Application of splenic T lymphocyte exosomes in establishing animal models of depression

Abstract

This invention provides the use of splenic T lymphocyte exosomes in establishing an animal model of depression. Exosomes from splenic T lymphocytes of stressed rats are injected into mice to establish an animal model of depression. This animal model of depression can be used for drug screening, efficacy evaluation, or diagnosis of depression, as well as for studying and revealing the pathogenesis of depression. The invention also experimentally demonstrates the important role of the IL4 / IL4Rα signaling pathway and the BDNF signaling pathway in depression. This invention has important implications for clinical research on depression.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a method for constructing a non-human animal model of depression. Background Art

[0002] Major depressive disorder (MDD) is a significant global health challenge, affecting approximately 20% of the population and ranking among the leading causes of disability. Looking ahead, the World Health Organization (WHO) projects an even more alarming outlook, predicting that by 2030, MDD will rise to become the leading cause of global disease burden. These statistics highlight a pressing issue: even with significant research efforts and advances in therapeutic interventions, a significant proportion of patients currently treated with antidepressants experience poor responses, with some responding partially or even ineffectively, and experiencing a range of side effects. This situation suggests significant gaps in our understanding of the molecular complexity of MDD.

[0003] The etiology, pathogenesis, and inducing factors of depression remain unclear. Therefore, appropriate animal models of depression are crucial for studying the pathogenesis of depression, screening drugs, and guiding clinical medication. A limitation of animal models is that their cognitive and emotional capacities differ from those of humans. However, in recent years, animal models of depression have been continuously refined, primarily employing stress, medication, and surgery to construct effective and effective animal models of depression, providing insights into the pathogenesis of depression and its clinical treatment.

[0004] Currently, commonly used animal models of depression include chronic stress models, unpredictable stress models, olfactory bulb resection models, post-stroke depression models, and models induced by drugs such as reserpine or glucocorticoids. Different models reflect different causes or mechanisms of depression. However, current animal models of depression still have various unresolved technical issues. For example, the use of chronic stress models can easily lead to animal adaptation; unpredictable stress models require long modeling times and high workloads; surgical models are complex and require certain technical skills from the experimenter; and drug models can lead to false positives due to poor drug specificity or other side effects that can affect subsequent experiments. Summary of the Invention

[0005] In view of this, in order to make up for the deficiencies of the prior art, the present invention is proposed.

[0006] A first aspect of the present invention provides a method for constructing a non-human animal model of depression, comprising administering exosomes of spleen T lymphocytes from a stressed second non-human animal to a first non-human animal.

[0007] In the present invention, descriptions such as "first" and "second" are used to distinguish different individuals, and do not represent the order of precedence, nor do they limit "first" and "second" to different types.

[0008] Furthermore, the depression includes but is not limited to major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia.

[0009] Furthermore, the depression is selected from major depressive disorder and anxious depression.

[0010] For purposes of this invention, a "non-human animal model" refers to a non-human animal that possesses or exhibits characteristics of a disease or condition. Use as an animal model refers to any use of an animal to study a disease or condition, such as to study progression or development or response to new or existing therapies.

[0011] In the present invention, non-human animals include non-human vertebrates, more preferably mammals, which refer to all members of the class Mammalia, such as cattle, horses, pigs, sheep, monkeys, pets (e.g., dogs, cats), or rodents. The term "rodent" refers to any and all members of the phylogenetic class Rodentia (e.g., mice, rats, squirrels, beavers, woodchucks, hamsters, guinea pigs, agouti), including all descendants derived therefrom.

[0012] Furthermore, the mammal is selected from mice and rats.

[0013] Furthermore, the second non-human animal is of the same species or a different species than the first non-human animal.

[0014] Furthermore, the second non-human animal is of a different species than the first non-human animal.

[0015] Furthermore, the first non-human animal includes but is not limited to mice, rats, rabbits, dogs, pigs, monkeys, and sheep.

[0016] Furthermore, the first non-human animal is selected from mice.

[0017] Furthermore, the second non-human animal includes but is not limited to mice, rats, rabbits, dogs, pigs, monkeys, and sheep.

[0018] Furthermore, the second non-human animal is selected from rats.

[0019] In the present invention, the types of stress stimulation may include but are not limited to: cold water immersion, food taboos, odor stimulation, and light cycle changes.

[0020] Furthermore, the method includes injecting spleen T lymphocyte exosomes from rats with chronic unpredictable mild stress into mice to obtain mice that exhibit depressive-like behavior.

[0021] Furthermore, the depressive-like behaviors include but are not limited to excessive sleeping, prolonged immobility, loss of interest, lack of energy, emotional reactivity, lead-like paralysis, depressed mood, and rapid eating.

[0022] Furthermore, injection methods include, but are not limited to, intradermal injection, subcutaneous injection, intraperitoneal injection, intramuscular injection, intravenous injection, and brain injection.

[0023] Furthermore, the injection method is selected from intravenous injection and brain injection.

[0024] Furthermore, the injection site is the tail and the lateral ventricle.

