Application of anti-VLA4 treatment in resisting depression by inhibiting migration of gamma delta T cells to central system

Abstract

The invention relates to the field of biological medicine, in particular to application of anti-VLA4 treatment in depression resistance by inhibiting gamma delta T cells from being migrated to a central system. The anti-VLA4 antibody is used for treating and reducing infiltration of gamma delta T cells to mouse meninx and brain parenchyma, it is revealed that anti-VLA4 inhibits migration of peripheral T cells and can block depression-like behaviors induced by CUMS, and a new strategy is provided for treating depression.

Description

Technical Field

[0001] The present 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 Art

[0002] 1. Interaction between γδ T cells and the central nervous system

[0003] γδ T cells are distributed in a variety of lymphoid and non-lymphoid tissues and are early responding cells for a variety of diseases. Previous studies have confirmed that they play an important role in infection, cancer, autoimmune diseases, and tissue maintenance. γδ T cells are T lymphocytes that express the γ and δ chains of T cell receptors and constitute the γδ T cell receptor (TCR). Like conventional αβT cells and B cells, γδ T cells use V, D, and J gene rearrangements to express various TCRs for antigen recognition. Although the number of γδ T cells in the blood and lymphoid tissues is small, they are abundant in barrier tissues, and their frequency in the blood increases dramatically during infection. Using 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, intestines, blood, and secondary lymphoid organs, depending on the developmental timing of γδ T cells before and after birth. Depending on the strength of TCR signaling during development, γδ T cells differentiate into two major effector subsets based on the type of cytokines produced: interferon-γ (IFN-γ) and interleukin 17 (IL-17) (γδ T17 cells). γδ T cells can participate in immediate immune responses because of their direct antigen recognition, widespread distribution, multiple ligands for γδ TCR, and expression of innate receptors. In fact, a large body of evidence suggests that γδ T cells play a key role in infection, tumors, autoimmunity, and immune surveillance.

[0004] Over the past few decades, the central nervous system (CNS) has traditionally been considered to be immune-privileged because it is protected by the blood-brain barrier (BBB), which is characterized by low expression of leukocyte adhesion molecules and tight junctions between brain capillary endothelial cells. However, increasing evidence suggests that the CNS and the immune system can directly interact. Studies have reported that meningeal T cells have been shown to secrete interleukin 4 (IL-4), interleukin 13 (IL-13), and IFN-γ, which play important roles in learning, long-term memory, and social behavior. More importantly, new evidence suggests 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 neuronal-immune interactions.

[0005] 2. γδ T cells play an important role in neurological diseases

[0006] Two outstanding studies by the Ribot team and the Kipnis team found 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 showed impaired short-term memory in the Y-maze and Morris water maze tests. γδ T cell-derived IL-17 can regulate the expression of neurotrophic factor (BDNF) in the hippocampus, which can regulate the synaptic plasticity of short-term memory-related neurons. On the other hand, compared with WT mice, TCRδ - / - Mice and WT mice injected with anti-TCRδ antibodies in their cerebrospinal fluid (CSF) showed reduced anxiety levels in the elevated plus maze test and open field, indicating that meningeal γδ17 T cells control the occurrence of anxiety-like behaviors. Together, these data suggest that meningeal γδT17 cells play a key role in short-term memory and anxiety-like behaviors. Recently, studies have shown that long-term stress can lead to intestinal dysbiosis and a decrease in the number of lactic acid bacteria in susceptible individuals, while promoting an increase in intestinal γδ T cells and γδ17 T cells, which migrate to the meninges and promote the occurrence of neuropathology and depressive behaviors. Compared with WT mice, TCRδ - / - Mice and WT mice injected with anti-TCRδ antibodies peripherally could block CSDS-induced social avoidance and depressive behaviors. In summary, it was shown that γδ T cells and the inflammatory factors they produced were involved in the behavior of mice.

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

[0008] Depression is a common clinical mental illness with high morbidity, high disability rate and high mortality rate. Depression has become the second largest burden disease in the world after cardiovascular disease, bringing a heavy burden to society and the economy. Although the pathological mechanism of depression has been widely reported, its specific pathogenesis has not yet been fully elucidated. In recent years, immune disorder-induced depression has received widespread attention. Clinical studies have shown that the proportion of peripheral blood T cells in patients with depression is increased and significantly correlated with the severity of depression. Animal experiments have found that the proportion of γδ T cell subpopulations in depression model mice is significantly increased; abnormal T cell activation leads to depressive-like behavior in mice. It can be seen that the disorder of T cells and the inflammatory factors they produce is closely related to depression.

