Use of lactobacillus reuteri for the preparation of a medicament for the treatment or / and prevention of parkinson's disease

Abstract

The application discloses application of lactobacillus reuteri in preparation of a medicine for treating or / and preventing Parkinson's disease. The application provides application of lactobacillus reuteri in preparation of a product for treating or / and preventing Parkinson's disease. The application also provides application of a bacterium agent with lactobacillus reuteri as an active ingredient in preparation of a product for treating or / and preventing Parkinson's disease. The application proves that, by using a Parkinson's disease (PD) mouse model induced by chronic rotenone, the application evaluates the influence of lactobacillus reuteri (Lactobacillus reuteri) on Parkinson's disease, proves that, L. reuteri treatment can reduce motor dysfunction and gastrointestinal dysfunction of Parkinson's disease mice. L. reuteri ​

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the application of Lactobacillus reuteri in the preparation of drugs for the treatment and / or prevention of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a progressive neurodegenerative disorder long characterized by typical motor dysfunctions, including tremor, rigidity, bradykinesia, and postural instability. These symptoms are associated with the loss of dopaminergic neurons and Lewy bodies formed by α-synuclein in the substantia nigra (SN). However, it is increasingly recognized that PD presents with heterogeneity, including significant nonmotor symptoms such as gastrointestinal (GI) dysfunction. Despite advances in our understanding of the disease, its etiology and pathogenesis remain unclear, and treatment options for PD remain limited. Therefore, exploring new therapies for PD from fresh perspectives is imperative. Notably, gastrointestinal dysfunction often precedes the onset of motor symptoms in Parkinson's disease (PD) patients, suggesting that PD may originate in the gut. Consistent with this, mounting evidence suggests a link between gut microbiota dysbiosis and the pathogenesis of PD. For example, Zachary et al. performed deep randomized sequencing of fecal DNA from 490 PD patients and 234 healthy controls. The results showed that over 30% of the microbiome species abundance were significantly altered in PD patients, indicating widespread dysbiosis. Furthermore, a recent meta-analysis of gut microbiome sequencing data revealed an abundance of *Femobacterium* species in PD patients. (Faecalibacterium) and Rochetomyces (Roseburia) The abundance of genera such as Akermania was significantly reduced, while the abundance of Akermania was significantly reduced. (Akkermansia) and Bilofilaria (Bilophila) The abundance of certain bacterial taxa increased. Recent studies have shown that gut microbiota imbalance can further lead to blood-brain barrier (BBB) ​​damage and neuroinflammation in the substantia nigra (SN). This may affect the pathogenesis of Parkinson's disease by regulating microbial metabolites such as serum short-chain fatty acids (SCFAs), modulating intestinal resident immune responses, or transmitting α-synuclein via the enteric nervous system. These studies indicate that the complex bidirectional communication between the gut and brain can be regulated by the gut microbiota, a mechanism known as the "microbiota-gut-brain axis." In summary, these findings suggest that gut microbiota imbalance plays a crucial role in the pathogenesis of Parkinson's disease. Currently, regulating the gut microbiota has become a hot topic in PD intervention research. Current studies largely focus on the overall effects of probiotic mixtures or multi-strain formulations on PD animal models and patients. Evidence suggests that compound probiotics can partially improve motor symptoms or regulate inflammatory responses. However, existing literature lacks research exploring the regulatory effects of single specific bacterial species on blood-brain barrier and intestinal barrier function in PD and their potential mechanisms. Given that single bacterial species have the advantages of clearly defined components and more easily traceable mechanisms compared to compound formulations, exploring the independent effects of specific single probiotics on barrier function will help to elucidate the target of probiotic action at a more precise level and provide new experimental evidence for developing PD adjunctive intervention strategies based on single bacterial species. Summary of the Invention

[0003] The technical problem solved by this invention is to provide new uses for Lactobacillus reuteri.

[0004] To address the aforementioned technical problems, the first aspect of this invention provides the application of *Lactobacillus reuteri*, specifically its use in the preparation of products for the treatment and / or prevention of Parkinson's disease.

[0005] The second aspect of the present invention provides the application of a microbial agent, wherein the active ingredient of the microbial agent is Lactobacillus reuteri, and the application is the use of the microbial agent in the preparation of products for the treatment and / or prevention of Parkinson's disease.

[0006] In the applications described above, the treatment and / or prevention of Parkinson's disease is manifested in at least one of the following: 1) Improve motor dysfunction; 2) Relieves gastrointestinal dysfunction; 3) Reduce intestinal epithelial damage; 4) Relieves or inhibits intestinal or systemic inflammation; 5) Reduces intestinal permeability; 6) Reduce the loss of dopaminergic neurons in the substantia nigra; 7) Alleviates the accumulation of α-synuclein in the substantia nigra; 8) Reduces blood-brain barrier dysfunction; 9) Maintaining the integrity of the blood-brain barrier; 10) Inhibits neuroinflammation in the substantia nigra; 11) Regulate the metabolism of taurine deoxycholic acid (TDCA) and / or taurine chenodeoxycholic acid (TCDCA).

[0007] In one embodiment, maintaining the integrity of the blood-brain barrier is manifested in reducing apoptosis of vascular smooth muscle cells in the substantia nigra.

[0008] In one embodiment, the inhibition of neuroinflammation in the substantia nigra manifests as a reduction in the loss of vascular smooth muscle cells (VSMCs) and a decrease in the number of IL-1α-positive cells.

[0009] In an embodiment of the present invention, Parkinson's disease is a Parkinson's disease model induced by rotenone.

[0010] In an embodiment of the present invention, *Lactobacillus reuteri* is *Lactobacillus reuteri* (… Lactobacillusreuteri )SHBCC D24591=DSM26866.

[0011] In the above text, "prevention" generally refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject.

[0012] The term "treatment" generally refers to methods implemented to achieve beneficial or desired clinical outcomes. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, narrowing of disease extent, stabilization of disease (i.e., cessation of disease progression), delay or slowing of disease progression, improvement or relief or remission of disease status (whether partial or complete remission), whether detectable or undetectable.

[0013] In some implementations, the prevention and / or treatment is relief.

[0014] The term "relief" as used above refers to reducing, inhibiting, weakening, mitigating, preventing, or stabilizing the development or progression of a disease.

[0015] The product mentioned above is a medicine.

[0016] In the above text, the microbial agent also includes a pharmaceutically acceptable carrier.

[0017] In the above text, the microbial agent may be a liquid or solid microbial agent. The dosage forms include, but are not limited to, tablets, capsules, pills, powders, granules, suspensions, emulsions, etc.

[0018] The pharmaceutically acceptable carrier may be any of the following: 1) a diluent, such as starch, dextrin, lactose, mannitol, microcrystalline cellulose, calcium sulfate, etc.; 2) a wetting agent or binder, such as water, glycerin, starch paste, sodium carboxymethyl cellulose, etc.; 3) a disintegrant, such as dried starch, alginate, etc.; 4) a lubricant, such as talc, silicon dioxide, hydrogenated vegetable oil, etc.

[0019] In the above text, "medicine" means any substance that provides therapeutic, preventative, and / or beneficial effects.

[0020] This invention demonstrates that, using a chronic rotenone-induced Parkinson's disease (PD) mouse model, the effects of supplementation with Lactobacillus reuteri (…) were evaluated. L. reuteriThe effect of ) on Parkinson's disease, as demonstrated, L. reuteri Treatment alleviated motor dysfunction and gastrointestinal disturbances in Parkinson's disease mice. Further mechanistic analysis revealed that it exerts a protective effect against blood-brain barrier impairment and neuroinflammation by regulating serum levels of microbial-derived bile acids. In summary, this study reveals… L. reuteri The ability to alleviate symptoms in a mouse model of Parkinson's disease through the microbiota-gut-brain axis provides a new treatment option for Parkinson's disease.

