A probiotic and creatine composition with antidepressant efficacy and uses thereof

Abstract

The application discloses a probiotic and creatine composition with an antidepressant effect and application thereof, and contains bifidobacterium and creatine, has small toxic side effects, long-term effects, and low prices, and has wide application prospects. The application finds that the content of creatine in feces increases, and the content of creatine in blood and brain decreases in the occurrence of depression; and finds that the abundance of pseudolongum bifidobacterium and adolescent bifidobacterium in feces decreases in the occurrence of depression. The combined use of pseudolongum bifidobacterium and creatine or adolescent bifidobacterium and creatine can significantly increase the antidepressant effect of creatine. Experimental research proves that the medicine containing effective amounts of bifidobacterium and creatine as active ingredients has small toxic side effects, long-term effects, and low prices in the prevention and treatment of depression.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a probiotic and creatine composition with antidepressant effects and its uses. Background Technology

[0002] Recent research indicates that at least 700 million people worldwide suffer from mental illness (approximately 300 million in my country); depression is a common disease globally, affecting about 3.8% of the population. Due to its high incidence, high disability rate, and severe suicidal tendency, depression brings endless suffering to patients and their families, seriously endangering human life and health, and having a significant negative impact on social harmony and stability, thus becoming a serious public health problem. Currently, clinically available antidepressants are mainly based on the monoamine neurotransmitter theory, such as monoamine oxidase inhibitors, serotonin and / or norepinephrine reuptake inhibitors, etc. These drugs have achieved some efficacy, but still face many serious challenges, such as slow onset of action, low efficacy, significant side effects, and high relapse rate. Therefore, finding antidepressants with rapid onset of action, high efficacy, long-lasting effect, and few side effects is of great significance for the clinical treatment of depression.

[0003] Creatine (Cr) is a substance synthesized from three amino acids: arginine, glycine, and methionine. Creatine supplementation can rapidly resynthesize ATP, providing energy for nerve cells. Creatine can be synthesized by the body itself or obtained from food. To maintain adequate creatine levels, the body needs to synthesize or ingest approximately 2 grams of creatine daily, with 50% obtained from food. Creatine in food mainly comes from meat and fish; vegetarian diets contain very little creatine. A cohort study of elderly Chinese men found that those following a vegetarian diet had a higher risk of depression, more severe depressive symptoms, and higher scores on the Geriatric Depression Scale; suggesting that a diet pattern that reduces creatine intake may be associated with the risk of depression. A clinical RCT study by the In Kyoon Lyoo research group at the University of Utah found that 8 weeks of creatine treatment significantly improved symptoms and functional network connectivity in core brain regions of patients with depression. However, creatine treatment for depression still faces challenges such as long duration of action and limited therapeutic effect.

[0004] Numerous studies have suggested that gut microbiota influence energy metabolism, higher brain functions, and behavior. The gut and central nervous system interact and influence each other through the "gut-brain axis." Studies in germ-free animals and animals infected with pathogenic bacteria, treated with probiotics, or treated with antibiotics have demonstrated that the gut microbiota plays a crucial role in regulating depression, anxiety, cognition, and autism. The gut microbiota modulates the gut-brain axis through multiple potential direct and indirect pathways, including endocrine (cortisol), immune (cytokines), neural pathways, and gut metabolites. Therefore, modulating the gut microbiota may be a controllable strategy offering novel potential therapies for complex central nervous system disorders. Bifidobacteria are important beneficial gut microbes. As physiologically beneficial bacteria, Bifidobacteria possess numerous important physiological functions for human health, including biological barrier function, nutritional role, anti-tumor effect, immune enhancement, improvement of gastrointestinal function, and anti-aging. Bifidobacteria play a vital role in inhibiting the growth of harmful bacteria in the human body, resisting pathogen infection, synthesizing vitamins needed by the body, promoting the absorption of substances in the intestines, and producing organic acids such as acetic acid, propionic acid, butyric acid, and lactic acid to stimulate intestinal peristalsis, promote defecation, prevent constipation, inhibit intestinal putrefaction, purify the intestinal environment, decompose carcinogens, and stimulate the human immune system, thereby improving disease resistance. However, there is still a lack of probiotic preparations with good antidepressant effects on the market. Therefore, developing a composition containing probiotics with good antidepressant effects is particularly urgent and necessary. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a probiotic and creatine composition with antidepressant effects and its uses.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] This invention first discovered that in a mouse model of depression, creatine levels increased in the feces of depressed mice, while creatine levels decreased in the blood and brain, and the abundance of *Bifidobacterium pseudolongum* in the feces also decreased. After transplanting the gut microbiota from the feces of depressed patients, the abundance of *Bifidobacterium pseudolongum* and *Bifidobacterium adolescentis* in the mouse feces was significantly reduced.

[0008] This invention creatively develops a novel combination of probiotics and creatine, and a novel antidepressant strategy. By combining Bifidobacterium pseudolongum strains with creatine or combining Bifidobacterium adolescentis strains with creatine, it was found that the two can synergistically enhance the antidepressant effect of creatine and promote the level of creatine in the blood.

[0009] Therefore, the object of the present invention is to provide a probiotic and creatine composition with antidepressant effects and its use, wherein the composition comprises Bifidobacterium pseudolongum and creatine, or Bifidobacterium adolescentis and creatine.

[0010] The aforementioned *Bifidobacterium pseudolongum* is *Bifidobacterium pseudolongum* GDMCC NO. 1.2852, and the aforementioned *Bifidobacterium adolescentis* is *Bifidobacterium adolescentis* GDMCC NO. 1.278.

[0011] A second objective of this invention is to provide a medicine or health food with antidepressant effects, comprising an effective amount of a combination of probiotics and creatine as active ingredients.

