Application of probiotic composition in preparation of medicine for preventing and / or treating Alzheimer's disease

Abstract

The invention provides application of a probiotic composition in preparation of a medicine for preventing and / or treating Alzheimer's disease, and belongs to the technical field of probiotic medical application. The probiotic composition disclosed by the invention is prepared from lactobacillus rhamnosus, bifidobacterium lactis and bifidobacterium bifidum. According to the probiotic composition disclosed by the invention, on the phylum level, the relative abundance of Verrucomicrobiota (verruca microflora phylum) is improved by the probiotic composition, and the relative abundance of the verrucomicrobiota (verruca microflora phylum) is improved by the probiotic composition; in the level of genus, the probiotic composition increases the relative abundance of the beneficial bacteria genus Akkermansia (Ackermania genus) and Bifidobacterium (Bifidobacterium genus), and in the level of genus Akkermania (Ackermania genus) and Bifidobacterium (Bifidobacterium genus); the behavioral cognition level can be remarkably improved, neuron injury is improved, and intestinal barrier injury is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of the medical use of probiotics, and particularly relates to the application of a probiotic composition in the preparation of a drug for preventing and / or treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is the most common type of dementia and is a fatal and disabling neurodegenerative disease, mainly characterized by progressive cognitive and memory decline. With the aging of the global population, the incidence and prevalence of AD are increasing year by year. More seriously, there is currently no effective treatment to stop the progression of AD. As AD progresses, its associated pathological features continue to increase. Two well-known pathological features include extracellular senile plaques formed by β-amyloid (Aβ) and intracellular neurofibrillary tangles (NFTs) formed by hyperphosphorylated tau protein. Therefore, a deep understanding of the pathogenesis of AD and the development of new disease-modifying treatment strategies are of crucial significance for slowing cognitive decline and improving the quality of life of AD patients.

[0003] Recent clinical and preclinical studies have highlighted the dysregulation of the gut microbiota as an important factor in the occurrence and progression of AD, which is mainly regulated through the "microbiota-gut-brain axis". Meanwhile, preclinical studies have shown that various intervention strategies targeting the gut microbiota, such as probiotics, prebiotics, antibiotics, and fecal microbiota transplantation (FMT), have the potential to alleviate cognitive impairment and pathological changes in AD mouse models. Among these interventions, probiotic therapy is considered a safe and effective innovative strategy for delaying the progression of AD. For example, it has been found that the combination of multiple probiotics (Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum) with selenium, or a probiotic mixture (Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium lactis, and Lactobacillus fermentum) can improve the Mini-mental State Examination (MMSE) scores and some metabolic indices of AD patients. The current research results suggest that probiotics may play a role in delaying AD, but their molecular mechanisms have not been fully elucidated. In addition, the optimal combination of different probiotics to achieve better therapeutic effects needs to be further explored. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide the application of a probiotic composition composed of Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum in the preparation of a drug for preventing and / or treating Alzheimer's disease.

[0005] To achieve the above-mentioned invention purposes, the present invention provides the following technical solutions:

[0006] The present invention provides an application of a probiotic composition in the preparation of a drug for preventing and / or treating Alzheimer's disease, and the probiotic composition is composed of Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum.

[0007] Preferably, the Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum are composed according to the viable cell count ratio of (1-2):(1-2):(1-2).

[0008] Preferably, the probiotic composition increases the Simpson index of the intestinal flora.

[0009] Preferably, the probiotic composition increases the relative abundance of Verrucomicrobia in the intestinal flora; the probiotic composition increases the relative abundances of Akkermansia and Bifidobacterium in the intestinal flora.

[0010] Preferably, the probiotic composition improves the behavioral and cognitive levels.

[0011] Preferably, the probiotic composition improves the neuronal damage in the cerebral cortex and hippocampus.

[0012] Preferably, the probiotic composition improves the intestinal barrier damage.

[0013] Preferably, the active ingredient of the drug is the probiotic composition, and the probiotic composition is composed of Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum.

[0014] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0015] Preferably, the dosage form of the drug includes capsules, granules, pills, and oral liquids.

