Use of active compounds for preventing, delaying or treating cognitive and / or motor dysfunction

Abstract

The invention provides application of a compound shown in a formula I or pharmaceutically acceptable salt thereof and / or hyodeoxycholic acid or pharmaceutically acceptable salt thereof in preparation of a medicine for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction. Muscle strength, athletic ability, active exploration ability and spatial learning and memory ability can be effectively improved, antioxidant pressure resistance and proinflammatory state can be improved, expression of neurotrophic factors is increased, and nerve injury repair is improved.

Description

Technical Field

[0001] The present invention relates to a method and a medicine for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction. Background Art

[0002] Cognitive dysfunction is mainly manifested by abnormalities in the brain's higher-level intellectual processing related to learning, memory, thinking and judgment, resulting in severe learning and memory disorders, accompanied by pathological changes such as aphasia, apraxia, agnosia or apraxia. Common diseases that cause cognitive impairment are dementia caused by various diseases. Motor dysfunction is mainly manifested by dysfunction in voluntary movement regulation while muscle strength, sensation and cerebellar function are not affected. Common movement disorders include Parkinson's disease. Studies have found that a variety of cognitive impairment diseases may be accompanied by movement disorders such as gait abnormalities, and movement disorders may also be accompanied by cognitive dysfunction. Cognitive dysfunction and motor dysfunction have become important health service issues and socioeconomic burdens, especially in the aging society.

[0003] Therefore, there is still much room for development of drugs for the prevention and treatment of cognitive dysfunction or motor dysfunction, especially the development of drugs with few side effects and safe and effective long-term use. Summary of the Invention

[0004] To address one of the aforementioned technical problems in the prior art, the present disclosure provides a drug and treatment method for preventing, delaying, or treating neurodegenerative changes or diseases characterized by cognitive dysfunction and / or motor dysfunction. Treatment with the drug disclosed herein can effectively enhance learning and cognitive function, muscle strength, and antioxidant capacity.

[0005] According to a first aspect of the present disclosure, there is provided a use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction in a subject, wherein the compound of Formula I has the following structure:

[0006]

[0007] Among them, R 1 and R 2 Each is independently selected from H, C1-C6 alkyl, C1-C6 alkenyl and C1-C6 alkynyl.

[0008] In some embodiments, R 1 and R 2 can be independently selected from H.

[0009] In some embodiments, R 1 and R 2Can be independently selected from branched or straight chain C1-C6 alkyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C5 alkyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C4 alkyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C3 alkyl. 1 and R 2 Each may be independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl or n-pentyl.

[0010] In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C6 alkenyl. In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C5 alkenyl. In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C4 alkenyl. In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C3 alkenyl. In some embodiments, R 1 and R 2 Each may be independently selected from ethenyl, propenyl, butenyl or pentenyl.

[0011] In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C6 alkynyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C5 alkynyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C4 alkynyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C3 alkynyl. 1 and R 2 Each may be independently selected from ethynyl, propenyl or butynyl.

[0012] In some embodiments, R1 and R 2 Can be the same or different.

[0013] In some embodiments, the salt is selected from an alkali metal salt, an alkaline earth metal salt, or a salt formed with an organic ligand. Preferably, the salt is selected from a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or a quaternary ammonium salt.

[0014] In some embodiments, provided is a use of sodium fumarate in the preparation of a medicament for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction in a subject.

[0015] In some embodiments, the subject has a neurodegenerative change or disease that causes cognitive dysfunction and / or motor dysfunction.

[0016] In some embodiments, the neurodegenerative changes include physiological natural aging or pathological aging with disease warning or risk. In this disclosure, physiological natural aging refers to the physiological degeneration process that occurs after maturity, while pathological aging, also known as premature aging, is a degenerative change caused by various exogenous factors (including various diseases or iatrogenic factors such as radiotherapy and chemotherapy).

[0017] In some embodiments, the neurodegenerative disease is manifested by cognitive dysfunction and / or motor dysfunction.

[0018] In some embodiments, the neurodegenerative disease may include, but is not limited to, one or more of: dementia with Lewy bodies (DLB), Alzheimer's disease (AD), senile dementia, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease dementia (Pick disease), frontotemporal dementia (FTD), vascular dementia, mixed dementia, or aging-related muscle strength and motor function decline.

[0019] In the present disclosure, the pathology of the subject suffering from the neurodegenerative disease manifests as cognitive dysfunction, motor dysfunction, or both.

[0020] In some embodiments, a subject suffering from a neurodegenerative disease with cognitive dysfunction only exhibits cognitive dysfunction or mainly exhibits cognitive dysfunction, such diseases including but not limited to Alzheimer's disease.

[0021] In some embodiments, a subject suffering from a neurodegenerative disease with movement dysfunction only exhibits movement dysfunction or mainly exhibits movement dysfunction, and such diseases include but are not limited to ALS, mild Parkinson's disease, moderate Parkinson's disease, etc.

[0022] In other embodiments, a subject suffering from a neurodegenerative disease with both cognitive dysfunction and motor dysfunction exhibits both cognitive dysfunction and motor dysfunction. Such diseases include but are not limited to severe Parkinson's disease, Lewy body dementia, and the like.

[0023] The compound of Formula I disclosed herein or a pharmaceutically acceptable salt thereof can be used to prevent, delay, or treat the above-mentioned neurodegenerative changes or diseases with cognitive dysfunction and / or motor dysfunction. In particular, the applicant has found that the drug of the present application has a better preventive, delaying, or therapeutic effect for neurodegenerative diseases with both cognitive dysfunction and / or motor dysfunction.

[0024] In some embodiments, the neurodegenerative changes or diseases are caused by genetic factors, immune factors, environmental factors and / or advanced physiological age.

[0025] In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof has at least one of the following uses: (1) improving and enhancing muscle strength and motor ability; (2) improving and enhancing active avoidance response ability, spatial learning and memory ability, and enhancing cognitive level; (3) improving and enhancing antioxidant capacity, pro-inflammatory state, and alleviating oxidative stress damage; (4) increasing the level of superoxide dismutase (SOD) in serum and decreasing the level of lactate dehydrogenase (LDH) in serum; (5) improving immunity; (6) reducing weight; (7) increasing the expression of neurotrophic factors; and (8) improving nerve damage repair.

[0026] According to a second aspect of the present disclosure, there is provided a use of hyodeoxycholic acid or a pharmaceutically acceptable salt thereof in combination for preparing a medicament for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction in a subject.

[0027] In some embodiments, the salt is selected from an alkali metal salt, an alkaline earth metal salt, or a salt formed with an organic ligand. Preferably, the salt is selected from a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or a quaternary ammonium salt.

[0028] In some embodiments, the subject has a neurodegenerative change or disease that causes cognitive dysfunction and / or motor dysfunction.

[0029] In some embodiments, the neurodegenerative changes include physiological natural aging or pathological aging with disease warning or risk. In this disclosure, physiological natural aging refers to the physiological degeneration process that occurs after maturity, while pathological aging, also known as premature aging, is a degenerative change caused by various exogenous factors (including various diseases or iatrogenic factors such as radiotherapy and chemotherapy).

[0030] In some embodiments, the neurodegenerative disease is manifested by cognitive dysfunction and / or motor dysfunction.

[0031] In some embodiments, the neurodegenerative disease may include, but is not limited to, one or more of: dementia with Lewy bodies (DLB), Alzheimer's disease (AD), senile dementia, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease dementia (Pick disease), frontotemporal dementia (FTD), vascular dementia, mixed dementia, or aging-related muscle strength and motor function decline.

[0032] In the present disclosure, the pathology of the subject suffering from the neurodegenerative disease manifests as cognitive dysfunction, motor dysfunction, or both.

[0033] In some embodiments, a subject suffering from a neurodegenerative disease with cognitive dysfunction only exhibits cognitive dysfunction or mainly exhibits cognitive dysfunction, such diseases including but not limited to Alzheimer's disease.

[0034] In some embodiments, a subject suffering from a neurodegenerative disease with movement dysfunction only exhibits movement dysfunction or mainly exhibits movement dysfunction, and such diseases include but are not limited to ALS, mild Parkinson's disease, moderate Parkinson's disease, etc.

[0035] In other embodiments, a subject suffering from a neurodegenerative disease with both cognitive dysfunction and motor dysfunction exhibits both cognitive dysfunction and motor dysfunction. Such diseases include but are not limited to severe Parkinson's disease, Lewy body dementia, and the like.

[0036] The compound of Formula I disclosed herein or a pharmaceutically acceptable salt thereof can be used to prevent, delay, or treat the above-mentioned neurodegenerative changes or diseases with cognitive dysfunction and / or motor dysfunction. In particular, the applicant has found that the drug of the present application has a better preventive, delaying, or therapeutic effect for neurodegenerative diseases with both cognitive dysfunction and / or motor dysfunction.

[0037] In some embodiments, the neurodegenerative changes or diseases are caused by genetic factors, immune factors, environmental factors and / or advanced physiological age.

[0038] In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof has at least one of the following uses: (1) improving and enhancing muscle strength and motor ability; (2) improving and enhancing active avoidance response ability, spatial learning and memory ability, and enhancing cognitive level; (3) improving and enhancing antioxidant capacity, pro-inflammatory state, and alleviating oxidative stress damage; (4) increasing the level of superoxide dismutase (SOD) in serum and decreasing the level of lactate dehydrogenase (LDH) in serum; (5) improving immunity; (6) reducing weight; (7) increasing the expression of neurotrophic factors; and (8) improving nerve damage repair.

[0039] According to a third aspect of the present disclosure, there is provided a use of a compound of Formula I or a pharmaceutically acceptable salt thereof in combination with hyodeoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction in a subject, wherein the compound of Formula I has the following structure:

[0040]

[0041] Among them, R 1 and R 2 Each is independently selected from H, C1-C6 alkyl, C1-C6 alkenyl and C1-C6 alkynyl.