[0009] Depression is characterized by a significant and persistent low mood. It belongs to the category of "depression" in traditional Chinese medicine. It is a disease caused by emotional discomfort and qi stagnation. The clinical treatment of depression is mainly to soothe the liver and relieve depression. Xiaoyaosan comes from the "Taiping Huimin Hejijufang" of the Song Dynasty. The whole prescription is composed of eight Chinese herbs: Bupleurum, White Peony, Angelica, Atractylodes, Poria, Zhigancao, Ginger, and Mint. It has the effects of soothing the liver and relieving depression, strengthening the spleen and nourishing the body. It is a representative prescription for soothing the liver, strengthening the spleen and relieving depression. The effect of Xiaoyaosan in treating depression has been widely recognized. The results of clinical trials conducted by multiple teams showed that Xiaoyaosan has a significant effect on treating patients with mild to moderate depression, and has fewer adverse reactions; a large number of basic studies have shown that Xiaoyaosan can significantly improve the depressive-like behavior of depressed animals. Clinical data show that compared with Western medicine antidepressant treatment, Xiaoyaosan has a more obvious improvement effect on HDRS and SDS scale scores.

[0010] The clinical treatment of depression is mainly to relieve depressive symptoms, supplemented by social activities and psychological counseling. Antidepressant drug therapy (such as selective serotonin reuptake inhibitors, tricyclic antidepressants and new drugs, etc.) and psychotherapy are currently the main treatments for depression. However, studies have found that clinical antidepressants are slow to take effect, have insufficient efficacy, and have certain adverse reactions to the cardiovascular system, endocrine system and central nervous system. Exploring the targets of antidepressant drugs and developing new, efficient and safe antidepressant targeted therapeutic drugs have important scientific significance and clinical application value. Summary of the invention

[0011] In view of this, the present invention provides an application of anti-VLA4 in inhibiting the migration of γδ T cells to the central nervous system in anti-depression, 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 CUMS-induced depressive-like behavior, which can provide a new strategy for treating depression.

[0012] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

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

[0014] In some specific embodiments of the present invention, the above-mentioned drug improves depression by any of the following:

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

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

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

[0018] In some specific embodiments of the present invention, the drug used 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 used above inhibits the migration of γδ T cells to the central nervous system by resisting VLA4.

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

[0021] In some specific embodiments of the present invention, the above-mentioned drug reduces the level of serum pro-inflammatory cytokines.

[0022] In some specific embodiments of the present invention, the serum proinflammatory 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 the VLA4 antagonist in preparing a medicine for improving depression.

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

[0025] In some specific embodiments of the present invention, the above-mentioned drug 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 used above inhibits the migration of γδ T cells to the central nervous system by resisting VLA4.

[0029] The present invention also provides a method for treating depression, comprising reducing γδ T cell therapy.

[0030] In some specific embodiments of the present invention, the reducing the proportion of γδ T cells in the above method comprises 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 comprises inhibiting the migration of γδ T cells to the central nervous system to treat depression.

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

[0036] In some embodiments of the invention, the above methods comprise administering a VLA4 antagonist.

[0037] In some specific embodiments of the present invention, the VLA4 antagonist of the above method comprises 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 the patient's γδ T cells.

