Use of mydgf in preventing and treating memory impairment and related diseases

Abstract

This invention discloses the application of MYDGF in the prevention and treatment of memory impairment and related diseases. This invention discloses that MYDGF shows significant differences in individuals with memory impairment. After administration of MYDGF, memory impairment can be prevented and memory impairment can be prevented and the death of newly generated neurons can be stopped.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of MYDGF in the prevention and treatment of memory impairment and related diseases. Background Technology

[0002] Chronic stress is a major risk factor for many human diseases in modern society, and the brain is a primary target of chronic stress. Increasing evidence suggests that prolonged exposure to stress conditions can affect learning and memory, decision-making, and emotional responses, and may even induce pathological processes such as Alzheimer's disease (AD) and depression (Oloiira TG, Chan RB, Bravo FV, et al. The impact of chronic stress on the rat brain lipidome[J]. Mol Psvchiatry, 2016, 21(1):80-88.). Prolonged chronic stress increases the risk of obesity, hypertension, heart disease, and digestive problems. Furthermore, it may be associated with weakened immune system function, leading to depression, impaired fertility, and memory impairment.

[0003] In today's fast-paced society, people face increasing work pressure, coupled with frequent natural disasters and public health events, leading to various forms of chronic stress. The World Health Organization estimates the prevalence of stress-related mental disorders at 22.1% (Chanson F, Van OM, Flaxman A, et al. New WHO prevalence estimates of mental disorders in conflict settings: a systematic review and meta-analysis[J]. Lancet, 2019, 394(10194):240-248.), exceeding the prevalence of mental disorders in the general population of my country (Huang Y, Wang Y, Wang H, et al. Prevalence of mental disorders in China: a cross-sectional epidemiological study[J]. Lancet Psychiatry, 2019, 6(3):211-224.). Continuous chronic stress may impair the body's learning and memory abilities.

[0004] In recent years, numerous studies have reported that chronic stress, by damaging the negative feedback balance of the HPA axis, activates hippocampal glucocorticoid receptors, increases neuronal metabolism, and reduces neuronal survival and regeneration. Furthermore, it influences long-term enhancement and cognitive function by promoting dendritic atrophy (McEwen BS, Magarinos AM. Stress and hippocampalplasticity: implications for the pathophysiology of affective disorders[J]. Human psychopharmacology. 2001, 16(S1): S7-S19.), thereby inducing neuropsychiatric disorders. The chronic restraint stress (CRS) model is a widely used common stress model for exploring the learning and memory impairments and depressive behaviors induced by chronic stress (Liu Y, Zhuang X, Gou L, et al. Protective effects of nizofenone administration on the cognitive impairments induced by chronic restraint stress in mice[J]. Pharmacology, biochemistry, and behavior. 2013, 103(3): 474-80.). Currently, there are no specific drugs for treating chronic stress, so there is an urgent need for anti-stress drugs to improve the condition, especially to restore cognitive function. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide drugs and methods for preventing and treating memory impairment.

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

[0007] The first aspect of the present invention provides the use of a reagent for detecting MYDGF in the preparation of products for detecting memory impairment or memory impairment-related diseases.

[0008] In some embodiments, the reagent includes an oligonucleotide probe that specifically recognizes the MYDGF gene, a primer that specifically amplifies the MYDGF gene, or a binding agent that specifically binds to the protein encoded by the MYDGF gene.

[0009] A second aspect of the present invention provides the application of a reagent for detecting MYDGF in the preparation of products for diagnosing memory impairment-related diseases.

[0010] In some embodiments, the reagent includes an oligonucleotide probe that specifically recognizes the MYDGF gene, a primer that specifically amplifies the MYDGF gene, or a binding agent that specifically binds to the protein encoded by the MYDGF gene.

[0011] In some implementations, the memory impairment-related diseases include depression and Alzheimer's disease.

[0012] A third aspect of the present invention provides a product for detecting memory impairment or memory impairment-related diseases, characterized in that the product comprises a chip, a reagent kit, or a nucleic acid membrane strip capable of detecting MYDGF expression levels.