[0016] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0017] The present invention uses a probiotic composition composed of three probiotics, which changes the composition of the intestinal microbiota in mice, and the Simpson index of the mice increases significantly; at the phylum level, the probiotic composition increases the relative abundance of Verrucomicrobiota; at the genus level, the probiotic composition increases the relative abundances of the beneficial genera Akkermansia and Bifidobacterium. The probiotic composition of the present invention can significantly improve the behavioral and cognitive levels of APP / PS1 mice, improve the neuronal damage in APP / PS1 mice, and significantly reduce the Aβ deposition in the cerebral cortex and hippocampus of APP / PS1 mice. The probiotic composition of the present invention can improve the intestinal barrier damage in APP / PS1 mice. Description of the Drawings

[0018] Figure 1 : Effects of mixed probiotics on the diversity and composition of the intestinal microbiota in APP / PS1 mice;

[0019] Figure 2 : Results and analysis of the water maze test and open field test in mice;

[0020] Figure 3 : Results of Nissl staining and Aβ immunofluorescence detection in the cerebral cortex and hippocampus of three groups of mice;

[0021] Figure 4 : Effects of mixed probiotics on the intestinal barrier damage in APP / PS1 mice. Detailed Embodiments

[0022] The present invention provides an application of a probiotic composition in the preparation of a drug for preventing and / or treating Alzheimer's disease, and the probiotic composition is composed of Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum. Preferably, the Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum are composed according to the viable count of (1-2):(1-2):(1-2). More preferably, the Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum are composed according to the viable count of 1:1:1.

[0023] The present invention uses a probiotic composition composed of three probiotics to explore its efficacy and molecular mechanism against AD in APP / PS1 mice. Taking age-matched C57BL / 6 mice as normal controls, three mixed probiotics are used at 1×10 9The dose of CFU per day was administered orally to 6.5-month-old APP / PS1 mice for 6 weeks. Subsequently, the present invention used 16S rRNA sequencing to analyze the intestinal flora structure and diversity of mice in each group. The results showed that the probiotic composition changed the intestinal microbiota composition of mice, and the Simpson index of mice increased significantly; at the phylum level, the probiotic composition increased the relative abundance of Verrucomicrobiota; at the genus level, the probiotic composition increased the relative abundances of the beneficial genera Akkermansia and Bifidobacterium.

[0024] The present invention evaluated the cognitive levels of APP / PS1 and C57BL / 6 mice through Morris water maze experiments and open field experiments. The results showed that the probiotic composition increased the number of times the mice crossed the platform area, and the time required for the first crossing of the platform was also shorter; it increased the number of times the mice stood, the duration in the central area, the frequency of entering the central area, and the moving distance in the central area, significantly improving the behavioral cognitive level of APP / PS1 mice.

[0025] The present invention further detected the changes in pathological features related to AD. The results showed that treatment with the probiotic composition could significantly increase the number of neurons in the cerebral cortex and hippocampus of APP / PS1 mice, thereby improving the neuronal damage in APP / PS1 mice, and could significantly reduce Aβ deposition in the cerebral cortex and hippocampus of APP / PS1 mice. At the same time, the HE staining results of the jejunum, ileum, and colon of the mice showed that the mice treated with the probiotic could significantly improve the intestinal barrier damage in APP / PS1 mice, and the expression of tight junction proteins (Claudin-1, Occludin, ZO-1) increased significantly. It was shown that treatment with the probiotic composition could significantly increase the expression of intestinal tight junction proteins in the jejunum of APP / PS1 mice, thereby further improving intestinal barrier damage.

[0026] The active ingredient of the drug of the present invention is a probiotic composition, and the probiotic composition is composed of Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum. Preferably, the Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum are composed according to the viable count ratio of (1-2):(1-2):(1-2). More preferably, the Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium bifidum are composed according to the viable count ratio of 1:1:1.

[0027] The drug of the present invention further comprises pharmaceutically acceptable excipients to ensure the convenient preparation and clinical application of the prepared pharmaceutical preparation. Preferably, the dosage forms of the drug of the present invention include capsules, granules, pills, and oral liquids. However, the dosage forms of the drug of the present invention are not limited thereto, and other achievable dosage forms are within the protection scope of the present invention.