[0012] Preferably, the drug or health food also includes a pharmaceutically acceptable carrier; the pharmaceutically acceptable carrier is a conventional pharmaceutical excipient or adjuvant.

[0013] Preferably, the drug contains 0.1% to 99.9% by weight of a combination of probiotics and creatine.

[0014] Preferably, the dosage form of the drug is a liquid dosage form or a solid dosage form.

[0015] Preferably, the liquid dosage form is a solution, suspension, or emulsion.

[0016] Preferably, the solid dosage form is a tablet, capsule, pill, powder for injection, sustained-release preparation, or microparticle delivery system.

[0017] The medicament of the present invention can be prepared according to methods known in the art. For this purpose, if desired, the active ingredient can be combined with one or more solid or liquid pharmaceutical excipients and / or adjuvants to form a suitable administration or dosage form for use as a medicine or health food.

[0018] Dosage forms can be liquid or solid. Liquid dosage forms include solutions, colloids, microparticles, emulsions, and suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, and lyophilized powder injections.

[0019] The dosage form further includes a freeze-drying protectant and / or prebiotics. The freeze-drying protectant includes any one or a combination of at least two of the following: skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol, or xylitol; the prebiotics include any one or a combination of at least two of the following: fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrophyllum, trachomatis polysaccharides, stachyose, polydextrose, α-lactalbumin, or lactoferrin.

[0020] To formulate unit-dose dosage forms into tablets, a wide variety of carriers known in the art can be used. Examples of carriers include: diluents and absorbents: starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; humectants and binders: water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrants, such as dried starch, alginate, agar powder, brown algae starch, etc. Sodium bicarbonate, citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid esters, sodium lauryl sulfonate, methylcellulose, ethylcellulose, etc.; disintegration inhibitors, such as sucrose, tristearate, cocoa butter, ammoniacal oil, etc.; absorption promoters, such as quaternary ammonium salts, sodium lauryl sulfate, etc.; lubricants, such as talc, silica, corn starch, stearates, boric acid, liquid paraffin, polyethylene glycol, etc.; tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0021] To formulate the drug delivery unit into pills, a wide variety of carriers known in the art can be used. Examples of carriers include, for instance, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste, etc.; and disintegrants such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.

[0022] To formulate the drug delivery unit into a suppository, a wide variety of carriers known in the art can be used, such as polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, etc.

[0023] To formulate suppositories into capsules, the active ingredient is mixed with the various carriers described above, and the resulting mixture is placed in hard gelatin capsules or soft capsules. Alternatively, the active ingredient can be formulated into microcapsules, suspended in an aqueous medium to form a suspension, or filled into hard capsules or formulated into injectable preparations for use.

[0024] In addition, excipients may be added to pharmaceutical formulations if necessary, such as any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

[0025] A third objective of this invention is to provide the use of Bifidobacterium pseudolongum and creatine, or Bifidobacterium adolescentis and creatine in combination, in the preparation of drugs or health foods with antidepressant effects.

[0026] The dosage of the drug of the present invention can vary within a certain range taking into account individual differences, such as the nature and severity of the disease to be prevented or treated, the sex, age, weight, personality and individual response of the patient or animal, the route of administration, the frequency of administration, etc.

[0027] This invention has discovered a probiotic and creatine composition with antidepressant effects through pharmacological and pharmacological experiments. The composition contains Bifidobacterium and creatine, has few side effects, long-lasting effects, and is inexpensive, and has broad application prospects. Attached Figure Description

[0028] Figure 1 Dynamic changes in creatine levels in cerebrospinal fluid, blood, and feces during the development of depression

[0029] Figure 2 Gut microbiota-mediated antidepressant effects of exogenous creatine

[0030] Figure 3 Bifidobacteria combined with creatine can enhance the antidepressant effect.

[0031] Figure 4 Bifidobacteria mediate the antidepressant effect of exogenous creatine by promoting the expression of creatine transporter protein in intestinal epithelial cells.

[0032] Figure 5 The combined intervention of Bifidobacterium and creatine reduced the depression scale scores and increased plasma creatine levels in patients. Detailed Implementation

[0033] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0034] The following will describe in detail, with reference to specific implementation methods, a probiotic and creatine composition with antidepressant effects and its uses.

[0035] Example 1

[0036] I. Detection of Creatine and Intestinal Microbiota

[0037] 1. Human Sample

[0038] This invention utilizes peripheral blood, stool, and cerebrospinal fluid samples from patients with depression and healthy controls recruited through Guangdong 39 Brain Hospital. All procedures were approved by the Ethics Committee of Guangdong 39 Brain Hospital (Approval No.: 2021-01-087). Informed consent was obtained from all participants before any research procedure began. In Clinical Cohort 1, 41 participants (26 patients with depression and 15 healthy controls) provided cerebrospinal fluid samples for creatine detection; in Clinical Cohort 2, 53 participants (31 patients with depression and 22 healthy controls) provided plasma samples, with a subset of 35 participants (18 patients with depression and 17 healthy controls) also providing stool samples for creatine detection; in Clinical Cohort 3, 48 participants (27 patients with depression and 21 healthy controls) provided stool samples for metagenomic sequencing. Samples that did not meet established quality standards were excluded. This study included patients aged 18 to 60 years with depression, diagnosed through a structured clinical interview using the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (SCID-5), and with a HAMD score >16 on all 17 items. Patients with a history of other mental illnesses, clinically significant neurological disorders, who had used antibiotics or probiotics within the past 30 days, had a positive pregnancy test, or were breastfeeding were excluded. No gender-based or socially gender-based differential analyses were performed. The entire study strictly adhered to the ethical guidelines of the Declaration of Helsinki.