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

[0040] The present invention discloses that in a study of a mouse model of depression induced by chronic unpredictable mild stress (CUMS), mice exhibited depressive-like behaviors such as decreased activity in the central area of ​​the open field, decreased sugar intake, and prolonged immobility in forced swimming and tail-suspended swimming, and CUMS induced an increase in the proportion of peripheral and central γδT cells in mice. Treatment with Xiaoyaosan can improve the depressive behavior of mice, reduce the proportion of γδ T cells, and thus improve neuroinflammation. Further, using γδ T cell knockout (TCRδ - / - ) mice found that mice lacking γδ T cells did not show depressive behavior after CUMS modeling. At the same time, neutralizing antibodies were used to deplete peripheral γδ T cells, and mice did not show depressive behavior after CUMS modeling. - / - After the mice were infused with WT mouse γδ T cells in vitro, the mice showed depressive behavior, indicating that γδ T cells promote the occurrence of depressive behavior, and Xiaoyaosan exerts antidepressant effects by inhibiting γδ T cells. The experiment showed that after treatment with VLA4 neutralizing antibodies, the sugar water preference rate of CUMS mice increased significantly, and the immobility time in the tail suspension test and forced swimming test was significantly reduced, indicating that anti-VLA4 significantly improved the depressive-like behavior of CUMS mice. Compared with control mice, the proportion of γδ T cells in the spleen, meninges and brain parenchyma of CUMS mice increased significantly, and the injection of anti-VLA4 antibodies could significantly inhibit the proportion of γδ T cells, indicating that anti-VLA4 exerts antidepressant effects by inhibiting the migration of γδ T cells to the central nervous system. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0042] Figure 1 Figure 4 shows the effect of Xiaoyaosan on CUMS-induced depressive behavior, where A shows the movement trajectory of mice in the open field test (OFT) displayed by video tracking software, B to D show the open field test, B shows the total movement distance of mice, C shows the residence time in the central area, D shows the number of times entering the central area, E shows the data statistics of the sugar preference test (SPT), F shows the immobility time of mice in the last 4 min of the forced swimming test (FST), XYS indicates Xiaoyaosan; ns indicates no difference; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001;

[0043] Figure 2 The results showed that Xiaoyaosan reduced the proportion of γδ T cells in the spleen of CUMS mice, where A showed the changes of splenic CD4+T cells and CD8+T cells detected by flow cytometry, and B showed the changes of splenic γδ T cells detected by flow cytometry, XYS indicated Xiaoyaosan; ns indicated no difference; * indicated P < 0.05; ** indicated P < 0.01; *** indicated P < 0.001;

[0044] Figure 3 The results showed that Xiaoyaosan reversed the increase in the proportion of γδ T cells in the spleen of CUMS mice and the increase in the level of IFN-γ derived from γδ T cells, where A showed the levels of IFN-γ and IL-17 produced by splenic CD4+T cells detected by flow cytometry, and B showed the levels of IFN-γ and IL-17 produced by splenic γδ T cells detected by flow cytometry, XYS indicated Xiaoyaosan; ns indicated no difference; * indicated P < 0.05; ** indicated P < 0.01;

[0045] Figure 4 The figure shows the effect of Xiaoyaosan on γδ T cells in meninges and brain tissues, where A shows γδ T cells marked by TCRδ / CD4 in brain tissues, B shows the proportion of γδ T cells in mouse brain tissues detected by flow cytometry, C shows γδ T cells marked by TCRδ / CD3 in meninges, and D shows the proportion of γδ T cells in mouse meninges detected by flow cytometry. XYS indicates Xiaoyaosan; ns indicates no difference; * indicates P < 0.05;

[0046] Figure 5Figure 2 shows the effect of Xiaoyaosan on the expression level of serum inflammatory factors in CUMS mice, where A shows the detection of serum inflammatory factors in mice, different colors represent different protein contents, from red to blue represents from high to low, B shows the effect of Xiaoyaosan 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 in CUMS mice were significantly upregulated, and these changes were reversed after Xiaoyaosan intervention, XYS represents Xiaoyaosan; ns represents no difference; * represents P < 0.05; ** represents P < 0.01; *** represents P < 0.001;

[0047] Figure 6 The results show that γδ T cells play an important role in CUMS-induced depressive behavior, where A shows the experimental flow chart, B to D show the open field test, B shows the total movement distance of the mouse, C shows the number of times entering the central area, D shows the residence time in the central area, E shows the data statistics of the sugar water preference test, F shows the immobility time of the mouse in the last 4 min in the tail suspension test (TST), G shows the immobility time of the mouse in the last 4 min in the forced swimming test (FST), NT means no treatment; ns means no difference; * means P < 0.05; ** means P < 0.01; *** means P < 0.001;

