Use of lithocholic acid as AMPK activator in pharmaceuticals and pharmaceutical composition thereof

Abstract

The present disclosure relates to the field of biopharmaceuticals, and in particular, to use of lithocholic acid (LCA) as an AMPK activator in pharmaceuticals and a pharmaceutical composition thereof. According to the present disclosure, the LCA is used as a new AMPK activator and can play a role in activating AMPK in various organs or tissues including the liver, kidney, muscle tissue, and the like of mammals, thereby achieving a significant prevention or relieving effect on various indications including high cholesterol, diabetes, and tumors, and providing a thought for developing new drugs and new therapeutic means.

Description

Use of lithocholic acid as an AMPK activator in medicine and pharmaceutical composition thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311668715X filed on December 7, 2023, and the entire text of the above-mentioned Chinese patent application is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of biomedicine, and in particular to the use of lithocholic acid as an AMPK activator in medicine and a pharmaceutical composition thereof. Background Art

[0004] Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a key molecule in regulating cellular and biological energy homeostasis. AMPK is composed of three distinct subunits, each with several isoforms: the α subunit (α1 or α2); the β subunit (β1 or β2); and the γ subunit (γ1, γ2, or γ3). There are a total of 12 possible heterotrimeric isoforms, each with its own tissue-specific distribution. Together, these isoforms form the AMPK complex, which is widely distributed throughout all tissues of the body. Furthermore, the functions of these 12 heterotrimeric AMPK forms are similar, or in other words, no distinct functions have been reported. Their differences lie solely in their tissue-specific distribution. Activation of any AMPK isoform leads to activation of the same downstream proteins.

[0005] Traditionally, AMPK activation is mediated by its allosteric activators—AMP / ADP and its analogs. AMP / ADP binds to the γ subunit of AMPK, causing structural changes in the holoenzyme, making it more susceptible to phosphorylation at threonine 172 on the α subunit (p-AMPKα) by its upstream kinase, leading to activation. In addition to allosteric activation by the γ subunit, the β subunit of AMPK can also be bound by metabolites such as glycogen, which can regulate its activity. Furthermore, recent research indicates that under certain conditions (such as glucose deprivation and metformin treatment), while AMPK is activated in vivo, the AMP / ATP ratio does not undergo drastic changes. Instead, the activity of the intracellular v-ATPase is inhibited, activating the lysosomal pathway and, in turn, AMPK.

[0006] However, existing research indicates that current drugs targeting AMPK suffer from numerous drawbacks, such as insufficient efficacy. The most established AMPK activators discovered so far are primarily used in the treatment of diabetes. For example, the widely used AMPK activator metformin has minimal side effects and, by activating AMPK, can significantly lower blood sugar and fatty liver levels, thereby alleviating diabetic symptoms. This makes it a significant advantage over other AMPK activators. However, due to its poor cell membrane permeability, metformin requires specific transporters for entry into cells. These transporters are only found in a few tissues, such as the liver, significantly limiting the drug's efficacy and the full potential of AMPK. Besides metformin, the drug currently furthest along in clinical trials is A-769662 (CAS No: 844499-71-4), which directly binds to the β subunit, allosterically activating AMPK. Compared to metformin, A-769662 has excellent cell membrane permeability, allowing it to enter most tissues and exhibits good persistence, enabling prolonged action. However, A-769662 is not suitable for oral administration, which significantly limits its application. Furthermore, recent reports have indicated that A-769662 has multiple targets beyond AMPK, a specificity that is problematic.

[0007] Currently, there is an urgent need to develop new drugs and technical means to activate AMPK. Summary of the Invention

[0008] The present disclosure aims to provide an AMPK activator that is easily absorbed, has significant efficacy, and can activate AMPK in multiple organs or tissues. The inventors of this disclosure have discovered for the first time that lithocholic acid, a secondary metabolite of bile acids, can activate AMPK in multiple organs and tissues, potentially enabling the development of new AMPK-activating drugs.

[0009] Based on this, in a first aspect, the present disclosure provides the use of lithocholic acid (LCA) or a soluble salt thereof as an AMPK activator, or its use in the preparation of an AMPK activator, in particular its activation effect on AMPK in the liver, kidney or muscle tissue of mammals.

[0010] In a second aspect, the present disclosure provides the use of lithocholic acid or a soluble salt thereof in the preparation of any of the following drugs:

[0011] (a) Drugs that inhibit cholesterol synthesis;

[0012] (b) drugs that reduce fatty acid synthesis;

[0013] (c) drugs for the prevention and / or treatment of diabetes;

[0014] (d) drugs for preventing and / or treating tumors;

[0015] (e) Drugs for enhancing the motor performance of mammals;

[0016] (f) Drugs that extend the lifespan of mammals.

[0017] In a third aspect, the present disclosure provides an AMPK activator comprising lithocholic acid or a soluble salt thereof.

[0018] In a fourth aspect, the present disclosure provides a pharmaceutical composition comprising lithocholic acid or a soluble salt thereof, or the AMPK activator described in the third aspect, and a pharmaceutically acceptable excipient; the pharmaceutical composition has at least one of the following effects:

[0019] (a) Drugs that inhibit cholesterol synthesis;

[0020] (b) drugs that reduce fatty acid synthesis;

[0021] (c) drugs for the prevention and / or treatment of diabetes;

[0022] (d) drugs for preventing and / or treating tumors;

[0023] (e) Drugs for enhancing the motor performance of mammals;

[0024] (f) Drugs that extend the lifespan of mammals.

