Use of an agent that inhibits expression of the igsf1 gene in the manufacture of a medicament for treating depression

Abstract

The application discloses application of a reagent for inhibiting Igsf1 gene expression in preparation of a medicine for treating depression, and the depression symptom can be effectively improved by silencing Igsf1 gene expression or blocking the function of the Igsf1 gene by using an antibody, and the normal physiological state is not affected. Therefore, the Igsf1 can be used as a target in preparation and screening of the medicine for treating depression, and has an important clinical application prospect.

Description

Technical Field

[0001] This invention relates to therapeutic drugs for depression, and more specifically, to the use of a reagent that inhibits the expression of the Igsf1 gene in the preparation of therapeutic drugs for depression. Background Technology

[0002] Depression is a mood-related disorder characterized by low mood and loss of interest. With the accelerated pace of modern life and increasing social pressure, its incidence is constantly rising, and it has become the second leading cause of death after cancer. Compared with the general population, people with depression have significantly shorter lifespans (with cardiovascular disease being the leading cause of death), significantly lower quality of life and cardiopulmonary health (defined as the cardiovascular and respiratory systems' ability to supply oxygen to skeletal muscles during sustained exercise), and significantly increased risks of type 2 diabetes, metabolic syndrome, hyperglycemia and hypertriglyceridemia, hypertension, and alcohol use disorders. Depression severely impacts patients' quality of life and places enormous pressure on global healthcare systems. Statistics show that in 2010, the economic burden of mental disorders globally was comparable to, or even higher than, that of cardiovascular disease, cancer, chronic respiratory diseases, and diabetes. According to estimates by the World Health Organization (WHO), by 2030, depression will become the largest disease burden globally. Therefore, controlling and preventing mental disorders such as depression, and alleviating the increasing economic and health burden, are currently key priorities for global healthcare systems.

[0003] Currently available antidepressants mainly include monoamine oxidase inhibitors (MAOIs), tricyclic antidepressants (TAOs), and selective serotonin reuptake inhibitors (SSRIs). Due to limitations in our understanding of the pathogenesis of depression, these drugs are not ideally effective, generally exhibiting problems such as long onset of action, strong side effects, and varying drug sensitivity among different populations. Therefore, in-depth research into the pathogenesis of depression and the search for novel and safe therapeutic targets are key issues in current clinical work on depression.

[0004] In recent years, researchers have discovered a significant correlation between abnormal blood-brain barrier function and the development of depression. The blood-brain barrier is composed of endothelial cells, pericytes, and astrocytes, with endothelial cells being the most crucial component. Severe disruption of vascular endothelial morphology and function is observed in the brain tissue of depressed mice. Further research into the functional abnormalities of endothelial cells in the pathogenesis of depression will contribute to the development of novel therapeutic targets for depression. Summary of the Invention

[0005] To address the issues of long onset of action, strong side effects, and differences in drug sensitivity among individuals associated with the aforementioned antidepressants, this invention provides an application of a reagent that inhibits Igsf1 gene expression in the preparation of drugs for treating depression. By silencing Igsf1 gene expression or blocking its function with antibodies, the symptoms of depression can be effectively improved, providing a new strategy for the development of antidepressants.

[0006] To achieve the above objectives, the present invention provides the use of Igsf1 as a target in the preparation of antidepressant drugs, wherein the drug is a drug that targets and inhibits or silences Igsf1, or an antibody that neutralizes and blocks the protein function of Igsf1.

[0007] The drug affects the expression of the Igsf1 gene or the function of its protein by acting directly on the Igsf1 gene or its protein.

[0008] In a preferred embodiment, interference or inhibition of Igsf1 gene expression is achieved by Igsf1 gene knockout or RNA interference, including but not limited to the use of siRNA, shRNA, microRNA, and interference plasmids that can produce siRNA or shRNA.

[0009] In a preferred embodiment of the present invention, the drug that neutralizes and blocks the protein function of Igsf1 includes, but is not limited to, antibodies against Igsf1 protein and specific antagonists of Igsf1 protein. For example, specific antagonists of Igsf1 protein include, but are not limited to, mutant proteins or peptides of Igsf1 protein, and expression genes or plasmids that produce mutant proteins or peptides of Igsf1 protein.

