Application of anti-VLA4 therapy in antidepressants by inhibiting the migration of γδ T cells to the central nervous system

Abstract

This invention relates to the field of biomedicine, and particularly to the application of anti-VLA4 therapy in antidepressant treatment by inhibiting the migration of γδ T cells to the central nervous system. This invention utilizes anti-VLA4 antibody treatment to reduce the infiltration of γδ T cells into the meninges and brain parenchyma of mice, revealing that anti-VLA4 inhibition of peripheral T cell migration can block CUMS-induced depressive-like behaviors, providing a novel strategy for the treatment of depression.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to anti-VLA4 antibody therapy that exerts an antidepressant effect by inhibiting the migration of γδ T cells to the central nervous system. Background Technology

[0002] (I) Interaction between γδ T cells and the central nervous system

[0003] γδ T cells are distributed in various lymphatic and non-lymphatic tissues and are early-responding cells in many diseases. Previous studies have confirmed their important role in infection, cancer, autoimmune diseases, and tissue maintenance. γδ T cells are T lymphocytes that express the γ and δ chains of the T cell receptor and constitute the γδ T cell receptor (TCR). Like conventional αβ T cells and B cells, γδ T cells utilize V, D, and J gene rearrangements to express various TCRs for antigen recognition. Although γδ T cells are few in number in blood and lymphatic tissues, they are abundant in barrier tissues, and their frequency in the blood increases dramatically during infection. Utilizing different V regions of the γδ TCR chain, different subsets of γδ T cells reside in the meninges, skin, lungs, liver, abdominal cavity, adipose tissue, uterus, tongue, intestine, blood, and secondary lymphatic organs, depending on the developmental sequence of γδ T cells before and after birth. Based on the intensity of TCR signaling during development, γδ T cells differentiate into two major effector subsets according to the types of cytokines they produce: interferon-γ (IFN-γ) and interleukin-17 (IL-17) (γδ T17 cells). γδ T cells can participate in immediate immune responses because they possess direct antigen recognition, widespread distribution, multiple ligands for the γδ TCR, and expression of innate receptors. In fact, substantial evidence suggests that γδ T cells play a crucial role in infection, tumorigenesis, autoimmunity, and immune surveillance.

[0004] For decades, the central nervous system (CNS) has been traditionally considered immune-privileged due to its protection by the blood-brain barrier (BBB), characterized by low expression of leukocyte adhesion molecules and tight junctions between brain capillary endothelial cells. However, mounting evidence suggests that the CNS and immune system can interact directly. Studies have reported that meningeal T cells secrete interleukin-4 (IL-4), interleukin-13 (IL-13), and IFN-γ, factors that play crucial roles in learning, long-term memory, and social behavior. More importantly, new evidence indicates that meningeal γδ T cells play a key role in maintaining nervous system homeostasis. These findings suggest that γδ T cells play a complex role in neuron-immune interactions.

[0005] (II) γδ T cells play an important role in nervous system diseases

[0006] Two outstanding studies by Ribot's and Kipnis's teams have revealed that meningeal γδ T cells can secrete IL-17 to regulate short-term memory and anxiety-like behavior. Mice lacking γδ T cells and IL-17 exhibited impaired short-term memory in the Y-maze and Morris water maze tests. γδ T cell-derived IL-17 modulates the expression of neurotrophic factor (BDNF) in the hippocampus, and BDNF regulates synaptic plasticity in neurons related to short-term memory. On the other hand, compared to WT mice, TCRδ... - / - WT mice injected with anti-TCRδ antibodies in their cerebrospinal fluid (CSF) showed reduced anxiety levels in the elevated cross maze and open field tests, indicating that meningeal γδ17 T cells control the occurrence of anxiety-like behaviors. In summary, these data suggest that meningeal γδT17 cells play a crucial role in short-term memory and anxiety-like behaviors. Recently, studies have shown that chronic stress can lead to gut microbiota dysbiosis and a decrease in lactobacilli in susceptible individuals, while simultaneously promoting an increase in gut γδ T cells and γδ17 T cells. These cells migrate to the meninges, promoting neuropathology and depressive behaviors. Compared to WT mice, TCRδ... - / - Mice and WT mice peripherally injected with anti-TCRδ antibodies blocked CSDS-induced social avoidance and depressive behaviors. In summary, this indicates that γδ T cells and the inflammatory factors they produce are involved in behavioral development in mice.

