Use of Anti-VLA4 therapy in antidepressant treatment by means of inhibiting γδt cell migration to central nervous system

Abstract

The present invention relates to the field of biomedicine, and particularly to the use of an anti-VLA4 therapy in antidepressant treatment by means of inhibiting γδT cell migration to the central nervous system. In the present invention, the anti-VLA4 antibody therapy is used to reduce γδT cell infiltration into the meninges and brain parenchyma of mice, revealing that the inhibition of peripheral T cell migration by anti-VLA4 can block CUMS-induced depressive-like behaviors, which provides a new strategy for treating depression.

Description

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

[0001] This application claims priority to Chinese Patent Application No. 202411807096.2, filed on December 10, 2024, entitled "Application of Anti-VLA4 Therapy in Antidepressant Treatment by Inhibiting the Migration of γδT Cells to the Central Nervous System", the entire contents of which are incorporated herein by reference. Technical Field

[0002] 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 γδT cells to the central nervous system. Background Technology

[0003] (I) Interaction between γδT cells and the central nervous system

[0004] γδT cells are distributed in various lymphatic and non-lymphatic tissues and are early-responding cells in many diseases. Previous studies have confirmed their important roles in infection, cancer, autoimmune diseases, and tissue maintenance. γδT cells are T lymphocytes that express the γ and δ chains of T cell receptors and constitute the γδT cell receptor (TCR). Like conventional αβT cells and B cells, γδT cells utilize V, D, and J gene rearrangements to express various TCRs for antigen recognition. Although γδT 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 γδTCR chain, different subsets of γδT cells reside in the meninges, skin, lungs, liver, peritoneum, adipose tissue, uterus, tongue, intestine, blood, and secondary lymphatic organs, depending on the developmental sequence of γδT cells before and after birth. Based on the intensity of TCR signaling during development, γδT cells differentiate into two major effector subsets according to the types of cytokines they produce: interferon-γ (IFN-γ) and interleukin-17 (IL-17) (γδT17 cells). γδT cells can participate in immediate immune responses because they possess direct antigen recognition, widespread distribution, multiple ligands for the γδTCR, and expression of innate receptors. In fact, substantial evidence suggests that γδT cells play a crucial role in infection, tumorigenesis, autoimmunity, and immune surveillance.

[0005] 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.

[0006] (II) γδT cells play an important role in nervous system diseases

[0007] Two outstanding studies by Ribot's and Kipnis's teams have revealed that meningeal γδT cells can secrete IL-17 to regulate short-term memory and anxiety-like behavior. Mice lacking γδT cells and IL-17 exhibited impaired short-term memory in the Y-maze and Morris water maze tests. γδT 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 test and open field test, 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 lactobacillus count in susceptible individuals, while simultaneously promoting an increase in gut γδT cells and γδ17T 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.

[0008] (III) γδT cells play a key role in depression

[0009] Depression is a common mental disorder clinically, characterized by high prevalence, high disability rate, and high mortality rate. Currently, there are approximately 350 million depression patients globally, and up to 700,000 patients commit suicide due to depression every year. Depression has become the second-largest burden disease globally after cardiovascular diseases, bringing a heavy burden to society and the economy. Although the pathological mechanisms of depression have been widely reported, its specific pathogenesis has not been fully elucidated yet. In recent years, the induction of depression by immune dysregulation has received extensive attention. Clinical studies have shown that the proportion of peripheral blood T cells in depression patients increases, and it is significantly correlated with the severity of depression. Animal experiments have found that the proportion of γδT cell subsets in depression model mice increases significantly; abnormal activation of T cells leads to depressive-like behaviors in mice. Thus, the imbalance of T cells and the inflammatory factors they produce is closely related to depression.

[0010] Depression is mainly clinically characterized by significant and persistent low mood, belonging to the category of "depressive syndrome" in traditional Chinese medicine, which is a disease syndrome caused by emotional discomfort and qi stagnation. The clinical treatment of depression mainly focuses on soothing the liver and relieving depression. Xiaoyao San originated from "Taiping Huimin Heji Jufang" in the Song Dynasty. The whole formula consists of eight traditional Chinese medicines: Bupleurum chinense, Paeonia lactiflora, Angelica sinensis, Atractylodes macrocephala, Poria cocos, roasted licorice, ginger, and Mentha haplocalyx, and has the effects of soothing the liver and relieving depression, strengthening the spleen and harmonizing the nutrient qi. It is a representative famous formula for soothing the liver, strengthening the spleen, and relieving depression. The effect of Xiaoyao San in treating depression has been widely recognized. The results of clinical trials carried out by multiple teams show that Xiaoyao San has a significant effect in treating patients with mild to moderate depression and fewer adverse reactions; a large number of basic studies have shown that Xiaoyao San can significantly improve the depressive-like behaviors of depressed animals. Clinical data show that compared with the antidepressant treatment of western medicines, Xiaoyao San has a more obvious improvement effect on the HDRS and SDS scale scores.

