WO2025242146A1 - Use of active compound in preventing, delaying, or treating cognitive and/or motor dysfunction - Google Patents

Abstract

The use of the compound represented by formula I or a pharmaceutically acceptable salt thereof, and/or hyodeoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing, delaying, or treating cognitive dysfunction and/or motor dysfunction, which can effectively enhance and improve muscle strength, motor function, active exploration ability, and spatial learning and memory ability, and can also ameliorate anti-oxidative stress ability and inflammation prone state, enhance the expression of neurotrophic factors, as well as improve nerve damage repair.

Description

Use of active compounds in the prevention, delay or treatment of cognitive and/or motor dysfunction Technical Field

This invention relates to methods and medicines for preventing, delaying or treating cognitive and/or motor dysfunction.

Background Technology

Cognitive impairment primarily manifests as abnormalities in higher cognitive processes related to learning, memory, and judgment, leading to severe learning and memory disorders, often accompanied by pathological changes such as aphasia, apraxia, agnosia, or apraxia. Common causes of cognitive impairment include various forms of dementia. Motor impairment primarily manifests as dysfunction in voluntary motor regulation, while muscle strength, sensation, and cerebellar function remain unaffected. Common motor disorders include Parkinson's disease. Research has found that many cognitive impairments may be accompanied by gait abnormalities and other motor impairments, while motor disorders can also be accompanied by cognitive abnormalities. Cognitive and motor impairments have become significant health service and socioeconomic burdens, particularly in aging societies.

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

Summary of the Invention

To address one of the aforementioned technical problems in the prior art, this disclosure provides pharmaceuticals and methods for preventing, delaying, or treating neurodegenerative changes or diseases characterized by cognitive and/or motor dysfunction. Pharmaceutical treatment according to this disclosure can effectively enhance learning and cognitive functions, muscle strength, and antioxidant capacity, among other things.

According to a first aspect of this disclosure, the use of a compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention, delay, or treatment of cognitive and/or motor dysfunction in a subject is provided, wherein the compound of Formula I has the following structure.

^R1 and ^R2 are each independently selected from H, _C1 - _C6 alkyl, _C1 - _C6 alkenyl and _C1 - _C6 alkynyl.

In some implementations, ^R1 and ^R2 can each be independently selected from H.

In some embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C6 alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C5 alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain C1- _C4 alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _{C1 - C3} alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or n-pentyl.

In some embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C6 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C5 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C4 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C3 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from vinyl, propenyl, butenyl, or pentenyl groups.

In some embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C6 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C5 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C4 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C3 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from acetylene, propenyl, or butynyl.

In some implementations, ^R1 and ^R2 may be the same or different.

In some embodiments, 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.

In some embodiments, the use of sodium fumarate in the preparation of a medicament for the prevention, delay, or treatment of cognitive and/or motor dysfunction in a subject is provided.

In some implementations, the subject has neurodegenerative changes or diseases that cause cognitive and/or motor dysfunction.

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

In some embodiments, the neurodegenerative disease presents with cognitive and/or motor dysfunction.

In some embodiments, the neurodegenerative disease may include, but is not limited to, one or more of the following: 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, frontotemporal dementia (FTD), vascular dementia, mixed dementia, or age-related muscle weakness and motor function decline.

In this disclosure, the pathology of subjects suffering from the aforementioned neurodegenerative disease is characterized by cognitive impairment, motor impairment, or both.

In some implementations, subjects with neurodegenerative diseases that cause cognitive impairment exhibit only or primarily cognitive impairment; such diseases include, but are not limited to, Alzheimer's disease.

In some implementations, subjects with neurodegenerative diseases that cause motor dysfunction exhibit only or primarily motor dysfunction, including but not limited to ALS, mild Parkinson's disease, and moderate Parkinson's disease.

In other implementations, subjects with neurodegenerative diseases that present with both cognitive and motor impairments exhibit both cognitive and motor impairments. These diseases include, but are not limited to, severe Parkinson's disease and Lewy body dementia.

The compounds represented by Formula I of this disclosure, or pharmaceutically acceptable salts thereof, can be used to prevent, delay, or treat the aforementioned neurodegenerative changes or diseases involving cognitive and/or motor dysfunction. In particular, the applicant has found that the medicament of this application has superior preventative, delaying, or therapeutic effects on neurodegenerative diseases involving both cognitive and motor dysfunction.

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

In some embodiments, the compound represented by 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, spatial learning and memory, and cognitive ability; (3) improving and enhancing antioxidant capacity, pro-inflammatory state, and alleviating oxidative stress damage; (4) increasing serum superoxide dismutase (SOD) levels and decreasing serum lactate dehydrogenase (LDH) levels; (5) improving immunity; (6) reducing weight; (7) increasing the expression of neurotrophic factors; and (8) improving nerve damage repair.

According to a second aspect of this disclosure, the use of porcine deoxycholic acid or a pharmaceutically acceptable salt thereof in combination with other ingredients in the preparation of a medicament for the prevention, delay, or treatment of cognitive and/or motor dysfunction in a subject is provided.

In some embodiments, 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.

In some implementations, the subject has neurodegenerative changes or diseases that cause cognitive and/or motor dysfunction.

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

In some embodiments, the neurodegenerative disease presents with cognitive and/or motor dysfunction.

In some embodiments, the neurodegenerative disease may include, but is not limited to, one or more of the following: 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, frontotemporal dementia (FTD), vascular dementia, mixed dementia, or age-related muscle weakness and motor function decline.

In this disclosure, the pathology of subjects suffering from the aforementioned neurodegenerative disease is characterized by cognitive impairment, motor impairment, or both.

In some implementations, subjects with neurodegenerative diseases that cause cognitive impairment exhibit only or primarily cognitive impairment; such diseases include, but are not limited to, Alzheimer's disease.

In some implementations, subjects with neurodegenerative diseases that cause motor dysfunction exhibit only or primarily motor dysfunction, including but not limited to ALS, mild Parkinson's disease, and moderate Parkinson's disease.

In other implementations, subjects with neurodegenerative diseases that present with both cognitive and motor impairments exhibit both cognitive and motor impairments. These diseases include, but are not limited to, severe Parkinson's disease and Lewy body dementia.

The compounds represented by Formula I of this disclosure, or pharmaceutically acceptable salts thereof, can be used to prevent, delay, or treat the aforementioned neurodegenerative changes or diseases involving cognitive and/or motor dysfunction. In particular, the applicant has found that the medicament of this application has superior preventative, delaying, or therapeutic effects on neurodegenerative diseases involving both cognitive and motor dysfunction.

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

In some embodiments, the compound represented by 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, spatial learning and memory, and cognitive ability; (3) improving and enhancing antioxidant capacity, pro-inflammatory state, and alleviating oxidative stress damage; (4) increasing serum superoxide dismutase (SOD) levels and decreasing serum lactate dehydrogenase (LDH) levels; (5) improving immunity; (6) reducing weight; (7) increasing the expression of neurotrophic factors; and (8) improving nerve damage repair.

According to a third aspect of this disclosure, the use of a compound of Formula I or a pharmaceutically acceptable salt thereof in combination with porcine deoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention, delay, or treatment of cognitive and/or motor dysfunction in a subject is provided, wherein the compound of Formula I has the following structure,

^R1 and ^R2 are each independently selected from H, _C1 - _C6 alkyl, _C1 - _C6 alkenyl and _C1 - _C6 alkynyl.

In some implementations, ^R1 and ^R2 can each be independently selected from H.

In some embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C6 alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C5 alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain C1- _C4 alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _{C1 - C3} alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or n-pentyl.

In some embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C6 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C5 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C4 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C3 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from vinyl, propenyl, butenyl, or pentenyl groups.

In some embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C6 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C5 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C4 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C3 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from acetylene, propenyl, or butynyl.