[0025] A second aspect of the present invention provides any of the following methods:

[0026] (1) A method for screening drug candidates for treating depression, the method comprising:

[0027] a) administering the agent to be screened to a non-human animal with depression prepared by the method according to the first aspect of the present invention;

[0028] b) Analyze and evaluate the therapeutic effects of the agents to be screened, and select agents that can significantly improve the depressive-like behavior of the mouse model;

[0029] Further, the drug candidates include, but are not limited to, selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, tricyclic drugs, tetracyclic drugs, serotonin modulators, and stimulants;

[0030] (2) A method for evaluating the efficacy of a drug for treating depression, the method comprising:

[0031] a) administering a drug to a non-human animal with depression prepared by the method according to the first aspect of the present invention;

[0032] b) evaluating the therapeutic effect of the drug on the depression;

[0033] (3) A method for studying the pathogenesis of depression, wherein the method uses a non-human animal with depression prepared by the method described in the first aspect of the present invention to study the pathogenesis of depression;

[0034] (4) A method for regulating IL-4 / IL4Rα expression levels, the method comprising regulating IL-4 / IL4Rα expression levels using stressed exosomes;

[0035] (5) A method for regulating NFIX expression levels, the method comprising regulating NFIX expression levels using stress exosomes;

[0036] (6) A method for regulating BDNF / TrkB expression levels, the method comprising regulating BDNF / TrkB expression levels using stress exosomes.

[0037] In the present invention, treatment refers to the improvement, prevention or reversal of a disease or condition or at least one identifiable symptom thereof. Further, the treatment refers to the improvement, prevention or reversal of at least one measurable physiological parameter associated with the disease or condition to be treated, and the parameter is not necessarily identifiable in mammals or recognized by mammals. Further, the treatment refers to the inhibition or slowing of the disease or disease process, and this inhibition or slowing can be physical, such as certain identifiable adverse symptoms. The "treatment" used in the present invention covers diseases in mammals, especially humans, including: (a) preventing the occurrence of diseases or conditions in individuals who are susceptible to the disease but have not yet been diagnosed with the disease. (b) Inhibiting the disease, such as blocking the development of the disease. Or (c) Alleviating the disease, such as alleviating the symptoms associated with the disease.

[0038] In the present invention, drug candidates can be obtained from a wide variety of sources, including but not limited to synthetic, naturally occurring or recombinantly produced molecules, including but not limited to selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, tricyclic drugs, tetracyclic drugs, serotonin modulators and stimulants. For example, random and directed synthesis of a variety of organic compounds and biomolecules, or natural compound libraries in the form of bacterial, fungal, plant or animal extracts, or natural or synthetic libraries and compounds modified by conventional chemical, physical or biochemical means, or directed or random chemical modification of known pharmacological agents, such as acylation, alkylation, esterification, amidation, etc., to generate structural analogs.

[0039] Furthermore, the evaluation indicators include but are not limited to animal behavioral tests, neurogenesis performance, and microglial cell status.

[0040] Furthermore, animal behavioral tests include, but are not limited to, tail suspension test, forced swim test, sucrose preference test, Y maze test, water maze test, open field test, elevated plus maze test, rotarod test, and chronic restraint.

[0041] In the present invention, the term "Y-maze test", also known as "YMaze", refers to a behavioral test based on the natural exploratory curiosity of animals. The spontaneous alternating behavior in this test is considered to reflect short-term spatial working memory. It utilizes the natural tendency of the test animals to explore new environments. During the test, the test animals need to remember the directions they have explored before each time they switch to explore a new direction. Therefore, the Y-maze test can well reflect the spatial working ability of the test animals. The Y-maze test is generally divided into a Y-maze alternating behavior test and an electrical stimulation Y-maze test. In a specific embodiment of the present invention, a Y-maze alternating behavior test is used.

[0042] In the present invention, the term "water maze test", also known as the "Morris water maze" or "MWM", is a test that forces experimental animals (rats and mice) to swim and learn to find a platform hidden in the water. It is mainly used to test the learning and memory ability of experimental animals for sense of spatial position and direction (spatial positioning). It is not only used to study the evaluation of brain region functions related to spatial learning and memory, but is also widely used in scientific research and computer-assisted teaching in multiple disciplines such as learning and memory, Alzheimer's disease, hippocampus / exohippocampus research, intelligence and aging, new drug development / screening / evaluation, pharmacology, toxicology, preventive medicine, neurobiology, animal psychology and behavioral biology. It has been widely recognized in the world and is the preferred classic test for medical schools to carry out behavioral research, especially learning and memory research.

[0043] In the present invention, the term "open field test," also known as the "open box test," is a method for evaluating the autonomous behavior, exploratory behavior, and stress level of test animals in a novel environment. The frequency and duration of certain behaviors of test animals in a novel environment reflect the autonomous and exploratory behavior of the test animals in the unfamiliar environment, while the frequency of urination and defecation reflects their stress level. Currently, the open field test is mainly used to observe the autonomous movement ability, exploratory behavior in a novel environment, stress level, mania, anxiety, and depression of test animals. Due to its simple operation, high feasibility, and accurate data recording, it has become a popular behavioral test method in the field of animal mental research.

[0044] In the present invention, the term "elevated plus maze test", also known as the "EPM test", refers to a test method for evaluating the anxiety response of rodents. Compared with the detection of anxiety behavior in mice caused by noxious stimulation (such as electrical stimulation, noise stimulation, dietary deprivation and exposure to predator odor, etc.), this test has the advantage of simple operation and can intuitively reflect the conditioned response of mice. The EPM test was developed on the basis of the elevated Y-shaped maze. The EPM consists of two open arms and two closed arms, which are crossed in a cross shape. The intersection is the central area, which is at a certain height from the ground. The principle is that when faced with new things (open arms), mice will be curious to explore, while they have a dark nature (closed arms), and a conflicting behavior of exploration and avoidance occurs between the two, resulting in anxiety. The height of the entire maze from the ground is equivalent to that of a human standing on the edge of a cliff, which easily causes animals to feel fear and anxiety. The anxiety behavior of mice can be evaluated by comparing the time and distance the mice stay in the open arms and closed arms.