[0011] 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 currently. However, studies have found that clinical antidepressant drugs have a slow onset 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 efficient, and safe antidepressant targeted therapeutic drugs have important scientific significance and clinical application value. Summary of the Invention

[0012] In view of this, the present invention provides the application of anti-VLA4 in inhibiting the migration of γδT cells to the central system in antidepressant treatment. Using anti-VLA4 antibody treatment to reduce the infiltration of γδT 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.

[0013] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0014] This invention provides the application of γδT cells as a target in the preparation of drugs to improve depression.

[0015] In some specific embodiments of the present invention, the drug described above improves depression by any of the following:

[0016] (i) Reduce the proportion of γδT cells in the spleen;

[0017] (ii) Reduce the proportion of γδT cells in the meninges;

[0018] (iii) Reduce the proportion of γδT cells in the brain parenchyma.

[0019] In some specific embodiments of the present invention, the drug described above improves depression by inhibiting the migration of γδT cells to the central nervous system.

[0020] In some specific embodiments of the present invention, the drug described above inhibits the migration of γδT cells to the central nervous system by inhibiting VLA4.

[0021] In some specific embodiments of the present invention, the target of the above application includes VLA4 expressed by γδT cells.

[0022] In some specific embodiments of the present invention, the drug used above reduces serum pro-inflammatory cytokine levels.

[0023] 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.

[0024] The present invention also provides the use of VLA4 antagonists in the preparation of medicaments for improving depression.

[0025] In some specific embodiments of the present invention, the VLA4 antagonist used above includes an anti-VLA4 antibody.

[0026] In some specific embodiments of the present invention, the drug described above improves depression by any of the following:

[0027] (I) Reduce the proportion of γδT cells in the meninges;

[0028] (II) Reduce the proportion of γδT cells in the brain parenchyma.

[0029] In some specific embodiments of the present invention, the drug described above inhibits the migration of γδT cells to the central nervous system by inhibiting VLA4.

[0030] The present invention also provides a method for treating depression, including reducing γδT cell therapy.

[0031] In some specific embodiments of the present invention, the reduction of the proportion of γδT cells in the above method includes any of the following:

[0032] (i) Reducing the proportion of γδT cells in the spleen;

[0033] (ii) Reducing the proportion of γδT cells in the meninges;

[0034] (iii) Reducing the proportion of γδT cells in the brain parenchyma.

[0035] In some specific embodiments of the present invention, the above method includes inhibiting the migration of γδT cells to the central nervous system for treating depression.

[0036] In some specific embodiments of the present invention, the above method includes improving depression by blocking VLA4 to inhibit the migration of γδT cells to the central nervous system.

[0037] In some specific embodiments of the present invention, the above method includes administering a VLA4 antagonist.

[0038] In some specific embodiments of the present invention, the VLA4 antagonist in the above method includes an anti-VLA4 antibody.

[0039] In some specific embodiments of the present invention, the above method includes the step of temporarily or permanently knocking out γδT cells of the patient.

[0040] The application of the present invention has the following effects:

[0041] 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 immobility time in forced swimming and tail suspension swimming, and CUMS induced an increase in the proportion of peripheral and central γδT cells in the mice. Treatment with Xiaoyaosan can improve the depressive behaviors of the mice, reduce the proportion of γδT cells, and further improve neuroinflammation. Further, using γδT cell knockout (TCRδ - / - ) mice, it was found that mice lacking γδT cells did not show depressive behaviors after CUMS modeling. At the same time, using neutralizing antibodies to deplete peripheral γδT cells and subjecting the mice to CUMS modeling, the mice also did not show depressive behaviors; further, for TCRδ - / -In vitro infusion of γδT cells from WT mice into mice resulted in depressive behavior, indicating that γδT cells promote depressive behavior, while Xiaoyao San exerts its antidepressant effect by inhibiting γδT cells. Experiments showed that treatment with VLA4 neutralizing antibodies significantly increased the sucrose preference rate in CUMS mice and significantly reduced immobility time in the tail suspension and forced swimming tests, indicating that anti-VLA4 significantly improved depressive-like behavior in CUMS mice. Compared with control mice, the proportion of γδT cells in the spleen, meninges, and brain parenchyma of CUMS mice was significantly increased, while injection of anti-VLA4 antibodies significantly inhibited the proportion of γδT cells, suggesting that anti-VLA4 exerts its antidepressant effect by inhibiting the migration of γδT cells to the central nervous system. Attached Figure Description