[0042] In some embodiments, R 1 and R 2 can be independently selected from H.

[0043] In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C6 alkyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C5 alkyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C4 alkyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C3 alkyl. 1 and R 2 Each may be independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl or n-pentyl.

[0044] In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C6 alkenyl. In some embodiments, R1 and R 2 Can be independently selected from branched or straight chain C1-C5 alkenyl. In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C4 alkenyl. In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C3 alkenyl. In some embodiments, R 1 and R 2 Each may be independently selected from ethenyl, propenyl, butenyl or pentenyl.

[0045] In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C6 alkynyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C5 alkynyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C4 alkynyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C3 alkynyl. 1 and R 2 Each may be independently selected from ethynyl, propenyl or butynyl.

[0046] In some embodiments, R 1 and R 2 Can be the same or different.

[0047] In some embodiments, the salt is selected from an alkali metal salt, an alkaline earth metal salt, or a salt formed with an organic ligand. Preferably, the salt is selected from a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or a quaternary ammonium salt.

[0048] In some embodiments, the compound represented by Formula I or a pharmaceutically acceptable salt thereof comprises sodium fumarate or dimethyl fumarate (DMF).

[0049] In some embodiments, the subject has a neurodegenerative change or disease that causes cognitive dysfunction and / or motor dysfunction.

[0050] In some embodiments, the neurodegenerative changes include physiological natural aging or pathological aging with disease warning or risk. In this disclosure, physiological natural aging refers to the physiological degeneration process that occurs after maturity, while pathological aging, also known as premature aging, is a degenerative change caused by various exogenous factors (including various diseases or iatrogenic factors such as radiotherapy and chemotherapy).

[0051] In some embodiments, the neurodegenerative disease is manifested by cognitive dysfunction and / or motor dysfunction.

[0052] In some embodiments, the neurodegenerative disease may include, but is not limited to, one or more of: dementia with Lewy bodies (DLB), Alzheimer's disease (AD), senile dementia, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease dementia (Pick disease), frontotemporal dementia (FTD), vascular dementia, mixed dementia, or aging-related muscle strength and motor function decline.

[0053] In the present disclosure, the pathology of the subject suffering from the neurodegenerative disease manifests as cognitive dysfunction, motor dysfunction, or both.

[0054] In some embodiments, a subject suffering from a neurodegenerative disease with cognitive dysfunction only exhibits cognitive dysfunction or mainly exhibits cognitive dysfunction, such diseases including but not limited to Alzheimer's disease.

[0055] In some embodiments, a subject suffering from a neurodegenerative disease with movement dysfunction only exhibits movement dysfunction or mainly exhibits movement dysfunction, and such diseases include but are not limited to ALS, mild Parkinson's disease, moderate Parkinson's disease, etc.

[0056] In other embodiments, a subject suffering from a neurodegenerative disease with both cognitive dysfunction and motor dysfunction exhibits both cognitive dysfunction and motor dysfunction. Such diseases include but are not limited to severe Parkinson's disease, Lewy body dementia, and the like.

[0057] The compound of Formula I disclosed herein or a pharmaceutically acceptable salt thereof can be used to prevent, delay, or treat the above-mentioned neurodegenerative changes or diseases with cognitive dysfunction and / or motor dysfunction. In particular, the applicant has found that the drug of the present application has a better preventive, delaying, or therapeutic effect for neurodegenerative diseases with both cognitive dysfunction and / or motor dysfunction.

[0058] In some embodiments, the neurodegenerative changes or diseases are caused by genetic factors, immune factors, environmental factors and / or advanced physiological age.

[0059] In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof has at least one of the following uses: (1) improving and enhancing muscle strength and motor ability; (2) improving and enhancing active avoidance response ability, spatial learning and memory ability, and enhancing cognitive level; (3) improving and enhancing antioxidant capacity, pro-inflammatory state, and alleviating oxidative stress damage; (4) increasing the level of superoxide dismutase (SOD) in serum and decreasing the level of lactate dehydrogenase (LDH) in serum; (5) improving immunity; (6) reducing weight; (7) increasing the expression of neurotrophic factors; and (8) improving nerve damage repair.

[0060] In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof and hyodeoxycholic acid or a pharmaceutically acceptable salt thereof are administered simultaneously or sequentially.

[0061] In some embodiments, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the hyodeoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.05-50), for example, 1:0.05, 1:0.1, 1:0.2, 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50 or any value therebetween. Preferably, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the hyodeoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.1-10). More preferably, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the hyodeoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.5-5). Further preferably, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the hyodeoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.5-2). In some specific embodiments, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the hyodeoxycholic acid or a pharmaceutically acceptable salt thereof is 1:1.

[0062] The inventors of the present invention surprisingly found through a large number of experiments that the combination of the compound represented by Formula I or a pharmaceutically acceptable salt thereof (e.g., sodium fumarate) and hyodeoxycholic acid has a good effect in preventing, delaying or treating cognitive dysfunction and / or motor dysfunction diseases.

[0063] According to a fourth aspect of the present disclosure, a composition for preventing or treating cognitive dysfunction and / or motor dysfunction in a subject is provided, the composition comprising a compound represented by Formula I or a pharmaceutically acceptable salt thereof and / or hyodeoxycholic acid or a pharmaceutically acceptable salt thereof,

[0064]

[0065] Among them, R 1 and R 2 Each is independently selected from H, C1-C6 alkyl, C1-C6 alkenyl and C1-C6 alkynyl.

[0066] In some embodiments, R 1 and R 2 can be independently selected from H.

[0067] In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C6 alkyl. In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C5 alkyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C4 alkyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C3 alkyl. 1 and R 2 Each may be independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl or n-pentyl.

[0068] In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C6 alkenyl. In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C5 alkenyl. In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C4 alkenyl. In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C3 alkenyl. In some embodiments, R 1 and R 2 Each may be independently selected from ethenyl, propenyl, butenyl or pentenyl.

[0069] In some embodiments, R 1 and R 2 Can be independently selected from branched or straight chain C1-C6 alkynyl. 1and R 2 Can be independently selected from branched or straight chain C1-C5 alkynyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C4 alkynyl. 1 and R 2 Can be independently selected from branched or straight chain C1-C3 alkynyl. 1 and R 2 Each may be independently selected from ethynyl, propenyl or butynyl.

[0070] In some embodiments, R 1 and R 2 Can be the same or different.

[0071] In some embodiments, the compound represented by Formula I or a pharmaceutically acceptable salt thereof comprises sodium fumarate or dimethyl fumarate (DMF).

[0072] In some embodiments, the salt is selected from an alkali metal salt, an alkaline earth metal salt, or a salt formed with an organic ligand. Preferably, the salt is selected from a sodium salt, a potassium salt, a calcium salt, a magnesium salt, or a quaternary ammonium salt.

[0073] In some embodiments, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the hyodeoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.05-50), for example, 1:0.05, 1:0.1, 1:0.2, 1:0.5, 1:0.8, 1:1, 1:1.2, 1:1.5, 1:1.8, 1:2, 1:2.5, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50 or any value therebetween.

[0074] Preferably, the mass ratio of the compound shown in Formula I or its pharmaceutically acceptable salt to the hyodeoxycholic acid or its pharmaceutically acceptable salt is 1:(0.1-10). More preferably, the mass ratio of the compound shown in Formula I or its pharmaceutically acceptable salt to the hyodeoxycholic acid or its pharmaceutically acceptable salt is 1:(0.5-5). Further preferably, the mass ratio of the compound shown in Formula I or its pharmaceutically acceptable salt to the hyodeoxycholic acid or its pharmaceutically acceptable salt is 1:(0.5-2). In some specific embodiments, the mass ratio of the compound shown in Formula I or its pharmaceutically acceptable salt to the hyodeoxycholic acid or its pharmaceutically acceptable salt is 1:1.

[0075] In some embodiments, the compound of Formula I or a pharmaceutically acceptable salt thereof is used in combination with hyodeoxycholic acid or a pharmaceutically acceptable salt thereof to reduce body weight, increase learning and cognitive functions, improve spatial discrimination, increase serum superoxide dismutase (SOD) levels, reduce serum lactate dehydrogenase (LDH) levels, enhance muscle strength, improve immunity, increase the expression of neurotrophic factors, improve nerve damage repair, etc.

[0076] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient may include, but is not limited to, one or more of a pharmaceutically acceptable carrier, a diluent, an adjuvant, and an excipient.

[0077] In some embodiments, the dosage form of the composition includes, but is not limited to, tablets, capsules, solutions, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, creams, sprays, patches, sustained-release preparations, controlled-release preparations, or targeted preparations.

[0078] In some embodiments, the composition can be administered, for example, by injection, oral administration, rectal administration, etc.

[0079] In some embodiments, the compound of Formula I and its pharmaceutically acceptable salts, and / or hyodeoxycholic acid and its pharmaceutically acceptable salts are formulated for administration in the same dosage form or in separate dosage forms.

[0080] In some embodiments, the composition includes the compound of Formula I or a pharmaceutically acceptable salt thereof, hyodeoxycholic acid, or a pharmaceutically acceptable salt thereof, and optional pharmaceutically acceptable excipients.

[0081] In some embodiments, the composition comprises fumaric acid or a pharmaceutically acceptable salt thereof, hyodeoxycholic acid, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

[0082] According to a fourth aspect of the present disclosure, a method for treating or preventing cognitive dysfunction and / or motor dysfunction in a subject is provided, the method comprising administering a preventively or therapeutically effective amount of the composition of the third aspect to a subject in need thereof.

[0083] According to a fifth aspect of the present disclosure, a DHX57 mutant gene is provided. Compared with the gene encoding human wild-type DHX57, the DHX57 mutant gene has a mutation to a stop codon at the encoding nucleotide corresponding to amino acid position 526 of human wild-type DHX57.