[0025] The present disclosure uses LCA as a new AMPK activator, which can activate AMPK in various organs or tissues including the liver, kidney and muscle tissue of mammals, thereby achieving significant prevention or relief effects on various indications including high cholesterol, diabetes and tumors, and providing ideas for the development of new drugs and new treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 shows the effect of LCA on AMPK activation in various cell lines;

[0028] FIG2 shows the experimental results of AMPK activation in model animals by LCA feeding;

[0029] Figure 3 shows the experimental results of LCA improving the health and lifespan of nematodes and fruit flies;

[0030] Figure 4 shows the experimental results of LCA improving the health level of elderly mice;

[0031] FIG5 shows the experimental results of LCA extending the lifespan of aged mice through AMPK. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in combination with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments.

[0033] (I) Definitions or terms

[0034] The following abbreviations shall apply to the relevant definitions or terms involved in this disclosure. Unless otherwise defined, all scientific and technical terms used herein have the meanings commonly understood by those of ordinary skill in the art. The following terms are provided below.

[0035] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked together by a phosphodiester bond. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0036] In the present disclosure, knockdown of DNA or RNA includes, but is not limited to, complete knockout and partial knockout. Complete knockout refers to reducing the level of the target DNA or target RNA or the level of the protein expressed by it to a level that is almost undetectable (in fact, in general, it is difficult to knock out 100% of the target DNA or target RNA). Partial knockout refers to a situation where the degree of knockout is greater than zero but less than complete knockout.

[0037] As used herein, the term "nucleic acid construct" is defined herein as a single-stranded or double-stranded nucleic acid molecule, preferably an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further comprises one or more operably linked regulatory sequences.

[0038] As used herein, "expression" refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0039] As used herein, the term "operably linked" refers to the functional spatial arrangement of two or more nucleotide regions or nucleic acid sequences. The "operably linked" can be achieved by means of genetic recombination.

[0040] As used herein, a "vector" is a replicable nucleic acid that, when transformed into an appropriate host cell, can express one or more heterologous proteins from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for the purpose of amplifying nucleic acids or for expressing / displaying polypeptides encoded by nucleic acids. Vectors are typically kept episomal, but can be designed to integrate genes or portions thereof into chromosomes of the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vectors are well known to those skilled in the art.

[0041] As used herein, vectors also include "viral vectors" or "viral vectors". Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as a vehicle or shuttle) exogenous genes into cells. For example, vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs) or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda phages or M13 phages and animal viruses, etc. The types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements that control expression.

[0042] As used herein, "expression vector" includes a vector capable of expressing DNA that is operably linked to a regulatory sequence such as a promoter region that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus or other vector, which, when introduced into an appropriate host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells, as well as expression vectors that remain episomal or that are integrated into the host cell genome.

[0043] As used herein, the term "host cell" refers to a cell that is used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, the following cell types, such as prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0044] As used herein, the term "disease and / or condition" refers to a physical condition of the subject that is associated with the diseases and / or conditions described in the present disclosure.

[0045] As used herein, "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged after treatment. Thus, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing the underlying disease and / or preventing the worsening of symptoms or progression of the disease. Treatment also includes any pharmaceutical use of the pharmaceutical compositions provided herein.

[0046] Unless otherwise specified in this disclosure, the term "improve, alleviate, relieve or treat" refers to the medical management of a patient for the purpose of curing, improving, stabilizing or preventing a disease, pathological state or condition. The term includes active therapy, which is treatment specifically aimed at improving a disease, pathological state or condition, and also includes etiological treatment, which is treatment aimed at removing the cause of the relevant disease, pathological state or condition. In addition, the term also includes palliative treatment, which is treatment designed to relieve symptoms rather than cure the disease, pathological state or condition; the term also includes preventive treatment, which is treatment aimed at minimizing or partially or completely inhibiting the development of the relevant disease, pathological state or condition; and supportive treatment, which is treatment used to supplement another specific therapy aimed at improving the relevant disease, pathological state or condition.

[0047] As used herein, "therapeutic effect" refers to an effect resulting from treatment of a subject that alters, typically ameliorates or improves the symptoms of a disease or condition, or cures the disease or condition.

[0048] As used herein, "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is the amount necessary to prevent, cure, ameliorate, arrest, or partially arrest the symptoms of a disease or condition.

[0049] As used herein and unless otherwise specified, the terms "comprises," "includes," "has," "contains," and their grammatical equivalents should generally be understood as open-ended and non-limiting, e.g., not excluding other unlisted elements or steps.

[0050] The "homology" mentioned in the present disclosure has a meaning generally recognized in the art, and the percentage of sequence identity between two nucleic acids or polypeptides or regions can be calculated using disclosed techniques. For example, sequence identity can be measured along the entire length of a polynucleotide or polypeptide or along a region of the molecule. Currently, there are many methods for measuring the identity between two polynucleotides or polypeptides that can be routinely selected by those skilled in the art according to actual needs.

[0051] Expression as used herein refers to the process of producing a polypeptide through transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, methods for measuring the amount of polypeptide produced by a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0052] The host cells described herein are cells that are used to receive, maintain, replicate, and amplify vectors. Host cells can also be used to express polypeptides encoded by the vectors. When the host cells divide, the nucleic acids contained in the vectors replicate, thereby amplifying the nucleic acids. The host cells can be eukaryotic or prokaryotic.

[0053] The vectors disclosed herein are replicable nucleic acids that, when transformed into appropriate host cells, can express one or more heterologous proteins from the vector. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, typically by restriction enzyme digestion and ligation. Vectors also include those that contain nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of polypeptides encoded by nucleic acids. Vectors typically remain episomal, but can be designed to integrate genes or portions thereof into chromosomes of the genome. Artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes, are also contemplated. The selection and use of such vectors are well known to those skilled in the art.

