Use of lactobacillus taiwanensis in the preparation of an antidepressant or anxiolytic drug

Abstract

The application relates to application of lactobacillus taiwanensis in preparation of an antidepressant or anxiolytic drug, and belongs to the technical field of biological medicine. The technical problem to be solved is that the existing antidepressant and anxiolytic drugs have defects such as slow effect, limited treatment response rate, obvious adverse reactions and easy recurrence, and the lactobacillus strains in the prior art have generally insufficient antidepressant and anxiolytic effects, and cannot meet the clinical demand for high-efficiency, safe and natural antidepressant and anxiolytic microecological preparations. The technical solution is characterized in that application of lactobacillus taiwanensis CICC 23148 and a composition thereof in preparation of an antidepressant or anxiolytic drug is provided.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Lactobacillus taiwanensis in the preparation of antidepressant or anti-anxiety drugs. Background Technology

[0002] Depression is a group of mental disorders characterized by depressed mood, loss of interest, and impaired cognitive function. It is characterized by high incidence, high relapse rate, and high disability rate, and has become a major challenge in the global public health field. Its pathogenesis is complex, involving multiple levels such as neurotransmitter imbalance, neuroinflammation, changes in neuroplasticity, and gut microbiota dysbiosis. Currently used antidepressants (such as SSRIs and SNRIs) still have drawbacks such as slow onset of action, limited treatment response rate, significant adverse reactions, and high relapse rate, making it difficult to meet clinical treatment needs.

[0003] In recent years, in-depth research on the "gut-brain axis" theory has revealed that gut microbiota and its metabolites play a key role in mood regulation and are closely related to the occurrence and development of depression. Gut microbiota achieves bidirectional communication with the central nervous system through multiple pathways, including neural, immune, endocrine, and metabolic pathways. Disruption of its structure can lead to impaired intestinal barrier function and aggravated inflammatory response, thereby affecting central nervous system function and inducing depressive or anxiety-like behaviors.

[0004] With breakthroughs in microecological research technology, probiotics have become a research hotspot in the fields of biomedicine and health. Currently, there is an increasingly urgent demand for natural, safe, and side-effect-free health interventions. Compared to chemical drugs, probiotics have significant advantages such as high safety, good tolerability, and low likelihood of developing drug resistance, perfectly meeting the public's health needs. Furthermore, the continuous improvement of cross-organ regulation theories such as the "gut-brain axis" further reveals the diverse values ​​of probiotics. They can not only effectively regulate intestinal homeostasis but also influence the host's overall physiological functions through multiple pathways. Their potential value, especially in areas such as mood regulation and immune regulation, is being continuously explored, driving probiotics to gradually extend from the traditional field of intestinal health conditioning to multiple fields such as neuropsychiatric diseases and metabolic diseases.

[0005] Existing research indicates that Lactobacillus bacteria, as one of the most representative probiotic groups, can improve depressive-like behaviors by regulating the balance of gut microbiota and producing beneficial metabolites, but the functions of different strains vary significantly.

[0006] Relevant patent documents retrieved: The publication, published in China (CN119331767A) on January 21, 2025, discloses that *Lactobacillus reuteri* CCFM1289 can alleviate anxiety-like and depression-like behaviors in depressed mice, relieve HPA axis hyperactivity, increase the level of brain-derived neurotrophic factor (BDNF) in the hippocampus, promote the secretion of 5-HTP and 5-HT in the brain, increase the content of indoleacetic acid in the colon, serum, and brain, improve intestinal flora metabolic imbalance, increase the mRNA levels of neuron-derived neuron 2, synaptophysin, and postsynaptic density protein 95 in the hippocampus, maintain the stability of neuronal synaptic function, activate the AHR signaling pathway, increase the expression of dicortin (DCX), and promote neurogenesis, thereby alleviating depressive mood.

[0007] Relevant non-patent literature retrieved: The journal or book title is "Graduate Thesis of Jiangnan University", the article title is "Study on the Relief of Depression-like Symptoms in Mice by Four Types of Lactobacillus", the publication date is 2022-06-01. This article discloses that the intervention of Lactobacillus paracasei 126L6, Lactobacillus paracasei CCFM1229 and Lactobacillus rhamnosus CCFM1228 can reduce anxiety and depression-related behaviors in mice induced by chronic stress.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The *Lactobacillus reuteri* CCFM1289 strain disclosed in patent document CN119331767A, while able to increase the percentage of open-arm dwell time in the elevated maze test and reduce the stationary time in the forced swimming test in mice to some extent, and simultaneously increase the relative protein expression level of AhR, its effect on improving depressive-like and anxiety-like behaviors and the AhR signaling pathway is still not significant. Specific evidence is as follows: Compared to the model group, after intervention with this strain, the percentage of open-arm dwell time in the elevated maze test in mice increased by only about 46.155%, the stationary time in the forced swimming test decreased by only about 21.21%, and the relative expression level of AhR increased by only 1.25 times. The improvement efficacy is limited and cannot meet the clinical demand for highly effective antidepressant probiotics.

[0009] Furthermore, according to the non-patent literature "Study on the Relief of Depressive-like Symptoms in Mice by Four Types of Lactobacillus," different Lactobacillus strains showed varying effects on improving anxiety and depression-related behaviors in mice, and the improvement effects were not significant. Specific evidence is as follows: In the behavioral tests described in this literature, the behavioral indicators of mice treated with each Lactobacillus strain showed no significant difference compared to the model group. This indicates that the aforementioned Lactobacillus strains did not significantly improve chronic stress-induced depressive-like and anxiety-like behaviors in mice, further confirming the technical deficiency of the generally insufficient antidepressant efficacy of existing Lactobacillus strains, and highlighting the necessity of screening for highly effective and specific antidepressant Lactobacillus strains.