[0039] 2. The specific steps for creating the mouse model are as follows:

[0040] 2.1 This invention employs the widely used Chronic Social Failure (CSDS) model. During the experiment, mice were placed in a cage with a CD1-aggressive mouse, ensuring physical contact between the two mice for 10 minutes, for a total of 10 days. After 10 minutes, the modeling mouse and the CD1 mouse were separated by a transparent, perforated plexiglass plate. The plate was placed in the middle of the cage, and for the next 24 hours, the test mouse was exposed to chronic stress in a threatening manner. The control group mice were kept in the same cage but with members of the same breed. The mice placed with the control group were rotated daily. After the stress period, the mice were housed separately, and a social interaction experiment was conducted 24 hours later for behavioral evaluation.

[0041] The social interaction experiment was conducted in an open field measuring 42 cm × 42 cm × 25 cm. The open field was divided into an interaction zone and two corners away from the interaction zone. At the start of the experiment, the mice were placed in the open field, and a 3 cm × 6 cm × 25 cm perforated transparent plastic box was placed in the interaction zone. In the absence of CD1 mice, their movement was tracked and recorded for 2.5 minutes, with the time spent in the interaction zone designated as T1. Then, in the presence of CD1 mice, their activity was tracked and recorded for 2.5 minutes, with the time spent in the interaction zone designated as T2. The time spent in the interaction zone and other indicators were analyzed and calculated using Ethvision software (Noldus). After each experiment, the instrument was cleaned with 70% ethanol solution to remove odor, and all behavioral tests were conducted in darkness. Mice with a T2 / T1 ratio less than 1 were classified as the depression group.

[0042] 2.2 Knockout mice of creatine transporter in intestinal epithelial cells

[0043] Vil1-Cre (strain number: 021504) and Slc6a8 loxP / loxP (Strain number: 020642) Mice were all purchased from Jackson Laboratory, USA. The VIL1 gene encodes Villin 1, an epithelial cell-specific Ca(2+)-regulated actin-modified protein expressed in intestinal epithelial cells. The Slc6a8 gene encodes a creatine transporter. When Vil1-Cre mice were bred with Slc6a8 mice containing the loxP site... loxP / loxP After hybridization in mice, sequence knockout between loxP sites mediated by Cre recombinase occurred in the intestinal epithelial cells, preventing the normal expression of creatine transporter protein in the mouse intestinal epithelial cells.

[0044] 3. Creatine detection method:

[0045] Creatine levels were measured using an Abcam creatine assay kit (catalog number: ab65339).

[0046] 4. Metagenomics

[0047] One μg of genomic DNA was taken from the sample and randomly fragmented into segments of approximately 350 bp using a Covaris ultrasonic disruptor. Library construction followed end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification. After library construction, the integrity and insert size of the library fragments were checked using AATI. If the results met expectations, the effective concentration of the library was accurately quantified using Q-PCR (effective concentration > 3 nM) to ensure library quality. After passing the library test, different libraries were pooled according to their effective concentration and the target data volume requirements before PE150 sequencing. The raw data obtained from the NovaSeq sequencing platform was preprocessed using FastP (https: / / github.com / OpenGene / fastp) to obtain clean data for subsequent analysis. The specific processing steps are as follows: a) If any sequencing read contains an adapter sequence, remove the paired read; b) If any sequencing read contains more than 50% of the bases in low-quality (Q<=5) bases, remove the paired read; c) If any sequencing read contains more than 10% of the bases in N bases, remove the paired read. MEGAHIT software was used to assemble and analyze the clean data. Assembly parameters were set as follows: -- presets meta-large (--end-toend,--sensitive, -I 200, -X 400 (Karlsson FH et al., 2013; Nielsen HB et al., 2014). The assembled scaffolds were then broken at N-joints to obtain scaffolds without N-joints. MetaGeneMark (http: / / topaz.gatech.edu / GeneMark / ) was used to predict the ORF of scaffolds (>=500bp) for each sample, and information shorter than 100 nt in the prediction results was filtered out. The ORF prediction results were then deredundant using CD-HIT software (http: / / www.bioinformatics.org / cd-hit / ) to obtain a non-redundant initial gene catalogue (here, the non-redundant continuous gene-encoding nucleic acid sequences are referred to as genes, parameters: -c 0.95, -G 0, -aS). 0.9, -g 1, -d 0.Bowtie2 was used to align the clean data of each sample to the initial gene catalogue, calculating the number of aligned reads for each gene in each sample. Alignment parameters: --end-to-end, --sensitive, -I 200, -X 400. Genes with <=2 reads in each sample were filtered out, resulting in the final gene catalogue (unigenes) for subsequent analysis. Abundance information for each gene in each sample was calculated based on the number of aligned reads and gene length. The unigenes were aligned with Micro_NR using DIAMOND software (https: / / github.com / bbuchfink / diamond / ), with parameters: blastp, -e 1e-5. Micro_NR consists of bacterial, fungal, archaea, and viral sequences extracted from the NCBI NR database (https: / / www.ncbi.nlm.nih.gov / ). For each sequence alignment, the result with an evalue less than or equal to the minimum evalue * 10 was selected. Since each sequence may have multiple alignment results, the LCA algorithm (applied to the systematic classification in MEGAN software) was used to determine the species annotation information for that sequence. Starting from the LCA annotation results and gene abundance table, abundance information and gene count tables for each sample at each taxonomic level (kingdom, phylum, class, order, family, genus, species) were obtained. The abundance of a species in a sample is equal to the sum of the abundances of genes annotated as belonging to that species; the gene count of a species in a sample is equal to the number of genes with a non-zero abundance among those annotated as belonging to that species. MetaGenomeSeq and LEfSe analyses were used to identify species with intergroup differences. MetaGenomeSeq analysis performed hypothesis testing at each taxonomic level to obtain p-values ​​and Q-values, while LEfSe analysis used the LEfSe software.

