Depression intestine-brain axis bionic chip for screening mental probiotics

Abstract

The invention provides an intestine-brain axis bionic chip for treating depression, which is used for screening mental probiotics. According to the platform, an intestine chip and a brain chip are connected in series, and an in-vitro model for simulating intestine-brain axis interaction of a depression patient is constructed. Human colonic adenocarcinoma cells are inoculated on the upper channel side of the intestinal chip, and intestinal flora of a depression patient is introduced to simulate an intestinal microenvironment; the upper channel of the brain chip is inoculated with human brain microvascular endothelial cells, the porous membrane side of the lower channel is inoculated with human brain astroglia cells, and the bottom side of the lower channel is inoculated with human brain microglia cells, so that the brain chip is used for simulating a blood brain barrier and a nervous system. Through series connection of the two chips, transmission of intestine-brain signals of a depression patient is simulated. The platform can be used for screening mental probiotics and evaluating the regulating effect of the mental probiotics on intestinal barriers, neuroinflammation, blood-brain barriers and neurotransmitters. Through 16S rRNA sequencing, immunofluorescence staining, metabonomics and other technologies, intestinal-brain axis function changes are analyzed, and a mental probiotic strain with anti-depression potential is screened. The invention provides a novel experimental platform and technical means, and innovative support can be provided for mental probiotic screening and depression treatment research.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a bionic chip for the gut-brain axis of depression for screening psychoactive probiotics. The platform can be used to simulate the gut-brain axis function of patients with depression, and provide a new experimental platform and technical means for the screening and functional verification of psychoactive probiotics. Background Art

[0002] Major Depressive Disorder (MDD) is a common mental disorder, characterized by long-term low mood, loss of interest, and decreased cognitive function. There are more than 350 million people suffering from depression worldwide, of which 21.3% are in China. Depression not only affects the quality of life of patients, but also significantly increases the risk of suicide and disability. Although there are many hypotheses such as neurotransmitter imbalance, inflammatory response, and genetic factors, the complex pathological mechanism of depression has not yet been fully understood, and there is an urgent need to develop more precise and effective treatment strategies.

[0003] In recent years, the role of the Microbiota-Gut-Brain Axis (MGB), as a bidirectional signaling pathway between the intestinal microbiota and the central nervous system, in the pathogenesis of depression has attracted widespread attention. Imbalance of intestinal microorganisms may lead to inflammatory responses and neurotransmitter imbalances, thereby affecting brain function and inducing depression. Therefore, regulating the intestinal microbiota is considered a potential therapeutic strategy to alleviate depression.

[0004] Psychobiotics are a type of probiotics that can regulate intestinal microbiota and improve mood and mental state. Studies have shown that some probiotics can relieve depressive symptoms by improving the structure of intestinal flora, increasing the abundance of beneficial bacteria, and regulating the balance of neurotransmitters. However, there is currently a lack of efficient and accurate screening technology, especially in simulating the complex interaction mechanism of the gut-brain axis, which still poses great challenges.

[0005] Organ chip technology provides a new experimental platform for the study of the gut-brain axis. This technology can accurately reproduce the interaction between the intestine and the nervous system in vitro and simulate the signal transmission between the intestinal microorganisms and the nervous system. Although some studies have tried to use organ chip technology to simulate the gut-brain axis, most of the existing models are single tissues and have not yet integrated the intestine and neurovascular units, and have failed to fully simulate the physiological functions of the gut-brain axis. Therefore, the development of a bionic chip that can fully reproduce the mechanism of action of the gut-brain axis has become an urgent need in the screening and research of psychoactive probiotics. This platform can effectively screen probiotics with psychoactive probiotic functions and provide technical support for the clinical treatment of depression. Summary of the invention

[0006] The present invention relates to a bionic chip for simulating the gut-brain axis (MGB) of depression, specifically, a model for simulating the gut-brain axis function of patients with depression in vitro is constructed by connecting a gut chip and a brain chip in series. The platform integrates intestinal flora, intestinal epithelium, blood-brain barrier and nervous system, and can effectively simulate the transmission process of gut-brain signals. It is mainly used in the research of depression and the screening and functional verification of psychoactive probiotics.