[0021] Abbreviation list α-synuclein; ASD, autism spectrum disorder; BBB, blood-brain barrier; BP, biological process; BSH, bile salt hydrolase; CC, cellular component; CFU, colony-forming unit; CNS, central nervous system; CMC, carboxymethyl cellulose; cPAS, combinatorial probe anchoring synthesis; DAB, 3,3′-diaminobenzidine; DNB, DNA nanospheres; DNBSEQ, DNA nanosphere sequencing; DCA, deoxycholic acid; DEG, differentially expressed gene; ELISA, enzyme-linked immunosorbent assay; FC, fold change; FMT, fecal microbiota transplantation; GI, gastrointestinal tract; GO, gene ontology; H&E / HE, hematoxylin and eosin staining; HRP, horseradish peroxidase; IHC, immunohistochemistry; IL-1 / IL-6 / IL-12, interleukin-1 / 6 / 12; LB, lysogenic broth; LATS2, large tumor suppressor kinase 2. LPS, lipopolysaccharide; MF, molecular function; MLN, mesenteric lymph node; MRS, Deman-Rogoza-Sharp medium; OTU, operational taxonomic unit; PBS, phosphate-buffered saline; PCA, principal component analysis; PD, Parkinson's disease; PLS-DA, partial least squares discriminant analysis; PQN, probability quotient normalization; qPCR, quantitative polymerase chain reaction; RNA-seq, RNA sequencing; RSEM, RNA-seq based on expectation-maximization algorithm; SCFA, short-chain fatty acid; SN, substantia nigra; SLFN5, Schlafen family member 5; STAT1, signal transducer and activator of transcription 1; TAGLN, transgel protein; TCDCA, taurine chenodeoxycholic acid; TDCA, taurine deoxycholic acid; TEM, transmission electron microscopy; TH, tyrosine hydroxylase; TSA, tyrosine signal amplification; VSMC, vascular smooth muscle cells.

[0022] Ethical approval and participation consent All animal experimental procedures were approved by the Experimental Animal Ethics Committee of Peking University Third Medical College (No.: SA2023109) and complied with the guidelines formulated by the Beijing Municipal Ethics Committee. Attached Figure Description

[0023] Figure 1 for L. reuteri Treatment can restore motor and gastrointestinal dysfunction. (A) Experimental design flowchart. (B) Mouse weight changes during the experiment. (C) Spinning stick test. (D) Pole climbing test. (E) Grip strength test. (F) Deadhesion test. (G) Representative trajectory diagram of the open field test. (H) Total distance traveled. (I) Total number of fecal particles. (J) Moisture content of fecal particles. (K) Time variation curve of fecal excretion over 20 minutes. (LM) Representative image of the colon and colon length. (N) Intestinal transit distance. For B–F and H–K, n = 18 per group; for MN, n = 5 per group. Data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by LSD post-hoc tests (BC, F, H, J, and MN), or the Kruskal-Wallis test followed by the Mann-Whitney U test (I and K). ###p<0.001 compared to the control group; *P<0.05, **P<0.01, ***P<0.001 compared to the rotenone group.

[0024] Figure 2 for L. reuteri Treatment can alleviate intestinal inflammation and intestinal barrier damage. (AB) Representative images of HE-stained colon (A, black arrows indicate inflammatory infiltration) and colonic histological score (B). (CE) Levels of inflammatory cytokines IL-1 (C), IL-6 (D), and TNF-α (E) in colonic samples. (F) Levels of lipopolysaccharide (LPS) endotoxin in the colon. (G) Representative electron micrographs of tight junctions in the colonic epithelium. (H) Culture positivity rate of translocated bacteria in the liver, spleen, and lymph nodes (MLNs). (I) Levels of LPS endotoxin in serum. (JL) Levels of inflammatory cytokines IL-1 (J), IL-6 (K), and TNF-α (L) in serum. For Figure B, n = 5 per group. For Figures CF and IL, n = 12 per group. For Figure H, n = 8 per group. Data are expressed as mean ± standard deviation. Statistical analysis was performed using the Kruskal-Wallies test, followed by the Mann-Whitney U test (B); one-way ANOVA, followed by the LSD post-hoc test (CF and IL); or Fisher's exact test (H). ## p < 0.01, ### p < 0.001 compared to the control group; ** p < 0.01, *** p < 0.001 compared to the rotenone group.

[0025] Figure 3 for L. reuteriTreatment can alleviate neuropathological changes associated with Parkinson's disease (PD) and blood-brain barrier (BBB) ​​dysfunction in the substantia nigra (SN). (A) Representative image of TH immunohistochemical (IHC) staining in the substantia nigra. (B) TH in the substantia nigra. + Number of neurons. (C) Representative image of α-syn immunohistochemical staining in the substantia nigra. (D) Optical density of α-syn staining in the substantia nigra. (E) Representative electron micrograph of tight junctions of the blood-brain barrier in the substantia nigra. For Figures B and D, n = 5 per group. Data are expressed as mean ± standard deviation. Statistical analysis was performed using the Kruskal-Wallis test followed by the Mann-Whitney U test (B), or one-way ANOVA followed by the LSD post-hoc test (D). ## p < 0.01, ### p < 0.001 compared with the control group; ** p < 0.01 compared with the rotenone group.

[0026] Figure 4 for L. reuteri Treatment affected gene expression and signaling pathways in the midbrain of mice induced by rotenone. (A) Volcano plot showing the effects of rotenone treatment on the midbrain expression and signaling pathways in mice. L. reuteri Differentially expressed genes (DEGs) in the brains of mice in the control group. Significant genes have been labeled. (B) Venn diagram showing the control group versus the rotenone group, and the rotenone group versus... L. reuteri Overlap of differentially expressed genes between groups. (C) Box plot of Slfn5 gene expression level. (DG) Selected gene ( Slfn5 , Hspa5 , Tipin and Zkscan2 The relative expression levels of (H) were compared between the control group and the rotenone group, and between the rotenone group and... L. fold changes in gene expression in the reuteri group. (IJ) Comparison of the control group and the rotenone group (I) and the rotenone group with... L. Dot plot of enriched pathways in reuteri group comparison (J). In RNA-seq data, n = 6 per group. In qPCR validation, n = 9 per group. Box plots: the midline represents the median; the boxes represent the interquartile range (range between the 25th and 75th percentiles); the whiskers represent 1.5 × interquartile range. Bar plots: data are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by LSD post-hoc test (CG). ## p < 0.01, ### p < 0.001 compared to the control group; ** P < 0.01, *** P < 0.001 compared to the rotenone group.

[0027] Figure 5Treatment with *Lactobacillus reuteri* can reduce the loss of vascular smooth muscle cells in the substantia nigra. (A) Proportion of different cell types in a midbrain sample. (BD) Proportion of VSMC (B), PC (C), and EC (D) cell types in the midbrain. (EG) VSMC marker genes ( Acta2 , Tagln and Myh11 (H) Representative immunofluorescence images of SM22α (red, smooth muscle marker), cleavage caspase-3 (green, apoptosis marker), and DAPI (blue, nucleus) staining in the substantia nigra. (I) Quantitative analysis of SM22α fluorescence intensity. (J) Quantitative analysis of cleavage caspase-3 fluorescence intensity. For AD, n = 6 per group. For EG, n = 9 per group. For HJ, n = 5 per group. Box plots: median; box size (interquartile range, 25th to 75th percentile); whiskers (1.5 × interquartile range). Bar plots: data are expressed as mean ± standard deviation. Statistical analysis was performed using the Kruskal-Wallis test followed by the Mann-Whitney U test (BD), or one-way ANOVA followed by the LSD post-hoc test (EG and IJ). ns, no statistical significance; ## p<0.01, ### p<0.001, compared with the control group; *P<0.05, **P<0.01, ***P<0.001, compared with the rotenone group.