[0010] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: The antidepressant activity of Lactobacillus strains varies greatly, making screening difficult: Existing literature has confirmed that most Lactobacillus strains have weak or no significant antidepressant effects, and the efficacy of Lactobacillus reuteri CCFM1289 is also limited. Moreover, the functional heterogeneity of different strains is strong, making it difficult to quickly identify specific strains with highly effective antidepressant activity through conventional screening methods. It is necessary to screen a large number of Lactobacillus strains in vitro and in vivo, which is time-consuming, labor-intensive, and has low screening efficiency. Summary of the Invention

[0011] The purpose of this invention is to provide: The application of Lactobacillus taiwanensis in the preparation of antidepressant or anti-anxiety drugs, and related technologies, aim to solve the shortcomings of existing antidepressant and anti-anxiety drugs, such as slow onset of action, limited treatment response rate, obvious adverse reactions and easy relapse. In addition, the antidepressant and anti-anxiety efficacy of existing Lactobacillus strains is generally insufficient, making it difficult to meet the clinical demand for highly effective, safe and natural antidepressant and anti-anxiety microecological preparations, or combinations thereof.

[0012] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0013] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0014] The definition of standard chemical terminology can be found in the reference "Microbiology", edited by Shen Ping and Chen Xiangdong, Higher Education Press.

[0015] Unless otherwise stated, conventional methods within the scope of the art, such as microbial culture, protein extraction, behavioral detection, and molecular biological detection, shall be used.

[0016] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0017] The terms "optional / arbitrary" or "optionally / arbitrarily" mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation. For example, according to the definition below: the drug contains any one or more of the following: a culture of *Lactobacillus taiwanensis*, inactivated bacteria, bacterial powder, metabolites, bacterial extracts, fermentation supernatant, or lyophilized bacterial preparation. Examples include: the drug containing a culture of *Lactobacillus taiwanensis*; or the drug containing inactivated bacteria of *Lactobacillus taiwanensis*; or the drug containing bacterial powder of *Lactobacillus taiwanensis*; or the drug containing fermentation supernatant and lyophilized bacterial preparation of *Lactobacillus taiwanensis*, etc.

[0018] The term "depressive-like behavior" used in this article refers to behavioral characteristics in mice that are similar to human symptoms of depression, including decreased interest, reduced activity, and sluggish response to stimuli. It is an important behavioral indicator for evaluating the effectiveness of antidepressants.

[0019] The term “chronic unpredictable mild stress (CUMS)” used in this article refers to an animal model construction method that continuously induces depressive-like behaviors in mice by randomly administering various mild stresses such as fasting, day-night reversal, and noise stimulation, which can simulate the pathogenesis of human depression.

[0020] The term "gut-brain axis" used in this article refers to the bidirectional regulatory network formed between the gut and the central nervous system through neural, endocrine, and immune pathways, which enables the transmission of gut signals to the central nervous system and participates in emotion regulation.

[0021] The term “aromatic hydrocarbon receptor (AHR)” as used in this article refers to a transcription factor that can be activated by specific ligands, participates in regulating immune responses, metabolic homeostasis and neural function, and its activation state is closely related to the improvement of depressive-like behavior.

[0022] The term "Taiwanese Lactobacillus" as used in this article refers to a Gram-positive probiotic strain with antidepressant and anti-anxiety activities, whose strain number is CICC 23148. It can improve depressive-like behaviors induced by chronic stress by producing specific metabolites (such as indole substances), regulating the balance of intestinal flora, and activating the AHR signaling pathway. Compared with existing lactobacillus strains, its antidepressant efficacy is more significant. It can be used to prepare antidepressant and anti-anxiety related drugs and is the core functional strain in this invention.

[0023] In a first aspect, the present invention provides: Lactobacillus taiwanensis ( Lactobacillus taiwanensis Its application in the preparation of antidepressant or anti-anxiety drugs.

[0024] Secondly, the present invention provides the use of a composition containing Lactobacillus taiwanensis in the preparation of an antidepressant or anti-anxiety medicament.

[0025] Among them are the technical features: Lactobacillus taiwanensis, compositions containing Lactobacillus taiwanensis, and pharmaceuticals.

[0026] Preferably, the Lactobacillus taiwanensis is Lactobacillus taiwanensis CICC 23148.

[0027] Preferably, the drug comprises any one or more of the following: culture of Lactobacillus taiwanensis, inactivated bacteria, bacterial powder, metabolites, bacterial extract, fermentation supernatant, and lyophilized bacterial preparation.

[0028] Specifically, the drug contains at least 1×10 6 CFU / dosage of Taiwan Lactobacillus.

[0029] Preferably, the drug contains 1×10 6 -1×10 12 CFU / dosage of Taiwan Lactobacillus.

[0030] More preferably, the drug contains 1×10 6 -1×10 7 1×10 7 -1×10 8 1×10 8 -1×10 9 1×10 9 -1×10 10 1×10 10 -1×10 11 Or 1×10 11 -1×10 12 CFU / dosage of Taiwan Lactobacillus.