[0013] In some embodiments, the chip includes a gene chip and a protein chip, the gene chip including an oligonucleotide probe against the MYDGF gene for detecting the transcriptional level of the MYDGF gene, and the protein chip including a specific binding agent for the MYDGF protein; the kit includes a gene detection kit and a protein detection kit, the gene detection kit including reagents or a chip for detecting the transcriptional level of the MYDGF gene, and the protein detection kit including reagents or a chip for detecting the expression level of the MYDGF protein.

[0014] In some embodiments, the kit includes reagents for detecting the expression level of the MYDGF gene or protein by RT-PCR, qRT-PCR, microarray detection, DNA blotting, in situ hybridization, or immunoblotting.

[0015] The fourth aspect of this invention provides the application of MYDGF in constructing models for predicting memory impairment / memory impairment-related diseases.

[0016] The fifth aspect of this invention provides the use of MYDGF in the preparation of a medicament for preventing and treating memory impairment in subjects.

[0017] In some embodiments, the drug includes a MYDGF promoter.

[0018] In some implementations, the promoter specifically promotes the expression level of MYDGF.

[0019] In some embodiments, the promoter is a MYDGF overexpression vector or MYDGF protein.

[0020] In some implementations, the memory impairment-related diseases include depression and Alzheimer's disease.

[0021] In some implementations, the drug prevents the death of newly formed neurons.

[0022] The sixth aspect of this invention provides the use of MYDGF in the preparation of medicaments for the prevention and treatment of diseases related to memory impairment in subjects.

[0023] In some embodiments, the drug includes a MYDGF promoter.

[0024] In some implementations, the promoter specifically promotes the expression level of MYDGF.

[0025] In some embodiments, the promoter is a MYDGF overexpression vector or MYDGF protein.

[0026] In some implementations, the memory impairment-related diseases include depression and Alzheimer's disease.

[0027] In some implementations, the drug prevents the death of newly formed neurons.

[0028] The seventh aspect of the present invention provides a medicament for preventing or treating memory impairment or memory impairment-related diseases, the medicament comprising a promoter of MYDGF.

[0029] In some implementations, the promoter specifically promotes the expression level of MYDGF.

[0030] In some embodiments, the promoter is a MYDGF overexpression vector or MYDGF protein.

[0031] In some implementations, the drug includes a pharmaceutically acceptable carrier.

[0032] The eighth aspect of the present invention provides a method for preventing the death of newly generated neurons by administering an effective amount of a MYDGF promoter.

[0033] In some implementations, the promoter specifically promotes the expression level of MYDGF.

[0034] In some embodiments, the promoter is a MYDGF overexpression vector or MYDGF protein.

[0035] In this invention, the method can be used for research and other non-therapeutic purposes.

[0036] The ninth aspect of this invention provides the application of MYDGF in screening candidate drugs for the prevention and treatment of memory impairment or memory impairment-related diseases.

[0037] In some implementations, the method for screening candidate drugs is as follows: treating a culture system expressing or containing the MYDGF gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the MYDGF gene or its encoded protein in the system; wherein, when the substance to be screened promotes the expression level or activity of the MYDGF gene or its encoded protein, the substance to be screened is a candidate drug for the prevention and treatment of memory impairment-related diseases.

[0038] The tenth aspect of this invention provides a method for screening candidate drugs for preventing and treating memory impairment-related diseases, the method comprising: treating a culture system expressing or containing the MYDGF gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the MYDGF gene or its encoded protein in the system; wherein, when the substance to be screened promotes the expression level or activity of the MYDGF gene or its encoded protein, the substance to be screened is a candidate drug for preventing and treating memory impairment-related diseases.

[0039] Advantages and beneficial effects of the present invention:

[0040] This invention is the first to discover that MYDGF is significantly downregulated in patients with memory impairment. By detecting the level of MYDGF, memory impairment or memory impairment-related diseases can be detected or diagnosed.

