Application of Apob gene in preparation of medicine for relieving depression

Abstract

The invention discloses application of an Apob gene in preparation of drugs for relieving depression, and belongs to the technical field of biological medicines. According to the present invention, it is found that the APOB protein of the brain region of the DMS is overexpressed, and the APOB protein has the anti-depression effect; when the Apob gene in the GABA energy neuron of the dorsal medial striatum (DMS) is knocked out, the mouse is correspondingly irritable and susceptible, which indicates that the Apob gene has a positive effect in dealing with depression.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to the application of the Apob gene in preparing a drug for alleviating depression. Background Art

[0002] Depression is a common mental disorder, often called the "common cold" of psychiatry. Its lifetime prevalence is as high as 10% to 20%, affecting 350 million people worldwide. Depression is characterized by high morbidity, high recurrence rates, and high disability rates. It is the leading cause of disability worldwide and a significant contributor to the global burden of disease. The biological etiology and pathogenesis of depression remain unclear, but it is generally believed to result from a combination of genetic and environmental factors. The pathogenesis of depression involves multiple highly complex and interconnected pathways, 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 a universal drug target for the unclear genetic etiology and pathogenesis of depression poses significant challenges to clinical treatment.

[0003] At present, there are more than 30 kinds of antidepressants in the domestic medical market. These antidepressants cover various mechanisms of action, but the difference in their curative effects on patients is not significant. There are still quite a number of patients who do not see obvious results after drug treatment, and the antidepressant effect of drug treatment is very limited. Currently, the antidepressants currently being used clinically almost all improve depressive symptoms by affecting the signal transmission of monoamine neurotransmitters (including serotonin and norepinephrine), and this drug target was discovered 30 years ago. The defect of this type of drug is that it can only relieve the symptoms of a small number of patients, and it takes weeks or even months to alleviate the symptoms of depression, and the side effects are serious.

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

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

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides the use of the Apob gene in preparing a drug for alleviating depression.

[0008] In a second aspect, the present invention provides use of an Apob gene expression vector in the preparation of a drug for alleviating depression.

[0009] In a third aspect, the present invention provides the use of APOB protein in the preparation of a drug for alleviating depression.

[0010] In a fourth aspect, the present invention provides the use of the Apob gene in screening drugs for alleviating depression. The Apob gene is used as the target of drugs or preparations, and drugs or preparations for alleviating depression are screened based on regulating the Apob gene. The screened drugs or preparations can promote the transcription or translation of the Apob gene, or can increase the expression or activity of the APOB protein.

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

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

[0013] Contains at least the following beneficial technical effects:

[0014] The present invention found 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 susceptible to stress, indicating that the Apob gene plays a positive role in coping with depression. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 See the figure for verification of the ink injection position in Example 1.

[0016] Figure 2 This is a diagram showing the results of the sugar water preference experiment in Example 1.

[0017] Figure 3 Figure 1 shows the results of the female mouse urine sniffing experiment in Example 1.

[0018] Figure 4 This is a diagram showing the experimental results of the forced swimming test in Example 1.

[0019] Figure 5 This is the viral expression diagram in Example 2.

[0020] Figure 6 This is a diagram of the FUST experiment results in Example 2.

[0021] Figure 7 This is the FST experimental result diagram in Example 2. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting 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 terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

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

[0027] Unless otherwise specified, the "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C.

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

[0029] Example 1

[0030] Mouse selection:

[0031] The mice used in this example were C57BL / 6 mice (Stock No: 000664) purchased from Jackson Laboratory and successfully maintained in our SPF laboratory animal center. The mice used in this example were adult male mice weighing 25 ± 5 g. All mice were housed in a 12-hour light and 12-hour dark environment with ample water and food supply, and the humidity of the housing environment was 50%-60%. The principle of a reasonable and optimal number of experimental mice was to minimize animal suffering during the experimental procedures, and the experimental animals were randomly grouped.

[0032] Experimental Project:

[0033] Chronic unpredictable stress (CUS): This method was performed according to published literature (Lei et al., Molecular Psychiatry, 2022; Wang et al., Journal of Psychiatric Research, 2021). Briefly, mice were exposed to two stimuli at irregular times daily for 2 weeks: restraint for 2 hours, tail clamping for 15 minutes, 24-hour continuous light exposure, tilted cage with wet bedding for 24 hours, 10 minutes of inescapable foot shock (0.3 mA current for 2 seconds, 16 seconds rest), and high platform placement for 30 minutes.

[0034] The sucrose preference test (SPT) examines an animal's ability to experience pleasure from natural rewards. Mice are acclimated for one week by placing water bottles on the food and water sides of their cages. Over a two-hour period (7:00 PM to 9:00 PM), sucrose consumption is measured. A 1% (w / v) sucrose solution is placed on the food side of the cage, while drinking water is placed on the contralateral side, allowing the mice to freely choose between drinking water and sucrose solution. Sugar preference is expressed as the percentage of sucrose solution consumption relative to total fluid consumption.