[0010] The aforementioned antidepressant and / or anti-anxiety medication may be in liquid or solid dosage form.

[0011] The liquid dosage form may be an injection, solution, suspension, emulsion, or aerosol.

[0012] The solid dosage form is a tablet, capsule, pill, powder for injection, sustained-release preparation or various microparticle delivery systems.

[0013] The purpose of this discovery is to provide a new drug screening method that uses Igsf1 as a therapeutic target for screening antidepressants.

[0014] Specifically, the present invention relates to the following technical solutions: This invention discloses a method for screening antidepressant drugs, which includes the step of screening Igsf1 as a therapeutic target.

[0015] Igsf1 is used as a therapeutic target to screen antidepressants, with the decrease or absence of Igsf1 gene expression level, or the inhibition or absence of Igsf1 protein function as screening indicators.

[0016] Through the above technical solution, the present invention achieves the following beneficial effects: This invention effectively improves the symptoms of depression by silencing the Igsf1 gene expression or blocking its function with antibodies, demonstrating that Igsf1 can serve as a target for treating depression, thus providing a new strategy for developing antidepressant drugs. Attached Figure Description

[0017] Figure 1 In Example 1 of this invention, immunofluorescence staining was used to identify the difference in IGSF1 protein expression in the brain endothelial cells of control mice and CUMS mice. Red fluorescent markers were used to label vascular endothelial CD31, green fluorescent markers were used to label IGSF1 protein, and blue-purple markers were used to label nuclear DAPI. Figure 2 In Example 2 of this invention, endothelial cell-specific knockout of Igsf1 in mice can effectively improve the depressive symptoms in CUMS mice. A is a strategy diagram of endothelial cell-specific knockout of Igsf1 mice, B is the knockout efficiency of Igsf1, C is a schematic diagram of experimental operation, and D is the mining behavior results of control mice and endothelial cell-specific knockout of Igsf1 mice. Figure 3 This is the mine behavior result of CUMS mice after Igsf1 antibody injection treatment in Example 3 of the present invention. Detailed Implementation

[0018] The specific embodiments of the present invention will be described in detail below with reference to examples. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention. Experimental methods not specified with specific conditions in the embodiments are generally performed under conventional conditions.

[0019] Example 1: Immunofluorescence staining revealed high expression of IGSF1 protein in the cerebral endothelial cells of depressed mice. 1. Establishment of a mouse model of depression: The CUMS model is a widely used method for studying depression in animal experiments. By randomly subjecting experimental animals to a series of unpredictable mild stressors, such as food or water deprivation, wet cages, noise exposure, temperature changes, and restraint, behavioral and physiological changes similar to those of depression can be induced, thereby helping researchers better understand the pathogenesis of depression and evaluate the effectiveness of drug treatment. 2. After CUMS modeling, brain tissues from control and CUMS mice were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with antigen after antigen retrieval. CD31-labeled cerebral vascular endothelial cells were used for localization observation. 3. Results: such as Figure 1 As shown, compared with healthy control mice, CUMS mice (depressed mice) have high expression of IGSF1 protein in their brain vascular endothelial cells. This suggests that IGSF1 in endothelial cells may play an important pathogenic role in the development of depression, and also suggests that IGSF1 protein in endothelial cells may be a novel therapeutic target for depression.

