Use of Lactobacillus sanfranciscous for preventing and / or treating attention deficit hyperactivity disorder

Abstract

The present invention belongs to the field of microorganisms, and more specifically, the present invention relates to Lactobacillus sanfranciscous ( Lactobacillus_ sanfranciscensis The present invention relates to the use of a microbial agent in the prevention, diagnosis and / or treatment of attention deficit hyperactivity disorder (ADHD), and a microbial preparation for the prevention and / or treatment of ADHD. This provides further evidence for the correlation between the gut microbiota and ADHD, and also provides new candidates for the treatment of ADHD.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and more specifically, the present invention relates to Lactobacillus sanfranciscous ( Lactobacillus_ sanfranciscensis ) in the preparation of a product for preventing and / or treating attention deficit hyperactivity disorder and a microbial preparation for preventing and / or treating attention deficit hyperactivity disorder. Background Art

[0002] Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder with a high prevalence worldwide. [1] , which is characterized by inattention, hyperactivity, and impulsivity, and places a heavy burden on many children and adolescents [2，3] While the etiology of ADHD is complex, researchers agree that genetic influences and environmental factors play a significant role in the development of the disorder. The importance of the gut microbiome in several psychiatric disorders has been demonstrated through the microbiome-gut-brain axis. [4，5] The human intestinal microbiome is composed of more than 1,000 bacterial species that participate in multiple functions such as metabolism, immune response, and synthesis. [6-8] , plays an important role in maintaining human health. However, when the intestinal microbiome is disturbed, it may cause various diseases. Existing studies have shown that the imbalance of intestinal microbiome is related to a variety of mental illnesses, including autism spectrum disorder, schizophrenia, bipolar depression [4，5，9] Over the past few years, about ten studies have explored the potential link between the gut microbiome and ADHD, but there's no consensus among researchers. [10-19] .

[0003] Therefore, there is an urgent need in this field to conduct more in-depth research on the intestinal microbiota of ADHD patients in order to provide a new solution for preventing and / or treating attention deficit hyperactivity disorder through intestinal microorganisms. Summary of the Invention

[0004] As mentioned above, most existing studies on the gut microbiota of ADHD patients are still focused on identifying differential bacteria at the genus or family level. Further in-depth studies of the gut microbiota of ADHD patients are urgently needed to understand the role of the gut microbiota in the pathogenesis of ADHD and provide a new approach to prevent and / or treat ADHD through gut microbiota.

[0005] In view of this, in a first aspect, the present invention provides Lactobacillus sanfranciscous ( Lactobacillus_ sanfranciscensis ) in the preparation of a product for preventing and / or treating attention deficit hyperactivity disorder.

[0006] In some embodiments, the attention deficit hyperactivity disorder is characterized by a lack of or a lower abundance of Lactobacillus sanfrancisco compared to a healthy individual.

[0007] In some embodiments, the product comprises a pharmaceutical.

[0008] In some embodiments, the product is administered gastrointestinally.

[0009] In some embodiments, the dosage forms of the drug include pills, powders, capsules, tablets, suspensions, and granules.

[0010] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0011] In a second aspect, the present invention provides a microbial preparation for preventing and / or treating attention deficit hyperactivity disorder, the microbial preparation comprising Lactobacillus sanfrancisco.

[0012] In some embodiments, the content of Lactobacillus sanfranciscosum is at least 1×10 8 CFU / dose.

[0013] In some embodiments, the microbial preparation is administered gastrointestinal tract.

[0014] In a third aspect, the present invention provides a method for preventing and / or treating attention deficit hyperactivity disorder, comprising administering Lactobacillus sanfranciscous or the microbial preparation of the second aspect of the present invention to a subject in need thereof.

[0015] In some embodiments, the attention deficit hyperactivity disorder is characterized by a lack of or a lower abundance of Lactobacillus sanfrancisco compared to a healthy individual.

[0016] In some embodiments, the Lactobacillus sanfranciscous is deposited in the American Type Culture Collection with the deposit number ATCC27651.

[0017] In some embodiments, the Lactobacillus sanfranciscous is isolated from the intestinal microbiota of a fecal sample of a healthy human.

[0018] In some embodiments, the Lactobacillus sanfranciscous is isolated from the intestinal microbiota of an autologous fecal sample of a subject.

