviating hippocampal neuronal damage in DEP-exposed mice; Figure 6 Figure 1 shows the results of improving lysosomal damage in DEP-exposed mice by IAA / AHR / Wnt9a / PLD3 axis for Lr bacterial colonization; where a represents the expression of Wnt9a and PLD3 proteins in mouse hippocampus; b represents the expression level of Wnt9a protein in mouse hippocampus; and c represents the expression level of PLD3 protein in mouse hippocampus. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0023] This invention provides a strain of Lactobacillus reuteri ( Limosilactobacillus_reuteri The use of Lactobacillus reuteri (Lr) in the preparation of medicaments or cognitive protective agents for the prevention, relief and / or treatment of cognitive impairment, wherein the strain number of Lactobacillus reuteri is ATCC 23272; and the cognitive impairment is cognitive impairment caused by exposure to environmental pollutants.

[0024] In one implementation, the *Lactobacillus reuteri* prevents, alleviates, and / or treats cognitive impairment by improving neuronal damage and / or repairing hippocampal neuronal structure. In one implementation, improving neuronal damage includes improving neuronal lysosomal damage.

[0025] As one implementation method, the prevention, mitigation, and / or treatment of cognitive impairment includes at least one of the following: 1) Enhance the exploratory abilities of patients with cognitive impairment; 2) Improve motor coordination in patients with cognitive impairment; 3) Improve the ability of patients with cognitive impairment to recognize new objects.

[0026] In one implementation, the environmental pollutants include air pollutants; the environmental pollutants include environmental neurotoxins.

[0027] In one embodiment, the air pollutant includes diesel engine exhaust particulate matter. In another embodiment, the particle size of the diesel engine exhaust particulate matter is less than 100 nm.

[0028] As one implementation, the cognitive impairment includes one or more of the following: decreased ability to explore new environments, weakened voluntary motor ability, and reduced ability to recognize new objects.

[0029] As one implementation method, the environmental pollutant exposure includes nasal inhalation.

[0030] This invention also provides the application of a strain of Lactobacillus reuteri in the preparation of a drug that activates the IAA / AHR / Wnt9a / PLD3 signaling pathway, wherein the strain number of Lactobacillus reuteri is ATCC 23272; the cognitive impairment is cognitive impairment caused by exposure to environmental pollutants.

[0031] In one implementation, the Lactobacillus reuteri metabolizes tryptophan to generate indoleacetic acid (IAA), thereby activating the IAA / AHR / Wnt9a / PLD3 signaling pathway.

[0032] In one embodiment, the dosage form of the drug includes an oral formulation or a gavage formulation.

[0033] In the efficacy verification process, the present invention was colonized in the intestines of a diesel exhaust particulate matter (DEP) exposure model mouse via gavage. Specifically, the DEP exposure model mice were gavaged at a dose of 200 μL / mouse once a day for 4 consecutive weeks.

[0034] In this invention, the method for constructing the DEP exposure model mouse is as follows: DEP particles with a particle size of less than 100 nm are administered to C57BL / 6 mice via nasal drip at a dose of 5 mg / kg, three times a week for four weeks. The improvement in cognitive function is evaluated through behavioral tests (open field test, new object recognition test) and pathological analysis (Nissl staining, immunohistochemistry).

[0035] In one embodiment, the drug is a live bacterial preparation; the concentration of Lactobacillus reuteri in the drug is 10. 8 CFU / mL, further increased to 1×10 8 CFU / mL. As one embodiment, the *Lactobacillus reuteri* in the drug exists in the form of a bacterial suspension. As one embodiment, the bacterial suspension of *Lactobacillus reuteri* is prepared by the following method: *Lactobacillus reuteri* ATCC 23272 strain is anaerobically cultured and centrifuged to prepare a bacterial suspension.

[0036] As one implementation method, the culture conditions for the Lactobacillus reuteri are as follows: using MRS medium containing 0.5% tryptophan by volume, cultured in an anaerobic environment at 37°C until the early exponential phase.