[0042] Figure 1 shows the effect of Xiaoyao San on CUMS-induced depressive behavior. A shows the movement trajectory of mice in 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 represents the dwell time in the central zone; D represents the number of times the mice entered the central zone; E shows the data statistics of the sugar water preference test (SPT); F shows 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 shows that Xiaoyao San reduced the proportion of γδT cells in the spleen of CUMS mice. In the figure, A shows the changes in CD4+ T cells and CD8+ T cells in the spleen as detected by flow cytometry, and B shows the changes in the proportion of γδT cells in the spleen as detected by flow cytometry. XYS represents Xiaoyao San; ns indicates no difference; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001.

[0044] Figure 3 shows that Xiaoyao San reversed the increase in the proportion of γδT cells in the spleen and the increase in IFN-γ levels derived from γδT cells in CUMS mice. In the figure, A shows the levels of IFN-γ and IL-17 produced by CD4+ T cells in the spleen as detected by flow cytometry, and B shows the levels of IFN-γ and IL-17 produced by γδT cells in the spleen as detected by flow cytometry. XYS represents Xiaoyao San; ns indicates no difference; * indicates P<0.05; ** indicates P<0.01.

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

[0046] Figure 5 shows the effect of Xiaoyao San on the expression levels of inflammatory factors in the serum of CUMS mice. A shows the detection of inflammatory factors in mouse serum, with different colors representing different protein levels, from red to blue representing high to low levels. 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 Xiaoyao San intervention. XYS represents Xiaoyao San; ns indicates no difference; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.

[0047] Figure 6 shows the important role of γδT 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 shows the behavioral evaluation of mice after γδT cell reinfusion. 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; E shows the proportion of γδT 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 shows that VLA4 antibody significantly improved depressive-like behavior in CUMS mice. In the figure, A shows the experimental flowchart, B shows the total movement distance of 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, and 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 shows that VLA4 antibody inhibits the migration of peripheral γδT cells to the central nervous system. In the figure, A shows the γδT cells detected by flow cytometry in the spleen, meninges and brain parenchyma; B shows the proportion of γδT cells in the spleen; C shows the proportion of γδT cells in the meninges; and D shows the proportion of γδT 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 γδT 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 γδT 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 γδT cells in the peripheral and central tissues of CUMS mice was significantly increased. Using γδT cell knockout (TCRδ)... - / - After establishing the CUMS model in mice, no depressive behavior was observed; further analysis of TCRδ... - / - When γδT cells from WT mice were reinfused into mice in vitro, the mice exhibited significant depressive behavior, suggesting that γδT 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 may 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 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 an actual value within plus or minus 10%, 5%, 1%, or 0.5% of a particular 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) γδT cells promote depressive-like behavior: First, in order to clarify the changes in γδT 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; γδT cell reinfusion control group: TCRδ cells infused with PBS - / - Mice + CUMS; WT mouse γδT cell reinfusion model group: TCRδ of WT mouse γδT cells reinfused - / - Mice + CUMS. Normal mice had free access to food and water. In the reinfusion group, γδT cells (1×10⁻⁶) from WT mice were transfused via 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), product 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 γδT cells

[0066] (1) In vitro expansion and culture of γδT 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 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 400 g for 7 minutes. The supernatant was discarded. The pellet was resuspended in erythrocyte lysis buffer and allowed to stand 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. After centrifugation at 400 g for 7 minutes, the supernatant was discarded. The pellet was resuspended in 10 mL of culture medium and mixed thoroughly. Cells were counted using a cell counting chamber. The appropriate volume of cell suspension was aspirated according to the required number of cells and centrifuged again. The supernatant was collected and refluxed with a solution containing 10%... Resuspend the cells in FBS medium and adjust the concentration to 2.5 × 10⁻⁶. 6 Cells / mL, store at 4°C in a refrigerator or on ice for later use to maintain cell viability.