[0028] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention.

[0029] In the specific embodiments of the present invention, Lactobacillus rhamnosus (batch number MXE20241020), Bifidobacterium lactis (batch number MXE20241029), and Bifidobacterium bifidum (batch number MXE20241029) are freeze-dried powders purchased from Shaanxi Yunqi Biotechnology Co., Ltd.

[0030] The data in the specific embodiments of the present invention are expressed as mean ± standard deviation (SD). One-way and two-way ANOVA and Kruskal-Wallis tests are used to test the significance between multiple groups, and then Tukey multiple comparison test and Dunn multiple comparison test are performed respectively. Data analysis is performed using GraphPad Prism 9.0 software.

[0031] In the following embodiments, unless otherwise specified, all are conventional methods.

[0032] The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0033] Example 1

[0034] 1. Experimental animals

[0035] Female APP / PS1 and C57BL / 6 mice at 24 weeks of age were purchased from Changzhou Cavens Experimental Animal Co., Ltd. According to the standard of 3 mice per cage, the mice were housed in the specific pathogen-free animal experiment center of Mianyang Central Hospital. The environmental conditions were room temperature 22 ± 2°C, relative humidity 55 ± 5%, and a 12-hour light / 12-hour dark cycle was maintained. All animal experiment procedures were carried out strictly in accordance with the institutional guidelines and the relevant regulations of the Council of the European Communities (86 / 609 / ECC). This experimental protocol has been approved by the Experimental Animal Ethics Committee of Mianyang Central Hospital (approval number: S20240205-01).

[0036] 2. Animal grouping and treatment

[0037] After two weeks of adaptive feeding, 6.5-month-old APP / PS1 mice were randomly divided into two groups: the APP / PS1 control group and the APP / PS1 + P3 group. Littermate wild-type C57BL / 6 mice were used as the normal control group (WT group), with 12 mice in each group. The APP / PS1 control group and the WT group were respectively given 200 μL of normal saline by gavage every day, while the APP / PS1 + P3 group received the above-mentioned mixed solution of the three probiotics (Lactobacillus rhamnosus, Bifidobacterium lactis, Bifidobacterium bifidum, dissolved in normal saline, and the dose of each probiotic was 1×10 9 CFU / mouse) by gavage. The gavage treatment was carried out for 6 weeks for subsequent experiments.

[0038] 3. Mouse feces collection and detection

[0039] The mice after the above gavage treatment were transferred to the biosafety cabinet in the operation room. The mice to be sampled were placed in a clean cage lined with sterile filter paper. Immediately after the mice defecated, fecal samples were collected. A total of 3 tubes of fecal samples were collected from each mouse, with 3 - 5 pellets in each tube. After quick-freezing in liquid nitrogen, they were stored in a -80°C refrigerator for later use. Note: When sampling different mice, new filter paper needs to be replaced, and behavioral experiments were carried out after sampling.

[0040] Twelve cases of the mouse fecal samples collected above were selected from each group, and the microbial group DNA was extracted using the Tiangen fecal microbiome DNA kit. Then, 1% agarose gel electrophoresis was used to evaluate the degradation and contamination of the detected DNA. Then, the purity of the DNA was detected using a Nanodrop spectrophotometer, and the concentration of the DNA was determined using Qubit 2.0. The qualified DNA samples were subjected to PCR amplification of the V3 - V4 variable region, and then the PCR products were used for library construction. The qualification of the samples after library construction was detected, and the qualified samples were sent to Novogene Co., Ltd. in Beijing for Illumina miSeq sequencing. The QIIME 2.0 data analysis software package was used to analyze the data downloaded from the machine.

[0041] The effects of the mixed probiotics on the intestinal flora diversity and composition of APP / PS1 mice are as Figure 1 shown. In the figure, (A) the difference in the α-diversity index Chao1 among the three groups; (B) the difference in the α-diversity index Shannon among the three groups; (C) the difference in the α-diversity index Simpson among the three groups; (D) the difference in the α-diversity index Pielou_e among the three groups; (E) the β-diversity analysis at the genus level; (F) the intestinal flora composition at the phylum level among the three groups; (G) the intestinal flora composition at the genus level among the three groups; (H) the differential species at the genus level among the three groups; (I) the non-linear discriminant analysis of the differential species between the APP / PS and APP / PS1 + P3 groups.