[0084] In some embodiments, the protein number of the human wild-type DHX57 is XP_054200443.1, wherein the 1st-550th amino acid sequence in the amino acid sequence of the human wild-type DHX57 is shown as SEQ ID NO: 5.

[0085] In some embodiments, compared with the coding gene of the human wild-type DHX57, the DHX57 mutant gene is mutated at the 1576th base C to T corresponding to the coding gene of the human wild-type DHX57.

[0086] In some embodiments, the nucleic acid reference sequence number of the human wild-type DHX57 is XM_054344468.1.

[0087] In some embodiments, the 1st-1600th nucleotide sequence in the nucleotide sequence of the human wild-type DHX57 is shown as SEQ ID NO: 6.

[0088] According to a sixth aspect of the present disclosure, a DHX57 mutant protein encoded by the mutant gene described in the present disclosure is provided.

[0089] In some embodiments, the amino acid sequence of the DHX57 mutant protein is shown as SEQ ID NO: 14.

[0090] According to a seventh aspect of the present disclosure, the use of the DHX57 mutant gene, the DHX57 mutant protein, or the detection reagent thereof described in the present disclosure is provided:

[0091] (1) for the diagnosis of cognitive dysfunction and / or motor dysfunction;

[0092] (2) for the preparation of a diagnostic reagent for cognitive dysfunction and / or motor dysfunction.

[0093] In some embodiments, the cognitive dysfunction and / or motor dysfunction is caused by neurodegenerative changes or diseases.

[0094] In some embodiments, the neurodegenerative changes include physiological natural aging or pathological aging, preferably pathological aging.

[0095] In some embodiments, the neurodegenerative diseases include one or more of Lewy body dementia (DLB), Alzheimer's disease (AD), senile dementia, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease dementia (Pick's disease), frontotemporal dementia (FTD), vascular dementia, mixed dementia, or muscle strength motor function decline associated with aging.

[0096] According to an eighth aspect of the present disclosure, a method for constructing a mouse model of cognitive dysfunction and / or motor dysfunction is provided, the method comprising mutating the coding nucleotide CAG of the 527th amino acid of the wild-type mouse Dhx57 gene to the stop codon TGA.

[0097] In some embodiments, the amino acid sequence from position 1 to position 550 of the amino acid sequence of the mouse wild-type DHX57 is as shown in SEQ ID NO:7.

[0098] In some embodiments, the Dhx57 gene information of the wild-type mouse is as follows: GenBank accession number: NM_001163759.1; Ensembl: ENSMUSG00000035051).

[0099] In some embodiments, the method comprises the steps of:

[0100] (1) Determine the target site based on the sequence of exon 6 of the mouse Dhx57 gene;

[0101] (2) synthesizing an sgRNA sequence according to the targeting site determined in step (1), and then connecting the synthesized sequence to the backbone vector to construct an sgRNA targeting vector, wherein the sgRNA sequence of the targeting site is: CAGGCAGTTTCAGATGAAACAGG (SEQ ID NO: 8);

[0102] (3) The sgRNA, CRISPR / Cas9, and donor oligonucleotides obtained by in vitro transcription were introduced into the fertilized eggs of donor female mice to obtain mouse fertilized eggs with Dhx57 point mutations;

[0103] (4) The obtained fertilized eggs are transplanted into the uterus of a surrogate mouse to obtain a mouse model.

[0104] According to a ninth aspect of the present disclosure, there is provided a method for screening a drug for preventing or treating cognitive dysfunction and / or motor dysfunction, the method comprising any one or more of the following:

[0105] (1) a step of performing drug screening using cells harboring a DHX57 gene mutation, wherein the nucleotide encoding the 526th amino acid of the DHX57 mutant gene is mutated to a stop codon compared to the wild-type DHX57, and preferably, the amino acid sequence from positions 1 to 550 of the amino acid sequence of the wild-type DHX57 is as shown in SEQ ID NO: 5;

[0106] (2) The step of using the mouse model obtained by the construction method disclosed in the present invention to perform drug screening.

[0107] In some embodiments, the cognitive dysfunction and / or motor dysfunction is caused by a neurodegenerative change or disease.

[0108] In some embodiments, the neurodegenerative changes include physiological natural aging or pathological aging, preferably pathological aging.

[0109] In some embodiments, the neurodegenerative disease comprises one or more of dementia with Lewy bodies (DLB), Alzheimer's disease (AD), senile dementia, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease dementia (Pick disease), frontotemporal dementia (FTD), vascular dementia, mixed dementia, or aging-related muscle strength and motor function decline. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] Figure 1 Shown are the changes in body weight of aged mice during drug administration.

[0111] Figure 2 The figures show the results of grip force (A) and rotarod test (BC) of aged mice after drug administration, where A shows the peak grip force, B shows the falling time, and C shows the distance traveled.

[0112] Figure 3 The results of the water maze test after drug administration to the aged mice are shown, where A shows the time to reach the target platform, B shows the total swimming distance, C shows the average swimming speed, D shows the time to reach the target platform for the first time, E shows the time spent on the target platform, F shows the number of times the target platform is passed, and G shows the swimming trajectory.

[0113] Figure 4 Shows the results of the Y-maze test on aged mice after drug administration.

[0114] Figure 5 The results of redox index detection in the serum of aged mice after administration are shown, where A is the result of SOD and B is the result of LDH.

[0115] Figure 6 Shown are the results of telomere length testing in aged mice after drug administration.

[0116] Figure 7 The figure shows the changes in blood cells in the peripheral blood and bone marrow of aged mice after administration, where A represents hematopoietic stem and progenitor cells, B represents myeloid cells, erythroid cells, macrophages and granulocytes, C represents the ratio of peripheral blood leukocytes, D represents the ratio of cells at each level, and E represents the ratio of myeloid cells to lymphocytes.

[0117] Figure 8 The immunostaining results of the hippocampus of brain sections of aged mice after drug administration are shown, wherein A is the immunostaining result and B is the quantitative statistical result of A.

[0118] Figure 9 The figure shows the results of immunofluorescence staining of nerve growth factor (BDNF) in the hippocampus of brain sections of aged mice after drug administration, wherein A is the fluorescence staining result and B is the quantitative statistical result of A.

[0119] Figure 10 The figures show the immunofluorescence staining results of axon growth-associated protein (GAP-43) in the hippocampus of brain slices of aged mice after drug administration (bar=500 μm), wherein A is the immunostaining result and B is the quantitative statistical result of A.

[0120] Figure 11 Shown are the results of organ tissue morphology-safety evaluation in aged mice after administration.

[0121] Figure 12 Schematic diagram of Dhx57 point mutations is shown.

[0122] Figure 13 Shown are the sequencing results of Dhx57 point mutation mice.

[0123] Figure 14 Shown are the results of a grip strength test in male mice with a Dhx57 point mutation.

[0124] Figure 15 Shown are the results of the rotarod test of Dhx57 point mutant mice, where A shows the average falling time and B shows the average distance traveled.

[0125] Figure 16 The results of the water maze test of Dhx57 point mutant mice are shown, where A shows the average swimming speed, B shows the time required to reach the target platform, C shows the number of platform crossings, D shows the time required to reach the target platform during the test, and E shows the time spent in the SW quadrant.

[0126] Figure 17 Shown are the head exploration times in the open field test of Dhx57 point mutant mice.

[0127] Figure 18 Shown are the results of senescence scoring for Dhx57 point mutant mice.

[0128] Figure 19 Shown are the survival curves of Dhx57 point mutant mice.

[0129] Figure 20Shown are the results of the rotarod test in Dhx57 point mutant mice after drug administration, where A shows the fall time and B shows the average stroke.

[0130] Figure 21 The results of the water maze test for Dhx57 point mutant mice after drug administration are shown, where A shows the time required to reach the target platform, B shows the total distance traveled on the first day, and C shows the number of times the platform was crossed.

[0131] Figure 22 Shown is the head exploration time in the open field test in Dhx57 point mutation mice after drug administration.

[0132] Figure 23 The cell proliferation and migration results of DHX57 cells are shown, where the control is non-knockout KGN cells.

[0133] Figure 24 The senescence characterization results of DHX57 knockout cells are shown, where blue indicates senescent cells, A is a β-galactosidase staining image, blue indicates senescent cells, B is the quantitative result of senescent cells, and C is the mRNA level expression detection of senescence markers P16 and P21 genes, * represents P < 0.05, and ** represents P < 0.01. DETAILED DESCRIPTION

[0134] At present, drugs for treating cognitive dysfunction diseases such as Alzheimer's disease are faced with the dilemma of insignificant effects, only delaying the process and having large side effects, and monoclonal antibody drugs relying on imports, limited application and high prices. In the present disclosure, fumaric acid itself is a precursor of L-malic acid in the tricarboxylic acid cycle (TCA), formed by the oxidation of succinic acid by succinate dehydrogenase, and its derivative dimethyl fumarate is already a marketed drug for the treatment of multiple sclerosis and psoriasis, etc., and the drug has undergone toxicological evaluation; hyodeoxycholic acid is also a natural bile acid present in the body, and hyodeoxycholic acid tablets are already on the market and are applicable to hyperlipidemia. Therefore, the combined use of these two naturally occurring substances in the body to treat cognitive impairment diseases has relatively few side effects and is relatively low in price.

[0135] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0136] definition

[0137] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0138] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0139] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0140] The term "alkyl" as used herein refers to a fully saturated straight or branched chain non-aromatic hydrocarbon. Examples of straight and branched C1-C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, amyl and octyl.

[0141] As used herein, the term "alkenyl" refers to a non-aromatic hydrocarbon containing at least one double bond.

[0142] As used herein, the term "alkynyl" refers to a non-aromatic hydrocarbon containing at least one triple bond.

