Application of lactobacillus johnsonii in preparation of medicine for treating depression
The drug prepared using Lactobacillus johnsonii VPI7960 improves the gut microbiota structure, solving the problems of large side effects and low efficacy of existing antidepressant drugs, significantly improving depressive symptoms and reducing hippocampal neuronal damage.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-19
- Publication Date
- 2026-04-03
AI Technical Summary
Existing medications for treating depression have problems such as significant side effects, low efficacy, and slow onset of action. Furthermore, the effects of probiotics on the central nervous system after improving gut microbiota structure have not been fully utilized.
Lactobacillus johnsonii VPI7960 was used as the active ingredient to prepare an oral drug formulation for the treatment of depression. It affects the central nervous system by improving the gut microbiota structure and reduces depressive-like behaviors.
It significantly improved depression-like behavior in depressed mice, shortened the time of tail wagging immobility and forced swimming floating, increased sucrose preference, and improved hippocampal neuronal damage, thus enhancing the therapeutic effect on depression.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically, to the use of Lactobacillus johnsonii in the preparation of a drug for treating depression. Background Technology
[0002] Depression is a common but serious mental health problem, primarily characterized by significant and persistent low mood. In recent years, the incidence of depression has been rising annually, currently affecting approximately 5% of adults worldwide each year. Conventional medications for treating depression mainly include selective serotonin reuptake inhibitors (SSRIs), which are effective in improving depressive symptoms, but suffer from significant side effects, lower efficacy rates, and relatively slow onset of action. Improving gut microbiota through probiotic supplementation, dietary optimization, or fecal microbiota transplantation can alleviate depressive symptoms.
[0003] The gut-brain axis is a complex bidirectional communication system connecting the gut microbiota and the central nervous system, facilitating information exchange between the gut and brain through multiple pathways, including neural, immune, endocrine, and metabolic pathways. The gut microbiota can influence the occurrence and development of depression through various mechanisms, such as microbial metabolites, immune regulation, and the expression and secretion of nerve growth factor. Increasing research indicates that certain bacterial strains or mixtures of bacteria have the potential to improve depressive and anxiety symptoms, with probiotics attracting particular attention due to their unique biological activities.
[0004] The beneficial effects of probiotics on gastrointestinal function are widely recognized, but new animal and clinical studies have revealed that alterations in the gut microbiota can have broad systemic effects. Affected non-gut sites include the immune, endocrine, cardiovascular, and central nervous systems. With the emergence of the gut-brain axis theory, there is increasing interest in the potential role of the gut microbiota in influencing central nervous system functions through the "microbiota-gut-brain axis." Probiotics have potential therapeutic benefits for various central nervous system disorders, such as anxiety, depression, autism spectrum disorders, and Parkinson's disease. Summary of the Invention
[0005] The purpose of this invention is to provide the application of Lactobacillus johnsonii in the preparation of a drug for treating depression.
[0006] A first aspect of the present invention provides the use of *Lactobacillus johnsonii* in the preparation of a medicament for treating depression; wherein the *Lactobacillus johnsonii* is *Lactobacillus johnsonii*. Lactobacillus johnsonii .
[0007] Furthermore, the aforementioned *Lactobacillus johnsonii* Lactobacillus johnsoniiSpecifically, it is Lactobacillus johnsonii VPI7960, which can be purchased from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, with accession number BNCC No.186384.
[0008] Furthermore, the aforementioned *Lactobacillus johnsonii* can alleviate depressive-like behavior and improve hippocampal neuronal damage.
[0009] Furthermore, the drug is a pharmaceutical preparation with Lactobacillus johnsonii as the active ingredient.
[0010] Furthermore, the pharmaceutical formulation contains 2×10 8 CFU of live Lactobacillus johnsonii. The Lactobacillus johnsonii is a bacterial suspension.
[0011] Furthermore, the pharmaceutical preparation is an oral pharmaceutical preparation. The dosage form of the pharmaceutical preparation is selected from one or more of the following: powder, tablet, granule, capsule, and solution.