[0007] In order to solve the above technical problems, the present invention provides the following solutions:

[0008] The present invention provides a depression gut-brain bionic chip, which consists of two dual-channel membrane chips connected in series. The dual-channel membrane chip includes an upper channel chamber, a lower channel chamber and a porous membrane located between the upper channel and the lower channel.

[0009] The two chips connected in series are respectively a gut chip and a brain chip.

[0010] The intestinal chip is a dual-channel sandwich chip on which human colon adenocarcinoma cells are inoculated on a porous membrane on the upper channel side, and intestinal flora from patients with depression are introduced to colonize the human colon adenocarcinoma cells in the upper channel; the culture medium of the upper channel of the intestinal chip is a modified microbial culture medium suitable for co-culture of human intestinal epithelial cells and intestinal microorganisms, and the culture medium of the lower channel of the intestinal chip is an endothelial cell culture medium containing endothelial cell growth factor.

[0011] The brain chip is inoculated with human brain astrocytes on the porous membrane on the lower channel side of the dual-channel sandwich chip, and human brain microglia on the basal side of the lower channel; human brain microvascular endothelial cells are inoculated on the porous membrane on the upper channel side of the sandwich chip. The culture medium of the upper channel of the brain chip is an endothelial culture medium infected by the intestinal flora of patients with depression that flows out of the lower channel of the intestinal chip, and the culture medium of the lower channel of the brain chip is a mixed culture medium suitable for human brain astrocytes and human brain microglia.

[0012] Furthermore, in the above technical solution, the porous membrane is made of PET and has a pore size of 1.2 μm.

[0013] Furthermore, in the above technical solution, the chip material is PDMS.

[0014] Furthermore, in the above technical solution, the chip channel dimensions are: 1.5 mm wide and 375 μm high.

[0015] Furthermore, in the above technical solution, the inoculation concentration of the original intestinal flora of depression patients in the depressive-like gut-brain axis chip is 1:100.

[0016] The present invention also provides the depression gut-brain bionic chip, which detects multiple cell states and functional disorders after the intestinal flora of depression patients is introduced.

[0017] The present invention also provides the depression gut-brain bionic chip as an application for exploring the anti-depressant effect of psychoactive probiotics by regulating the gut-brain axis at the multi-organ level.

[0018] The present invention also provides a method for constructing an in vitro model of the gut-brain axis of depression based on an organ chip, and the specific process is as follows:

[0019] (1) Construction of gut-brain axis biomimetic chip

[0020] Construction of Intestine Chip: Caco-2 suspension (6×10 5 cells / mL) were inoculated into the channel, cultured in a cell culture incubator at 37°C for 2 h, and then the injection pump was connected to the inlet of the upper and lower channels of the chip at a flow rate of 100 μL / h, and perfusion culture was carried out in a 37°C incubator for 24 h.

[0021] Construction of brain chip: Human brain astrocytes U-118MG (~5×10 6 cells / mL) were inoculated on the PET membrane side of the lower channel and allowed to adhere to the PET membrane surface under static conditions. Two hours later, the lower channel was washed with fresh culture medium to remove unattached U-118MG cells. Then HMC3 cells (~1×10 6 cells / mL) were seeded at the bottom of the lower channel, and hCMEC / D3 (~1×10 6 cells / mL) were inoculated into the upper channel. After the cells attached, the culture medium was injected into the upper and lower layers of the chip at a constant flow rate (100 μL / h) using a syringe pump. The syringe pump was connected to the entrance of the upper and lower channels of the chip at a flow rate of 100 μL / h, and the perfusion culture was carried out in a 37°C incubator for 24 hours.

[0022] The depression gut-brain axis chip was constructed by transporting the effluent from the blood side of the intestinal chip to the blood side of the brain chip through a pipeline at a flow rate of 100 μL / h and perfusion culture in a 37°C incubator for 48 hours.