[0037] As one embodiment, the method for preparing the bacterial suspension of Lactobacillus reuteri includes the following steps: (1) The frozen Lactobacillus reuteri ATCC 23272 strain was inoculated into MRS liquid medium and activated to obtain activated bacterial solution; (2) The activated bacterial solution was inoculated into MRS medium containing 0.5% tryptophan at a volume ratio of 1:50 and cultured to the logarithmic growth phase to obtain the culture product, OD 600 =0.6~0.8; (3) Centrifuge the expanded culture product, collect the precipitate, and obtain bacterial cells; (4) The bacterial cells are resuspended to obtain a bacterial suspension.

[0038] As one implementation method, the activation culture conditions are: anaerobic culture at 37℃ for 24 hours; the expansion culture conditions are: anaerobic culture at 37℃; the centrifugation program is: centrifugation at 4℃ and 5000 rpm for 5 minutes; the resuspension reagents include PBS buffer, further, sterile PBS buffer, and even further, sterile phosphate buffer at pH 7.4; the MRS medium is prepared by adding 49.3 g of MRS medium powder to 1000 mL of distilled water, autoclaving at 121℃ for 15 minutes before use; the anaerobic culture uses a 2.5L anaerobic gas-generating bag and an anaerobic culture box to maintain the anaerobic environment; further, the 2.5L anaerobic gas-generating bag is purchased from Mitsubishi Corporation, Japan, product number: C-1; the anaerobic culture box product number: C-31; the OD of the expansion culture product... 600 =0.6~0.8; the tryptophan was purchased from Sigma-Aldrich, catalog number: T0254.

[0039] The preparation method of the present invention can stably prepare high-concentration bacterial suspensions of Lactobacillus reuteri.

[0040] In one embodiment, the drug is a unit-dose formulation; based on mice, the unit-dose formulation is prepared to facilitate administration at a dose of 200 μL / mouse.

[0041] This invention reveals for the first time that *Lactobacillus reuteri* ATCC 23272 is a functional probiotic capable of specifically regulating the gut microbiota-metabolic axis (tryptophan-IAA pathway). Its target is clearly defined: by metabolizing tryptophan to generate indoleacetic acid (IAA), it activates the IAA / AHR / Wnt9a / PLD3 signaling pathway, thereby improving DEP-induced neuronal damage and cognitive impairment. *Lactobacillus reuteri* ATCC 23272 significantly enhances exploratory ability, motor coordination, and novel object recognition in model mice, and repairs hippocampal neuronal structure. This invention provides a novel microecological regulation strategy for intervening in the neurotoxicity of environmental pollutants, overcoming the shortcomings of traditional neuroprotective drugs such as poor targeting and difficulty in penetrating the blood-brain barrier, and providing a safe and efficient microbial solution for the prevention and treatment of cognitive impairment.

[0042] In summary, the *Lactobacillus reuteri* ATCC 23272 of this invention has a clearly defined function and can exert neuroprotective effects through specific metabolic regulation to address cognitive impairment caused by environmental pollutants such as DEP. This invention provides an innovative microbial solution for neurodegenerative diseases caused by environmental pollutants, featuring a clear mechanism, simple preparation, and high safety, and has significant potential for clinical translation.

[0043] To further illustrate the present invention, the application of a strain of Lactobacillus reuteri provided by the present invention in the preparation of medicaments for the prevention, relief and / or treatment of cognitive impairment is described in detail below with reference to the accompanying drawings and embodiments, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0044] The main materials used in the embodiments of the present invention and their sources are as follows: SPF-grade C57BL / 6J mice (for establishing a mouse model of cognitive impairment): Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. The method for establishing the mouse model of cognitive impairment was as follows: each mouse inhaled 10 μg of DEP daily for four weeks.

[0045] Ultrasonic cleaner: Saif (China) Co., Ltd., Model: SB-320D-6L.

[0046] JEM-2100 Transmission Electron Microscope: JEOL Ltd., Model: JEM-2100.

[0047] Scanning electron microscope: JEOL Ltd., model: JCM-7000.

[0048] Various models of pipettes: Eppendorf AG.

[0049] Electronic balance: Shanghai Ohaus International Trading Co., Ltd., Model: Q / SGYM1008.

[0050] Open field experimental setup: Beijing Zhongshi Technology Co., Ltd.

[0051] Sealing film: Defan Instruments, Model: AM-PM996.

[0052] SMART3.0 Video Behavioral Analysis System: Beijing Zhongshi Technology Co., Ltd., Item No.: 1056000.

[0053] Sliced ​​paraffin (58-60°C): Leica GmbH, Germany, item number: P100928-500g.