In some implementations, ^R1 and ^R2 may be the same or different.

In some embodiments, 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.

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

In some implementations, the subject has neurodegenerative changes or diseases that cause cognitive and/or motor dysfunction.

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

In some embodiments, the neurodegenerative disease presents with cognitive and/or motor dysfunction.

In some embodiments, the neurodegenerative disease may include, but is not limited to, one or more of the following: 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, frontotemporal dementia (FTD), vascular dementia, mixed dementia, or age-related muscle weakness and motor function decline.

In this disclosure, the pathology of subjects suffering from the aforementioned neurodegenerative disease is characterized by cognitive impairment, motor impairment, or both.

In some implementations, subjects with neurodegenerative diseases that cause cognitive impairment exhibit only or primarily cognitive impairment; such diseases include, but are not limited to, Alzheimer's disease.

In some implementations, subjects with neurodegenerative diseases that cause motor dysfunction exhibit only or primarily motor dysfunction, including but not limited to ALS, mild Parkinson's disease, and moderate Parkinson's disease.

In other implementations, subjects with neurodegenerative diseases that present with both cognitive and motor impairments exhibit both cognitive and motor impairments. These diseases include, but are not limited to, severe Parkinson's disease and Lewy body dementia.

The compounds represented by Formula I of this disclosure, or pharmaceutically acceptable salts thereof, can be used to prevent, delay, or treat the aforementioned neurodegenerative changes or diseases involving cognitive and/or motor dysfunction. In particular, the applicant has found that the medicament of this application has superior preventative, delaying, or therapeutic effects on neurodegenerative diseases involving both cognitive and motor dysfunction.

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

In some embodiments, the compound represented by 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, spatial learning and memory, and cognitive ability; (3) improving and enhancing antioxidant capacity, pro-inflammatory state, and alleviating oxidative stress damage; (4) increasing serum superoxide dismutase (SOD) levels and decreasing serum lactate dehydrogenase (LDH) levels; (5) improving immunity; (6) reducing weight; (7) increasing the expression of neurotrophic factors; and (8) improving nerve damage repair.

In some embodiments, the compound represented by Formula I or a pharmaceutically acceptable salt thereof is administered simultaneously or sequentially with porcine deoxycholic acid or a pharmaceutically acceptable salt thereof.

In some embodiments, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic 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 between them. Preferably, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.1-10). More preferably, the mass ratio of the compound represented by Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.5-5). Even more preferably, the mass ratio of the compound represented by Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.5-2). In some specific embodiments, the mass ratio of the compound represented by Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic acid or a pharmaceutically acceptable salt thereof is 1:1.

Through extensive experiments, the inventors of this invention have surprisingly discovered that the combination of the compound represented by Formula I or a pharmaceutically acceptable salt thereof (e.g., sodium fumarate) and porcine deoxycholic acid has a good effect on the prevention, delay, or treatment of cognitive and/or motor dysfunction diseases.

According to a fourth aspect of this disclosure, a composition is provided for the prevention or treatment of cognitive and/or motor dysfunction in a subject, said composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and/or deoxycholic acid or a pharmaceutically acceptable salt thereof.

^R1 and ^R2 are each independently selected from H, _C1 - _C6 alkyl, _C1 - _C6 alkenyl and _C1 - _C6 alkynyl.

In some implementations, ^R1 and ^R2 can each be independently selected from H.

In some embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C6 alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C5 alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain C1- _C4 alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _{C1 - C3} alkyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, or n-pentyl.

In some embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C6 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C5 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C4 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C3 alkenyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from vinyl, propenyl, butenyl, or pentenyl groups.

In some embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C6 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C5 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C4 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from branched or straight-chain _C1 - _C3 ynyl groups. In some specific embodiments, ^R1 and ^R2 may each be independently selected from acetylene, propenyl, or butynyl.

In some implementations, ^R1 and ^R2 may be the same or different.

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

In some embodiments, 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.

In some embodiments, the mass ratio of the compound represented by Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic 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 between them.

Preferably, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic 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 porcine deoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.5-5). Even more preferably, the mass ratio of the compound of Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic 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 porcine deoxycholic acid or a pharmaceutically acceptable salt thereof is 1:1.

In some embodiments, the compound represented by Formula I or a pharmaceutically acceptable salt thereof, when used in combination with porcine deoxycholic acid or a pharmaceutically acceptable salt thereof, can reduce body weight, increase learning and cognitive function, improve spatial discrimination, increase serum superoxide dismutase (SOD) levels, decrease serum lactate dehydrogenase (LDH) levels, enhance muscle strength, improve immunity, increase the expression of neurotrophic factors, and improve nerve damage repair, etc.

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 pharmaceutical carrier, diluent, adjuvant, and excipient.

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 formulations, controlled-release formulations, or targeted formulations.

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

In some embodiments, the compound represented by Formula I and its pharmaceutically acceptable salts, and/or porcine deoxycholic acid and its pharmaceutically acceptable salts, are formulated for administration in the same dosage form or in separate dosage forms.

In some embodiments, the composition comprises a compound of Formula I or a pharmaceutically acceptable salt thereof, deoxycholic acid, or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

In some embodiments, the composition includes fumaric acid or a pharmaceutically acceptable salt thereof, deoxycholic acid or a pharmaceutically acceptable salt thereof, and optionally a pharmaceutically acceptable excipient.

According to a fourth aspect of this disclosure, a method for treating or preventing cognitive and/or motor dysfunction in a subject is provided, the method comprising administering to a subject in need a preventative or therapeutically effective amount of the composition described in the third aspect.

According to a fifth aspect of this disclosure, a DHX57 mutant gene is provided, wherein, compared with the coding gene for human wild-type DHX57, the DHX57 mutant gene has a mutated stop codon at the coding nucleotide corresponding to amino acid 526 of human wild-type DHX57.

In some embodiments, the protein number of the human wild-type DHX57 is XP_054200443.1, wherein the amino acid sequence of the human wild-type DHX57 from position 1 to position 550 is as shown in SEQ ID NO:5.

In some embodiments, the DHX57 mutant gene is mutated to T at base C at position 1576, corresponding to the encoding gene of human wild-type DHX57, compared to the encoding gene of human wild-type DHX57.

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

In some embodiments, the nucleotide sequence of the human wild-type DHX57 from position 1 to position 1600 is as shown in SEQ ID NO:6.

According to a sixth aspect of this disclosure, a DHX57 mutant protein is provided, said DHX57 mutant protein being encoded by the mutant gene described in this disclosure.

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

According to the seventh aspect of this disclosure, the uses of the DHX57 mutant gene, the DHX57 mutant protein, or a detection reagent thereof described herein are provided:

(1) Used for the diagnosis of cognitive and/or motor dysfunction;

(2) Diagnostic reagents for the preparation of cognitive and/or motor dysfunction.

In some implementations, the cognitive and/or motor dysfunction is caused by neurodegenerative changes or diseases.

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

In some embodiments, the neurodegenerative disease includes one or more of the following: 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, frontotemporal dementia (FTD), vascular dementia, mixed dementia, or age-related muscle weakness and motor function decline.

According to the eighth aspect of this disclosure, a method for constructing a mouse model of cognitive 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.

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

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

In some embodiments, the method includes the following steps:

(1) The target site was determined based on the sequence of exon 6 of the mouse Dhx57 gene.

(2) Synthesize an sgRNA sequence according to the target site determined in step (1), and then connect the synthesized sequence with the backbone vector to construct an sgRNA target vector, wherein the sgRNA sequence of the target site is: CAGGCAGTTTCAGATGAAACAGG (SEQ ID NO:8).

(3) The sgRNA, CRISPR/Cas9 and donor oligonucleotides obtained by in vitro transcription were introduced into donor female mouse zygotes to obtain mouse zygotes with Dhx57 point mutation.

(4) The obtained fertilized eggs were transplanted into the uterus of a surrogate mother mouse to obtain a mouse model.