[0069] Culture of γδT 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 γδT 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 Vγ1γδ T cells.

[0070] γδT cell reinfusion: Purified WT mouse γδT cells (1×10⁻⁶) were resuspended in sterile PBS solution. 6 TCRδ (cells / mL) was anesthetized by ether inhalation. - / - In mice, γδT 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 mice within 5 minutes to evaluate their motor ability. 2) Forced swimming test (FST): Measured the immobility time of the mice after 4 minutes of swimming within 5 minutes. 3) Sucrose preference test (SPT): Measured the preference rate of mice for drinking sucrose water within 1 hour. 4) Tail suspension test (TST): Measured the immobility time of the mice after 4 minutes of tail suspension within 5 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 cycle, 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 to detect the proportion of γδT cells in spleen and brain tissue: Fresh spleen and brain tissue from mice were prepared into cell suspensions. The cells were stained with staining solutions prepared using fluorescent 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 γδT 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 CUMS-induced depression model mice.

[0084] Based on the CUMS model, the antidepressant effect of Xiaoyao San was evaluated through open field test, sucrose preference test, and forced swimming test. The results showed that Xiaoyao San effectively increased the time spent in the central region of CUMS mice (Figure 1A, Figure 1C), the number of times mice entered the central region (Figure 1A, Figure 1D), and the sucrose preference rate (Figure 1E), and reduced the immobility time of mice in forced swimming (Figure 1F), significantly improving the depressive behavior of 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 is significantly correlated with the severity of depression. Animal experiments have found that T cell responses and the proportion of T cell subsets change in mice with 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 CUMS mice was significantly reduced, while treatment with Xiaoyao San significantly increased the proportion of CD4+ T cells, restoring it to normal levels (Figure 2A). Compared with the control group, there was no significant difference in the proportion of CD8+ T cells in the spleen of CUMS mice (Figure 2A). However, compared with the control group, the proportion of γδ T cells in the spleen of CUMS mice was significantly increased, and treatment with Xiaoyao San effectively reversed this change, restoring it to normal levels (Figure 2B). This suggests that Xiaoyao San may improve depressive behavior in mice by inhibiting γδ T cells. Based on the TCR signal intensity during development, γδT cells differentiate into two main effector subsets according to the types of cytokines they produce: interferon-γ (IFN-γ) and interleukin-17 (IL-17) (γδT17 cells). Therefore, the changes in IFN-γ and IL-17 derived from γδT cells were further examined. The results showed that compared with the Control group, there was no significant difference in IFN-γ and IL-17 produced by CD4+ T cells in the spleen of the CUMS group (Figure 3A); compared with the Control group, the levels of IFN-γ and IL-17 produced by γδT cells in the spleen of CUMS mice were significantly increased, and Xiaoyao San treatment effectively reversed the IFN-γ level derived from γδT cells, restoring it to normal levels, but had no effect on IL-17 derived from γδT cells (Figure 3B).

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

[0088] Further examination of γδT 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 γδT cells in the brain parenchyma and meninges of CUMS mice was significantly increased. Xiaoyao San treatment reversed this change (Figure 4), indicating that Xiaoyao San has a good effect on improving depression, and its antidepressant mechanism is closely related to the regulation of peripheral and central γδT 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 kit. The results showed that the production of serum pro-inflammatory cytokines in CUMS mice was increased, particularly IFN-γ, IL-1β, IL-6, MIP-1α, and MIP-1β, while treatment with Xiaoyao San restored them to normal levels (Figure 5).