[0007] (III) γδ T cells play a key role in depression

[0008] Depression is a common mental illness characterized by high morbidity, high disability rate, and high mortality rate. It has become the second leading cause of disease burden globally, second only to cardiovascular disease, imposing a heavy burden on society and the economy. Although the pathological mechanisms of depression have been widely reported, their specific pathogenesis remains incompletely understood. In recent years, the induction of depression by immune dysregulation has received widespread attention. Clinical studies have shown that the proportion of peripheral blood T cells is elevated in patients with depression and is significantly correlated with the severity of depression. Animal experiments have found a significant increase in the proportion of γδ T cell subsets in mice with depression; abnormal T cell activation leads to depressive-like behavior in mice. Therefore, it is evident that dysregulation of T cells and their produced inflammatory factors is closely related to depression.

[0009] Depression is mainly characterized by significant and persistent low mood. It belongs to the category of "depressive syndrome" in traditional Chinese medicine and is a disease caused by emotional discomfort and qi stagnation. The clinical treatment of depression mainly focuses on soothing the liver and relieving depression. Xiao Yao San originated from "Taiping Huimin He Ji Ju Fang" in the Song Dynasty. The whole formula consists of eight traditional Chinese medicines: Bupleurum chinense, Paeonia lactiflora, Angelica sinensis, Atractylodes macrocephala, Poria cocos, roasted Glycyrrhiza uralensis, Zingiber officinale, and Mentha haplocalyx. It has the effects of soothing the liver and relieving depression, strengthening the spleen and regulating the nutrient qi, and is a representative famous formula for soothing the liver, strengthening the spleen, and relieving depression. The effect of Xiao Yao San in treating depression has been widely recognized. The results of clinical trials conducted by multiple teams show that Xiao Yao San has a significant effect in treating patients with mild to moderate depression and fewer adverse reactions; a large number of basic studies indicate that Xiao Yao San can significantly improve the depressive-like behaviors of depressed animals. Clinical data show that compared with the antidepressant treatment of western medicines, Xiao Yao San has a more obvious improvement effect on the HDRS and SDS scale scores.

[0010] The clinical treatment of depression mainly focuses on relieving depressive symptoms, supplemented by social activities and psychological counseling. Antidepressant drug treatment (such as selective serotonin reuptake inhibitors, tricyclic antidepressants, and new drugs, etc.) and psychotherapy are the main treatment methods for depression at present. However, studies have found that clinical antidepressant drugs have a slow onset of action and insufficient efficacy, and have certain adverse reactions on the cardiovascular system, endocrine system, and central nervous system. Exploring the action targets of antidepressant drugs and developing new, highly effective, and safe antidepressant targeted therapy drugs have important scientific significance and clinical application value. Summary of the Invention

[0011] In view of this, the present invention provides the application of anti-VLA4 in inhibiting the migration of γδ T cells to the central system in antidepressant treatment. Using anti-VLA4 antibody treatment to reduce the infiltration of γδ T cells into the meninges and brain parenchyma of mice to treat depression, and anti-VLA4 inhibiting the migration of peripheral T cells can block the depressive-like behaviors induced by CUMS, which can provide a new strategy for treating depression.

[0012] In order to achieve the above invention purpose, the present invention provides the following technical solutions:

[0013] The present invention provides the application of γδ T cells as a target in the preparation of drugs for improving depression.

[0014] In some specific embodiments of the present invention, the drugs in the above application improve depression through any of the following:

[0015] (i), reducing the proportion of γδ T cells in the spleen;

[0016] (ii), reducing the proportion of γδ T cells in the meninges;

[0017] (iii), reducing the proportion of γδ T cells in the brain parenchyma.

[0018] In some specific embodiments of the present invention, the drug described above improves depression by inhibiting the migration of γδ T cells to the central nervous system.

[0019] In some specific embodiments of the present invention, the drug described above inhibits the migration of γδ T cells to the central nervous system by inhibiting VLA4.

[0020] In some specific embodiments of the present invention, the target of the above application includes VLA4 expressed by γδ T cells.

[0021] In some specific embodiments of the present invention, the drug used above reduces serum pro-inflammatory cytokine levels.

[0022] In some specific embodiments of the present invention, the serum pro-inflammatory cytokines used above include at least one of IL-1β, IL-6, MIP-1α, and MIP-1β.

[0023] The present invention also provides the use of VLA4 antagonists in the preparation of medicaments for improving depression.

[0024] In some specific embodiments of the present invention, the VLA4 antagonist used above includes an anti-VLA4 antibody.