[0048] Figure 7 Figure 2 shows the behavioral evaluation of mice after γδ T cell infusion, where A shows the experimental flow chart, B shows the data statistics of the sugar water preference experiment, C shows the immobility time of mice in the last 4 min in the forced swimming test, D shows the immobility time of mice in the last 4 min in the tail suspension test, and E shows the proportion of γδ T cells in the spleen, blood and meninges 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 the depressive-like behavior of CUMS mice, where A shows the experimental flow chart, B shows the total movement distance of mice in the open field test, C shows the data statistics of the sugar water preference test, D shows the immobility time of mice in the last 4 minutes of the forced swimming test, and E shows the immobility time of mice in the last 4 minutes of the tail suspension test. * indicates P < 0.05; ** indicates P < 0.01;

[0050] Fig. 9 The results show that VLA4 antibody inhibits the migration of peripheral γδ T cells to the central nervous system, where 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 DESCRIPTION

[0051] The present invention discloses the application of VLA4 in antidepressant by inhibiting the migration of γδ T cells to the central nervous system. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0052] The present invention proposes the use of γδ T cells in inducing depression. In the chronic unpredictable mild stress (CUMS) depression mouse model, the mice showed obvious depressive-like behavior, and the proportion of γδ T cells in the peripheral and central tissues of CUMS mice increased significantly. - / - ) mice constructed CUMS model did not show depressive behavior; further investigation of TCRδ - / - After mice were infused with WT mouse γδ T cells in vitro, the mice showed significant depressive behavior. γδ T cells can be used as a target for treating depression.

[0053] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

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

[0055] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the application is still operable. In addition, two or more steps or actions can be performed simultaneously.

[0056] The use of any and all examples or exemplary language such as "for example" or "including" herein is intended only to better illustrate the present application and is not intended to limit the scope of the present application. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the present application.

[0057] In addition, the numerical ranges and parameters used to define the present application are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally refers to the actual value 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 the present invention are all common commercial products and can be purchased from the market.

[0059] The present invention will be further described below in conjunction with 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 mice with depression, 8-week-old SPF-level C57BL / 6J male mice were numbered according to their weight using a random number table and randomly divided into the following groups: 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 - / - Mice were grouped as follows: TCRδ - / - Mouse control group: TCRδ - / - Mouse; TCRδ - / - Mouse model group: TCRδ - / - Mice + CUMS; γδ T cell infusion control group: TCRδ infused with PBS - / - Mice + CUMS; WT mouse γδ T cell infusion model group: TCRδ of WT mouse γδ T cells infused - / - Mice + CUMS. The mice in the normal group were free to eat and drink. The mice in the re-infusion group were injected with γδ T cells (1×10 6 cells) and PBS solution (100 μL) were returned to TCRδ - / - Mice, for 4 weeks.

[0064] (2) Anti-VLA4 therapeutic effect: 8-week-old SPF-level C57BL / 6J male 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 (InVivoMAbanti-mouse / human VLA-4 (CD49d), Catalog No.: 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 in PBS to their working concentrations. The control group mice were intraperitoneally injected with an equal volume of PBS solution at the same time.

[0065] 2. In vitro culture and transfusion 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; after thorough mixing, add to a 48-well plate, 100 μL / well; after sealing with sealing film, place in a 37°C incubator for 2 hours, or incubate in a 4°C refrigerator overnight.

[0068] Preparation of spleen single cell suspension: fresh spleen of wild-type mice was taken, placed in sterile RPMI-1640 medium (containing 100 units of penicillin, 100 μg of streptomycin, and 50 μM of β-ME), and transferred to a sterile culture dish in the cell room clean bench; the spleen was gently ground into the culture medium with a sterile glass slide; it was filtered into a 15 mL centrifuge tube through a 40 μm Cell Strainer, centrifuged at 400 g for 7 minutes, and the supernatant was discarded; the precipitate was resuspended with red blood cell lysis buffer, and allowed to stand at room temperature for 5 minutes to fully lyse the red blood cells; 10 mL of culture medium was added to terminate the red blood cell lysis, and after mixing, it was filtered into a new 15 mL centrifuge tube through a 40 μm Cell Strainer; after centrifugation at 400 g for 7 minutes, the supernatant was discarded, and the precipitate was resuspended with 10 After resuspending in 100 mL of culture medium, mix thoroughly; count with a cell counting plate; pipette the corresponding volume of cell suspension according to the required number of cells, centrifuge again; take the supernatant and resuspend the cells in culture medium containing 10% FBS to adjust the concentration to 2.5×10 6 cells / mL and place in a 4°C refrigerator or on ice for later use to maintain cell activity.