[0054] (II) Detailed technical plan

[0055] In a specific embodiment, in the first aspect, the present disclosure provides the use of lithocholic acid or a soluble salt thereof as an AMPK activator, or its use in the preparation of an AMPK activator. The molecular structural formula of the lithocholic acid is shown in formula (I), and the soluble salt of the lithocholic acid includes salts formed by lithocholic acid and cations, such as ammonium salts, alkali metal salts (including sodium salts, lithium salts and potassium salts), and alkaline earth metal salts (including calcium salts and magnesium salts). It can also be a salt formed by lithocholic acid and an organic base, such as an organic amine, benzathines, dicyclohexylamine, hydrabamines formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glucamide, and tert-butylamine. It can also be a salt formed with an amino acid, such as arginine salts, lysine salts, and aspartate salts.

[0056] In alternative embodiments, the AMPK activator activates AMPK in the liver, kidney, or muscle tissue of a mammal.

[0057] Based on the first aspect that the organs in which lithocholic acid activates AMPK include the liver and kidney of mammals, and the tissues include muscle tissue, the second aspect of the present disclosure provides the use of lithocholic acid or a soluble salt thereof in the preparation of any of the following drugs:

[0058] (a) Drugs that inhibit cholesterol synthesis;

[0059] (b) drugs that reduce fatty acid synthesis;

[0060] (c) drugs for the prevention and / or treatment of diabetes;

[0061] (d) drugs for preventing and / or treating tumors;

[0062] (e) Drugs for enhancing the motor performance of mammals;

[0063] (f) Drugs that extend the lifespan of mammals.

[0064] In an optional embodiment, the content of lithocholic acid or a soluble salt thereof in the drug is 0.00001 wt% to 1 wt%, preferably 0.001 wt% to 1 wt%, and more preferably 0.01 wt% to 0.1 wt%.

[0065] In an optional embodiment, the drug may further contain at least one other AMPK activator, including but not limited to metformin, A-769662, betaine, coenzyme Q10, PQQ (pyrroloquinoline quinone) or lipoic acid.

[0066] In an optional embodiment, the drug further comprises a pharmaceutically acceptable carrier or excipient. The dosage form of the drug includes one or more of an injection, oral liquid, suspension, emulsion, extract, powder, granule, suppository, aerosol, granule, gel, transdermal patch, sustained-release agent, enteric solvent, osmotic pump, tablet, capsule, microcapsule, microsphere, nanoparticle, liposome or capsule. Those skilled in the art can determine the selection of different drug dosage forms according to different administration requirements, and then determine the selection of specific carriers or excipients.

[0067] Based on the first aspect, the present disclosure provides an AMPK activator, wherein the AMPK activator contains lithocholic acid or a soluble salt thereof.

[0068] On the other hand, the present disclosure further provides a pharmaceutical composition comprising an effective dose of lithocholic acid or a soluble salt thereof, or the AMPK activator according to claim 8, and pharmaceutically acceptable excipients;

[0069] The pharmaceutical composition has at least one of the following effects:

[0070] (a) Drugs that inhibit cholesterol synthesis;

[0071] (b) drugs that reduce fatty acid synthesis;

[0072] (c) drugs for the prevention and / or treatment of diabetes;

[0073] (d) drugs for preventing and / or treating tumors;

[0074] (e) Drugs for enhancing the motor performance of mammals;

[0075] (f) Drugs that extend the lifespan of mammals.

[0076] In an optional embodiment, the content of lithocholic acid or a soluble salt thereof is 0.00001 wt% to 1 wt%, preferably 0.001 wt% to 1 wt%, and more preferably 0.01 wt% to 0.1 wt%.

[0077] In an optional embodiment, the pharmaceutical composition further contains at least one other AMPK activator, including but not limited to metformin, A-769662, betaine, coenzyme Q10, PQQ (pyrroloquinoline quinone) or lipoic acid.

[0078] In an optional embodiment, the dosage form of the pharmaceutical composition includes one or more of injection, oral liquid, suspension, emulsion, extract, powder, granule, suppository, aerosol, granule, tablet, capsule, microcapsule, microsphere, nanoparticle, liposome or capsule.

[0079] In an optional embodiment, the auxiliary material includes a cosolvent, and the cosolvent is preferably sodium carboxymethylcellulose (CMC), dimethyl sulfoxide (DMSO), 2-hydroxypropyl-β-cyclodextrin (HP-CD) or corn oil.

[0080] In another aspect, the present disclosure provides lithocholic acid or a soluble salt thereof, the aforementioned AMPK activator, or the aforementioned pharmaceutical composition, for use in:

[0081] activating AMPK, preferably, activating AMPK in the liver, kidney or muscle tissue of a mammal;

[0082] Inhibit cholesterol synthesis;

[0083] Reduce fatty acid synthesis;

[0084] prevention and / or treatment of diabetes;

[0085] Prevention and / or treatment of tumors;

[0086] Improving the motor performance of a mammal; and / or

[0087] Extending mammalian lifespan.

[0088] In another aspect, the present disclosure provides a method for activating AMPK, inhibiting cholesterol synthesis, reducing fatty acid synthesis, preventing and / or treating diabetes, preventing and / or treating tumors, improving mammalian athletic ability, and / or extending mammalian lifespan, comprising administering to a cell or individual a therapeutically effective amount of lithocholic acid or a soluble salt thereof, the aforementioned AMPK activator, or the aforementioned pharmaceutical composition. In some embodiments, the AMPK activation is activating AMPK in the liver, kidney, or muscle tissue of a mammal.

[0089] The following describes some embodiments of the present disclosure in detail with reference to the accompanying drawings. In the absence of conflict, the following embodiments and features therein may be combined with each other.