[0012] Furthermore, the pharmaceutical formulation may also include other pharmaceutically acceptable carriers or excipients.
[0013] Furthermore, the pharmaceutical preparation further includes one or more of the following: excipients, binders, diluents, disintegrants, fillers, wetting agents, absorption promoters, surfactants, adsorbent carriers, and lubricants.
[0014] Furthermore, the excipient is selected from one or more of lactose, microcrystalline cellulose, mannitol, and starch;
[0015] The adhesive is selected from one or more of the following: methylcellulose, ethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, hydroxypropylcellulose, and hydroxypropyl methylcellulose;
[0016] The diluent is selected from one or more of the following: calcium sulfate, glucose binder, dextrin, monosaccharide or polysaccharide, kaolin, sugar alcohol, and microcrystalline cellulose;
[0017] The disintegrant is selected from one or more of the following: dry starch, sodium carboxymethyl starch, low-substituted cellulose, polyvinylpyrrolidone, sodium carboxymethyl cellulose, microcrystalline cellulose, potassium polyacrylaldehyde, sodium glycolate starch, and low-substituted hydroxypropyl cellulose.
[0018] In a second aspect, the present invention provides a pharmaceutical preparation for treating depression, wherein *Lactobacillus johnsonii* is the active ingredient, specifically *Lactobacillus johnsonii* VPI 7960. This is available from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, accession number BNCC No. 186384.
[0019] The advantages of this invention are:
[0020] This invention provides the application of *Lactobacillus johnsonii* in the preparation of drugs for treating depression. *Lactobacillus johnsonii* can be used as an adjunct drug in the treatment of depression. Through the establishment of a mouse model of depression and experiments involving group feeding with *Lactobacillus johnsonii*, behavioral evaluations of mice in the depressed group and the probiotic group were compared in sucrose preference, open field test, tail suspension test, and forced swimming test. It was found that gavage administration of *Lactobacillus johnsonii* significantly improved sucrose preference (P < 0.001), significantly shortened the immobility time of tail swaying in mice (P < 0.0001), significantly shortened the immobility time of forced swimming (P < 0.001), and significantly increased the central distance traveled by mice in the open field test (P < 0.01). This demonstrates that *Lactobacillus johnsonii* can alleviate depressive-like behaviors in depressed mice. Microscopic examination of hippocampal sections from mice revealed that the state of pyramidal cells in the CA1 region of the hippocampus was significantly improved after administration of *Lactobacillus johnsonii* compared to that of depressed mice. The pyramidal cells were neatly arranged, and Nissl bodies were clearly visible within their cells, demonstrating that *Lactobacillus johnsonii* can improve hippocampal neuronal damage in depressed mice. Attached Figure Description
[0021] Figure 1 This is a schematic diagram showing the results of the sugar water preference (Mean±SD, n=10) of mice in each experimental group.
[0022] Figure 2 This is a schematic diagram showing the results of the time (Mean±SD, n=10) during the tail suspension test for mice in each experimental group.
[0023] Figure 3 This is a schematic diagram showing the central zone distance (Mean±SD, n=10) of mice in each experimental group during the open field experiment.
[0024] Figure 4 This is a schematic diagram showing the results of the time mice in each experimental group remained floating still during the forced swimming experiment (Mean±SD, n=10).
[0025] Figure 5 This is a schematic diagram of the Nissl staining results (×20) of the ipsilateral hippocampal CA1 region of a mouse. Detailed Implementation
[0026] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.
[0027] Example 1: Lactobacillus johnsonii improves depressive-like behavior in mice
[0028] 1. Establishment and animal grouping of a chronic, unpredictable, mild stress model
[0029] Thirty male C57BL / 6J mice, aged 6-8 weeks, were used as experimental animals. After one week of acclimatization, the animals were randomly divided into three groups of 10 mice each: a blank control group, a depression model group, and a probiotic group.
[0030] The probiotic group uses Lactobacillus johnsonii, specifically Lactobacillus johnsonii VPI 7960, purchased from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, with accession number BNCC No. 186384.