[0019] The beneficial effects of the present invention are: by studying the composition of the intestinal microbiota of ADHD patients at the bacterial species level, further evidence is provided for the correlation between the intestinal microbiota and ADHD, thereby proposing the use of probiotics in the preparation of products for the prevention and / or treatment of attention deficit hyperactivity disorder, and providing new candidates for the prevention and treatment of ADHD. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other implementation plans can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a brief flow chart of the zebrafish experimental groups and experiments.

[0022] Figure 2 These are photos of bacterial growth in different groups.

[0023] Figure 3 It is a statistical chart of the development indicators of each group of juvenile fish (including mortality and deformity rates).

[0024] Figure 4 This is a trajectory diagram of the zebrafish's spontaneous activity detected at 5 dpf.

[0025] Figure 5 This is a comparison of the autonomous activity speed of zebrafish in the Lactobacillus sanfrancisco colonization group (MC) and other groups (* p <0.05, ** p <0.01).

[0026] Figure 6 It is a marker of noradrenergic nerves in all groups of zebrafish dβh and dopaminergic neuron markers th Quantitative gene expression (* p <0.05, *** p <0.001).

[0027] Figure 7 Comparison of the spontaneous activity speed of zebrafish in the atomoxetine-treated group (TO) and other groups (5 dpf, *** p <0.001).

[0028] Figure 8 This is a comparison of the movements of the San Francisco Lactobacillus treatment group (FMT-AA) and the control group (FMT-AC). The right picture is a schematic diagram of the trajectory, and the left picture is a statistical result diagram ( *p<0.05 , **p<0.01 ).

[0029] Figure 9 This is the 5-CSRTT test results of the San Francisco Lactobacillus treatment group (FMT-AA) and the control group (FMT-AC) ( *p ＜0.05, **p＜0.01 ).

[0030] Figure 10 This is the Beta diversity (PCoA) analysis result of each group of mice and wild-type mice (WT) after microbiota transplantation and treatment.

[0031] Figure 11 This figure shows the results of Lactobacillus abundance analysis in each group of mice and wild-type mice (WT) after microbiota transplantation and treatment. DETAILED DESCRIPTION

[0032] The present invention will be described clearly and completely below in conjunction with the embodiments and accompanying drawings of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments that can be obtained by a person of ordinary skill in the art based on the embodiments of the present invention fall within the scope of protection of the present invention.

[0033] Unless otherwise indicated, all terms used herein should be understood as having ordinary meanings known in the art. Other more specific definitions of certain terms used in this application are described below and are intended to be uniformly applied throughout the specification and the scope of the invention patent application, unless the definitions explicitly listed otherwise provide a broader definition. Throughout the specification and the scope of the invention patent application, the word "comprise" and its variants are not intended to exclude other technical features, additives, components or steps. In addition, the word "comprise" covers the situation of "consisting of...". By reading the specification, other objects, advantages and features of the present invention will become apparent to those skilled in the art, or can be learned by practicing the present invention. In addition, the present invention covers all possible combinations of the specific and particular embodiments described herein.

[0034] In this specification and the accompanying claims, the singular indefinite article terms ("a", "an") and the definite article terms ("the") include plural referents. Unless the context clearly indicates otherwise. The indefinite article term "a" (or "an") and the terms "one or more" and "at least one" are used interchangeably herein. In addition, "and / or" as used herein should be taken as explicitly disclosing that each of the two specified features or components has or does not have the other. Thus, the term "and / or" (e.g., "A and / or B") used in phrases herein is intended to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" (e.g., "A, B, and / or C") used in phrases is intended to cover each of the following: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure relates. Units, prefixes, and symbols are expressed in their International System of Units (SI) accepted form. Numerical ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects or aspects of this disclosure, which can be obtained by reference to the entire specification as a whole. Therefore, the terms defined directly below are more fully defined by reference to the entire specification.