[0090] The experimental materials and main reagents used in the following examples are as follows:

[0091] (1) Cell lines

[0092] Mouse embryonic fibroblasts (MEFs) are immortalized MEFs, which are isolated from mouse embryos and then transfected with SV40T antigen to immortalize the cells. For construction methods, see Lei Y, Methods Mol Biol. 2013; 1031: 59-64. Generation and culture of mouse embryonic fibroblasts.

[0093] Human embryonic kidney cells HEK293T (cat. CRL-321) were purchased from ATCC.

[0094] Mouse primary hepatocytes are primary cells isolated from mouse liver.

[0095] Mouse primary muscle cells are primary cells isolated from mouse muscle tissue.

[0096] Drosophila melanogaster late embryonic stage cells (S2, cat. CRL-1963) were purchased from ATCC.

[0097] (2) Culture medium and drugs

[0098] DMEM medium (Dulbecco's modified Eagle's medium, Gibco, cat.11965), Opti-MEM Reduced Serum Medium (Gibco, cat.31985070), William's E Medium (cat.12551032), and Ham's F-10 medium (cat.11550043) were purchased from Thermofisher.

[0099] Lithocholic acid (LCA, cat. L6250), 2-hydroxypropyl-β-cyclodextrin, cat. C0926, paraquat (cat. #36541), iron(II) sulfate heptahydrate (FeSO4), cat. #F8633

[0100] (3) Primary antibody used in Western blot

[0101] Rabbit anti-phospho-AMPKα-T172 (Rabbit 548 anti-phospho-AMPKα-T172, cat#2535) was purchased from Cell Signaling Technology.

[0102] Anti-AMPKα (cat#2532, diluted 1:1000 for immunoblotting) was purchased from Cell Signaling Technology.

[0103] Anti-phospho-ACC-Ser79 (cat. #3661) was used for immunoblotting at a dilution of 1:1000 and was purchased from Cell Signaling Technology.

[0104] Anti-ACC (cat#3662, diluted 1:1000 for immunoblotting) was purchased from Cell Signaling Technology.

[0105] Anti-GAPDH (cat#5174, diluted 1:1000 for immunoblotting) was purchased from Cell Signaling Technology.

[0106] Mouse anti-OXPHOS (anti-total oxidative phosphorylation (OXPHOS) complex, cat# ab110413, diluted 1:5000 for immunoblotting) was purchased from Abcam.

[0107] Anti-β-ACTIN (cat#A5316, diluted 1:1000 for immunoblotting) was purchased from Sigma.

[0108] Rabbit anti-tubulin (cat#10068-1-AP, diluted 1:1000 for immunoblotting) was purchased from Proteintech.

[0109] Mouse anti-MHCIIa (anti-MHCIIa, cat.# SC-71, used at 6 μg / mL) was purchased from Developmental Studies Hybridoma Bank.

[0110] Anti-MHCIIb (cat #BF-F3, used at 6 μg / mL) was purchased from Developmental Studies Hybridoma Bank.

[0111] Anti-MHCI (cat.#C6B12, used at 6 μg / mL) was purchased from Developmental Studies Hybridoma Bank.

[0112] (4) Secondary antibodies used in Western blotting:

[0113] HRP-conjugated goat anti-mouse IgG (cat. 115-035-003).

[0114] HRP-conjugated goat anti-rabbit IgG (cat. 111-035-003).

[0115] All were purchased from Jackson ImmunoResearch.

[0116] (5) Fluorescent secondary antibodies used for immunofluorescence staining

[0117] Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 conjugated TM 594, cat.# A-21145, used at 1:200).

[0118] Alexa Fluor 488-linked goat anti-mouse IgM antibody (Goat anti-Mouse IgM (Heavy chain). Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 488, cat.# A-21042, used at 1:200).

[0119] Goat anti-Mouse IgG1 Cross-Adsorbed Secondary Antibody, Alexa Fluor 647 TM 647, cat.#A-21240, used at a dilution of 1:200).

[0120] All were purchased from Thermo Scientific.

[0121] (6) Histochemistry experiments

[0122] Hematoxylin solution (cat. 03971) and eosin Y solution (cat. 318906) were purchased from Sigma.

[0123] (7) Experimental animals

[0124] Wild-type mice, 4-5 weeks old males, were purchased from Shanghai Slack.

[0125] AMPK knockout mice: AMPKα1 F / F (#014141) and AMPKα2 F / F Mice (#014142) were purchased from Jackson Laboratory. Two strains of FLOX mice were crossed and AMPKα1 / 2 was purified. F / F Mice were isolated and hybridized with mck-cre mice expressing muscle-specific CRE protein (a protein that targets loxP sequences to achieve genome splicing effects), and AMPK muscle-specific knockout mice were obtained after purification.

[0126] Drosophila strains were purchased from BDSC and VDRC. Act5C-GAL4 is a wild-type control fruit fly, and Act5C-GAL4>AMPKa RNAi is an AMPK knockout strain, which was prepared in the laboratory.

[0127] Nematode strains were purchased from the Caenorhabditis Genetics Center, where N2 is a wild-type nematode and aak-2 is an AMPK knockout nematode.

