Use of adiponectin and adiponectin receptor agonists in treatment of amyotrophic lateral sclerosis

Abstract

The invention provides application of adiponectin and an adiponectin receptor agonist in treatment of amyotrophic lateral sclerosis. The invention finds that the plasma adiponectin level of an ALS patient is related to the inflammatory factor level, and finds that AdipoR1 and AdipoR2 in the ALS patient are remarkably up-regulated and are related to M1 type macrophage activation. Experiments prove that the adiponectin receptor stimulant can relieve the proinflammatory response of macrophages, regulate the polarization of the macrophages and protect neurons, and the adiponectin receptor stimulant can postpone the occurrence of dyskinesia of ALS patients and prolong the lifetime of the ALS patients. The invention provides a new strategy for diagnosis and treatment of ALS, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of adiponectin and adiponectin receptor agonists in the treatment of amyotrophic lateral sclerosis. Background Art

[0002] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Approximately two-thirds of typical ALS patients have a spinal form of the disease (limb onset) and present symptoms related to local muscle weakness and wasting, where the onset of symptoms can start distally or proximally in the upper and lower extremities. Gradually, spasticity may occur in the weakened and atrophied limbs, affecting hand dexterity and gait. Patients with bulbar-onset ALS typically present dysarthria and difficulty swallowing solids or liquids. Limb symptoms can occur almost simultaneously with bulbar symptoms, and in the vast majority of cases, limb symptoms develop within 1 to 2 years. Paralysis is progressive, and bulbar-onset cases die of respiratory failure within 2 to 3 years, while limb-onset ALS cases die of respiratory failure within 3 to 5 years.

[0003] Due to the variability and complexity of the ALS disease itself and the lack of standard and clinically meaningful effective treatments, the treatment of ALS remains a challenge for clinicians. In addition, the efficacy of known drugs is limited and decreases over time.

[0004] Therefore, there is a continuing need to identify drugs with higher efficacy for the treatment of ALS. Summary of the Invention

[0005] In view of this, in order to make up for the deficiencies of the prior art, the present invention is specifically proposed.

[0006] The first aspect of the present invention provides any one of the following applications:

[0007] (1) The application of adiponectin or an adiponectin receptor agonist in the preparation of a drug for treating ALS or symptoms caused by ALS or inhibiting the progression of said symptoms;

[0008] (2) The application of adiponectin or an adiponectin receptor agonist in promoting neuron proliferation or reducing neuronal apoptosis.

[0009] In the present invention, adiponectin mainly exerts its biological functions through adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2).

[0010] In the present invention, adiponectin receptor agonists are a class of compounds or drugs that can mimic or enhance the action of adiponectin. Currently, some small-molecule compounds and adiponectin-derived polypeptides have been found to target and activate adiponectin receptors, inducing downstream signaling cascades similar to those of adiponectin (such as AMPK, p38MAPK, and PPARα, etc.), and exerting biological effects similar to those of adiponectin. These adiponectin receptor agonists include synthetic small-molecule compounds such as AdipoRon and AdipoAI, etc., adiponectin-derived polypeptides such as ADP355, ALY688, ADP399, ADP-1, APNpI, APNpII, KS23, 5-mer peptide, Pep70, PEG-BHD 1028, JT003, Tyr-Pro dipeptide, YPG, and YPP tripeptide, etc., flavonoid substances such as GTDF and Tiliroside, etc., and some plant proteins such as Osmotin, etc. Among them, AdipoRon is an orally active adiponectin receptor agonist that can bind to AdipoR1 and AdipoR2, and its binding constants (Kd) are 1.8 μM and 3.1 μM respectively; JT003 is a dual agonist of AdipoR1 / AdipoR2, which can simultaneously stimulate the signaling pathways mediated by AdipoR1 and AdipoR2.

[0011] Furthermore, the adiponectin receptor agonist is selected from AdipoRon.

[0012] In the present invention, ALS is an adult-onset neurodegenerative disease and a progressive fatal disease, which is characterized by the selective death of motor neurons in the motor cortex, brainstem, and spinal cord. Patients diagnosed with ALS develop a progressive muscle phenotype, which is characterized by spasticity, hyperreflexia or hyporeflexia, fasciculations, muscle atrophy, and paralysis. These motor impairments are caused by muscle denervation resulting from the loss of motor neurons. The main pathological features of ALS include the degeneration of the corticospinal tract and a large loss of lower motor neurons (LMNs) or anterior horn cells, the degeneration and loss of Betz cells and other pyramidal cells in the primary motor cortex, and reactive gliosis in the motor cortex and spinal cord.

[0013] Furthermore, examples of the symptoms caused by ALS include, but are not limited to, decreased respiratory function, speech disorders, dysphagia, limb motor dysfunction, etc. ALS or the symptoms caused by it should be interpreted as widely as possible according to the technical knowledge in the relevant technical field, and should not be limited by the differences in the names of the diseases.

[0014] Approximately 10% of ALS cases have a family history of the disease, and these patients are referred to as familial ALS (fALS) or hereditary patients, typically with an autosomal dominant Mendelian inheritance pattern and high penetrance. The remaining (approximately 90%-95%) are classified as sporadic ALS (sALS) as they have no documented family history and are thought to be caused by other risk factors, including environmental factors, genetic polymorphisms, somatic mutations, and possible gene-environment interactions. In most cases, familial (or hereditary) ALS is inherited as an autosomal dominant disorder, but there are pedigrees with autosomal recessive and X-linked inheritance and incomplete penetrance.

[0015] In the present invention, amyotrophic lateral sclerosis includes gene mutation type amyotrophic lateral sclerosis.

[0016] Furthermore, the genes include, but are not limited to, SOD1, TARDBP, FUS / TLS, C9orf72, ALS2, SETX, VAPB, ANG.

[0017] Furthermore, the gene is selected from SOD1.

[0018] In the present invention, SOD1 is one of the three human superoxide dismutases identified and characterized in mammals: copper-zinc superoxide dismutase (Cu / ZnSOD or SOD1), manganese superoxide dismutase (MnSOD or SOD2), and extracellular superoxide dismutase (ECSOD or SOD3). SOD1 is a 32 kDa homodimer of a 153-residue polypeptide, with each subunit having a copper and a zinc binding site, encoded by the SOD1 gene (NCBI reference sequence NM_000454.4) on human chromosome 21. SOD1 catalyzes the conversion of superoxide anion (O2-) to molecular oxygen (O2) and hydrogen peroxide (H2O2) at the bound copper ion. The intracellular concentration of SOD1 is high (ranging from 10 to 100 μM), accounting for 1% of the total protein content in the central nervous system (CNS). The protein is located not only in the cytoplasm of eukaryotic cells but also in the nucleus, lysosomes, peroxisomes, and the intermembrane space of mitochondria. Currently, at least 170 different mutations distributed throughout the 153-amino acid SOD1 polypeptide have been found to cause ALS, and an updated list can be found in the ALS Online Genetic Database (ALSOD) (Wroe R et al., Amyotroph Lateral Scler., 2008, 9, 249-250).