[0041] This invention is the first to discover that MYDGF can be used for the prevention and treatment of memory impairment or memory disorders. Using MYDGF can effectively prevent and treat memory impairment and inhibit the death of newly formed neurons. Attached Figure Description

[0042] Figure 1 This is a diagram showing the expression of cytokines.

[0043] Figure 2 These are the results of the water maze experiment. 2A shows the results of the water maze test for the first 4 days; 2B shows the results of the number of times the typhoon entered the NW area after the typhoon was removed on the 5th day; and 2C shows the results of the time spent in the NW area after the typhoon was removed on the 5th day.

[0044] Figure 3 This is a diagram showing the effect of MYDGF on newly formed neurons in the hippocampus. In the diagram, 3A is a neuronal cell image detected by immunofluorescence, and 3B is a statistical diagram of neuronal cells. Detailed Implementation

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Otherwise, certain terms used herein have the meanings set forth in this specification.

[0046] In this invention, the terms "memory impairment," "memory disorder," "learning disability," "learning and memory disorder," and "cognitive disorder" are used interchangeably and should be understood to include any cognitive disorder or condition. Non-limiting examples of such cognitive disorders or conditions include Attention Deficit Disorder (ADD), Attention Deficit Hyperactivity Disorder (ADHD), dyslexia, age-related memory impairment and learning disabilities, amnesia, mild cognitive impairment, cognitive impairment-associated non-dementia pre-Alzheimer's disease, autism, dystonia and Tourette syndrome, dementia, age-related cognitive decline, cognitive deterioration, moderate mental impairment, age-related mental deterioration, conditions affecting brainwave intensity and / or brain glucose utilization, tension, anxiety disorders, concentration and attention disorders, mood deterioration, general cognitive and mental health, neurodegenerative diseases, hormonal imbalances, depression, or any combination thereof.

[0047] In this invention, the term "differential expression" refers to a difference in the expression levels of one or more biomarkers of the present invention in a sample, compared to the expression levels of the same one or more biomarkers of the present invention in a second sample, by measuring the amount or level of mRNA. "Differential expression" may also include a measurement of the protein encoded by the biomarker of the present invention in the sample or sample group, compared to the protein expression level or level in the second sample or sample group. Differential expression can be determined by methods as described herein and understood by those skilled in the art. The term "differential expression" or "change in expression level" refers to an increase or decrease in the measurable expression level of a given biomarker in a sample, compared to the measurable expression level of a given biomarker in a second sample, by measuring the amount of RNA and / or protein. The term "differential expression" or "change in expression level" may also refer to an increase or decrease in the measurable expression level of a given biomarker in a sample group, compared to the measurable expression level of a biomarker in a second sample group.

[0048] This invention includes any method available in the art for detecting the expression of intrinsic genes described herein. “Detection of expression” means determining the amount or presence of the RNA transcript of an intrinsic gene or its expression product. Methods for detecting the expression of intrinsic genes disclosed herein, i.e., gene expression profiling, include methods based on multinucleotide hybridization analysis, multinucleotide sequencing, immunohistochemistry, and proteomics. These methods typically detect the expression product (e.g., mRNA) of the intrinsic genes described herein. In preferred embodiments, PCR-based methods, such as reverse transcription PCR (RT-PCR), and array-based methods, such as microarrays, are used. “Microarray” refers to an ordered arrangement of hybridizable array elements, such as, for example, polynucleotide probes, on a matrix. The term “probe” refers to a molecule capable of selectively binding to a particularly intended target biomolecule, such as a nucleotide transcript or protein encoded by or corresponding to an intrinsic gene. Probes can be synthesized by those skilled in the art or can be derived from suitable biopreparations. Probes can be specifically designed to be labeled. Examples of molecules that can be used as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0049] In this invention, the binding agent that specifically binds to the protein encoded by the MYDGF gene includes, for example, a receptor for the MYDGF protein, a lectin that binds to the MYDGF protein, an antibody against the MYDGF protein, a peptide body against the MYDGF protein, a bispecific dual binding agent, or a bispecific antibody. Specific examples of the specific binding agent are peptides, peptide mimics, aptamers, spiegelmers, darpins, ankylosing spicules, Kunitz domains, antibodies, single-domain antibodies, and monovalent antibody fragments. In a specific embodiment of this invention, the specific binding agent is a MYDGF-specific antibody.

