Use of apob gene in preparation of medicine for relieving depression

Abstract

This invention discloses the application of the Apob gene in the preparation of drugs to alleviate depression, belonging to the field of biomedical technology. This invention discovers that overexpression of APOB protein in the dorsomedial striatum (DMS) brain region has an antidepressant effect; knockout of the Apob gene in DMS GABAergic neurons makes mice more susceptible to stress, demonstrating the positive role of the Apob gene in addressing depression.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of the Apob gene in the preparation of drugs to alleviate depression. Background Technology

[0002] Depression is a common mental disorder, often referred to as the "common cold of psychiatry," with a lifetime prevalence of 10%–20%, affecting 350 million people worldwide. Characterized by high incidence, high recurrence, and high disability rates, depression is the leading cause of disability globally and a significant contributor to the global disease burden. The biological causes and pathogenesis of depression are not yet fully understood, but it is generally believed to result from the combined effects of genetic and environmental factors. The pathogenesis of depression involves multiple highly complex and interconnected pathways, primarily including monoamines, the HPA axis, neurotrophic factors and neurogenesis, excitatory and inhibitory neurotransmission, mitochondrial dysfunction, epigenetics, inflammation, the opioid system, myelination, and the gut-brain axis. The lack of clarity regarding the genetic etiology and pathological mechanisms of depression, along with the absence of universally applicable drug targets, poses a significant challenge to clinical treatment.

[0003] Currently, there are over 30 types of antidepressants on the domestic medical market. These antidepressants cover various mechanisms of action, but their curative effects on patients are not significantly different. A considerable number of patients experience little effect after drug treatment, indicating that drug therapy has very limited efficacy in treating depression. Almost all antidepressants currently used clinically improve depressive symptoms by affecting the signaling of monoamine neurotransmitters (including serotonin and norepinephrine), the target of which was discovered 30 years ago. The drawback of this class of drugs is that they can only alleviate symptoms in a small number of patients, requiring weeks or even months to reduce depressive symptoms, and they also have serious side effects.

[0004] The Apob gene plays a central role in lipid metabolism, and its mutations can lead to a variety of genetic diseases. However, there is currently no research on the relationship between the Apob gene and depression. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide the application of the Apob gene in the preparation of drugs to relieve depression. The Apob gene described in this invention has a significant regulatory effect on depression and has a significant positive effect on the preparation or screening of drugs to relieve depression.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides the application of the Apob gene in the preparation of drugs to alleviate depression.

[0008] Secondly, this invention provides the application of the Apob gene expression vector in the preparation of drugs to alleviate depression.

[0009] Thirdly, this invention provides the application of APOB protein in the preparation of drugs for relieving depression.

[0010] Fourthly, this invention provides the application of the Apob gene in screening drugs to alleviate depression. The Apob gene is used as a target of drugs or preparations. Based on the regulation of the Apob gene, drugs or preparations to alleviate depression are screened. The screened drugs or preparations can promote the transcription or translation of the Apob gene, or can increase the expression or activity of APOB protein.

[0011] Fifthly, the present invention provides a drug for relieving depression, comprising one or more of the Apob gene, Apob gene expression vector, and APOB protein.

[0012] Preferably, the drug also contains pharmaceutically acceptable excipients.

[0013] It contains at least the following beneficial technical effects:

[0014] This invention discovered that overexpression of APOB protein in the dorsomedial striatum has an antidepressant effect; knockout of the Apob gene in GABAergic neurons of the dorsomedial striatum (DMS) makes mice more susceptible to stress, indicating that the Apob gene plays a positive role in coping with depression. Attached Figure Description

[0015] Figure 1 The figure shows the verification of the ink injection location in Example 1.

[0016] Figure 2 The figure shows the results of the sugar water preference experiment in Example 1.

[0017] Figure 3 The image shows the results of the smelling experiment on female mouse urine in Example 1.

[0018] Figure 4 The image shows the experimental results of the forced swimming experiment in Example 1.

[0019] Figure 5 This is a diagram of virus expression in Example 2.

[0020] Figure 6 The figure shows the FUST experimental results in Example 2.

[0021] Figure 7 The figure shows the FST experimental results in Example 2. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25±2℃.

[0028] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0029] Example 1

[0030] Mouse selection:

[0031] The mice used in this embodiment were C57BL / 6 mice (Stock No: 000664) purchased from Jackson Laboratory and successfully bred at our SPF Laboratory Animal Center. The mice used in this embodiment were adult males weighing 25±5g. All mice were housed in an environment with 12 hours of light and 12 hours of darkness, with ample water and food supply, and an ambient humidity of 50%-60%. Following the principle of optimal and reasonable mouse numbers to minimize animal suffering during the experimental procedures, all experimental animals were randomly assigned to groups.

[0032] Experimental Projects:

[0033] Chronic unpredictable stress (CUS): This method was based on published literature (Lei et al., Molecular Psychiatry, 2022; Wang et al., Journal of Psychiatric Research, 2021). In brief, mice were given two types of stimulation daily at non-fixed times for two weeks: restraint for 2 hours, tail clamping for 15 minutes, continuous light exposure for 24 hours, 24-hour tilted cage with wet bedding, 10 minutes of unavoidable foot shocks (0.3mA current for 2 seconds, with a 16-second interval), and a high platform for 30 minutes.