[0019] In the present invention, some examples of mutations in SOD1 in ALS include, but are not limited to, mutations of Q22L, E21K, G, F20C, N19S, G16A, S, VI4M, S, G12R, G10G, V, R, L8Q, V, V7E, C6G, F, V5L, A4T, V, S in exon 1; mutations of T54R, E49K, H48R, Q, V47F, A, H46R, F45C, H43R, G41S, D, G37R in exon 2; mutations of D76Y, V, G72S, C, L67R, P66A, N65S, S59I, S in exon 3; mutations of DI24G, V, V118L, InSAAAAC, L117V, T116T, R115G, G114A, I113T, F, I112M, T, G108V, L106V, F, S106L, delTCACTC, I104F, D101G, Y, H, N, E100G, K, I99V, V97L, M, D96N, V, A95T, V, G93S, V, A, C, R, D, D90V, A, A89T, V, T88delACTGCTGAC, V87A, M, N86I, S, D, K, G8SR, S, L84V, F, H80R in exon 4; mutations of I151T, S, I149T, V148I, G, G147D, R, C146R, stop, A145T, G, L144F, S, G141E, stop, A140A, G, N139D, K, H, N, G138E:T137R, S134N, E133V, delGAA, insTT, E132insTT, G127R, InsTGGG, L126S, delITT, stop, D126, delTT in exon 5. These mutations are mainly single amino acid substitutions (i.e., missense mutations), although deletions, insertions, and C-terminal truncations also occur. Different SOD1 mutations show different geographical distribution profiles. For example, among all Americans with ALS caused by SOD1 gene mutations, approximately half have a specific mutation, Ala4Val (or A4V). The A4V mutation is usually associated with more severe signs and symptoms. So far, the I113T mutation is the most common mutation in the UK. In Europe, the most prevalent mutation is the D90A substitution.

[0020] Further, the mutation type of the SOD1 is selected from SOD1 G93A 。

[0021] In the present invention, "treatment" refers to the amelioration, prevention, or reversal of a disease or disorder or at least one distinguishable symptom thereof. Further, the treatment refers to the amelioration, prevention, or reversal of at least one measurable physiological parameter related to the disease or disorder to be treated, and such parameter is not necessarily recognizable in or recognized by a mammal. Further, the treatment refers to the inhibition or slowing down of a disease or disease course, and such inhibition or slowing down can be physical, such as certain distinguishable adverse symptoms. "Treatment" as used in the present invention encompasses diseases of mammals, particularly humans, including: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not been diagnosed with the disease; (b) inhibiting the disease, such as arresting the development of the disease; or (c) alleviating the disease, such as reducing the symptoms associated with the disease.

[0022] In the present invention, the effect of treatment can be judged by behavioral assessment and / or neurological scoring. Further, the behavioral assessment includes, but is not limited to, the rotarod test.

[0023] In the present invention, "promote" means allowing neuronal cells to enhance their ability to proliferate. For example, the viability or proliferation rate of neuronal cells can include an enhancement of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 98%.

[0024] In the present invention, "inhibit" means allowing neuronal cells to reduce their ability to apoptose. For example, the apoptosis ratio or apoptosis rate of neuronal cells can include a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more than 98%.

[0025] The second aspect of the present invention provides a pharmaceutical composition, which comprises at least one active ingredient selected from adiponectin or an adiponectin receptor agonist and a pharmaceutically acceptable carrier and / or excipient thereof.

[0026] In the present invention, the active ingredient is administered in a therapeutically effective amount. The term "therapeutically effective amount" refers to the level or amount of the target agent that does not produce significant negative or adverse side effects: (1) delaying or preventing the onset of ALS; (2) slowing down or preventing the progression, exacerbation, or deterioration of one or more symptoms of ALS; (3) ameliorating the symptoms of ALS; (4) reducing the severity or incidence of ALS; or (5) curing ALS. A therapeutically effective amount can be administered before the onset of ALS for preventive purposes. Alternatively, a therapeutically effective amount can be administered after the onset of ALS for therapeutic or maintenance therapeutic purposes.

[0027] In the present invention, "pharmaceutically acceptable carriers and / or excipients" include but are not limited to diluents, binders, surfactants, humectants, adsorbent carriers, lubricants, disintegrants, emulsifiers, bioavailability enhancers, suspending agents, sweeteners, flavoring agents, coloring agents, excipients, preservatives, solubilizers, dispersants, and / or wetting agents. Among them, diluents include but are not limited to lactose, sodium chloride, glucose, urea, starch, water; binders include but are not limited to starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and its salts, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; surfactants include but are not limited to polyoxyethylene sorbitan fatty acid esters, sodium dodecyl sulfate, monoglyceride stearate, cetyl alcohol; humectants include but are not limited to glycerol, starch; adsorbent carriers include but are not limited to starch, lactose, bentonite, silica gel, kaolin, saponite; lubricants include but are not limited to zinc stearate, glycerol monostearate, polyethylene glycol, talc, calcium stearate, magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, sucrose monolaurate, sodium lauryl sulfate, magnesium lauryl sulfate, sodium dodecyl sulfate.

[0028] In the present invention, the administration method of the pharmaceutical composition can refer to the administration situation of edaravone injection. When the pharmaceutical composition of the present invention is used for the treatment of ALS, for example, the administration method described in WO2020 / 091036 (or US Patent Publication No. 2020 / 268712) can be adopted, or the administration method of clinically used edaravone injection can be adopted. The administration method can be, for example, daily administration or intermittent administration. The intermittent administration is discontinuous administration, that is, for example, a unit (a cycle composed of an administration period and a drug withdrawal period) is repeated 2 times or more (for example, 2 to 24 times), preferably 3 times or more (for example, 3 to 18 times), more preferably 4 times or more (for example, 4 to 12 times). When the administration period and the drug withdrawal period are repeated as a unit 2 times or more, the last period is the drug withdrawal period. In this case, the last drug withdrawal period can be set, or the last drug withdrawal period can be not set. That is, for example, when the administration period and the drug withdrawal period are repeated as a unit 2 times, this can be the case of "administration period, drug withdrawal period, administration period, drug withdrawal period", or the case of "administration period, drug withdrawal period, administration period" without setting the last drug withdrawal period.

