Application of spleen T lymphocyte exosome in construction of depression animal model

Abstract

The invention provides an application of a spleen T lymphocyte exosome in construction of a depression animal model, a stress rat spleen T lymphocyte exosome is injected into a mouse body to construct the depression animal model, and the depression animal model can be used for depression drug screening, drug efficacy evaluation or diagnosis, and can be used for treating depression. And the method can also be used for researching and revealing pathogenesis of depression. The invention also proves that the IL4 / IL4R alpha signal channel and the BDNF signal channel play an important role in depression through experiments. The invention has important significance for clinical research of depression.

Description

Technical Field

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

[0002] Globally, major depressive disorder (MDD) is a huge health challenge, affecting approximately 20% of the population and is one of the leading causes of disability. Looking ahead, the World Health Organization (WHO) predicts an even more worrying outlook, predicting that by 2030, MDD will rise to the top of the global burden of disease. These statistics highlight a pressing issue: even after extensive research efforts and advances in therapeutic interventions, a large proportion of patients currently treated with antidepressants experience poor responses, including partial or even no effects, accompanied by a range of side effects. This situation indicates a major gap in our understanding of the molecular complexity of MDD.

[0003] The etiology, pathogenesis and inducing factors of the disease are still unclear, and a reasonable animal model of depression is particularly important for studying the pathogenesis of depression, drug screening and guiding clinical medication. The defect of animal models is that their cognitive and emotional abilities are different from those of humans, but in recent years, animal models of depression have been continuously improved, mainly using stress, drugs, surgery and other modeling methods to build reasonable and effective animal models of depression, providing a reference for the study of the pathogenesis of depression and clinical treatment.

[0004] At present, the more commonly used animal models of depression include chronic stress modeling, unpredictable stress modeling, olfactory bulb resection model, post-stroke depression model, reserpine or glucocorticoid drug modeling, etc. Different models reflect different causes or mechanisms of depression. However, the current animal models of depression still have various unresolved technical problems, such as: the use of chronic stress modeling makes animals prone to adaptability; the use of unpredictable stress modeling takes a long time and requires a lot of work; the use of surgical modeling has a complex preparation process and certain technical requirements for experimental personnel; the use of drug modeling may cause false positives due to poor drug targeting, or the drug may have other side effects that 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, the method comprising administering exosomes of spleen T lymphocytes of 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 a sequence of precedence or 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] In the present invention, "non-human animal model" refers to a non-human animal that has or displays 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 (such as 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, shrews, voles, groundhogs, hamsters, guinea pigs, agouti), including any 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 of 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, depression, 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 a drug candidate 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 the expression level of NFIX, the method comprising regulating the expression level of NFIX 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 distinguishable 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 mitigation of a disease or disease course, and this inhibition or mitigation can be physical, such as certain distinguishable 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 generated 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 behavior tests, neurogenesis performance, and microglial cell status.

[0040] Furthermore, animal behavioral tests include, but are not limited to, tail suspension test, forced swim test, sugar water 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" is also called "YMaze", which refers to a behavioral test based on the natural exploration curiosity of animals. The spontaneous alternation behavior in the test is considered to reflect short-term spatial working memory. It utilizes the nature of the test animals to explore new environments. During the test, the test animals need to remember the previously explored directions each time they switch to explore new directions. 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 alternation behavior test and an electrical stimulation Y-maze test. In a specific embodiment of the present invention, a Y-maze alternation behavior test is used.

[0042] In the present invention, the term "water maze test" is also called "Morris water maze", "MWM", which is a test that forces experimental animals (rats, 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 to 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 the fields of 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" is also called "open box test", which is a method for evaluating the autonomous behavior, exploratory behavior and tension of test animals in a novel environment. The frequency and duration of certain behaviors of test animals in a novel environment reflect the autonomous behavior and exploratory behavior of test animals in a strange environment, and the number of urination and defecation reflects their tension. At present, the open field test is mainly used to observe the autonomous movement ability, exploratory behavior in a novel environment, tension, mania, anxiety and depression behavior of test animals. Due to its simple operation, high feasibility, accurate data recording and other characteristics, it has become a popular behavioral test method in the field of animal spirit research.