[0042] In the α-diversity analysis, there were no significant differences in the Chao1, Shannon, and Pielou_e indices among the three groups. However, compared with the APP / PS1 group of mice, the Simpson indices of the WT and APP / PS1+P3 groups of mice were significantly increased (A-D). Non-metric multidimensional scaling analysis showed that there was a certain distance in the microbial communities among the three groups, indicating that the intestinal microbial communities of APP / PS1 mice treated with mixed probiotics had changed to some extent (E). Further analysis of the effects of mixed probiotic treatment and saline treatment on the composition of the intestinal microbial communities of mice was carried out. Taxonomic analysis was performed on the operational taxonomic units obtained from the preliminary sequencing data, revealing the changes in the intestinal microbial composition of the three groups of mice, and showing the overall distribution of the top ten phyla and genera levels of the WT, APP / PS1, and APP / PS1 groups of mice, respectively. At the phylum level, compared with the APP / PS1 group of mice, the relative abundances of Verrucomicrobiota in the WT and APP / PS1+P3 groups of mice were higher, and there were no significant differences in the relative abundances of other major phyla (F). At the genus level, the relative abundances of the top 10 bacteria were significantly different among the three groups. For example, compared with the APP / PS1 group, the relative abundances of the beneficial genera Akkermansia and Bifidobacterium in the WT group and the APP / PS+P3 group of mice were higher (G). (H) shows the heatmap of the relative abundances of the top 10 genus-level bacteria among the three groups, and (I) shows the species with significant differences among the three groups annotated by linear discriminant analysis. Generally speaking, the above results indicate that mixed probiotic treatment can change the intestinal microbial diversity and composition of APP / PS1 mice.

[0043] 4. Morris water maze test and open field test

[0044] The Morris water maze experiment is mainly used to detect the spatial learning and memory abilities of mice. One day before the experiment, the mice were transported to the water maze experiment room in advance to allow them to adapt to the environment. The water maze experiment is divided into two stages. The first stage is called the place navigation experiment, which is carried out continuously for 5 days. First, an appropriate amount of water is injected into the maze, and the water temperature is controlled at 22±2°C. Four entry points are selected, and the maze is divided into four quadrants, with different marks pasted on each quadrant. The platform is placed in the third quadrant, 1 cm below the water surface, and the position of the platform remains unchanged during the experiment. Titanium dioxide is added to the water and stirred evenly to make the platform invisible. Using the midpoints of the pool walls of the four quadrants as the entry points, the mice are placed into the water facing the pool wall, and the time it takes for the mice to find the platform within 60 s is recorded as the escape latency. If the platform is not found within 60 s, the mice are guided to the platform position and allowed to stay for 10 s, and the escape latency is recorded as 60 s. The second stage is the spatial exploration experiment. On the 6th day of the experiment, the platform is removed, and the mice are placed into the water on the opposite side of the original platform. The water maze software is used to record the number of times the mice cross the platform, the retention time in the platform quadrant, the movement distance, the movement speed, and the movement trajectory within 60 s, and corresponding analyses are performed.

[0045] The open field experiment is mainly used to detect the emotion-related behaviors of mice, such as activity level, anxiety behavior, and exploratory behavior. The open field experiment equipment mainly consists of a white three-dimensional opaque box with a size of 45 cm×45 cm×45 cm and behavior analysis software. Before the experiment, the mice were transported to the experimental environment in advance to adapt for 1 h. Then, the corresponding parameters are set in the behavior analysis software. Each mouse is placed in the box with its back to one side wall of the box and allowed to freely explore for 10 min. The total walking distance of the mice, the activity time in the central area, the frequency and activity distance of entering the central area, and the number of standing behaviors of the mice are recorded, and corresponding analyses are performed.