[0045] In this application, the "tail suspension test," also known as the "TST," is a classic method for rapidly evaluating the efficacy of antidepressants, stimulants, and sedatives. The principle is to utilize the fact that mice, after being suspended by their tails, attempt to escape but are unable to do so, resulting in them giving up their struggle and entering a unique state of depressive immobility. This typical "immobility" reflects a state known as "behavioral despair." This behavioral despair model is similar to depression and is sensitive to most antidepressants. Its efficacy is significantly correlated with clinical efficacy, making it widely used in the initial screening of antidepressants. During the test, the duration of immobility is recorded to reflect the depressive state. Antidepressants and stimulants can significantly shorten or alter this period of immobility.

[0046] As used herein, the "forced swim test," also known as the "FST," is a behavioral despair test that places animals in a confined environment (such as water), where they desperately struggle to escape but are unable to do so. This creates an unavoidable, oppressive environment. After a period of time, the animals exhibit a typical "immobility." Observing and recording a series of parameters during this period can be used to evaluate the effects of depressants and antidepressants. In the forced swim test, the presence of hind limb movement is typically used as a criterion for evaluating the effects of depressants and antidepressants.

[0047] In the present invention, the term "sugar water preference test", also known as "SPT", is a behavioral test widely used to evaluate the emotional state of experimental animals (such as mice and rats). Its basic principle is to evaluate the emotional state of animals by measuring their relative preference for sugar water and ordinary water. It is mainly used to detect the happiness level and depression symptoms of animals. This test is based on an observed phenomenon: healthy animals usually prefer sweet food and drinks, while melancholic or depressed animals may have a reduced preference for sweet substances. SPT can be used as an effective tool to evaluate the depressive symptoms of experimental animals. At the same time, SPT is also widely used to evaluate the efficacy of antidepressant drugs and observe whether the animal's preference for sugar water is restored after drug treatment.

[0048] In the present invention, the term "chronic restraint" is also called "CRS" and "chronic restraint stress test", which refers to placing the test animals in an adjustable cylindrical restraint made of a transparent material with ventilation holes, restraining them for a fixed length of time each day, and depriving them of food and water during the daily stress phase. Chronic restraint can be selected with different time intensity and stress intensity according to the test requirements. The time intensity includes 1, 2, 3, 6 hours per day, etc., and the stress intensity includes 1, 3, 7, 14, 21 and 28 consecutive days. Because the animals are restrained in a confined space for a long time, they initially show anxiety and irritability and try to escape. Eventually, when they feel that there is no hope of escape, they develop symptoms similar to human anhedonia, weight loss, reduced appetite, despair, fatigue, etc., which can be improved by antidepressant drugs, and other behavioral assessment tests are performed subsequently. In a specific embodiment of the present invention, the time intensity of chronic restraint is 6 hours per day for 10 consecutive days. After the model is established, a tail suspension test is performed to test the depressive-like behavior of the mice.

[0049] In this study, the rotarod test assesses an animal's motor coordination and balance by forcing it to run on a rotating, accelerating rod. The animal is placed on a rotating rod with a steady acceleration, and the time it takes to fall off the rod, known as its latency, is measured. This metric can be used to assess an animal's motor learning and coordination abilities, and further analyze the effects of drugs, diseases, or other interventions on its motor function.

[0050] Furthermore, the animal behavior test is selected from the forced swim test, the sugar water preference test, the open field test, and the rotarod test.

[0051] In the present invention, the promoter increases protein expression level or protein activity, and can also increase protein expression level by knock-in.

[0052] Furthermore, the promoter includes but is not limited to nucleic acid molecules, carbohydrates, liposomes, small molecule chemical drugs, antibody drugs, polypeptides, proteins or interfering lentiviruses.

[0053] In the present invention, the term nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). As equivalents, nucleic acids also include DNA or RNA analogs generated from nucleotide analogs and, where applicable, single-stranded (sense or antisense) and double-stranded polynucleotides. Liposomes refer to small vesicles composed of various types of lipids, phospholipids, and / or surfactants that can be used to deliver drugs to mammals. Antibodies include intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, all of which fall within the scope of protection of the present invention as long as they exhibit the desired antigen-binding activity.

[0054] The third aspect of the present invention provides any of the following applications:

[0055] (1) Use of the non-human animal model of depression prepared by the method described in the first aspect of the present invention in screening drug candidates for treating depression;

[0056] (2) Use of the non-human animal model of depression prepared by the method described in the first aspect of the present invention in evaluating the therapeutic effect of drugs for treating depression;

[0057] (3) Application of the non-human animal model of depression prepared by the method described in the first aspect of the present invention in studying the pathogenesis of depression;

[0058] (4) Application of stress exosomes in regulating IL-4 / IL4Rα expression levels;

[0059] (5) Application of IL-4 / IL4Rα signaling pathway promoters in the preparation of drugs for the treatment of depression; (6) Application of IL-4 / IL4Rα in the preparation of drugs for the treatment of depression;

[0060] (7) Application of BDNF / TrkB signaling pathway promoters in the preparation of drugs for the treatment of depression.