[0050] This invention provides the application of MYDGF in constructing computational models for predicting memory impairment or memory impairment-related diseases. As those skilled in the art will know, the steps of associating biomarker levels with a certain probability or risk can be implemented and realized in various ways. Preferably, the measured concentrations of the biomarker and one or more other biomarkers are mathematically combined, and the combined value is associated with the underlying diagnostic question. The measured biomarker values ​​can be combined using any suitable existing mathematical method.

[0051] This invention provides the application of MYDGF in the preparation of medicaments for the prevention and treatment of memory impairment or memory impairment-related diseases, and the medicaments for the prevention and treatment of memory impairment or memory impairment-related diseases, wherein the medicaments include MYDGF promoters. The promoters refer to any substance that can increase the activity of MYDGF protein, improve the stability of the MYDGF gene or protein, upregulate the expression of MYDGF protein, increase the effective duration of MYDGF protein action, or promote the transcription and translation of the MYDGF gene. These substances can be used in this invention as substances useful for upregulating MYDGF, thereby being used for the prevention and treatment of memory impairment or memory impairment-related diseases. For example, the promoters include nucleic acid promoters and protein promoters. The promoters include, but are not limited to, vectors overexpressing MYDGF, MYDGF protein, or its active peptides.

[0052] This invention provides a medicament for preventing and treating memory impairment or memory impairment-related diseases, the medicament comprising an effective amount of MYDGF promoter.

[0053] In some implementations, the promoter specifically promotes the expression level of MYDGF.

[0054] In some embodiments, the promoter is a MYDGF overexpression vector or MYDGF protein.

[0055] While the drug can be administered alone, it is preferably administered as a pharmaceutical formulation comprising at least one active ingredient, such as MYDGF, and one or more pharmaceutically acceptable carriers. Each carrier must be "acceptable," meaning it is compatible with the other components of the formulation and will not cause harm to the patient.

[0056] Formulations include preparations suitable for oral, rectal, nasal, topical (including transdermal, oral, and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), and pulmonary administration. These preparations can be conveniently presented in unit dosage forms and can be prepared by any method well-known in the pharmaceutical field. Such methods involve the step of binding the active ingredient with a carrier constituting one or more excipients. Generally, the preparation method involves homogeneously and tightly binding the active ingredient with a liquid carrier or a finely separated solid carrier, or both, and then shaping the product as necessary.

[0057] The formulations of the present invention suitable for oral administration can exist as discrete units, such as capsules, buffers, or tablets, each unit containing a predetermined amount of the active ingredient; as powders or granules; as solutions or suspensions in aqueous or non-aqueous liquids; or as oil-in-water emulsions or water-in-oil emulsions. The active ingredient may also be in the form of pellets, pastes, or granules.

[0058] Tablets can be made by compression or molding and may optionally contain one or more excipients. Compressed tablets can be prepared by compressing a free-flowing active ingredient (such as powder or granules) in a suitable machine, optionally mixed with a binder (e.g., povidone, gelatin, hydroxypropyl methylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium glycolate starch, crospovidone, crospovidone), surfactant, or dispersant. Molded tablets can be prepared by molding a mixture of powdered compounds wetted with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored and may be formulated to provide a slow or controlled release of the active ingredient therein using, for example, hydroxypropyl methylcellulose in varying proportions to provide a desired release profile. Tablets may optionally be coated with an intestinal coating for release in the intestinal portion other than the stomach.