[0046] The results and analyses of the Morris water maze experiment and the open field experiment of mice are as Figure 2 shown. In the figure, (A) Representative swimming trajectory diagrams of mice in each group during the water maze test stage; (B) Escape latency of mice in each group during the water maze training stage; (C) Number of times mice in each group cross the platform during the water maze test stage; (D) Average time for mice in each group to cross the platform during the water maze test stage; (E) Swimming speed of mice in each group during the water maze test stage; (F) Total swimming distance of mice in each group during the water maze test stage; (G) Representative movement trajectory diagrams of mice in each group during the open field experiment; (H) Total movement distance of mice in each group during the open field experiment; (I) Average number of standing times of mice in each group during the open field experiment; (J) Duration of mice in each group in the central area during the open field experiment; (K) Frequency of mice in each group entering the central area during the open field experiment; (L) Movement distance of mice in each group in the central area during the open field experiment.

[0047] During the 5-day exploration phase of the water maze experiment, mice in each group gradually learned to find the hidden platform. As shown in (B) of the figure, the time taken by the WT group and the APP / PS1+P3 group to find the hidden platform was significantly shorter than that of the APP / PS1 group. On the sixth day, the platform was removed to evaluate the memory ability of mice in each group within 60 seconds. The representative swimming trajectories of mice in each group within 60 seconds are shown in (A) of the figure. During the test phase, the number of times mice in the WT group and the APP / PS1+P3 group crossed the platform area was significantly more than that of the APP / PS1 group, and the time required for the first crossing of the platform was also shorter. In addition, there were no significant statistical differences in the average swimming speed and total swimming distance among the three groups of mice. (G) in the figure is the representative trajectory map of each group of mice during the open field experiment. When the three groups of mice performed the open field experiment, there was no significant difference in the total activity distance. However, the number of standing times, the duration in the central area, the frequency of entering the central area, and the moving distance in the central area of mice in the WT group and the APP / PS1+P3 group were higher than those of mice in the APP / PS1 group. The above results indicate that the treatment with three mixed probiotics can significantly improve the behavioral and cognitive level of APP / PS1 mice.

[0048] 5. Anatomical Sampling

[0049] Sampling by cardiac perfusion: Randomly select 6 mice from each group for sampling by cardiac perfusion, and collect the brain tissue and intestinal tissue of the mice for subsequent pathological analysis. Anesthetize the mice in each group with 10% chloral hydrate and fix the anesthetized mice well; use a scalpel to cut open the chest cavity, find the position of the heart, carefully insert the needle of the infusion set into the left ventricle to avoid piercing, slowly inject normal saline at 37°C to make the blood aggregation in the right auricle more obvious, then cut open the right auricle, and quickly inject 50 mL of normal saline at 37°C to wash out the blood from the right auricle; then quickly perfuse a sufficient amount of 4% paraformaldehyde at 4°C from the left ventricle to completely fix the tissues in the mouse body until the stomach of the mouse becomes hard, then quickly dissect the brain tissue on ice and place it in 4% paraformaldehyde, and fix it in a 4°C refrigerator overnight for later use. At the same time, dissect the gastrointestinal tract of the mice, separate the jejunum, ileum, and colon tissues of each mouse, and store them in 4% paraformaldehyde for testing.

[0050] Ordinary anatomical sampling: The remaining 6 mice in each group were sampled anatomically in an ordinary way. Blood, brain tissues (cerebral cortex and hippocampus), and intestinal tissues of the mice were collected for subsequent molecular biology experiments. Blood specimens of each mouse were collected by ophthalmic blood collection. The blood specimens were placed at room temperature and allowed to clot naturally for 1 h without shaking or vibration. Then, the blood specimens were centrifuged at 3000 rpm at 4 °C for 20 min, and the supernatant was transferred to a new cryotube and stored in aliquots at -80 °C in a low-temperature refrigerator for later use. The brain tissues of the mice after blood collection were quickly dissected and separated on ice, and the cerebral cortex and hippocampus of the mice were isolated, placed in a cryotube, frozen quickly in liquid nitrogen, and then stored at -80 °C in the refrigerator for standby. At the same time, the jejunum, ileum, and colon tissues of the mice were dissected and separated, placed in labeled cryotubes, frozen quickly in liquid nitrogen, and stored at -80 °C in the refrigerator for later use.