[0028] Figure 6 for L. reuteri Treatment can suppress neuroinflammation in the substantia nigra (SN). (A) Heatmap showing gene expression of inflammatory cytokines in the midbrain. (BC) Showing expression of inflammatory cytokine genes in the midbrain. Il1a (B) and Il12a (C) Box plot of expression level. (D) Midbrain Il12a Relative mRNA expression levels. (EG) Levels of inflammatory cytokines IL-1 (E), IL-6 (F), and TNF-α (G) in midbrain samples. (H) Representative immunofluorescence images showing the staining of SM22α (red, smooth muscle marker), IL-1α (green, pro-inflammatory cytokine), and DAPI (blue, nucleus) in the substantia nigra. (I) Quantitative analysis of SM22α fluorescence intensity. (J) IL-1α in the substantia nigra. +Cell count. For AC, n = 6 per group. For D, n = 9 per group. For EG, n = 12 per group. For HJ, n = 5 per group. Regarding box plots: the midline represents the median; the boxes represent the interquartile range (between the 25th and 75th percentiles); the whiskers represent 1.5 × interquartile range. Regarding bar plots: data are expressed as mean ± standard deviation. Statistical analysis was performed using the Kruskal-Wallies test followed by the Mann-Whitney U test (BD), or one-way ANOVA followed by the LSD post-hoc test (EG and IJ). ns, no statistical significance; # p < 0.05, ## p < 0.01, ### p < 0.001, compared with the control group; * p < 0.05, ** p < 0.01, *** p < 0.001, compared with the rotenone group.

[0029] Figure 7 for L. reuteri Treatment significantly altered the gut microbiota and serum metabolite profile. (AB) Alpha diversity analysis based on Chao1 (A) and Simpson (B) indices. (C) Beta diversity of different groups based on Adonis analysis. (D) Principal coordinate analysis (PCoA) plot of beta diversity of different groups. (E) Circos plot showing the distribution of microbial species in different groups. (F) PLS-DA plot of metabolomics lineage. (G) Volcano plot of differentially abundant metabolites in serum of mice in the rotenone and L. reuteri groups. (H) Venn diagram showing the overlap of differentially abundant metabolites in the three groups. (I) Heatmap showing hierarchical clustering of differentially abundant metabolites in the three experimental groups. (J) Top 20 differentially abundant metabolites enriched by the KEGG pathway. (KL) Quantitative analysis of TDCA (K) and TCDCA (L) in serum. (MN) Correlation between vascular smooth muscle cell (VSMC) proportion and TDCA (M) and TCDCA (N) levels. For AE, n = 5 per group. For FN, n = 6 per group. For box plots: the midline represents the median; the box represents the interquartile range (between the 25th and 75th percentiles); the whisker represents 1.5 × interquartile range. For bar plots: data are expressed as mean ± standard deviation. Statistical analysis was performed using the Kruskal-Wallis test followed by the Mann-Whitney U test (AB), one-way ANOVA followed by the LSD post-hoc test (KL), or Spearman correlation test (MN). *P < 0.05, **P < 0.01, compared with the rotenone group.

[0030] Figure 8 for L. reuteri Potential mechanisms of the gut-microbiota axis in the protective effects of treatment. (A) The network diagram shows the association between gene expression (yellow nodes) and serum metabolites (dark green nodes), with red and blue edges representing positive and negative correlations, respectively. (BC) Lats2 The expression of TDCA (B) and TCDCA (C) levels were negatively correlated. (D) Heatmap of the correlation between TDCA and TCDCA abundance and other experimental results. Color gradients represent Spearman correlation coefficients. (EF) TDCA abundance was negatively correlated with tumbler test performance (E) and fecal water content percentage (F). For BF, statistical significance was calculated using the Spearman correlation test. *P<0.05, **P<0.01. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0033] The *Lactobacillus reuteri* in the following examples is *Lactobacillus reuteri* (… Lactobacillusreuteri SHBCCD24591=DSM26866, purchased from Shanghai Center for Preservation of Biotechnology (SHMCC), hereinafter referred to as... L. reuteri .

[0034] The methods in the following embodiments are as follows: 1. Five different behavioral experiments To assess motor function, five different behavioral experiments were conducted every hour, as follows: 1) Rotating rod experiment Motor coordination was assessed using a rotating rod test. Mice were placed on a rotating rod device at a speed of 5 to 40 rpm for 300 seconds, and the dwell time was recorded. Each mouse was tested three times at one-hour intervals. The average of the three tests was taken as the dwell time.

[0035] 2) Pole Climbing Experiment Motor balance and agility were assessed using a vertical wooden pole (50 cm long and 3 cm in diameter) with a wooden ball fixed to the top. Before the test, mice were trained to climb back into their cages from the top of the pole. Performance was scored from 0 to 5, with 0 being the lowest and 5 the highest. Each mouse underwent three trials, with one hour between each trial. The average of the three trials was used as the performance score.

[0036] 3) Adhesive label removal test Each mouse was placed in a clean, transparent cage and allowed to acclimatize for 3 minutes. A circular adhesive tag was then applied to its front paw with gentle pressure. The time required for the mouse to completely remove the tag was recorded to assess its sensory perception and motor responses. Each mouse underwent three consecutive tests, with 30-minute intervals between each test. The average of the three tests was taken as the removal time.

[0037] 4) Grip strength test During the test, mice were instructed to grasp a horizontal rope with a diameter of 5 mm using their front paws. Scores were given on a scale of 0 to 5 based on the following criteria: 0 points, slipping off the rope within 10 seconds; 1 point, grasping the rope with only one front paw; 2 points, grasping the rope with both front paws; 3 points, grasping the rope with one hind paw; 4 points, grasping the rope with both hind paws; 5 points, attempting to reach the end of the rope. Each mouse underwent three independent trials, with one hour between each trial. The average of the three trials was used as the performance score.

[0038] 5) Open field experiment The movement and exploratory behavior of mice were analyzed in an open-field test chamber (40 × 40 cm). Mice were placed in the center of the enclosure and allowed to explore freely for five minutes. Their activities, including total distance traveled, time spent in the central and peripheral areas, average speed, number of area transitions, and number of times they entered the central area, were recorded using a SMART 3.0 video system (model 64088, RWD LifeScience Co., Ltd.). Data were processed and analyzed using SMART 3.0 advanced software (SMARTSUPER, Panlab, Spain). The test area was thoroughly cleaned with ethanol between each experiment, and the experiments were conducted in a quiet environment.

[0039] 2. Gastrointestinal function assessment 1) Fecal particle output After a 2-hour fast, each mouse was transferred to a clean, transparent plastic cage for observation for 2 hours. Fecal pellets were then collected and counted. The wet weight of fresh feces was recorded, and the dry weight was measured after drying at 85°C for 24 hours. The moisture content of the fecal pellets was calculated based on the difference between the wet and dry weights. To assess intestinal motility, feces were collected over 20 minutes, and the number of fecal pellets was recorded every 5 minutes.