According to a ninth aspect of this disclosure, a method for screening drugs for the prevention or treatment of cognitive and/or motor dysfunction is provided, the method comprising any one or more of the following:

(1) The step of using cells with DHX57 gene mutation for drug screening, wherein, compared with wild-type DHX57, the DHX57 mutant gene has a stop codon mutated at the 526th amino acid corresponding to human wild-type DHX57. Preferably, the amino acid sequence of the first to the 550th amino acid sequence of human wild-type DHX57 is as shown in SEQ ID NO:5.

(2) Steps for drug screening using mouse models obtained by the construction method of this disclosure.

In some implementations, the cognitive and/or motor dysfunction is caused by neurodegenerative changes or diseases.

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

In some embodiments, the neurodegenerative disease includes one or more of the following: 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, frontotemporal dementia (FTD), vascular dementia, mixed dementia, or age-related muscle weakness and motor function decline.

Attached Figure Description

Figure 1 shows the weight changes of aged mice during the drug administration process.

Figure 2 shows the results of gripping force (A) and rotarod motion experiments (B-C) in aged mice after drug administration, where A shows the peak gripping force, B shows the fall time, and C shows the distance traveled.

Figure 3 shows the results of the water maze test in aged mice after administration of the drug. A shows the time to reach the target, B shows the total swimming distance, C shows the average swimming speed, D shows the time to reach the target position for the first time, E shows the time spent on the target, F shows the number of times the target was passed, and G shows the swimming trajectory.

Figure 4 shows the results of the Y-maze test in aged mice after administration of the drug.

Figure 5 shows the results of redox index detection in serum of aged mice after drug administration, where A represents the result of SOD and B represents the result of LDH.

Figure 6 shows the telomere length detection results after administration to aged mice.

Figure 7 shows the changes in blood cells in peripheral blood and bone marrow of aged mice after drug administration. A represents hematopoietic stem and progenitor cells, B represents myeloid cells, erythroid cells, macrophages, and granulocytes, C represents the proportion of peripheral blood leukocytes, D represents the proportion of cells at each level, and E represents the proportion of myeloid cells and lymphocytes.

Figure 8 shows the immunostaining results of brain slices from the hippocampus of aged mice after drug administration, where A represents the immunostaining results and B represents the quantitative statistical results of A.

Figure 9 shows the immunofluorescence staining results of nerve growth factor (BDNF) in the hippocampus of brain sections from aged mice after drug administration, where A represents the fluorescence staining results and B represents the quantitative statistical results of A.

Figure 10 shows the immunofluorescence staining results (bar = 500 μm) of axonal growth-associated protein (GAP-43) in the hippocampus of brain slices from aged mice after drug administration, where A is the immunostaining result and B is the quantitative statistical result of A.

Figure 11 shows the organ and tissue morphology-safety evaluation results of aged mice after drug administration.

Figure 12 shows a schematic diagram of the Dhx57 point mutation.

Figure 13 shows the sequencing results of the Dhx57 point mutant mouse.

Figure 14 shows the results of the grip test in male mice with the Dhx57 point mutation.

Figure 15 shows the results of the rotarod exercise experiment in Dhx57 point mutant mice, where A shows the average fall time and B shows the average distance traveled.

Figure 16 shows the results of the water maze test in Dhx57 point mutant mice, where A shows the average swimming speed, B shows the time required to reach the target platform, C shows the number of times the platform was crossed, D shows the time required to reach the target platform during the test, and E shows the time spent in the SW quadrant.

Figure 17 shows the head exploration time in the open field experiment of Dhx57 point mutant mice.

Figure 18 shows the aging score results of Dhx57 point mutant mice.

Figure 19 shows the survival curves of Dhx57 point mutant mice.

Figure 20 shows the results of the rotarod exercise experiment in Dhx57 point mutant mice after administration, where A shows the fall time and B shows the average distance traveled.

Figure 21 shows the results of the water maze test in Dhx57 point mutant mice after administration, 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.

Figure 22 shows the head exploration time in the open field experiment after administration to Dhx57 point mutant mice.

Figure 23 shows the cell proliferation and migration results of DHX57 cells, where Control is the non-knockout KGN cell.

Figure 24 shows the senescence characterization results of DHX57 knockout cells, where blue represents senescent cells, A is a β-galactosidase staining image, blue represents 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, ** represents P<0.01.

Detailed Implementation

Current treatments for cognitive impairment diseases such as Alzheimer's disease face challenges including limited efficacy, slowing disease progression, and significant side effects. Monoclonal antibody drugs are also limited by import restrictions and high prices. In this disclosure, fumaric acid, a precursor of L-malate in the tricarboxylic acid cycle (TCA), is formed by the oxidation of succinate by succinate dehydrogenase. Its derivative, dimethyl fumarate, is already a marketed drug used to treat multiple sclerosis and psoriasis, and has undergone toxicological evaluation. Deoxycholic acid is also a naturally occurring bile acid, and deoxycholic acid tablets are already marketed for hyperlipidemia. Therefore, the combined use of these two naturally occurring substances to treat cognitive impairment diseases offers relatively fewer side effects and lower costs.

To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

definition

Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

As used herein, the term "alkyl" refers to a fully saturated straight-chain or branched non-aromatic hydrocarbon. Examples of straight-chain and branched _C1 - _C6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl.

As used in this article, the term "alkenyl" refers to a non-aromatic hydrocarbon containing at least one double bond.

As used in this article, the term "alkynyl" refers to a non-aromatic hydrocarbon containing at least one triple bond.

As used herein, the term "pharmaceutically acceptable" means that it can be administered to humans and/or other animals as subjects without producing excessive adverse reactions or side effects (such as toxicity, irritation, allergic reactions, etc.). The term "excipient" refers to auxiliary materials that coexist with the active ingredient in a pharmaceutical preparation without producing excessive adverse reactions or side effects, including carriers, osmotic pressure regulators, pH adjusters, diluents, disintegrants, excipients, solubilizers, stabilizers, preservatives, etc. The term "pharmaceuticalally acceptable excipient" refers to a highly safe excipient suitable for a specific pharmaceutical preparation and routinely used in pharmaceutical practice. The term "carrier" includes, but is not limited to, liposomes, liposomes, polymer micelles, nanostructured lipid carriers, solid lipid nanocarriers, mesoporous silica nanoparticles, etc.

As used herein, the term “pharmaceutically acceptable salt” may include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands (e.g., quaternary ammonium salts).

The term "hyodesoxycholic acid (HDCA)" used in this article originally refers to a cholanic acid extracted from porcine bile. It is a steroidal acid primarily found in mammalian bile, with the chemical name 3α,6α-dihydroxy-5β-cholanic acid. The differences between various bile acids are minimal, mainly differing in the presence or absence of hydroxyl groups at positions 3, 7, and 12. Bile acids act as physiological cleansers, aiding in the dissolution, absorption, and transport of fats and steroids in the gastrointestinal tract and liver. They are also steroidal amphiphilic molecules derived from cholesterol metabolism, regulating bile distribution and lipid secretion. They are crucial for the absorption of dietary fats and vitamins and participate in regulating enzyme function in cholesterol homeostasis. Bile acids are recycled through the enterohepatic axis formed by the liver, bile ducts, small intestine, and portal vein. Studies have shown that porcine deoxycholic acid (HDCA) can significantly reduce the cell necrosis and apoptosis rates and significantly improve cell survival rates in nerve cells subjected to hypoxia-hypoglycemia-reoxygenation injury, indicating that HDCA has a significant anti-hypoxia-hypoglycemia-reoxygenation effect on nerve cells. Furthermore, HDCA, a secondary hydrophilic bile acid formed by intestinal flora in the small intestine, can prevent the formation of gallstones in mice. Research has found that HDCA 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, bronchitis, viral upper respiratory tract infections in children, and indigestion caused by hepatobiliary diseases; it also has certain inhibitory effects on Bordetella pertussis, Corynebacterium diphtheriae, and Staphylococcus aureus.