[0025] In some specific embodiments of the present invention, the drug described above improves depression by any of the following:

[0026] (I) Reduce the proportion of γδ T cells in the meninges;

[0027] (II) Reduce the proportion of γδ T cells in the brain parenchyma.

[0028] In some specific embodiments of the present invention, the drug described above inhibits the migration of γδ T cells to the central nervous system by inhibiting VLA4.

[0029] The present invention also provides a method for treating depression, including therapy that reduces γδ T cells.

[0030] In some specific embodiments of the present invention, the reduction of the γδ T cell ratio in the above method includes any of the following:

[0031] (i) Reduce the proportion of γδ T cells in the spleen;

[0032] (ii) Reduce the proportion of γδ T cells in the meninges;

[0033] (iii) Reduce the proportion of γδ T cells in the brain parenchyma.

[0034] In some specific embodiments of the present invention, the above method includes treating depression by inhibiting the migration of γδ T cells to the central nervous system.

[0035] In some specific embodiments of the present invention, the above method includes improving depression by blocking VLA4 to inhibit the migration of γδ T cells to the central nervous system.

[0036] In some specific embodiments of the present invention, the above method includes administering a VLA4 antagonist.

[0037] In some specific embodiments of the present invention, the VLA4 antagonist of the above method includes an anti-VLA4 antibody.

[0038] In some specific embodiments of the present invention, the above method includes the step of temporarily or permanently knocking out γδ T cells of the patient.

[0039] The application of the present invention has the following effects:

[0040] The present invention reveals that: in the study of a mouse model of depression induced by chronic unpredictable mild stress (CUMS), the mice showed depressive-like behaviors such as reduced activity in the central area of the open field, reduced sugar water intake, and prolonged immobile time in the forced swimming and tail suspension swimming tests. And CUMS induced an increase in the proportion of peripheral and central γδ T cells in the mice. Treatment with Xiaoyao San can improve the depressive behaviors of the mice, reduce the proportion of γδ T cells, and further improve neuroinflammation. Further, using γδ T cell knockout (TCRδ - / - ) mice, it was found that mice lacking γδ T cells did not show depressive behaviors after CUMS modeling. At the same time, using neutralizing antibodies to deplete peripheral γδ T cells and subjecting them to CUMS modeling, the mice also did not show depressive behaviors. Further, after in vitro re-infusion of γδ T cells from WT mice into TCRδ - / - mice, the mice showed depressive behaviors. It indicates that γδ T cells promote the occurrence of depressive behaviors, and Xiaoyao San plays an antidepressant role by inhibiting γδ T cells. Experiments show that after treatment with VLA4 neutralizing antibody, the sugar water preference rate of CUMS mice was significantly increased, and the immobile time in the tail suspension test and forced swimming test was significantly reduced, indicating that anti-VLA4 significantly improved the depressive-like behaviors of CUMS mice. Compared with control mice, the proportion of γδ T cells in the spleen, meninges and brain parenchyma of CUMS mice was significantly increased, and injection of anti-VLA4 antibody could significantly inhibit the proportion of γδ T cells, indicating that anti-VLA4 plays an antidepressant role by inhibiting the migration of γδ T cells to the central nervous system. Description of the Drawings

[0041] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0042] Figure 1 The study investigated the effect of Xiaoyao San on CUMS-induced depressive behavior. A shows the movement trajectory of mice during the Open Field Test (OFT) as displayed by video tracking software; B to D show the open field test results, with B representing the total movement distance of the mice; C representing the time spent in the central zone; D representing the number of times the mice entered the central zone; E showing the data from the Sugar Water Preference Test (SPT); and F showing the immobility time of mice in the last 4 minutes of the Forced Swimming Test (FST). XYS represents Xiaoyao San; ns indicates no difference; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0043] Figure 2 The results showed that Xiaoyao San reduced the proportion of γδ T cells in the spleen of CUMS mice. In Figure A, flow cytometry analysis showed changes in CD4+ and CD8+ T cells in the spleen; in Figure B, flow cytometry analysis showed changes in the proportion of γδ T cells in the spleen. XYS represents Xiaoyao San; ns indicates no difference; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0044] Figure 3 The study showed that Xiaoyao San reversed the increase in the proportion of γδ T cells in the spleen and the increase in IFN-γ levels derived from γδ T cells in CUMS mice. In Figure A, flow cytometry was used to detect the levels of IFN-γ and IL-17 produced by CD4+ T cells in the spleen; in Figure B, flow cytometry was used to detect the levels of IFN-γ and IL-17 produced by γδ T cells in the spleen. XYS represents Xiaoyao San; ns indicates no difference; * indicates P < 0.05; ** indicates P < 0.01.