[0059] Suitable formulations for topical oral administration include lozenges containing active ingredients on a flavored base, typically sucrose and gum arabic or scutellaria baicalensis; and tablets containing active ingredients on an inert base (such as gelatin and glycerin, or sucrose and gum arabic).

[0060] The pharmaceutical compositions for topical administration according to the present invention can be formulated as ointments, creams, suspensions, emulsions, powders, solutions, pastes, gels, sprays, aerosols or oils.

[0061] Formulations suitable for topical ocular application also include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, particularly in an aqueous solvent used in the formulation.

[0062] Formulations suitable for nasal administration, wherein the carrier is a solid, including, for example, coarse powder having a particle size in the range of about 20 to about 500 micrometers, which is administered as a dry powder or rapidly inhaled through a nasal passage from a powder container near the nose in an inhaler device. The carrier is a suitable formulation for an application of a liquid (e.g., a nasal spray, nasal drops, or an aerosol via a nebulizer), including an aqueous or oil solution of the formulation.

[0063] Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation isotonic with the blood of the intended subject; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickeners, as well as liposomes or other microparticle systems designed to target the compound to blood components or one or more organs. Formulations may be presented in single-dose or multi-dose sealed containers (e.g., ampoules and vials) and may be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier (e.g., water for injection) immediately before use. Temporary injectable solutions and suspensions may be prepared from sterile powders, granules, and tablets of the types described above.

[0064] In this invention, pharmaceutically acceptable carriers include, but are not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulation agents, excipients, fillers, binders, sprays, transdermal absorbents, humectants, disintegrants, absorption enhancers, surfactants, colorants, flavoring agents, adsorbents, etc.

[0065] Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). In some embodiments, a sterile phosphate-buffered saline is used as a pharmaceutically acceptable diluent.

[0066] It should be understood that, in addition to the ingredients specifically mentioned above, the medicaments of the present invention may include other formulations commonly used in the art. Considering the type of the formulation, for example, formulations suitable for oral administration may include other formulations as sweeteners, thickeners and flavoring agents.

[0067] It will also be apparent to those skilled in the art that the effective dose of the medicament of the present invention will vary depending on the desired effect. Therefore, those skilled in the art can readily determine the optimal dose to be administered, and the optimal dose will vary depending on the specific compound used, the route of administration, the strength of the formulation, and the progression of the disease condition. Furthermore, factors relevant to the specific patient receiving treatment, including the patient's age, weight, diet, and timing of administration, will necessitate adjustments to the dose to an appropriate therapeutic level.

[0068] According to the specific implementation plan, an effective amount or effective dose refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, disorder, or condition to be treated or related symptoms; (ii) reducing the duration of the disease, disorder, or condition to be treated or related symptoms; (iii) preventing the development of the disease, disorder, or condition to be treated or related symptoms; (iv) causing the resolution of the disease, disorder, or condition to be treated or related symptoms; (v) preventing the development or recurrence of the disease, disorder, or condition to be treated or related symptoms. (vi) to prevent recurrence of the treated disease, disorder, or condition or related symptoms; (vii) to reduce hospitalization of subjects suffering from the treated disease, disorder, or condition or related symptoms; (viii) to reduce the length of hospital stay of subjects suffering from the treated disease, disorder, or condition or related symptoms; (ix) to improve the survival of subjects suffering from the treated disease, disorder, or condition or related symptoms; (xi) to suppress or reduce the treated disease, disorder, or condition or related symptoms in subjects; and / or (xii) to enhance or improve the preventive or therapeutic effects of another therapy.

[0069] The effective therapeutic dose or dosage can vary depending on various factors, such as the disease, disorder, or condition to be treated, the route of administration, the target site, the subject's physiological state (including, for example, age, weight, and health), whether the subject is a human or an animal, other drugs administered, and whether the treatment is preventative or therapeutic.