[0031] A chronic depression model was established using the internationally recognized CUMS protocol. The blank control group received no stimulation other than behavioral evaluation. The depression model group and the probiotic group received chronic, unpredictable, mild stress stimulation once daily for 7 weeks, with each stimulus not repeated within a week. The stress factors used in the experiment included: restraint for 4 hours, intermittent electric shocks for 30 minutes, aggression for 20 minutes, climbing ice for 10 minutes, horizontal shaking for 15 minutes, a 45° tilted cage for 24 hours, and water deprivation for 24 hours.
[0032] Blank control group: Received no CUMS stimulation, and were administered PBS solution by gavage once daily from week 7 after modeling until the end of the experiment. Depression model group: Received CUMS stimulation and were administered PBS solution by gavage once daily from week 7 after modeling until the end of the experiment. Probiotic group: Received 2×10⁻⁶ probiotics by gavage during the CUMS modeling process, starting from week 7 after modeling. 8 0.2 mL of live CFU lactobacillus was administered by gavage once daily until the end of the experiment.
[0033] Preparation of PBS solution for bacteria: Take 200 μL of bacterial culture and add it to 10 mL of MRS liquid medium for amplification culture. After culturing for 1 day, centrifuge at 3000 rpm for 10 min, discard the medium, wash three times with sterile PBS, and resuspend in 10 mL of sterile PBS. Transfer 10 μL of the resuspended bacterial culture to a cell counting plate and count the bacteria under a microscope. Based on the counting results, dilute the bacterial culture with PBS to a concentration of 2 × 10⁻⁶. 8 CFU bacterial suspension. Use within 1 hour after dilution.
[0034] 2. Behavioral evaluation methods
[0035] (1) Sugar Water Preference Test (SPT): This test is used to assess an animal's ability to experience pleasure. Mice were trained with sugar water for 48 hours before the test. Two bottles of 1% sugar water were placed in each cage and maintained for 24 hours. Then, one bottle of 1% sugar water and the other bottle of pure water were placed in each cage and maintained for 12 hours, with the positions being switched once in the middle. The sugar water preference test was then conducted on mice in the 7th and 9th weeks after modeling. Before the test, mice were fasted for 24 hours. During the test, mice were given one bottle of 1% sugar water and one bottle of pure water at the same time. The test was conducted for 12 hours, with the positions of the sucrose solution and pure water being switched once in the middle. The weights of the sugar water and pure water before and after the test were measured. The weights of the sugar water consumed and the total weight consumed were calculated. The percentage of sugar water consumed to the total weight consumed was the sugar water preference percentage.
[0036] (2) Tail Suspension Test (TST): Used to evaluate the animals' despair behavior and feelings of helplessness. The TST was conducted in weeks 7 and 9 of modeling, from 3 pm to 5 pm each day. Adhesive tape was attached to the tail of the mouse about 2 cm from the end, and the mouse was suspended on a support, about 15 cm from the ground. The mouse's behavior was recorded by a camera for 6 minutes, and the duration of stillness in the last 4 minutes was analyzed using behavioral analysis software. Stillness was defined as the mouse giving up struggling and hanging in the air. The mice were returned to their cages after the TST.
[0037] (3) Open Field Test (OFT): Used to evaluate spontaneous activity and exploratory behavior of animals in unfamiliar environments. The OFT was conducted in weeks 7 and 9 of modeling, with tests performed daily from 3 PM to 5 PM. Before the test, the bottom of the observation box was wiped clean with 75% alcohol. The homemade observation box (50cm×50cm×50cm) was placed in a uniformly lit area, allowing mice to explore freely for 5 minutes. A camera fixed to the horizontal bar above the box recorded the entire process of the mouse's activity. After the experiment, the bottom of the box was wiped clean again with 75% alcohol. The video of the mouse's activity trajectory was analyzed using the behavioral analysis software Visutrack. During the analysis, the bottom area of the box was divided into a 16-grid system, with the four grids not touching the four sides designated as the central area, and the other 12 grids as the surrounding areas. The distance the mouse traveled in the central area during this period was used as the evaluation index.