[0045] Figure 4 The effects of Xiaoyao San on γδ T cells in the meninges and brain tissue are shown in Figure A, where TCRδ / CD4-labeled γδ T cells in brain tissue are displayed; Figure B shows the proportion of γδ T cells in mouse brain tissue as detected by flow cytometry; Figure C shows TCRδ / CD3-labeled γδ T cells in the meninges; and Figure D shows the proportion of γδ T cells in mouse meninges as detected by flow cytometry. XYS represents Xiaoyao San; ns indicates no difference; * indicates P < 0.05.

[0046] Figure 5The effect of Xiaoyao San on the expression levels of inflammatory factors in the serum of CUMS mice was shown in Figure A. A shows the detection of inflammatory factors in mouse serum, with different colors representing different protein levels (red to blue indicating high to low levels). Figure B shows the effect of Xiaoyao San on inflammatory factors in the serum of CUMS mice. The expression levels of IFN-γ, IL-1β, IL-6, RANTES, MIP-1α, and MIP-1β were significantly upregulated in CUMS mice, and these changes were reversed after intervention with Xiaoyao San. XYS represents Xiaoyao San; ns indicates no difference; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0047] Figure 6 This study demonstrates the important role of γδ T cells in CUMS-induced depressive behavior. A shows the experimental flowchart; B to D show the open field test; B represents the total movement distance of the mice; C represents the number of times they entered the central zone; D represents the time spent in the central zone; E shows the data statistics of the sucrose preference test; F shows the immobility time of mice in the last 4 minutes of the tail suspension test (TST); G shows the immobility time of mice in the last 4 minutes of the forced swimming test (FST); NT indicates no treatment; ns indicates no difference; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0048] Figure 7 The behavioral evaluation of mice after γδ T cell reinfusion is shown in Figure A, where A shows the experimental flowchart, B shows the data statistics of the sucrose preference test, C shows the immobility time of mice in the last 4 minutes of the forced swimming test, D shows the immobility time of mice in the last 4 minutes of the tail suspension test, and E shows the proportion of γδ T cells in the spleen, blood, and meninges as detected by flow cytometry. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0049] Figure 8 The results show that VLA4 antibody significantly improved depressive-like behavior in CUMS mice. A shows the experimental flowchart; B shows the total distance moved by mice in the open field experiment; C shows the data statistics of the sucrose preference experiment; D shows the immobility time of mice in the last 4 minutes of the forced swimming experiment; E shows the immobility time of mice in the last 4 minutes of the tail suspension experiment. * indicates P < 0.05; ** indicates P < 0.01.

[0050] Figure 9 VLA4 antibody inhibits the migration of peripheral γδ T cells to the central nervous system. In this study, A shows the detection of γδ T cells in the spleen, meninges, and brain parenchyma by flow cytometry; B shows the proportion of γδ T cells in the spleen; C shows the proportion of γδ T cells in the meninges; and D shows the proportion of γδ T cells in the brain parenchyma. * indicates P < 0.05; ** indicates P < 0.01. Detailed Implementation

[0051] This invention discloses the application of VLA4 in antidepressant therapy by inhibiting the migration of γδ T cells to the central nervous system. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired effect. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0052] This invention proposes the application of γδ T cells in inducing depression. In a mouse model of chronic unpredictable mild stress (CUMS) depression, mice exhibited significant depressive-like behavior, and the proportion of γδ T cells in the peripheral and central tissues of CUMS mice was significantly increased. Using γδ T cell knockout (TCRδ)... - / - After establishing the CUMS model in mice, no depressive behavior was observed; further analysis of TCRδ... - / - When γδ T cells from WT mice were reinfused into mice in vitro, the mice exhibited significant depressive behavior, suggesting that γδ T cells could serve as a target for the treatment of depression.

[0053] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0054] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0055] It should be understood that the order of steps or the sequence of actions is not important as long as this application remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0056] The use of any and all instances or exemplary language such as “e.g.” or “include” in this document is intended merely to better illustrate the application and does not constitute a limitation on the scope of the application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of this application.

[0057] Furthermore, the numerical ranges and parameters used to define this application are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise explicitly stated, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately." Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0058] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in this invention are all commercially available products and can be purchased from the market.

[0059] The present invention will be further illustrated below with reference to the embodiments.