[0070] In this invention, “prevention” of a symptom or disease means (1) preventing the development of symptoms or disease in a susceptible or unexhibited subject; (2) suppressing a disease or preventing its development or recurrence; or (3) improving or causing the disease or symptom to subside. As understood in the art, “prevention” is a method for obtaining beneficial or desired results (including clinical results). For the purposes of this technology, beneficial or desired results may include, but are not limited to, the reduction or improvement of one or more symptoms, the reduction of the severity of a symptom (including disease), the stabilization (i.e., non-deterioration) of a symptom (including disease), the delay or mitigation of a symptom (including disease), the progression, improvement or mitigation (whether partial or complete) of a symptom (including disease), state or remission, whether detectable or not.

[0071] In this invention, "subject" refers to any animal, preferably a mammal, and most preferably a human. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, and more specifically, humans, such as those with memory impairment, depression, or Alzheimer's disease.

[0072] The present invention further provides a method for screening candidate drugs for preventing and treating memory impairment or memory impairment-related diseases. The method includes treating a culture system expressing or containing the MYDGF gene or its encoded protein with a substance to be screened; and detecting the expression or activity of the MYDGF gene or its encoded protein in the system; wherein, when the substance to be screened promotes the expression level or activity of the MYDGF gene or its encoded protein, the substance to be screened is a candidate drug for preventing and treating memory impairment or memory impairment-related diseases.

[0073] In this invention, the method further includes: further testing the candidate drug obtained in the above steps to see its effect on inhibiting memory impairment or memory impairment-related diseases. If the tested compound has a significant inhibitory effect on memory impairment or memory impairment-related diseases, then the candidate drug is a candidate drug for the prevention and treatment of memory impairment or memory impairment-related diseases.

[0074] The culture system includes (but is not limited to) cell systems, subcellular systems, solution systems, tissue systems, organ systems, or animal systems (such as animal models, preferably non-human mammalian animal models, such as mice, rabbits, sheep, monkeys, etc.).

[0075] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0076] Example 1: Expression of MYGDF in individuals with memory impairment

[0077] A previously filed national invention patent, "Method for constructing and applying a mouse model of CD317-induced depression and memory impairment (Patent No.: 202011100708.6, authorized on 2021-9-27)," discovered that long-term clearance of pDC by CD317 neutralizing antibodies leads to depression and memory impairment. Using the method described in the patent, a mouse model of depression and memory impairment was constructed. Whole-cell high-throughput sequencing was performed on brain tissue from mice treated with control IgG, CD317 neutralizing antibodies, and CD317 neutralizing antibodies combined with eleutheroside E (EE) to detect genes showing significant differences. The detection steps are as follows:

[0078] I. Total RNA extraction using the Trizol method

[0079] 1. Collect cells into clean EP tubes, wash once with pre-cooled PBS buffer, centrifuge and discard the supernatant;

[0080] 2. Add 500 μl Trizol, pipette and invert to mix for 15 seconds to lyse the cells, and let stand at room temperature for 5 minutes;

[0081] 3. Add 100 μl of chloroform, mix vigorously by inverting, vortex for 10 seconds, and let stand at room temperature for 10 minutes.

[0082] Centrifuge at 4.4℃ and 12,000 rpm for 15 min. At this time, the solution in the EP tube will separate into three layers: upper, middle and lower. Transfer the upper aqueous phase layer to a new EP tube, being careful not to aspirate the middle or lower layer to avoid contaminating the RNA.

[0083] 5. Add an equal volume of isopropanol (about 200 μl), gently invert to mix, and let stand at room temperature for 15 min;

[0084] Centrifuge at 6.4℃ and 12,000 rpm for 10 min, discard the supernatant, and you will see a small white precipitate at the bottom of the test tube, which is RNA. Add 1 ml of pre-cooled 75% ethanol (prepared with DEPC water) to wash the precipitate.

[0085] Centrifuge at 7.4℃ and 12,000 rpm for 10 min, discard the supernatant, and open the lid to allow the remaining ethanol to evaporate completely;

[0086] 8. Add 30µl of DEPC water, vortex to mix, measure the RNA concentration, and store in a -80℃ freezer or for subsequent experiments.