[0029] The withdrawal period refers to a period during which no pharmaceutical composition is administered for, for example, several consecutive days (such as 2 or 3 days) or more, preferably a period of 7 or 14 consecutive days without administering the pharmaceutical composition. The administration period is, for example, 14 days. In this case, the administration can be carried out for 14 consecutive days, or for 10 days within the 14-day period. The 10 days out of 14 days refer to any 10 days within the consecutive 14 days. The 10 days of administration can be 10 consecutive days, or 10 non-consecutive days with one or more non-administration periods (such as a period of 1 to 4 days) in between. The administration period can be selected as a preferred period by observing the patient's condition. More specifically, for example, the following method can be adopted: first provide an initial 14-day administration period, then provide an initial 14-day withdrawal period, and then repeat the 10-day administration period within 14 days and the subsequent 14-day withdrawal period. There is no specific limit on the number of repetitions of the 10-day administration period within 14 days and the 14-day withdrawal period. For example, it can be one or more times (such as 1 to 23 times), preferably two or more times (such as 2 to 17 times), and more preferably three or more times (such as 3 to 11 times). In this repetition, a final withdrawal period can be set or not set.

[0030] During the administration period, there is no limit on the number of daily administrations, and the preferred number of administrations can be selected while observing the patient's condition. Considering the patient's burden, the number of administrations is preferably 1, 2, or 3 times, more preferably 1 or 2 times, and even more preferably 1 time.

[0031] In the present invention, the administration route of the pharmaceutical composition is not particularly limited and may be oral administration or parenteral administration. Additionally, bolus administration and sustained release administration are also possible. Sustained release administration is preferred. In the case of sustained release administration, intravenous administration by infusion, transdermal administration, oral administration using sublingual tablets, oral and rectal administration using sustained release preparations, oral or intragastric administration using suspensions, etc. can be used. Intravenous administration by infusion or oral or intragastric administration using suspensions is preferred. In the case of multiple administrations of the suspension to a subject, the administration may be only intragastric administration, only oral administration, a combination of intragastric and oral administrations, or may be converted from oral administration to intragastric administration or from intragastric administration to oral administration. Generally, intragastric administration refers to directly administering the pharmaceutical composition to the stomach without passing through the oral cavity or esophagus. This is a measure for dealing with the situation where a patient cannot ingest necessary nutrients through the oral cavity due to dysphagia or the like. Thus, for example, in cases where more effective treatment is required according to the symptoms, the administration can be converted from oral administration to intragastric administration. In addition, in the case of intragastric administration, for example, tube feeding (also referred to as enteral administration or gastroenteral administration) can be used. Specific examples include administration through a gastrostomy catheter or a nasogastric catheter, or a combination of both, or conversion from one to the other. When performing bolus administration by injection or intravenous administration by infusion, it is preferred to use, for example, the injectables described in Japanese Patent Application Laid-Open No. S63-132833, Japanese Patent Application Laid-Open No. 2011-62529 (or U.S. Patent Application Publication No. 2011 / 68037). When administering orally or intragastrically, it is preferred to use, for example, the suspensions described in WO2020 / 091036 (or U.S. Patent Application Publication No. 2020 / 0268712).

[0032] In the present invention, the pharmaceutical composition can be used in combination with other drugs for treating ALS to treat a subject suffering from ALS.

[0033] Furthermore, the other drugs for treating ALS include, but are not limited to, riluzole, edaravone, the combination of dextromethorphan / quinidine, or laquinimod.

[0034] In the present invention, the severity of ALS (hereinafter referred to as ALS severity) can be grade 1, grade 2, grade 3, grade 4, or grade 5.

[0035] Grade 1: Generally able to do housework and work.

[0036] Grade 2: Have difficulty doing housework and work, but generally able to live independently (take care of oneself) in daily life.

[0037] Grade 3: Unable to independently complete one or more of eating, excretion, or activities, and need help in daily life.

[0038] Grade 4: Respiratory distress, difficulty in expectoration or dysphagia occurs.

[0039] Grade 5: Tracheotomy, non-oral nutritional intake (tube feeding, total parenteral nutrition, etc.) or use of a ventilator is required.

[0040] The third aspect of the present invention provides a method for promoting neuron proliferation or reducing neuron apoptosis in vitro, the method comprising promoting neuron proliferation or reducing neuron apoptosis in vitro by administering adiponectin or an adiponectin receptor agonist.

[0041] The fourth aspect of the present invention provides the use of a reagent for detecting AdipoR1 and / or AdipoR2 and their expression products in the preparation of a product for diagnosing ALS.

[0042] In the present invention, the term "diagnosis" refers to the discovery, judgment or recognition of the health status or condition of an individual based on one or more symptoms, data or other information related to the individual. Diagnosis can usually be carried out between a diseased patient and a normal human body, or between different disease subtypes of a diseased patient. In the present invention, the diagnosis of ALS includes diagnosing individuals without ALS and individuals with ALS, or diagnosing the SOD1 G93A subtype of ALS.

[0043] Furthermore, the reagent includes an oligonucleotide probe specifically recognizing AdipoR1 and / or AdipoR2, a primer specifically amplifying AdipoR1 and / or AdipoR2, a binder specifically binding to the protein encoded by AdipoR1 and / or AdipoR2, or a chip specifically analyzing AdipoR1 and / or AdipoR2.

[0044] Furthermore, the reagent further includes a detectable label. A detectable label refers to a composition capable of generating a detectable signal indicating the presence of a target polynucleotide in a sample to be assayed. Suitable labels include but are not limited to radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Thus, a label is any composition capable of being detected by a device or method, including but not limited to spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, chemical detection devices or any other suitable device. Further, the label can be visually detected without the aid of a device.

[0045] In the present invention, radioisotopes include but are not limited to 3 H, 14 C, 35 S, 125 I, 131I. Enzymes include, but are not limited to, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase. Fluorescent molecules include, but are not limited to, FITC, rhodamine, lanthanide phosphors.

[0046] In the present invention, the term "probe" refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as the "target polynucleotide") through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe can be directly or indirectly labeled. Hybridization methods include, but are not limited to: solution phase, solid phase, mixed phase, or in situ hybridization assays.

[0047] In the present invention, the term "primer" refers to an oligonucleotide that can specifically anneal to an RNA or DNA locus adjacent to the target sequence and serve as an initiation primer for DNA synthesis under suitable conditions, under which the synthesis of primer extension products is induced, for example, in the presence of nucleotides and a polymerization inducer such as DNA-dependent DNA polymerase, as well as suitable temperature, pH, metal concentration, and salt concentration. Typically, a pair of amplification primers, also known as a "primer pair", is used in a PCR reaction, including an "upstream" or "forward" primer and a "downstream" or "reverse" primer, which define the region of RNA or DNA to be amplified.

[0048] In the present invention, the term "amplification" refers to a method of replicating a part of a nucleic acid by applying, for example, any of various primer extension reactions. Exemplary primer extension reactions include, but are not limited to, PCR. Unless explicitly stated, "amplification" refers to a single replication, or arithmetic, logarithmic, or exponential amplification.