[0061] A fourth aspect of the present invention provides the use of splenic T lymphocyte exosomes in constructing a non-human animal model of depression.

[0062] Furthermore, the non-human animal model refers to a non-human animal that has or exhibits characteristics of a disease or condition.

[0063] Furthermore, the non-human animal is a mammal.

[0064] Furthermore, the mammals include but are not limited to mice, rats, rabbits, dogs, pigs, monkeys, and sheep.

[0065] Furthermore, the mammal is selected from mice and rats.

[0066] A fifth aspect of the present invention provides the use of a reagent for detecting NFIX and its expression products in exosomes in the preparation of a product for diagnosing depression.

[0067] Furthermore, the reagents include oligonucleotide probes that specifically recognize NFIX in exosomes, primers that specifically amplify NFIX in exosomes, binding agents that specifically bind to proteins encoded by NFIX in exosomes, or chips that specifically analyze NFIX in exosomes.

[0068] Furthermore, the reagent is selected from primers that specifically amplify NFIX in exosomes.

[0069] Furthermore, the depression includes but is not limited to major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia.

[0070] Furthermore, the depression is selected from major depressive disorder and anxious depression.

[0071] A sixth aspect of the present invention provides a product for diagnosing depression, comprising a reagent capable of detecting the expression level of NFIX in exosomes.

[0072] Furthermore, the product also includes a chip, a kit or a nucleic acid membrane strip.

[0073] Furthermore, the chip includes a gene chip and a protein chip.

[0074] Furthermore, the gene chip includes an oligonucleotide probe targeting NFIX in exosomes for detecting the transcription level of NFIX in exosomes.

[0075] Furthermore, the protein chip includes a specific binding agent for NFIX protein in exosomes.

[0076] Furthermore, the kit includes reagents for detecting the expression level of NFIX gene or protein in exosomes by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, immunoblotting, and mass spectrometry.

[0077] Furthermore, the kit includes instruments or reagents for processing samples.

[0078] Furthermore, the kit also includes instructions or labels, positive controls, negative controls, buffers, adjuvants or solvents; the instructions or labels indicate that the kit is used to detect depression.

[0079] Furthermore, the sample includes but is not limited to cells, tissues, blood, urine, saliva or mucus.

[0080] Furthermore, the sample is selected from blood.

[0081] Furthermore, the depression includes but is not limited to major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia.

[0082] Furthermore, the depression is selected from major depressive disorder and anxious depression.

[0083] The present invention has the following advantages and beneficial effects:

[0084] This invention provides the use of splenic T lymphocyte exosomes in establishing an animal model of depression. Exosomes from splenic T lymphocytes of stressed rats are injected into mice to establish an animal model of depression. This animal model of depression can be used for drug screening, efficacy evaluation, or diagnosis of depression, as well as for studying and revealing the pathogenesis of depression. The invention also experimentally demonstrates the important role of the IL4 / IL4Rα signaling pathway and the BDNF signaling pathway in depression. This invention has important implications for clinical research on depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 Figures 1 and 2 show the experimental results of the effects of exosomes from splenic T lymphocytes of stressed rats on mice. Figure a shows the results of depression-like symptoms induced by injection of CUMS exosomes into healthy mice; Figure b shows the results of the rotarod test in healthy mice injected with CUMS exosomes; Figures c and d show the results of a significant reduction in Doublecortin (DCX)-positive cells after exposure to CUMS exosomes; Figure e shows the results of the exosome contents and depressive characteristics of mice after enzyme treatment; Figure f shows the results of detecting DiI-labeled exosomes in the hippocampus using a two-photon laser scanning microscope; Figures g and h show the results of red fluorescence in the cytoplasm of microglia.

[0086] Figure 2 Figure 1 is the result of CUMS exosomes inducing neuroinflammation in vivo and in vitro, among which, Figure a, Figure b, Figure c, and Figure d are the morphological images of microglia after exposure to CUMS exosomes; Figure e, Figure f, and Figure g are the enrichment pathways of differentially expressed genes IL4, H2-Q2, BDNF, IL10, IL6, Six3, and Tnfsf8 in the CUMS-exosome group and their correlation with the immune system response and neurotrophic factor activity; Figure h is the result of real-time fluorescence quantitative PCR; Figure i is the result of IL4 protein level decreased in the hippocampus after injection of CUMS exosomes; Figure j is the result of IL4Rα protein level decreased in the hippocampus after injection of CUMS exosomes; Figure k is the flow cytometry result of primary microglia; Figure l is the result of CD45 + CD11b +Figure 2 shows the results of microglial cells internalizing DiI-labeled exosomes; Figures m, n, and o show the results of CD45 + CD11b + Figure 2 shows the results of microglial activation; Figure p shows the results of decreased IL4 protein levels after CUMS-exosome treatment; Figure q shows the results of decreased IL4Rα protein levels after CUMS-exosome treatment.

[0087] Figure 3 These are experimental results showing the effects of intracerebroventricular injection of CUMS exosomes on mice, where Figure a shows the depressive-like behavior of mice after intracerebroventricular injection of CUMS exosomes; Figures b, c, d, e, f, and g show the reduced IL4 signaling and neurogenesis, as well as increased microglial activation in the hippocampus.