The term "prevention" as used in this article refers to preventive treatment of subclinical disease states, aimed at reducing the probability of clinical disease states occurring. "Prevention" can be divided into (a) primary prevention and (b) secondary prevention. Primary prevention is defined as treatment of subjects who have not yet presented with a clinical disease state, while secondary prevention is defined as prevention of the recurrence of the same or similar clinical disease state.

As used in this article, the term "treatment" refers to the treatment of a disease, symptom, or condition, including: 1) suppressing the development of a disease, symptom, or condition; and/or, 2) delaying or alleviating a disease, symptom, or condition.

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

“Cognitive impairment” refers to a decline in cognitive ability relative to a healthy individual, such as an age-matched healthy individual, or relative to the individual’s abilities 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).

"Motor dysfunction" refers to a decline in motor ability/skill relative to a healthy individual, such as an age-matched healthy individual, or relative to that 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).

Neurodegeneration is the gradual loss of neuronal structure and function, including neuronal death and glial cell balance, which can lead to cognitive or motor impairments. Among other causes, age (Alzheimer's disease (AD), Parkinson's disease (PD)) or gene mutations affecting CNS cell function (Huntington's disease, early-onset AD or PD, amyotrophic lateral sclerosis (ALS)) can cause neurodegenerative diseases.

Alzheimer's disease (AD) is a chronic, progressive, degenerative neurodegenerative disease that occurs in old age and pre-old age, characterized by progressive memory loss, cognitive impairment, and personality changes. It is divided into two types: familial Alzheimer's and sporadic. Familial Alzheimer's is further divided into early-onset and late-onset types.

Vascular dementia (VaD) is a group of clinical syndromes characterized by impairment of higher neurocognitive functions, resulting from brain tissue damage caused by cerebrovascular diseases, including ischemic, hemorrhagic, and acute/chronic ischemic-hypoxic cerebrovascular diseases. It mainly includes multiple infarct dementia, multiple lacunar dementia, cerebral amyloid angiopathy, cerebral hypoperfusion dementia, and hemorrhagic dementia.

Mixed dementia refers to a condition that includes both Alzheimer's disease and vascular dementia or other types of dementia. Other types of dementia mainly include fronto-temporal dementia (FTD), dementia of Lewy body (DLB), Parkinson's disease dementia, Huntington's disease dementia, and corticobasal degeneration.

In this disclosure, fumaric acid is a precursor of L-malate in the tricarboxylic acid cycle (TCA), formed by the oxidation of succinate by succinate dehydrogenase, and deoxycholic acid is also a naturally occurring bile acid. Therefore, using these two naturally occurring substances to treat cognitive impairment and delay aging does not produce adverse reactions, is safe, and has few side effects.

The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

Example

Example 1. Method for treating cognitive and motor dysfunction in aged mice with drug administration.

1. Animal Model: Sixteen 20-month-old wild-type C57 mice were selected. These aged mice served as a model, exhibiting typical cognitive and motor dysfunctions, such as weakened muscle strength and balance, reduced memory and spatial exploration abilities, and decreased antioxidant capacity.

2. Grouping: The mice were randomly divided into two groups based on littermates and body weight: a control group (n=8, 2 males and 6 females) and a group treated with sodium fumarate and deoxycholic acid (SF+HDCA) (n=8, 2 males and 6 females). The sodium fumarate and deoxycholic acid group received 3 mg orally once daily via gavage, calculated at 100 mg/kg/day body weight (e.g., 30 g body weight). Sodium fumarate and deoxycholic acid were dissolved in 0.08% (8 mg/100 ml water) hydroxymethyl cellulose. The control group received 0.08% hydroxymethyl cellulose orally once daily via gavage. The treatment lasted for 28 days.

3. Weigh the patient before and after administering the medication.

4. After the administration of the drug, behavioral tests were performed on the mice.

5. Sacrifice the mice, collect blood, and use the serum for AD marker detection and redox index (superoxide dismutase (SOD) and lactate dehydrogenase (LDH)) detection; extract genomic DNA from the blood clots for telomere length detection.

6. Sacrifice the mice and collect the following tissues: brain, hippocampus, liver, spleen, kidney, peripheral blood, and bone marrow. Bone marrow and peripheral blood are used to analyze blood immune indicators; tissue sections of the brain and hippocampus are stained to detect Alzheimer's disease (AD) and aging-related indicators; liver, spleen, and kidneys are used to evaluate drug safety, etc.

See Examples 2-9 for details.

Example 2. Weight changes during drug administration

Mice body weight was measured weekly during drug administration, and statistical analysis was performed after 28 days (see Figure 1). The results of multivariate ANOVA showed that the differences at each time point were statistically significant; there was an interaction between time and grouping. A statistically significant difference was observed between the two groups at week 2 of drug administration (P = 0.033).

Example 3. Experiment to test muscle strength and motor balance

3.1 Grip force measurement

The grip strength testing instrument was placed horizontally. After starting the instrument, the mouse was placed on the gripping board. The mouse's tail was grasped and gently pulled backward. Once the mouse had a firm grip on the gripping board, a uniform force was applied and pulled backward until the mouse released its grip. The instrument recorded the mouse's maximum grip strength. Each mouse was tested three times, and the average value was taken as the measurement result to evaluate the mouse's muscle strength.

The results are shown in Figure 2A, which shows that the muscle strength of the mice was significantly enhanced after administration, and the difference was statistically significant (P = 0.013).

3.2 Rotating bar test

Used to assess motor coordination and balance in rodents. Mice must maintain balance on a rotating bar. The instrument records the time (delay) required for the animal to fall from the bar at different speeds or with continuous acceleration (from 4 to 40 rpm), the speed of the bar at the time of fall, and the distance the animal travels.

The results are shown in Figures 2B and 2C, which show that the mice's motor balance was enhanced after drug administration, with a longer fall delay time and a longer movement distance.

Example 4. Behavioral Indicator Detection

Administration method: Same as in Example 1.

4.1. Morris Water Maze Experiment

The water maze used in this embodiment consisted of a circular water tank with a diameter of 120 cm, filled with tap water, with the platform placed 1-2 cm below the water surface. Both room temperature and water temperature were controlled at 22℃. Different shapes were affixed along the walls of the water tank as spatial reference cues. Note: The positions of objects within the room must remain fixed throughout the experiment. A camera was installed above the maze to record the mouse's swimming tracks, ensuring the actual positions matched the virtual positions in the computer image acquisition software. Animal groups, swimming time (60s), and platform dwell time (10s) were set in the program. During the data collection experiment, the mouse was placed in the maze at one of four points (north, south, east, west) facing the water tank wall.

4.1.1 Learning and Training Experiment: The experiment was conducted for 5 days, with each animal receiving 4 training sessions per day. Each time, the animal was placed in the pool from different entry points, head facing the pool wall, using a semi-random selection of starting positions. The platform was located in the southwest quadrant. This design prevented the animal from learning a specific right-turn or left-turn sequence to locate the platform, using each of the four starting positions each day (see Table 1). The time required for the animal to climb onto the platform from entry into the water was recorded as the latency period; the animal's swimming trajectory in the water was also recorded as a basis for analyzing the strategies employed by the animal when searching for its target. After climbing onto the platform, the animal was allowed to stand on it for 10 seconds; if the animal did not find the platform or fail to climb onto it within 60 seconds, it was led to stand on the platform for 10 seconds before being removed. The average escape latency period (for those who did not climb onto the platform, the latency period was calculated as 60 seconds), swimming distance, swimming speed, and swimming time in the outer ring area were statistically analyzed for each animal at the four entry points each day, and fluctuation curves were plotted to observe trends.