[0035] As mentioned above, previous studies of the gut microbiome in patients with ADHD have confirmed a close association between the gut microbiome and the development of ADHD, but most have focused on identifying differentially expressed bacteria at the genus or family level. Therefore, further investigation of the gut microbiome in patients with ADHD is urgently needed to provide new strategies or evidence for the prevention and / or treatment of ADHD through gut microbial intervention. To address this gap, the inventors employed strict inclusion and exclusion criteria and comprehensively analyzed the gut microbiome composition in stool samples from all participants using shotgun metagenomic sequencing to explore the differences and roles of the gut microbiome in patients with ADHD and healthy controls (HCs). The inventors recruited 188 participants, including 94 drug-naive patients diagnosed with ADHD and 94 HCs. Metagenomic sequencing of stool samples was performed by the Beijing Genomics Institute using the MGISEQ 2000 platform and PE150 mode. Compared to other sequencing methods (such as 16S rRNA sequencing), shotgun metagenomic sequencing provides more reliable microbial information and extends taxonomic resolution to the species level.

[0036] Subsequently, this study used linear discriminant analysis (LEfSe) and statistical analysis of metagenomic maps (STAMP) algorithms to distinguish and analyze the intestinal microorganisms in different experimental groups. LEfSe and STAMP are two analytical methods used to distinguish and locate different intestinal microorganisms between different experimental groups. LEfSe identifies microbial taxa with statistically significant differences, while STAMP provides more statistical tests and effect size measurements. The combination of these two methods can provide a more comprehensive and in-depth analysis. This study used LEfSe analysis to identify microbial taxa with significant differences between the two groups, and then used STAMP analysis to verify the statistical significance of different taxa and calculate the effect size.

[0037] By combining the results of LEfSe and STAMP analysis, the inventors identified seven bacterial species with significant differences between the ADHD and HC groups, five of which were significantly more abundant in the HC group, including Lactobacillus sanfranciscous ( Lactobacillus sanfranciscensis ), Pseudomonas xylella (Paraprevotella xylaniphila) , Bacteroides (Bacteroides zoogleoformans), Bacteroides heparinolyticus (Bacteroides heparinolyticus), Rumen-dwelling Prevotella (Prevotella ruminicola) , two species were significantly more abundant in the ADHD group, including Haemophilus parainfluenzae ( Haemophilus parainfluenzae ), Streptococcus salivarius ( Streptococcus salivarius Although five strains were significantly more abundant in the HC group compared to the ADHD group, four of them are anaerobic and cannot survive in water for long periods of time. Only Lactobacillus sanfranciscous (a facultative anaerobe) could survive under aerobic conditions.

[0038] Based on these experimental results, the inventors further transplanted a candidate strain (Lactobacillus sanfranciscous), found to be low in abundance in ADHD patients, into zebrafish and mouse models for in vivo experiments. The study found that supplementation with Lactobacillus sanfranciscous corrected the hyperactive behavior in zebrafish (GF group). Furthermore, a human gut microbiota deficient in Lactobacillus sanfranciscous could induce hyperactive behavior in mice, and this behavior could be eliminated by supplementation with Lactobacillus sanfranciscous. This confirms the relationship between ADHD and the abundance of Lactobacillus sanfranciscous in the gut microbiome, thus completing the present invention.

[0039] Therefore, in a first aspect, the present invention provides Lactobacillus sanfranciscous ( Lactobacillus_ sanfranciscensis ) in the preparation of a product for preventing and / or treating attention deficit hyperactivity disorder.

[0040] The inventors discovered that Lactobacillus sanfrancisco has the effect of treating and preventing ADHD, and further discovered the relationship between ADHD and the abundance of Lactobacillus sanfrancisco in intestinal microorganisms, thus proving that ADHD can be treated and prevented by supplementing Lactobacillus sanfrancisco. In the study, the inventors tested Lactobacillus sanfrancisco isolated from the intestinal microbiota of fecal samples of several healthy control groups and Lactobacillus sanfrancisco deposited in the American Type Culture Collection with the deposit number ATCC27651 ( Lactobacillus_sanfranciscensis ) (Weiss and Schillinger 1984, DSM 20451 T , Leibniz Institute DSMZ, Germany), found no significant difference in the efficacy of Lactobacillus sanfranciscous from different sources. Since Lactobacillus sanfranciscous is a common probiotic in the human gut, those skilled in the art will appreciate that Lactobacillus sanfranciscous from other sources can achieve similar effects. This disclosure is not limited to a specific Lactobacillus sanfranciscous strain for the diagnosis, treatment, and prevention of ADHD, but rather explores the relationship between ADHD and the abundance of Lactobacillus sanfranciscous in the gut microbiome.