[0060] Example

[0061] 1. Animal grouping and drug intervention

[0062] (1) γδ T cells promote depressive-like behavior: First, in order to clarify the changes in γδ T cells in depressed mice, 8-week-old SPF grade C57BL / 6J male mice were numbered by weight using a random number table and randomly grouped as follows: normal control group: WT mice; model group: WT mice + CUMS.

[0063] Second, to verify the role of γδ T cells in depression, B6.129P2-Tcrdtm1Mom / J, i.e., TCRδ, was used. - / - The mice were grouped as follows: TCRδ - / - Mouse control group: TCRδ - / - Mice; TCRδ - / - Mouse model group: TCRδ - / - Mice + CUMS; γδ T cell reinfusion control group: TCRδ cells infused with PBS - / - Mice + CUMS; WT mouse γδ T cell reinfusion model group: TCRδ of WT mouse γδ T cells reinfused - / - Mice + CUMS. Normal mice had free access to food and water. In the reinfusion group, γδ T cells (1×10⁻⁶) from WT mice were transfused via the tail vein once weekly. 6 Cells) and PBS solution (100 μL) were reinfused into TCRδ - / - Mice, for 4 weeks.

[0064] (2) Anti-VLA4 therapeutic effect: Eight-week-old SPF-grade male C57BL / 6J mice were randomly divided into the following groups: normal control group: WT mice + PBS; model group: WT mice + CUMS + PBS; treatment group: WT mice + CUMS + anti-VLA4 (InVivoMAb anti-mouse / human VLA-4 (CD49d), catalog number: BE0071, brand: BioXCell). During the modeling process, mice were intraperitoneally injected with anti-VLA4 (200 μg) every 3 days. All antibodies were stored in a refrigerator and diluted to their working concentration in PBS. Control group mice were intraperitoneally injected with an equal volume of PBS solution at the same time.

[0065] 2. In vitro culture and reinfusion of γδ T cells

[0066] (1) In vitro expansion and culture of γδ T cells

[0067] Antibody-coated cell culture plates: Take RPMI-1640 medium and add purified anti-mouse TCR Vγ4 or Vγ1 mAb to a final concentration of 10 μg / mL; mix thoroughly and add to 48-well plates, 100 μL / well; seal with sealing film and incubate at 37°C for 2 hours or at 4°C overnight.

[0068] Preparation of spleen single-cell suspension: Fresh spleens from wild-type mice were placed in sterile RPMI-1640 medium (containing 100 units of penicillin, 100 μg of streptomycin, and 50 μM of β-ME) and transferred to sterile culture dishes in a clean bench in the cell culture room. The spleen was gently ground onto a sterile glass slide and added to the culture medium. The suspension was filtered through a 40 μm Cell Strainer into a 15 mL centrifuge tube and centrifuged at 400g for 7 minutes. The supernatant was discarded. The pellet was resuspended in erythrocyte lysis buffer and incubated at room temperature for 5 minutes to fully lyse the erythrocytes. 10 mL of culture medium was added to stop erythrocyte lysis. After mixing, the suspension was filtered through a 40 μm Cell Strainer into a new 15 mL centrifuge tube and centrifuged at 400g for 7 minutes. The supernatant was discarded. The pellet was resuspended in 10 mL of erythrocyte lysis buffer and incubated at room temperature for 5 minutes to fully lyse the erythrocytes. 10 mL of culture medium was added to stop erythrocyte lysis. The suspension was then filtered through a 40 μm Cell Strainer into a new 15 mL centrifuge tube and centrifuged at 400g for 7 minutes. The supernatant was discarded. The pellet was resuspended in 10 mL of erythrocyte lysis buffer and centrifuged at 10 μm of erythrocyte lysis buffer. After resuspending the cells in mL of culture medium, mix thoroughly; count the cells using a cell counting chamber; pipette the appropriate volume of cell suspension according to the required cell number, and centrifuge again; collect the supernatant, resuspend the cells in culture medium containing 10% FBS, and adjust the concentration to 2.5 × 10⁻⁶ cells / mL. 6 Cells / mL, store at 4°C in a refrigerator or on ice for later use to maintain cell viability.