[0044] In the present invention, the term "elevated plus maze test" is also called "EPM test", which refers to a test method for evaluating the anxiety response of rodents. Compared with the detection of anxiety behavior of mice caused by noxious stimulation (such as electrical stimulation, noise stimulation, dietary deprivation and exposure to predator odor, etc.), the test has the advantages of simple operation and can intuitively reflect the conditioned response of mice. The EPM test is 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, and the intersection is the central area, which is at a certain height from the ground. The principle is that mice will be curious to explore when facing new things (open arms), and they have a dark nature (closed arms), and there is a conflict between exploration and avoidance 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 have a fearful and uneasy mentality. The anxiety behavior of mice can be evaluated by comparing the retention time and distance of mice in the open arms and closed arms.

[0045] In the present invention, the term "tail suspension test" is also called "TST", which is a classic method that can quickly evaluate the efficacy of antidepressants, stimulants, and sedatives. The principle is to use the fact that mice attempt to escape after being suspended by their tails but cannot escape, so they give up struggling and enter a unique state of depression and immobility. This typical "immobility state" reflects a so-called "behavioral despair state". This behavioral despair model is similar to depression and is sensitive to most antidepressants. Moreover, its efficacy is significantly correlated with clinical efficacy, so it is widely used in the preliminary selection of antidepressants. During the test, the depressive state is reflected by recording the immobility time of the animals. Antidepressants and stimulants can significantly shorten and change their immobility state.

[0046] In the present invention, the term "forced swim test" is also called "FST", which is a behavioral despair test method, by placing an animal in a confined environment (such as water), the animal desperately struggles in the environment to try to escape but cannot escape, thereby providing an unavoidable oppressive environment, after a period of time, the animal shows a typical "immobility state", and a series of parameters in the process of the test animal producing a desperate immobility state are observed and recorded, which can be used to evaluate the effects of depressants and antidepressants. In the forced swim test, the activity of the animal's hind limbs is usually used as the basis for judgment.

[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). The 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 depressive 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 transparent material with ventilation holes, restraining for a fixed length of time every day, and fasting and water deprivation during the daily stress stage. Chronic restraint can be selected according to the test requirements. Different time intensity and stress intensity, time intensity are 1, 2, 3, 6 hours per day, etc., and stress intensity can be selected for 1, 3, 7, 14, 21 and 28 consecutive days. Animals are restrained in a confined space for a long time, and initially show anxiety and irritability, and try to escape. Eventually, when they feel hopeless to escape, they develop symptoms similar to human anhedonia, weight loss, reduced diet, despair, fatigue, etc., which can be improved by antidepressant drugs, and other behavioral evaluation tests are performed later. In a specific embodiment of the present invention, the time intensity selected for chronic restraint is 6 hours per day for 10 consecutive days. After the modeling is completed, a tail suspension test is performed to test the depressive-like behavior of mice.

[0049] In the present invention, the rotarod test is to evaluate the motor coordination and balance of the test animal by forcing it to run on a rod that rotates at an accelerated speed. The test animal is placed on a rotating rod with a stable acceleration, and the test records the time it takes to fall from the rod, i.e., the latency. Through this indicator, the motor learning ability and coordination ability of the animal can be detected, and the effects of drugs, diseases or other interventions on its motor function can be further analyzed.

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

[0051] In the present invention, the promoter increases the protein expression level or protein activity, and can also increase the 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 single-stranded (sense strand or antisense strand) and double-stranded polynucleotides when applicable. 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 cover complete monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed by at least two complete antibodies, and antibody fragments, as long as they exhibit the desired antigen binding activity, they all fall within the scope of protection of the present invention.

[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, the product comprising a reagent capable of detecting the expression level of NFIX in exosomes.

[0072] Furthermore, the product also includes a chip, a test 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] Further, 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] The present invention provides an application of spleen T lymphocyte exosomes in constructing an animal model of depression. The present invention injects spleen T lymphocyte exosomes of stressed rats into mice to construct an animal model of depression. The animal model of depression can be used for drug screening, efficacy evaluation or diagnosis of depression, and can also be used to study and reveal the pathogenesis of depression. The present invention also experimentally proves the important role of IL4 / IL4Rα signaling pathway and BDNF signaling pathway in depression. The present invention is of great significance for clinical research on depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] Figure 1 Figure 1 is an experimental result of the effect of splenic T lymphocyte exosomes from stressed rats on mice, where a is the result of CUMS exosomes injected into healthy mice to induce depressive-like symptoms; b is the result of rotarod test injected into healthy mice; c and d are the results of a significant reduction in Doublecortin (DCX)-positive cells after exposure to CUMS exosomes; e is the result of the exosome contents and depressive characteristics of mice after enzyme treatment; f is the result of two-photon laser scanning microscopy to detect DiI-labeled exosomes in the hippocampus; g and h are 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 the 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 +Microglia internalized DiI-labeled exosomes; Figures m, n, and o are the results of CD45 + CD11b + Figure 1 is the result of microglia activation; Figure p is the result of IL4 protein level decrease after CUMS-exosome treatment; Figure q is the result of IL4Rα protein level decrease after CUMS-exosome treatment.