[0128] Example 1: LCA can activate AMPK in various cells

[0129] 1. LCA can activate AMPK in various cells

[0130] This experiment used WB to examine the changes in AMPK phosphorylation levels in cells. Cells in the logarithmic growth phase were plated at 2×10 5Cells were seeded into each well of a 6-well plate and cultured in a 37°C, 5% CO2 incubator. After 24 hours of culture, the LCA drug stock solution was diluted with the culture medium of each cell line to obtain a solution of the specified concentration. The stock solution was discarded from the 6-well plate and the prepared drug solution was added. The DMSO group served as a blank control. After drug addition, the plate was transferred to a 37°C CO2 incubator and cultured for another 2 hours. After adding cell lysis buffer containing dephosphatase inhibitors and protease inhibitors and ultrasonic centrifugation, total cell protein was extracted and the protein concentration was determined by BCA method. 30 μg protein sample was taken for SDS-polyacrylamide gel electrophoresis and transferred to the membrane. The membrane was blocked in sequence and incubated with p-AMPKα antibody (i.e. rabbit 548 anti-phospho-AMPKα-T172) (1:1000), AMPKα antibody (i.e. anti-AMPKα) (1:1000), p-ACC antibody (i.e. anti-phospho-ACC-Ser79) (1:1000), ACC antibody (i.e. anti-ACC) (1:1000), etc. at 4°C overnight, rinsed with TBST for 5 minutes and 3 times, and then incubated with the corresponding horseradish peroxidase-labeled secondary antibody (1:5000) for 2 hours. The membrane was washed with TBST for 5 minutes and 3 times, then treated with developer and developed by exposure machine for chemiluminescence image acquisition, thereby measuring the AMPK activation of cells by LCA.

[0131] FIG1 shows the effect of LCA on AMPK activation in various cell lines, wherein:

[0132] Figure 1(a) shows the AMPK activation effect of LCA on mouse embryonic fibroblasts (MEFs). As can be seen, after 4 hours of treatment with 1 μM LCA, the accumulation of LCA in MEFs was approximately 0.08 nmol / mg protein. Simultaneously, the phosphorylation levels of AMPKα and ACC in the cells increased significantly, demonstrating AMPK activation. However, the AMP / ATP and ADP / ATP ratios did not change significantly, indicating that the cellular energy status remained unchanged.

[0133] Figure 1(b) shows the AMPK activation effect of LCA on human embryonic kidney (HEK293T) cells. After 4 hours of treatment with 1 μM LCA, the phosphorylation levels of AMPKα and ACC in the cells increased significantly, demonstrating AMPK activation. However, the AMP / ATP and ADP / ATP ratios did not change significantly, indicating no significant change in the cellular energy status.

[0134] Figure 1(c) shows the AMPK activation effect of LCA on late-stage Drosophila melanogaster embryonic cells (S2). After 4 hours of treatment with 1 μM LCA, the accumulated amount of LCA in S2 cells was approximately 0.1 nmol / mg protein. Simultaneously, AMPKα phosphorylation levels increased significantly, demonstrating AMPK activation. However, the AMP / ATP and ADP / ATP ratios remained unchanged, indicating no significant change in the cellular energy status.

[0135] Figure 1(d) shows the AMPK activation effect of LCA on primary mouse muscle cells. After 4 hours of treatment with 1 μM LCA, the phosphorylation levels of AMPKα and ACC in primary mouse muscle cells increased significantly, demonstrating AMPK activation. However, the AMP / ATP and ADP / ATP ratios did not change significantly, indicating no significant change in the cellular energy status.

[0136] Figure 1(e) shows the activation effect of LCA on primary mouse hepatocytes. After treating the cells with 1 μM LCA for 4 hours, the phosphorylation levels of AMPK and ACC in the primary mouse hepatocytes increased significantly, indicating that AMPK was activated. However, the AMP / ATP ratio in the cells did not change significantly, indicating that the cellular energy status did not change significantly.

[0137] From the above, it can be seen that LCA can be used as a potential AMPK agonist with a wide range of activation effects, and on this basis, its physiological effects are further verified.

[0138] 2. LCA feeding can activate AMPK in model animals

[0139] This study investigated changes in AMPK phosphorylation in mice fed LCA. The mice were fed a 1g / l aqueous solution of LCA (coated with cyclodextrin HP-CD) directly as drinking water. After one week of drinking, the mice were sacrificed and samples collected.

[0140] Mouse tissue Western blotting (WB) sample preparation involves anesthetizing mice, rapidly opening the abdominal cavity, clamping the liver lobes and muscle tissue with liquid nitrogen clamps, breaking off and collecting the frozen tissue samples, and placing them in liquid nitrogen. Add 1000 μL of cell lysis buffer containing dephosphatase and protease inhibitors to 50 mg of frozen tissue. The tissue is thoroughly disrupted using a tissue homogenizer. After ultrasonic centrifugation, total cellular protein is extracted. Protein concentration is determined using the BCA assay, and 30 μg of protein sample is subjected to SDS-polyacrylamide gel electrophoresis to measure the level of AMPK activation in mouse tissues by LCA.

[0141] Figure 2(a) shows the accumulation of LCA and AMPK phosphorylation in nematodes after LCA feeding. 24 hours after LCA feeding, the accumulated LCA concentration in the nematodes was approximately 0.14 nmol / mg protein, while AMPK was significantly activated. The AMP / ATP and ADP / ATP ratios did not change significantly.

[0142] Figure 2(b) shows AMPK phosphorylation levels in Drosophila larvae fed LCA. As can be seen, after LCA feeding, the concentration of LCA in the larvae was 0.07 nmol / mg protein, while AMPK was significantly activated. However, the AMP / ATP and ADP / ATP ratios in the larvae did not change significantly.

[0143] Figure 2(c) shows AMPK phosphorylation levels in Drosophila adults fed LCA. As can be seen, after LCA feeding, the concentration of LCA in Drosophila adults reached 0.05 nmol / mg protein, and AMPK was significantly activated, while the AMP / ATP and ADP / ATP ratios in the adults did not change significantly.

[0144] Figure 2(d) shows the LCA levels in serum and skeletal muscle of mice fed LCA. It can be seen that after long-term LCA feeding, the LCA level in serum of mice was approximately 1 μM, and the LCA level in skeletal muscle was approximately 0.04 nmol / mg protein at two different time points.