[0035] Forced swimming test (FST): Mice were placed in a cylindrical glass container (25 cm long, 16 cm high, filled with 15 cm of water at 24°C). The video was recorded for 6 minutes, and the duration of immobility for the last 4 minutes was recorded. Immobility was defined as the mouse floating on the surface of the water, with only slight movements to maintain breathing and keep the head above water.

[0036] Female urine sniffing test (FUST): Cotton swabs were placed in the mice's cages one hour before the test to acclimate them and eliminate their novelty and fear of cotton swabs. The test was conducted in a dimly lit room with a light intensity of 3 lux and was divided into two phases. A cotton swab soaked in 80 μl of pure water and another 80 μl of urine from an estrous female mouse (vaginal smear test) were placed in the mice's cages. The sniffing time of the cotton swabs was recorded over three minutes, with 45 minutes between each phase.

[0037] Insertion of injection cannula: Mice were anesthetized with intraperitoneal injection of sodium pentobarbital solution (40 mg / kg), fixed on a brain stereotaxic apparatus with ear bars, placed in a prone position, a midline incision was made on the head, the periosteum was peeled off, the skull surface was exposed, the anterior and posterior fontanelles were at the same level, and the left and right were adjusted to keep them level. The drilling position was determined according to the brain atlas, and a hole was drilled in the skull above the brain area with a drill. The cannula used coordinates: DMS: (AP: +0.65mm; ML: ±1.4mm; DV: -2.0mm). The depth of the cannula implantation was 1mm higher than the depth of the brain area. After the skull was drilled, the medical cannula was implanted and fixed with dental cement. After the behavioral test was completed, ink was injected at the cannula position through the catheter, and the brain tissue was sliced and the injection site was observed under a microscope to see if it was accurate. Mice with incorrect injection sites were excluded. After the mice were injected with drugs through the cannula, ink was injected to verify the position. Figure 1 .

[0038] Experimental methods:

[0039] For each experiment, mice were divided into three groups: one control group received 0.9% saline; the second group received CUS plus 0.9% saline; and the third group received CUS plus APOB protein injection (APOB protein was diluted in 0.9% saline to a concentration of 0.05 μg / μl, and 0.5 μl was injected into each DMS region). Recombinant Apolipoprotein B (APOB) (RPC003Mu01, Cloud-Clone Corp., Wuhan) was injected into the DMS region of the brain at a concentration of 0.5 μl per side. Mice underwent CUS and DMS microinjection for 14 days, and then underwent experimental procedures as described above. The detailed experimental schedule for the 14-day CUS treatment is shown in Table 1.

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

[0041]

[0042] Experimental results:

[0043] After 14 days, the mice were tested for depressive behavior, and the results were as follows:

[0044] (1) Sugar water preference experiment Figure 2 : After 14 days of CUS, mice showed depressive behavior with reduced preference for sugar water, while APOB protein injection during CUS reversed the depressive behavior of mice with reduced preference for sugar water.

[0045] (2) Female rat urine sniffing test Figure 3 After 14 days of CUS, mice showed depressive behavior of smelling reduced female urine, while APOB protein injection during CUS reversed the depressive behavior of mice smelling reduced female urine.

[0046] (3) Forced swimming test Figure 4 After 14 days of CUS, mice showed depressive behavior with increased immobility time, while APOB protein injection during CUS reversed the depressive behavior of mice with increased immobility time.

[0047] Example 2

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

[0049] After the Apob interfering virus was injected, behavioral tests were performed. In the FUST experiment, mice injected with AAV-Vgat-Apob shRNA-GFP showed depressive behavior. Figure 6 , the Apob gene was specifically knocked out in the GABAergic neurons of the DMS brain region. In the FUST experiment, AAV-Vgat-Apob shRNA-GFP knockout mice showed depressive behavior.

[0050] 7) After Apob interfering virus injection, behavioral assays were performed. In the FST experiment, mice injected with AAV-Vgat-Apob shRNA-GFP showed depressive behavior. Figure 7 The Apob gene was specifically knocked out in the GABAergic neurons of the DMS brain region. In the FST experiment, AAV-Vgat-Apob shRNA-GFP knockout mice showed depressive behavior.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of Apob gene in the preparation of drugs for alleviating depression.

2. Application of Apob gene expression vector in the preparation of drugs for alleviating depression.

3. Application of Apob protein in the preparation of drugs for alleviating depression.

4. The use of the Apob gene in screening drugs for alleviating depression is characterized in that: The Apob gene is used as the target of drugs or preparations, and drugs or preparations that relieve depression are screened based on regulating the Apob gene. The screened drugs or preparations can promote the transcription or translation of the Apob gene, or increase the expression or activity of the Apob protein.

5. A drug for relieving depression, characterized in that: It includes one or more of Apob gene, Apob gene expression vector and Apob protein.

6. The drug according to claim 5, characterized in that The medicine further contains pharmaceutically acceptable excipients.

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