[0088] Figure 4 Figures 1 and 2 are experimental results showing that IL4 deficiency causes depressive-like symptoms in mice, among which, Figures a and b are detailed proteomic analysis results of exosomes from spleen T lymphocytes of CUMS rats; Figures c, d, and e are results showing that upregulated transcription factors NFIX and NFYC play a role in regulating IL4 expression; Figures f and g are results showing increased NFIX expression in serum exosomes from CUMS-treated rats and mice; Figures h and i are results showing that NFIX levels in serum exosomes or T lymphocyte-derived serum exosomes from patients with major depressive disorder (MDD) were increased before treatment and decreased after treatment; Figure j is results showing that gender does not affect the changes in NFIX levels in serum exosomes or T lymphocyte-derived serum exosomes from patients with major depressive disorder (MDD) after treatment; Figures k, l, m, and n are results showing that injection of IL4 protein into the hippocampus leads to reversal of depressive behavior and neurogenesis damage induced by CUMS exosomes; Figures o, p, q, and r are results showing that IL4 injection changes CUMS- Figure s, t, u, and v show that IL4 alleviates CUMS-exosome-induced microglial activation and restores the IL4 / IL4Rα signaling pathway.

[0089] Figure 5Figure 3 is a structural diagram of the research on the key mechanism of CUMS-exosome-induced depressive-like behavior, among which, Figure a is the result of adeno-associated virus (AAV) increasing IL4 levels; Figure b is the microglial cell-specific result of AAV; Figure c is the result that IL4 overexpression successfully alleviates the depressive symptoms induced by CUMS exosomes, and silencing of IL4Rα in microglial cells in the hippocampus reverses the antidepressant effect of IL4; Figures d, e, f, and g are the results that inhibition of IL4Rα affects the branching and neurogenesis of microglial cells in the hippocampus of AAV-IL4 mice after exposure to CUMS-exosomes; Figure h is the behavioral test result of the healthy control group exosomes having no effect on mice overexpressing IL4 or inhibiting IL4Rα; Figures i and j are the results of using the Cre / Flex system by injecting β-globin-FLEX-EGFP-MIR155 (mcs)-WPRE-PA AAV into CX3CR1 Figure 1 shows the results of knocking down IL4Rα expression in microglia in the hippocampus of Cre mice; Figure k shows that knocking down IL4Rα in microglia in the hippocampus eliminated the antidepressant effect of IL4; Figures l and m show that knocking down IL4Rα in microglia in the hippocampus impairs neurogenesis; Figures n and o show that inhibiting IL4Rα in the hippocampus of CX3CR1 Cre mice effectively offsets the inhibitory effect of IL4 on microglial branching after exposure to CUMS-exosomes.

[0090] Figure 6 Figures 3 and 4 show the results of a study on the mechanism by which the IL4 signaling pathway regulates depressive behavior initiated by CUMS exosomes. Figures a, b, and c show the results of a significantly insufficient expression of BDNF in the hippocampus of mice treated with CUMS exosomes; Figures d and e show that the BDNF-trkb signaling pathway in microglia decreased after exposure to CUMS exosomes, while IL4 treatment restored this decrease; Figures f, g, h, and i show that reducing microglial IL4Rα in the hippocampus offsets the effect of IL4 in enhancing the BDNF-trkb signaling pathway.

[0091] Figure 7Figures a, b, c, d, e, f, g, and h show that depressive behavior was alleviated and defects in neurogenesis and microglial activation were effectively resolved after BDNF protein was injected into the dentate gyrus of the hippocampus; Figures i and j show that the introduction of BDNF normalized the changes in microglial IL-6, IL-10, and iNOS levels induced by CUMS exosomes; Figures k and l show that the BDNF / TrkB signaling pathway in microglial cells damaged by CUMS exosomes was restored after BDNF administration; Figures m, n, and o show that the BDNF / TrkB signaling pathway in microglial cells damaged by CUMS exosomes in vitro was restored after BDNF administration.

[0092] Figure 8 Diagram of the signaling pathway mediating CUMS exosome-induced depressive symptoms in mice.

[0093] Figure 9 Figure 1 is the NFIX diagnostic efficacy verification result diagram, where Figure a is the ROC result diagram of the training set; Figure b is the ROC result diagram of the verification set. DETAILED DESCRIPTION

[0094] The above disclosure generally describes the present invention. A more complete understanding can be obtained by referring to the following specific examples. The purpose of describing these examples is merely to illustrate and is not intended to limit the scope of the present invention. Permutations of form and equivalent substitutions are considered to be circumstances that may suggest or provide convenience. Although specific terms are used herein, the purpose of these terms is descriptive and not for limitation.

[0095] Example 1 Exosomes from T lymphocytes in the spleen of stressed rats induce a depressive-like phenotype in mice

[0096] To minimize animal sacrifice, we obtained spleen exosomes from rats subjected to chronic unpredictable mild stress (CUMS) and healthy control (HC) rats.

[0097] After three weeks of CUMS, the affected rats displayed clear depressive features: compared with controls, they spent less time in the central area of ​​the open field test (OFT), exhibited longer periods of immobility in the forced swim test (FST), and had a reduced preference for sucrose.

[0098] Subsequently, we isolated T lymphocytes from the spleens of the two groups of rats. Flow cytometry confirmed that CD3 + The purity of T lymphocytes was as high as 97.3%.