[0031] More preferably, the drug contains at least 1×10 7 -1×10 8 Or 1×10 8 -1×10 9 CFU / dosage of Taiwan Lactobacillus.

[0032] Specifically, the drug also includes pharmaceutically acceptable excipients or nutritional additives.

[0033] Preferably, the excipients include any one or more of the following: binders, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, or osmotic pressure regulators.

[0034] Preferably, the nutritional additives include one or more of dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

[0035] Preferably, the dosage form of the antidepressant includes any one or more of capsules, tablets, granules, powders, oral liquids, suspensions, emulsions, and injections.

[0036] Preferably, the drug exerts its antidepressant or anti-anxiety effects by regulating the aryl hydrocarbon receptor (AHR) signaling pathway through the gut-brain axis, repairing the intestinal mucosal barrier, and reducing intestinal inflammatory damage.

[0037] The present invention has at least the following beneficial effects: Compared with existing technologies, the present invention has better technical effects in improving anxiety-like behavior, depression-like behavior, and activating the AHR signaling pathway in mice.

[0038] According to experimental tests, the Lactobacillus taiwanensis CICC 23148 ​​used in this invention increased the open-arm dwell time of mice in the elevated cruciate maze by 46.155% compared to the model group in the prior art, and increased it by 61.54% compared to the model group.

[0039] According to experimental tests, the Lactobacillus taiwanensis CICC 23148 ​​used in this invention improves the static time in the forced swimming experiment by 21.21% compared to the model group in the prior art, and reduces it by 60% compared to the model group.

[0040] According to experimental tests, the Lactobacillus taiwanensis CICC 23148 ​​used in this invention increased the relative expression level of AhR by 1.25 times compared to the model group in the prior art, and increased it by about 2.4 times to 4 times compared to the model group.

[0041] Considering the possibility of this invention entering other countries, this invention also provides the following technical solutions: This invention provides a method for treating depression or anxiety, the method comprising using the Lactobacillus taiwanensis of this invention, wherein the Lactobacillus taiwanensis is Lactobacillus taiwanensis CICC 23148.

[0042] Specifically, the method includes administering an effective amount of the present invention, *Lactobacillus taiwanensis*, to the subject.

[0043] Preferably, the subjects include mammals.

[0044] More preferably, the subject is a human being.

[0045] Preferably, the effective amount is at least 1×10⁻⁶. 6 CFU / dosage of Taiwan Lactobacillus.

[0046] More preferably, the effective amount is 1×10⁻⁶. 6 -1×10 12 CFU / dosage of Taiwan Lactobacillus. Attached Figure Description

[0047] Figure 1 This is a flowchart of animal experiments.

[0048] Figure 2 The results are from animal behavioral experiments; where A is the time the mouse remained still in the tail suspension test (s); B is the time the mouse remained still in the forced swimming test (s); C is the total distance the mouse traveled in the open field test (mm); and D is the percentage of time the mouse spent in the open arm of the elevated cross maze test (%). p <0.01, p <0.001, p <0.0001.

[0049] Figure 3 Images of HE staining in mouse colons; where A is an HE staining image of the colon of mice in the CUMS group; B is... L. taiwanensis HE staining of the colon of mice in the group.

[0050] Figure 4 The image shows the results of a Western blot experiment on mouse hippocampus tissue. p <0.001.

[0051] Figure 5 The image shows the results of a Western blot experiment on mouse colon tissue. p <0.001. Detailed Implementation

[0052] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0053] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0054] Example 1: Culture of Lactobacillus taiwanense CICC 23148 This invention is based on Lactobacillus taiwanensis (Taiwan Lactobacillus). Lactobacillus taiwanensis CICC 23148 ​​was purchased from the China Industrial Microbial Culture Collection Center. This invention uses it for in vitro culture and functional verification experiments.

[0055] 1. Culture medium preparation Bacterial culture was performed using MRS liquid medium. 27 g of MRS medium powder was weighed and dissolved in 500 mL of deionized water. The solution was heated and stirred until completely dissolved, then dispensed into glass culture flasks. The flasks were then autoclaved at 121°C for 15 min and cooled before use. All glassware, pipette tips, and related consumables used in the experiment were autoclaved and handled under aseptic conditions in a laminar flow hood.

[0056] 2. Activation culture of bacterial strains The Lactobacillus taiwanense CICC 23148 ​​bacterial powder was revived under aseptic conditions. First, an appropriate amount of sterile culture medium was added to the lyophilized bacterial tube to dissolve the bacterial powder. Then, the bacterial suspension was transferred to a sterile test tube containing 4 mL of MRS liquid culture medium and mixed thoroughly.

[0057] The culture was then placed in a 37°C incubator and incubated for 24-48 hours. Once the culture medium became noticeably turbid, it indicated that the strain had been successfully revived and entered the growth phase.

[0058] 3. Growth curve of the strain To monitor the growth status of the bacterial strain, the growth curve of the strain was measured. The activated bacterial solution was inoculated into fresh MRS liquid medium at a ratio of 1:100 and cultured in a constant temperature shaker at 37°C and 230 rpm.

[0059] During the culture process, 200 μL samples were taken every 2 hours, and the OD600 value was measured using a multi-functional microplate reader. Three replicates were set up for each time point. The monitoring was carried out continuously for 18 hours, and the growth curve of the strain was plotted to determine its growth pattern and logarithmic growth phase.