[0054] Feather blade: Leica GmbH, Germany, part number: A35 blade.

[0055] Paraffin slicer: Leica GmbH, Germany, part number: 2265.

[0056] Cover glass mm: Jiangsu Shitai Experimental Equipment Co., Ltd., Item No.: 10212450C.

[0057] Cover glass Jiangsu Shitai Experimental Equipment Co., Ltd., Item No.: 10212424C.

[0058] Glass slide: Jiangsu Shitai Experimental Equipment Co., Ltd., Item No.: 188105W.

[0059] Dyeing vat: Shanghai Jinzhong Instruments.

[0060] Scissors and tweezers: Shanghai Jinzhong Instruments.

[0061] Fully automated slice scanning system: Leica GmbH, Germany.

[0062] Low-power microscope: Leica GmbH, Germany.

[0063] Refrigerator: Midea Group.

[0064] Microwave oven: Midea Group.

[0065] Refrigerator: Midea Group.

[0066] Ordinary carbon support membrane: Zhongke Instrument Co., Ltd.

[0067] Centrifuge tubes of various models: Selected Biotechnology Co., Ltd.

[0068] Enzyme-free EP tubes and pipette tips: Axygen, USA.

[0069] Insect needles: Taobao.

[0070] New and old objects in the new object experiment: Taobao.

[0071] 2.5L Anaerobic Gas Generating Bag: Mitsubishi Corporation, Japan, Item No.: C-1.

[0072] 2.5L Anaerobic Culture Box: Mitsubishi Corporation, Japan, Product No.: C-31.

[0073] Tryptophan: Sigma-Aldrich, catalog number: T0254.

[0074] MRS: Solarbio Biotechnology Co., Ltd., Product No.: M8540.

[0075] PBS: Hyclone Inc., USA, catalog number: SH30256.01.

[0076] Diesel particulate matter: National Institute of Standards and Technology, catalog number: NIST2975.

[0077] Physiological saline: Solarbio Biotechnology Co., Ltd., Product No.: IN9000.

[0078] Sliced ​​paraffin (58-60°C): Leica GmbH, Germany, item number: P100928-500g.

[0079] Xylene: Beijing Innocare Technology Co., Ltd. Reagent Website, Product No.: A62975.

[0080] Anhydrous ethanol: Beijing Innocare Technology Co., Ltd. Reagent Website, Product No.: G00004.

[0081] Neutral resin: Solarbio Biotechnology Co., Ltd., Product No.: G8590.

[0082] Hematoxylin-eosin staining kit: Beyotime Biotechnology Co., Ltd., catalog number: C0105M.

[0083] Hematoxylin staining solution: Beyotime Biotechnology Co., Ltd., product number: C0107.

[0084] Hydrochloric acid: Beijing Innocare Technology Co., Ltd. Reagent Website, Product No.: A04558.

[0085] Ammonia solution: Beijing Innocare Technology Co., Ltd. Reagent Website, Product No.: A04558.

[0086] Hydrogen peroxide solution (30%): Shanghai Aladdin Biochemical Technology Co., Ltd., Product No.: H112515.

[0087] MRS culture medium preparation: Add 49.3 g of this product to 1000 mL of distilled water, heat to dissolve, dispense, autoclave at 121℃ for 15 min, and store at 4℃; Lactobacillus reuteri ( Limosilactobacillus_reuteri The Lr (ATCC23272) was purchased from Baosai Biotechnology. The Lr was cultured in MRS medium under anaerobic conditions at 37°C.

[0088] Metabolite extraction and lyophilization of bacterial culture supernatant (for preparing subsequent nasal drop reagents in DEP-exposed mouse models): Overnight cultured bacteria were inoculated at a ratio of 1:50 into bacterial MIS medium containing 0.5% tryptophan and anaerobically incubated to the early exponential phase. The bacteria were removed from the anaerobic bag and centrifuged at 5,000 rpm for 5 min at room temperature. The supernatant was collected (using 15 mL EP tubes, aliquoted into 4 mL tubes) and immediately frozen at -80°C. After the supernatant was completely frozen, the caps of the EP tubes were opened, the tube openings were sealed with aluminum foil, and secured with rubber bands (note that 3-5 holes should be punched in the sealed foil to ensure gas flow). The processed EP tubes containing the supernatant were transferred to a lyophilizer for lyophilization.