[0145] Figure 2(e) shows the AMPK phosphorylation levels in muscle tissue of LCA-fed mice. It can be seen that after LCA feeding, the phosphorylation levels of AMPK and ACC in the skeletal muscle of aged mice increased significantly, while the ratios of AMP / ATP and ADP / ATP in muscle did not change significantly.

[0146] From the above, we can see that directly feeding LCA can activate AMPK in the muscle tissue of elderly mice, and LCA can achieve the biological effects of AMPK on model organisms.

[0147] Example 2: LCA can improve the health and lifespan of nematodes and fruit flies

[0148] This example investigates the beneficial effects of LCA feeding in two non-vertebrate animal models: nematodes and fruit flies.

[0149] 1. LCA improves the health of nematodes and extends their lifespan, an effect that depends on the presence of AMPK.

[0150] To count the lifespan of nematodes, the nematodes were cultured to the L4 stage and then transferred to agar medium for counting. 12 / mL, with free access to food, add the diluted bacterial solution dropwise to NGM medium containing 50 mg / mL ampicillin and 50 mg / mL kanamycin (add 350 μL of bacterial solution to a 35 mm dish). If medication is required, simply add the drug to the medium during preparation. Transfer nematodes every two days, and record the number of surviving and deceased nematodes during transfer.

[0151] The nematode pharyngeal sucking rate, or swallowing rate, is assessed by the number of contraction-relaxation cycles of the nematode pharyngeal terminal bulb within 1 minute. Briefly, synchronized nematodes were cultured to the L4 stage, after which LCA was administered. One-day-old nematodes were then picked and placed on a new NGM plate containing E. coli. After incubation at room temperature for 10 minutes, the contraction-relaxation cycle of each nematode terminal bulb was recorded for 4 consecutive minutes using Capture software (v.2021.1.13, Capture Visualisation) on a stereomicroscope (M165FC, Leica) through a 63x objective lens, and the average contraction-relaxation cycle per minute was calculated using Aimersoft Video Editor software (v.3.6.2.0, Aimersoft).

[0152] To assess nematode resistance to oxidative stress, synchronized nematodes were cultured to the L4 stage and then treated with LCA. One day after LCA treatment, 20 worms were transferred to NGM plates containing 15 mM FeSO₄. The nematodes were then cultured on these plates at 20°C, and the number of live and dead nematodes was counted every hour.

[0153] Measuring NAD in C. elegans using HPLC-MS + After rinsing the nematodes with M9 buffer containing Triton X-100, the cells were centrifuged at 100 g for 5 seconds to remove the bacterial suspension. The nematodes were then vortexed with 1 mL of pre-chilled methanol solution to lyse the nematodes. 1 mL of chloroform and 400 μL of ultrapure water containing an internal standard were added and vortexed. The supernatant was then centrifuged at 15,000 g for 15 minutes, collected, lyophilized, reconstituted with 50% acetonitrile, and analyzed by mass spectrometry.

[0154] Nematode RT-PCR method: Approximately 1000 nematodes were harvested in 15 mL of M9 buffer containing 0.05% Triton X-100 (v / v) and centrifuged at 1000 g for 2 minutes. The pellet was then washed twice with 1 mL of M9 buffer to remove bacteria and lysed with 1 mL of TRIzol. The nematodes were then frozen in liquid nitrogen, thawed at room temperature, and freeze-thawed twice. The nematode lysate was then allowed to stand at room temperature for 5 minutes, mixed with 0.2 mL of chloroform, and shaken vigorously for 15 seconds. RNA was then precipitated with 450 μL of isopropanol and centrifuged at 12,000 g for 30 minutes at 4°C. The RNA pellet was washed twice with 75% ethanol and twice with 100% ethanol, then dissolved in 20 μL of DEPC-treated water. RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo). Approximately 1 μg of RNA was diluted to 10 μL with DEPC-treated water, heated at 65°C for 5 minutes, and then immediately placed on ice. Random primer mix, enzyme mix, and 5× RT buffer (all from ReverTra Ace qPCR RT Master Mix) were then added to the RNA solution and incubated at 37°C for 15 minutes, followed by incubation at 98°C for 5 minutes on a thermal cycler. Reverse-transcribed cDNA was quantified using Maxima SYBR Green / ROX qPCR Master Mix on a LightCycler 480II system (Roche) using the following program: initial denaturation at 95°C for 10 minutes; denaturation at 95°C for 10 seconds, followed by annealing and extension at 65°C for 30 seconds each, for 45 cycles. mRNA levels were then calculated using the comparative ΔΔct method using LightCycler software (v.96 1.1, Roche; this software was used for all subsequent qPCR experiments). Among them, the oxidative phosphorylation genes of nematodes are nuo-6, nuaf-1, sdha-1, sdhb-1, cco-1, cco-2, atp-3, nduo-1, nduo-2, nduo-4, nduo-5, ctb-1, ctc-1, ctc-2, and atp-6.

[0155] To measure OCR in C. elegans, 15 to 25 nematodes were washed, suspended in 200 μL of M9 buffer, plated into a Seahorse XF 96-well plate, and cultured overnight in a CO2-free incubator. Respiratory chain inhibitors were used at concentrations of 10 μM FCCP and 40 mM sodium azide. Data were collected and analyzed using Wave 2.6.1 Desktop software (Agilent Technologies).