[0069] Culture of γδ T cell subsets: Add purified anti-mouse CD28 (1 μg / mL) and rmIL-2 (2 ng / mL) to the cell suspension; remove the coated antibody in the 48-well plate; after the cell suspension is fully mixed, add it to the 48-well plate, 500 μL / well, and place it in a 37℃ water-jacketed CO2 incubator for culture, which is set as day 0; after 48 hours, collect all the cells, centrifuge at 4℃ 400g for 7 minutes, and discard the supernatant; resuspend the precipitate in fresh culture medium containing 10% FBS and rmIL-2 (2 ng / mL) and continue to culture; thereafter, replace the fresh culture medium every 1.5 days for expanded culture until the 6th day. Usually, obviously activated γδ T cells begin to appear on the 3rd to 4th day of culture, and the cell concentration must be adjusted to 0.7×10 6 ~1×10 6 On day 6, approximately 1×10 cells / mL can be obtained from each spleen. 8 Activated spleen cells, of which approximately 20% to 60% are corresponding Vγ4 or Vγ1 γδ T cells.

[0070] γδ T cell transfusion: Resuspend the purified WT mouse γδ T cells (1×10 6 cell / mL), and TCRδ was anesthetized by ether inhalation - / - Mice were injected with γδ T cells from WT mice into TCRδ - / - In mice.

[0071] 3. Behavioral testing of mouse behavioral changes

[0072] The open field test, sucrose preference test, forced swimming test and tail suspension test were used to detect the depressive behavior of mice. All tests were carried out in the same room, under the same lighting and temperature. The behavioral test process was recorded by a camera, and all behavioral test data were analyzed by the small animal behavior trajectory tracking and analysis system (NOLDUS EthoVision XT, the Netherlands). 1) Open field test (OFT): The total movement distance of mice within 5 min was tested to evaluate the exercise ability of mice. 2) Forced swimming test (FST): The immobility time of mice within 4 min after swimming for 5 min. 3) Sucrose preference test (SPT): The preference rate of mice to drink sucrose water within 1 h was tested. 4) Tail suspension test (TST): The immobility time of mice within 4 min after tail suspension for 5 min was tested. The above behavioral tests were used to evaluate the depressive behavior of mice.

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

[0074] One to two different mild stressors were given every day for 4 weeks. The stressors included: day and night reversal, fasting from food and water (12 h), 45° tilted cage (3 h), empty cage (12 h), wet bedding (21 h), ice water swimming (5 min), restraint (2 h) and tail clamping (2 min).

[0075] 5. Flow cytometry

[0076] (1) Detection of the proportion of γδ T cells in spleen and brain tissue by flow cytometry: Fresh spleen and brain tissue of mice were obtained and prepared into cell suspensions respectively. Surface staining was performed using 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 to detect the level of IFN-γ produced by γδ T cells in spleen and brain tissue: Fresh spleen and brain tissue of mice were obtained and prepared into cell suspensions. After counting, the cells were resuspended in a culture medium containing 10% FBS and the cell concentration was adjusted to about 5×10 6 cells / mL, add 50 ng / mL phorbol ester, 1 g / mL ionomycin, Golgi-Plug (containing BrefeldinA, 1:1000), mix well and add to 48-well plate, stimulate in 37℃ incubator for 4~6h. After stimulation, add fluorescently labeled anti-CD45, anti-CD3, anti-TCRδ, anti-IFN-γ and other flow cytometry antibodies for staining, resuspend the cells in PBS, and analyze by flow cytometry.

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

[0079] Mice were anesthetized with sodium pentobarbital, and then perfused. The meningeal tissue of the brain was taken and placed in a bacterial culture dish. The meningeal tissue was digested with 1 mg / mL collagenase VIII (containing 10 µg / mL DNase I) and digested in a 37°C water bath for 30 min. The digested suspension was filtered with a 100 µm filter, centrifuged at 300g at 4°C, and the supernatant was discarded. 1 mL of red blood cell lysis buffer was added to resuspend the cells, and the red blood cells were lysed at room temperature for 5 min. The cells were filtered again with a 100 µm filter, centrifuged at 300g at 4°C, and the supernatant was discarded. 500 µL PBS was added to resuspend and wash the cells, centrifuged at 1400 rpm, and the supernatant was discarded. 200 µL PBS was added to resuspend. After flow antibody staining, the cells were detected on a flow cytometer.