[0089] Example 1: Construction of a DEP-exposed mouse model Animal model construction includes the following steps: (1) Animal handling: Healthy adult male C57BL / 6 mice (18-22 g) aged 6-8 weeks were selected from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Nanjing, China) and were specific pathogen-free (SPF) grade. The housing environment met the requirements and was an SPF grade laboratory with an ambient temperature of 20±2℃, relative humidity of 60±10%, and a light cycle of 12 h light / 12 h dark. The required number of mice was 5 per individual ventilated cage (IVC). According to the experimental requirements (when there were no special dietary requirements), water and food were ensured to be freely available, and the bedding was changed 2-3 times a week to ensure that the living environment of the mice was clean and comfortable. (2) DEP exposure: 20 mice in the control group and 20 mice in the exposure group. The control group mice were injected with 10 μL of PBS through the nasal cavity, and the DEP exposure group mice were injected with 10 μL of prepared DEP solution through the nasal cavity once a day. That is, each mouse inhaled 10 μg of DEP per day for four weeks. (3) Model validation: After the exposure period ended, the open field test (OFT) and the new object recognition test (NOR) were used to investigate the neurobehavioral changes in mice; H&E staining, Nissl staining and immunohistochemical staining were used to observe the effects of DEP exposure on neurons and glial cells.

[0090] like Figure 1 As shown, a is the trajectory of the mouse in the box; b is the total distance the mouse moves in the box (mm); c is the time the mouse stays in the area around the box (s); d is the time the mouse stays on the central platform of the box (s). The experimental steps of this open field experiment are as follows: (1) The open field experiment is set with a length, width and height of 30cm and a bottom surface composed of 16 squares of equal area (7.5cm x 7.5cm). The ambient light is kept dim during the experiment. A camera is set up above to record the movement of the animals in real time; (2) Each mouse is taken out after acclimatizing in the open field for 2 minutes before the experiment. Before each replacement of the mouse, urine and feces must be wiped off with a paper towel and the box must be wiped with alcohol; (3) Each mouse is tested once during the experiment, and each test lasts for 5 minutes; (4) Record the total distance the mouse travels and the time it stays in the central area.

[0091] like Figure 2As shown, a is the trajectory of the mouse in the box; b is the recognition index (%) of the mouse for the new object. The experimental steps of this part of the new object recognition experiment are as follows: (1) Experimental preparation: 3 days before the start of the experiment, the experimenters contacted the mice for 1-2 minutes every day to prevent the mice from having a stress response during the experiment; before the start of the experiment, the new object recognition box without objects was sprayed with 75% alcohol and water and wiped clean with paper towels; before the start of the experiment, the mice were moved to the experimental room equipped with the new object recognition device to adapt for half an hour; the behavioral video recording device was adjusted, the camera was facing the new object recognition device, and all the space in the box was within the recording range, and the recording screen was as parallel as possible to the edge of the screen to ensure that the video clarity was normal and the video could be recorded normally; (2) First stage, training stage: two cylindrical objects of the same color and shape were placed on the ground 5-8 cm away from the wall of the experimental device box, and the bottom of the objects was fixed with double-sided tape to prevent them from moving; the experimental model mice were gently taken out of the breeding cage and placed in the middle of the new object recognition box, allowing the mice to explore freely in the box for 5 minutes. min; After recording, take the mouse out and place it in a temporary feeding cage, and give it water and food in time; After the mouse is taken out, spray the new object identification box with 75% alcohol and water and wipe it with dry paper towels in turn to eliminate odor and ensure that the box does not reflect light in the video; This stage lasts for 2 days; (3) Second stage, test stage: The second stage can start one day after the end of the first stage. Generally, the longer the time, the higher the memory requirement for the model mouse; replace one of the old objects in the second stage with a new object. The color and shape of the new object are different from the old object. Place the new object on the ground 5-8 cm away from the wall of the experimental device box. Fix the bottom of the new object with double-sided tape to prevent it from moving; after adjusting the recording parameters and screen, gently take the experimental mouse out of the breeding cage and place it in the center of the new object recognition box. Let the mouse explore freely for 5 / 10 min; the experimental personnel in the experimental room retreat to a position where the mouse cannot see and click to record the video. The recording time is 5 / 10 cm. Save the recorded video; after the recording is completed, take the mouse out and place it in a temporary breeding cage. Give it water and food in time; after the mouse is taken out, spray the new object recognition box with 75% alcohol and water and wipe it with dry paper towels in turn to eliminate the odor and ensure that the box does not reflect light in the video; continue the behavioral test of the next mouse; (4) after the video recording is completed, use the SMART3.0 video behavioral analysis system to perform trajectory analysis, calculate the time when the mouse comes into contact with the new object and the old object, and calculate the new object recognition index (recognition). The new object recognition index (RI) indicates that the more time a mouse spends around a new object, the better its cognitive ability. The formula for calculating the new object recognition index is: RI = Time spent around the new object / (Time spent around the new object + Time spent around the old object) × 100%. The higher the index, the stronger the mouse's cognitive ability.