[0051] Nissl staining is mainly used to detect neuronal damage. Three brains from each group of fixed tissues were taken out, dehydrated, embedded in paraffin, and sectioned. First, dewax the paraffin sections to water: Dewax in environmental protection dewaxing agent (1), environmental protection dewaxing agent (2), and environmental protection dewaxing agent (3) for 10 min each in sequence, and then pass through absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 min each. Wash with distilled water 3 times. Then place the sections in a preheated 1% toluidine blue aqueous solution at 50 °C and stain in an incubator at 56 °C for 20 min, and wash clean with distilled water. Then differentiate with 95% alcohol or 0.1% glacial acetic acid, controlled under the microscope until the Nissl bodies are clearly shown. Finally, dehydrate quickly with absolute ethanol, clear with environmental protection clearing agent, mount the slides, and examine under the microscope. Observe the number of neurons in the cerebral cortex and hippocampus in the selected fields of view under the microscope, and perform semi-quantitative analysis using Image J software.

[0052] Immunofluorescence detection:

[0053] (1) Dewax the paraffin sections of the brain tissues to water: Dewax in environmental protection dewaxing agent (1), environmental protection dewaxing agent (2), and environmental protection dewaxing agent (3) for 10 min each in sequence, and then pass through absolute ethanol, 95% ethanol, and 75% ethanol for 5 min each. Wash with distilled water 3 times, 3 min each time, and then soak and wash.

[0054] (2) Antigen retrieval (microwave retrieval): Add antigen retrieval solution (citric acid, pH 6.0) into the retrieval box. Place the dewaxed and hydrated tissue sections into the retrieval box, and the volume of the retrieval solution should immerse the sections. Cover the lid loosely, put the retrieval box with the sections to be retrieved into the microwave oven, adjust the power of the microwave oven to the high-fire gear, and set the time to 3 minutes. After the first round of retrieval, take the retrieval box out of the microwave oven. After the retrieval solution naturally cools to room temperature (25 - 30 °C, about 1 hour), repeat the retrieval operation of the first round to perform the second and third rounds of retrieval (the cooling interval time after each retrieval is about 1 hour). After the three rounds of retrieval are completed and cooled, take the sections out of the retrieval solution and wash them 3 times with distilled water, 5 minutes each time.

[0055] (3) Circling: After circling with a histochemical pen, place the sections in TBST.

[0056] (4) Blocking: Drop 10% serum from the same source as the secondary antibody and incubate at 37 °C for 30 minutes.

[0057] (5) Incubating the primary antibody: Drain the serum, dilute the primary antibody solution with 10% serum to prepare the primary antibody working solution, and drop 50 - 100 μl (depending on the tissue size) of the primary antibody working solution on each section, and incubate overnight at 4 °C.

[0058] (6) Incubating the secondary antibody: Take out the sections from the refrigerator the next day, place them at room temperature for 15 minutes (rewarming), wash 3 times with TBST, and then soak 3 times, 3 minutes each time. Dilute the secondary antibody solution with TBST to prepare the secondary antibody working solution, and drop 50 - 100 μl (depending on the tissue size) of the secondary antibody working solution on each section, and incubate at 37 °C for 45 minutes. Wash 3 times with TBST, and then soak 3 times, 3 minutes each time.

[0059] (7) Nuclear staining: Remove TBST, drop 50 - 100 μl (depending on the tissue size) of DAPI working solution (prepared with DAPI stock solution at 1:500) on each section, stain the nucleus for 5 minutes in the dark, and then wash with TBST.

[0060] (8) Mounting: Mount with a fluorescence mounting medium and store in the dark at 4 °C.

[0061] (9) Microscopic examination: Examine under a microscope, collect and analyze images, and perform fluorescence intensity analysis using Image J software.