[0087] Figure 3 These are experimental results of 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 reduction in IL4 signaling and neurogenesis, as well as the increase in microglial activation in the hippocampus.

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

[0089] Figure 5Figure 1 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 microglia-specific result of AAV; Figure c is the result of IL4 overexpression successfully alleviating the depressive symptoms induced by CUMS exosomes, and silencing of IL4Rα in microglia in the hippocampus reversed the antidepressant effect of IL4; Figures d, e, f, and g are the results of IL4Rα inhibition affecting the branching and neurogenesis of microglia 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 result of knocking down the expression of microglia IL4Rα in the hippocampus of Cre mice; Figure k shows that knocking down IL4Rα in microglia in the hippocampus eliminated the antidepressant effect of IL4; Figure l and Figure m show that knocking down IL4Rα in microglia in the hippocampus impairs neurogenesis; Figure n and Figure 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 1 and 2 are research results on the mechanism by which IL4 signaling pathway regulates depressive behavior initiated by CUMS exosomes, wherein Figures a, b, and c show that the expression of BDNF is significantly insufficient in the hippocampus of mice treated with CUMS exosomes; Figures d and e show that the BDNF-trkb signaling pathway of microglia decreases after exposure to CUMS exosomes, while IL4 treatment can restore 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 are the results of the relief of depressive behavior and the effective resolution of defects in neurogenesis and microglial activation after injection of BDNF protein into the dentate gyrus of the hippocampus; Figures i and j are the results of the normalization of changes in microglial IL-6, IL-10, and iNOS levels induced by CUMS exosomes by the introduction of BDNF; Figures k and l are the results of the restoration of vitality of the BDNF / TrkB signaling pathway of microglia damaged by CUMS exosomes after BDNF administration; Figures m, n, and o are the results of the restoration of vitality of the BDNF / TrkB signaling pathway of microglia damaged by CUMS exosomes in vitro after BDNF administration.

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

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

[0094] The disclosure above generally describes the present invention. A more complete understanding can be obtained with reference to the following specific embodiments. The purpose of describing these embodiments is only for illustration and is not intended to limit the scope of the present invention. Form changes and equivalent substitutions are considered to be circumstances that may suggest or confer convenience. Although specific terms are used in this article, the purpose of these terms is descriptive rather than limiting.

[0095] Example 1 Exosomes from spleen T lymphocytes 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, affected rats displayed clear depressive features: compared with controls, they spent less time in the central zone in 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, we extracted exosomes from the culture medium. Nanoparticle tracking analysis showed that the diameter of most exosome particles was within the peak range of 100 nm, indicating that their size was consistent with the typical exosome characteristics. Western immunoblot analysis 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 in 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. In addition, we found that neither HC exosomes nor CUMS exosomes induced a peripheral immune response, as leukocyte and hs-CRP levels did not change.

[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 the 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 after 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 idea in neuroscience is that exosomes can cross the blood-brain barrier (BBB) ​​and affect 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 by confocal microscopy, we found that exosomes were distributed in the dorsal and ventral regions of the hippocampus. Notably, higher concentrations were observed in the dentate gyrus, with obvious red fluorescence in the cytoplasm of microglia in this region ( Figure 1 g, h). A closer look at the hippocampus region revealed worrying 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 after CUMS-exosome introduction 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 presented 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. The enriched pathways of these differentially expressed genes were related to immune system response 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 application—and given the known relationship between IL4 dysregulation and depression—protein levels of IL4 and its receptor IL4Rα were measured using western blotting. 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 h of culture, these cells internalized DiI-labeled exosomes, indicating that endocytosis occurred ( Figure 2 l). After CUMS exosome treatment, these cells showed significant activation, with increased CD68 expression compared with 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 lateral ventricle injection study showed that mice receiving CUMS exosomes exhibited depressive-like behaviors ( Figure 3 In addition, we noted 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 depressive-like symptoms in mice