[0049] In the present invention, the term "binding agent" refers to a protein (protein, protein-like, or protein-containing) molecule or a part thereof that can bind to a membrane protein using specific intermolecular interactions. Binding agents for proteins are, for example, receptors for proteins, lectins that bind proteins, antibodies against proteins, peptidebodies against proteins, bispecific dual binding agents, or bispecific antibodies. More specifically, the term "binding agent" refers to a polypeptide, more specifically a protein domain. A suitable protein domain is an element of the overall protein structure that is self-stabilizing and folds independently of the rest of the protein chain and is generally referred to as a "binding domain". The length of such binding domains varies between about 25 amino acids up to 500 amino acids and more. Many binding domains can be classified as folds and are recognizable, identifiable, 3-D structures. Some folds are very common in many different proteins such that they are given specific names.

[0050] Furthermore, the reagent is selected from binders that specifically bind to the proteins encoded by AdipoR1 and / or AdipoR2.

[0051] Furthermore, when detecting AdipoR1 and / or AdipoR2 and their expression products in a subject, it is possible to detect only AdipoR1 or AdipoR2 and their expression products, or to detect both AdipoR1 and AdipoR2 and their expression products simultaneously.

[0052] Furthermore, when a subject is determined to have ALS, the level of AdipoR1 or its expression product is higher than that of AdipoR2. Similarly, it is also possible to determine whether a subject has ALS based on the level of AdipoR1 or its expression product in the subject being higher than that of AdipoR2.

[0053] The fifth aspect of the present invention provides a product for diagnosing ALS, and the product includes a reagent capable of detecting the expression levels of AdipoR1 and / or AdipoR2.

[0054] In the present invention, "expression level" refers to the amount, accumulation or rate of biomarker molecules or genomes. The expression level can be represented, for example, by the following: the amount or synthesis rate of messenger RNA (mRNA) encoded by a gene, the amount or synthesis rate of a polypeptide or protein encoded by a gene, or the amount or synthesis rate of a biomolecule accumulated in a cell or biological fluid. The term "expression level" refers to the absolute amount or relative amount of a molecule in a sample determined under steady-state or non-steady-state conditions.

[0055] Furthermore, the product further includes a chip, a kit or a nucleic acid membrane strip.

[0056] Furthermore, the chip includes a gene chip and a protein chip.

[0057] Furthermore, the gene chip includes oligonucleotide probes for detecting the transcriptional levels of AdipoR1 and / or AdipoR2 that are specific for AdipoR1 and / or AdipoR2.

[0058] Furthermore, the protein chip includes specific binders for AdipoR1 and / or AdipoR2 proteins.

[0059] Furthermore, the kit includes reagents for detecting the expression levels of AdipoR1 and / or AdipoR2 genes or proteins by RT-PCR method, qRT-PCR method, biochip detection method, Southern blotting method, in situ hybridization method, immunoblotting method, mass spectrometry method.

[0060] Further, the kit further comprises reagents for detecting the expression levels of AdipoR1 and / or AdipoR2 proteins by flow cytometry.

[0061] Further, the kit includes instruments or reagents for processing samples.

[0062] Further, the sample includes cells, tissues, blood, urine, saliva or mucus.

[0063] Further, the sample is selected from cells.

[0064] Further, the cells include monocytes or macrophages.

[0065] The sixth aspect of the present invention provides any one of the following applications:

[0066] (1) The application of adiponectin or an adiponectin receptor agonist in regulating inflammatory cytokines;

[0067] (2) The application of adiponectin or an adiponectin receptor agonist in regulating macrophage polarization.

[0068] Further, the inflammatory cytokines include IL-1β, IL-2, IL-8, IL-6, TNF-α, IL-4, IL-10.

[0069] Further, the adiponectin or an adiponectin receptor agonist has a significant negative correlation with the expression levels of IL-1β, IL-2, IL-8, IL-6, TNF-α. When the expression level of the adiponectin or an adiponectin receptor agonist increases, the expression levels of IL-1β, IL-2, IL-8, IL-6, TNF-α decrease. When the expression level of the adiponectin or an adiponectin receptor agonist decreases, the expression levels of IL-1β, IL-2, IL-8, IL-6, TNF-α increase.

[0070] Further, the adiponectin or an adiponectin receptor agonist has a significant positive correlation with the expression levels of IL-4, IL-10. When the expression level of the adiponectin or an adiponectin receptor agonist increases, the expression levels of IL-4, IL-10 increase. When the expression level of the adiponectin or an adiponectin receptor agonist decreases, the expression levels of IL-4, IL-10 decrease.

[0071] The seventh aspect of the present invention provides a computer-based method for diagnosing ALS, comprising:

[0072] Obtaining data: obtaining the expression level data of AdipoR1 and / or AdipoR2 in a sample of a subject to be tested.

[0073] Processing data: Input the expression level data of AdipoR1 and / or AdipoR2 into the constructed model, which predicts whether a subject is an ALS patient based on the AdipoR1 and / or AdipoR2 expression data.

[0074] Output result: Output whether the subject to be tested is an ALS patient.

[0075] Furthermore, the sample includes cells, tissues, blood, urine, saliva or mucus.

[0076] Furthermore, the sample is selected from cells.

[0077] Furthermore, the cells include monocytes or macrophages.

[0078] The eighth aspect of the present invention provides a computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, it implements the method for diagnosing ALS described in the seventh aspect of the present invention.

[0079] The ninth aspect of the present invention provides a device for computer-aided diagnosis of ALS, which includes a memory and a processor. The memory is used to store program instructions. The processor is used to call the program instructions, and when the program instructions are executed, it implements the method for diagnosing ALS described in the seventh aspect of the present invention.

[0080] Advantages and beneficial effects of the present invention:

[0081] The present invention provides the application of adiponectin and adiponectin receptor agonists in the treatment of ALS. The present invention discovers that there is a correlation between the plasma adiponectin level and the inflammatory factor level in ALS patients, and also discovers that AdipoR1 and AdipoR2 are significantly up-regulated in ALS patients and are related to the activation of M1 macrophages. The present invention proves through experiments that adiponectin receptor agonists can reduce the pro-inflammatory response of macrophages, regulate macrophage polarization, protect neurons, and adiponectin receptor agonists can delay the occurrence of motor dysfunction in ALS patients and prolong their survival period. The present invention provides a new strategy for the diagnosis and treatment of ALS and has good clinical application prospects. Description of the drawings

[0082] Figure 1It is a graph showing the expression results of AdipoR1 and AdipoR2 in monocytes and macrophages of ALS patients. Among them, Figure A is a graph showing the expression results of AdipoR1 on the surface of classical monocytes (CD14++CD16-) detected by flow cytometry; Figure B is a graph showing the expression results of AdipoR2 on the surface of classical monocytes (CD14++CD16-) detected by flow cytometry; Figure C is a graph showing the expression results of AdipoR1 and AdipoR2 on the surface of monocytes (CD14++CD16-) of ALS patients; Figure D is a graph showing the expression results of AdipoR1 and AdipoR2 on the surface of macrophages induced by monocytes detected by immunocytochemistry.