[0069] Culture of γδ T cell subsets: Purified anti-mouse CD28 (1 μg / mL) and rmIL-2 (2 ng / mL) were added to the cell suspension; the antibody coating in the 48-well plates was removed; after thorough mixing of the cell suspension, 500 μL / well was added to each 48-well plate and incubated in a 37°C water-jacketed CO2 incubator, designated as day 0; after 48 hours, all cells were collected, centrifuged at 400g for 7 minutes at 4°C, and the supernatant was discarded; the pellet was resuspended in fresh medium containing 10% FBS and rmIL-2 (2 ng / mL) and cultured again; thereafter, the medium was replaced with fresh medium every 1.5 days for expansion culture until day 6. Typically, significantly activated γδ T cells appear on days 3-4 of culture, after which the cell concentration should be adjusted to 0.7 × 10⁻⁶ cells / well. 6 ~1×10 6 cells / mL. On day 6, each spleen typically yields approximately 1 × 10⁶ cells / mL. 8 Activated spleen cells, of which approximately 20%–60% are corresponding Vγ4 or Vγ1 γδ T cells.

[0070] γδ T cell reinfusion: Purified WT mouse γδ T cells (1×10⁻⁶) were resuspended in sterile PBS solution. 6 (cells / mL), TCRδ was anesthetized by ether inhalation. - / - In mice, γδ T cells from WT mice were reinfused into TCRδ cells via tail vein injection. - / - In mice.

[0071] 3. Behavioral testing of changes in mouse behavior

[0072] Depressive behavior in mice was assessed using the open field test, sucrose preference test, forced swimming test, and tail suspension test, all conducted in the same room under the same lighting and temperature. The behavioral tests were recorded by a camera, and the data were analyzed using a small animal behavior tracking and analysis system (NOLDUS EthoVision XT, Netherlands). 1) Open field test (OFT): Measured the total distance moved by the mouse within 5 minutes to evaluate its motor ability. 2) Forced swimming test (FST): Measured the immobility time of the mouse after swimming for 5 minutes, followed by 4 minutes. 3) Sucrose preference test (SPT): Measured the preference rate of the mouse for drinking sucrose water within 1 hour. 4) Tail suspension test (TST): Measured the immobility time of the mouse after suspending its tail for 5 minutes, followed by 4 minutes. These behavioral tests were used to evaluate depressive behavior in mice.

[0073] 4. Establishment of a mouse model of chronic unpredictable mild stress (CUMS)

[0074] One to two different mild stressors were applied daily for four weeks to establish the model. The stressors included: reversed day and night, fasting and water restriction (12 hours), 45° tilted cage (3 hours), empty cage (12 hours), damp bedding (21 hours), swimming in ice water (5 minutes), restraint (2 hours), and tail clamping (2 minutes).

[0075] 5. Flow cytometry detection

[0076] (1) Flow cytometry detection of the proportion of γδ T cells in spleen and brain tissue: Fresh spleen and brain tissue of mice were taken and cell suspensions were prepared. The surface was stained with staining solution prepared with fluorescent group antibodies such as anti-CD45, anti-CD3 and anti-TCRδ. After staining, the cells were resuspended in PBS and analyzed by flow cytometry.

[0077] (2) Flow cytometry detection of IFN-γ levels produced by γδ T cells in spleen and brain tissue: Fresh spleen and brain tissue from mice were collected, and cell suspensions were prepared separately. After counting, the cells were resuspended in a culture medium containing 10% FBS to adjust the cell concentration to approximately 5 × 10⁻⁶ cells / mL. 6 Cells / mL were mixed with 50 ng / mL phorbol ester, 1 g / mL iomycin, and Golgi-Plug (containing Brefeldin A, 1:1000), and then added to 48-well plates. The plates were incubated at 37°C for 4–6 h. After stimulation, fluorescently labeled anti-CD45, anti-CD3, anti-TCRδ, and anti-IFN-γ antibodies were added for staining. Cells were resuspended in PBS and analyzed by flow cytometry.

[0078] (3) Flow cytometry detection of meningeal tissue cells and cytokines

[0079] Mice were anesthetized with sodium pentobarbital, and then perfused to collect meningeal tissue from the brain. The meningeal tissue was digested with 1 mg / mL collagenase VIII (containing 10 µg / mL DNase I) in a 37°C water bath for 30 min. The digested suspension was filtered through a 100 µm filter, centrifuged at 300g at 4°C, and the supernatant was discarded. The cells were resuspended in 1 mL of erythrocyte lysis buffer and lysed at room temperature for 5 min. The cells were then filtered again through a 100 µm filter, centrifuged at 300g at 4°C, and the supernatant was discarded. The cells were resuspended in 500 µL of PBS, washed, centrifuged at 1400 rpm, and the supernatant was discarded. The cells were then resuspended in 200 µL of PBS. After flow cytometry antibody staining, the cells were analyzed using a flow cytometer.