[0023] (2) Functional testing

[0024] The integrity of the intestinal barrier was evaluated by immunofluorescence staining of tight junction protein ZO-1 and Lucifer yellow permeability test.

[0025] The integrity of the blood-brain barrier of the brain chip was detected by immunofluorescence staining of platelet endothelial cell adhesion factor 1CD31 and tight junction protein ZO-1. The permeability of the blood-brain barrier of the brain chip was monitored by FITC-labeled dextran. The activation status of the two glial cells was detected by astrocyte-specific markers (GFAP, S100β) and microglia-specific markers (IBA1).

[0026] (3) Introducing the intestinal flora of patients with depression

[0027] The original intestinal flora suspension of depression patients was resuspended in a modified microbial culture medium at a ratio of 1:100, and after anaerobic aeration treatment for 12 hours, it was introduced into the upper channel of the Intestinal Chip and adsorbed to the surface of Caco-2 cells. The upper channel is a modified intestinal microbial culture medium. After deoxygenated gas is introduced, it is perfused and cultured at a flow rate of 100 μL / h through a syringe pump.

[0028] (4) Construction of a gut-brain bionic chip for depression

[0029] After the intestinal flora of depression patients was introduced into the intestinal chip, the effluent from the blood side of the lower channel of the intestinal chip was transported to the blood side of the upper channel of the brain chip through a pipeline and perfused and cultured in a 37°C incubator at a flow rate of 100 μL / h for 48 hours.

[0030] (5) Characterization of depression gut-brain bionic chip

[0031] After the introduction of the intestinal flora of patients with depression, the changes in the integrity of the intestinal barrier can be evaluated by the fluorescent yellow permeability test; the effluent from the channel on the intestinal chip is collected for 16s rRNA detection to analyze the species abundance and composition of the intestinal flora. After the effluent of the intestinal chip is perfused into the brain chip, the changes in the integrity of the blood-brain barrier part of the brain chip are evaluated by the tight junction protein ZO-1. The activation status of the two types of glial cells is detected by astrocyte-specific markers (GFAP, S100β) and microglia-specific markers (IBA1).

[0032] (6) Screening of psychoactive probiotics

[0033] Psychoactive probiotics from different sources were introduced to observe their effects in the gut-brain axis model and to evaluate their effects on neuroinflammation and blood-brain barrier by regulating intestinal flora.

[0034] Inoculation and culture of psychoactive probiotics: Psychoactive probiotic strains with antidepressant potential were selected and inoculated into the intestinal chip channel to co-culture with the intestinal flora of patients with depression. A constant flow rate (100 μL / h) was used through a syringe pump to simulate the interaction between intestinal microorganisms and epithelial cells.

[0035] Evaluation of the gut-brain axis effect: Psychobiotics may affect the intestinal barrier and gut-brain axis signaling by regulating the intestinal microbiota and its metabolites. The changes in intestinal barrier integrity were evaluated by fluorescent yellow permeability test, and the diversity and abundance of intestinal flora were analyzed by 16S rRNA sequencing to further screen out probiotic strains that can improve intestinal barrier function or affect neural function. In the brain chip, the integrity of the blood-brain barrier was detected by ZO-1 immunofluorescence staining, and the neuroinflammatory state was evaluated by GFAP and IBA1 labeling of astrocytes and microglia to detect the effect of probiotics on the activation state of neurons. .

[0036] Evaluation of the effects of psychoactive probiotics: By observing the gut-brain axis model, the effects of probiotics on improving neurochemistry and cellular function in the depression model were evaluated, and psychoactive probiotics with significant antidepressant effects were further screened.

[0037] Compared with the prior art, the present invention has achieved the following beneficial technical effects:

[0038] (1) The present invention provides a depression gut-brain bionic chip for screening psychoactive probiotics, which can simulate the interaction between the intestinal flora and the brain of patients with depression, and provides an experimental basis for the screening of psychoactive probiotics.

[0039] (2) The present invention provides an innovative method for screening psychoactive probiotics, which can be used to explore the interaction mechanism among multiple organs and verify the antidepressant effect.