[0099] After culture, exosomes were extracted from the culture medium. Nanoparticle tracking analysis revealed that the majority of exosome particles had a diameter within a peak range of 100 nanometers, indicating a size consistent with typical exosome characteristics. Western immunoblotting confirmed the presence of exosome-specific proteins, including ALIX, HSP70, CD63, and TSG101. Exosomes were further observed by transmission electron microscopy at ×7000 magnification.

[0100] We evaluated whether exosomes derived from splenic T lymphocytes of stressed rats could affect the depressive-like phenotype of naive animals. Interestingly, administration of CUMS-derived exosomes into healthy mice via tail vein injection induced depressive-like symptoms, similar to the behaviors observed in the OFT, FST, and tail suspension test (TST). Figure 1 a). In contrast, HC-derived exosomes did not induce such depressive-like behaviors ( Figure 1 a). Rotarod test showed that CUMS exosomes did not impair the motor activity of mice ( Figure 1 b), suggesting that the reduced activity observed in OFT reflects a lack of motivation rather than a motor impairment. Furthermore, we found that neither HC nor CUMS exosomes induced a peripheral immune response, as leukocyte and hs-CRP levels remained unchanged.

[0101] Furthermore, given the association between decreased neurogenesis and stress-induced depression, we assessed hippocampal neurogenesis in mice. After exposure to CUMS exosomes, a significant decrease in Doublecortin (DCX)-positive cells was observed in the dentate gyrus ( Figure 1 c, d). To ensure that the observed effects were not due to contaminants co-eluted with exosomes, we treated the exosomes with proteinase K and RNase, as exosomes are known to be resistant to degradation by these enzymes. The results showed that the exosome contents and depressive features of the mice were not affected by enzyme treatment, emphasizing the key role of exosomes in inducing depressive-like symptoms ( Figure 1 e).

[0102] Example 2 CUMS exosomes induce neuroinflammation in vivo and in vitro

[0103] A common concept in neuroscience is that exosomes can cross the blood-brain barrier (BBB) ​​and influence physiological and pathological processes. To track their journey, exosomes were labeled with DiI and then injected into mice via the tail vein for 24 hours. Subsequent two-photon laser scanning microscopy detected these DiI-labeled exosomes in the hippocampus ( Figure 1f). By observing their distribution through confocal microscopy, we found that exosomes were distributed in the dorsal and ventral regions of the hippocampus. Notably, a higher concentration was observed in the dentate gyrus, where there was obvious red fluorescence in the cytoplasm of microglia ( Figure 1 g, h). A closer look at the hippocampus revealed concerning neuroinflammatory signatures after various treatments. After CUMS exosome exposure, microglia exhibited an active morphology, with more cells and shorter branches ( Figure 2 a–d). Interestingly, the behavior, microglial activation, and neurogenesis of mice following CUMS-exosome administration were similar to those of mice directly exposed to CUMS.

[0104] Using RNA sequencing, we delved into the entire transcriptome profile of the hippocampus. The CUMS-exosome group exhibited 76 differentially expressed genes—46 upregulated and 30 downregulated. Crucially, genes such as IL4, H2-Q2, BDNF, and IL10 were downregulated, while IL6, Six3, and Tnfsf8 were enhanced in the CUMS-exosome group. Pathways enriched for these differentially expressed genes were associated with immune system responses and neurotrophic factor activity ( Figure 2 e–g). Real-time quantitative PCR further confirmed these findings, showing that IL4 and IL10 decreased, and iNOS and IL6 increased after CUMS or CUMS-exosome exposure ( Figure 2 h). Given the significant decrease in IL4 mRNA after CUMS-exosome administration—and given the known association between IL4 dysregulation and depression—protein levels of IL4 and its receptor IL4Rα were measured using Western immunoblotting. Both were found to be decreased in the hippocampus after CUMS-exosome administration ( Figure 2 i, j).

[0105] In in vitro experiments, primary microglia were cultured. Flow cytometry confirmed that more than 90% of the cells were CD45 + CD11b + Microglia ( Figure 2 k). After 24 hours of culture, these cells internalized DiI-labeled exosomes, indicating that endocytosis occurred ( Figure 2 l). After treatment with CUMS exosomes, these cells showed significant activation, with increased CD68 expression compared to the control group ( Figure 2 m–o). Similarly, in cultured microglia, the protein levels of IL4 and its receptor IL4Rα decreased after CUMS-exosome treatment ( Figure 2p, q). To verify that the systemic effects of exosome administration stem from their ability to cross the blood-brain barrier and directly affect the CNS, we injected CUMS exosomes into the lateral ventricles of mice. The results of the intracerebroventricular injection study showed that mice receiving CUMS exosomes exhibited depressive-like behaviors ( Figure 3 Furthermore, we observed decreased IL4 signaling and neurogenesis, as well as increased microglial activation in the hippocampus ( Figure 3 bg), which is consistent with the results of peripheral administration of CUMS exosomes.