[0040] 2) Measurement of intestinal transit distance and colon length Evans blue solution (Sigma-Aldrich) was dissolved in 1.5% sodium carboxymethyl cellulose (CMC-Na, Sigma-Aldrich). Thirty minutes before sacrifice, mice were administered 0.3 mL of 2.5% Evans blue solution orally by gavage to assess intestinal transit. The distance from the pylorus to the furthest point reached by Evans blue was then recorded as the intestinal transit distance. Additionally, the distance from the terminal cecum to the anus was measured as the total colonic length.

[0041] 3. Hematoxylin-eosin (HE) staining Five mice were randomly selected from each group, and their colon tissue was processed for histological analysis. Colon samples were embedded in paraffin, cut into 5 μm thick sections, and stained with hematoxylin and eosin as previously described. Histological evaluation was performed by two independent researchers in a blinded manner. Tissue damage was scored according to the following criteria: 0, no damage; 1, lymphoepithelial lesions; 2, focal ulcers or superficial mucosal erosions; 3, extensive mucosal damage involving the deep layers of the intestinal wall. Inflammatory cell infiltration was assessed according to the following criteria: 0, very few inflammatory cells in the lamina propria; 1, increased inflammatory cells in the lamina propria; 2, inflammatory cell infiltration extending into the submucosa; 3, transmural inflammatory cell infiltration. The total histological score was calculated by summing the scores for tissue damage and inflammatory cell infiltration. Each section was evaluated in five randomly selected fields of view.

[0042] 4. Enzyme-linked immunosorbent assay (ELISA) Enzyme-linked immunosorbent assay (ELISA) was performed using kits purchased from Shanghai Jianglai Industrial Co., Ltd., China, to detect mouse interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and lipopolysaccharide (LPS) endotoxins. The assays were performed according to the manufacturer's instructions. Protein concentrations were quantified using a standard curve generated from target proteins of known concentrations.

[0043] 5. Transmission electron microscope Mice were anesthetized and perfused with 0.9% saline. Brain and colon tissues were then dissected and removed, and cut into 1 mm³ cubes. These samples were fixed with 4% paraformaldehyde at 4°C for 4 hours, followed by post-fixation with 1% osmium tetroxide at room temperature for 2 hours. Subsequently, the tissues were dehydrated via an ethanol gradient, embedded in resin, and polymerized by baking at 60°C for 48 hours. Ultrathin sections of 60 nm thickness were prepared using an ultramicrotome. Transmission electron microscopy (HITACHI HT7700, Japan) was used to analyze the ultrastructure of the barriers in the brain and colon.

[0044] 6. Bacterial translocation detection At sacrifice, spleen, liver, and mesenteric lymph node (MLN) samples were aseptically collected from each mouse. Each tissue sample was homogenized in 0.9% sterile saline at a 1:9 ratio (0.1 g tissue to 0.9 mL saline). 100 μL of the suspension was then inoculated onto LB agar plates. The plates were then incubated aerobically at 37°C for 24 hours. Finally, bacterial translocation was detected by counting and analyzing colony-forming units (CFU).

[0045] 7. Immunohistochemical staining Midbrain tissue from five randomly selected mice in each group was paraffin-embedded and sectioned. Immunohistochemical (IHC) analysis was then performed according to previously described methods. Briefly, sections were dewaxed, rehydrated, and blocked with 3% bovine serum albumin. Sections were then incubated overnight at 4 °C with primary antibodies including anti-α-syn (1:1500, Servicebio, Wuhan, China) and anti-tyrosine hydroxylase (TH, 1:1500, Servicebio). After thorough washing, sections were incubated for two hours at room temperature with horseradish peroxidase (HRP)-labeled secondary antibodies (goat anti-rabbit IgG or goat anti-mouse IgG, 1:200, Servicebio). Positive cells were stained using DAB substrate (Servicebio), and images were taken using a Carl Zeiss microscope (Jena, Germany). The number or expression level of positive cells was analyzed using ImageJ software (version 1.52).

[0046] 8. RNA sequencing and bioinformatics analysis Total RNA was extracted from tissues using TRIzol (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and purified by precipitation with chloroform and isopropanol. RNA quality and quantity were assessed using an Agilent 2100 Bioanalyzer. Total RNA samples were then separated using magnetic beads coated with oligomers (dT). The isolated mRNA was fragmented and converted to cDNA via a two-step synthetic process involving first- and second-strand reactions. The cDNA underwent end repair, addition of a single "A" nucleotide, and aptamer ligation. The library was amplified by PCR and quality-controlled. Subsequently, the library product was denatured and circularized. Single-stranded circular DNA molecules were replicated into DNA nanospheres (DNBs) using rolling circle amplification. DNBs were loaded into patterned nanoarrays and sequenced using combinatorial probe anchoring synthesis (cPAS) on the DNBSEQ high-throughput platform (G400 platform, BGI Genomics Shenzhen, Shenzhen, China).

[0047] Sequencing data were processed and filtered using SOAPnuke, specifically including: (1) removing reads containing sequencing adapters; (2) removing reads with more than 20% low-quality bases (base quality ≤ 15); and (3) discarding reads with more than 5% unknown bases ('N'). The processed reads were then stored in FASTQ format. Bioinformatics analysis included aligning reads to a reference genome using HISAT2, aligning clean reads using Bowtie2, and quantifying gene expression using RSEM (v1.3.1). Differentially expressed genes (DEGs) were identified using DESeq2 with a threshold of FDR < 0.1 and |log2FC| > 0.5. GO and KEGG pathway enrichment analyses were performed using Phyper, and the results were corrected using multiple validation (Q value ≤ 0.05). To deconvolve the batch RNA sequencing (RNA-seq) data, the BayesPrism algorithm (https: / / github.com / Danko-Lab / BayesPrism) was used, based on the processed single-cell data of mouse midbrain tissue published on the Cell BLAST website (https: / / cblast.gao-lab.org / ).

[0048] 9. Quantitative polymerase chain reaction (PCR) detection Total RNA was extracted from tissues using the TRIZOL method. Subsequently, cDNA synthesis and quantitative polymerase chain reaction (qPCR) detection of various genes were performed using the RevertAid First StrandcDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) and the Hieff™ qPCR SYBR® Green Master Mix (No Rox Plus) 11201ES (Yeasen BioTechnologies, Shanghai, China). Primer pairs used for amplification are listed in Table 1. qPCR amplification and detection were performed on a Quantagne q225 real-time PCR system (Kubo Tech Co., Shanghai, China). Finally, 2... -ΔΔCt Algorithm analysis of relative mRNA expression levels. Table 1 lists the paired primers used for quantitative PCR (qPCR).