[0080] 6. Statistical Analysis

[0081] Each experiment was repeated at least 3 times or more independently. All experimental data were expressed as mean ± SEM, and GraphPad Prism was used to analyze the experimental data. All experimental data were tested for normality. One-way ANOVA was used to analyze the differences between groups for experimental data that met the normal distribution. Nonparametric independent sample analysis was used for experimental data that did not meet the normal distribution. P < 0.05 was considered statistically significant.

[0082] Effect example

[0083] 1. Xiaoyaosan significantly improved the depressive behavior of CUMS-induced depression model mice

[0084] Based on the CUMS model, the antidepressant effect of Xiaoyaosan was evaluated by open field test, sugar water preference test and forced swimming test. The results showed that Xiaoyaosan effectively increased the time CUMS mice stayed in the central area ( Figure 1 A in Figure 1 C in the above), the number of times entering the central area ( Figure 1 A in Figure 1 D) and sugar water preference rate ( Figure 1 E), reduced immobility time in mice during forced swimming ( Figure 1 F), significantly improved the depressive behavior of mice.

[0085] 2. Xiaoyaosan reduces the proportion of γδ T cells in the spleen of CUMS mice

[0086] Immune disorders induce depression and have received widespread attention. Clinical studies have shown that the proportion of peripheral blood T cells in patients with depression is increased and significantly correlated with the severity of depression. Animal experiments have found that the T cell response of depression model mice has changed, and the proportion of T cell subsets has changed; abnormal T cell activation leads to depressive-like behavior in mice. Flow cytometry was used to detect changes in T lymphocytes in the spleen. The results showed that compared with the Control group, the proportion of CD4+T cells in the spleen of the CUMS group was significantly reduced, while Xiaoyaosan treatment significantly increased the proportion of CD4+T cells and restored it to normal levels ( Figure 2 A in the figure); 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 figure); Compared with the Control group, the proportion of γδ T cells in the spleen of CUMS mice increased significantly, and Xiaoyaosan treatment effectively reversed this change and restored it to normal levels ( Figure 2 B in the figure) indicates that Xiaoyaosan may improve the depressive behavior of mice by inhibiting γδ T cells. According to the strength of TCR signals during development, γδ T cells differentiate into two main effector subsets according to the type of cytokines produced: 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 detected. 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 3 A in the figure); compared with the Control group, the levels of IFN-γ and IL-17 produced by spleen γδ T cells in CUMS mice were significantly increased, and Xiaoyaosan treatment effectively reversed the level of IFN-γ derived from γδ T cells and restored it to normal levels, but had no effect on IL-17 derived from γδ T cells ( Figure 3 B in the figure).

[0087] 3. Xiaoyaosan reversed the increase in the number of γδ T cells in the meninges and brain tissues of CUMS mice

[0088] Further detection of γδ T cells in the meninges and brain parenchyma of CUMS mice showed consistent results 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 increased significantly, and Xiaoyaosan treatment reversed this change ( Figure 4 ), indicating that Xiaoyaosan has a good effect in improving depression, and its antidepressant mechanism is closely related to the regulation of peripheral and central γδ T cells.

[0089] 4. Xiaoyaosan reduces the expression level of serum inflammatory factors in CUMS mice