[0040] (3) The depression gut-brain bionic chip provided by the present invention can be used to screen and evaluate psychoactive probiotics through a series of biological experimental methods, including detection of cell proliferation, cytokine secretion, nerve conduction and barrier function.

[0041] (4) The organ chip technology used in the present invention takes into account both low cost and high-throughput experiments, and provides a convenient in vitro detection platform for simulating the dynamic changes of the gut-brain microenvironment, which is suitable for the screening of psychoactive probiotics, drug development and research on the gut-brain axis mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the implementation of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor.

[0043] Figure 1 Schematic diagram of the depression gut-brain bionic chip of the present invention. A is a schematic diagram of the intestinal unit, B is a schematic diagram of the brain unit, and C is a schematic diagram of the depression gut-brain axis bionic chip;

[0044] Figure 2 The immunofluorescence staining characterization diagram of the brain chip of the present invention. A is a confocal microscopy image of hCMEC / D3 cells labeled with CD31 and ZO-1, B is a confocal microscopy image of U-118MG cells labeled with GFAP and s100β, and C is a confocal microscopy image of HMC3 cells labeled with IBA1;

[0045] Figure 3 This is a test diagram of the permeability performance of the brain chip of the present invention.

[0046] Figure 4 The functional test diagram of the intestinal chip of the present invention. A is the immunofluorescence staining characterization, and B is the permeability test of the intestinal chip;

[0047] Figure 5 The intestinal microbiota of depression patients introduced into the intestinal chip of the present invention; A is an immunofluorescence staining image without intestinal microbiota, and B is an immunofluorescence staining image of intestinal microbiota of depression patients introduced;

[0048] Figure 6 The metabolic characteristics of the lateral effluent of the brain and its related KEGG enrichment pathways were introduced into the depression gut-brain axis chip of the present invention. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0050] The purpose of the present invention is to provide a depression gut-brain axis bionic chip for screening psychoactive probiotics to solve the deficiencies of the prior art in simulating the gut-brain axis function of patients with depression and in screening and functional verification of psychoactive probiotics. In order to make the above-mentioned purposes, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0051] Example 1

[0052] Construction of a gut-brain axis biomimetic chip.

[0053] First, a 100 μg / mL collagen solution was coated on the PET membrane surface of the upper channel to form a collagen layer. Then, a Caco-2 cell suspension (6×10 5cells / mL) were inoculated into the collagen-coated channel and incubated in a 37°C incubator for 2 hours to promote the attachment and growth of cells on the porous membrane. The culture medium of the upper channel adopts a modified microbial culture medium suitable for co-culture of intestinal flora and intestinal epithelial cells, and the specific composition includes basal culture medium MEM, 20% fetal bovine serum (FBS), 1% glutamine, 1mg / mL pectin, 1mg / mL mucin, 5μg / mL heme and 0.5μg / mL vitamin K1. The culture medium of the lower channel is endothelial cell culture medium, which includes 93% basal culture medium, 5% fetal bovine serum (FBS) and 1% endothelial cell culture additive. After that, the injection pump was connected to the upper and lower channel inlets of the chip, the flow rate was set to 100μL / h, and continuous perfusion culture was carried out in a 37°C incubator for 48h.

[0054] Before inoculating cells, the chip was sterilized by ultraviolet irradiation overnight and then pre-coated with 400 μg / mL collagen IV and 100 μg / mL fibronectin at 37°C for 24 hours. 6 Cells / chip) were seeded on the PET membrane side of the lower channel and allowed to adhere to the PET membrane surface under static conditions. Two hours later, the lower channel was washed with fresh culture medium to remove unattached U-118MG cells. HMC3 cells (~1×10 6 cells / chip) were seeded at the bottom of the lower channel, and hCMEC / D3 (~1×10 6 After the cells attached, the culture medium was injected into the upper and lower layers of the chip at a constant flow rate (100 μL / h) using a syringe pump, and perfusion culture was performed for 24 hours to form a brain chip containing a blood-brain barrier.