[0092] Compared with the control group, the novel object recognition index of mice in the DEP-exposed group was significantly reduced. However, Lr bacterial colonization effectively improved the novel object recognition index of DEP-exposed mice.

[0093] Example 2: Preparation of a cognitive function protective agent for Lactobacillus reuteri A method for preparing a cognitive function protective agent based on Lactobacillus reuteri includes the following steps: (1) Preparation of MRS culture medium: Take 49.3 g of MRS and add it to 1000 mL of distilled water, heat, dissolve, dispense, autoclave at 121℃ for 15 min, and store at 4℃ for later use; (2) Lactobacillus reuteri ( Limosilactobacillus_reuteri Lr (ATCC23272) was purchased from Baosai Biotechnology. Lr was cultured in MRS medium under anaerobic conditions at 37°C. (3) Extraction and lyophilization of bacterial culture supernatant (for evaluating the intervention effect of lyophilized bacterial agents): Bacteria cultured overnight were inoculated into bacterial MIS medium containing 0.5% tryptophan at a volume ratio of 1:50 and anaerobic incubated until the early exponential phase. Bacteria were removed from the anaerobic bag and centrifuged at 5,000 rpm for 5 minutes at room temperature. The centrifuged bacterial cells were resuspended in sterile phosphate buffer at pH 7.4 to obtain a concentration of 1×10⁻⁶. 8 CFU / mL bacterial suspension. Remove the supernatant (using 15 mL EP tubes, aliquoting 4 mL into each tube) and immediately freeze at -80°C. After the supernatant is completely frozen, open the cap of the EP tube, wrap the opening with aluminum foil, and secure it with a rubber band (make sure to poke 3-5 holes in the sealed foil to ensure airflow). Transfer the treated EP tubes containing the supernatant to a freeze dryer for lyophilization.

[0094] Example 3 Evaluation of the effect of microbial agent intervention Includes the following steps: (1) The model mice obtained in Example 1 were divided into a model group and a probiotic gavage group, using the probiotic prepared in Example 2 at a concentration of 1×10⁻⁶. 8 The probiotic gavage group was administered CFU / mL bacterial suspension by gavage, with each mouse receiving 200 μL of gavage, for a total of 4 weeks; the model group was administered an equal volume of physiological saline by gavage. (2) Behavioral experiments were used to detect the effects of gut microbiota and DEP exposure on the ability of GF mice to explore new environments, their ability to move independently, and their ability to recognize new objects. Nissl staining and immunohistochemical staining were used to observe the effects of gut microbiota and DEP exposure on neurons, microglia, and astrocytes in GF mice.

[0095] like Figure 3As shown, a) represents the mouse's movement trajectory within the enclosure; b) represents the total distance the mouse traveled within the enclosure (mm); c) represents the time the mouse spent in the area surrounding the enclosure (s); and d) represents the time the mouse spent on the central platform of the enclosure (s). *L. erinaceus* bacterial colonization effectively increased the total distance traveled by DEP-exposed mice within the enclosure and the time spent on the central platform area, while decreasing the time spent in the outer periphery of the enclosure. In conclusion, *L. erinaceus* bacterial colonization can effectively restore the ability of DEP-exposed mice to explore novel environments, and this protective ability may be related to the production of IAA by *L. erinaceus*.

[0096] like Figure 4 As shown, a) represents the movement trajectory of the mouse within the box; b) represents the mouse's recognition index (%) of novel objects. Compared with the control group, the novel object recognition index of mice in the DEP-exposed group was significantly reduced. Lr bacterial colonization effectively improved the novel object recognition index of DEP-exposed mice, suggesting that Lr bacterial colonization can alleviate cognitive impairment in DEP-exposed mice.