4.1.2 Memory test experiment: Remove the platform from its original location, put the animal into the pool from an entry point, and record the time it takes for the animal to explore and swim past the original platform location for the first time, the number of times it explores and swims past the original platform location area within 60 seconds (i.e., the number of times it passes through the loop), and the swimming time of the animal in the target quadrant where the platform is located.

Table 1. Starting position of the water maze space

The results of the water maze test are shown in Figure 3. After 5 days of training and 1 day of testing, the mice administered the drug showed significantly enhanced learning and memory abilities. Specifically, the time to reach the target platform was significantly shorter than that of the control group, with a significant difference observed from day 4 of training (P = 0.003) (Figure 3A). Conversely, the swimming distance of the control group was significantly longer than that of the drug-treated group (P = 0.03) (Figure 3B), and the swimming speeds were similar between the two groups (Figure 3C). This indicates that the longer time to reach the destination was not due to abnormal limb movement in the control group, i.e., unwillingness or inability to swim, but rather to the stronger learning ability of the drug-treated mice. The test after removing the platform showed that the drug-treated group had a shorter time to reach the platform on the first attempt than the control group (Figure 3D), a longer time spent at the platform (Figure 3E), and passed the target platform more times (Figure 3F). The swimming trajectory of the mice during the test (60s) is shown in Figure 3G (where ○ represents the platform position). Overall, this demonstrates that the memory ability of the mice was significantly enhanced after drug administration.

4.2. Y-maze Experiment

Y-maze alternation behavior test and parameters

The Y-maze test is a behavioral test based on the natural exploratory curiosity of mice. The spontaneous alternation behavior in this test is considered to reflect short-term spatial working memory. It utilizes the test animal's natural tendency to explore new environments. During the experiment, the test animal needs to remember the previously explored directions each time it changes direction to explore a new one. Therefore, the Y-maze test can effectively reflect and measure the animal's spatial working ability.

The Y-maze consists of three arms of equal length (50cm x 18cm x 35cm), with an angle of 120 degrees between each pair of arms. Each arm has a movable partition at its center. A mouse is placed at the end of any arm of the Y-maze and allowed to explore freely for 8 minutes. A video system records the animal's behavioral changes over these 8 minutes, recording the following indicators: ① Total number of entries: The number of times the animal entered an arm (each entry is defined as all four of the mouse's feet entering an arm); ② An alternation: Entering all three arms of the Y-maze consecutively once; ③ The number of maximum alternations: Total number of entries - 2. The score for spontaneous alternation behavior = Total number of alternations / Maximum number of alternations * 100%.

The results of the Y-maze experiment are shown in Figure 4. The number of spontaneous alternations in the drug-treated group increased significantly, indicating that the spatial exploration ability of the mice was significantly enhanced after drug administration.

Example 5. Determination of Oxidative Stress Levels

The oxidative stress hypothesis posits that a decrease in the body's antioxidant capacity weakens its ability to scavenge free radicals, leading to oxidative damage to biomolecules and an imbalance between cellular oxidation and antioxidant functions, resulting in oxidative stress and consequently disease and aging. Oxygen free radicals act on unsaturated fatty acids in lipids, generating lipid peroxides, including malondialdehyde (MDA); antioxidant defense systems that scavenge free radicals include superoxide dismutase (SOD). Lactate dehydrogenase (LD or LDH) is a nicotinamide adenine dinucleotide (NAD)-dependent kinase. LDH is an important enzyme system in glycolysis, catalyzing the reductive oxidation reaction between propionic acid and L-lactate, and also catalyzing related α-keto acids. Oxidative stress has been reported to be associated with aging and Alzheimer's disease. Therefore, this example investigated the effect of sodium fumarate plus deoxycholic acid on oxidative stress in aged mice. The administration method was the same as in Example 1, and the redox indicators SOD and LDH in mouse serum were measured 28 days after administration (according to the Beyotime reagent kit instructions).

The results are shown in Figures 5A and 5B, respectively, indicating that the SOD concentration in the treatment group was significantly increased and the LDH concentration was significantly decreased compared with the control group. This suggests that the combined administration of sodium fumarate and porcine deoxycholic acid can significantly increase the antioxidant capacity of mice, reduce glycolysis, and improve the redox state in the blood of mice.

Example 6. Detection of granule length after drug administration in mice

Every time a cell divides, chromosomes are replicated. However, the enzymes responsible for replication cannot completely reach the chromosome ends, so a small portion of the chromosome ends is lost during each replication process. Telomeres provide a small amount of extra chromosome as a buffer, protecting vital genetic information from loss. In other words, telomeres lose a little length with each cell division. When telomeres can no longer shorten, the cell dies because it can no longer divide. Telomeres are therefore called the "biological clock" by scientists, and telomere shortening is considered a biological marker of cellular aging. Measuring telomere length can indirectly reflect biological age.

In this embodiment, mice were euthanized 28 days after drug 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 telomere repeat sequences to single-copy genes (36B4). When designing primers, telomere primers should be designed for repetitive sequences (forward: 5′-CGG TTT GTT TGG GTT TGG GTT TGG GTT TGG GTT TGG GTT-3′ (SEQ ID NO:1), reverse: 5′-GGC TTG CCT TAC CCT TAC CCT TAC CCT TAC CCT TAC CCT-3′ (SEQ ID NO:2)); primers for the single-copy internal reference gene 36B4 (RPLP0) should be designed across introns to avoid interference with 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 (telomere primer final concentration 900 nM, 36B4 primer 300 nM), and DNA template (10-20 ng). Amplification programs for telomeres and 36B4 were run separately (95℃ 10 min pre-denaturation, 40 cycles: 95℃ 15 s, 60℃ 15 s, 72℃ 30 s). The T/S ratio was calculated using the ΔΔCt method (T/S = 2^[Ct(telomere) - Ct(36B4)]). A standard curve (gradually diluted DNA) was set to verify amplification efficiency (90%-110%), and melting curves were analyzed to ensure primer specificity. The experiment was repeated three times to avoid DNA degradation and primer dimer interference.

The results are shown in Figure 6. It can be seen that the mitochondrial length of mice after drug administration is longer than that of the control group.

Example 7. Changes in blood cells in peripheral blood and bone marrow of mice after drug administration

After euthanizing mice, peripheral blood and bone marrow were collected separately. Flow cytometry was used to analyze various blood cell types. The steps were as follows: sample collection and processing, antibody staining, and instrumental analysis. For bone marrow collection, the femur and tibia were separated after euthanasia. The bone marrow cavity was washed with pre-cooled PBS to obtain a cell suspension. Bone fragments were removed by passing the suspension through a 70μm filter. After centrifugation (300×g, 5 minutes), the supernatant was discarded, and ACK buffer was added to lyse red blood cells for 35 minutes. The cells were washed twice with PBS for later use. For peripheral blood, 50μl was collected via the tail vein into an anticoagulant tube. Red blood cells were directly lysed and washed. Before antibody staining, the cell density was adjusted to 1× ^10⁶ –1×10⁷ ^. Cells/mL, add anti-mouse CD16/32 antibody to block Fc receptor for 10 minutes, then incubate with fluorescently labeled antibody at the concentration specified in the instructions for 20-30 minutes in the dark (CD45: panleukocyte 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/neutrophils); granulocytes: Gr1 (Ly6G/Ly6C complex), CD11b; hematopoietic stem cells/progenitor cells (bone marrow): hematopoietic stem cells (HSC): CD34-, cKit (CD117 ⁺ ), Sca1 ⁺ (LSK phenotype: Lineage ^- Sca1 ⁺⁾ cKit ⁺ ); progenitor cells: CD34 ⁺ , CD16/32 (FcγR), washed twice with PBS to remove unbound antibodies. During flow cytometry, the instrument channels were set according to the fluorescent dyes (FITC, PE, APC), and compensation was adjusted using a single-stain control. Fragmentation was excluded using FSC (cell size) and SSC (granularity) thresholds. For data analysis, viable FSC/SSC cells were first selected, and CD45 ⁺ leukocytes were screened. Further subpopulation analysis was performed, such as CD3 ⁺ T cell typing (CD4 ⁺ /CD8 ⁺ ), CD19 ⁺ B cells, and CD11b ⁺ myeloid cells (Ly6G ⁺ neutrophils, Ly6C ⁺ monocytes). Finally, the data was analyzed using FlowJo software.