[0049] 10. Immunofluorescence staining Five mice were randomly selected from each group, fully sedated, and then perfused thoroughly with 0.9% saline, followed by perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. Colon and brain tissues were dissected and immediately placed in 4% paraformaldehyde for 24 hours, then in 4% paraformaldehyde containing 30% sucrose. Brain and colon tissues were paraffin-embedded and sectioned into 5 μm thick sections. Immunofluorescence staining was performed according to methods used in previous studies. Briefly, sections were first subjected to antigen retrieval using sodium citrate solution (pH 6.0). After blocking with 3% bovine serum albumin (Servicebio), sections were incubated overnight at 4°C with primary antibody. The primary antibodies used were: anti-smooth muscle protein 22-α (SM22α; 1:250, Servicebio), anti-cleavage caspase-3 (1:4000, Cell Signaling Technology, Danvers University, MD, USA), and anti-IL-1α (1:1000, Abcam, Cambridge University, UK). Secondary antibodies used subsequently included goat anti-rabbit IgG-HRP (1:500, Servicebio) and tyramine signal amplification (TSA) antibodies, containing iF488-tyramine (1:500, Servicebio) and FITC-tyramine (1:500, Servicebio), respectively. Cell nuclei were detected using DAPI solution. Representative images were captured using a fluorescence microscope (Nikon Eclipse C1, Tokyo, Japan). The number of positive cells or expression levels were analyzed using ImageJ software (version 1.52). 11. Fecal DNA Extraction and Metagenomic Sequencing At week 8, mice were randomly selected from each group for microbial community sequencing. Each mouse was placed individually in a sterile cage, and 6–8 fresh fecal particles were collected and immediately stored at -80°C within 30 minutes. DNA was extracted using the MagPure Stool DNA KF Kit B (BGI-Shenzhen) according to the manufacturer's instructions. After extraction, library preparation was performed using the MGIEAsy Universal DNA Library Preparation Kit (BGI-Shenzhen). DNA nanospheres (DNBs) containing multiple copies of DNA were generated using high-intensity DNA nanochip technology and loaded onto a patterned nanoarray. Sequencing was performed on the DNBSEQ-2000 platform (BGI-Shenzhen), generating 150-base paired reads (PEs). The raw data were preprocessed using SOAPnuke v.2.2.1 and mapped to the host genome using SOAP2 to remove host read sequences. High-quality read sequences were assembled de novo using MEGAHIT, excluding fragments shorter than 300 bp. Gene prediction was performed using MetaGeneMark, with redundancy reduced using CD-HIT. Gene abundance was quantified using Salmon, and protein sequences were aligned with functional databases using DIAMOND for annotation. Taxonomic annotation was performed by Kraken LCA, and abundance profiles were constructed by Bracken.

[0050] 12. Serum metabolomics analysis Mouse serum samples were thawed at 4°C and treated with an extraction reagent containing an internal standard (methanol:acetonitrile:water = 4:2:1, v / v). Samples were incubated at -20°C for two hours, followed by centrifugation at 25,000 × g for 15 minutes at 4°C. The supernatant was dried, reconstituted with methanol, and centrifuged again. After quality control, metabolite analysis was performed using a WatersUPLC I-Class Plus (Waters, USA) and a QTRAP 6500 Plus (SCIEX, USA) mass spectrometer. Identification was performed using Compound Discoverer 3.3 software (Thermo Fisher Scientific) and databases such as bmdb, mzcloud, and chemspider.

[0051] Bioinformatics analysis included data preprocessing using Probability Quotient Normalization (PQN) and Quality Control-Based Robust LOESS Signal Correction (QC-RLSC) to standardize and correct for batch effects. Principal Component Analysis (PCA) was used for quality control and sample distribution analysis. Partial Least Squares Discriminant Analysis (PLS-DA) was employed to identify significant metabolites, with thresholds set as fold change (FC) > 1.5 or < 1 / 1.5, p-value < 0.01, and variable importance in projection (VIP) > 1. Metabolites were annotated using the human metabolome database and the KEGG pathway database. 13. Statistical Analysis Statistical analysis was performed using GraphPad Prism 8 software. Data are expressed as mean ± standard deviation (SD). For normally distributed data, one-way ANOVA was used with post-hoc tests combining least significant difference (LSD) for multiple comparisons; Stielike t-tests were used for comparisons between two groups. For non-normally distributed data, nonparametric tests were used. Spearman correlation analysis was performed where appropriate. A p-value <0.05 was considered statistically significant.

[0052] Example 1: Application of Lactobacillus reuteri in the preparation of drugs for the treatment and / or prevention of Parkinson's disease one, L. reuteri Experimental design for treating Parkinson's disease 1. Discovery of Lactobacillus reuteri Given the significant neuroprotective effect of fecal microbiota transplantation (FMT) in a mouse model of Parkinson's disease (PD) found in previous studies, we characterized changes in common probiotics after FMT treatment. Following FMT, the relative abundance of Lactobacillus significantly increased, while the abundance of Bifidobacterium did not change significantly. Further OTU analysis revealed that *Lactobacillus reuteri* was the dominant Lactobacillus species in the fecal samples. These results indicate... ， Lactobacillus reuteri has potential as a probiotic for the treatment of Parkinson's disease.

[0053] 2. L. reuteri Treatment of Parkinson's disease mouse model Eight-week-old male C57BL / 6J mice, weighing 20–22 g, were purchased from the Department of Laboratory Animal Science, Peking University School of Medicine (Beijing, China). The mice were then placed under standard conditions (temperature 22 ± 2°C, humidity 50–60%) for a 7-day acclimatization period (12-hour light-dark cycle), during which they had free access to food and water. All animal experimental procedures were approved by the Laboratory Animal Ethics Committee of Peking University Third Medical College (No. SA2023109) and complied with the guidelines established by the Beijing Municipal Ethics Committee.

[0054] L. reuteri Anaerobic culture was carried out in de Man, Rogosa, and Sharpe (MRS) medium at 37°C in an environment consisting of 90% N2, 5% CO2, and 5% H2. The cultures were then centrifuged, washed, and resuspended in sterile phosphate-buffered saline (PBS, 0.01 M, pH 7.4) to obtain a bacterial suspension. The bacterial suspension was then stored... 80°C until used.

[0055] By inoculating serially diluted cultures onto LB agar plates and counting colonies, the effects on... L. reuteri The colony-forming units (CFU) were quantified.

[0056] Animal experimental design, such as Figure 1 As shown in Figure A, the specific experiment is as follows: 1) Establishment of a mouse model of Parkinson's disease A total of 54 mice were randomly divided into three groups: control group, rotenone group, and... L. The reuteri group. For use in the rotenone group and... L. In the reuteri group, a mouse model of Parkinson's disease was induced by oral administration of a fresh rotenone solution (CAS No. 83-79-4, Sigma-Aldrich, St. Louis, Missouri, USA) at a dose of 30 mg / kg body weight once daily via gastric tube for four weeks. The rotenone solution was obtained by dissolving rotenone in a 4% carboxymethyl cellulose (CMC; Sigma-Aldrich) solution containing 1.25% chloroform (Beijing Chemical Plant, Beijing, China).

[0057] 2) Drug therapy The following treatment trial will be conducted 4 weeks after oral administration of rotenone solution: L. reuteri Group: Each mouse was administered 200 μL of live [drug] via gastric administration daily. L. reuteri Bacterial suspension, dosage 2 × 10 8 CFU, lasting four weeks.

[0058] Control group: Each mouse was given an equal volume of sterile PBS via the stomach daily.

[0059] Rotenone group: Each mouse was given an equal volume of sterile PBS via gastric administration daily.

[0060] two, L. reuteri Treatment can improve motor dysfunction and gastrointestinal disorders. 1. Weight measurement During the experiment described above (day 0 was the day the mice were orally administered rotenone), all mice were weighed every two days.

[0061] The results of the test on changes in mouse body weight are as follows: Figure 1 As shown in Figure B, the mice in the rotenone group experienced a significant decrease in body weight after rotenone induction. L. reuteri The group that received the medication significantly recovered their body weight.