[0020] Example 2: Conditional knockout of Igsf1 in endothelial cells effectively alleviates depressive symptoms in mice. 1. Constructing mice with endothelial cell-specific Igsf1 knockout: such as Figure 2 As shown in Figure A, Cyagen Biosciences was commissioned to construct Igsf1. fl / fl In mice, the loxp sites were designed flanking the first exon of Igsf1, thereby inducing Igsf1... fl / fl Mice were bred with Tekcre mice to produce Tekcre mice. + Igsf1 fl / fl Mouse. Tek is widely expressed in endothelial cells, therefore Igsf1 expression can be specifically knocked out in endothelial cells; 2. Enrichment and verification of knockout efficiency in mouse endothelial cells: (1) Euthanized control mice (Tek cre + Igsf1 + / + ) and Igsf1-specific knockout mice (Tek cre + Igsf1 fl / fl The cerebral cortex of mice was isolated and cut into small pieces, then soaked in DMEM medium containing type I collagenase and type I DNase and digested at 37°C for 30 min. (2) After digestion was terminated, the tissue was ground and filtered through a 70 μm cell sieve to form a single cell suspension. After centrifugation, washing and resuspending, Biolegend anti-mouse CD45 Biotin (catalog number 103103) was added and stained at 4°C for 30 min. (3) After centrifuging and washing the cells, add Biolegend's magnetic Streptavidin Nanobeads (catalog number 480016), incubate at 4°C for 15 min, wash and resuspend, and then use a magnet (catalog number 480173) to negatively select the cells to obtain CD45. - Cell suspension; (4) After washing and reselecting the cells, add Biolegend's anti-mouse CD31 Biotin (catalog number 102404) and stain at 4°C for 30 min; (5) After centrifuging and washing the cells, add magnetic Streptavidin Nanobeads, incubate at 4°C for 15 min, wash and resuspend, and then use a magnet to positively select the cells to obtain CD45. - CD31 + Endothelial cell suspension; (6) After adding PBS, centrifuge and wash the endothelial cells, discard the supernatant, add loading buffer to the cell pellet, and lyse at 95°C for 5 min; (7) Using a 15-well 10% SDS-PAGE gel, after electrophoresis-transfer-blocking-incubation with primary antibody (IGSF1, β-actin)-incubation with secondary antibody, exposure is performed, as shown. Figure 2 As shown in Figure B, Igsf1 is knocked out efficiently in endothelial cells, and this mouse can be used for further research. 3. The above-mentioned control mice and Igsf1-specific knockout mice were used to induce the CMUS model. After 4 weeks, the mouse behavioral experiment was carried out: In this embodiment, the Open Field Test (OFT) was used as the quantitative standard to track and record the behavior of the mice. The shorter the distance of the mouse's movement migration, the shorter the distance of movement in the central area, and the shorter the time spent in the central area, the more severe the depression was. 4. Results: such as Figure 2 As shown, CUMS modeling leads to depressive symptoms in mice; however, knocking out Igsf1 in endothelial cells can significantly alleviate these symptoms.

[0021] Example 3: Igsf1 antibody treatment effectively improved depressive symptoms in mice. 1. Screening for IgSf1-targeting antibodies using hybridoma cells: (1) Purification of Igsf1 protein: construct pet-28a-Igsf1 plasmid, transform it into BL21-DE3 competent cells, amplify the bacterial culture, add IPTG to induce protein expression, harvest the bacterial cells, lyse them and purify the protein using a nickel column, and obtain Igsf1 protein after elution, concentration and ultrafiltration replacement. (2) Immunization of mice: The above Igsf1 protein was mixed with Sigma's complete Freund's adjuvant in a water-in-oil state and then subcutaneously immunized mice. Afterwards, booster immunizations were performed every 7-10 days. (3) Euthanize mice, grind their spleens to obtain a single cell suspension, wash them and fuse them with sp20 cells with polyethylene glycol, and then screen them to obtain hybridoma cells that can secrete specific Igsf1, and purify the antibody to obtain an antibody targeting Igsf1. 2. Antibody treatment: C57BL / 6 mice were divided into a control group and a CUMS modeling group. During the modeling process, Igsf1 antibody was injected once a week. After 4 weeks, mouse behavioral experiments were conducted. 3. Results: such as Figure 3 As shown, CUMS modeling leads to depressive symptoms in mice. When CUMS mice are injected with Igsf1 antibody to block its function, the depressive symptoms are significantly improved. This indicates that Igsf1 antibody targeted therapy can effectively improve the depressive symptoms in mice, and Igsf1 can serve as a new target for drug development in the future treatment of depression.

[0022] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0023] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0024] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. The application of a reagent that inhibits Igsf1 gene expression in the preparation of drugs for treating depression.

2. The application according to claim 1, characterized in that, The drug is either a drug that targets and inhibits or silences Igsf1, or an antibody that neutralizes and blocks the protein function of Igsf1.

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