The results showed that the number of hematopoietic stem cells and the proportion of myeloid cells increased during aging in mice. In mice treated with sodium fumarate and porcine deoxycholic acid, analysis of the proportions of various cell types in the bone marrow and peripheral blood revealed a decrease in hematopoietic stem and progenitor cells (Figure 7A). Although there was no statistically significant difference, the proportion of leukocytes in peripheral blood was reduced, particularly the proportion of myeloid and lymphocytes (Figures 7B-7E), indicating that combined medication can improve the pro-inflammatory state in aging mice.

Example 8. Immunostaining of hippocampal region in brain sections of mice after drug administration

The hippocampus is associated with learning and memory functions; therefore, this embodiment performs histological examination on the mouse brain from Example 1. The mouse skull was pried open, the whole brain was harvested, dehydrated with 20% sucrose, fixed with paraformaldehyde, and then embedded in OCT to prepare frozen sections; or fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned.

IBA-1 is a marker of microglia/macrophages in the brain and other tissues, and is a neuroinflammatory marker. Alzheimer's disease patients often have immune activation of hippocampal microglia. In this example, IBA-1 is immunostained.

Paraffin sections were dewaxed and hydrated sequentially, followed by antigen retrieval, blocking of endogenous peroxidase, and blocking of nonspecific binding: First, the sections were dewaxed by immersing them in xylene I and II for 10 minutes each, followed by hydration with graded ethanol (100% to 70%) and washing with distilled water; antigen retrieval was performed using sodium citrate buffer (pH 6.0), which was microwaved to boiling and then maintained on low heat for 10-15 minutes, followed by cooling and washing with _PBS ; then, endogenous enzymes were blocked by incubation with 3% _H2O2 at room temperature for 10 minutes, followed by blocking with 5% normal serum for 30 minutes; diluted primary antibody was added and incubated overnight at 4°C or at room temperature for 1-2 hours, followed by washing with PBS and incubation with HRP-labeled secondary antibody at room temperature for 30-60 minutes; DAB chromogenic solution was used for color development in the dark (time controlled under a microscope, usually 30 seconds to 5 minutes), followed by hematoxylin counterstaining for 1-3 minutes after stopping with distilled water, followed by differentiation with hydrochloric acid alcohol, blueing with tap water, dehydration with graded ethanol, clearing with xylene, and mounting with neutral resin for observation (positive signals appear brownish-yellow, and the nucleus is blue).

The results, as shown in Figures 8A-8B, revealed that the combined drug administration reduced the expression of IBA-1 in the hippocampus of mice, indicating that the combined drug administration can reduce immune stress.

Example 9. Immunofluorescence staining of neural growth factor (BDNF) in the hippocampus of mouse brain sections after drug administration.

BDNF is a neurogenic growth factor that binds to its receptor TrkB and mediates the development and function of nerve cells. This example describes immunofluorescence staining of BDNF in the hippocampus of mouse brain tissue. Frozen sections were fixed with 4% paraformaldehyde at room temperature for 15-20 minutes, followed by thorough washing with PBS. Permeabilization with 0.1%-0.5% Triton X-100 was performed for 5-15 minutes, followed by washing with PBS. Incubation with 5% BSA blocking solution at room temperature for 30-60 minutes was performed to reduce non-specific binding. The primary antibody was diluted with blocking solution and used to cover the sample. The sample was incubated overnight at 4°C or for 1-2 hours at room temperature. After washing, the secondary fluorescent antibody was added and incubated in the dark for 1 hour, followed by washing three times with PBST. DAPI nuclear staining was performed for 5-10 minutes. Finally, the slides were mounted with anti-quenching mounting medium and stored in the dark. During observation, the microscope channel should be selected according to the fluorescent dye, and images should be acquired at different time intervals to avoid cross-contamination and quenching.

The results, as shown in Figures 9A-9B, revealed that the combined drug administration significantly increased the expression of BDNF in the hippocampus, indicating that the combined drug administration can improve the growth, development, and function of nerve cells in aging mice.

Example 10. Immunofluorescence staining of axonal growth-associated protein (GAP-43) in the hippocampus of mouse brain sections after drug administration.

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

The results, as shown in Figures 10A-10B, revealed that the drug significantly upregulated the expression of GAP43, indicating that the combined drug therapy could improve the repair of nerve damage in aging mice.

Example 11. Evaluation of drug administration safety

Mice were sacrificed after 28 days of drug administration, and their internal organs were dissected for morphological observation. No obvious abnormalities were found in the drug-treated group. Further morphological observation of liver, spleen, kidney, testis, or ovary was performed to determine the safety of the drug. The results showed that no morphological abnormalities were found in these organs after combined drug administration (Figure 11).

This disclosure utilizes an aged (20-month-old) mouse model to verify that the combination of the compound represented by Formula I and/or its pharmaceutically acceptable salt with porcine deoxycholic acid, administered orally and by gavage for 28 days, resulted in weight loss, enhanced muscle grip strength, improved rotarod movement ability, improved performance in the Morris water maze and Y-maze tests for learning and memory, telomere shortening, enhanced antioxidant indicators, reduced myeloid/lymphatic ratio in the blood system, and improved immunity. These results indicate that fumaric acid or its derivatives, porcine deoxycholic acid, or a combination of both, have significant therapeutic effects in improving cognitive impairment and delaying aging.

Example 12. Construction of DHX57 point mutant mice

The inventors discovered a nonsense mutation in the DExH-box helicase 57 (DHX57) in a clinical premature ovarian failure family through whole-exome sequencing. Specifically, in the coding region of DHX57 (XM_054344468.1), position 1576 of the C-codon was mutated to T (c.C1576T), and position 526 of the arginine (R, CGA) was mutated to the stop codon TGA. The conservation of this mutation site in humans and other species was then analyzed. Position 1579 of the coding region of the mouse Dhx57 gene (NM_001163759.1) corresponds to position 1576 of the human DHX57 gene. In terms of amino acid sequence, position 527 of the mouse DHX57 glutamine (Q) corresponds to position 526 of the human arginine (R). Based on this, Dhx57 point mutant mice were generated.

The amino acid sequence of human wild-type DHX57, from position 1 to position 550, is shown in SEQ ID NO:5:

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

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

A C57BL/6J mouse model with a Dhx57 point mutation was constructed using CRISPR/Cas-mediated genome engineering. Figure 12 shows a schematic diagram.

The steps are as follows:

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

Design gRNA targeting vector and donor oligonucleotide Oligo (with targeting sequence, flanked by 120bp or 150bp homologous sequences respectively).

The sequence of the gRNA is as follows:

The sequence of the donor oligonucleotide is as follows:

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

Cas9 mRNA and gRNA, generated through in vitro transcription, were co-injected, along with donor oligonucleotides, into the nuclei of fertilized egg cells obtained from the uterus of a donor female mouse 3.5 days post-mating. The fertilized eggs, injected with RNA and donor Oligo, were then transplanted into the uterus of a surrogate mother mouse; after successful transplantation, the mice were born as F0 mice.

Around one week old, the toes of the mice were clipped for PCR identification of F0 mice. Positive mice were selected and crossed with 8-week-old mice to obtain F1 mice. The positive F1 female mice were crossed with F1 male mice to obtain F2 mice, and the obtained F2 generation mice were identified by PCR.