[0083] Figure 2 It is a graph showing the results of AdipoRon inhibiting macrophage inflammation and regulating macrophage polarization.

[0084] Figure 3 It is a graph showing the results of AdipoRon promoting neuron proliferation and reducing apoptosis. Among them, Figures A, B, E, and F are graphs showing the results of AdipoRon promoting neuron proliferation; Figures C, D, G, and H are graphs showing the results of reducing neuron apoptosis.

[0085] Figure 4 It is a graph showing the effects of AdipoRon on the rotarod test, neurological score, onset time, and survival time of SOD1 G93A transgenic mice. Among them, Figure A is a graph showing the effect of AdipoRon on the rotarod test of SOD1 G93A transgenic mice; Figure B is a graph showing the effect of AdipoRon on the neurological score of SOD1 G93A transgenic mice; Figure C is a graph showing the effect of AdipoRon on the onset time of SOD1 G93A transgenic mice; Figure D is a graph showing the effect of AdipoRon on the survival time of SOD1 G93A transgenic mice. Detailed implementation methods

[0086] The following further illustrates the present invention in conjunction with embodiments. The following description is only for the preferred embodiments of the present invention, and does not limit the present invention in other forms. Any person skilled in the art may use the disclosed technical content to make equivalent changes into equivalent embodiments. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0087] Embodiment

[0088] I. Materials and methods

[0089] 1. Chemical drugs

[0090] The adiponectin receptor agonist (AdipoRon, catalog number HY-15848) was purchased from MedChemExpress, LPS (catalog number L4391) was purchased from sigma-Aldrich, and IL-4 (catalog number 200-04) was purchased from Peprotech.

[0091] 2. Research subjects

[0092] All ALS patients were recruited from the Department of Neurology, the Second Hospital of Hebei Medical University from February 2023 to April 2024 and diagnosed using the revised El Escorial criteria. The exclusion criteria were: 1) Respiratory insufficiency at the time of enrollment (FVC < 80% or score of < 4 for dyspnea, orthopnea, or respiratory insufficiency in ALSFRS-R); 2) Severe dysarthria resulting in inability to communicate; 3) Comorbid severe neurological or psychiatric diseases; 4) Patients with acute or chronic inflammatory diseases (such as acute pneumonia and rheumatoid arthritis). Age- and sex-matched healthy individuals served as normal controls.

[0093] 3. Collection of basic information and disease-related information of research subjects

[0094] For ALS patients, the following data were collected and recorded: disease duration, site of onset (limb / medulla), disease severity (ALSFRS-R), and disease progression rate (δFS: [48 - ALSFRS-R] / disease duration). For all participants, age, sex, height, and weight were collected and recorded.

[0095] 4. Collection of plasma specimens

[0096] Eighty-two ALS patients and 25 normal controls had venous blood samples collected on an empty stomach in the early morning and collected into EDTA vacuum blood collection tubes, and then immediately centrifuged at 2000 × g for 15 minutes at room temperature. After centrifugation, the plasma was taken out, aliquoted, and frozen at -80°C for further use.

[0097] 5. Detection of human adipokines, inflammatory cytokines, and chemokines

[0098] The cryopreserved plasma was assayed for adipokines, inflammatory cytokines, and chemokines by enzyme-linked immunosorbent assay (ELISA). Plasma samples from ALS patients and normal controls were quantitatively analyzed using an ELISA kit (Bioswamp, Wuhan, China), and the detected indicators included adiponectin, interleukin-1β, interleukin-2, interleukin-8, interleukin-6, tumor necrosis factor-α, interleukin-4, interleukin-10, interleukin-13, transforming growth factor-β, CXC chemokine ligand 13, CXC chemokine ligand 10, chemokine C-X3-C-motif ligand 1, and chemokine ligand 2. The optical density was measured at 450 nm using an ELISA analyzer (AMR-100, Hangzhou, China), and the concentrations of plasma adipokines, inflammatory cytokines, and chemokines were calculated.

[0099] 6. Flow cytometry

[0100] 200 μL of whole blood was stained on ice in the dark for 15 minutes with cell surface antigen-specific antibodies CD14-APC (1:100, BD Biosciences, 555399) and CD16-perCY-5.5 (1:100, BD Biosciences, 560717), and 2 mL of red blood cell lysis buffer (BD Biosciences, 349202) was added to completely lyse red blood cells for 20 minutes. Then, it was centrifuged at 500×g for 5 minutes with phosphate-buffered saline (PBS) and washed twice. After washing with PBS, 500 μL of fixation and permeabilization solution (BD Biosciences, 554722) was added and incubated at room temperature for 20 minutes. Then, it was centrifuged at 500×g for 5 minutes with PBS and washed twice. Anti-AdipoR1 (1:400, Santa Cruz, sc-518030) and anti-AdipoR2 antibody (1:400, Santa Cruz, sc-514045) were added to the cell suspension and incubated for 30 minutes. After incubation, fluorescein-5-isothiocyanate (FITC)-conjugated secondary antibody goat anti-mouse IgG (H+L) (1:1000, Protein tech, SA00003-1) was added and incubated again for 20 minutes. Finally, the cells were washed with PBS and resuspended for flow cytometry analysis.

[0101] 7. Isolation of peripheral blood mononuclear cells (PBMCs)

[0102] Add human peripheral blood lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd., LTS1077) into a high-speed centrifuge tube (Tianjin Haoyang Biological Products Technology Co., Ltd., 601002), centrifuge at 200×g for 2 minutes at room temperature, then add the peripheral blood sample and centrifuge at 800×g for 30 minutes. Aspirate the middle mononuclear cell layer into a new centrifuge tube and centrifuge at 300×g for 13 minutes. After aspirating the supernatant, the cells are retained for subsequent experiments.

[0103] 8. In vitro culture and polarization induction of macrophages

[0104] Resuspend the previously prepared PBMCs with the medium, which contains RPMI-1640 medium (Gibco, C11875500BT), 1% penicillin-streptomycin (Gibco, 15140122), 10% fetal bovine serum (CellMax, SA211.02) and macrophage colony-stimulating factor (M-CSF) (Pepro Tech, 300-25-10). Seed the cells in a 48-well plate and change the medium every 3 days. On the seventh day of cell culture, add 100 ng / ml lipopolysaccharide (LPS) (Sigma-Aldrich, L4391) and 20 ng / ml recombinant human IL-4 (Pepro tech, 200-04) respectively to induce the cells into M1 and M2 phenotypes. Meanwhile, an adiponectin receptor agonist (AdipoRon) (MedChemExpress, HY-15848) with a concentration of 50 μM is also added to study its effect on macrophage inflammation.