[0087] II. cDNA Library Construction and Sequencing

[0088] 1 µg of total RNA was used in subsequent experiments, and a cDNA library was prepared using the NEBNext Ultra™ RNA Library Prep kit. Sequencing was performed using the Illumina HiSeq platform, generating 150 bp end-paired sequences. The sequence read value for each gene was calculated using HTSeq v0.6.0, and the FPKM (Fragments per Kilobase of transcript sequence per Millions base pairs sequenced) for each gene was calculated. For gene i, the calculation formula is as follows: FPKM i =X i *10 9 / (l i N). X i The raw sequencing read of gene i; l i , the sum of the lengths of all exons of gene i; N: the expression level of all genes in the sample, also known as sequencing depth.

[0089] III. Results

[0090] The results are as follows Figure 1 As shown, MYGDF mRNA levels were reduced in individuals with depression and memory impairment, and the reduction in MYDGF mRNA levels was reversed by eleutheroside E (EE), which maintains memory.

[0091] Example 2: The effect of MYGDF on memory impairment

[0092] I. Experimental Grouping

[0093] Eight-week-old C57 BL / 6 mice were randomly divided into three groups: one group was restricted for 8 hours a day (9:00-17:00) for 14 consecutive days (Restraint group); another group received intranasal administration of MYDGF (0.5 mg / mouse / day; Restraint+MYDGF group) before restriction; and the third group was not restricted, but its food and water were removed only when the other mice were restricted each day (Ctrl group).

[0094] Methods of restraint:

[0095] The restraint device is a modified 50 mL centrifuge tube (in addition to several small holes drilled in the tube wall, there should also be a small hole at the tip and cap to facilitate the mouse's breathing and tail movement). When restraining, the restraint tube should be placed at a 45° angle on the cage, and the mouse's tail should pass through the hole in the cap of the restraint tube to prevent the tail from breaking due to prolonged curling, which could lead to the mouse's death. After the restraint period, return the mouse to its original cage and allow it to drink water and be fed normally.

[0096] II. Water Maze Experiment

[0097] 1. Preparations before the experiment:

[0098] 1) Pour water into the water maze, ensuring the water level is approximately 1 cm above the platform. Maintain a suitable water temperature (21-22℃ for mice). If the water temperature is too low, use a water heater to add hot water to the water maze and adjust it to a suitable temperature. Bring the animals into the laboratory 30 minutes before the experiment to familiarize them with the environment.

[0099] 2) Turn on the experimental computer and observe whether the light distribution on the water surface is uniform through the monitoring system. Since turning on the lights will cause glare, the indoor lights should be turned off and the curtains opened.

[0100] 3) Check that the markers are in a fixed position and that they cannot be moved throughout the several days of the experiment.

[0101] 2. Formal Experiment:

[0102] The numbered mice were placed into the water maze one by one for the experiment. The specific procedure was as follows:

[0103] 1) Remove the mouse from its cage and place it facing the pool wall into the water maze quickly; do not throw it directly in. Select entry points in all four quadrants, and test each entry point for each group, for a total of four rounds (alternating between clockwise and counterclockwise, from closest to furthest from the platform, daily). The moment the mouse enters the water, immediately tighten the enclosure to prevent light and human interference. Then quickly press the record button.

[0104] 2) Set the recording time to 60 seconds. When the animal reaches the platform and stays there for 30 seconds, it is considered to have found the platform, and the recording will stop automatically. If the mouse does not swim to the platform within the specified time, guide the animal to the platform and let it stay there for 30 seconds to familiarize itself with the surrounding environment before taking it away. If the mouse reaches the platform within the specified time, it should also stay there for 10 seconds to familiarize itself with the platform before taking it away.

[0105] 3) After testing one bird, dry it with a towel before testing the next bird. After one round, wait 30 minutes before starting the next round, for a total of four rounds.

[0106] 4) Positioning and navigation experiment (to test its learning ability) for 4 days. On the 5th day, the platform is removed, which is the space exploration experiment (to test its memory ability). The opposite quadrant of the quadrant where the platform is located is selected as the entry point into the water. This is done only once, and the video recording time is 60 seconds.