[0107] To identify exosome contents that regulate IL4 expression in the hippocampus, we performed a detailed proteomic analysis of exosomes from splenic T lymphocytes from 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 the results 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 also apply 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 levels after treatment ( Figure 4 h, i). Notably, gender 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 with 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 CUMS-exosome-induced depressive behavior and neurogenesis impairment ( Figure 4k-n). The introduction of IL4 also normalized the abnormal levels of IL4, IL-6, IL-10, and iNOS induced by exosomes. In addition, 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 s-v). Western immunoblot 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 evaluate the direct role of microglial activation in the development of depressive-like behaviors induced by CUMS-exosomes, we inhibited microglial activation using minocycline. Our analysis showed that treatment of exosomes with minocycline did not result in significant alterations in protein or RNA concentrations, implying that minocycline did not significantly affect the exosome cargo itself. However, minocycline significantly alleviated the depressive symptoms associated with exosomes. Furthermore, minocycline reduced the hyperactivation of microglia in the hippocampus of CUMS-exosome-treated mice and promoted neurogenesis. These findings strongly suggest that microglial activation triggered by IL4 deficiency in the hippocampus is a key factor in CUMS-exosome-induced depressive behaviors.

[0108] Example 4 IL4 / IL4Rα signaling in hippocampal microglia: a key mechanism for CUMS-exosomes to 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 IL4 expression was primarily associated with microglia under both conditions, while IL4Rα was associated with microglia to varying degrees. To further explore this relationship, we increased IL4 levels using adeno-associated virus (AAV), 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 shRNA under the CD68 promoter (β-globin-CD68p-EGFP-MIR155(mcs)-WPRE-PA). The specificity of this AAV for microglia has been demonstrated in previous studies. We quantified the expression of AAV in microglia versus other cell types by co-immunofluorescence staining of EGFP / Iba1, confirming the microglial specificity of AAV, as shown in Figure 2. Figure 5 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 branching after exposure to CUMS-exosomes ( Figure 5 n, o).

[0110] Overall, 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 explored 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 independent samples ( Figure 6 ac). In addition, we found that the BDNF-TrkB signaling pathway in microglia decreased after CUMS exosome exposure, while 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), indicating 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 the defects in neurogenesis and microglial activation were effectively resolved ( Figure 7 Moreover, 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 the BDNF signaling pathway is critical for the protective effects of IL4 against depressive behaviors, we used K252a to inhibit the TrkB pathway. The results clearly showed that K252a abolished the antidepressant effects of IL4 in mice exposed to CUMS-exosomes. In addition, K252a had adverse effects on neurogenesis and microglial activation, as shown by changes in the number of hippocampal dcx-positive cells and changes in microglial branch length after AAV-IL4 treatment in the context of CUMS-exosomes. As expected, administration of K252a alone resulted in depressive-like behaviors, reduced neurogenesis within the hippocampus, and enhanced microglial activation in mice. These results reinforce the important role of BDNF signaling in the emergence of a depressive-like phenotype. Unexpectedly, in mice exposed to CUMS exosomes, we observed that MAM did not abolish the antidepressant effects of IL4. Although subcutaneous injection 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 spleen 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 NFIX diagnostic efficacy verification

[0114] ROC curves generated by Medcalc software were used to evaluate the potential of NFIX as a marker for distinguishing MDD patients from healthy subjects.

[0115] In order 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. Fig. 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 were ( Fig. 9 b) The AUC value was 0.790 (95%CI = 0.552–0.937), indicating that NFIX can effectively distinguish MDD patients from healthy subjects. These results suggest that NFIX is a candidate marker for the diagnosis of MDD.

[0116] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also 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 to a first non-human animal exosomes from spleen T lymphocytes of a stressed second non-human animal; Preferably, the depression includes major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia; Preferably, the depression is selected from major depressive disorder and anxious depression.

2. The method according to claim 1, characterized in that The non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition; Preferably, the non-human animal is a mammal; Preferably, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep; Preferably, the mammal is selected from mice and rats.

3. The method according to claim 1, characterized in that The second non-human animal is of the same species or of a different species than the first non-human animal; Preferably, the second non-human animal is of a different species than the first non-human animal; Preferably, the first non-human animal includes mice, rats, rabbits, dogs, pigs, monkeys, and sheep; Preferably, the first non-human animal is selected from mice; Preferably, the second non-human animal includes mice, rats, rabbits, dogs, pigs, monkeys, and sheep; Preferably, the second non-human animal is selected from rats.