[0143] The term "pharmaceutically acceptable" as used herein refers to an agent that can be administered to humans and / or other animals as subjects and does not produce excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). The term "excipient" refers to an auxiliary material that is present in the pharmaceutical preparation at the same time as the active ingredient and does not produce excessive adverse reactions or side effects, including carriers, osmotic pressure regulators, pH regulators, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, etc. The term "pharmaceutically acceptable excipient" refers to a high-safety excipient that is suitable for a specific pharmaceutical preparation and is routinely used in pharmacy. The term "carrier" includes, but is not limited to, liposomes, ethosomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, mesoporous silica nanoparticles, etc.

[0144] The term "pharmaceutically acceptable salt" used herein may include alkali metal salts (eg, sodium salts or potassium salts), alkaline earth metal salts (eg, calcium salts or magnesium salts), and salts formed with suitable organic ligands (eg, quaternary ammonium salts).

[0145] The term "hyodeoxycholic acid (HDCA)" used in this article refers to a bile acid originally extracted from pig bile. It is a steroid acid found primarily in mammalian bile and is chemically known as 3α,6α-dihydroxy-5β-cholanoic acid. The differences between different bile acids are minimal, primarily distinguished by the presence or absence of hydroxyl groups at positions 3, 7, and 12. Bile acids are physiological detergents that aid in the breakdown, absorption, and transport of fats and steroids in the gastrointestinal tract and liver. Bile acids are also steroidal amphiphilic molecules derived from cholesterol metabolism. They regulate bile distribution and lipid secretion, are important for the absorption of dietary fats and vitamins, and participate in the function of enzymes regulating cholesterol homeostasis. Bile acids are recycled through the hepatobiliary axis formed by the liver, bile duct, small intestine, and portal vein. Studies have shown that hyodeoxycholic acid can significantly reduce the cell necrosis rate and apoptosis rate of neurons damaged by hypoxia, glucose deprivation and reoxygenation, and significantly increase the cell survival rate, indicating that hyodeoxycholic acid has a significant effect in resisting neuronal hypoxia, glucose deprivation and reoxygenation injury. In addition, hyodeoxycholic acid is a secondary hydrophilic bile acid formed by intestinal flora in the small intestine, which can prevent the formation of gallstones in mice. Studies have found that hyodeoxycholic acid not only inhibits intestinal cholesterol absorption, but also exerts other anti-atherosclerotic effects. HDCA has lipid-lowering, antispasmodic and expectorant effects, and can be used clinically to treat hyperlipidemia, atherosclerosis, tracheitis, pediatric viral upper respiratory tract inflammation, and indigestion caused by hepatobiliary diseases; it has a certain inhibitory effect on pertussis bacteria, diphtheria bacteria, Staphylococcus aureus, etc.

[0146] As used herein, the term "prevention" refers to the prophylactic treatment of a subclinical disease state, intended to reduce the probability of the clinical disease state occurring. "Prevention" can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment of a subject who has not yet developed a clinical disease state, while secondary prevention is defined as preventing a secondary occurrence of the same or similar clinical disease state.

[0147] As used herein, the term "treat" refers to the treatment of a disease, symptom, or condition, including: 1) inhibiting the development of the disease, symptom, or condition; and / or, 2) delaying or alleviating the disease, symptom, or condition.

[0148] Biomarkers mentioned herein, such as superoxide dismutase (SOD), malondialdehyde (MDA), lactate dehydrogenase (LDH), etc., can be detected using methods generally known in the art. Detection methods generally encompass methods for quantifying the levels of biomarkers in a sample (quantitative methods). It is generally known to those skilled in the art which of the following methods are suitable for qualitative and / or quantitative detection of biomarkers. Samples can be conveniently assayed, for example, using immunoassays for proteins, such as ELISA, RIA, etc., which are commercially available.

[0149] "Cognitive impairment" means a decrease in cognitive ability relative to a healthy individual, e.g., an age-matched healthy individual, or relative to the individual's ability at an earlier time point, e.g., 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10 years or earlier.

[0150] "Motor dysfunction" means a decrease in motor ability / skill relative to a healthy individual, e.g., an age-matched healthy individual, or relative to the individual's ability at an earlier time point, e.g., 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years, 5 years, or 10 years or earlier.

[0151] Neurodegeneration is the progressive loss of neuronal structure and function, including neuronal death and glial cell homeostasis, which can lead to cognitive impairment or motor disorders. Neurodegenerative diseases can be caused, among other things, by aging (Alzheimer's disease (AD), Parkinson's disease (PD)) or genetic mutations that affect the function of CNS cells (Huntington's disease, early-onset AD or PD, amyotrophic lateral sclerosis (ALS)).

[0152] Alzheimer's disease (AD), also known as Alzheimer's disease, is a chronic, progressive degenerative brain disorder that occurs in the elderly and pre-elderly stages. It is characterized by progressive memory loss, cognitive impairment, and personality changes. There are two types of Alzheimer's disease: hereditary and sporadic. Hereditary Alzheimer's disease is further divided into early-onset and late-onset forms.

[0153] Vascular dementia (VaD) is a clinical syndrome characterized by impairment of higher-level neurocognitive function, caused by cerebrovascular disease, including ischemic, hemorrhagic, acute and chronic ischemic-hypoxic cerebrovascular damage, and ischemic-hypoxic cerebrovascular disease. It primarily includes multi-infarct dementia, multi-lacunar dementia, cerebral amyloid angiopathy, cerebral hypoperfusion dementia, and hemorrhagic dementia.

[0154] Mixed dementia refers to a combination of senile dementia, vascular dementia or other types of dementia. Other types of dementia mainly include frontotemporal dementia (FTD), dementia of Lewy bodies (DLB), Parkinson's disease dementia, Huntington's disease dementia, corticobasal degeneration and other types of dementia.

[0155] In the present disclosure, fumaric acid itself is a precursor of L-malic acid in the tricarboxylic acid cycle (TCA), formed by the oxidation of succinate by succinate dehydrogenase. Hyodeoxycholic acid is also a naturally occurring bile acid. Therefore, the use of these two naturally occurring substances in the body to treat cognitive dysfunction and delay aging does not produce adverse reactions, is safe, and has minimal side effects.

[0156] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0157] Example

[0158] Example 1. Method for selecting aged mice for drug administration to treat cognitive dysfunction and motor dysfunction

[0159] 1. Animal Model: Sixteen 20-month-old wild-type C57 mice were selected. Aged mice, as a model, exhibit typical cognitive and motor impairments, such as decreased muscle strength and balance, weakened memory and spatial exploration abilities, and decreased antioxidant capacity.

[0160] 2. Grouping: The mice were randomly divided into two groups based on littermate and body weight: a control group of 8 mice (2 males, 6 females) and a sodium fumarate and hyodeoxycholic acid (SF+HDCA) combination group of 8 mice (2 males, 6 females). The sodium fumarate and hyodeoxycholic acid combination group received 100 mg / kg / day of body weight by oral gavage once daily, 3 mg per day (e.g., 30 g body weight). Sodium fumarate and hyodeoxycholic acid were each dissolved in 0.08% (8 mg / 100 ml water) hydroxymethylcellulose. The control group received 0.08% hydroxymethylcellulose by oral gavage once daily for a total of 28 days.

[0161] 3. Weigh body weight before and after administration.

[0162] 4. After the administration, the mouse behavioral test was performed.

[0163] 5. Mice were killed and blood was collected. The serum was used for the detection of AD markers and redox indicators (superoxide dismutase (SOD) and lactate dehydrogenase (LDH)). Genomic DNA was extracted from the blood clots and telomere length was detected.

[0164] 6. Sacrifice the mice and obtain the following samples: brain, hippocampus, liver, spleen, kidney, peripheral blood, and bone marrow. Bone marrow and peripheral blood are used to analyze immune markers; brain and hippocampus tissue sections are stained to detect AD and aging-related markers; liver, spleen, and kidney tissues are used to evaluate drug safety.

[0165] See Examples 2-9 for details.

[0166] Example 2. Body weight changes during drug administration

[0167] The body weight of mice was measured once a week during the administration process, and statistical analysis was performed 28 days after administration. Figure 1 The results of multivariate analysis of variance showed statistically significant differences at each time point; there was an interaction between time and group. A statistically significant difference was observed between the two groups at week 2 of treatment (P = 0.033).

[0168] Example 3. Muscle strength and movement balance ability testing experiment

[0169] 3.1 Grip strength measurement

[0170] Place the grip strength tester horizontally and start the instrument. Place a mouse on the grip plate. Grasp the mouse's tail and gently pull backward. Once the mouse has a firm grip on the grip plate, pull evenly backward until the mouse releases its claws. The instrument then records the mouse's maximum grip strength. Perform three measurements on each mouse, and the average value is used to assess muscle strength.

[0171] See the results Figure 2 A, shows that the muscle strength of mice was significantly enhanced after drug administration, with a statistically significant difference (P=0.013).

[0172] 3.2 Rotarod test

[0173] This test assesses motor coordination and balance in rodents. Mice must balance on a rotating rod. The instrument records the time (latency) it takes for the animal to fall from a rod that rotates at different speeds or is continuously accelerated (from 4 to 40 rpm), the speed of the rod at the time of fall, and the distance the animal travels.

[0174] See the results Figure 2 B and Figure 2 C, shows that after drug administration, the mice's movement balance ability is enhanced, the falling delay time is longer, and the movement distance is longer.

[0175] Example 4. Behavioral index detection

[0176] Method of administration: Same as Example 1.