[0060] 4. Strain expansion culture The activated bacterial culture was inoculated at a rate of 2 mL into a 250 mL sterile Erlenmeyer flask containing 100 mL of MRS liquid medium. The flask was then incubated at 37 °C and 230 rpm for 12 h with shaking to obtain sufficient bacterial culture for subsequent experiments.

[0061] 5. Frozen storage of bacterial strains To preserve the bacterial strain for long-term preservation, the logarithmic growth phase bacterial culture will be cryopreserved. First, the cultured bacterial culture will be centrifuged at 4℃ and 4000 rpm for 10 min, the supernatant will be discarded and the bacterial sludge will be collected.

[0062] The bacterial sludge was then resuspended in fresh MRS medium and cultured for 10-20 hours to ensure the strain was in the logarithmic growth phase. The bacterial suspension was then mixed with 50% glycerol preservation solution at a 1:1 ratio to achieve a final glycerol concentration of 20%-30%. After thorough mixing, the mixture was dispensed into sterile cryovials, labeled with the strain name and storage date, and stored at -80°C.

[0063] Example 2: Efficacy evaluation of Lactobacillus taiwanensis CICC 23148 ​​on CUMS model mice treated with Abx. 1. Laboratory animals Healthy, specific pathogen-free (SPF) C57BL / 6 mice (male, 6 weeks old) were purchased from Nanjing Jicui Pharmaceutical Co., Ltd. and housed at the Experimental Animal Center of Zhejiang University City College. Mice in each group had free access to food and water and were kept under a 12-hour light / dark cycle. The room temperature was maintained at (22 ± 2.5) ℃ and 55 ± 5% relative humidity, and the conditions were kept free of specific pathogens.

[0064] This experiment followed the Guidelines for the Management and Use of Laboratory Animals of Zhejiang University City College and was approved by the Laboratory Animal Welfare and Ethics Review Committee of Zhejiang University City College, with ethics approval number 24040.

[0065] 2. Experimental Methods 2.1 Drug Preparation The *Lactobacillus taiwanense* CICC 23148 ​​bacterial suspension in the logarithmic growth phase was centrifuged at 4°C and 8000 rpm. The supernatant was discarded, and the suspension was resuspended in sterile PBS solution and thoroughly mixed to obtain a *Lactobacillus taiwanense* suspension. Then, it was further centrifuged at 10... 8 Mice were administered the drug at a concentration of CFU / mouse via gavage. The drug was to be taken fresh daily.

[0066] 2.2 Establishment of animal models and drug grouping intervention (1) Antibiotic (Abx) treatment to construct pseudo-germ-free mice To investigate the role of gut microbiota in disease progression, this invention constructed a pseudo-germ-free mouse model by administering a broad-spectrum antibiotic cocktail via gavage.

[0067] The preferred method is to prepare antibiotic suspensions containing ampicillin (100 mg / kg / animal), metronidazole (100 mg / kg / animal), neomycin (100 mg / kg / animal), and vancomycin (50 mg / kg / animal). All antibiotics are dissolved in sterile phosphate-buffered saline (PBS, pH 7.4) and sterilized by filtration through a 0.22 μm filter membrane. The suspensions should be prepared and used immediately.

[0068] All 6-week-old SPF-grade male C57BL / 6 mice were acclimatized to a standard specific pathogen-free (SPF) environment for one week before being treated with Abx. During model establishment, mice received the above-mentioned antibiotic cocktail via oral gavage once daily, with a gavage volume of 200 μL per mouse. This treatment continued for 14 days.

[0069] (2) Establishment of the CUMS depression model This invention establishes a mouse model of depression using a chronic, unpredictable, mild stress method. All SPF-grade male C57BL / 6 mice were acclimatized to standard housing conditions for one week, followed by a 21-day CUMS program. The stress protocol included seven different mild stress stimuli: restraint, horizontal rocking, ice water swimming, foot shocks, cage tilting, audio-visual stimulation, and damp bedding. One stimulus was randomly applied daily, with each stimulus appearing three times throughout the program, and the same stimulus was not applied on consecutive days to maintain the unpredictability of the stress and prevent the animals from developing adaptations.

[0070] The methods of stress response are as follows: a. Restraint: Mice were placed in a restraint device with ventilation holes at fixed time intervals to restrict their turning but avoid chest and abdominal compression. Each restraint lasted for 3 hours. The room temperature was (22 ± 2)℃ and the humidity was (50 ± 10)%. The mice were immediately returned to their cages after the operation. The control group was only moved by grasping. Blinding and ethical monitoring were maintained throughout the process.

[0071] b. Shaking: The mice, along with their cages, were placed on a horizontal shaking table and shaken at a constant speed of 120±10 rpm and an amplitude of 2 cm for 15 minutes. This was done 1-2 times per week and was randomly interspersed with other stress environments for a total of 3 weeks. The control group was only moved without shaking.

[0072] c. Ice water swimming: Place mice in a 50ml beaker with water at approximately 4±1℃ and a depth of 15cm. The mice's toes should not touch the bottom of the container. Each mouse should swim for 5 minutes.

[0073] d. Electric shock to the soles of the feet: Place the mice in an electric shock box with a current of 0.3A. The mice adapt for 5 seconds, followed by 10 seconds of auditory and visual stimulation, with a 5-second interval between each 10-second electric shock. This is repeated 10 times. The total duration is 5 minutes per mouse.