[0055] The effluent from the blood side of the intestinal chip was collected and added to the blood side of the brain chip through a syringe pump. The flow rate was set to 100 μL / h, and continuous perfusion culture was carried out in a 37°C incubator for 48 h to realize the construction of the intestinal-brain chip.

[0056] Functional testing of the gut-brain axis biomimetic chip.

[0057] The barrier integrity of the intestinal chip was evaluated by ZO-1 immunofluorescence staining and fluorescent yellow permeability test, and the intestinal lateral effluent was collected for analysis of changes in bacterial species composition and abundance. The blood-brain barrier integrity of the brain chip was detected by CD31 and ZO-1 immunofluorescence staining, and its permeability was monitored using FITC-labeled dextran. In addition, astrocyte (GFAP, S100β) and microglia (IBA1) markers were used to detect the activation state of the two glial cells. By collecting brain lateral effluent for non-targeted metabolomics analysis, the effects of small molecule metabolites crossing the blood-brain barrier on brain cell function were explored.

[0058] Example 2

[0059] Collection of gut microbial samples from patients with depression.

[0060] The experiment recruited patients with depression based on the DSM-IV-TR criteria and the 17 items in the Hamilton Depression Rating Scale (HDRS-17). All subjects were evaluated by two experienced psychiatrists in the Department of Neurology of the hospital. HDRS-17>24 was defined as depression; at the same time, attention was paid to excluding patients with depression who had one or more confounding factors, such as pre-existing physical or mental illness, patients who had undergone drug treatment and / or abuse of drugs; and female patients who were pregnant, breastfeeding, and menstruating were also excluded. Accordingly, individuals with mental illness or any family history of mental illness could not be included in the healthy control group; patients with depression and healthy controls who were taking antibiotics and other drugs were also excluded. Finally, this project study plans to screen 5 patients with depression aged 18-65 years old and with junior high school education or above, with a moderate ratio of men to women. This study will be conducted after the review and approval of the Ethics Committee and after all subjects have signed the completed informed consent form.

[0061] Construction and characterization of a biomimetic chip platform for the gut-brain axis in depression.

[0062] The intestinal flora of patients with depression were introduced into the gut-brain chip to construct a bionic chip for the depression gut-brain axis. When inoculating the intestinal flora, the original intestinal flora suspension of patients with depression was resuspended in a modified microbial culture medium at a ratio of 1:100, and introduced into the upper channel of the intestinal chip after anaerobic aeration treatment for 12 hours. After standing for 30 minutes, after the intestinal bacteria were adsorbed on the mucus layer produced by Caco-2 cells, the MEM culture medium was replaced with a modified intestinal microbial culture medium and N2 containing 5% CO2 was introduced for aeration and deoxygenation, and perfused into the top channel, thereby achieving co-culture of intestinal microorganisms and Caco-2 cells in the intestinal unit.

[0063] The flow of liquid was achieved by transporting the effluent from the blood side of the intestinal chip (i.e., the lower channel of the intestinal chip) to the blood side of the brain chip (i.e., the upper channel of the brain chip) through a pipeline. The culture was continuously run in a 37°C, 5% CO2 incubator for 48 hours. The perfusion fluid was collected at the outlet of each channel at regular intervals during the culture period. After the culture was completed, the microfluidic chip was disassembled, and the intestinal microorganisms and cells in each channel were collected and stored at -80°C. The performance of the platform was characterized and analyzed according to the following research methods.

[0064] The analysis mainly includes: 1) using targeted and non-targeted metabolomics and other technologies to detect and analyze substances such as short-chain fatty acids and neurotransmitters in the perfusion fluid; 2) using enzyme-linked immunosorbent assay (ELISA) to detect and analyze components such as cytokines in the perfusion fluid; 3) using CCK-8 method and flow cytometry to evaluate the activity of each cell and the expression level of surface markers; 4) using transcriptome sequencing technology (RNA-seq), real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and tandem mass spectrometry tag (TMT) quantitative proteomics and other technologies to identify the differential genes and proteins of cells; 5) using immunofluorescence and sodium fluorescein detection methods to characterize the integrity and permeability of the intestine and blood-brain barrier; 6) using 16S rRNA sequencing technology, metagenomics and metaproteomics technologies to analyze the species composition and gene function of the intestinal flora.