[0062] The results of Nissl staining and Aβ immunofluorescence detection in the cerebral cortex and hippocampus of three groups of mice are as Figure 3As shown in the figure. In the figure, (A) Nissl staining of the cerebral cortex and hippocampus of three groups of mice (scale bar is 25 μm); (B) Immunofluorescence detection of Aβ in the cerebral cortex and hippocampus of three groups of mice (scale bar is 50 μm). The results showed that compared with the APP / PS1 group of mice, probiotic treatment could significantly increase the number of neurons in the cerebral cortex and hippocampus of APP / PS1 mice, thus improving the neuronal damage in APP / PS1 mice. At the same time, the immunofluorescence detection results of Aβ deposition in the cerebral cortex and hippocampus of the three groups of mice showed that compared with the WT and APP / PS1+P3 groups of mice, the APP / PS1 group of mice had the most Aβ deposition in both the cerebral cortex and hippocampus, while probiotic treatment could significantly reduce Aβ deposition in the cerebral cortex and hippocampus of APP / PS1 mice. The above results indicate that probiotic treatment can significantly improve the neuronal damage in the cerebral cortex and hippocampus of APP / PS1 mice and improve the pathological characteristics of APP / PS1 mice.

[0063] Hematoxylin-eosin staining: Collect the empty, ileum, and colon tissues and fix them in 10% neutral buffered formalin. Subsequently, embed the tissues in paraffin, cut 5-μm thick sections and place them on glass slides. Dewax the above paraffin sections to water: Dewax in environmental protection dewaxing agent (1), environmental protection dewaxing agent (2), and environmental protection dewaxing agent (3) for 10 minutes each in sequence, then pass through absolute ethanol, 95% ethanol, 85% ethanol, and 75% ethanol for 5 minutes each, and rinse with tap water for 1 minute. Stain with hematoxylin staining solution (Harris) for 4 minutes, wash with tap water for 2 minutes until there is no excess staining solution on the section. Differentiate with 0.8% hydrochloric acid alcohol for 2 seconds, rinse with tap water, or use lithium carbonate aqueous solution for bluing, and then wash with water for 2 minutes. Stain with eosin staining solution (alcohol-soluble) for 20 seconds, do not wash with water, directly transfer to 95% ethanol for color adjustment for 5 seconds, transfer to absolute ethanol (1) and absolute ethanol (2) for dehydration for 2 minutes. Transparent with environmental protection transparent agent, seal, and examine under the microscope.

[0064] Immunohistochemical analysis: Immunohistochemistry (IHC) was used to analyze the expression and distribution of tight junction proteins in the jejunum, ileum, and colon of mice.

[0065] (1) Dewax the paraffin sections to water: Dewax in environmental protection dewaxing agent (1), environmental protection dewaxing agent (2), and environmental protection dewaxing agent (3) for 10 minutes each in sequence, then pass through absolute ethanol, 95% ethanol, and 75% ethanol for 5 minutes each. Wash with distilled water 3 times, 3 minutes each time, and then soak and wash.

[0066] (2) Antigen retrieval (microwave retrieval): Add antigen retrieval solution (citric acid, pH 6.0) into the retrieval box. Place the dewaxed and hydrated tissue sections into the retrieval box, and the volume of the retrieval solution should immerse the sections. Cover the lid loosely and put the retrieval box with the sections to be retrieved into the microwave oven. Adjust the power of the microwave oven to high fire and set the time to 3 minutes. After the first round of retrieval, take the retrieval box out of the microwave oven. Wait for the retrieval solution to cool naturally to room temperature (25 - 30 °C, about 1 hour), then repeat the retrieval operation of the first round to perform the second and third rounds of retrieval (the cooling interval time after each retrieval is about 1 hour). After the three rounds of retrieval are completed and cooled, take the sections out of the retrieval solution and wash them 3 times with distilled water, 5 minutes each time.

[0067] (3) Block endogenous peroxidase: After washing, immerse the retrieved sections in 3% H 2 O 2 for 30 minutes at room temperature in the dark, and then wash with distilled water.

[0068] (4) Draw a circle: After drawing a circle with a histochemical pen, place the sections in TBST.

[0069] (5) Blocking: Drop 10% serum from the same source as the secondary antibody and incubate at room temperature for 30 minutes.

[0070] (6) Incubate the primary antibody: Discard the serum, dilute the primary antibody solution with 10% serum to prepare the primary antibody working solution. Drop 50 - 100 μl (depending on the size of the tissue) of the primary antibody working solution on each section and incubate overnight at 4 °C.