[0105] 9. Immunocytochemistry

[0106] After the above stimulation and intervention, the culture medium was removed and fixed with 4% paraformaldehyde-PBS solution. Then the cells were permeabilized with PBS solution containing 0.1% Triton-X100 for 15 minutes and blocked with PBS solution containing 10% donkey serum at room temperature for 1 hour, and incubated with the primary antibody overnight. After that, the cells were washed three times with PBS solution, and then incubated with the corresponding secondary antibody at room temperature for 1 hour. Then the cells were washed three times with PBS and observed using a confocal fluorescence microscope (Oberkochen, LSM900). The primary and secondary antibodies used in this study are as follows: CD68 (1:500, Abcam, ab31630), F4 / 80 (1:200, Abcam, ab6640), MHC-II (1:200, Abcam, ab23990), CD206 (1:100, R&D Systems, AF2535), IL-1B (1:200, proteintech, 16806-1-AP), IL-4 (1:200, Biolegend, 504101), AdipoR1 (1:400, Santa Cruz, sc-518030), AdipoR2 (1:400, Santa Cruz, sc-514045), donkey anti-mouse IgG H&L 647 (1:1000, Abcam, ab150111), donkey anti-rabbit IgG H&L 594 (1:100, Invitrogen, A21207), donkey anti-rat IgG H&L 488 (1:1000, Invitrogen, A21208), donkey anti-goat IgG H&L 649 (1:11000, Invitrogen, A25447).

[0107] 10. Neuron cell line, cell culture

[0108] NSC34 hybrid cells generated by the fusion of embryonic mouse spinal cord motor neurons and mouse neuroblastoma cells were stably transfected with green fluorescent protein (GFP)-empty vector (E), GFP-human SOD1 G93A (hSOD1 G93A) plasmid, and stored in liquid nitrogen. When the experiment was carried out, the cell line was taken out from liquid nitrogen and rapidly resuscitated, and then cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, C11995500BT) containing 10% FBS and 1% P-S. The cells were cultured in an environment of 37 °C and 5% CO2, and the culture medium was changed every 2 days.

[0109] 11. Detection of neuron cell proliferation and apoptosis

[0110] NSC-34-E and NSC34-hSOD1 G93A cells were seeded into 96-well plates and supplemented with the supernatants of different groups of macrophages. Annexin V red dye (Sartorius, 4641) was added to each well at a ratio of 1:200 and analyzed by the Incucyte live cell imaging system (Sartorius). Live cell images were captured every 1 hour and 30 minutes for a total of 72 hours of monitoring.

[0111] 12. Animal models and treatments

[0112] SOD1 G93A transgenic mice and age-matched wild-type (WT) littermates were bred from male hemizygous carriers (B6SJL-Tg(SOD1-G93A)1Gur / J) (purchased from Jackson Laboratory) and female B6SJL / F1 hybrids. The animals were housed in a temperature- and humidity-controlled environment with a 12 / 12 h light / dark cycle and provided with specific pathogen-free rodent food and sterile water. The genotypes of SOD1 G93A transgenic mice were identified by PCR. Animal groups: SOD1 G93A vehicle group, SOD1 G93A AdipoRon group.

[0113] 13. Behavioral assessment

[0114] 13.1 Rotarod test

[0115] The rotarod test was started at week 12 and measured once a week. After week 17, it was measured twice a week. The 5 days before the experiment were the adaptation and learning stages for transgenic mice. The mice were placed on a stationary rotarod, which started at 2 r / min and reached 30 r / min within 3 min. The time the mice stayed on the rotarod was recorded. Each mouse was measured three times, with a 30-min interval between each measurement, and the longest stay time was recorded.

[0116] 13.2 Neurological score

[0117] The neurological score was started at week 12 and evaluated once a day. Scoring: 0 points: When the mouse was suspended by the tail, the hind limbs were fully extended, away from the side midline, and the mouse could maintain this posture for 2 seconds. 1 point: Collapse or partial collapse of the legs towards the lateral midline or tremors of the hind limbs occurred during suspension. 2 points: The toes were bent at least 2 times when walking 12 inches, or any part of the foot dragged along the bottom of the cage or table. 3 points: Rigid paralysis or minimal joint movement, and the foot could not produce forward movement. 4 points: When the mouse was tilted, it could not return to the upright position within 30 seconds.

[0118] 13.3 Onset period and survival period

[0119] A mouse was recorded as having a disease onset when it could not hold on for 3 minutes in three consecutive rotarod tests or the neurological score reached 1 point. The mice were monitored daily starting from 120 days of age. The experimental endpoint was determined when the neurological score reached 4 points and / or a weight loss of more than 15% was observed.

[0120] 14. Statistical analysis

[0121] The Shapiro-Wilk test was used to test for normality. Continuous variables with a normal distribution were expressed as mean ± standard deviation, and non-normal distributions were expressed as median (interquartile range). Categorical variables were expressed as proportions. The Student t-test and Mann-Whitney U test were used to compare the baseline data, adipokines, and inflammatory cytokines between ALS patients and the control group. In addition, the Spearman rank correlation coefficient test was used for correlation analysis. All statistical analyses were performed using SPSS 22, and P ≤ 0.05 was considered statistically significant.

[0122] II. Experimental results

[0123] 1. Demographic characteristics, plasma adiponectin, inflammatory cytokine, and chemokine levels in ALS patients and the control group

[0124] This study included 82 ALS patients and 25 controls. The ALS group consisted of 51 males and 31 females, with a mean age of 60.53 ± 10.33 years and a BMI of 23.24 (5.36). The control group consisted of 14 males and 11 females, with a mean age of 57.13 ± 7.16 years and a BMI of 26.11 (3.7). There were no significant differences in age and gender between the two groups (Table 1). The levels of plasma adiponectin and inflammatory cytokines in ALS patients and controls are shown in Table 1. Compared with the control group, the plasma adiponectin concentration in ALS patients was significantly decreased (P < 0.05). In this study, we measured the levels of pro-inflammatory cytokines, anti-inflammatory cytokines, and chemokines in plasma respectively. In terms of pro-inflammatory cytokines, the levels of IL-1β, IL-2, IL-8, IL-6, and TNF-α in ALS patients were significantly higher than those in the control group (P < 0.05). In terms of anti-inflammatory cytokines, there was only a significant difference in the level of IL-10 between the two groups (IL-4: 462.66 (175.12) vs. 498.45 (258.32), P = 0.480; IL-13: 224.64 (118.33) vs. 229.15 (109.61), P = 0.524; TGF-β: 492.98 (257.67) vs. 468.71 (268.67), P = 0.982). There was no difference in the chemokine levels between ALS patients and the control group, including CXCL13 (193.43 (77.21) vs. 184.19 (89.44), P = 0.381), CXCL10 (452.52 (163.9) vs. 425.73 (196.15), P = 0.749), CX3CL1 (3.08 (1.37) vs. 3.05 (1.18), P = 0.965), CCL2 (357.08 (152.56) vs. 360.52 (115.18), P = 0.988).