[0177] Morris water maze test

[0178] The water maze used in this embodiment includes a circular water tank with a diameter of 120 cm, filled with tap water, and the platform is placed 1-2 cm below the water surface. The room temperature and water temperature are both controlled at 22°C. Different shapes are posted along the walls of the water tank as spatial reference clues. Note: The position of objects in the room must remain fixed throughout the experiment. A camera is installed above the maze to record the traces of mice swimming in the water maze, and the actual position is unified with the virtual position in the computer image acquisition software. Set the animal group in the program, set the animal swimming time (60s) and the platform stay time (10s). During the acquisition experiment, the mouse is placed in the maze facing one of the four points (north, south, east, and west) on the water tank wall.

[0179] 4.1.1 Learning and Training Experiment: This experiment was conducted over five days, with each animal undergoing four training sessions per day. Each time, the animal was placed into the pool from a different entry point, facing the pool wall. The starting position was semi-randomly selected, with the platform located in the southwest quadrant. This design prevented the animals from learning a specific right- or left-turn sequence to locate the platform. Each of the four starting positions was used daily (see Table 1). The time required for the animal to climb onto the platform from entry was recorded as the latency. The animal's swimming trajectory was also recorded to analyze the target search strategy. After climbing onto the platform, the animal was allowed to stand on it for 10 seconds. If the animal failed to find the platform or climb onto it within 60 seconds, it was guided to the platform and allowed to stand for 10 seconds before being removed. The average escape latency (latency for those who failed to climb onto the platform was calculated as 60 seconds), swimming distance, swimming speed, and time spent swimming in the outer ring were calculated for each animal across the four entry points each day. Fluctuations were plotted to analyze trends.

[0180] 4.1.2 Memory test: The platform was removed from its original location and the animal was placed into the pool through a water entry point. The time it took for the animal to first explore and swim past the original platform location, the number of times it explored and swam past the original platform location within 60 s (i.e., the number of loops), and the time the animal spent swimming in the target quadrant where the platform was located were recorded.

[0181] Table 1 Starting position of water maze space

[0182] sky first Second time The third time Fourth time 1 north East southeast northwest 2 southeast north northwest East 3 northwest southeast East north 4 East northwest north southeast 5 north southeast East northwest 6 (test) northeast / / /

[0183] The results of the water maze test are shown in Figure 3 It was found that after 5 days of training and 1 day of testing, the learning and memory ability of the mice after drug administration was significantly enhanced, specifically, the time to reach the target platform was significantly shorter than that of the control group, and there was a significant difference from the 4th day of training (P=0.003) ( Figure 3 A), while the swimming distance of the control group was significantly longer than that of the drug-treated group (P=0.03) ( Figure 3 B), and the swimming speed was similar between the two groups ( Figure 3C), indicating that the longer time required to reach the destination was not due to abnormal limb movement of the control group, that is, unwillingness or inability to swim, but rather that the drug-treated mice had a stronger learning ability. The test after removing the platform found that the drug-treated group took less time to reach the platform position for the first time than the control group ( Figure 3 D), the time spent on the platform becomes longer ( Figure 3 E), and passes through the target platform more times ( Figure 3 F), swimming trajectory of mice during the test (60s) Figure 3 G (where ○ indicates the location of the platform). Overall, it shows that the memory ability of mice is significantly enhanced after drug administration.

[0184] 4.2. Y-maze test

[0185] Y-maze alternation behavior test and parameters

[0186] The Y-maze test is a behavioral test based on the natural exploratory curiosity of mice. The spontaneous alternation behavior in this test is believed to reflect short-term spatial working memory. It exploits the animal's natural tendency to explore new environments. During the experiment, the animal needs to remember the previously explored direction each time it switches to a new direction. Therefore, the Y-maze test is a good indicator of an animal's spatial working memory.

[0187] The Y-maze consists of three arms of equal length (50 cm x 18 cm x 35 cm), each with a 120-degree angle between them and a movable central partition. Mice were placed at the end of any arm of the Y-maze and allowed to freely explore for 8 minutes. A video camera recorded the animal's behavior for 8 minutes, recording the following indicators: ① Total number of entries: the number of times the animal entered an arm (one entry was defined as entering an arm with all four legs); ② Alternation: the number of consecutive entries into all three arms of the Y-maze. ③ Maximum number of alternations: total number of arm entries minus 2. Spontaneous alternation score = total number of alternations / maximum number of alternations * 100%.

[0188] The results of the Y-maze experiment are shown in Figure 4 , showing that the number of spontaneous alternations in the drug-administered group increased significantly, which indicates that the spatial exploration ability of mice was significantly enhanced after drug administration.

[0189] Example 5. Determination of oxidative stress levels

[0190] The oxidative stress hypothesis posits that a reduction in the body's antioxidant components leads to a weakened ability to scavenge free radicals, which in turn causes oxidative damage to biomacromolecules, an imbalance between cellular oxidation and antioxidant function, and oxidative stress, which in turn leads to disease and aging. Oxygen free radicals act on unsaturated fatty acids in lipids to generate lipid peroxides, including malondialdehyde (MDA); the antioxidant defense system that scavenges free radicals includes superoxide dismutase (SOD), etc. Lactate dehydrogenase (LD or LDH) is a type of nicotinamide adenine dinucleoside (NAD)-dependent kinase. LDH is one of the important enzymes in glycolysis, catalyzing the redox reaction between propionate and L-lactic acid, and also catalyzing related α-keto acids. It has been reported that oxidative stress is associated with aging and Alzheimer's disease. Therefore, this example tested the effect of sodium fumarate plus hyodeoxycholic acid on oxidative stress in aged mice. The administration method was the same as in Example 1, and the redox indicators SOD and LDH were measured in the mouse serum 28 days after administration (according to the kit instructions of Biyuntian Company).

[0191] The results are shown in Figure 5 Figures A and 5B show that the SOD concentration in the treatment group was significantly increased, while the LDH concentration was significantly decreased compared to the control group. This indicates that the combined use of sodium fumarate and hyodeoxycholic acid can significantly increase the antioxidant capacity of mice, reduce glycolysis, and improve the redox state in the blood of mice.

[0192] Example 6. Detection of telomere length in mice after drug administration

[0193] Every time a cell in the body divides, chromosomes are replicated. However, the enzymes responsible for replication cannot fully reach the ends of the chromosomes, so a small amount of chromosome ends are lost during each replication process. Telomeres provide a small amount of extra chromosomes as a buffer, protecting important genetic information from being lost. In other words, each time a cell divides, the telomeres lose a little length. When the telomeres can no longer shorten, the cell dies due to its inability to divide. Scientists have therefore dubbed telomeres the "clock of life," and telomere shortening is considered a biological marker of cellular aging. Measuring telomere length can indirectly reflect biological age.

[0194] In this example, mice were sacrificed 28 days after administration and blood was collected. Genomic DNA was extracted from the blood clots. Telomere length was assessed by comparing the relative copy number ratio (T / S ratio) of the telomeric repeat sequence (Telomere) to the single copy gene (36B4). When designing primers, the telomeric primers targeted the repetitive sequence (forward: 5'-CGG TTT GTT TGG GTT TGG GTT TGG GTT TGGGTT TGG GTT-3' (SEQ ID NO: 1), reverse: 5'-GGC TTG CCT TAC CCT TAC CCT TAC CCT TACCCT TAC CCT-3' (SEQ ID NO: 2)). Primers for the single-copy internal reference gene 36B4 (RPLP0) were designed to span the intron to avoid interference from genomic DNA (forward: 5'-ACT GGT CTA GGA CCC GAG AAG-3' (SEQ ID NO: 3), reverse: 5'-TCA ATG GTG CCT CTG GAG ATT-3' (SEQ ID NO: 4)). The qPCR reaction system (20 μL) contained SYBR Green Master Mix, primers (final concentration 900 nM for telomere primers, 300 nM for 36B4 primers), and DNA template (10-20 ng). Telomere and 36B4 amplification protocols were run separately (95°C for 10 minutes, initial denaturation, 40 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 30 seconds). T / S ratios were calculated using the ΔΔCt method (T / S = 2^[Ct(telomere) - Ct(36B4)]). A standard curve (gradient dilutions of DNA) was established to verify amplification efficiency (90%-110%), and melting curves were analyzed to ensure primer specificity. Experiments were repeated three times to minimize DNA degradation and primer-dimer interference.

[0195] See the results Figure 6 It can be seen that the telomere length of mice after drug administration is longer than that of the control group.

[0196] Example 7. Changes in peripheral blood and bone marrow blood cells in mice after administration

[0197] After the mice were killed, peripheral blood and bone marrow were collected, and various types of blood cells were detected by flow cytometry. The steps are as follows: sample collection and processing, antibody staining and instrument analysis. When collecting bone marrow, the femur and tibia were separated after the mice were killed, and the bone marrow cavity was rinsed with pre-cooled PBS to obtain a cell suspension. The broken bones were removed through a 70μm filter, centrifuged (300×g, 5 minutes), and the supernatant was discarded. After adding ACK buffer to lyse red blood cells for 35 minutes, the cells were washed twice with PBS for later use; 50μl of peripheral blood was collected from the tail vein into an anticoagulant tube, and the red blood cells were directly lysed and washed. The cell density needs to be adjusted to 1×10 before antibody staining. 6 ~1×107 cells / mL, anti-mouse CD16 / 32 antibody was added to block Fc receptors for 10 minutes, and then fluorescent-labeled antibodies were incubated in the dark for 20-30 minutes according to the concentration specified in the instructions (CD45: pan-leukocyte marker (distinguishing leukocytes from non-leukocytes); lymphocyte subsets: T cells: CD3ε, CD4, CD8α; B cells: CD19, B220 (CD45R), IgM, IgD; NK cells: NK1.1 (C57BL / 6 strain), CD49b (DX5); myeloid cells: monocytes / macrophages: CD11b, F4 / 80, Ly6C, Ly6G (distinguishing monocytes from neutrophils); granulocytes: Gr1 (Ly6G / Ly6C complex), CD11b; hematopoietic stem / progenitor cells (bone marrow): hematopoietic stem cells (HSC): CD34-, cKit (CD117 + ), Sca1 + (LSK phenotype: Lineage - Sca1 + cKit + ); progenitor cells: CD34 + , CD16 / 32 (FcγR)), and washed twice with PBS to remove unbound antibodies. During flow cytometry, set the instrument channel according to the fluorescent dye (FITC, PE, APC), adjust the compensation by single-stained control, and use FSC (cell size) and SSC (granularity) thresholds to exclude debris; when analyzing data, first circle the FSC / SSC live cell population and screen for CD45 + Leukocytes, further subpopulation analysis such as CD3 + T cell typing (CD4 + / CD8 + ), CD19 + B cells and CD11b+ myeloid cells (Ly6G + Neutrophils, Ly6C + Monocytes), and the data were finally analyzed by FlowJo software.