[0071] (7) Incubate the secondary antibody: Take out the sections from the refrigerator the next day, place them at room temperature for 15 minutes (rewarming), wash 3 times with TBST, and then wash 3 times, 3 minutes each time. Dilute the secondary antibody solution with TBST to prepare the secondary antibody working solution. Drop 50 - 100 μl (depending on the size of the tissue) of the secondary antibody working solution on each section and incubate at 37 °C for 45 minutes. Wash 3 times with TBST, and then wash 3 times, 3 minutes each time.

[0072] (8) DAB color development: Remove TBST, drop 50 - 100 μl (depending on the size of the tissue) of freshly prepared DAB working solution on each section, observe under the microscope, time, and terminate the reaction with tap water.

[0073] (9) Stain the nucleus: Stain the nucleus with hematoxylin for 1 minute, wash, differentiate with hydrochloric acid alcohol for 1 - 2 seconds, wash, blue with the blueing solution for several seconds and then wash, and observe the coloring of the cell nucleus under the microscope.

[0074] (10) Mount the slides: Mount the slides with an environmentally friendly mounting medium and air dry. Store at room temperature in a dry and cool place.

[0075] (11) Microscopic examination: Examine under the microscope and collect and analyze the images.

[0076] The effects of mixed probiotics on intestinal barrier damage in APP / PS1 mice are as follows Figure 4 shown in the figure. In the figure, (A) HE staining of the jejunum, ileum, and colon of three groups of mice (scale bar is 100 μm); (B) Immunohistochemical expression of tight junction proteins (Claudin-1, Occludin, ZO-1) in the jejunum of three groups of mice (scale bar is 100 μm).

[0077] The study found that intestinal barrier function and the corresponding inflammatory response may be involved in the occurrence and development of Alzheimer's disease. HE staining results of the jejunum, ileum, and colon of three groups of mice showed that compared with WT and APP / PS+P3 group mice, the intestinal mucosal layer of APP / PS1 mice was atrophied, crypts were lost, and villi were partially disrupted. Thus, it can be seen that probiotic-treated mice can significantly improve intestinal barrier damage in APP / PS1 mice. Immunohistochemical analysis of the jejunum was performed to evaluate the expression of intestinal tight junction proteins. The results showed that compared with APP / PS1 group mice, the expression of tight junction proteins (Claudin-1, Occludin, ZO-1) in the jejunum of WT group and probiotic-treated APP / PS1+P3 group mice was significantly increased. It is indicated that treatment with mixed probiotics can significantly increase the expression of intestinal tight junction proteins in the jejunum of APP / PS1 mice, thereby further improving intestinal barrier damage.

[0078] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of a probiotic composition in the preparation of a drug for preventing and / or treating Alzheimer's disease, characterized in that: The probiotic composition consists of Lactobacillus rhamnosus, Bifidobacterium lactis and Bifidobacterium bifidum.

2. The use according to claim 1, characterized in that: The lactobacillus rhamnosus, bifidobacterium lactis and bifidobacterium bifidum are composed according to the viable bacterial count of (1-2): (1-2): (1-2).

3. The use according to claim 1, characterized in that: The probiotic composition improves the Simpson index of intestinal flora.

4. The use according to claim 1, characterized in that: The probiotic composition increases the relative abundance of Verrucomicrobia in the intestinal flora; the probiotic composition increases the relative abundance of Akkermansia and Bifidobacterium in the intestinal flora.

5. The use according to claim 1, characterized in that: The probiotic composition improves behavioral cognitive levels.

6. The use according to claim 1, characterized in that: The probiotic composition improves neuronal damage in the cerebral cortex and hippocampus.

7. The use according to claim 1, characterized in that: The probiotic composition improves intestinal barrier damage.

8. The use according to claim 1, characterized in that: The active ingredient of the medicine is a probiotic composition, which consists of Lactobacillus rhamnosus, Bifidobacterium lactis and Bifidobacterium bifidum.

9. The use according to claim 1, characterized in that: The drug also includes pharmaceutically acceptable excipients.

10. The use according to claim 1, characterized in that: The dosage forms of the medicine include capsules, granules, pills and oral liquid.

Images