[0125] Table 1 Demographic data, plasma adiponectin, inflammatory factors, and chemokine levels in ALS patients and normal controls

[0126]

[0127] Note: Except for adiponectin and CX3CL1 with the unit of ng / ml, the units of the remaining inflammatory factors are pg / ml.

[0128] 2. Correlation analysis of plasma adiponectin with inflammatory cytokines and chemokines in ALS patients

[0129] We performed Spearman correlation analysis on adiponectin and inflammatory cytokines in 82 ALS patients. We found that plasma adiponectin was significantly negatively correlated with the levels of IL-1β (r = -0.619, P < 0.001), IL-2 (r = -0.506, P < 0.001), IL-8 (r = -0.464, P < 0.001), IL-6 (r = -0.564, P < 0.001), and TNF-α (r = -0.682, P < 0.001), while it was significantly positively correlated with the levels of IL-4 (r = 0.262, P = 0.017) and IL-10 (r = 0.489, P < 0.001) (Table 2). There was no correlation between adiponectin and the detected chemokines (CXCL13: r = -0.060, P = 0.595; CXCL10: r = -0.145, P = 0.193; CX3CL1: r = -0.060, P = 0.595) (Table 2).

[0130] Table 2 Correlation analysis of plasma adiponectin levels with inflammatory factors and chemokine levels in ALS patients.

[0131]

[0132] 3. Expression of AdipoR levels in monocytes and monocyte-induced macrophages in the blood of ALS patients

[0133] A total of 30 ALS patients and 15 controls were included. Flow cytometry results showed that compared with the control group, the expression levels of AdipoR1 and AdipoR2 on monocytes in ALS patients were increased ( Figure 1 A, Figure 1 B). At the same time, their plasma adiponectin levels were lower than those of the control group (Table 3). In addition, compared with AdipoR2, the expression level of AdipoR1 in monocytes of ALS patients was higher ( Figure 1 C). Subsequently, we successfully differentiated the monocytes of the above-mentioned ALS patients and healthy controls into macrophages in vitro. Using immunocytochemistry (ICC) to detect the expression of AdipoR1 and AdipoR2 on macrophages of ALS patients, our results showed that under basal conditions, the expression levels of AdipoR1 and AdipoR2 on macrophages of ALS patients were higher than those of the control group ( Figure 1 D).

[0134] Table 3 Plasma adiponectin levels in ALS patients and the expression levels of AdipoR1 and AdipoR2 on the surface of classical monocytes (CD14++CD16-).

[0135]

[0136] 4. AdipoRon inhibits macrophage inflammation and regulates macrophage polarization

[0137] To investigate the effects of AdipoR on macrophage inflammation and polarization, we first evaluated the expression levels of the typical pro-inflammatory M1-related cytokine IL-1β, the M1 marker MHC-II, the anti-inflammatory M2-related cytokine IL-10, and the M2 marker CD206 in macrophages from ALS patients. The results of ICC showed that, compared with the control group, the macrophages from ALS patients had increased expression of IL-1β and MHC-II, while there was no difference in the expression of IL-10 and CD206. This may indicate that the increased expression of AdipoR1 and AdipoR2 is related to the activation of M1 macrophages. Then, we treated the macrophages from ALS patients with an adiponectin analogue: the adiponectin receptor agonist (AdipoRon), and observed the changes in IL-1β, MHC-II, IL-10, and CD206 in the macrophages. We found that, compared with the untreated group, the macrophages from ALS patients intervened with AdipoRon had decreased expression of IL-1β and MHC-II, increased expression of IL-10 and CD206, and at the same time, the expression of AdipoR1 and AdipoR2 was downregulated ( Figure 2 ). Therefore, we speculated that AdipoRon could regulate the polarization of macrophages from the M1 phenotype to the M2 phenotype.

[0138] To further reveal the effect of AdipoRon on macrophage polarization, we constructed M1 and M2 macrophage models. The macrophages from ALS patients were divided into four groups: the untreated group, the LPS-stimulated group, the LPS + AdipoRon-treated group, and the IL-4 repolarized (LPS + IL-4) group, and the changes in the expression of IL-1β, MHC-II, IL-10, and CD206 in the macrophages were detected. The results showed that, compared with the untreated group, the LPS-stimulated group significantly upregulated the expression levels of IL-1β and MHC-II. After administration of AdipoRon, this upward trend was reversed, showing decreased expression of IL-1β and MHC-II and increased expression of IL-10 and CD206, and this was similar to the results of the IL-4 repolarized group ( Figure 2 ). This indicates that AdipoRon can regulate macrophage polarization, promote the phenotypic transformation of macrophages from M1 to M2, and thus reduce inflammation.

[0139] 5. AdipoRon promotes neuronal proliferation and reduces apoptosis

[0140] Inflammation is crucial for the survival of neurons, and chronic systemic inflammation can lead to neuronal death, neuronal axonal injury, and neuromuscular junction dysfunction. Since AdipoRon can reduce the pro-inflammatory response of macrophages, we further co-cultured the supernatant of macrophages from ALS patients with NSC34-E and NSC34-hSOD1 G93A cells. We investigated the effects of AdipoRon-treated macrophages on neuronal proliferation and apoptosis by live cell imaging technology. Our results showed that, compared with the untreated group, the supernatant of macrophages treated with AdipoRon significantly enhanced neuronal proliferation ( Figure 3 A, Figure 3 B, Figure 3 E, Figure 3 F) and reduced apoptosis ( Figure 3 C, Figure 3 D, Figure 3 G, Figure 3 H). In summary, our study demonstrated that AdipoRon could inhibit pro-inflammatory macrophages and play a role in protecting motor neurons.

[0141] 6. AdipoRon can delay the onset of motor dysfunction in SOD1 G93A mice and extend their survival

[0142] To investigate whether AdipoRon has a therapeutic effect on the ALS transgenic mouse model, we grouped the mice as follows: SOD1 G93A-vehicle group (G93A-Veh), SOD1 G93A-AdipoRon (G93A-AdipoRon) group. Intraperitoneal injection was performed on the mice at 60 days of age, and the control group used an equal volume of normal saline as the vehicle to eliminate the influence of the intervention method. Quantification was performed by the rotarod test and neurological scoring, and the results were as Figure 4 shown. Compared with the untreated G93A-Veh mice, the latency to fall in the rotarod test of G93A-AdipoRon mice was significantly prolonged, and the neurological score was lower ( Figure 4 A, Figure 4 B). And AdipoRon treatment significantly prolonged the onset time of hindlimb motor dysfunction (median, 108 days vs 116 days; P < 0.0001) and survival (median, 123 days vs 133.5 days; P < 0.0001) in SOD1 G93A mice ( Figure 4 C, Figure 4 D).