[0034] Sucrose preference test (SPT): This test examines an animal's ability to derive pleasure from natural rewards. Mice are placed in water bottles on both the food and water sides of their cages for a week to acclimate. The test then measures the mice's sucrose consumption over two hours (7:00 PM - 9:00 PM). A 1% (w / v) sucrose solution is placed on the food side of the cage, while drinking water is placed on the opposite side, allowing the mice to freely choose between the drinking water and the sucrose solution. Sucrose preference is expressed as a percentage of total fluid consumption relative to the amount of sucrose solution consumed.

[0035] Forced swimming test (FST): Mice are placed in a cylindrical glass container (25 cm long, 16 cm high, filled with 15 cm of water at 24°C), and videotaped for 6 minutes. The time the mice remain still for the last 4 minutes is also recorded. A still state is defined as the mouse floating on the water surface with only slight movements to maintain breathing and keep its head above water.

[0036] Female urine sniffing test (FUST): One hour before the test, cotton swabs were fixed in the mice's cages to allow them to acclimatize and eliminate any novelty or fear associated with the swabs. The test was conducted in a dimly lit room with a light intensity of 3 lux and was divided into two phases. One swab was placed with 80 μl of pure water, and the other with 80 μl of urine from a female mouse in estrus (for vaginal smear testing). The sniffing time of the mice was recorded for three minutes at each phase, with a 45-minute interval between each phase.

[0037] Injection cannula insertion: Mice were anesthetized by intraperitoneal injection of sodium pentobarbital solution (40 mg / kg), fixed to a stereotaxic instrument with an ear rod, placed in a prone position, and a midline incision was made in the head. The periosteum was dissected to expose the skull surface, ensuring the anterior and posterior fontanelles were at the same level, and adjustments were made to maintain horizontal alignment. The drilling location was determined based on brain atlases, and a hole was drilled in the skull above the brain region. The cannula was inserted using the following coordinates: DMS: (AP: +0.65 mm; ML: ±1.4 mm; DV: -2.0 mm). The cannula was inserted 1 mm deeper than the brain region. After drilling the skull, the medical cannula was inserted and fixed with dental cement. After behavioral testing, ink was injected into the insertion site through the cannula. Brain tissue sections were prepared, and the injection site was observed under a microscope to verify accuracy. Mice with incorrect injection sites were excluded. The location was verified by injecting ink after administering the drug into the cannula. Figure 1 .

[0038] Experimental methods:

[0039] Mice were divided into three groups for each experiment: Group 1 was the control group, injected with 0.9% saline; Group 2 received CUS + 0.9% saline; and Group 3 received CUS + APOB protein injection (APOB protein was diluted with 0.9% saline to a concentration of 0.05ug / ul, and 0.5ul was injected into each DMS brain region) Recombinant Apolipoprotein B (APOB) (RPC003Mu01, Cloud-clone corp.wuhan). Mice underwent microinjection into the CUS and DMS brain regions for 14 days, followed by the experiments as described above. The specific experimental schedule for CUS during the 14-day period is shown in Table 1.

[0040] Table 1. Schedule of 14-day CUS in mice

[0041]

[0042] Experimental results:

[0043] Fourteen days later, depressive behavior was assessed in the mice, and the results are as follows:

[0044] (1) See the sugar water preference experiment. Figure 2 Mice subjected to CUS for 14 days exhibited depressive behavior with decreased sucrose preference, but injection of APOB protein during CUS reversed this depressive behavior.

[0045] (2) The smelling test of female mouse urine is shown in Figure 3 Mice subjected to CUS for 14 days exhibited depressive behavior characterized by reduced olfactory smell of female mice urine. However, injection of APOB protein during CUS reversed this depressive behavior.

[0046] (3) Forced swimming test see Figure 4 Mice subjected to CUS for 14 days exhibited depressive behavior with increased immobility time, but injection of APOB protein during CUS reversed this depressive behavior.

[0047] Example 2

[0048] Apob gene knockout: AAV-Vgat-Apob shRNA-GFP virus was injected directly into the DMS brain region of C57 mice. AAV-Vgat-NC-shRNA-GFP was used as a control virus. Viral expression is shown in [the table below]. Figure 5 .

[0049] Following Apob interference virus injection, behavioral assessments were conducted. In the FUST experiment, mice injected with AAV-Vgat-Apob shRNA-GFP knockout mice exhibited depressive behavior. Figure 6 In the FUST experiment, AAV-Vgat-Apob shRNA-GFP knockout mice exhibited depressive behavior after specific knockout of the Apob gene in GABAergic neurons of the DMS brain region.

[0050] 7) After Apob interference virus injection, behavioral assessments were performed. In the FST experiment, mice injected with AAV-Vgat-Apob shRNA-GFP knockout mice exhibited depressive behavior. (See...) Figure 7 In the FST experiment, AAV-Vgat-Apob shRNA-GFP knockout mice exhibited depressive behavior after specific knockout of Apob gene in GABAergic neurons of the DMS brain region.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of an ApoB protein for the preparation of a medicament for alleviating depression, characterized in that, The model number of the Apob protein is RPC003Mu01; The concentration of the Apob protein is 0.05 ug / ul.

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