[0041] In an experiment using a mouse model that is closer to the human intestinal microbiome, the inventors divided the mice into three groups: a group colonized with healthy bacteria (FMT-H), an ADHD sample group colonized with high-abundance pathogenic bacteria (Haemophilus parainfluenzae and Streptococcus salivarius) (FMT-B), and an ADHD sample group colonized with low-abundance probiotics (Lactobacillus sanfranciscous deficiency) (FMT-A). Both colonization with high-abundance pathogenic bacteria and low-abundance probiotics caused mice to have hyperactive behavior, but after supplementing the two groups of mice with Lactobacillus sanfranciscous, only the hyperactive behavior of the mice in the low-abundance probiotic group disappeared, while there was no change in the high-abundance pathogenic bacteria group.

[0042] In the present invention, Lactobacillus sanfranciscous can be used to prepare various products for preventing and / or treating attention deficit hyperactivity disorder, including medicines.

[0043] Because Lactobacillus sanfranciscous is derived from the intestinal microbiome, products prepared using it are preferably administered via the gastrointestinal tract. Therefore, any product type or pharmaceutical dosage form suitable for gastrointestinal administration is suitable for use in the present invention. In some embodiments, the pharmaceutical dosage forms include pills, powders, capsules, tablets, suspensions, and granules.

[0044] In some embodiments, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

[0045] In a second aspect, the present invention provides a microbial preparation for preventing and / or treating attention deficit hyperactivity disorder, wherein the microbial preparation comprises Lactobacillus sanfranciscous ( Lactobacillus_sanfranciscensis ).

[0046] In this article, the term "probiotics" refers to live microbial preparations made from normal microorganisms or substances that promote microbial growth. In other words, any preparation that promotes the growth and reproduction of normal microorganisms and inhibits the growth and reproduction of pathogenic bacteria is called a "probiotic."

[0047] In some embodiments, the content of Lactobacillus sanfranciscosum is at least 1×10 8 CFU / dose.

[0048] In some embodiments, the microbial preparation is administered gastrointestinal tract.

[0049] Those skilled in the art will appreciate that the above descriptions related to attention deficit hyperactivity disorder in the first aspect of the present invention are all applicable to the second aspect of the present invention, and therefore will not be repeated here.

[0050] The present invention will be further described below by the following examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the materials and reagents used in the following examples are all commercially available. Example

[0051] Materials and methods

[0052] 1. Zebrafish Experimental Design

[0053] All zebrafish experiments were conducted using wild-type AB zebrafish. Zebrafish were maintained at a density of 8-10 per liter at 28°C. Sterile zebrafish larvae were generated by exploiting the axenic development of zebrafish embryos.

[0054] To investigate how the microbiome influences behavior, juvenile fish were divided into five groups, 200 each: germ-free (GF), conventionally housed (CV), conventionally housed with sterile treatment (CVZ), microbial colonization (MC), and atomoxetine-treated (TO). The CV group was maintained in standard zebrafish culture medium without any sterile treatment, while the GF, CVZ, MC, and TO groups were sterile-treated. After sterile treatment, embryos were collected into cell culture flasks containing either sterile zebrafish culture medium (GF, MC, and TO groups) or standard zebrafish culture medium (CVZ group). At 3.5 days post-fertilization (p.i.), germ-free zebrafish in the MC group were placed in culture medium containing Lactobacillus sanfranciscous. Behavioral assays were performed on day 5 post-fertilization (p.i.). On the day of the behavioral assay, the TO group was treated with 10 μmol / L atomoxetine for 1 hour before undergoing behavioral analysis along with the other groups. Figure 1 The grouping and brief process of the experiment are shown.

[0055] 2. Sterile Derivation of Zebrafish Embryos

[0056] Refer to existing methods for culturing germ-free zebrafish

[24] Table 1 shows the timeline for establishing germ-free zebrafish embryos. On the afternoon of the day before spawning (Day -1), healthy adult zebrafish females and males were placed at opposite ends of a separate breeding cage. On Day 0, at the onset of the light cycle, females and males were transferred to sterile water. The females and males were allowed to chase each other for spawning, and fertilized eggs were collected within 60 minutes to ensure that all fertilized eggs were at the same developmental stage. Fertilized eggs were transferred to culture dishes containing antibiotic medium (containing 250 mg / mL amphotericin B, 5 μg / mL kanamycin, and 100 μg / mL ampicillin) and surface-sterilized for 6 hours. Subsequently, the eggs were surface-sterilized by rinsing with 0.1% polyvinylpyrrolidone-iodine (PVP-I) and 0.003% sodium hypochlorite, respectively. The fertilized eggs were then placed in sterile cell culture flasks (with a 0.2 μm filter cap to filter out airborne microorganisms and maintain a sterile environment) and incubated at 28°C. Check the sterility and viability of embryos daily.