[0062] 2. Five different behavioral experiments During the aforementioned experiment, gastrointestinal function assessments and behavioral experiments were conducted in week 4 (with the day of oral administration of rotenone recorded as day 0) and week 8, respectively. The five different behavioral experimental methods are the same as Method 1 above, including the stick spinning test for assessing motor coordination, the pole climbing test for assessing motor balance and agility, the sticky sticker removal test for measuring sensory perception and motor response, the grip strength test for measuring limb muscle strength, and the open field test for examining motor activity and exploratory behavior. The results are as follows Figure 1 As shown in Figure 1H, compared with the control group, the rotenone group mice had shorter dwell time on the rotating rod, lower scores on the standing rod test, decreased limb muscle strength, delayed response to sticker removal, reduced motor activity, and increased anxiety-like behavior (all P < 0.001, Figure 1CH). L. reuteri Treatment significantly alleviated motor dysfunction in the stick-twirling test (P<0.001, Figure 1C) and the pole-climbing test (P<0.01, Figure 1 D) Grip strength test (P<0.05, Figure 1 E) Peeling test of sticky substances (P<0.05, Figure 1 F) and open field experiments (both P < 0.001, Figure 1 In the GH group, treatment with L. reuteri significantly alleviated motor dysfunction compared to the rotenone group. 3. Assessment of gastrointestinal dysfunction During the above-mentioned experiment, gastrointestinal function was assessed in week 4 (the day of oral administration of rotenone was recorded as day 0) and week 8 (using the same method as in method 2 above). The results were as follows: Compared with the control group, the rotenone group showed significant gastrointestinal dysfunction, with fewer fecal particles (P<0.001). Figure 1 I), the water concentration in feces decreased (P<0.001, Figure 1 J), bowel movement frequency decreased (P<0.001, Figure 1 K), shortened colon length (P<0.001, Figure 1 LM) and shortened intestinal transit distance (P<0.001, Figure 1 N). In contrast, compared to the rotenone group, L.The gastrointestinal function of mice in the reuteri group was improved, as evidenced by an increase in the number of fecal particles (P<0.001). Figure 1 I) Increased fecal water content (P<0.001, Figure 1 J) Increased bowel movement frequency (P<0.001, Figure 1 K), colon length (P<0.001, Figure 1 LM) and intestinal transit distance (P<0.001, Figure 1 All (N) have improved.

[0063] In summary, these data collectively indicate that L. Reuteri treatment significantly alleviated rotenone-induced weight loss, motor dysfunction, and gastrointestinal disorders. three, L. reuteri Treatment can alleviate intestinal inflammation and intestinal barrier damage. To further explore L. The potential mechanism of reuteri treatment in restoring gastrointestinal dysfunction was investigated in week 8 of the aforementioned experiment (day 0 was defined as the day rotenone was administered orally), during which all mice were sacrificed and colon samples were collected. Figure 1 A), for further analysis.

[0064] 1. Relieves intestinal inflammation HE staining was performed according to method 3 described above to assess immune infiltration and epithelial damage in the colon. The results showed that rotenone administration significantly induced intestinal inflammation and epithelial damage (P<0.01). Figure 2 AB), and L. The HE histological score of mice in the reuteri group was significantly lower (P<0.01). Figure 2 AB). ELISA analysis was performed according to method 4 described above. The results showed that, compared with the control group, the levels of IL-1, IL-6, and TNF-α in the colon of mice in the rotenone group were significantly increased (all P < 0.001). Figure 2 CE). In comparison L. reuteri Treatment significantly reduced the levels of these three inflammatory cytokines (all P < 0.001, Figure 2C-E). Furthermore, ELISA analysis showed that rotenone-induced LPS levels were elevated in the colon of mice (P < 0.001), while... L. reuteri Treatment reduced LPS levels (rotenone group, 369.3 ng / L); L. reuteri Group, 330.2 ng / L; P<0.001 (Figure 2F). In summary, these data indicate that L. reuteri The administration of this drug can suppress intestinal inflammation. 2. Intestinal permeability testing To determine whether intestinal inflammation is related to increased intestinal permeability, the structure and function of the intestinal barrier were further measured.

[0065] Transmission electron microscopy (TEM) analysis of colon samples performed according to method 5 above showed that the colonic epithelial function of mice treated with rotenone was impaired, manifested as sparse microvilli, reduced and fragmented electron-dense material, and enlarged intercellular spaces. Figure 2 G). However, through L. reuteri After treatment, the epithelial cells exhibited orderly arranged microvilli and continuous tight junctions. Figure 2 G).

[0066] A bacterial translocation experiment was performed on colon samples according to method 6 described above to assess intestinal permeability.

[0067] The results are as follows Figure 2 As shown in H, after rotenone induction, translocated microorganisms were detected in sterile organs (such as liver (87.5%), spleen (75%), and mesenteric lymph nodes (MLNs) (87.5%) (all P < 0.01), while L. Reuteri treatment significantly inhibited microbial translocation in these organs (liver, 25%; spleen, 12.5%; MLNs, 12.5%; all P < 0.01). Furthermore, serum LPS levels were elevated in mice in the rotenone group (P < 0.001), while... L. Reuteri treatment reduced LPS levels (rotenone group, 374.0 ng / L); L. The reuteri group had a concentration of 311.1 ng / L; P < 0.001. Figure 2 I), indicating L. Reuteri treatment reduced the increase in intestinal permeability.

[0068] Further ELISA analysis showed that rotenone-induced increases in the systemic levels of IL-1, IL-6, and TNF-α in mouse serum were all observed (all P < 0.001), but... L. All decreased after reuteri treatment (all P < 0.001). Figure 2 JL). In summary, these results indicate that L. reuteri Administration of this drug can reduce intestinal epithelial damage, decrease intestinal permeability, and alleviate rotenone-induced intestinal and systemic inflammation.

[0069] Four, L. reuteri Treatment can reduce neuropathological changes in the substantia nigra (SN) associated with Parkinson's disease (PD) and blood-brain barrier (BBB) ​​dysfunction. The neuropathological features associated with Parkinson's disease include the loss of dopaminergic neurons in the substantia nigra and the accumulation of α-synuclein, which are closely related to the progressive motor dysfunction observed in Parkinson's disease patients. The levels of TH and α-syn in the substantia nigra of mice in each group sacrificed in week 8 of the above experiment were detected using the aforementioned immunohistochemical analysis method 7.

[0070] The results showed that dopaminergic (TH) levels in the substantia nigra of mice in the rotenone group were significantly increased. + The number of neurons was significantly reduced, less than one-tenth of the number found in the substantia nigra of control mice (P<0.01, Figures 3A-B). However, L. reuteri Treatment significantly restored the loss of dopaminergic neurons (P<0.01, Figures 3A-B). Furthermore, the optical density of α-syn in the substantia nigra of mice induced by rotenone was almost twice that of the median in the substantia nigra of the control group (P<0.001), while... L. reuteri Treatment significantly alleviated the accumulation of α-syn (P<0.01, Figure 3 CD). Impaired blood-brain barrier (BBB) ​​function increases the brain's exposure to circulating harmful toxins and inflammatory cytokines, which is closely related to the pathogenesis of Parkinson's disease (PD). Therefore, this study evaluated the structure and function of the blood-brain barrier.

[0071] Transmission electron microscopy (TEM) analysis of the substantia nigra using method 5 described above showed that the tight junctions in the blood-brain barrier of mice in the rotenone group were disordered, and endothelial cells were damaged. Figure 3 E). However, L. reuteri The administration of the drug significantly protected the blood-brain barrier structure, making it similar to the blood-brain barrier structure of the control group mice (Figure 3E).

[0072] In summary, these data collectively indicate that L. reuteri Treatment can reduce the loss of dopaminergic neurons, accumulation of α-synuclein, and blood-brain barrier dysfunction in the substantia nigra (SN) of mice treated with rotenone.