[0198] The results showed that the number of hematopoietic stem cells and the proportion of myeloid cells increased during the aging process of mice. In mice treated with sodium fumarate and hyodeoxycholic acid, the proportion of various cells in the bone marrow and peripheral blood was analyzed and it was found that the number of hematopoietic stem cells decreased ( Figure 7 A), although there was no statistical difference, the ratio of peripheral blood leukocytes decreased, especially the ratio of myeloid cells to lymphocytes ( Figure 7 B-7E), indicating that the combined drug treatment can improve the pro-inflammatory state of aged mice.

[0199] Example 8. Immunostaining of the hippocampus in brain sections of mice after drug administration

[0200] The hippocampus is associated with learning and memory, so this example performed histological testing on the mouse brains described in Example 1. The mouse skulls were pried open, and the whole brains were removed. The brains were dehydrated with 20% sucrose, fixed with paraformaldehyde, and then embedded in OCT for frozen sections. Alternatively, the brains were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned.

[0201] IBA-1 is a marker of microglia / macrophages in the brain and other tissues and is a neuroinflammatory marker. AD patients are often accompanied by immune activation of hippocampal microglia. In this example, immunostaining of IBA-1 was performed.

[0202] Paraffin sections were sequentially dewaxed and hydrated, followed by antigen retrieval, endogenous peroxidase blocking, and nonspecific binding. Sections were first dewaxed by immersion in xylene I and II for 10 minutes each, hydrated with graded ethanol (100% to 70%), and then washed with distilled water. Antigen retrieval was performed with sodium citrate buffer (pH 6.0), microwaved to boiling, then maintained on low heat for 10-15 minutes. After cooling, the sections were washed with PBS. Endogenous enzymes were then blocked with 3% H₂O₂ for 10 minutes at room temperature, followed by 5% normal serum blocking for 30 minutes. Diluted primary antibodies were added dropwise and incubated at 4°C overnight or at room temperature for 1-2 hours. After washing with PBS, HRP-conjugated secondary antibodies were added and incubated at room temperature for 30-60 minutes. Development was performed with DAB colorimetric solution in the dark (control the time under a microscope, usually 30 seconds to 5 minutes). After terminating with distilled water, the sections were counterstained with hematoxylin for 1-3 minutes, differentiated with hydrochloric acid and alcohol, and blued with tap water. Dehydrated with graded ethanol, transparentized with xylene, and mounted with neutral gum for observation (positive signals appeared brownish-yellow, with blue nuclei).

[0203] The results are as follows Figure 8 As shown in A-8B, it was found that combined drug use reduced the expression of IBA-1 in the hippocampus of mice, indicating that combined drug use can reduce immune stress.

[0204] Example 9. Immunofluorescence staining of nerve growth factor (BDNF) in the hippocampus of brain slices of mice after drug administration

[0205] BDNF is a neurogenic growth factor that binds to its receptor TrkB and can mediate the development and functional mechanism of nerve cells. In this example, immunofluorescence staining of BDNF was performed on the hippocampus of mouse brain tissue. Frozen sections were fixed with 4% paraformaldehyde at room temperature for 15-20 minutes, and then washed thoroughly with PBS. Permeabilize with 0.1%-0.5% Triton X-100 for 5-15 minutes, and then wash with PBS. Incubate with 5% BSA blocking solution at room temperature for 30-60 minutes to reduce nonspecific binding. The primary antibody was diluted with blocking solution and covered with the sample, incubated at 4°C overnight or at room temperature for 1-2 hours, and then washed with PBST three times. Incubate with DAPI nuclear staining for 5-10 minutes, and finally seal with antifade sealing agent and store in the dark. When observing, it is necessary to select the microscope channel according to the fluorescent dye, and collect images in time periods to avoid bleed-through and quenching.

[0206] The results are as follows Figure 9 As shown in A-9B, it was found that the combined use of drugs could significantly increase the expression of BDNF in the hippocampus, indicating that the combined use of drugs could improve the growth, development and function of nerve cells in aging mice.

[0207] Example 10. Immunofluorescence staining of axon growth-associated protein (GAP-43) in the hippocampus of brain slices of mice after drug administration

[0208] GAP-43 is a key protein involved in axonal growth and synaptic remodeling, and is involved in nerve damage repair. This example detected the expression of GAP43 in the hippocampus of brain tissue after drug administration. The staining method was the same as in Example 8.

[0209] The results are as follows Figure 10 As shown in A-10B, it was found that the expression of GAP43 could be significantly upregulated after drug administration, indicating that combined drug administration can improve the repair of nerve damage in aging mice.

[0210] Example 11. Drug administration safety evaluation

[0211] After 28 days of drug administration, the mice were killed and the morphology of the internal organs was observed after dissection. No obvious abnormalities were found in the drug group. The liver, spleen, kidney, testicle or ovary were further taken for morphological observation to determine the safety of the drug. The results showed that after the combined use of the drug, no morphological abnormalities were found in these organs ( Figure 11 ).

[0212] The present disclosure verifies that the compound represented by Formula I and / or its pharmaceutically acceptable salt is combined with hyodeoxycholic acid, and the oral gavage treatment is performed for 28 days, so as to make the body weight of the mouse reduced, the muscle grip strength enhanced, the rotarod motor ability enhanced, the learning and memory morris water maze experiment and spatial discrimination Y maze experiment improved, the telomere shortened, the antioxidant index enhanced, the myeloid / lymphoid ratio of the blood system reduced, and the immunity improved. These results show that fumaric acid or its derivative, hyodeoxycholic acid or the combination of the two has a significant effect of treating cognitive dysfunction and delaying aging.

[0213] Example 12. Construction of DHX57 point mutation mouse

[0214] The inventors found that there is a nonsense mutation of DExH-box helicase 57 (DHX57) in a clinical premature ovarian failure family by whole exome sequencing. The 6th exon of the coding region of DHX57 (XM_054344468.1) is mutated from C to T at position 1576 (c.C1576T), and the arginine (R, CGA) at position 526 is mutated to a stop codon TGA. Subsequently, the conservation of the mutation site in humans and other species was analyzed. The 1579th position of the coding region of mouse Dhx57 gene (NM_001163759.1) corresponds to the 1576th position of human DHX57. In the amino acid sequence, the 527th glutamine (Q) of mouse DHX57 corresponds to the 526th arginine (R) of human. Thus, a Dhx57 point mutation mouse at this site was prepared.

[0215] wherein the 1st-550th amino acid sequence in the amino acid sequence of the wild-type DHX57 of human is shown in SEQ ID NO: 5:

[0216] (SEQ ID NO:5).

[0217] The nucleotide sequence from position 1 to position 1600 of the nucleotide sequence of the human wild-type DHX57 is shown in SEQ ID NO: 6:

[0218]

[0219] The amino acid sequence from positions 1 to 550 of the amino acid sequence of mouse wild-type DHX57 is shown in SEQ ID NO: 7:

[0220] (SEQ ID NO:7).

[0221] A C57BL / 6J mouse model with a Dhx57 point mutation was constructed using CRISPR / Cas-mediated genome engineering technology. Figure 12 A schematic diagram is shown.

[0222] Here are the steps:

[0223] The mouse Dhx57 (DExHBox Helicase 57) gene (GenBank accession number: NM_001163759.1; Ensembl: ENSMUSG00000035051) is located on mouse chromosome 17.

[0224] Design gRNA targeting vector and donor oligonucleotide (with targeting sequence and 120bp or 150bp homologous sequence on both sides).

[0225] Among them, the sequence of gRNA is:

[0226] CAGGCAGTTTCAGATGAAACAGG (SEQ ID NO:8).

[0227] The sequence of the donor oligonucleotide is:

[0228] AGATACGACAGGCCGGCAAAGTCCTTATTTGCTGAGAACAGTAAGATCTGCAGGCAGTTTTGAA TGAAACAGGTACTGTTTAAGGGCCAGCGCCACCTCCCTCAGGTCTGGAGCCTACTCTTC (SEQ ID NO: 9)

[0229] The Q527X (CAG→TGA) mutation site was introduced into exon 6 in the donor oligonucleotide by homology-directed repair.

[0230] Cas9 mRNA and gRNA generated by in vitro transcription are co-injected, along with donor oligonucleotides, into the nuclei of fertilized eggs obtained from the uterus of donor female mice 3.5 days after mating. The fertilized eggs injected with RNA and donor oligonucleotides are then transplanted into the uterus of surrogate female mice. Once the fertilized eggs are transplanted, the mice are born and designated as F0 mice.

[0231] At around one week of age, the toes of the mice were clipped and PCR-generated to identify F0 mice. Positive mice were screened and crossed with 8-week-old mice to obtain F1 mice. F1 female mice that tested positive were crossed with F1 male mice to obtain F2 mice, and the resulting F2 mice were then subjected to PCR-generated identification.