[0080] 6. Statistical Analysis

[0081] Each experiment was performed at least three times independently. All experimental data are expressed as mean ± standard error (mean ± SEM) and analyzed using GraphPad Prism. Normality was tested for all experimental data. For normally distributed data, one-way ANOVA was used to analyze differences between groups; for non-normally distributed data, independent samples analysis using nonparametric tests was used. P < 0.05 was considered statistically significant.

[0082] Example of effect

[0083] 1. Xiaoyao San significantly improved depressive behavior in a CUMS-induced depression model mouse.

[0084] The antidepressant effect of Xiaoyao San was evaluated using the CUMS model through open field test, sucrose preference test, and forced swimming test. Results showed that Xiaoyao San effectively increased the time CUMS mice spent in the central region (…). Figure 1 A in Figure 1 (C) Number of times entering the central area ( Figure 1 A in Figure 1 D) and sugar water preference rate ( Figure 1 E), reducing the immobility time of mice during forced swimming ( Figure 1 The F in the formula significantly improved depressive behavior in mice.

[0085] 2. Xiaoyao San reduced the proportion of γδ T cells in the spleen of CUMS mice.

[0086] Immune dysregulation-induced depression has received widespread attention. Clinical studies have shown that the proportion of peripheral blood T cells is elevated in patients with depression, and this elevation is significantly correlated with the severity of depression. Animal experiments have revealed altered T cell responses and changes in the proportion of T cell subsets in mouse models of depression; abnormal T cell activation leads to depressive-like behavior in mice. Flow cytometry analysis of T lymphocytes in the spleen showed that, compared with the control group, the proportion of CD4+ T cells in the spleen of the CUMS group was significantly reduced, while treatment with Xiaoyao San significantly increased the proportion of CD4+ T cells, restoring it to normal levels. Figure 2 (A) Compared with the Control group, there was no significant difference in the proportion of CD8+ T cells in the spleen of the CUMS group ( Figure 2 (A in the text); Compared with the Control group, the proportion of γδ T cells in the spleen of CUMS mice was significantly increased, and Xiaoyao San treatment effectively reversed this change, restoring it to normal levels ( ). Figure 2 (B in the text) indicates that Xiaoyao San may improve depressive behavior in mice by inhibiting γδ T cells. Based on TCR signal intensity during development, γδ T cells differentiate into two major effector subsets according to the types of cytokines they produce: interferon-γ (IFN-γ) and interleukin-17 (IL-17) (γδ T17 cells). Therefore, the changes in IFN-γ and IL-17 derived from γδ T cells were further examined. The results showed no significant difference in IFN-γ and IL-17 production by CD4+ T cells in the spleen of the CUMS group compared to the Control group. Figure 3 (A) Compared with the Control group, the levels of IFN-γ and IL-17 produced by splenic γδ T cells in CUMS mice were significantly increased. Treatment with Xiaoyao San effectively reversed the IFN-γ levels derived from γδ T cells, restoring them to normal levels, but had no effect on IL-17 derived from γδ T cells. Figure 3 (B in the middle).

[0087] 3. Xiaoyao San reversed the increase in the number of γδ T cells in the meninges and brain tissue of CUMS mice.

[0088] Further examination of γδ T cells in the meninges and brain parenchyma of CUMS mice revealed results consistent with those in the peripheral spleen. Compared with the control group, the proportion of γδ T cells in the brain parenchyma and meninges of CUMS mice was significantly increased, and Xiaoyao San treatment reversed this change. Figure 4 This indicates that Xiaoyao San has a good effect on improving depression, and its antidepressant mechanism is closely related to the regulation of peripheral and central γδ T cells.

[0089] 4. Xiaoyao San reduced the expression levels of serum inflammatory factors in CUMS mice.

[0090] To investigate the effects of Xiaoyao San on serum pro-inflammatory cytokines in CUMS mice, serum pro-inflammatory cytokines were detected using a multiplex luminex assay kit. The results showed that serum pro-inflammatory cytokines production was increased in CUMS mice, particularly IFN-γ, IL-1β, IL-6, MIP-1α, and MIP-1β, while treatment with Xiaoyao San restored these levels to normal. Figure 5 ).

[0091] 5. γδ T cells play a key role in depression.