[0038] (4) Forced Swimming Test (FST): Used to evaluate desperate behavior in animals. The FST was conducted in weeks 7 and 9 of modeling, from 3 PM to 5 PM daily. Warm water (approximately 25°C) was placed in a 1000mL beaker to a depth of approximately 800mL, and each animal was separated by an opaque partition. After placing the mice in the water, a camera recorded their behavior for 6 minutes, and behavioral analysis software was used to analyze the mice's immobility time for the following 4 minutes. Immobility was defined as the mouse ceasing to struggle in the water, floating, and exhibiting only slight limb movements to keep its head afloat. After each mouse finished swimming, it was dried, and fresh water was provided before testing the next mouse.
[0039] (5) Statistical analysis: GraphPad Prism8 (GraphPad Software, USA) was used for statistical analysis and graphing. One-way ANOVA was used to compare multiple groups of data, and Tukey test was used to further compare the groups pairwise.
[0040] 3. Results show
[0041] (1) such as Figure 1 As shown, Figure 1 This is a schematic diagram showing the results of the sugar water preference (Mean±SD, n=10) of mice in each experimental group. P < 0.001, compared with the blank control group, ### P < 0.001, compared with the model group. After 7 weeks of stimulation in the model group and 2 weeks of gavage treatment in the probiotic group, the percentage of sucrose preference in the depression model group was significantly lower than that in the blank control group (P < 0.001); while the percentage of sucrose preference was significantly higher in the probiotic group than in the depression model group (P < 0.001). This indicates that gavage with Lactobacillus johnsonii can improve anhedonia behavior in CUMS mice.
[0042] (2) such as Figure 2 As shown, Figure 2 This is a schematic diagram showing the results of the time (Mean±SD, n=10) during the tail suspension test for mice in each experimental group. P < 0.01, compared with the blank control group, #### P < 0.0001, compared with the model group. Compared with the blank control group, 7 weeks of modeling stimulation significantly increased the tail suspension immobility time of mice in the depression model group (P < 0.01); compared with the model group, the probiotic group significantly shortened the tail suspension immobility time of mice (P < 0.0001). These results indicate that gavage administration of Lactobacillus johnsonii can improve the CUMS-induced prolongation of tail suspension immobility time in mice.
[0043] (3) such as Figure 3 As shown, Figure 3 This is a schematic diagram showing the central zone distance (Mean±SD, n=10) of mice in each experimental group during the open field experiment. P < 0.001, compared with the blank control group, ## P < 0.01, compared with the model group. Compared with the blank control group, the central distance of mice in the model group was significantly shortened (P < 0.001); compared with the depression model group, the central distance of mice in the probiotic group was significantly increased (P < 0.01). This indicates that Lactobacillus johnsonii gavage can significantly improve the CUMS-induced shortening of central distance in mice.
[0044] (4) such as Figure 4 As shown, Figure 4 This is a schematic diagram showing the results of the time mice in each experimental group remained floating still during the forced swimming experiment (Mean±SD, n=10). P < 0.05, compared with the blank control group, ### P < 0.001, compared with the model group. Compared with the blank control group, the immobility time of mice in the depression model group was significantly prolonged (P < 0.05); compared with the depression model group, the probiotic group significantly shortened the immobility time of mice in the swimming activity (P < 0.001). These results indicate that *Lactobacillus johnsonii* can improve the CUMS-induced prolonged immobility time in forced swimming in mice.