[0106] Example 3 IL4 deficiency-driven microglial activation: key to depression-like symptoms in mice

[0107] To identify the exosome contents that regulate IL4 expression in the hippocampus, we performed a detailed proteomic analysis of exosomes from splenic T lymphocytes of CUMS rats. This analysis revealed significant changes in the exosome protein profile, with 91 proteins upregulated and 90 proteins downregulated in the exosomes of stressed rats compared to controls ( Figure 4 a, b). These changes suggest that modified exosomal protein content may play a key role in mediating depressive-like behaviors. Further bioinformatics analysis showed that upregulated transcription factors NFIX and NFYC may play a role in regulating IL4 expression ( Figure 4 To confirm this, we performed luciferase assays and showed that overexpression of NFYC or NFIX significantly reduced IL4 expression in HEK 293T and BV2 cells ( Figure 4 d, e). In addition, we found increased NFIX expression in serum exosomes from CUMS-treated rats and mice ( Figure 4 f, g). Importantly, our findings are also applicable to the human setting, as we observed elevated NFIX levels in serum exosomes or T lymphocyte-derived serum exosomes from patients with major depressive disorder (MDD) before treatment and decreased after treatment ( Figure 4 h, i). Notably, sex did not influence these effects ( Figure 4 j). Interestingly, T lymphocyte-derived serum exosomes from MDD patients had a greater effect on upregulating NFIX expression compared to serum exosomes from the same patients. Given that CUMS-exosome injection resulted in a significant decrease in depressive-like behavior and hippocampal IL4 levels, we injected IL4 protein directly into the hippocampus. This intervention resulted in a reversal of the depressive behavior and neurogenesis impairment induced by CUMS exosomes ( Figure 4k-n). IL4 administration also normalized the abnormal levels of IL4, IL-6, IL-10, and iNOS induced by exosomes. Furthermore, IL4 injection altered microglia in the hippocampus of CUMS-exosome-treated mice, resulting in a reduced number of cells and longer branches ( Figure 4 o - r). Supportive in vitro results showed that IL4 alleviated CUMS-exosome-induced microglial activation and restored the IL4 / IL4Rα signaling pathway ( Figure 4 Western blot analysis further confirmed the restoration of IL4 / IL4Rα signaling in the IL4-treated group compared with the CUMS exosome group ( Figure 4 u, v). To assess the direct role of microglial activation in the development of depressive-like behaviors induced by CUMS-exosomes, we used minocycline to inhibit microglial activation. Our analyses showed that treatment of exosomes with minocycline did not result in significant changes in protein or RNA concentrations, suggesting that minocycline did not significantly affect the exosome cargo itself. However, minocycline significantly alleviated exosome-associated depressive symptoms. Furthermore, minocycline reduced microglial hyperactivation and promoted neurogenesis in the hippocampus of CUMS-exosome-treated mice. These findings strongly suggest that microglial activation triggered by IL4 deficiency in the hippocampus is a key factor in the CUMS-exosome-induced depressive behaviors.

[0108] Example 4 IL4 / IL4Rα signaling in hippocampal microglia: a key mechanism by which CUMS-exosomes induce depressive-like behavior

[0109] The alpha chain of the IL4 receptor (IL4Rα) is essential for IL4 signaling. We aimed to understand how IL4 / IL4Rα signaling in hippocampal microglia affects depressive symptoms in mice following CUMS-exosome injection. Our initial studies focused on the sources of IL4 and IL4Rα, analyzing their expression in microglia and other cell types under in vivo and in vitro conditions. Our results showed that in both conditions, IL4 expression was primarily associated with microglia, while IL4Rα was associated with microglia to varying degrees. To further explore this relationship, we used adeno-associated virus (AAV) to increase IL4 levels, which resulted in increased IL4 protein levels ( Figure 5a). To specifically target IL4Rα expression in wild-type mouse hippocampal microglia, we used an AAV vector encoding a shRNA under the CD68 promoter (β-globin-CD68p-EGFP-MIR155(mcs)-WPRE-PA). This AAV has been shown to be specific for microglia in previous studies. We quantified AAV expression in microglia and other cell types by co-immunofluorescence staining with EGFP / Iba1, confirming the microglial specificity of AAV. Figure 5 As shown in B. Overexpression of IL4 successfully alleviated the depressive symptoms induced by CUMS exosomes, as shown in the OFT, FST, and TST test results ( Figure 5 c). However, silencing of IL4Rα in microglia in the hippocampus reversed the antidepressant effect of IL4 ( Figure 5 c), indicating that these effects are mediated through microglial IL4Rα signaling. Furthermore, inhibition of IL4Rα affected microglial branching and neurogenesis in the hippocampus of AAV-IL4 mice after exposure to CUMS-exosomes ( Figure 5 d-g). In addition, behavioral tests showed that HC exosomes had no effect on mice overexpressing IL4 or inhibiting IL4Rα ( Figure 5 h). To more specifically target microglial IL4Rα in the hippocampus, we used the Cre / Flex system to knock down microglial IL4Rα expression by injecting β-globin-FLEX-EGFP-MIR155(mcs)-WPRE-PA AAV into the hippocampus of CX3CR1 Cre mice ( Figure 5 i, j). Overexpression of IL4 alleviated depressive symptoms in these mice; however, knockdown of IL4Rα in microglia in the hippocampus abolished the antidepressant effect of IL4 ( Figure 5 k) and impaired neurogenesis ( Figure 5 l, m). Furthermore, inhibition of IL4Rα in the hippocampus of CX3CR1 Cre mice effectively counteracted the inhibitory effect of IL4 on microglial outgrowth after exposure to CUMS-exosomes ( Figure 5 n, o).

[0110] Collectively, these findings strongly support that the IL4 / IL4Rα signaling pathway in hippocampal microglia plays a key role in CUMS-exosome-induced depressive symptoms in mice.