[0092] The above experimental results indicate that γδ T cells are involved in the occurrence of depressive behavior. To further explore the role of γδ T cells in the occurrence of depressive behavior, a mouse model of depression was established using CUMS, and γδ T cell knockout mice (TCRδ) were used. - / - ) and TCRδ - / - Mouse CUMS modeling ( Figure 6 (A) The behavior of mice was evaluated using the OFT, SPT, FST, and TST behavioral tests, respectively. The results showed that compared with control mice, CUMS mice did not show a significant change in movement distance in the open field test, but entered the central area less frequently and spent less time in the central area. Figure 6 B in Figure 6 (D in the text); CUMS mice showed decreased sucrose preference and significantly increased immobility time in forced swimming and tail suspension tests, exhibiting marked depressive-like behavior; while even with combined CUMS modeling after γδ T cell knockout, mice did not exhibit depressive behavior (D in the text). Figure 6 E~ Figure 6 The presence of G in the data indicates that γδ T cells play a crucial role in the development of depressive behavior. To further confirm the role of γδ T cells in depression, γδ T cells (1 × 10⁻⁶) from WT mice were separately analyzed. 6 Cells) and PBS (100 μL) were reinfused into TCRδ - / - mice ( Figure 7 (A) and simultaneously combined with the CUMS model, the behavior of mice was evaluated using OFT, SPT, FST, and TST behavioral tests, respectively. Flow cytometry results showed successful γδ T cell reinfusion ( Figure 7 (E), behavioral experimental results showed that the TCRδ of reinfused γδ T cells - / - Mice showed a lower preference for saccharide solution compared to mice that received PBS. Figure 7 In the B section, the immobility time in forced swimming and tail suspension tests increased ( Figure 7 C and D in the study showed obvious depressive behavior, indicating that γδ T cells play a damaging role in the development of depression.

[0093] 6. Anti-VLA4 antibodies exert their antidepressant effect by inhibiting the migration of γδ T cells.

[0094] Previous experimental results showed that the proportion of γδ T cells in the meninges and brain tissue of mice with depression was increased. Further analysis using TCRδ... - / - Mice have demonstrated that γδ T cells promote the development of depression, suggesting that γδ T cells can serve as a target for antidepressant effects. VLA4 neutralizing antibody treatment can inhibit the migration of T cells to the central nervous system. To investigate whether anti-VLA4 antibodies can improve depressive-like behavior in mice, 8-week-old SPF-grade male C57BL / 6J mice were randomly divided into the following groups: normal control group: WT mice + PBS; model group: WT mice + CUMS + PBS; treatment group: WT mice + CUMS + anti-VLA4. During the modeling process, mice were intraperitoneally injected with anti-VLA4 every 3 days, while control mice were intraperitoneally injected with an equal volume of PBS at the same time. After the modeling was completed, the behavior of the mice was evaluated using OFT, SPT, FST, and TST behavioral tests. Figure 8 (A in the text). The results showed that, compared with the control mice, the CUMS mice did not show a significant change in the distance they moved in the open field experiment, indicating that the mice had normal motor abilities. Figure 8 (B in the text); the sucrose preference rate was reduced in the CUMS group mice ( Figure 8 C), in the forced swimming and tail suspension tests, showed a significant increase in immobility time, exhibiting marked depressive-like behavior. Figure 8 (D and E in the text); while after anti-VLA4 treatment, the sucrose preference rate of mice increased significantly, and the immobility time in the tail suspension test and forced swimming test was significantly reduced (D and E in the text). Figure 8 C- Figure 8 The results (E in the text) indicate that anti-VLA4 treatment significantly improved depressive-like behavior in CUMS mice. Further flow cytometry analysis of the proportion of γδ T cells in the spleen, meninges, and brain parenchyma showed that compared to Control mice, the proportion of γδ T cells in the spleen, meninges, and brain parenchyma of CUMS mice was significantly increased, while injection of VLA4 neutralizing antibody significantly inhibited the proportion of γδ T cells (E in the text). Figure 9 The above experimental results indicate that anti-VLA4 treatment exerts its antidepressant effect by inhibiting the migration of γδ T cells to the central nervous system.

[0095] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of a VLA4 antagonist for the preparation of a medicament for ameliorating CUMS-induced depression, characterized in that, The VLA4 antagonist is an anti-VLA4 antibody.

2. Use according to claim 1, wherein The drug improves depression by any of the following: (I) reducing the proportion of γδ T cells in the meninges; (II) reducing the proportion of γδ T cells in the brain parenchyma.

3. Use according to claim 1 or 2, characterized in that, The drug inhibits the migration of γδ T cells to the central nervous system by blocking VLA4.

Images