[0073] five, L. reuteri Effects of treatment on midbrain transcriptome profile In order to clarify L. The possible mechanism of the neuroprotective effect of reuteri treatment in a mouse model of Parkinson's disease was investigated by RNA sequencing of the midbrain containing the substantia nigra (SN) in each group that was sacrificed at week 8 in the above experiment.

[0074] Compared with the control group, a total of 148 differentially expressed genes (DEGs, FDR < 0.1 and |log2FC| > 0.5) were identified in the midbrain of mice in the rotenone group. Among them, 91 genes were significantly upregulated (e.g., Uvssa , Aire and Cd46 ), 57 genes were significantly downregulated (e.g. Hspa1a , Hspa1b and Slfn5 Further analysis shows that... In L. reuteri Following treatment, rotenone-treated mice showed increased expression of six genes in the midbrain (e.g., Slfn5 , Hspa1a and Hspa1b ), the expression of 8 genes was reduced (e.g. Gm40367 , Rsc1a1 and Tac2 (Figure 4A). A total of 7 genes showed differential expression in both groups. Figure 4 B). It is worth noting that among the genes mentioned above, Hspa1a , Hspa1b , Hspa5 and Slfn5 It was significantly downregulated in mice in the rotenone group, but... L. Reuteri treatment significantly upregulated ( Figure 4 C). In contrast, the remaining genes ( Gm40367 , Tipin and Zkscan2 The opposite expression trend is observed, indicating that these genes are related to... L. reuteri Treatment is closely related to the protective effect against the progression of Parkinson's disease (PD).

[0075] Further qPCR analysis according to Method 9 verified the changes in the expression levels of these genes in the three groups (Figure 4D-G).

[0076] like Figure 4 As shown in H, more differentially expressed genes (DEGs) with similar expression patterns were also identified in these three groups. For example, in the rotenone group mice... Cd209b , Eno1b and Gm45521 The expression level was lower than that of the control group and L. reuteri group ( Figure 4 H). In addition, genes ( such as Gprc5d , LOC100504180 and Spata31d1b Upregulated in the rotenone group ( Figure 4 H).

[0077] In order to predict L. Functional enrichment analysis was performed to investigate the underlying mechanisms of the neuroprotective effects of reuteri treatment. KEGG pathway enrichment analysis revealed that differentially expressed genes (DEGs) between the control and rotenone groups were associated with the following pathways: protein processing in the endoplasmic reticulum, lipid and atherosclerosis, α-linolenic acid metabolism, and linoleic acid metabolism. Figure 4 I). Meanwhile, in comparing the rotenone group with... L. When genes were expressed among the reuteri groups, other pathways, including antigen processing and presentation, vascular smooth muscle contraction, and tight junctions, were also enriched. Figure 4 J). In addition, gene ontology (GO) enrichment analysis was performed, along with results for biological processes (BP), cellular components (CC), and molecular functions (MF).

[0078] Overall, these findings support the view that... L. reuteri Effects of drug administration on the midbrain transcriptome profile of rotenone-induced Parkinson's disease mice.

[0079] six, L. reuteri Treatment can reduce apoptosis of VSMCs in SN. Interestingly, the KEGG pathway analysis mentioned above revealed that the "vascular smooth muscle contraction" pathway was associated with the rotenone group. L. Enrichment of differentially expressed genes (DEGs) between reuteri groups suggests that vascular smooth muscle cells (VSMCs), a key component of the blood-brain barrier (BBB), may be involved in [the process]. L. The protective effect of reuteri intervention.

[0080] To further explore L. The effect of reuteri administration on the proportion of midbrain cell types was analyzed using the BayesPrism algorithm to deconvolve the batch RNA-seq data from the groups sacrificed at week 8 in the above experiment. On average, neuroendocrine cells (NendC, 62.2%), mature neurons (mNEUR, 14.3%), oligodendrocytes (OLG, 8.5%), astrocyte-specific progenitor cells (ARP, 7.9%), astrocytes (ASC, 3.5%), and vascular smooth muscle cells (VSMCs, 2.2%) were the predicted major cell types in the midbrain samples. Figure 5 A). Further differential analysis showed that the proportions of vascular smooth muscle cells (VSMCs, P = 0.003), pericytes (PCs, 0.020), endothelial cells (ECs, P = 0.022), monocytes (MNCs, P = 0.014), oligodendrocytes (OLGs, P = 0.019), and tanni cells (TNCs, P = 0.029) differed significantly among the three groups. Figure 5 Notably, rotenone-induced vasoconstriction significantly reduced the proportion of smooth muscle cells (VSMCs) in the midbrain of mice (P<0.01), but in mice receiving rosinone-induced vasoconstriction... L. The level significantly increased after reuteri intervention (P<0.01). Figure 5 B). Furthermore, the expression of VSMC marker genes in different groups sacrificed at week 8 of the above experiment was investigated using qPCR analysis according to method 9. The results showed... L. The expression of these genes was significantly increased after reuteri treatment (Acta2, P<0.001; Tagln, P<0.01; Myh11, P<0.05; Figure 5 EG).

[0081] Reports indicate that inhibiting vascular smooth muscle cell (VSMC) apoptosis is crucial for promoting cell survival, maintaining vascular integrity, and protecting blood-brain barrier (BBB) ​​function.

[0082] To further verify the changes in VSMCs in the SN and to explore the role of VSMC apoptosis in the underlying mechanism, this study used SM22α as a VSMC marker and cleavage-type Caspase-3 as an apoptosis marker, and performed immunofluorescence staining according to Method 10.

[0083] The results were as follows: In the SN of mice in the rotenone group, small arterial walls were damaged and SM22α expression was decreased (P<0.01). Figure 5 HI), indicating significant vascular damage. In contrast, L. reuteri The treatment effectively restored the vascular integrity of these small arteries (P<0.01). Figure 5 HI). Furthermore, in the rotenone group, the expression of cleaved caspase-3 was significantly increased in SM22α-positive cells (P<0.001, Figures 5H and 5J). Conversely, L. reuteri Treatment significantly inhibited the expression of cleavage-type caspase-3 in these cells (P<0.05, Fig. 5H and 5J).

[0084] These data indicate that L. reuteri Treatment may maintain the integrity of the blood-brain barrier (BBB) ​​by reducing rotenone-induced apoptosis of vascular smooth muscle cells (VSMCs) in mouse SNs.

[0085] seven, L. reuteri Treatment can suppress neuroinflammation in the substantia nigra (SN). Blood-brain barrier (BBB) ​​dysfunction is associated with neuroinflammation. Differential expression analysis was performed on 24 neuroinflammation-related cytokines in mice from different groups sacrificed at week 8 in the aforementioned experiment.

[0086] The results showed that the expression of Il1a and Il12a in the midbrain of mice in the rotenone group was upregulated, while L. Reuteri intervention suppressed their expression. Figure 6AC). Subsequently, qPCR validation using method 9 showed that the expression of both Il1a and Il12a was increased in the rotenone group. Il 1a, P<0.05; Il 12a, P<0.001), but in L. reuteri All decreased after treatment (all P<0.001); Figure 6 D). ELISA analysis using method 4 showed that ，L. Administration of reuteri significantly inhibited the levels of pro-inflammatory cytokines in the brain of mice in the rotenone group, including IL-1, IL-6 and TNF-α (all P<0.001); Figure 6 EG). Furthermore, immunofluorescence staining using method 10 showed that rotenone treatment significantly increased the number of IL-1α-positive cells surrounding damaged vascular smooth muscle cells (VSMCs) in the substantia nigra (SN) of mice (P<0.01). L. reuteri The intervention significantly reduced the number of these cells (P<0.05, Figure 6H-J). In summary, these results indicate that L. reuteri Treatment may reduce VSMC loss and decrease the number of IL-1α positive cells, thereby inhibiting neuroinflammation in the substantia nigra of mice treated with rotenone.