[0057]

[0058] To confirm the microbial status of each group, daily testing was performed to ensure sterility and to characterize the microbiota of the zebrafish larvae groups. 10 μL of culture medium from each group's culture flask was inoculated on a nutrient agar (LB) plate and cultured overnight at 28°C. In addition, before the behavioral assay, 50 zebrafish larvae from each group were ground under sterile conditions and evenly spread on nutrient agar plates for overnight culture to further confirm sterility. The results showed that no bacteria grew on the plates of the GF group, while bacterial growth was observed on the plates of the CV and CVZ groups ( Figure 2 ).

[0059] The developmental indicators of each group of juveniles were then evaluated before behavioral testing ( Figure 3 ), no difference was found, which shows that behavioral detection is feasible.

[0060] 3. Strain Cultivation

[0061] The inventors isolated Lactobacillus sanfrancisco from the intestinal microbiota of fecal samples from several healthy control groups. The isolation steps are briefly described as follows: the mixed feces were diluted and monoclonal bacteria were cultured using MRS agar medium, a selective medium commonly used for lactic acid bacteria culture. The initial screening was completed by observing the morphology of the monoclonal culture, and then the culture was cultured in MRS medium at 30°C. The culture was confirmed by rRNA sequencing. In addition to isolating Lactobacillus sanfrancisco from the intestinal microbiota of fecal samples from healthy control groups, the inventors also tested Lactobacillus sanfrancisco, which is deposited in the American Type Culture Collection with the deposit number ATCC27651 ( Lactobacillus_sanfranciscensis ) (Weiss and Schillinger 1984, DSM 20451 T , Leibniz Institute DSMZ, Germany) and purchased from Bioscibio.

[0062] Lactobacillus sanfranciscous was cultured in MRS medium at 30°C. The culture medium was centrifuged at 8000 rpm for 5 minutes, the cells were collected, and washed twice with sterile PBS. The bacterial concentration was 1 × 10 5 When selecting the concentration of Lactobacillus sanfranciscous, due to the lack of relevant literature on the colonization of Lactobacillus sanfranciscous in zebrafish larvae, the inventors referred to the relevant literature on the colonization of other strains in zebrafish larvae and adopted 1´10 5 to 1´10 8 The results showed that there was no significant difference in the effect of Lactobacillus sanfrancisco from different sources. The concentration of Lactobacillus sanfrancisco was 1´10, which had the least effect on the development of zebrafish larvae. 5 For the sake of experimental consistency, Lactobacillus sanfrancisco purchased from Baosai Biotechnology Co., Ltd. was used in the examples of this application. Since Lactobacillus sanfrancisco is a common probiotic in the human intestine, those skilled in the art will understand that Lactobacillus sanfrancisco from other sources can also achieve similar effects and are included in the scope of the present invention.

[0063] 4. Behavioral Detection

[0064] At 5 dpf, 96 juveniles from each group were randomly selected for behavioral analysis. Juveniles were placed into each well of a 96-well plate (four groups of 24 juveniles were placed in each 96-well plate for a total of four trials). Movement speed and distance were analyzed over time using a zebrafish behavioral analysis system (DanioVision with EthoVision XT 14.1324, Noldus, Netherlands). The first 15 minutes of the measurement period were an acclimatization phase, followed by 45 minutes of recorded movement and speed. This was repeated three times.