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

[0092] The above experimental results indicate that γδT cells are involved in the occurrence of depressive behavior. To further explore the role of γδT cells in the occurrence of depressive behavior, a mouse model of depression was established using CUMS, and γδT cell knockout mice (TCRδ-) were used. / -) and TCRδ- / - Mice were modeled using a combined CUMS model (Figure 6, A), and their behavior was evaluated using OFT, SPT, FST, and TST behavioral tests. Results showed that compared to Control mice, CUMS mice did not show significant changes in movement distance in the open field test, but the number of times they entered the central area and the duration of their stay in the central area were reduced (Figure 6, B-D). CUMS mice exhibited a decreased sugar water preference rate and significantly increased immobility time in the forced swimming and tail suspension tests, demonstrating marked depressive-like behavior. However, even with combined CUMS modeling, mice with knocked-out γδT cells did not exhibit depressive behavior (Figure 6, E-G), indicating that γδT cells play a crucial role in the development of depressive behavior. To further confirm the role of γδT cells in depression, γδT cells (1×10⁻⁶) from WT mice were... 6 Cells) and PBS (100 μL) were reinfused into TCRδ- / Mice (A in Figure 7), combined with the CUMS model, had their behavior evaluated using OFT, SPT, FST, and TST behavioral tests. Flow cytometry results showed successful γδT cell reinfusion (E in Figure 7), and behavioral experiments showed that the TCRδ of the reinfused γδT cells was... - / - Compared with PBS infusion, mice showed a lower sugar water preference rate (Figure 7, B) and increased immobility time in forced swimming and tail suspension experiments (Figure 7, C and D), exhibiting obvious depressive behavior, indicating that γδT cells play a damaging role in the development of depression.

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

[0094] Previous experimental results showed that the proportion of γδT cells in the meninges and brain tissue of mice with depression was increased. Further analysis using TCRδ... - / -Mice have demonstrated that γδT cells promote the development of depression, suggesting that γδT 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). The results showed that compared with control mice, CUMS mice did not show significant changes in movement distance in the open field test, indicating normal motor ability (Figure 8, B). CUMS mice exhibited decreased sucrose preference (Figure 8, C) and significantly increased immobility time in the forced swimming and tail suspension tests, demonstrating marked depressive-like behavior (Figure 8, D and E). However, after anti-VLA4 treatment, the sucrose preference significantly increased, and the immobility time in the tail suspension and forced swimming tests significantly decreased (Figure 8, C-E). This indicates that anti-VLA4 treatment significantly improved the depressive-like behavior in CUMS mice. Further flow cytometry analysis of the proportion of γδT cells in the spleen, meninges, and brain parenchyma revealed a significantly increased proportion of γδT cells in the spleen, meninges, and brain parenchyma compared to control mice. Injection of VLA4 neutralizing antibodies significantly inhibited the proportion of γδT cells (Figure 9). These experimental results demonstrate that anti-VLA4 treatment exerts its antidepressant effect by inhibiting the migration of γδT 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. Application of γδT cells as a target in the preparation of drugs to improve depression.

2. The application as described in claim 1, characterized in that, The drug improves depression by any of the following: (i) Reduce the proportion of γδT cells in the spleen; (ii) Reduce the proportion of γδT cells in the meninges; (iii) Reduce the proportion of γδT cells in the brain parenchyma.

3. The application as described in claim 1 or 2, characterized in that, The drug improves depression by inhibiting the migration of γδT cells to the central nervous system.

4. The application as described in claim 3, characterized in that, The drug inhibits the migration of γδT cells to the central nervous system by blocking VLA4.

5. The application as described in any one of claims 1 to 4, characterized in that, The target includes VLA4 expressed on γδT cells.

6. Application of VLA4 antagonists in the preparation of drugs to improve depression.

7. The application as described in claim 6, characterized in that, The VLA4 antagonist includes an anti-VLA4 antibody.

8. The application as described in claim 6 or 7, characterized in that, The drug improves depression by any of the following: (I) Reduce the proportion of γδT cells in the meninges; (II) Reduce the proportion of γδT cells in the brain parenchyma.

9. The application as described in any one of claims 6 to 8, characterized in that, The drug inhibits the migration of γδT cells to the central nervous system by blocking VLA4.

10. A method for improving depression, characterized in that, Treatment targeting γδT cells and / or using VLA4 antagonists.

11. The method as described in claim 10, characterized in that, Improve depression by any of the following: (i) Reduce the proportion of γδT cells in the spleen; (ii) Reduce the proportion of γδT cells in the meninges; (iii) Reduce the proportion of γδT cells in the brain parenchyma.

12. The method as described in claim 10, characterized in that, It can improve depression by inhibiting the migration of γδT cells to the central nervous system.

13. The method as described in claim 10, characterized in that, Improving depression by blocking VLA4 to inhibit the migration of γδT cells to the central nervous system.

14. The method as described in claim 10, characterized in that, The target includes VLA4 expressed on γδT cells.

15. The application as described in claim 1 or 6, or the method as described in claim 10, characterized in that, The depression mentioned is CUMS-induced depression.

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