[0048] 5. Bifidobacterium detection

[0049] After collecting mouse feces, fecal microbiota DNA was extracted according to the instructions of the DNA extraction kit (zymoresearch). DNA concentration was measured using NanoDrop. Real-time quantitative PCR was performed using SYBR premix (TaKaRa) and primers on an ABI 7500 Real-Time PCR system. Primers were designed to target the 16S ribosomal subunits of *Bifidobacterium pseudolongum*, *Bifidobacterium adolescentis*, and all bacteria.

[0050] II. Experimental Protocol for Drug Efficacy and Pharmacological Effects

[0051] The composition of this invention consists of *Bifidobacterium pseudolongum* (accession number GDMCC NO. 1.2852) and creatine, or *Bifidobacterium adolescentis* (accession number GDMCC NO. 1.278) and creatine. *Bifidobacterium pseudolongum* (accession number GDMCC NO. 1.2852) is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO. 1.2852. *Bifidobacterium adolescentis* (accession number GDMCC NO. 1.278) is also deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC NO. 1.278.

[0052] 1. Antibiotic gavage treatment

[0053] SPF-grade C57BL / 6J mice aged 7-8 weeks received continuous antibiotic treatment for 7 days, administered a mixture of vancomycin (50 mg / kg), neomycin (100 mg / kg), and metronidazole (100 mg / kg) by gavage every 12 hours daily. Ampicillin (1 mg / mL) was continuously administered via drinking water concurrently. Control group (treatment mimic) mice were administered an equal volume of plain drinking water by gavage at the same schedule. All behavioral experiments were performed 24 hours after the last gavage.

[0054] 2. Microbial transplantation

[0055] SPF-grade C57BL / 6J mice aged 7-8 weeks were given antibiotic pretreatment via gavage for 7 consecutive days, followed by a 2-day interval, and then administered 200 μL of a solution containing *Bifidobacterium pseudolongum* (10... 8 Or 10 9 (10 live bacteria) or Bifidobacterium adolescentis (10) 8 Or 10 9 Mice were colonized with a bacterial solution containing 10 live bacteria; control mice were simultaneously administered an equal volume of PBS by gavage. On day 14 post-transplantation, all mice underwent behavioral tests. Additionally, 7-8 week old germ-free C57BL / 6J mice were orally administered 200 μL of a solution containing *Bifidobacterium pseudolongum* (10 live bacteria). 9 The bacterial solution containing 1 live bacteria was used to complete the colonization of the bacterial community.

[0056] 3. Fecal microbiota transplantation

[0057] Fecal samples (0.1 g each) were collected from five randomly selected male patients with major depressive disorder (MDD, n=5) and five healthy male controls. Each sample was suspended in 1.5 mL of sterile PBS and mixed to prepare the donor sample pool. Seven- to eight-week-old SPF-grade C57BL / 6J mice were pretreated with antibiotics by gavage for seven consecutive days. Two days later, intestinal colonization was achieved by oral administration of 100 μL of a mixed bacterial suspension from either the MDD patient or the healthy control group. Control group (treatment-simulated group) mice were administered an equal volume of sterile PBS by gavage. All mice were housed separately in sterile isolators according to their treatment group. Behavioral tests were performed on day 14 post-microbiota transplantation.

[0058] 4. Creatine treatment

[0059] 7-8 week old SPF-grade C57BL / 6J mice were orally administered 200 μL of a solution containing creatine monohydrate (at doses of 10, 100, 500, or 1000 mg / kg) via gavage. Control group mice were administered an equal volume of normal drinking water via gavage. One hour after gavage, forced swimming or tail suspension tests were performed to assess depressive-like behaviors.

[0060] 5. Creatine isotope detection

[0061] On day 14 after colonization, 7-8 week old germ-free C57BL / 6J mice were orally administered 200 μL of a solution containing isotopically labeled creatine monohydrate (creatine-d5 (monohydrate), deuterated creatine monohydrate) via gavage at a dose of 100 mg / kg. One hour after gavage, mouse plasma was collected, and the concentration of isotopically labeled creatine monohydrate was determined by liquid chromatography-mass spectrometry (LC / MS). The chromatographic conditions were as follows: a Waters UPLC BEH Amide column (2.1 × 100 mm, 1.7 μm) was used at a column temperature of 55 ℃, with an injection volume of 5 μL. Mobile phase A consisted of an aqueous solution containing 25 mM ammonium acetate and 25 mM ammonium hydroxide, and mobile phase B consisted of acetonitrile. The gradient elution program was as follows: 0–1 min, 85% B; 1–12 min, 65% B; 12–12.1 min, 40% B; 12.1–15 min, 40% B; 15–15.1 min, 85% B; 15.1–20 min, 85% B; flow rate 0.3 mL / min. Mass spectrometry conditions were: electrospray ionization (ESI) source, ion source temperature 600 °C, ion source voltage -4500 V or 5500 V, curtain gas 20 psi, nebulizer gas and auxiliary gas both 60 psi. Multiple reaction monitoring (MRM) mode was used for scanning. Raw data were processed using SCIEX OS software for peak alignment, retention time correction, and peak area extraction. The concentration of creatine in plasma was calculated based on the standard curve.