[0111] Microglial IL4 signaling regulates depressive phenotypes through BDNF signaling. We next investigated how their IL4 signaling regulates depressive behaviors initiated by CUMS exosomes. Transcriptome analysis revealed that BDNF expression was significantly underrepresented in the hippocampus of mice treated with CUMS exosomes ( Figure 2 e), which was confirmed in an independent sample ( Figure 6 ac). In addition, we found that BDNF-TrkB signaling in microglia decreased after CUMS exosome exposure, and IL4 treatment restored this decrease ( Figure 6 A notable observation is that reducing microglial IL4Rα in the hippocampus counteracts the effect of IL4 on enhancing BDNF-TrkB signaling ( Figure 6 fi), suggesting that the microglial IL4 signaling pathway may regulate depressive behavior through the BDNF signaling pathway. To confirm the key role of BDNF, we injected BDNF protein into the dentate gyrus of the hippocampus. The results were significant: depressive behavior was alleviated, and defects in neurogenesis and microglial activation were effectively resolved ( Figure 7 Furthermore, the introduction of BDNF normalized the changes in microglial IL-6, IL-10, and iNOS levels induced by CUMS exosomes ( Figure 7 i, j). Further analysis showed that the microglial BDNF / TrkB signaling pathway, which was impaired by CUMS exosomes, was restored after BDNF administration ( Figure 7 k, l). Similar results were observed in vitro ( Figure 7 mo).

[0112] To determine whether BDNF signaling is crucial for the protective effects of IL4 against depressive behaviors, we used K252a to inhibit the TrkB pathway. Results clearly demonstrated that K252a abolished the antidepressant effects of IL4 in mice exposed to CUMS exosomes. Furthermore, K252a adversely affected neurogenesis and microglial activation, as demonstrated by changes in the number of DCX-positive cells and microglial branch length in the hippocampus following AAV-IL4 treatment in the context of CUMS exosomes. As expected, administration of K252a alone resulted in depressive-like behaviors, decreased neurogenesis within the hippocampus, and enhanced microglial activation in mice. These results reinforce the crucial role of BDNF signaling in the emergence of depressive-like phenotypes. Unexpectedly, we observed that MAM did not abolish the antidepressant effects of IL4 in mice exposed to CUMS exosomes. Although subcutaneous administration of MAM did abolish the pro-neurogenic effects of IL4 in these mice, MAM had no effect on the effects of IL4 on microglial activation or BDNF signaling. These findings suggest that defects in neurogenesis are independent of CUMS splenic exosome-induced depression in mice. In conclusion, this study highlights the crucial role of microglial IL4 signaling in mediating CUMS exosome-induced depressive symptoms in mice through the BDNF-centric pathway. Figure 8 ).

[0113] Example 5 Verification of NFIX diagnostic efficacy

[0114] The receiver operating characteristic (ROC) curve generated by Medcalc software was used to evaluate the potential of NFIX in distinguishing MDD patients from healthy controls.

[0115] To verify whether NFIX is specific for MDD, we drew a ROC curve to test and verify whether NFIX can distinguish MDD patients from healthy people. One of the ROC curves included 10 MDD patients and 10 healthy volunteers. The results ( Figure 9 a) showed an AUC value of 0.760 (95% CI = 0.520–0.920); another ROC curve included 10 MDD patients and 10 healthy volunteers, and the results ( Figure 9 b) The AUC value was 0.790 (95% CI = 0.552–0.937), indicating that NFIX can effectively distinguish MDD patients from healthy controls. These results suggest that NFIX is a candidate biomarker for the diagnosis of MDD.

[0116] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a non-human animal model of depression, characterized in that: The method comprises administering exosomes derived from splenic T lymphocytes of chronic unpredictable mildly stressed rats to a non-human animal selected from a mouse or a rat.

2. The method according to claim 1, characterized in that The depression includes major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia.

3. The method according to claim 2, characterized in that The depression is selected from major depressive disorder and anxious depression.

4. The method according to claim 1, wherein The non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition.

5. The method according to claim 1, wherein The method comprises injecting exosomes derived from spleen T lymphocytes of rats subjected to chronic unpredictable mild stress into mice to obtain mice exhibiting depressive-like behavior.

6. The method according to claim 5, characterized in that The depressive-like behaviors include prolonged immobility, loss of interest, low mood, and lack of energy.

7. The method according to claim 5, characterized in that The injection method is intravenous injection or brain injection.

8. The method according to claim 5, characterized in that The injection sites are the tail and the lateral ventricle.

9. A method for screening drug candidates for treating depression, characterized in that: The method comprises: a) administering the agent to be screened to a non-human animal with depression prepared by the method according to any one of claims 1 to 8; b) Analyze and evaluate the therapeutic effects of the agents to be screened, and select agents that can significantly improve the depressive-like behavior of the mouse model.

10. The method according to claim 9, characterized in that The drug candidates include selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, tricyclic drugs, tetracyclic drugs, serotonin modulators and stimulants.

11. Use of the non-human animal model of depression prepared by the method according to any one of claims 1 to 8 in screening drug candidates for treating depression.

12. Use of exosomes derived from splenic T lymphocytes of rats subjected to chronic unpredictable mild stress in constructing a non-human animal model of depression, wherein the non-human animal is selected from mice or rats.

13. The use according to claim 12, characterized in that The non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition.

Images