[0107] Precautions:

[0108] 1. Ensure that the position of the marked object does not change within the same experiment.

[0109] 2. Do not arbitrarily change the position of other experimental items in the laboratory.

[0110] 3. Maintain a quiet environment in the laboratory as much as possible during the experiment.

[0111] 4. During the water maze experiment, it is best for researchers to avoid using perfumes or other items with pungent odors.

[0112] 3. Statistical Analysis

[0113] 1) When there is a platform, statistical analysis is performed based on three indicators: time to reach the platform, distance, and speed.

[0114] 2) When there is no platform, four indicators are used: compare the quadrant where the shuttle platform is located and the number and time of shuttle platform visits.

[0115] 3) Data analysis was performed using SPSS software with various repeated analysis.

[0116] 4. Results

[0117] The results showed that in the first 4 days, the learning speed of mice in the Resttraint group was slower than that in the Ctrl group (P values ​​for 4 days were 0.805, 0.016, 0.266, and 0.025, respectively), while the learning speed of mice in the Resttraint+MYDGF group was faster than that in the Resttraint group (P values ​​for 4 days were 0.979, 0.314, 0.025, and 0.008, respectively), similar to that in the Ctrl group. After the platform was removed on the 5th day, mice in the Resttraint group entered the platform quadrant less often than those in the Ctrl group, while mice in the Resttraint+MYdGF group showed an increasing trend in the number of times they entered the platform quadrant compared to the Resttraint group, and the time spent entering the platform quadrant was prolonged. Figure 2 This indicates that MYDGF plays a role in maintaining memory.

[0118] III. Immunofluorescence assay for detecting newly formed neurons

[0119] 1. Immunofluorescence detection

[0120] 1) Frozen sections of mouse hippocampus were placed at room temperature for 5-10 minutes to allow them to warm up, and then washed three times with PBS for 5 minutes each time.

[0121] 2) Pour in ice-cold methanol to ensure the volume is sufficient to submerge the tissue, and fix at -20℃ for 1 h.

[0122] 3) After the time is up, remove the slides and wash them three times with PBS for 5 minutes each time.

[0123] 4) Carefully draw circles around the patch with a histochemistry pen, then add an appropriate amount of 3% BSA-TritonX-100-PBS blocking solution and block at room temperature for 1~2 h.

[0124] 5) Dilute the DCX antibody (CST, 4604) 1:100 with blocking solution, drop it onto the tissue, and incubate overnight in a display cabinet at 4°C.

[0125] 6) Take out the slides the next day and decide whether to recover the primary antibody based on the number of times they have been used. Then wash with PBS 3 times, 5 minutes each time.

[0126] 7) Add TRITC-labeled goat anti-rabbit fluorescent secondary antibody (Zhongshan Jinqiao) and stain at 37℃ for 1 h.

[0127] 8) Wash with PBS 3 times, 5 min each time.

[0128] 9) Observe the results using a fluorescence microscope within one week after adding the mounting medium.

[0129] 2. Results

[0130] The results are as follows Figure 3 The results showed that the number of DCX+ neurons, which reflects the number of newly generated neurons, was indeed reduced in the Resttraint group mice, while the Resttraint+MydGF group mice had more newly generated neurons than the Resttraint group mice. This indicates that MYDGF prevents the death of newly generated neurons in the hippocampus and has the function of maintaining memory.

[0131] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. The use of a MYDGF overexpression vector or MYDGF protein in the preparation of a drug for treating memory impairment-related diseases, wherein the memory impairment-related diseases are depression.

2. Use according to claim 1, characterized in that, The drug prevents the death of newly formed neurons.

3. A method for preventing the death of newly formed neurons in vitro, characterized in that, Apply an effective amount of a MYDGF promoter, wherein the MYDGF promoter is a MYDGF overexpression vector or MYDGF protein.

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