[0062] 6. Forced swimming test

[0063] The forced swimming test is a commonly used animal behavioral experiment to evaluate the efficacy of antidepressants. This test involves mice being forced to swim or float aimlessly in water where they cannot escape. The time the animal remains still during the experiment reflects its depressive state, and antidepressants can significantly shorten or alter this state. The forced swimming test is conducted in a transparent glass cylinder (45 cm high, 19 cm in diameter) filled with 23 cm of water at a temperature of 22-25°C. The test lasts for 6 minutes. After the test begins, the mice are gently removed from their cages and quickly placed into the forced swimming tank. The animals are allowed to swim for 6 minutes, and the time they remain still during the last 4 minutes is recorded. After the test, the mice are removed from the water, dried with a towel, and returned to their original cages.

[0064] 7. Tail Suspension Test

[0065] The tail suspension test is a classic and rapid method for evaluating the efficacy of antidepressants. The principle is that mice, after being suspended by their tails, attempt to escape but fail, thus giving up and entering a specific state of depressive immobility. The duration of immobility is recorded to reflect the depressive state, and antidepressants can significantly shorten or alter this state. In the tail suspension test, the posterior third of the mouse's tail is secured with tape and suspended from a support, with the head 15 cm above the table surface, and video is taken. After 6 minutes, the immobility time in the last four minutes (3-6 minutes) is statistically analyzed using small animal behavioral analysis software.

[0066] 8. Sugar Water Preference Experiment

[0067] Animals were housed individually and allowed to acclimatize to the new environment for at least 3-5 days. For 48 hours prior to the experiment, each animal was given two identical bottles containing plain drinking water to eliminate positional preference. After 24 hours, the two bottles were swapped, and water was provided for another 24 hours. For the first 24 hours before the experiment, one bottle was replaced with a 1% sucrose solution, while the other remained plain water. Animals were allowed free choice of which to drink. At the start of the test, each animal was given two pre-weighed bottles of 1% sucrose solution and plain water. After 24 hours of free drinking, the bottles were swapped again, and the process continued for another 24 hours. After the test, both bottles were removed, weighed accurately, and the percentage of sucrose solution consumed was calculated.

[0068] 9. Detection of the antidepressant effect of creatine in the CSDS depression model

[0069] To investigate the antidepressant effect of creatine in a depression model, 7-8 week old SPF-grade C57BL / 6J mice were divided into three groups: Group 1 received oral administration of drinking water containing creatine monohydrate (3 g / kg / day) for 14 days before and during CSDS modeling. This dose was calculated from the maximum safe daily intake of creatine for healthy adults. Group 2 received oral administration of normal drinking water for 14 days before and during CSDS modeling. Group 3 served as the control group, receiving oral administration of normal drinking water without any other treatment.

[0070] 10. Detection of the antidepressant effects of Bifidobacteria and creatine in the CSDS depression model.

[0071] To investigate the antidepressant effects of Bifidobacterium and creatine in a depression model, 7-8 week old SPF-grade C57BL / 6J mice were divided into three groups after CSDS modeling: the first group of mice were orally administered drinking water containing creatine monohydrate (3 g / kg / day) and were orally administered 200 μL of PBS solution containing Bifidobacterium daily (10 μL / kg / day). 8 The first group of mice received oral administration of creatine monohydrate dissolved in drinking water (3 g / kg / day). The second group of mice received oral administration of normal drinking water. After 14 consecutive days, social avoidance and saccharin preference tests were performed.

[0072] 11. Clinical Trials

[0073] This study recruited 16 healthy participants from Zhujiang Hospital of Southern Medical University. Healthy participants included in this study met the following criteria: a) age between 18 and 60 years (inclusive); b) BMI between 18 and 28 kg / m²; e) right-handed; f) current Hamilton Depression Rating Scale (17 items) score <7. The main exclusion criteria for all participants were as follows: 1) diagnosis of depression, bipolar disorder, schizophrenia, or schizophrenic affective disorder by SCID-5; 2) previous or current diagnosis of acute kidney injury (AKI), chronic kidney disease (CKD), or end-stage renal disease (ESRD); 3) history of epilepsy; 4) previous diagnosis of creatine deficiency syndrome or presence of creatine deficiency syndrome; 5) use of antibiotics within the past 30 days; 6) use of probiotics within the past 30 days; 7) positive pregnancy test; 8) breastfeeding. After collecting baseline blood samples, 16 healthy individuals were randomly assigned to receive either creatine or a combination of creatine and B. adolescentis. The creatine dose was 3 grams daily, and the B. adolescentis dose was 2 x 10 grams daily. 8 One live bacteria. Orally administered daily for 4 weeks. Changes in plasma creatine levels relative to baseline were assessed as clinical outcome measures.

[0074] This study recruited 38 patients with depression from Zhujiang Hospital of Southern Medical University. Patients with depression included in this study met the following criteria: a) age between 18 and 60 years (inclusive); b) BMI between 18 and 28 kg / m²; c) diagnosed with depression according to the SCID-5 assessment; d) current Hamilton Depression Rating Scale (17 items) score >16; e) right-handed. The main exclusion criteria for all participants were as follows: 1) diagnosed with bipolar disorder, schizophrenia, or schizoaffective disorder according to the SCID-5 assessment; 2) previous or current diagnosis of acute kidney injury, chronic kidney disease, or end-stage renal disease; 3) history of epilepsy; 4) significant suicide risk; 5) previous diagnosis of creatine deficiency syndrome or presence of creatine deficiency syndrome; 6) use of antibiotics within the past 30 days; 7) use of probiotics within the past 30 days; 8) positive pregnancy test; 9) breastfeeding. After baseline blood sample collection and Hamilton Depression Rating Scale (HAMD) scores, participants were randomly assigned to receive the following treatment: B. adolescentis (containing 2×10⁻⁶ mmol / L). 8 CFU (B. adolescentis) in combination with creatine (containing 3 grams of creatine), or placebo. The creatine dose is 3 grams daily, and B. adolescentis is 2 x 10 grams daily. 8 One live bacteria. Take daily for 4 weeks. All patients with depression in this clinical study received selective serotonin reuptake inhibitors (SSRIs) and agreed to undergo HAMD assessment. Baseline changes in HAMD score and plasma creatine level were assessed as clinical outcome measures.