[0074] e. Cage tilting: The rearing cages were tilted 45° along their long axis and fixed for 12 hours. The angle and time errors were ≤2° and ±10 min, respectively. The control group was only horizontally shifted. The entire process maintained unpredictability and single-cage variable control.

[0075] f. Acoustic and light stimulation: 120 dB, 4 kHz pure tone and 500 lx white light were simultaneously stimulated for 30 min in a soundproof box; 5 min of adaptation before stimulation was given, and the animals were immediately returned to their cages after stimulation. The control group was only moved to a dark and quiet box. Stress response was recorded throughout the process and ethical monitoring was conducted.

[0076] g. Moist bedding: Soak the mouse bedding in water to create a moist environment for the mice.

[0077] All mice simultaneously received the CUMS program starting in the second week after Abx treatment.

[0078] (3) Drug group intervention After one week of acclimatization, the mice were randomly divided into two groups according to their body weight using a random number table: Blank control group (CON group, n = 9): administered physiological saline by gavage.

[0079] Model group (CUMS group, n = 9): Received the CUMS procedure and were given saline via gavage daily after stress starting from the second week of CUMS modeling.

[0080] Taiwan Lactobacillus Group ( L. taiwanensis Group (n = 9): The CUMS treatment group was the same as the model group, and the group was given Lactobacillus taiwanense CICC 23148 ​​suspension by gavage daily after the second week of stress.

[0081] The dosage was calculated at 0.2 mL / 10 g body weight, administered once daily for 2 weeks. All gavage procedures were completed within 1 hour after the stress response ended to control for time-related errors. The animal experiment flowchart is shown below. Figure 1 As shown.

[0082] 2.3 Behavioral Experiments (1) Tail suspension test The tail suspension test was used to assess depressive-like behavior in mice under inescapable stress. During the experiment, the mouse's tail was secured to a horizontal bar approximately 50 cm above the ground using medical tape, suspending it in an inverted position. The mouse's head was kept approximately 15-30 cm above the table surface. The entire experiment was video-recorded for 6 minutes. After excluding the initial 1-minute adaptation period, the immobility time of the mice in the following 5 minutes was recorded. Immobility was defined as the mouse ceasing to struggle, its body drooping, with only slight limb movements to maintain respiration. After each mouse was tested, the restraint device and related contact areas were cleaned with 75% alcohol. Behavioral data were automatically analyzed using EthoVision XT software.

[0083] (2) Forced swimming experiment The forced swimming test was used to evaluate depressive-like behavior and antidepressant effects in mice. The experiment was conducted in a transparent cylindrical container (approximately 50 cm high and 14 cm in diameter) filled with approximately 20 cm of clean water at a temperature maintained at 25 ± 1 °C. Mice were placed alone in the container and videotaped continuously for 6 minutes. After removing the first minute of acclimatization, the immobile time in the following 5 minutes was recorded. Immobility was defined as the mouse ceasing active struggling and performing only minimal movements to keep its head above water. Immediately after the experiment, the mice were removed, dried, and placed in a warm environment for recovery.

[0084] (3) Open field experiment The open field test was used to assess the autonomous movement and exploratory behavior of mice in a new environment. The experimental setup was an open rectangular box (50 cm × 50 cm × 40 cm) with non-reflective monochromatic inner walls and a virtual division of the bottom into a central and peripheral area. The experiment was conducted under quiet, moderately lit conditions. Mice were gently placed in the center of the box and allowed free movement for 10-15 minutes, with their movement trajectory recorded by a top-mounted camera system. Key analytical parameters included total movement distance, time spent in the central area, and number of squares traversed. After the experiment, the mice were removed, and the box was cleaned with 75% alcohol to remove odor interference. Behavioral data were automatically collected and analyzed using EthoVision XT software.

[0085] (4) Elevated cross maze experiment The elevated cruciate maze test was used to assess anxiety-like behaviors in mice. The maze consisted of two opposing open arms (without lateral walls) and two opposing closed arms (with lateral walls) that intersected vertically in the central area, with the entire setup approximately 50 cm above the ground. The experiment was conducted in a quiet, uniformly lit environment. Mice were gently placed in the central area of ​​the maze with their heads facing the open arms and allowed to explore freely for 5-10 minutes, with their behavior recorded by a top-mounted camera system. The primary analytical parameters included the percentage of entries into the open arms (entry defined as all four limbs entering the arm) and the percentage of time spent in the open arms relative to the total exploration time. After each mouse's test, the maze surface was wiped with a 75% alcohol solution to eliminate odor interference. The behavioral videos were automatically identified and analyzed using EthoVisionXT software.

[0086] 2.4 Sample Collection After the animal behavioral tests are completed, samples are collected sequentially. The specific steps are as follows: (1) Collection of mouse fecal samples Fecal samples were collected using the natural defecation method. In short, mice were gently held and their abdomens were lightly massaged to induce natural defecation. Freshly expelled fecal particles were collected using sterile centrifuge tubes, with at least three particles collected from each mouse. Samples were immediately stored at -80°C for subsequent analysis.

[0087] (2) Collection of peripheral blood samples from mice Blood samples were collected via ocular blood collection. Mice were anesthetized, and their eyeballs were quickly enucleated, with blood collected in centrifuge tubes treated with heparin sodium anticoagulation. The blood samples were immediately centrifuged at 4500 rpm for 10 minutes at 4°C. After centrifugation, the supernatant was carefully aspirated and collected as plasma, aliquoted, and stored at -20°C.