[0065] 5. Real-time PCR of Noradrenergic and Dopaminergic Neural Marker Gene Expression

[0066] Real-time PCR was used to detect noradrenergic neuron markers in zebrafish of each group. dβh and dopaminergic neuron markers th Quantitative gene expression was performed. ADHD is associated with dysfunction of monoamine neurons, including norepinephrine and dopamine neurons. Tyrosine hydroxylase (TH) is a key enzyme in the synthesis of dopamine using tyrosine as a substrate, while dopamine β-hydroxylase (DβH) catalyzes the conversion of DA to NE. Therefore, the expression levels of TH and DβH can be used as markers of monoamine neuron activity. Total RNA was extracted using the Trizol method. β-actin was used as an internal control. Specific primer sequences are shown in Table 2.

[0067]

[0068] 6. Atomoxetine treatment

[0069] Atomoxetine is an effective clinical treatment for ADHD. During experimental preparation, atomoxetine powder (TOCRIS Company, the United States) was dissolved in ddH2O. Prior to behavioral testing, zebrafish larvae in the TO group were transferred to a 10 μmol / L atomoxetine solution for one hour before behavioral analysis with the other groups.

[0070] 7. Mouse Experiment Design

[0071] Four-week-old male C57BL / 6J mice (n=60), weighing 9 to 13 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. and housed at 22 to 23°C with a 12-h light / dark cycle. After a one-week acclimation period, the mice were given free access to food and water. Subsequently, the mice were orally administered a mixture of four antibiotics (ABX) for 7 days: ampicillin (200 mg / kg / day, Solarbio Cat#A6920), vancomycin (100 mg / kg / day, VIANEX SA PLANTC), neomycin (200 mg / kg / day, SIGMA Cat# N6386), and metronidazole (200 mg / kg / day, SIGMA Cat# M1547). [25，26] This regimen resulted in an enlarged cecum and a significant reduction in the gut microbiota in mice (as demonstrated by fecal 16S rRNA analysis).

[0072] Subsequently, the mice were divided into groups and received fecal microbiota transplants (FMTs) from different sources: a group colonized with healthy bacteria (FMT-H, n=13), a group colonized with high abundance of pathogenic bacteria (Haemophilus parainfluenzae and Streptococcus salivarius) and ADHD samples (FMT-B, n=18), and a group colonized with low abundance of probiotics (Lactobacillus sanfranciscous-deficient) and ADHD samples (FMT-A, n=20). Lactobacillus sanfranciscous (FMT-BA / FMT-AA group, 1x10^8 cells / mouse / day), butyrate (FMT-BB / FMT-AB group, 20 mg / 0.5 ml), or PBS (FMT-BC / FMT-AC group) were added to the drinking water of mice in the FMT-B and FMT-A groups, respectively. Because there was no previous reference for direct colonization with Lactobacillus sanfranciscous, the inventors referred to Lactobacillus reuteri ( Lactobacillus reuteri ) colonization concentration (Sgritta et al., 2019). After two rounds of colonization, each mouse was subjected to behavioral analysis using the open field test (open field test box volume 40 × 40 × 25 cm). In addition, the 5-CSRTT was used to evaluate the attention and impulsivity levels of the mice. Compared with mice with unrestricted food intake, mice were deprived of food to maintain 85% of their body weight. This task involves training mice to recognize the location of visual stimuli in order to obtain food rewards. Training is carried out in seven consecutive stages, each with specific criteria that mice must meet for two consecutive days before entering the next stage.

[37] . Typical training lasted 30 minutes or 100 trials, whichever came first. In correct trials, the latency of the correct response was defined as the time interval from the onset of visual stimulation to the time when the subject put their nose into the cue hole, which can reflect the attention level of the mouse. If the mouse poked its nose into an unlit hole, it was recorded as an incorrect response. If the mouse failed to indicate the cue location within the specified time limit, it was recorded as an omission. In addition, a nose poke that occurred before the cue was presented was classified as a premature response, which can reflect the impulsiveness level of the mouse. Throughout the experiment, the weight changes of mice were monitored before and after ABX treatment and before and after bacterial colonization in each group. In addition, fecal 16S rRNA of mice was monitored at baseline, after antibiotic treatment, after transplantation and after treatment.