[0156] 2. LCA improves the health and lifespan of fruit flies

[0157] For the lifespan experiment, newly hatched fruit flies were divided into male and female groups under mild anesthesia with carbon dioxide gas. Two hundred males and females were then randomly transferred into control and LCA groups, with 10 tubes per group containing 20 flies per tube. For the LCA-treated group, 100 μL of 100 μM HP-LCA (or 100 μL of HP-CD for the control group) was added to the surface of BDSC standard cornmeal medium or CSY medium. Flies were transferred every three days, and the number of dead flies was recorded until all flies had died.

[0158] To test the ability of Drosophila to resist oxidative stress, newly hatched flies were fed either an HP-CD diet or an HP-LCA diet (n=200, 20 flies per tube). On day 30 of each diet, flies from all groups were transferred to separate tubes containing filter paper soaked in either 20 mM paraquat or 5% H2O2 in a 5% glucose solution. The number of flies that died was recorded every 2 hours until the last fly died. For cold and heat stress, flies were exposed to either cold (4°C) or heat (37°C), and mortality was recorded every 2 hours. For starvation treatment, flies were transferred to tubes containing only 5% agar, and mortality was recorded every 2 hours.

[0159] Measuring NAD in Drosophila using CE-MS + Twenty adult fruit flies were snap-frozen in liquid nitrogen and ground. The cells were then vortex-lyzed with 1 mL of pre-chilled methanol containing an internal standard. 1 mL of chloroform and 400 μL of ultrapure water were added and vortexed. The cells were then centrifuged at 15,000 g for 15 minutes, and the upper aqueous phase was collected, lyophilized, reconstituted, and analyzed by mass spectrometry.

[0160] For the detection of mitochondrial (mt) and nuclear (n) DNA levels in Drosophila, flies were transferred to 1.5 mL EP tubes and snap-frozen in liquid nitrogen. They were then ground and lysis buffer (10 μl of 10 mg mL lysate was added to 1 mL of lysis buffer) was added to the fly tissues. -1 Proteinase K was added, and Drosophila DNA was quantified using the Maxima SYBR Green / ROX qPCR Master Mix on a LightCycler 480II system (Roche) using the following program: initial denaturation at 95°C for 5 minutes, followed by denaturation at 95°C for 10 seconds, annealing at 60°C for 20 seconds, and extension at 72°C for 20 seconds, for a total of 40 cycles. The mtDNA:nDNA ratio was then calculated using the comparative ΔΔct method.

[0161] Drosophila RT-PCR method: About 20 adults were used for each sample. After anesthetizing the adults, they were transferred to a 1.5 mL Eppendorf tube, then quickly frozen in liquid nitrogen and homogenized with a pellet pestle. After homogenization, the tubes were dissolved in 1 mL of TRIzol solution at room temperature for 5 minutes, and then centrifuged at 12,000 g for 15 minutes at 4°C. About 900 μL of the supernatant (without the lipid layer) was transferred to an RNase-free tube and then mixed with 200 μL of chloroform. After vigorous vortexing for 15 seconds, the tubes were centrifuged at 12,000 g for 15 minutes at 4°C, and then about 450 μL of the upper aqueous layer was transferred to an RNase-free tube. Then, 450 μL of isopropanol was added to precipitate the RNA, followed by centrifugation at 12,000 g for 30 minutes at 4°C. RNA was obtained, and subsequent operations were similar to those for nematode qPCR. Among them, the Drosophila oxidative phosphorylation-related genes are CG5389, CG18809, CG17856, CG9762, and CG9172.

[0162] 3. Experimental Results

[0163] Figure 3a shows the survival curve of LCA-fed nematodes. LCA feeding significantly extends the lifespan of wild-type nematodes, but has no lifespan-extending effect in AMPK-knockout nematodes.

[0164] Figure 3(b) shows the lifespan curve of Drosophila fed with LCA. Feeding LCA can extend the lifespan of control flies, and the lifespan extension effect caused by LCA is dependent on the presence of AMPK.

[0165] Figure 3c shows that the swallowing rate of nematodes fed with LCA was significantly increased, but this effect was not seen in AMPK knockout nematodes.

[0166] Figure 3d shows that the antioxidant capacity of nematodes was significantly improved after LCA feeding, and there was no significant change in this capacity after AMPK knockout.

[0167] Figure 3e, f show that the ability of flies to resist oxidative stress was improved after feeding LCA and treated with paraquat and H2O2, but there was no change after AMPK knockdown.

[0168] Figure 3g to i show that feeding LCA increases the survival ability of fruit flies under relatively extreme conditions such as cold, heat and starvation, while the survival ability is not improved in AMPK knockdown fruit flies.

[0169] Figure 3j shows the NAD in nematodes and fruit flies after feeding LCA + The levels were significantly increased, whereas there was no change after AMPK knockout.

[0170] Figure 3 (k, l) shows the mtDNA / nDNA levels and expression levels of oxidative phosphorylation-related genes in nematodes and flies after feeding LCA. After LCA treatment, the expression levels of mitochondrial-related genes in nematodes and flies increased significantly, indicating better mitochondrial function, while this beneficial effect was no longer seen when AMPK was missing.

[0171] Figure 3 (m) shows that the mitochondrial function of nematodes becomes stronger after feeding LCA, while this phenomenon does not occur in AMPK knockout nematodes.

[0172] Example 3: LCA can improve the health of elderly mice

[0173] This example investigates the beneficial effects of LCA on the motor performance and health of elderly mice. Eighteen-month-old male mice were used as experimental subjects. After being fed drinking water containing 1 g / l of LCA for two months, the mouse model was tested.