The primer sequences for PCR are shown below:

Dhx57 forward primer: CTTTGTTAGTGGTGTTATCCCTTGCC (SEQ ID NO: 10)

Dhx57-Reverse Primer: TGCATCAGATATTCCTAGAACATTTGG (SEQ ID NO:11)

As shown in Figure 13, in positive mice (CAG>TGA), the CAG mutation encoding arginine at position 527 of the Dhx57 gene is replaced by the stop codon TGA, resulting in the nucleotide sequence shown below:

5'-TGAGAACAGTAAGATCTG CAGGCAGTTT TGA ATGAAACAGG TACTGTTTAAGGGCCAGCG-3'(SEQ ID NO:12)

The Dhx57 gene with the Q527X mutation in the obtained F2 generation positive mice with point mutations (i.e., Dhx57 point mutation mice) encodes a polypeptide with the amino acid sequence shown in SEQ ID NO:13:

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

Example 13. Phenotypic identification of Dhx57 point mutant mice

Behavioral experiments were conducted on the Dhx57 point mutant mice that were about 11 months old, prepared in Example 12.

1. Experimental Grouping

Based on genotype, they are classified as wild-type (WT), heterozygous (heter), and homozygous (homo).

2. Aging score

The body weight, body temperature, and organ systems of the mice were observed and scored according to the methods in the literature (J Gerontol A Biol Sci Med Sci. 2014).

3. Survival curve

Record the birth and death (or serious illness) dates of mice and plot survival curves.

4. Behavioral experiments

4.1 Grip test: Performed according to 3.1 of Example 3.

4.2 Rotating bar experiment: Performed according to 3.2 of Example 3.

4.3 Morris water maze experiment: conducted according to 4.1 of Example 4.

4.4 Open field experiment: Observe autonomous activities and exploratory behaviors, and record the distance to the wall, the number of times the head is extended, and the time spent exploring the head.

One hour before the test, transfer the experimental animals to the testing room to acclimatize to the new environment. Adjust the camera height to ensure the entire area of the enclosure is visible on the computer screen. Set the experimental parameters and record the experiment date, animal grouping, and number in the operating software. Before the test, spray the enclosure with 75% alcohol and wipe it clean with paper towels to ensure it is clean and odorless. During the experiment, hold the mouse by the base of its tail (about 1/3 of the way down) and remove it from its cage, ensuring it faces away from the experimenter and avoiding any stressful handling. Quickly and gently place the animal in the center of the enclosure and simultaneously activate the video acquisition and analysis system to automatically record the animal's activity in the open space. The experimenter should immediately stand at least 1.5 meters away to observe, or leave the behavioral testing room to another room where the animal can be observed. The experiment is typically set for 15 minutes. After the experiment, stop and save the recording, remove the animal from the enclosure, and return it to its cage. After each test, remove the excrement from the previous animal and deodorize with 75% alcohol. Allow the test chamber to dry and become odorless before proceeding with the next animal. Export the data for statistical analysis.

5. Experimental Results

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

The results of the grip test of Dhx57 point mutant mice are shown in Figures 15A-15B. The results show that the average fall time and the distance traveled by Dhx57 point mutant mice are shorter.

The results of the water maze experiment in Dhx57 point mutant mice are shown in Figures 16A-16E. The results show that the training curves are significantly different from those of wild type; the number of steps through the platform is reduced, the time required to enter is longer, and the time to visit the quadrants is shorter.

Figure 17 shows the open field test results of Dhx57 point mutant mice. The results indicate that the head exploration time of Dhx57 point mutant mice is significantly shortened.

Furthermore, the aging score results of Dhx57 point mutant mice are shown in Figure 18, indicating that Dhx57 point mutant mice have a significantly elevated aging index. The survival curve results of Dhx57 point mutant mice are shown in Figure 19, indicating that the survival rate of Dhx57 point mutant mice is significantly lower than that of wild-type mice.

The above behavioral experiments show that mice with the Dhx57 point mutation have significant cognitive and motor dysfunction.

Example 14: The therapeutic effects of fumaric acid and porcine deoxycholic acid on Dhx57 point mutant mice

The effects of drug administration on the behavioral performance of Dhx57 point mutant mice were assessed using the same method as in Example 11.

1. Experimental Grouping

Animal model: Male mice, including wild-type, heterozygous, and homozygous Dhx57 point mutant mice. The ratio of each genotype in the control group and the treatment group was 1:1.

The treatment group received a combination of fumaric acid and porcine deoxycholic acid (Fu+HDCA) for 15 weeks. The administration method involved adding Fu+HDCA to the feed, with 0.8g of fumaric acid and 0.8g of porcine deoxycholic acid per kilogram of feed. Based on an average mouse weight of 40g, each mouse consumed approximately 5g of feed per day.

Control group: No medication administered.

2. Behavioral experiments

2.1 Rotating bar experiment: Performed according to 3.2 of Example 3.

2.2 Morris water maze experiment: conducted according to 4.1 of Example 4.

2.3 Open field experiment: Performed according to 4.4 of Example 13.

3. Experimental Results

The results of the rotarod experiment in Figures 20A-20B show that the fall time and distance of the mice in the drug-treated group were prolonged, indicating that the drug can enhance muscle strength.

The Morris water maze experiment results in Figures 21A-21C show that during the learning phase, the mice in the drug-treated group swam a shorter distance on the first day, indicating improved exploration efficiency. After training, there were significant differences in swimming paths between the drug-treated and control groups. Furthermore, the number of times mice crossed platforms increased in the drug-treated group after training.

The open field experiment results in Figure 22 show that, compared with the control group, the distance of the drug-treated group to the wall was shortened and the number of extensions increased, indicating that the exploration ability was enhanced after drug administration and the animals were more able to extend.

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

Example 15 Construction of DHX57 knockout cells

A DHX57 knockout human ovarian granulosa cell line (KGN) was prepared using the CRISPR-Cas9 system.

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

The proliferation and migration of DHX57 knockout KGN cells were detected by RCTA method. The results are shown in Figure 23. The results show that the proliferation and migration of DHX57 knockout cells were significantly slowed down.

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

Cell proliferation assay involves seeding cells into E-Plates pre-coated with microelectrodes. By monitoring changes in electrode impedance in real time (converted into cell index, CI value), the assay dynamically reflects cell adhesion and proliferation. The assay is continuously monitored for several days, and the data is presented as a CI-time curve.

Cell migration detection was performed using a CIM-Plate (with a microporous membrane at the bottom of the upper chamber). Serum-free cell suspension was added to the upper chamber, and culture medium containing chemokines (e.g., 10% fetal bovine serum) was added to the lower chamber. The instrument continuously monitored changes in electrode impedance in the lower chamber (reflecting the number of migrating cells), and migration ability was quantified using the migration index (MI). Before the experiment, cell density needed to be optimized (e.g., 5 × 10³ to 1 × 10⁴ cells per well) to ensure a linear response of the electrode signal. Background subtraction (for cell-free wells) was performed during data analysis, and dynamic curves were generated using software (e.g., RTCA or xCELLigence) to compare the proliferation rate or migration efficiency of different treatment groups.

Cell senescence was detected by β-galactosidase staining, and the results are shown in Figures 24A-24B. The results show that KGN cells exhibited a senescent state after DHX57 was knocked out.

The steps for detecting cell senescence using β-galactosidase (SA-β-gal) are as follows: After washing cells with PBS, fix them with a fixative (e.g., 2% formaldehyde/0.2% glutaraldehyde) at room temperature for 5-10 minutes, then wash with PBS to remove the fixative. Incubate the cells with a staining solution (containing 1 mg/mL X-gal, 5 mmol/L potassium ferrocyanide, 5 mmol/L potassium ferricyanide, 150 mmol/L NaCl, 2 mmol/L MgCl2, citrate-phosphate buffer, pH 6.0) at 37°C in the dark for 12-24 hours. The highly active β-galactosidase in senescent cells will catalyze the hydrolysis of X-gal to generate a blue precipitate. After staining, wash with PBS to remove residual dye, observe under a microscope and count the blue positive cells (senescent cells). Young cells show no obvious staining.