[0232] The primer sequences for PCR are as follows:

[0233] Dhx57-Forward Primer: CTTTGTTAGTGGTGTTATCCCTTGCC (SEQ ID NO: 10)

[0234] Dhx57-reverse primer: TGCATCAGATATTCCTAGAACATTTGG (SEQ ID NO: 11)

[0235] like Figure 13 As shown, the CAG encoding arginine at position 527 of the Dhx57 gene of the positive mouse (CAG>TGA) is mutated to the stop codon TGA, that is, the nucleotide sequence is as follows:

[0236] 5'-TGAGAACAGTAAGATCTG CAGGCAGTTT TGA ATGAAACAGGTACTGTTTAAGGGCCAGCG-3'(SEQ ID NO:12)

[0237] The Dhx57 gene with the Q527X mutation of the obtained F2 generation positive mouse with a point mutation (i.e., Dhx57 point mutation mouse) encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 13:

[0238] (SEQ ID NO: 13).

[0239] The human DHX57 gene with the DHX57 point mutation encodes a polypeptide having the amino acid sequence shown in SEQ ID NO: 14:

[0240] MSSSVRRKGKPGKGGGKGSSRGGRGGRSHASKSHGSGGGGGGGGGGGGGNRKASSRIWDDGDDFCIFSESRRPSRPSNSNISKGESRPKWKPKAKVPLQTLHMTSENQEKVKALLRDLQEQ DADAGSERGLSGEEEDDEPDCCNDERYWPAGQEPSLVPDLDPLEYAGLASVEPYVPEFTVSPFAVQKLSRYGFNTERCQAVLRMCDGDVGASLEHLLTQCFSETFGERMKISEAVNQISLD ECMEQRQEEAFALKSICGEKFIERIQNRVWTIGLELEYLTSRFRKSKPKESTKNVQENSLEICKFYLKGNCKFGSKCRFKHEVPPNQIVGRIERSVDDSHLNAIEDASFLYELEIRFSKDH KYPYQAPLVAFYSTNENLPLACRLHISEFLYDKALTFAETSEPVVYSLITLLEEESEIVKLLTNTHHKYSDPPVNFLPVPSRTRINNPACHKTVIPNNSFVSNQIPEVEKASESEESDEDD GPAPVIV ENESYVNLKKKISKRYDWQAKSVHAENGKICKQF (SEQ ID NO: 14).

[0241] Example 13. Phenotypic Identification of Dhx57 Point Mutation Mice

[0242] A behavioral experiment was performed on the Dhx57 point mutant mice of about 11 months old prepared in Example 12.

[0243] 1. Experimental Grouping

[0244] According to genotype, they are divided into wild type (WT), heterozygous (heter), and homozygous (homo).

[0245] 2. Aging score

[0246] The body weight, body temperature, and organ systems of the mice were observed and scored according to the literature (JGerontol A Biol Sci Med Sci. 2014).

[0247] 3. Survival Curve

[0248] The birth and death (or serious illness) dates of the mice were recorded and a survival curve was drawn.

[0249] 4. Behavioral Experiments

[0250] 4.1 Grip strength test: Conducted according to 3.1 of Example 3.

[0251] 4.2 Rotarod test: Conducted according to 3.2 of Example 3.

[0252] 4.3 Morris water maze test: Conducted according to 4.1 of Example 4.

[0253] 4.4 Open field test: Observe spontaneous activity and exploratory behavior, record the distance of clinging, the number of stretching and the time of head exploration.

[0254] One hour before the test, the experimental animals are transported to the test room to adapt to the new environment. Adjust the height of the camera to ensure that all areas of the box can be seen on the computer screen. Adjust the experimental parameters and record the test date, animal grouping and number in the operation software. Before the test, spray 75% alcohol and wipe clean with a paper towel to ensure that the experimental box is clean and odorless. During the test, hold the mouse tail root 1 / 3 from the feeding cage, pay attention to the back to the experimenter, avoid violent operation and other factors that cause animal stress. Place the experimental animal quickly and gently in the central area of the experimental box, start the video collection and analysis system at the same time, and automatically record the activity of the experimental animal in the open field. The experimenter immediately stands 1.5 m away to observe, or leaves the behavior test room to another room where the animal can be observed. The experimental time is usually set to 15 min. After the experiment, stop and save the video, take the animal out of the experimental box, and then put it back into the feeding cage. After each test, remove the excrement of the previous animal and remove the odor with 75% alcohol. After the box is dry and odorless, the next animal experiment is carried out. Export the data for statistical analysis.

[0255] 5. Experimental results

[0256] The grip strength test results of Dhx57 point mutation mice are shown in Figure 14 The results show that the grip strength of Dhx57 point mutation mice is reduced.

[0257] The grip strength test results of Dhx57 point mutation mice are shown in Figure 15 The results show that the average fall time of Dhx57 point mutation mice is shortened and the travel distance is shortened.

[0258] The water maze test results of Dhx57 point mutation mice are shown in Figure 16 A-16E, the results show that the training curve shows significant difference compared with wild type; the number of crossing the platform is reduced, the time required to enter is longer, and the quadrant access time is shorter.

[0259] The open field test results of Dhx57 point mutation mice are shown in Figure 17As shown, the results showed that the head exploration time was significantly shortened in Dhx57 point mutant mice.

[0260] In addition, the aging score results of Dhx57 point mutation mice were as follows Figure 18 The results show that Dhx57 point mutant mice have a significantly increased aging index. The survival curve of Dhx57 point mutant mice is shown in Figure 2. Figure 19 As shown, the results showed that the survival rate of Dhx57 point mutant mice was significantly lower than that of wild-type mice.

[0261] The above behavioral experiments showed that mice with Dhx57 point mutation had obvious cognitive and motor dysfunction.

[0262] Example 14 Therapeutic Effects of Fumaric Acid and Hyodeoxycholic Acid on Dhx57 Point Mutation Mice

[0263] The effect of drug administration on the behavioral performance of Dhx57 point mutation mice was evaluated in the same manner as in Example 11.

[0264] 1. Experimental Grouping

[0265] Animal model: Male mice, including wild-type, heterozygous, and homozygous Dhx57 point mutation mice. The ratio of each genotype between the control group and the treatment group was 1:1

[0266] Treatment group: Fumaric acid and hyodeoxycholic acid (Fu+HDCA) were co-administered for 15 weeks. Fu+HDCA was added to the feed at a rate of 0.8g fumaric acid and 0.8g hyodeoxycholic acid per kilogram of feed. Based on an average mouse weight of 40g, each mouse consumed approximately 5g of feed per day.

[0267] Control group: no drug administration group.

[0268] 2. Behavioral Experiments

[0269] 2.1 Rotarod test: Perform according to 3.2 of Example 3.

[0270] 2.2 Morris water maze test: performed according to 4.1 of Example 4.

[0271] 2.3 Open field test: Perform according to 4.4 of Example 13.

[0272] 3. Experimental Results

[0273] Figure 20 The results of the A-20B rotarod test showed that the falling time and stroke of the mice in the drug-treated group were prolonged, indicating that muscle strength can be enhanced after drug administration.

[0274] Figure 21Results from the A-21C Morris water maze test showed that during the learning phase, mice in the drug-treated group swam a shorter distance on the first day, suggesting improved exploratory efficiency. After training, significant differences in swimming paths were observed between the drug-treated and control groups. Furthermore, the drug-treated group experienced an increase in the number of platform crossings after training.

[0275] Figure 22 The results of the open field test showed that compared with the control group, the distance of the drug-treated group to the wall was shortened and the number of elongation times increased, indicating that the exploration ability was enhanced after drug administration and the animals were more stretched.

[0276] These results suggest that Fu+HDCA may improve motor coordination and cognitive function by regulating metabolic or neuroprotective pathways.

[0277] Example 15 Construction of DHX57 knockout cells

[0278] A DHX57 knockout human ovarian granulosa cell line (KGN) was generated using the CRISPR-Cas9 system.

[0279] Using bioinformatics tools, a highly specific sgRNA (TTCTCTACTATAACAGGTGC, SEQ ID NO: 15) was designed to target gene exon regions and avoid off-target effects. The sgRNA targets the vicinity of the aforementioned DHX57 point mutation site. The sgRNA and a viral vector expressing the Cas9 nuclease were co-transfected into cells. The Cas9-sgRNA complex specifically recognizes and binds to the target site of the genome, resulting in gene frameshift or functional domain disruption. After 48-72 hours of cell culture, successfully transfected cells were selected using puromycin, isolated and expanded by limiting dilution to obtain monoclonal cell lines. Finally, the editing efficiency of the target gene and the loss of protein expression were verified by Sanger sequencing, T7E1 digestion, or Western blot.

[0280] The proliferation and migration of KGN cells with DHX57 knockout were detected by RCTA method. Figure 23 As shown, the results showed that the proliferation and migration of DHX57 knockout cells were significantly slowed down.

[0281] The steps for detecting cell proliferation and migration using RCTA (real-time label-free dynamic cell analysis, xCELLigence) are as follows:

[0282] Cell proliferation assay: Cells are seeded into an E-Plate pre-coated with microelectrodes. Real-time monitoring of changes in electrode impedance (converted to cell index, CI value) dynamically reflects cell attachment and proliferation. Monitoring is continued for several days, and the data are presented as a CI-time curve.

[0283] Cell migration assays use a CIM-Plate (microporous membrane at the bottom of the upper chamber). Serum-free cell suspension is added to the upper chamber, and culture medium containing chemokines (such as 10% fetal bovine serum) is added to the lower chamber. The instrument continuously monitors the impedance changes of the lower chamber electrode (reflecting the number of migrating cells) and quantifies the migration ability through the migration index (MI). Cell density should be optimized before the experiment (e.g., 5×10 cells per well). 3 ~1×10 4 To ensure the linear response of the electrode signal, the background (cell-free wells) should be subtracted during data analysis, and dynamic curves should be generated by software (such as RTCA or xCELLigence) to compare the proliferation rate or migration efficiency of different treatment groups.