[0075] III. Experimental Results

[0076] like Figure 1 As shown, this project collected cerebrospinal fluid (15 depressed patients, 26 healthy controls), plasma (31 depressed patients, 22 healthy controls), and stool samples (18 depressed patients, 17 healthy controls) from depressed patients and healthy individuals. The tests revealed increased creatine levels in the stool of depressed patients and decreased creatine levels in plasma and cerebrospinal fluid. Figure 1 A, B). Increased fecal creatine levels and decreased plasma and cerebrospinal fluid creatine levels were also found in a mouse model of social frustration depression (6 mice per group). Figure 1 (C, D). This finding reveals a systemic imbalance in creatine metabolism during the development of depression, characterized by intestinal creatine accumulation and peripheral and central creatine deficiency. The results suggest that gut-brain axis-mediated creatine metabolism disorders may play an important role in the pathogenesis of depression.

[0077] like Figure 2As shown in AG, this study systematically evaluated the antidepressant potential of exogenous creatine. Male C57BL / 6J mice were used in the experiment. Standardized behavioral tests were performed 60 minutes after a single oral administration of different doses (0, 10, 100, 500, and 1000 mg / kg) of creatine monohydrate. Figure 2 A). The results showed that doses of 100, 500, and 1000 mg / kg significantly shortened the immobility time in the forced swimming test (FST) and tail suspension test (TST), and there were no significant differences among the three dose groups, suggesting that simply increasing the dose of creatine could not further enhance its antidepressant effect. Figure 2 B and C). To further validate the findings, the study employed a CSDS model. Experimental mice received creatine monohydrate pre-intervention (3 g / kg / day) or normal drinking water for 24 consecutive days before stress induction and during modeling. Control mice (Ctrl) were normal mice and received no treatment. Figure 2 D). Behavioral analysis showed that creatine pretreatment effectively alleviated CSDS-induced social avoidance behavior and improved the decline in sucrose preference (D). Figure 2 (EG). The above results indicate that exogenous creatine has a significant antidepressant effect.

[0078] like Figure 2 As shown in HJ, to elucidate the necessity of gut microbiota in the antidepressant effect of creatine, this study conducted a microbiota intervention experiment ( Figure 4 H). First, SPF-grade male C57BL / 6J mice were administered a 7-day broad-spectrum antibiotic (ABX) via gavage to purge the intestinal flora, and then orally administered 100 mg / kg creatine monohydrate 60 minutes before the behavioral test. Results showed that normal SPF mice performed better in the forced swimming test (FST). Figure 2 I) and the tail suspension test (TST) Figure 2 The immobility time in the control group was significantly shorter than that in the control group, while ABX treatment to eliminate gut microbiota blocked the antidepressant effect of creatine, manifested as an increase in immobility time; these results suggest that gut microbiota plays a key role in the antidepressant-like effect of exogenous creatine. To further investigate the influence of gut microbiota from different sources on the creatine effect, this study conducted a fecal microbiota transplantation (FMT) experiment (…). Figure 2 SPF-grade male C57BL / 6J mice treated with ABX were randomly divided into three groups, receiving fecal microbiota transplanted from patients with depression, fecal microbiota from healthy controls, or PBS, respectively. Fourteen days after transplantation, mice were treated with 100 mg / kg creatine monohydrate. Behavioral results showed that mice transplanted with the depressive microbiota group showed improved FST (fecal microbiota transplantation). Figure 2 L) and TST Figure 2The immobility time in the M group was not significantly different from that in the PBS control group; however, the immobility time in the healthy microbiota transplantation group was significantly shorter than that in both the depressive microbiota group and the PBS control group (p<0.05), indicating that healthy microbiota colonization can specifically restore the antidepressant effect of creatine. Further examination was conducted on the changes in plasma creatine concentration in each group of mice 60 minutes after oral administration of 100 mg / kg creatine. The results showed that plasma creatine levels in normal SPF mice increased approximately 4-fold from baseline, while this increase was inhibited in the ABX treatment group (p<0.05). Figure 2 O). Plasma creatine levels in mice transplanted with depressive gut microbiota were significantly lower than in normal SPF mice (p<0.01), while colonization with healthy gut microbiota restored them to near-normal levels. Figure 2 The above results collectively construct a regulatory axis of "gut microbiota → creatine absorption → antidepressant effect," providing a new therapeutic strategy for microbiota-targeted intervention in depression.

[0079] like Figure 3 As shown in AD, Bifidobacteria in the stool of depressed patients are disordered, with decreased abundance of Bifidobacterium pseudolongum and Bifidobacterium adolescentis. Figure 3 A, B). The abundance of *Bifidobacterium pseudolongum* and *Bifidobacterium adolescentis* in the feces of depressed mice was also reduced, indicating that the abundance of *Bifidobacterium pseudolongum* and *Bifidobacterium adolescentis* is disordered in the occurrence of depression. Figure 3 C). Furthermore, after transplantation of *Bifidobacterium pseudolongum* into germ-free mice, oral administration of 100 mg / kg of isotope-labeled creatine (C) was performed. Figure 3 D). One hour later, a significant increase in isotope-labeled creatine levels was found in the blood of mice transplanted with *Bifidobacterium pseudolongum*. Figure 3 D). These results indicate that Bifidobacteria can promote the absorption of exogenous creatine.