[0073] 8.16S rRNA gene analysis

[0074] DNA extraction: Transfer approximately 100-200 mg of each meta-sample to a centrifuge tube containing grinding beads and 1 mL of ATL / PVP-10 buffer; homogenize the sample using a grinder and incubate at 65°C for 20 minutes; centrifuge the lysate at 14,000 × g for 5 minutes, and transfer the supernatant to a new centrifuge tube; add 0.6 mL of PCI buffer and vortex for 15 seconds to achieve protein precipitation; centrifuge the mixture at 18,213 × g for 10 minutes, and transfer the resulting supernatant to a deep-well plate containing magnetic beads and binding solution (600 μL wash buffer (containing magnetic beads) + 20 μL proteinase K + 5 μL RNase A), wash successively with wash buffer 1 (700 μL), wash buffer 2 (700 μL), and wash buffer 3 (700 μL), and finally elute with 100 μL elution buffer; collect the purified DNA in a 1.5 mL tube for further analysis. Amplicon Library Construction and Sequencing: The 16S rRNA gene consists of nine variable regions and ten conserved regions. Variable regions, such as V3–V4, were amplified to characterize bacterial phylogeny and community diversity. Sequencing primers were designed targeting these regions and combined with fusion primers for amplification and high-throughput sequencing. Library Construction: Genomic DNA was quantified and amplified by PCR using region-specific primers. Amplified products were purified and quality tested using standard protocols. Sequencing libraries were prepared and validated before sequencing on the Illumina platform to ensure high accuracy and coverage. Raw sequencing reads were processed to generate clean data: primer and adapter sequences were trimmed using cutadapt v2.6. Low-quality reads were removed using a sliding window method (30 bp window, average quality <20). Reads shorter than 75% of their original length after trimming were discarded. Reads containing ambiguous bases (n) or low-complexity sequences (≥10 consecutive identical bases) were removed (software used: iTools Fqtools fqcheck v0.25, cutadapt v2.6, readfq v1.0). Amplicon sequence variants (ASVs) with 100% sequence similarity were generated using the DADA2 method in Qiime2. This workflow included importing filtered paired-end reads using Qiime tools. Denoising and feature table construction were performed using Qiime DADA2 denoising paired reads. The feature table was exported using Qiime tools for downstream analysis. Representative sequences for each OTU were classified against a reference database using RDP Classifier v2.2 with a confidence threshold of 0.6.

[0075] After obtaining the genus-level ASVs, the data were normalized to ensure comparability between samples. Downstream analyses of the normalized data were then performed using R4.4.1. Alpha diversity analysis: Indicators such as the Shannon and Simpson indices were calculated to assess diversity within the sample, reflecting richness and evenness. Beta diversity analysis: Principal coordinate analysis (PCoA) based on the Bray-Curtis distance was used to visualize differences in microbial community composition between different taxa. Genus-level bar charts: The relative abundance of genera was calculated and visualized as stacked bar charts to display the microbial composition of samples and groups. Target genus comparison: The relative abundance of target genera (e.g., Lactobacillus) between groups was statistically compared using the Wilcoxon rank sum test, highlighting significant differences.

[0076] 9. Behavioral Data Analysis

[0077] Behavioral data were collected using a video tracking system (video behavior analysis software Ethovision XT, Noldus Information Technology, USA). Statistical analysis was performed using Prism 10.1.2 with unpaired t-tests. Python 3.9 (available from https: / / github.com / sraorao / animapp_desktop) was used.

[29] Create a model diagram.

[0078] Experimental results

[0079] 1. Supplementation with Lactobacillus sanfranciscous corrects hyperactive behavior in a zebrafish model

[0080] according to Figure 4 、 Figure 5 As shown in Table 3, at 5 dpf, the GF group showed hyperactive behavior compared with the CV and CVZ groups. To confirm whether the hyperactive behavior of zebrafish in the GF group is a phenotype of ADHD, the inventors used real-time PCR to detect noradrenergic neural markers in zebrafish in the CV, CVZ, and GF groups. dβh and dopaminergic neuron markers th Quantitative expression of . Figure 6 It can be seen that although there was no significant difference in th expression among the groups, the expression of dβh in the GF group zebrafish was significantly downregulated compared with the other groups. This indicates that the activity of the noradrenal system in the GF group germ-free zebrafish is reduced.

[0081] Next, the inventors tried to treat the GF group with the ADHD drug (atoxetine) to determine the effectiveness of atomoxetine in correcting the behavior of zebrafish. At 5 dpf, zebrafish in the TO group were immersed in 10 μmol / L atomoxetine for 1 hour before behavioral analysis. Figure 7 The locomotor activity of the TO group was significantly decreased compared with the GF group, but was not significantly different from that of the CV group, indicating that atomoxetine was effective in the TO group.