[0097] like Figure 5 As shown, Nissl staining was performed on brain tissue sections of model mice. The experimental steps for this part are as follows: (1) Drying: Place the brain tissue sections that need to be dewaxed in the oven in advance at 65℃ for 1 h; (2) Gradient dewaxing: Take the sections out of the oven and perform gradient dewaxing in the following order and time (xylene, 15 min; xylene, 15 min; anhydrous ethanol, 2 min; anhydrous ethanol, 2 min; 95% ethanol, 2 min; 70% anhydrous ethanol, 2 min; distilled water, 2 min); (3) Nissl staining: Stain with Nissl staining solution for 15 min (if it is a frozen section, due to the thickness of the tissue, the temperature and staining time need to be increased, generally around 45 min); rinse once with distilled water (note that you should not stay in the distilled water, just rinse); (4) After quickly absorbing the water on the section rack with filter paper, place the section in 95% ethanol for about 2 s; (5) Dehydration: Soak in 95% ethanol for 2 min, soak in anhydrous ethanol for 2 s. min; (6) Mounting: Mount with neutral resin (the concentration of neutral resin should not be too high, dilute with xylene, and air bubbles are likely to occur when using too high a concentration of neutral resin for mounting). Under the microscope, the cells show mottled blue-purple staining; (7) Scanning tissue sections: Scan and photograph the mouse tissue pathological sections using a fully automated section scanning system. Lr bacterial colonization alleviated DEP-exposed mouse hippocampal neuronal damage. Compared with the control group, the hippocampal neurons in the DEP-exposed group mice were more disordered, the number of layers was reduced, and the Nissl staining color was lighter. Lr bacterial colonization can effectively restore DEP-induced neuronal damage and has certain preventive and therapeutic effects.

[0098] like Figure 6As shown, Lr bacterial colonization improves neuronal lysosomal damage in DEP-exposed mice through the IAA / AHR / Wnt9a / PLD3 axis. Compared with the control group, DEP exposure significantly reduced the expression of Wnt9a and PLD3 proteins in hippocampal neurons, while Lr bacterial colonization increased the expression levels of Wnt9a and PLD3 proteins in DEP-exposed mice.

[0099] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The use of a strain of Lactobacillus reuteri in the preparation of a medicament or cognitive protectant for the prevention, relief, and / or treatment of cognitive impairment, characterized in that, The strain number of the Lactobacillus reuteri is ATCC 23272; the cognitive impairment is cognitive impairment caused by exposure to environmental pollutants.

2. The application according to claim 1, characterized in that, The environmental pollutants include air pollutants; the environmental pollutants include environmental neurotoxins.

3. The application according to claim 2, characterized in that, The air pollutants include particulate matter from diesel engine exhaust.

4. The application according to claim 1, characterized in that, The prevention, mitigation, and / or treatment of cognitive impairment includes at least one of the following: 1) Enhance the exploratory abilities of patients with cognitive impairment; 2) Improve motor coordination in patients with cognitive impairment; 3) Improve the ability of patients with cognitive impairment to recognize new objects.

5. The application according to claim 1, characterized in that, The cognitive impairment includes one or more of the following: decreased ability to explore new environments, weakened voluntary motor ability, and reduced ability to recognize new objects.

6. The application according to claim 1, characterized in that, The mode of exposure to the environmental pollutants includes nasal inhalation.

7. The application of a strain of Lactobacillus reuteri in the preparation of drugs that activate the IAA / AHR / Wnt9a / PLD3 signaling pathway, characterized in that, The strain number of the Lactobacillus reuteri is ATCC 23272; the cognitive impairment is cognitive impairment caused by exposure to environmental pollutants.

8. The application according to any one of claims 1 to 7, characterized in that, The dosage forms of the drug include oral formulations or oral administration formulations.

9. The application according to claim 8, characterized in that, The concentration of Lactobacillus reuteri in the drug is 10. 8 CFU / mL.

10. The application according to claim 8 or 9, characterized in that, The drug is a unit dose formulation; based on mice, the unit dose formulation is prepared to facilitate administration at a dose of 200 μL / mouse.

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