[0090] In order to study the effect of Xiaoyaosan on serum proinflammatory cytokines in CUMS mice, multiplex luminex kit was used to detect serum proinflammatory cytokines in mice. The results showed that the production of serum proinflammatory cytokines in CUMS mice increased, especially IFN-γ, IL-1β, IL-6, MIP-1α and MIP-1β, while Xiaoyaosan treatment 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 γδ T cells are involved in the occurrence of depressive behavior. In order to further explore the role of γδ T cells in the occurrence of depressive behavior, CUMS was used to establish a depression mouse model, γδ T cell knockout mice (TCRδ - / - ) and TCRδ - / - Mouse composite CUMS model ( Figure 6 The behavior of mice was evaluated by OFT, SPT, FST and TST behavioral tests. The results showed that compared with the control mice, the movement distance of CUMS mice in the open field test did not change significantly, the number of times they entered the central area decreased and the time they stayed in the central area was also reduced ( Figure 6 B~ Figure 6 D in the figure); CUMS mice showed a decreased preference for sugar water, and significantly increased immobility time in the forced swimming test and tail suspension test, showing obvious depressive-like behavior; however, after knocking out γδ T cells, mice did not show depressive behavior even when combined with CUMS modeling ( Figure 6 E~ Figure 6 G in the figure), indicating that γδ T cells play a key role in the occurrence of depressive behavior. To further confirm the role of γδ T cells in depression, γδ T cells (1×10 6 cells) and PBS (100 μL) were then infused back into TCRδ - / - Mouse ( Figure 7 A in the figure), and the CUMS model was combined, and the behavior of mice was evaluated by OFT, SPT, FST and TST behavioral tests. Flow cytometry results showed that the γδ T cell infusion was successful ( Figure 7 E), behavioral experiments showed that the TCRδ of γδ T cells was injected back into the body. - / - The preference rate of mice for sugar water decreased compared with the PBS injection ( Figure 7 B), increased immobility time in the forced swim and tail suspension tests ( Figure 7 C and D), showing obvious depressive behavior, indicating that γδ T cells play a damaging role in the occurrence of depression.

[0093] 6. Anti-VLA4 antibodies exert antidepressant effects 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 tissues of depression model mice increased. - / - Mice have demonstrated that γδ T cells promote the occurrence of depression, indicating that γδ T cells can be used as an antidepressant target. VLA4 neutralizing antibody treatment can inhibit the migration of T cells to the central nervous system. In order to study whether anti-VLA4 antibodies can improve the depressive-like behavior of mice, 8-week-old SPF-level C57BL / 6J male 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, and mice in the control group were intraperitoneally injected with an equal volume of PBS at the same time. After the modeling, the behavior of the mice was evaluated using OFT, SPT, FST and TST behavioral tests ( Figure 8 The results showed that compared with the control mice, the movement distance of the CUMS mice in the open field test did not change significantly, indicating that the mice had normal motor ability ( Figure 8 B); The sucrose preference rate of mice in the CUMS group was reduced ( Figure 8 C), the immobility time in the forced swimming test and the tail suspension test increased significantly, showing obvious depressive-like behavior ( Figure 8 D and E in the figure); after anti-VLA4 treatment, the mice's sugar water preference rate increased significantly, and the immobility time in the tail suspension test and forced swimming test was significantly reduced ( Figure 8 C- Figure 8 E); indicating that anti-VLA4 treatment significantly improved the depressive-like behavior of CUMS mice. Flow cytometry was used to detect the proportion of γδ T cells in the spleen, meninges and brain parenchyma. The results showed that compared with Control mice, the proportion of γδ T cells in the spleen, meninges and brain parenchyma of CUMS mice was significantly increased, and the injection of VLA4 neutralizing antibodies could significantly inhibit the proportion of γδ T cells ( Fig. 9 ). The above experimental results indicate that anti-VLA4 treatment exerts antidepressant effects by inhibiting the migration of γδ T cells to the central nervous system.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of γδ T cells as targets in the preparation of drugs to improve depression.

2. The use according to claim 1, characterized in that The drug improves depression by any of the following: (i) Reduce the proportion of γδ T cells in the spleen; (ii) reduce the proportion of γδ T cells in the meninges; (iii) Reduce the proportion of γδ T cells in the brain parenchyma.

3. The use according to claim 1 or 2, characterized in that: The drug improves depression by inhibiting the migration of γδ T cells to the central nervous system.

4. The use according to claim 3, characterized in that The drug inhibits the migration of γδ T cells into the central nervous system by blocking VLA4.

5. The use according to any one of claims 1 to 4, characterized in that: The targets include VLA4 expressed by γδ T cells.

6. Use of VLA4 antagonists in the preparation of drugs for improving depression.

7. The use according to claim 6, characterized in that The VLA4 antagonists include anti-VLA4 antibodies.

8. The use according to claim 6 or 7, characterized in that The drug improves depression by any of the following: (I) Reduce the proportion of γδ T cells in the meninges; (II) Reduce the proportion of γδ T cells in the brain parenchyma.

9. The use according to any one of claims 6 to 8, characterized in that The drug inhibits the migration of γδ T cells into the central nervous system by blocking VLA4.

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