In addition, other aging markers (such as p16 and p21) were used for comprehensive assessment. The results in Figure 24C show that the expression levels of p16 and p21 mRNA in DHX57 knockout cells were significantly upregulated.

The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims (13)

The use of the compound represented by Formula I or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention, delay, or treatment of cognitive and/or motor dysfunction in a subject, wherein the compound represented by Formula I has the following structure,
^R1 and ^R2 are each independently selected from H, _C1 - _C6 alkyl, _C1 - _C6 alkenyl and _C1 - _C6 alkynyl. The use of porcine deoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the prevention, delay or treatment of cognitive and/or motor dysfunction in subjects. The use of the compound represented by Formula I or a pharmaceutically acceptable salt thereof in combination with porcine deoxycholic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention, delay, or treatment of cognitive and/or motor dysfunction in subjects, wherein the compound represented by Formula I has the following structure,
^R1 and ^R2 are each independently selected from H, _C1 - _C6 alkyl, _C1 - _C6 alkenyl and _C1 - _C6 alkynyl. According to claim 1 or 3, ^R1 and ^R2 are each independently selected from H or _C1 - _C6 alkyl groups; Preferably, ^R1 and ^R2 are each independently selected from H and _C1 - _C5 alkyl groups; more preferably, ^R1 and ^R2 are 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. The application according to any one of claims 1-4 is characterized in that the subject has neurodegenerative changes or diseases of cognitive dysfunction and/or motor dysfunction; Preferably, the neurodegenerative changes include physiological natural aging or pathological aging, and more preferably pathological aging; Preferably, the neurodegenerative disease presents with cognitive and/or motor dysfunction. More preferably, the neurodegenerative disease includes one or more of the following: 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, frontotemporal dementia (FTD), vascular dementia, mixed dementia, or age-related muscle weakness 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 represented by 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 cognitive level; (3) improving and enhancing antioxidant capacity, pro-inflammatory state, and alleviating oxidative stress damage; (4) increasing serum superoxide dismutase (SOD) level and decreasing serum lactate dehydrogenase (LDH) level; (5) improving immunity; (6) reducing weight; (7) increasing the expression of neurotrophic factors; and (8) improving nerve damage repair. The application according to any one of claims 3-5 is characterized in that the compound represented by formula I or a pharmaceutically acceptable salt thereof is administered simultaneously or sequentially with porcine deoxycholic acid or a pharmaceutically acceptable salt thereof; Preferably, the mass ratio of the compound represented by Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.05-50), more preferably 1:(0.1-10), more preferably 1:(0.5-5), further preferably 1:(0.5-2), and even more preferably 1:1. A composition for preventing, delaying, or treating cognitive and/or motor dysfunction in a subject, characterized in that the composition comprises a compound of formula I or a pharmaceutically acceptable salt thereof and/or deoxycholic acid or a pharmaceutically acceptable salt thereof.
^R1 and ^R2 are each independently selected from H, _C1 - _C6 alkyl, _C1 - _C6 alkenyl and _C1 - _C6 alkynyl; Preferably, ^R1 and ^R2 are each independently selected from H or _C1 - _C6 alkyl groups. More preferably, ^R1 and ^R2 are each independently selected from H and _C1 - _C5 alkyl groups. More preferably, ^R1 and ^R2 are 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 alkali metal salts, alkaline earth metal salts, or salts formed with organic ligands; more preferably, the salt is selected from sodium salts, potassium salts, calcium salts, magnesium salts, or quaternary ammonium salts. Preferably, in the composition, the mass ratio of the compound represented by Formula I or a pharmaceutically acceptable salt thereof to the porcine deoxycholic acid or a pharmaceutically acceptable salt thereof is 1:(0.05-50), more preferably 1:(0.1-10), more preferably 1:(0.5-5), and even more preferably 1:(0.5-2); More preferably, the composition further includes pharmaceutically acceptable excipients. More preferably, the pharmaceutically acceptable excipients include one or more of pharmaceutical carriers, diluents, adjuvants, and excipients; More preferably, the composition is a tablet, capsule, solution, granule, pill, powder, ointment, pill, suspension, powder, injection, suppository, cream, spray, patch, sustained-release preparation, controlled-release preparation, or targeted preparation. A method for treating or preventing cognitive and/or motor dysfunction in a subject, the method comprising administering to a subject in need a preventative or therapeutically effective amount of the composition of any one of claims 7-9. A DHX57 mutant gene, characterized in that, compared with the coding gene of human wild-type DHX57, the DHX57 mutant gene has a stop codon mutated at amino acid position 526 of human wild-type DHX57; preferably, the amino acid sequence of amino acids 1-550 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 from C to T at the 1576th base corresponding to the coding gene of human wild-type DHX57. Preferably, the nucleotide sequence of the nucleotide sequence of human wild-type DHX57 from position 1 to position 1600 is as shown in SEQ ID NO:6. A DHX57 mutant protein, characterized in that the DHX57 mutant protein is encoded by the mutant gene as described in claim 9; Preferably, the amino acid sequence of the mutant protein is shown in SEQ ID NO:14. Uses of the DHX57 mutant gene of claim 9, the DHX57 mutant protein of claim 10, or their detection reagents: (1) Used for the diagnosis of cognitive and/or motor dysfunction; (2) Diagnostic reagents for the preparation of cognitive and/or motor dysfunction; Preferably, the cognitive and/or motor dysfunction is caused by neurodegenerative changes or diseases; Preferably, the neurodegenerative changes include physiological natural aging or pathological aging, and more preferably pathological aging; Preferably, the neurodegenerative disease includes one or more of the following: 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, frontotemporal dementia (FTD), vascular dementia, mixed dementia, or age-related muscle weakness and motor function decline. A method for constructing a mouse model of cognitive and/or motor dysfunction, the method comprising mutating the nucleotide encoding amino acid CAG at amino acid position 527 of the wild-type mouse Dhx57 gene to the stop codon TGA; Preferably, the amino acid sequence of the wild-type mouse DHX57 from position 1 to position 550 is as shown in SEQ ID NO:7; Preferably, the method includes the following steps: (1) The target site was determined based on the sequence of exon 6 of the mouse Dhx57 gene. (2) Synthesize an sgRNA sequence according to the target site determined in step (1), and then connect the synthesized sequence with the backbone vector to construct an sgRNA target vector, wherein the sgRNA sequence of the target site is: CAGGCAGTTTCAGATGAAACAGG (SEQ ID NO:8). (3) sgRNA, CRISPR/Cas9 and donor oligonucleotides obtained by in vitro transcription were introduced into donor female mouse zygotes to obtain mouse zygotes with Dhx57 point mutation; (4) The obtained fertilized eggs were transplanted into the uterus of a surrogate mother mouse to obtain a mouse model. A method for screening drugs for the prevention or treatment of cognitive and/or motor dysfunction, characterized in that the method comprises any one or more of the following: (1) The step of using cells with DHX57 gene mutation for drug screening, wherein, compared with wild-type DHX57, the DHX57 mutant gene has a stop codon mutated at the 526th amino acid corresponding to human wild-type DHX57. Preferably, the amino acid sequence of the first to the 550th amino acid sequence of human wild-type DHX57 is as shown in SEQ ID NO:5. (2) The step of using the mouse model obtained in claim 12 for drug screening; Preferably, the cognitive and/or motor dysfunction is caused by neurodegenerative changes or diseases; Preferably, the neurodegenerative changes include physiological natural aging or pathological aging, and more preferably pathological aging; Preferably, the neurodegenerative disease includes one or more of the following: 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, frontotemporal dementia (FTD), vascular dementia, mixed dementia, or age-related muscle weakness and motor function decline.