[0284] The senescence of cells was detected by β-galactosidase staining. Figure 24 As shown in A-24B, the results showed that KGN cells exhibited a senescent state after DHX57 knockout.

[0285] The steps for detecting cell senescence using β-galactosidase (SA-β-gal) are as follows: After washing with PBS, cells are fixed with a fixative (such as 2% formaldehyde / 0.2% glutaraldehyde) at room temperature for 5-10 minutes, and the fixative is removed by washing with PBS. The cells are then incubated with a staining solution (containing 1 mg / mL X-gal, 5 mmol / L potassium ferrocyanide, 5 mmol / L potassium ferrocyanide, 150 mmol / L NaCl, 2 mmol / L MgCl2, and pH 6.0 citric acid-phosphate buffer) at 37°C in the dark for 12-24 hours. The highly active β-galactosidase in senescent cells catalyzes the hydrolysis of X-gal to form a blue precipitate. After staining, the cells are washed with PBS to remove residual dye, and blue-positive cells (senescent cells) are observed and counted under a microscope. Young cells show no obvious staining.

[0286] In addition, a comprehensive judgment is made in combination with other aging markers (such as p16 and p21). Figure 24 The results of Figure C showed that the expression levels of P16 and P21 mRNA in DHX57 knockout cells were significantly upregulated.

[0287] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction in a subject, wherein: The compound shown in Formula I has the following structure, Among them, R 1 and R 2 Each is independently selected from H, C1-C6 alkyl, C1-C6 alkenyl and C1-C6 alkynyl.

2. Use of hyodeoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction in a subject.

3. Use of a compound of formula I or a pharmaceutically acceptable salt thereof in combination with hyodeoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction in a subject, wherein: The compound shown in Formula I has the following structure, Among them, R 1 and R 2 Each is independently selected from H, C1-C6 alkyl, C1-C6 alkenyl and C1-C6 alkynyl.

4. The use according to claim 1 or 3, wherein R 1 and R 2 Each independently selected from H or C1-C6 alkyl; Preferably, R 1 and R 2 are each independently selected from H and C1-C5 alkyl, more preferably, R 1 and R 2 each independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl and n-pentyl; and / or The salt is selected from alkali metal salts, alkaline earth metal salts or salts formed with organic ligands. Preferably, the salt is selected from sodium salts, potassium salts, calcium salts, magnesium salts or quaternary ammonium salts.

5. The use according to any one of claims 1 to 4, characterized in that The subject has a neurodegenerative change or disease with cognitive dysfunction and / or motor dysfunction; Preferably, the neurodegenerative changes include physiological natural aging or pathological aging, preferably pathological aging; Preferably, the neurodegenerative disease has manifestations of cognitive dysfunction and / or motor dysfunction, more preferably, the neurodegenerative disease includes one or more of dementia with Lewy bodies (DLB), Alzheimer's disease (AD), senile dementia, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease dementia (Pick disease), frontotemporal dementia (FTD), vascular dementia, mixed dementia or aging-related muscle strength and motor function decline; Preferably, the neurodegenerative changes or diseases are caused by genetic factors, immune factors, environmental factors and / or advanced physiological age; Preferably, the compound of formula I or a pharmaceutically acceptable salt thereof has at least one of the following uses: (1) improving and enhancing muscle strength and motor ability; (2) improving and enhancing active avoidance reaction ability, spatial learning and memory ability, and enhancing cognitive level; (3) improving and enhancing antioxidant capacity, pro-inflammatory state, and alleviating oxidative stress damage; (4) enhancing the level of superoxide dismutase (SOD) in serum and lowering the level of lactate dehydrogenase (LDH) in serum; (5) improving immunity; (6) reducing weight; (7) increasing the expression of neurotrophic factors; and (8) improving nerve damage repair.

6. The use according to any one of claims 3 to 5, characterized in that The compound represented by formula I or a pharmaceutically acceptable salt thereof and hyodeoxycholic acid or a pharmaceutically acceptable salt thereof are administered simultaneously or sequentially; Preferably, the mass ratio of the compound represented by formula I or a pharmaceutically acceptable salt thereof to the hyodeoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and further preferably 1:

1.

7. A composition for preventing, delaying or treating cognitive dysfunction and / or motor dysfunction in a subject, characterized in that The composition comprises the compound represented by formula I or a pharmaceutically acceptable salt thereof and / or hyodeoxycholic acid or a pharmaceutically acceptable salt thereof, Among them, R 1 and R 2 Each is independently selected from H, C1-C6 alkyl, C1-C6 alkenyl and C1-C6 alkynyl; Preferably, R 1 and R 2 are each independently selected from H or C1-C6 alkyl, More preferably, R 1 and R 2 are each independently selected from H and C1-C5 alkyl, More preferably, R 1 and R 2 each independently selected from H, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl and n-pentyl; Preferably, the salt is selected from an alkali metal salt, an alkaline earth metal salt or a salt formed with an organic ligand, preferably, the salt is selected from a sodium salt, a potassium salt, a calcium salt, a magnesium salt or a quaternary ammonium salt; Preferably, in the composition, the mass ratio of the compound represented by Formula I or a pharmaceutically acceptable salt thereof to the hyodeoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.05-50), preferably 1:(0.1-10), more preferably 1:(0.5-5), and further preferably 1:(0.5-2); More preferably, the composition further comprises a pharmaceutically acceptable excipient, Further preferably, the pharmaceutically acceptable excipients include one or more of a pharmaceutical carrier, a diluent, an adjuvant and an excipient; Further preferably, the composition is in the form of tablets, capsules, solutions, granules, pills, powders, ointments, pills, suspensions, powders, injections, suppositories, creams, sprays, patches, sustained-release preparations, controlled-release preparations or targeted preparations.

8. A method for treating or preventing cognitive dysfunction and / or motor dysfunction in a subject, the method comprising administering to a subject in need thereof a preventively or therapeutically effective amount of the composition of any one of claims 7 to 9.

9. A DHX57 mutant gene, characterized in that: Compared with the coding gene of human wild-type DHX57, the coding nucleotide of the DHX57 mutant gene corresponding to the 526th amino acid of human wild-type DHX57 is mutated into a stop codon. Preferably, the amino acid sequence of positions 1 to 550 in the amino acid sequence of human wild-type DHX57 is as shown in SEQ ID NO:

5. Preferably, compared with the coding gene of human wild-type DHX57, the DHX57 mutant gene is mutated to T at the 1576th base C corresponding to the human wild-type DHX57 coding gene. Preferably, the nucleotide sequence from position 1 to position 1600 in the nucleotide sequence of human wild-type DHX57 is as shown in SEQ ID NO:

6.

10. A DHX57 mutant protein, characterized in that The DHX57 mutant protein is encoded by the mutant gene according to claim 9; Preferably, the amino acid sequence of the mutant protein is shown in SEQ ID NO:

14.

11. Use of the DHX57 mutant gene according to claim 9, the DHX57 mutant protein according to claim 10, or a detection reagent thereof: (1) For the diagnosis of cognitive dysfunction and / or motor dysfunction; (2) for the preparation of diagnostic reagents for cognitive dysfunction and / or motor dysfunction; Preferably, the cognitive dysfunction and / or motor dysfunction is caused by neurodegenerative changes or diseases; Preferably, the neurodegenerative changes include physiological natural aging or pathological aging, preferably pathological aging; Preferably, the neurodegenerative disease includes one or more of dementia with Lewy bodies (DLB), Alzheimer's disease (AD), senile dementia, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease dementia (Pick disease), frontotemporal dementia (FTD), vascular dementia, mixed dementia or aging-related muscle strength and motor function decline.

12. A method for constructing a mouse model of cognitive dysfunction and / or motor dysfunction, the method comprising mutating the nucleotide encoding amino acid CAG at position 527 of the wild-type mouse Dhx57 gene to the stop codon TGA; Preferably, the amino acid sequence from position 1 to position 550 of the amino acid sequence of the mouse wild-type DHX57 is as shown in SEQ ID NO: 7; Preferably, the method comprises the following steps: (1) Determine the target site based on the sequence of exon 6 of the mouse Dhx57 gene; (2) synthesizing an sgRNA sequence according to the targeting site determined in step (1), and then connecting the synthesized sequence to the backbone vector to construct an sgRNA targeting vector, wherein the sgRNA sequence of the targeting site is: CAGGCAGTTTCAGATGAAACAGG (SEQ ID NO: 8); (3) The sgRNA, CRISPR / Cas9, and donor oligonucleotides obtained by in vitro transcription were introduced into the fertilized eggs of donor female mice to obtain mouse fertilized eggs with Dhx57 point mutations; (4) The obtained fertilized eggs are transplanted into the uterus of a surrogate mouse to obtain a mouse model.

13. A method for screening drugs for preventing or treating cognitive dysfunction and / or motor dysfunction, characterized in that: The method includes any one or more of the following: (1) a step of performing drug screening using cells harboring a DHX57 gene mutation, wherein the nucleotide encoding the 526th amino acid of the DHX57 mutant gene is mutated to a stop codon compared to the wild-type DHX57, and preferably, the amino acid sequence from positions 1 to 550 of the amino acid sequence of the wild-type DHX57 is as shown in SEQ ID NO: 5; (2) a step of performing drug screening using the mouse model obtained according to claim 12; Preferably, the cognitive dysfunction and / or motor dysfunction is caused by neurodegenerative changes or diseases; Preferably, the neurodegenerative changes include physiological natural aging or pathological aging, preferably pathological aging; Preferably, the neurodegenerative disease includes one or more of dementia with Lewy bodies (DLB), Alzheimer's disease (AD), senile dementia, Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease dementia (Pick disease), frontotemporal dementia (FTD), vascular dementia, mixed dementia or aging-related muscle strength and motor function decline.

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