[0088] (3) Mouse perfusion fixation and tissue sampling Mice intended for histomorphological analysis (such as staining and sectioning) underwent cardiac perfusion fixation after blood was collected from the eyeballs. The procedure is briefly described below: The mice were fixed in a supine position, the thoracic cavity was opened to expose the heart, and a small incision was made at the right atrial appendage as an outflow tract. A cannula was inserted through the apex of the heart, and approximately 100 mL of pre-cooled physiological saline was perfused at a uniform rate until the outflow was clear to rinse away the blood. Then, approximately 50 mL of 4% paraformaldehyde (PFA) fixative was perfused for in situ fixation. After perfusion, the whole brain and colon tissue were carefully removed. The whole brain and colonic segments were immediately immersed in 4% PFA fixative and stored overnight at 4°C to complete fixation. For the colon tissue, it was gently rinsed with pre-cooled PBS to remove any residual feces from the lumen.

[0089] (4) Collection of tissue samples from specific brain regions and colon After blood was collected from the eyeballs of the remaining mice, they were quickly decapitated and their entire brains were removed. On an ice tray, based on anatomical landmarks from mouse brain atlases, the prefrontal cortex, hippocampus, cerebral cortex, and thalamus were meticulously separated. The tissues from each brain region were aliquoted and immediately rapidly frozen in liquid nitrogen, then transferred to -80°C for long-term storage. Simultaneously, the colon was removed from the abdominal cavity, rinsed with pre-cooled PBS on ice to remove intestinal contents, and then rapidly frozen in liquid nitrogen, subsequently stored in an ultra-low temperature freezer at -80°C.

[0090] 2.5 HE staining (1) Sample dewaxing The slides were baked in a 65°C oven for 30 minutes to remove moisture from the gap between the slides and the tissue, thereby enhancing the adhesion between the tissue and the slide. The slides were then immersed in dewaxing agent (I) and dewaxing agent (II) for 10 minutes each to completely remove the paraffin components from the tissue. After dewaxing, the slides were placed in 100% ethanol (I) and 100% ethanol (II) for 5 minutes each to wash away residual xylene and prepare for the subsequent hydration step.

[0091] (2) Gradient hydration of dewaxed samples The sections were immersed in anhydrous ethanol (twice, 5 minutes each) and 75% ethanol (5 minutes) in sequence to allow the tissue to gradually adapt to the water environment; finally, they were rinsed with running water for 3 minutes to remove the ethanol and fully hydrate the tissue.

[0092] (3) Sample staining The sections were first stained with hematoxylin for 3 minutes to make the DNA and RNA in the cell nucleus appear blue-purple; after rinsing with running water for 3 minutes, they were differentiated with 0.5% hydrochloric acid alcohol for 6 seconds to remove non-specifically adsorbed hematoxylin on the cytoplasm and cell membrane; the differentiation reaction was terminated by rinsing with running water for 3 minutes again; then, they were blued with 0.5% ammonia water for 2 minutes to change the color of the cell nucleus from reddish-purple to a bright blue-purple; after rinsing with running water for 2 minutes, they were stained with eosin for 1 minute to make the cytoplasm and extracellular matrix appear pink to red.

[0093] After staining, dehydration is performed: the sections are immersed in 95% ethanol (twice, 2 minutes each) and anhydrous ethanol (twice, 2 minutes each) to gradually remove water from the tissue and prepare for the clearing step.

[0094] (4) The sample is cleared after dehydration. Immerse the sections in dewaxing agent (I) and dewaxing agent (II) for 5 minutes each to make the tissue transparent, which will facilitate subsequent mounting and observation.

[0095] (5) Sealing Place an appropriate amount of neutral resin mounting medium in the center of the tissue section, carefully cover it with a coverslip to avoid air bubbles, allow it to dry at room temperature, and then observe and acquire images under a microscope.

[0096] 2.6 Western blot experiment (1) Extraction of total protein and determination of protein concentration Frozen mouse hippocampal and colon tissues were removed from the -80°C freezer and placed on ice. A working lysis buffer was prepared by adding protease inhibitors and phosphatase inhibitors (1:50) to the RIPA lysis buffer. The lysis buffer was added at a volume ratio of approximately 5:1 to tissue, and the samples were mechanically homogenized using a homogenizer under ice bath conditions. The homogenized samples were incubated on a shaker at 4°C for 15–30 min, followed by centrifugation at 12,000–14,000 g for 10–15 min at 4°C. The supernatant was collected as the total protein solution, and protein concentration was determined using a BCA protein quantification kit.

[0097] (2) Preparation of sample protein Dilute the total protein solution to 2 mg / mL with PBS and mix with 2× protein loading buffer at a 1:1 volume ratio. Heat the mixture at 95°C for 10 min to fully denature the protein. The denatured protein sample can be used directly for electrophoresis or stored at -80°C for later use.

[0098] (3) Electrophoresis Separating and stacking gels were prepared using the Vazyme One-Step PAGE Gel Fast Preparation Kit (10%). After gel polymerization, the gels were mounted in an electrophoresis tank, and 1×SDS-PAGE electrophoresis buffer was added. 10 μL of protein sample was added to each well, and 5 μL of pre-stained protein marker was added to both wells. Electrophoresis was performed at 80 V for approximately 30 min (stacking stage), then the temperature was increased to 135 V and electrophoresis continued until bromophenol blue migrated to the bottom of the separating gel.