[0045] Example 2: Effects of Lactobacillus johnsonii on hippocampal neurons in depressed mice
[0046] Nissl staining of mouse hippocampal tissue sections: After behavioral evaluation, mice were fasted for 12 hours after the last drug administration. One mouse from each group was selected, and the anesthetic was instilled into the eyeball of the side from which blood was to be collected. After blood was collected from the eyeball, the mice were euthanized by cervical dislocation. The chest was quickly opened to fully expose the heart. A needle was inserted into the left ventricle, the right atrial appendage was cut open, the abdominal aortic vein was clamped, and 30 mL of PBS was injected for rapid pressure perfusion to flush out the blood until the fluid flowing from the right atrial appendage was basically clear. Perfusion was then stopped, at which point the mouse's lungs, eyeballs, and paws were observed to be white. Then, 30 mL of 4% paraformaldehyde was rapidly perfused under pressure. Perfusion was stopped when the muscles of the mouse's upper limbs and head became rigid, and the mouse was decapitated and the brain was removed. After coronal section sampling, the brain tissue was fixed in 4% paraformaldehyde for 48 hours as soon as possible. After fixation, the tissue was dehydrated sequentially with ethanol from low to high concentrations: 50% ethanol (2h) → 75% ethanol (2h) → 85% ethanol (2h) → 95% ethanol (1h) → 95% ethanol (2h) → anhydrous ethanol (1h) → anhydrous ethanol (1.5h). It was then cleared in xylene in two separate baths (1h and 1h). The cleared brain tissue was then embedded in paraffin wax with a melting point of 52-60℃, and transferred to molten paraffin for further embedding in two separate baths (1h and 2h). Approximately 2mm of paraffin was retained around the brain tissue. Excess paraffin was trimmed off with a blade, and the tissue was sectioned to a thickness of 5μm. The slides were spread at 45℃ and then baked in a 60℃ slide oven for 2 hours. Paraffin sections were dewaxed in xylene for 5 minutes each in the first, second, and third baths, followed by 5 minutes each in anhydrous ethanol, 95% ethanol, 85% ethanol, and 75% ethanol, and then rinsed with tap water for 1 minute. The sections were then placed in a 1% toluidine blue solution and stained at 56°C for 20 minutes, followed by rinsing with distilled water. After differentiation in 70% ethanol for approximately 1 minute, differentiation was performed with 95% ethanol, controlled under a microscope until Nissl bodies were clearly visible. The sections were rapidly dehydrated in anhydrous ethanol. Xylene was then used for clearing and mounting.
[0047] The results are as follows Figure 5 As shown, Figure 5 This is a schematic diagram (×20) of Nissl staining results in the CA1 region of the ipsilateral hippocampus of mice. A is a schematic diagram of the results in the blank control group; B is a schematic diagram of the results in the CUMS depression model group; C is a schematic diagram of the results in the probiotic group. Compared with the blank control group, the hippocampal CA1 region pyramidal cells in the depression model group mice were disordered, with obvious nuclear pyknosis, increased intercellular spaces, and reduced or completely absent Nissl bodies within the cells. Compared with the depression model group, the hippocampal CA1 region pyramidal cells in the Lactobacillus johnsonii group mice were neatly arranged, and Nissl bodies within the cells were clearly visible, similar to the blank control group, suggesting that Lactobacillus johnsonii has antidepressant activity against CUMS-induced depression in mice.
[0048] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.
Claims
1. The use of *Lactobacillus johnsonii* in the preparation of a drug for treating depression, characterized in that... The Lactobacillus johnsonii mentioned above is Lactobacillus johnsonii. Lactobacillus johnsonii .
2. The application according to claim 1, characterized in that, The drug is a pharmaceutical preparation with Lactobacillus johnsonii as the active ingredient.
3. The application according to claim 2, characterized in that, The pharmaceutical preparation mentioned is an oral pharmaceutical preparation.
4. The application according to claim 2, characterized in that, The dosage form of the pharmaceutical preparation is selected from one or more of the following: powder, tablet, granule, capsule, and solution.
5. The application according to claim 2, characterized in that, The pharmaceutical preparation further includes one or more of the following: excipients, binders, diluents, disintegrants, fillers, wetting agents, absorption promoters, surfactants, adsorbent carriers, and lubricants.
6. The application according to claim 1, characterized in that, The aforementioned Lactobacillus johnsonii reduces depressive-like behavior and improves hippocampal neuronal damage.
7. A pharmaceutical preparation for treating drug-induced liver injury, characterized in that, It contains Lactobacillus johnsonii Lactobacillus johnsonii It is an active ingredient.