4. The method according to claim 1, characterized in that: The method comprises injecting spleen T lymphocyte exosomes of rats with chronic unpredictable mild stress into mice to obtain mice with depressive-like behavior; Preferably, the depressive-like behavior includes prolonged immobility, loss of interest, low mood, and lack of energy; Preferably, the injection method is intravenous injection or brain injection; Preferably, the injection site is the tail or lateral ventricle.

5. Any of the following methods: (1) 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 4; 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; Preferably, the drug candidates include selective serotonin reuptake inhibitors, serotonin and norepinephrine reuptake inhibitors, tricyclic drugs, tetracyclic drugs, serotonin modulators and stimulants; (2) A method for evaluating the efficacy of a drug for treating depression, characterized in that the method comprises: a) administering a drug to a non-human animal with depression prepared by the method according to any one of claims 1 to 4; b) evaluating the therapeutic effect of the drug on the depression; (3) A method for studying the pathogenesis of depression, characterized in that the method comprises using a non-human animal with depression prepared by the method according to any one of claims 1 to 4 to study the pathogenesis of depression; (4) A method for regulating IL-4 / IL4Rα expression levels, characterized in that the method comprises using stressed exosomes to regulate IL-4 / IL4Rα expression levels; (5) A method for regulating the expression level of NFIX, characterized in that the method comprises using stress exosomes to regulate the expression level of NFIX; (6) A method for regulating BDNF / TrkB expression levels, characterized in that the method comprises using stress exosomes to regulate BDNF / TrkB expression levels.

6. The method according to claim 5, characterized in that The evaluation indicators include animal behavior tests, neurogenesis performance, and microglial cell status; Preferably, the animal behavior test includes tail suspension test, forced swimming test, sugar water preference test, Y maze test, water maze test, open field test, elevated plus maze test, rotarod test, chronic restraint; Preferably, the animal behavioral test is selected from the group consisting of forced swimming test, sugar water preference test, open field test, and rotarod test.

7. Any of the following applications: (1) Use of a non-human animal model of depression prepared by the method according to any one of claims 1 to 4 in screening drug candidates for the treatment of depression; (2) Use of a non-human animal model of depression prepared by the method according to any one of claims 1 to 4 in evaluating the therapeutic effect of a drug for treating depression; (3) Use of the non-human animal model of depression prepared by the method according to any one of claims 1 to 4 in studying the pathogenesis of depression; (4) Application of stress exosomes in regulating IL-4 / IL4Rα expression levels; (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; (7) Application of BDNF / TrkB signaling pathway promoters in the preparation of drugs for the treatment of depression.

8. Application of splenic T lymphocyte exosomes in constructing non-human animal models of depression; Preferably, the non-human animal model refers to a non-human animal that has or displays characteristics of a disease or condition; Preferably, the non-human animal is a mammal; Preferably, the mammals include mice, rats, rabbits, dogs, pigs, monkeys, and sheep; Preferably, the mammal is selected from mice and rats.

9. Application of reagents for detecting NFIX and its expression products in exosomes in the preparation of products for diagnosing depression; Preferably, 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; Preferably, the reagent is selected from primers that specifically amplify NFIX in exosomes; Preferably, the depression includes major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia; Preferably, the depression is selected from major depressive disorder and anxious depression.

10. A product for diagnosing depression, characterized in that: The product includes a reagent capable of detecting the expression level of NFIX in exosomes; Preferably, the product further comprises a chip, a kit or a nucleic acid membrane strip; Preferably, the chip includes a gene chip and a protein chip; Preferably, the gene chip comprises an oligonucleotide probe targeting NFIX in exosomes for detecting the transcription level of NFIX in exosomes; Preferably, the protein chip comprises a specific binding agent for NFIX protein in exosomes; Preferably, 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, or mass spectrometry; Preferably, the kit includes an instrument or reagent for processing a sample; Preferably, the sample comprises cells, tissue, blood, urine, saliva or mucus; Preferably, the sample is selected from blood; Preferably, the depression includes major depressive disorder, unipolar depression, treatment-resistant depression, resistant depression, anxious depression, bipolar depression, and dysthymia; Preferably, the depression is selected from major depressive disorder and anxious depression.

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