[0065] Example 3

[0066] Screening and functional validation of psychoactive probiotics.

[0067] This experiment used the gut-brain axis bionic chip platform to screen and functionally verify Bacillus licheniformis as a psychoactive probiotic. Bacillus licheniformis (Bl) is considered to have the potential to improve symptoms of depression, and its effects in improving intestinal barrier, immune regulation and neurotransmitter levels were verified.

[0068] The mixture of the original intestinal flora inoculum and Bacillus licheniformis was resuspended in an aerated and deoxygenated intestinal flora culture medium and added to the intestinal chip. The amount of Bacillus licheniformis was calculated based on the dosage of two capsules per adult for 200 g of colon content, which was about 2.5×10 6 Pieces / mL.

[0069] Evaluation of intestinal barrier function: After the experiment, immunofluorescence staining was performed to detect the expression of tight junction protein ZO-1 as an indicator of intestinal barrier integrity. The expression intensity of ZO-1 was quantified by the image analysis software ImageJ to evaluate the repair effect of Bacillus licheniformis on intestinal barrier function. To further quantitatively analyze intestinal permeability, the permeability coefficient was determined using fluorescent yellow reagent to evaluate whether Bacillus licheniformis could reduce intestinal permeability and thus improve intestinal barrier function.

[0070] Immune factor and neurotransmitter detection: During the experiment, samples of the upper and lower effluent were collected regularly, and the concentrations of neurotransmitters (such as 5-HT) and immune cytokines (such as IL-10, IL-6, IL-1β, TNF-α) were detected using ELISA kits. These tests help evaluate whether Bacillus licheniformis affects the gut-brain axis and relieves depressive symptoms by regulating the levels of immune factors and neurotransmitters.

[0071] Analysis of intestinal flora and metabolites: After the experiment, intestinal flora samples were collected and subjected to 16S rRNA high-throughput sequencing to analyze the diversity and composition changes of intestinal flora and evaluate the effects of Bacillus licheniformis on intestinal flora. At the same time, upper and lower layer effluent samples were collected for non-targeted metabolome analysis to reveal whether Bacillus licheniformis produces beneficial metabolites by regulating intestinal metabolic activities, thereby affecting the function of the gut-brain axis.

Claims

1. A bionic chip for the depression gut-brain axis for screening psychoactive probiotics, characterized in that: The platform includes: a pair of dual-channel membrane chips connected in series, which are used to simulate the interaction between intestinal flora, intestinal epithelium, blood-brain barrier and nervous system. The chip platform can simulate the function of the gut-brain axis in patients with depression and provide technical support for the screening and functional verification of psychoactive probiotics; The system includes a gut chip and a brain chip connected in series; The intestinal chip is composed of a porous membrane located on the upper channel side of a dual-channel sandwich chip, on which human intestinal epithelial cells derived from human colon adenocarcinoma cells are inoculated; intestinal flora of depression patients are introduced into the upper channel of the intestinal chip so that the flora colonizes on the human intestinal epithelial cells; the culture medium of the upper channel of the intestinal chip is a modified microbial culture medium suitable for co-culture of human intestinal epithelial cells and intestinal microorganisms; the culture medium of the lower channel of the intestinal chip is an endothelial cell culture medium containing endothelial cell growth factor; The brain chip is composed of a porous membrane located on the lower channel side of a double-channel sandwich chip, human brain astrocytes are inoculated on the porous membrane side of the lower channel, and human brain microglia are inoculated on the basal side of the lower channel; human brain microvascular endothelial cells are inoculated on the porous membrane located on the upper channel side of the sandwich chip; the culture medium of the upper channel of the brain chip is an endothelial culture medium that flows out of the lower channel of the intestinal chip and is infected by the intestinal flora of patients with depression; the culture medium of the lower channel of the brain chip is a mixed culture medium suitable for human brain astrocytes and human brain microglia.