[0087] eight, L. reuteri The treatment significantly altered the gut microbiota and serum metabolic profile. To further explore and supplement L. reuteri The mechanism of the neuroprotective effect was investigated by obtaining fecal microbial community profiles through metagenomic sequencing of mice from different groups sacrificed at week 8 in the above experiment, and obtaining serum metabolomics data through liquid chromatography-mass spectrometry (LC-MS) technology, as described in method 11. The results are as follows: First, compared with the rotenone group, the relative abundance of *Lactobacillus reuteri* in the *Lactobacillus reuteri* group was significantly increased (P<0.05), confirming successful colonization and enrichment of the target strain. In terms of metagenomics, α-diversity analysis showed no difference in microbial species richness or diversity among the three groups (Figures 7A-B). Further β-diversity analysis based on Bray-Curtis distance indicated that rotenone-induced and... L. reuteri Intervention led to differentiation of bacterial community structure (Figures 7C-D). Furthermore, the top 15 gut microbial genera in each group (e.g., ...) Duncaniella , Akkermansia , Muribaculum and BacteroidesThe distribution of bacteria in each group showed significant differences. Furthermore, the relative abundance of bacterial species in each group was also shown in the figure. Figure 7 E). Including Duncaniella dubosii , Akkermansia muciniphila and Muribaculum sp. Significant taxa, including TLL-A4, exhibit distinguishable variations among the groups.

[0088] These results collectively prove L. reuteri Treatment can affect rotenone-induced gut microbiota dysbiosis.

[0089] In the non-targeted metabolomics analysis, the PLS-DA score plot showed a clear separation between the three groups, indicating significant metabolic differences. Figure 7 F). Subsequently, differential metabolite analysis was performed to identify specific metabolic alterations. Thresholds were set for FC > 1.5 or < 1 / 1.5, P < 0.01, and VIP > 1. A total of 65 differential metabolites were identified in the comparison between the control group and the rotenone group, indicating that rotenone exposure led to significant serum metabolic disturbances. L. reuteri When comparing the group with the rotenone group, 216 differentially expressed metabolites were identified, among which... L. The reuteri group has 138 upregulations and 78 downregulations. Figure 7 G). The Venn diagram shows that a total of 92 metabolites co-occurred in at least two comparison groups ( Figure 7 H). Overlapping metabolites were filtered from the Venn diagram and further visualized as a heatmap. Figure 7 I). Furthermore, functional enrichment analysis showed that these differentially expressed metabolites were mainly enriched in butyrate metabolism, indole diterpenoid alkaloid biosynthesis, the cGMP-PKG signaling pathway, and vascular smooth muscle contraction pathways. Figure 7 J). To further explore the possible mediators L. reuteri Metabolites with neuroprotective effects were studied, with a focus on those that are susceptible to... L. reuteri Treatment modulates differential abundance metabolites. Two conjugated bile acids—taurodeoxycholic acid (TDCA) and taurochenodeoxycholic acid (TCDCA)—were elevated in the rotenone group, but... L. reuteri The levels of these two bile acids were significantly reduced after treatment (all P < 0.01, Figure 7 K-L). Simultaneously, the serum levels of these two bile acids were negatively correlated with the proportion of vascular smooth muscle cells (VSMCs) in the midbrain (…). Figure 7 (MN), indicating that these two metabolites are involved in the regulation of VSMC survival.

[0090] Overall, these results indicate that L. reuteriThe treatment alleviated symptoms of rotenone-induced Parkinson's disease in mice by regulating the metabolism of bile acids such as TDCA and TCDCA. IX. Gut microbiota-gut-brain axis L. Potential mechanisms of protection in reuteri therapy To further elucidate how serum bile acids regulate gut-brain interactions, association analysis was performed to identify significant correlations between serum metabolites and other variables. First, regularized correlation analysis (CCA) was used to obtain a correlation matrix between metabolites and midbrain genes. To focus on the most relevant correlations, the correlations were analyzed based on the rotenone group and... L. The p-values ​​used in the reuteri group comparisons identified the top 20 species, genes, and metabolites. Subsequently, the correlation network between serum metabolites and midbrain gene expression was visualized by highlighting connections with absolute correlation coefficients > 0.8. Figure 8 A). Specifically Lats2 The expression values ​​were compared with TDCA (r = -0.8322, P = 0.0013); Figure 8 B) and TCDCA (r = -0.8252, P = 0.0016; Figure 8 C) shows a negative correlation. Furthermore... Slfn5 Significant associations were found with the abundance of TDCA (r = -0.7902, P = 0.0033) and TCDCA (r = -0.7622, ​​P = 0.0055). Furthermore, correlations between TDCA, TCDCA, and other experimental results were determined. Heatmaps showed that these two bile acids were negatively correlated with behavioral experimental results, gastrointestinal function experimental results, the number of dopaminergic neurons, and the expression of vascular smooth muscle cell (VSMC) markers, while they were positively correlated with α-syn expression, colonic histological score, and IL-1 levels in serum, colon, and midbrain. Figure 8 D). Specifically, TDCA levels were correlated with performance in the tumbler test (r = -0.8049, P = 0.0024); Figure 8 E) and fecal moisture content (r = -0.8345, P = 0.0012; Figure 8 F) showed a significant negative correlation. Similarly, a negative correlation was observed between TCDCA levels and the results of these two experiments (rotary rod experiment: r = -0.7944, P = 0.0030; fecal water content: r = -0.8064, P = 0.0023). In summary, these correlation analyses provide L. The protective effects of reuteri intervention provide new insights into the gut-brain axis mechanism mediated by serum metabolites such as TDCA and TCDCA.

[0091] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The application of Lactobacillus reuteri, characterized by: The application is the use of Lactobacillus reuteri in the preparation of products for the treatment and / or prevention of Parkinson's disease.

2. The application of the microbial agent, characterized in that: The active ingredient of the bacterial agent is Lactobacillus reuteri, and the application is the use of the bacterial agent in the preparation of products for the treatment and / or prevention of Parkinson's disease.

3. The application according to claim 1 or 2, characterized in that: The treatment and / or prevention of Parkinson's disease is manifested in at least one of the following: 1) Improve motor dysfunction; 2) Relieves gastrointestinal dysfunction; 3) Reduce intestinal epithelial damage; 4) Relieves or inhibits intestinal or systemic inflammation; 5) Reduces intestinal permeability; 6) Reduce the loss of dopaminergic neurons in the substantia nigra; 7) Alleviates the accumulation of α-synuclein in the substantia nigra; 8) Reduces blood-brain barrier dysfunction; 9) Maintaining the integrity of the blood-brain barrier; 10) Inhibits neuroinflammation in the substantia nigra; 11) Regulate the metabolism of taurine deoxycholic acid and / or taurine cholic acid.

4. The application according to any one of claims 1-3, characterized in that: The treatment and / or prevention mentioned are for relief.

5. The application according to any one of claims 1-4, characterized in that: The product is a medicine.

6. The application according to any one of claims 2-5, characterized in that: The microbial agent also includes a pharmaceutically acceptable carrier.

Images