[0099] (4) Transfer (wet transfer method) After electrophoresis, remove the gel and activate the PVDF membrane in anhydrous ethanol for approximately 30 seconds. Assemble the transfer clips in the order of "sponge-filter paper-gel-PVDF membrane-filter paper-sponge". Dilute the transfer buffer from 10× stock solution to 1× and add 10% ethanol. Transfer conditions are constant current 330 mA for 1 h. After transfer, block the PVDF membrane in rapid blocking buffer at room temperature for 15 min.

[0100] (5) Antibody incubation and development After blocking, the PVDF membrane was washed 1-2 times with TBST, and the membrane strips were cut according to the molecular weight of the target protein. The primary antibody was diluted according to the manufacturer's instructions, and the membrane was incubated overnight at 4°C in the primary antibody solution. The next day, the membrane was washed 3 times with TBST (10 min / wash), and then the HRP-labeled secondary antibody was diluted with 5% skim milk TBST solution and incubated at room temperature for 2 h. After incubation, the membrane was washed 6 times with TBST (10 min / wash). The chemiluminescence working solution (A:B=1:1) was prepared according to the ECL kit instructions, and uniformly added to the surface of the PVDF membrane. The protein band signals were acquired and analyzed using a chemiluminescence imaging system.

[0101] 2.7 Data Statistics and Analysis All experimental data were statistically processed using GraphPad Prism 10.1.2 software. Differences among multiple groups were compared using one-way ANOVA, and t-tests were used to compare two groups. Results are expressed as mean ± standard deviation. p < 0.05 was considered statistically significant. p <0.05, p <0.01, p <0.001, p <0.0001, ns indicates p >0.05.

[0102] 3. Experimental Results 3.1 Lactobacillus taiwani CICC 23148 ​​improves depressive-like behavior in CUMS model mice To evaluate the effects of various interventions on the behavioral phenotypes of CUMS-induced depression model mice, this invention systematically evaluated the antidepressant and anti-anxiety effects of Lactobacillus taiwanensis CICC 23148 ​​using four classic behavioral testing methods: tail suspension test, forced swimming test, elevated cross maze test, and open field test.

[0103] The results of the forced swimming experiment and the tail suspension experiment showed a consistent trend. Figure 2 (AB in the text). The resting time of mice in the CUMS group was approximately 200 seconds, while... L. taiwanensis The resting time in the group decreased to approximately 135 seconds. All drug interventions significantly reduced the resting time in mice. pThe value <0.0001 indicates that treatment with Lactobacillus taiwanensis CICC 23148 ​​can effectively alleviate CUMS-induced depressive-like hopelessness behavior.

[0104] The open field test is used to assess the spontaneous activity and exploratory behavior of mice. Figure 2 (C) The total movement distance of the CUMS model group mice was approximately 8000 mm, which was significantly lower than the normal activity level. L. taiwanensis The total distance of group DE increased to approximately 11,500 mm. Statistical analysis shows that... L. taiwanensis The difference between the group and the CUMS group was statistically significant. p <0.01). This result indicates that intervention with Lactobacillus taiwanensis CICC 23148 ​​can restore the spontaneous activity ability of CUMS mice to a certain extent and improve their bradykinesia symptoms.

[0105] Anxiety-like behavior test results showed ( Figure 2 In the D group, the percentage of time spent in the open arm was significantly reduced, only about 3%-4%, indicating that the model mice exhibited a significant anxiety-like phenotype. L. taiwanensis After intervention, the percentage of time mice spent in the open arm increased significantly, reaching approximately 13%. p <0.0001). This result suggests that intervention with Lactobacillus taiwanensis CICC 23148 ​​can effectively improve CUMS-induced anxiety-like behavior and enhance the exploratory tendency of mice.

[0106] In summary, the behavioral test results consistently indicate that the CUMS model successfully induced typical depressive and anxiety-like behavioral phenotypes in mice, manifested as increased hopeless behavior, intensified anxiety-like behavior, and reduced spontaneous activity. Intervention with *Lactobacillus taiwanensis* CICC23148 ​​effectively reversed these behavioral abnormalities, demonstrating significant efficacy in antidepressant and anti-anxiety treatments.

[0107] 3.2 Lactobacillus taiwanense CICC 23148 ​​improves intestinal damage in CUMS model mice To systematically evaluate the effects of CUMS on the colonic tissue structure of mice and the protective effects of different interventions, the colonic tissue of mice in each group was examined histopathologically using hematoxylin-eosin staining, and the morphological changes of the colonic mucosa, submucosa and muscularis were systematically observed under an optical microscope.

[0108] HE staining results showed that ( Figure 3In group A), the colonic tissue of mice in the CUMS model group maintained a basically intact overall structure, but mild pathological changes were observed. The mucosal layer was uneven in thickness and relatively thin, with slightly widened intercellular spaces, partial loss of epithelial integrity, irregular and distorted crypt structures, and a slightly sparse arrangement. The crypt depth in some areas appeared to be decreasing, suggesting impaired mucosal barrier function. A small number of inflammatory cells were scattered and infiltrated within the lamina propria. These results indicate that the CUMS model induced mild atrophy and damage to the mucosal layer and a slight inflammatory response, suggesting that chronic stress has an adverse effect on the integrity of the intestinal mucosal barrier.