[0080] like Figure 3 As shown in EG, in the forced swimming experiment, 10 8 CFU / day or 10 9 After administration of Bifidobacterium pseudolongum for 10 days, the immobility time of mice showed a decreasing trend, but no significant difference was observed; 8 CFU / day or 10 9 Following administration of Bifidobacterium adolescentis, the immobility time of mice decreased, indicating a mild antidepressant effect; however, after 10 days... 8 CFU / day or 10 9 No significant difference was observed between the two dose groups per day. Figure 3 E). In the tail suspension experiment, 10 8 CFU / day or 10 9 After administration of Bifidobacterium pseudolongum for 10 days, the immobility time of mice showed a decreasing trend, but no significant difference was observed; 8 CFU / day or 10 9Following administration of Bifidobacterium infantis for 10 days, the immobility time of mice decreased, indicating an antidepressant effect; however, after 10 days... 8 CFU / day or 10 9 No significant difference was observed between the two dose groups per day. Figure 3 F). In the open field test, 10 8 CFU / day or 10 9 No significant change was observed in the total distance of mice after 1 day of Bifidobacterium bifidum administration. Figure 3 G). The above results suggest that Bifidobacterium alone has a potential antidepressant effect, but the effect is not ideal.

[0081] like Figure 3 As shown in HJ, in the forced swimming experiment, Bifidobacterium pseudolongum (10) was administered in combination. 8 After administration of CFU and creatine (100 mg / kg), the immobility time of mice was significantly shorter than that of mice given creatine and Bifidobacterium pseudolongum alone, suggesting that its antisuppressive effect was superior to that of Bifidobacterium pseudolongum or creatine alone. Figure 3 H). Combined administration of Bifidobacterium adolescentis (10 8 After administration of CFU and creatine (100 mg / kg), the immobility time of mice was significantly shorter than that of mice given creatine and Bifidobacterium adolescentis alone, suggesting that its antisuppressive effect was superior to that of Bifidobacterium adolescentis or creatine alone. Figure 3 H). In the tail suspension experiment, Bifidobacterium pseudolongum (10) was administered in combination. 8 After administration of CFU and creatine (100 mg / kg), the immobility time of mice was significantly shorter than that of mice given creatine and Bifidobacterium pseudolongum alone, suggesting that its antisuppressive effect was superior to that of Bifidobacterium pseudolongum or creatine alone. Figure 3 I); combined administration of Bifidobacterium adolescentis (10) 8 After administration of CFU and creatine (100 mg / kg), the immobility time of mice was significantly shorter than that of mice given creatine and Bifidobacterium adolescentis alone, suggesting that its antisuppressive effect was superior to that of Bifidobacterium adolescentis or creatine alone. Figure 3 I). In the open field experiment, no significant difference was observed in the total distance traveled by the mice in each group ( Figure 3 J). For example Figure 3 As shown in KN, this study further validated the sustained effect of the combined intervention in CSDS model mice. CSDS model mice were divided into three groups: a combined treatment group (Bifidobacterium + creatine), a creatine-only group, and a model control group. Behavioral assessments were performed 14 days after the intervention. Figure 3 K). The results showed that creatine-only treatment after establishing a depression model was less effective, with no significant improvement in social avoidance behavior and sucrose preference; while combined treatment was more effective in reversing CSDS-induced social avoidance behavior. Figure 3 L) and decreased preference for sugary drinks ( Figure 3 M), and significantly increased plasma creatine levels (M).Figure 3 The above results indicate that the combined use of Bifidobacterium pseudolongum, Bifidobacterium adolescentis, and creatine has a significant antidepressant-like effect, which is better than the combined use of creatine and the bacteria alone, demonstrating a significant synergistic effect.

[0082] like Figure 4 As shown in AD, compared with mice transplanted with healthy donor microbiota, mice transplanted with depressed donor microbiota showed a significant decrease in the mRNA expression of creatine transporter (Slc6a8) in intestinal epithelial cells. Figure 4 A). SPF C57BL / 6 mice were treated with *Bifidobacterium pseudolongum* 14 days before CSDS modeling and during the stress period. The results showed that *Bifidobacterium pseudolongum* effectively inhibited the CSDS-induced decrease in creatine transporter (Slc6a8) levels in intestinal epithelial cells. Figure 4 (C, D). These results collectively indicate that *Bifidobacterium pseudolongum* can promote the expression of creatine transporters in intestinal epithelial cells. Figure 4 As shown in EL, this study constructed mice with intestinal epithelial cell-specific Slc6a8 knockout (Vil1-cre; Slc6a8). loxp / Y (VcKO for short), and conduct behavioral assessments on them ( Figure 4 E). The results showed that, without creatine supplementation, there was no significant difference in immobility time in FST and TST between VcKO mice and control (Ctrl) mice. Figure 4 F and G). However, after oral administration of 100 mg / kg creatine monohydrate for 1 hour, the immobility time in FST and TST of control mice was significantly lower than that of VcKO mice (F and G). Figure 4 F and G). Plasma creatine levels showed no significant difference between the two groups of mice at baseline; however, 60 minutes after oral administration of 100 mg / kg creatine, the plasma creatine levels in the control group mice were significantly higher than those in the VcKO mice (F and G). Figure 4 H). Subsequently, control and VcKO mice were given oral creatine 14 days before CSDS modeling and during the stress period, followed by behavioral testing. Figure 4 The results showed that creatine significantly alleviated social avoidance behavior and decreased sugar water preference induced by CSDS, while this effect was significantly weakened in VcKO mice. Figure 4 In summary, experiments using mouse models of depression and fecal microbiota transplantation in patients with depression showed that depression reduces the expression of the creatine transporter Slc6a8 in intestinal epithelial cells, and Bifidobacteria may promote the absorption of exogenous creatine by upregulating the expression of the creatine transporter Slc6a8 in intestinal epithelial cells, thereby mediating and enhancing its antidepressant effect.