[0082] The inventors tried to colonize GF zebrafish with the different bacterial strains found in clinical samples and found that after colonization with Lactobacillus sanfrancisco, the GF group's spontaneous activity speed decreased, with no significant difference from the CV and CVZ groups, and hyperactive behavior disappeared, indicating that Lactobacillus sanfrancisco colonization may help improve the hyperactive behavior of ADHD. Based on the experimental results of the MC group, the inventors found that supplementation with Lactobacillus sanfrancisco corrected the hyperactive behavior of the GF group ( Figure 4 、 Figure 5 and Table 3).

[0083]

[0084]

[0085] 2. Effects of Lactobacillus sanfranciscous Supplementation on a Mouse Model Colonized with Human Gut Microbiota

[0086] To further validate the effects of Lactobacillus sanfranciscoensis, the authors used a mouse model that more closely resembles the human gut microbiome to verify their results. First, five-week-old male mice were given antibiotics (ABX), and then the mice were divided into groups and received fecal microbiota transplants from different sources.

[30] : Healthy individuals (FMT-H), ADHD patients with high abundance of Haemophilus parainfluenzae and Streptococcus salivarius (FMT-B), and ADHD patients with low abundance of Lactobacillus sanfranciscous (FMT-A).

[0087] Results showed that compared with FMT-H mice, mice in the FMT-A / FMT-B groups exhibited significant increases in locomotion (FMT-B: F = 1.78, P = 0.031, n = 18; FMT-A: F = 1.89, P = 0.044, n = 20). In the first 5 minutes of the open field test, there were no statistically significant differences in the time spent in the center zone among the three groups, indicating no statistically significant differences in anxiety levels. Furthermore, in the 5-CSRTT, mice in the FMT-A group showed a significant decrease in attention, as evidenced by a prolonged mean latency to correct responses and a decrease in correct responses (t = 3.615, P = 0.0025; t = 2.275, P = 0.0380, n = 18), while there were no statistically significant differences in impulsive behavior (t = 0.225, P = 0.8247, n = 18).

[0088] Next, to explore the therapeutic potential of probiotic metabolites (such as butyrate) and Lactobacillus sanfranciscous, both groups received sodium butyrate and Lactobacillus sanfranciscous. The results showed that butyrate treatment did not significantly improve the movement of either group, while supplementation with Lactobacillus sanfranciscous alleviated the hyperactive behavior of the FMT-A group mice ( Figure 8 ), and at the same time, effectively alleviated the attention deficit of FMT-A group mice, such as Figure 9 As shown in the figure, the average correct response latency of mice in the FMT-A group was significantly reduced (t=2.466, P=0.0333, n=12), and the correct response rate showed an upward trend (t=1.898, P=0.0869, n=12).

[0089] Furthermore, the inventors performed 16S rRNA analysis on fecal samples collected from transplanted and treated mice. The results of β-diversity analysis determined by PCoA showed that the FMT-H mouse group exhibited a microbial community composition that was most similar to that of the WT mouse group. Other groups, such as FMT-A and FMT-AA, showed different community structures ( Figure 10 ). Figure 11 An analysis of the abundance of Lactobacillus in the different groups is provided in . Taken together, the results of this study suggest that a reduction in Lactobacillus sanfranciscous in the gut microbiota can lead to ADHD-like symptoms, while supplementation with Lactobacillus sanfranciscous in an ADHD model with low Lactobacillus sanfranciscous abundance can effectively alleviate ADHD-like symptoms.

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Claims

1. Lactobacillus sanfranciscous ( Lactobacillus_sanfranciscensis ) in the preparation of a medicament for preventing and / or treating attention deficit hyperactivity disorder, wherein the prevention and / or treatment of attention deficit hyperactivity disorder includes improving the hyperactive behavior of an individual, wherein the deposit number of the Lactobacillus sanfranciscous is ATCC27651. The method according to claim 1 , wherein the drug is administered via the gastrointestinal tract.

3. The use according to claim 1 or 2, wherein the dosage form of the drug is pills, powders, capsules, tablets, suspensions or granules.

4. The method according to claim 1 or 2, wherein the drug further comprises a pharmaceutically acceptable carrier or excipient.

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