[0143] Conclusion

[0144] There is a correlation between the plasma adiponectin level and the inflammatory factor level in ALS patients. In monocytes and induced macrophages in the peripheral blood of ALS patients, adiponectin receptor 1 and adiponectin receptor 2 are significantly upregulated and are related to the activation of M1 macrophages. Treatment with adiponectin receptor agonists can reduce the pro-inflammatory response of macrophages, regulate macrophage polarization, and protect neurons. Adiponectin receptor agonists can delay the occurrence of motor dysfunction in SOD1 G93A mice and extend their survival period.

[0145] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Any of the following applications: (1) Use of adiponectin or an adiponectin receptor agonist in the preparation of a drug for treating amyotrophic lateral sclerosis or symptoms caused by amyotrophic lateral sclerosis or inhibiting the progression of said symptoms; Preferably, the symptoms include decreased respiratory function, speech disorders, dysphagia or limb movement dysfunction; (2) Application of adiponectin or adiponectin receptor agonists in promoting neuronal proliferation or alleviating neuronal apoptosis.

2. The use according to claim 1, characterized in that: The adiponectin receptors include AdipoR1 and AdipoR2; Preferably, the adiponectin receptor agonist comprises AdipoRon, AdipoAI, ADP355, ALY688, ADP399, ADP-1, APNpI, APNpII, KS23, 5-mer peptide, Pep70, PEG-BHD 1028, JT003, Tyr-Pro dipeptide, YPG tripeptide, YPP tripeptide, GTDF, Tiliroside or Osmotin; Preferably, the adiponectin receptor agonist is selected from AdipoRon; Preferably, the amyotrophic lateral sclerosis includes gene mutation amyotrophic lateral sclerosis; Preferably, the genes include SOD1, TARDBP, FUS / TLS, C9orf72, ALS2, SETX, VAPB, ANG; Preferably, the gene is selected from SOD1; Preferably, the mutation types of SOD1 include SOD1 G93A 、SOD1 A4V 、SOD1 G37R 、SOD1 D90A 、SOD1 L84V 、SOD1 I113T ; Preferably, the mutation type of SOD1 is selected from SOD1 G93A .

3. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises at least one active ingredient of adiponectin or adiponectin receptor agonist and a pharmaceutically acceptable carrier and / or excipient thereof; Preferably, the adiponectin receptor agonist comprises AdipoRon, AdipoAI, ADP355, ALY688, ADP399, ADP-1, APNpI, APNpII, KS23, 5-mer peptide, Pep70, PEG-BHD 1028, JT003, Tyr-Pro dipeptide, YPG tripeptide, YPP tripeptide, GTDF, Tiliroside or Osmotin; Preferably, the adiponectin receptor agonist is selected from AdipoRon.

4. A method for promoting neuronal proliferation or reducing neuronal apoptosis in vitro, characterized in that: The method comprises promoting neuronal proliferation or reducing neuronal apoptosis in vitro by administering adiponectin or an adiponectin receptor agonist; Preferably, the adiponectin receptor agonist comprises AdipoRon, AdipoAI, ADP355, ALY688, ADP399, ADP-1, APNpI, APNpII, KS23, 5-mer peptide, Pep70, PEG-BHD 1028, JT003, Tyr-Pro dipeptide, YPG tripeptide, YPP tripeptide, GTDF, Tiliroside or Osmotin; Preferably, the adiponectin receptor agonist is selected from AdipoRon.

5. Use of reagents for detecting AdipoR1 and / or AdipoR2 and their expression products in the preparation of products for diagnosing amyotrophic lateral sclerosis; Preferably, the reagents include oligonucleotide probes that specifically recognize AdipoR1 and / or AdipoR2, primers that specifically amplify AdipoR1 and / or AdipoR2, binding agents that specifically bind to proteins encoded by AdipoR1 and / or AdipoR2, or chips that specifically analyze AdipoR1 and / or AdipoR2; Preferably, the agent is selected from a binding agent that specifically binds to a protein encoded by AdipoR1 and / or AdipoR2; Preferably, the level of AdipoR1 or its expression product is higher than that of AdipoR2.

6. A product for diagnosing amyotrophic lateral sclerosis, characterized in that: The product includes a reagent capable of detecting the expression level of AdipoR1 and / or AdipoR2; Preferably, the product further comprises a chip, a kit or a nucleic acid membrane strip; Preferably, the chip includes a gene chip and a protein chip; Preferably, the gene chip comprises oligonucleotide probes targeting AdipoR1 and / or AdipoR2 for detecting the transcription level of AdipoR1 and / or AdipoR2; Preferably, the protein chip comprises a specific binding agent for AdipoR1 and / or AdipoR2 protein; Preferably, the kit comprises reagents for detecting the expression level of AdipoR1 and / or AdipoR2 gene or protein by RT-PCR, qRT-PCR, biochip detection, Southern blotting, in situ hybridization, immunoblotting, or mass spectrometry; Preferably, the kit includes an instrument or reagent for processing a sample; Preferably, the sample comprises cells, tissue, blood, urine, saliva or mucus; Preferably, the sample is selected from cells; Preferably, the cells comprise monocytes or macrophages.

7. Any of the following applications: (1) Application of adiponectin or adiponectin receptor agonists in regulating inflammatory cytokines; Preferably, the inflammatory cytokines include IL-1β, IL-2, IL-8, IL-6, TNF-α, IL-4, and IL-10; Preferably, the adiponectin or adiponectin receptor agonist is significantly negatively correlated with the expression levels of IL-1β, IL-2, IL-8, IL-6, and TNF-α; Preferably, the adiponectin or adiponectin receptor agonist is significantly positively correlated with the expression levels of IL-4 and IL-10; (2) Application of adiponectin or adiponectin receptor agonists in regulating macrophage polarization.

8. A computer-based method for diagnosing amyotrophic lateral sclerosis, characterized in that: include: Obtaining data: obtaining the expression level data of AdipoR1 and / or AdipoR2 in the sample of the tested subject; Processing data: inputting the expression level data of AdipoR1 and / or AdipoR2 into the constructed model, wherein the model predicts whether the subject is an amyotrophic lateral sclerosis patient based on the expression data of AdipoR1 and / or AdipoR2; Output result: Output whether the subject to be tested is a patient with amyotrophic lateral sclerosis; Preferably, the sample comprises cells, tissue, blood, urine, saliva or mucus; Preferably, the sample is selected from cells; Preferably, the cells comprise monocytes or macrophages.

9. A computer-readable storage medium, characterized in that: The computer-readable storage medium stores a computer program, and when the computer program is executed by a processor, the method for diagnosing amyotrophic lateral sclerosis according to claim 8 is implemented.

10. A computer-aided diagnosis device / apparatus for amyotrophic lateral sclerosis, characterized in that: The device / apparatus comprises a memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method for diagnosing amyotrophic lateral sclerosis described in claim 8 is implemented.

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