[0109] L. taiwanensis Histopathological observation of the colon tissue of the group of mice showed that ( Figure 3 In the B group, the intestinal wall layers were intact and morphologically sound. The mucosal epithelial cells were neatly and densely arranged, with regular cell morphology, oval nuclei, fine chromatin, and clearly defined nucleoli. The crypts were well-developed, tightly packed, and of appropriate depth, with smooth crypt surfaces and no dilation or distortion. A rich capillary network was visible in the lamina propria, with minimal inflammatory cell infiltration and clear tissue structure. Goblet cells were abundant and plump, indicating good intestinal mucus barrier function. No obvious pathological changes were observed in the submucosa; the muscular layer was intact, uniform in thickness, and the muscle fibers followed a consistent course. Compared with the CUMS model group, L. taiwanensis The intervention significantly improved the histological structure of the colonic mucosa, reduced mucosal atrophy, inhibited inflammatory cell infiltration, and resulted in an overall tissue morphology close to that of normal controls, indicating that... L. taiwanensis It has a good protective effect against CUMS-induced colonic tissue damage.

[0110] 3.3 Lactobacillus taiwani CICC 23148 ​​improves depressive-like behavior in CUMS mice by activating the AHR signaling pathway. (1) Effects of Lactobacillus taiwanensis CICC 23148 ​​on the expression of AHR signaling pathway-related proteins in hippocampus of CUMS mice To further explore the possible mechanism by which Lactobacillus taiwanensis CICC 23148 ​​improves depressive-like behavior in CUMS mice, the expression level of AHR in the hippocampus of mice was detected by Western blot, and GAPDH was used as an internal reference protein for gray value normalization analysis.

[0111] result Figure 4 The results show that, compared to the CUMS model group, L. taiwanensis In the hippocampal tissue of the group, the AHR protein bands were significantly enhanced, and the grayscale analysis results showed that its expression level was significantly increased. p <0.05). The above results indicate that Lactobacillus taiwanensis CICC 23148 ​​can significantly promote the expression of AHR signaling pathway-related proteins in hippocampal tissue.

[0112] (2) Effects of Lactobacillus taiwanensis CICC 23148 ​​on the expression of AHR signaling pathway-related proteins in colon tissue of CUMS mice To further observe changes in the AHR signaling pathway in intestinal tissue, the expression level of AHR in mouse colon tissue was detected. Western blot results showed that, compared with the CUMS group, L. taiwanensis The expression of AHR protein in the colon tissue of the group showed an increasing trend (p<0.05), and the gray value analysis results showed that its expression level was higher than that of the CUMS group. Figure 5 Overall, the results suggest that Lactobacillus taiwanensis CICC 23148 ​​can also promote the expression of proteins related to the AHR signaling pathway in colon tissue.

[0113] Based on the antibiotic (Abx) pretreated CUMS depression model mice, this invention systematically evaluated the antidepressant effect of Lactobacillus taiwanensis CICC 2314, and further explored its possible mechanism of action from two levels: intestinal barrier structure and AHR signaling pathway.

[0114] Overall, the results indicate that *Lactobacillus taiwanensis* CICC 2314 can improve depressive-like behavior in CUMS mice to some extent, accompanied by restoration of intestinal tissue structure and upregulation of AHR signaling pathway-related protein expression. These results suggest that *Lactobacillus taiwanensis* CICC 2314 may play an important role in the improvement of CUMS-induced depressive-like behavior by activating the AHR signaling pathway and participating in the regulation of intestinal barrier function and brain function. These results provide new research ideas for further novel antidepressant intervention strategies based on the gut-brain axis.

[0115] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Lactobacillus taiwanense ( Lactobacillus taiwanensis Its application in the preparation of antidepressant or anti-anxiety drugs is characterized by, The Lactobacillus taiwanensis mentioned is Lactobacillus taiwanensis CICC 23148.

2. Contains Lactobacillus taiwanensis ( Lactobacillus taiwanensis The use of the composition of ) in the preparation of antidepressant or anti-anxiety medicines, characterized in that, The Lactobacillus taiwanensis mentioned is Lactobacillus taiwanensis CICC 23148.

3. The application according to any one of claims 1-2, characterized in that, The drug contains any one or more of the following: culture of Lactobacillus taiwanensis, inactivated bacteria, bacterial powder, metabolites, bacterial extract, fermentation supernatant, and lyophilized bacterial preparation.

4. The application according to any one of claims 1-2, characterized in that, The drug contains at least 1×10 6 CFU / dose of Lactobacillus taiwani.

5. The application according to claim 4, characterized in that, The drug contains 1×10 6 -1×10 12 CFU / dose of Lactobacillus taiwani.

6. The application according to any one of claims 1-2, characterized in that, The drug also includes pharmaceutically acceptable excipients or nutritional additives.

7. The application according to claim 6, characterized in that, The excipients are selected from any one or more of the following: adhesives, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, solubilizers, and osmotic pressure regulators.

8. The application according to claim 6, characterized in that, The nutritional additives mentioned are selected from one or more of the following: dietary fiber, prebiotics, protein, lipids, minerals, and vitamins.

9. The application according to any one of claims 1-2, characterized in that, The dosage forms of the drug are capsules, tablets, granules, powders, oral liquids, suspensions, emulsions, or injections.

10. The application according to any one of claims 1-2, characterized in that, The drug exerts its effects by regulating the aryl hydrocarbon receptor (AHR) signaling pathway through the gut-brain axis, repairing the intestinal mucosal barrier, and reducing intestinal inflammatory damage.

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