[0083] like Figure 5As shown in A and B, after 28 consecutive days of administration of creatine or Bifidobacterium adolescentis combined with creatine, plasma creatine levels significantly increased. The plasma creatine levels in healthy individuals receiving Bifidobacterium adolescentis combined with creatine were significantly higher than those receiving creatine alone. Figure 5 A, B). The above results indicate that *Bifidobacterium adolescentis* can effectively promote the level of creatine in human blood. (For example...) Figure 5 As shown in CG, after 28 consecutive days of administration of Bifidobacterium adolescentis combined with creatine to depressed patients, the depression scale scores of depressed patients decreased significantly more than those in the placebo group. Figure 5 D, E). Depressed patients receiving Bifidobacterium adolescentis combined with creatine showed a significantly higher increase in plasma creatine levels than those receiving placebo alone. Figure 5 F, G). The above results indicate that Bifidobacterium adolescentis and creatine can effectively reduce depression scale scores in depressed patients and restore creatine levels in the blood of depressed patients.

[0084] This invention discovers a probiotic and creatine composition with antidepressant effects, containing bifidobacteria and creatine, which has few toxic side effects, long-lasting effects, low price, and broad application prospects.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A probiotic and creatine composition with antidepressant effects, characterized in that, The composition comprises Bifidobacterium pseudolongum and creatine, or Bifidobacterium adolescentis and creatine.

2. The composition according to claim 1, characterized in that, The aforementioned *Bifidobacterium pseudolongum* is *Bifidobacterium pseudolongum* GDMCC NO. 1.2852, and the aforementioned *Bifidobacterium adolescentis* is *Bifidobacterium adolescentis* GDMCC NO. 1.

278.

3. A drug or health food with antidepressant effects, characterized in that, The active ingredient includes an effective amount of the probiotic and creatine composition as described in claim 1 or 2.

4. The drug or health food according to claim 3, characterized in that, The drug or health food also includes a pharmaceutically acceptable carrier; the carrier is a conventional drug excipient or adjuvant.

5. The drug or health food according to claim 3, characterized in that, The drug contains a combination of probiotics and creatine at a weight of 0.1% to 99.9%.

6. The drug or health food according to claim 3, characterized in that, The dosage form of the drug is a liquid dosage form or a solid dosage form. Preferably, the liquid dosage form is a solution, suspension or emulsion. Preferably, the solid dosage form is a tablet, capsule, pill, powder for injection, sustained-release preparation or microparticle delivery system.

7. The drug or health food according to claim 6, characterized in that, The liquid dosage form can be a solution, colloidal, microparticle, emulsion, or suspension.

8. The drug or health food according to claim 3, characterized in that, The drug further includes a lyophilization protectant and / or prebiotics. Preferably, the lyophilization protectant includes any one or a combination of at least two of the following: skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol, or xylitol. The prebiotics include any one or a combination of at least two of the following: fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, inulin, spirulina, arthrophyllum, trachomatis polysaccharides, stachyose, polydextrose, α-lactalbumin, or lactoferrin.

9. The drug or health food according to claim 3, characterized in that, The aforementioned drug is formulated into tablets and widely utilizes various carriers known in the art. Preferably, the carrier includes diluents and absorbents such as starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and aluminum silicate. The carrier also includes humectants and binders such as water, glycerin, polyethylene glycol, ethanol, propanol, starch paste, dextrin, syrup, honey, glucose solution, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and polyvinylpyrrolidone. The carrier includes disintegrants, such as dried starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc.; the carrier includes disintegration inhibitors, such as sucrose, tristearate, cocoa butter, ammoniacal oil, etc.; the carrier includes absorption promoters, such as quaternary ammonium salts, sodium dodecyl sulfate, etc.; the carrier includes lubricants, such as talc, silica, corn starch, stearate, boric acid, liquid paraffin, polyethylene glycol, etc. Preferably, the drug is a tablet, formulated as a coated tablet, such as a sugar-coated tablet, a film-coated tablet, an enteric-coated tablet, or a double-layer tablet or a multilayer tablet; Preferably, the drug is in the form of pills, and various carriers known in the art can be widely used. These carriers include diluents and absorbents, such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, gelucire, kaolin, talc, etc.; they also include binders, such as gum arabic, tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste, or flour paste; and they include disintegrants, such as agar powder, dried starch, alginate, sodium dodecyl sulfate, methylcellulose, ethylcellulose, etc. Preferably, the drug is a suppository, which can widely use various carriers known in the art, such as polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and semi-synthetic glycerides. Preferably, the drug is a capsule, in which the active ingredient is mixed with the various carriers mentioned above, and the resulting mixture is placed in a hard gelatin capsule or a soft capsule. Alternatively, the active ingredient can be made into a microcapsule and suspended in an aqueous medium to form a suspension. It can also be filled into a hard capsule or made into an injectable preparation for application. Preferably, excipients may also be added to the pharmaceutical formulation, such as any one or a combination of at least two of the following: fillers, binders, wetting agents, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, pH regulators, antioxidants, antibacterial agents, or buffers.

10. The use of Bifidobacterium pseudolongum and creatine, or Bifidobacterium adolescentis and creatine in combination, in the preparation of drugs or health foods with antidepressant effects.

Images