2. The depression gut-brain axis bionic chip platform according to claim 1, wherein: The improved microbial culture medium specifically comprises a basic culture medium MEM, 20% fetal bovine serum (FBS), 1% glutamine, 1 mg / mL pectin, 1 mg / mL mucin, 5 μg / mL heme and 0.5 μg / mL vitamin K1.

3. The depression gut-brain axis bionic chip platform according to claim 1, wherein: The mixed culture medium comprises a basic culture medium MEM, 10% fetal bovine serum (FBS) and a basic culture medium DMEM, 10% fetal bovine serum (FBS) in a 1:1 ratio.

4. The depression gut-brain axis bionic chip platform according to claim 1, characterized in that: The upper and lower layers of the chip include polydimethylsiloxane (PDMS).

5. The depression gut-brain axis bionic chip platform according to claim 1, characterized in that: The porous membrane is a PET porous membrane.

6. The depression gut-brain axis bionic chip platform according to claim 1, characterized in that: The pore size of the porous membrane is 1.2 μm.

7. A method for screening psychoactive probiotics using a depression gut-brain axis bionic chip platform, characterized in that: The following steps are involved: S1: Through the dual-channel membrane chip, intestinal flora and psychoactive probiotics are inoculated in the upper channel of the intestinal chip, and intestinal epithelial cells are cultured in the upper channel to simulate the intestinal microenvironment of patients with depression; S2: Introduce the intestinal flora inoculum of patients with depression into the channel on the Intestinal Chip where intestinal epithelial cells are cultured to simulate the microbial ecological environment in the intestine and ensure the normal interaction of the intestinal microbial community; S3: Through the microfluidic system, anaerobic intestinal flora culture medium and oxygenated cell culture medium are continuously perfused to maintain the stable physiological state of the gut-brain axis bionic chip and ensure the normal co-culture environment of the intestinal flora and intestinal epithelial cells; S4: Through the double-channel membrane chip, human brain astrocytes and human brain microglia are inoculated in the lower channel of the brain chip, and human brain microvascular endothelial cells are inoculated on the porous membrane on the upper channel side to simulate the function of the blood-brain barrier and reproduce the interaction between the nervous system and the intestinal microenvironment; S5: The construction of a bionic chip for the gut-brain axis of depression is achieved by transporting the effluent from the blood side of the intestinal chip to the blood side of the brain chip through a pipeline. S6: Mix the intestinal flora inoculum and psychoactive probiotics in a predetermined ratio and perfuse them into the constructed depression gut-brain axis bionic chip. Based on the above experimental steps, further evaluate the regulatory effect of psychoactive probiotics on intestinal barrier function, immune factors, and neurotransmitters, analyze the regulatory effect of psychoactive probiotics on intestinal flora, and then explore its potential clinical application value in depression.

8. The method according to claim 7, wherein: The intestinal flora is collected from patients with depression and is used to simulate the intestinal microenvironment.

9. The depression gut-brain axis bionic chip according to claim 7, characterized in that: The platform uses an intestinal flora culture medium that has been treated with aeration and deoxygenation to resuspend the original intestinal flora inoculum at a resuspension ratio of 1:100, and further performs aeration and deoxygenation treatment in an anaerobic environment for 12 hours.

10. The depression gut-brain axis bionic chip according to claim 7, characterized in that: The psychoactive probiotics are probiotics that can regulate intestinal microbiota and improve depression symptoms. Bacillus licheniformis (Bl) is administered to 200 g of colon content at a dose of two capsules each time, and the converted bacterial concentration is about 2.5×10 6 Pieces / mL.

11. The method according to claim 7, wherein: The evaluation of the effects of psychoactive probiotics includes analyzing intestinal barrier function by immunofluorescence staining, analyzing immune factor and neurotransmitter levels by ELISA, analyzing intestinal flora diversity and structure by 16S rRNA sequencing, and analyzing differential metabolites by non-targeted metabolomics.

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