[0174] Mouse tissue RT-PCR experimental method. The gastrocnemius muscle was dissected immediately after the mouse was sacrificed, and the gastrocnemius muscle was cut into small pieces with a side length of approximately 2 mm and soaked in RNAprotect tissue reagent (1 mL for every 100 mg of tissue) at room temperature for 24 hours. The tissue was then placed in 1 mL of TRIzol solution for culture, subjected to three rounds of freeze-thaw cycles, and finally homogenized. After homogenization, centrifuge at 12,000 g for 15 minutes at 4°C, and then transfer 900 μL of the clear supernatant (not the lipid layer above) to an RNase-free test tube. Then add 200 μL of chloroform to the supernatant, vortex vigorously for 15 seconds, then add 450 μL of isopropanol to precipitate RNA, and then centrifuge at 12,000 g for 30 minutes (4°C). The RNA precipitate was washed twice with 75% ethanol and 100% ethanol, and then dissolved with 20 μL of DEPC water. After obtaining RNA, the subsequent qPCR method is as follows:

[0175] RNA concentration was determined using a NanoDrop 2000 spectrophotometer (Thermo). Approximately 1 μg of RNA was diluted to 10 μL with DEPC-treated water, heated at 65°C for 5 minutes, and then immediately placed on ice. Random primer mix, enzyme mix, and 5× RT buffer (all from ReverTra Ace qPCR RT Master Mix) were then added to the RNA solution and incubated at 37°C for 15 minutes, followed by incubation at 98°C for 5 minutes on a thermal cycler. Reverse-transcribed cDNA was quantified using the Maxima SYBR Green / ROX qPCR Master Mix on a LightCycler 480II system (Roche) using the following program: initial denaturation at 95°C for 10 minutes; denaturation at 95°C for 10 seconds, followed by annealing and extension at 65°C for 30 seconds each, for 45 cycles. mRNA levels were then calculated using the comparative ΔΔct method using LightCycler software (v.96 1.1, Roche).

[0176] The muscle damage repair capacity of mice was evaluated by injecting cardiotoxin (CTX) at a concentration of 20 μM into the mice via tail vein injection in a volume of 50 μl to cause muscle damage. After 7 days, muscle samples were taken from the mice for staining and observation. The muscle regeneration capacity of the mice was determined by observing the muscle morphology and the fluorescence intensity of the muscle regeneration marker protein PAX-7.

[0177] Mouse muscle HE staining and muscle morphology experimental method: Experimental mice were sacrificed and liver and muscle samples were obtained to detect the expression of target proteins. Immunoblotting was used to verify the expression levels of each protein in the liver and muscle of experimental group mice. Mouse muscles were taken for HE staining experiments to observe the muscle morphology of the experimental and control groups. The method is as follows:

[0178] Mice were killed by cervical dislocation and the abdominal cavity was quickly opened. The muscle tissue of the mouse was fixed in 4% paraformaldehyde at room temperature for 24 hours, and then the fixed tissue was transferred to a tissue embedding box and washed with running tap water for 12 hours. The muscle tissue needed to be immersed in 70% alcohol for 12 hours. Then, it was immersed in 70% ethanol, 80% ethanol, and 95% ethanol for 1 hour respectively. The fixed tissue was dehydrated in anhydrous ethanol for 1 hour twice, and then immersed in 50% xylene for 30 minutes, followed by two immersions in xylene for 15 minutes each; then the tissue was immersed in paraffin twice for 1 hour each. The dehydrated tissue was embedded in paraffin on a Histore Arcadia paraffin embedding machine (Leica). Embedded tissue blocks were sectioned, and 3-μm-thick paraffin sections were attached to adhesive microscope slides and dried. The tissues were then rehydrated in the following order: xylene at 70°C for 10 minutes, xylene at 70°C for 10 minutes, anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and 50% ethanol for 5 minutes, followed by a brief placement in water. The sections were stained in hematoxylin for 8 minutes and then rinsed in running water for 5 minutes. The sections were then differentiated in 1% hydrochloric acid-alcohol solution for 30 seconds, rinsed in tap water for 1 minute, and then blued with 0.2% ammonia solution. Finally, the sections were rinsed in running water for 1 minute. The sections were then stained with eosin solution for 30 seconds. The stained sections were dehydrated in 70% ethanol for 5 minutes, then dehydrated twice in 95% ethanol for 5 minutes each, dehydrated twice in anhydrous ethanol for 5 minutes each, and immersed in xylene twice for 15 minutes each. The stained sections were mounted with Canada balsam and the tissue samples were observed under a microscope.

[0179] Mouse muscle fiber type immunofluorescence samples were as shown above. Sections were fixed in 4% paraformaldehyde for 10 minutes and then rinsed with PBS for 5 minutes at room temperature. After incubation with PBST (PBS supplemented with 5% (v / v) Triton X-100) for 10 minutes, sections were blocked with BSA solution (PBS containing 5% (m / v) BSA) for 30 minutes at room temperature. Muscle fibers were stained with MHCIIb antibody (6 μg / mL, diluted in BSA solution) at 4°C overnight and then rinsed three times with PBS at room temperature for 5 minutes each. Sections were then incubated with Alexa Fluor 488-linked goat anti-mouse IgM antibody (1:200, diluted in BSA solution) at room temperature for 1 hour, then rinsed three times with PBS for 5 minutes each, incubated with 4% paraformaldehyde for 2 minutes, and rinsed twice with PBS for 5 minutes each. All processes were performed at room temperature. The sections were then incubated with anti-MHC I antibody (6 μg / mL, diluted in BSA solution) at room temperature for 3 hours, washed three times with PBS buffer for 5 minutes each, and then incubated with Alexa Fluor 594-conjugated goat anti-mouse IgG2b antibody (1:200). The sections were then washed three times with PBS buffer at room temperature for 5 minutes each. After fixing with 4% paraformaldehyde for 2 minutes and rinsing twice with PBS at room temperature for 5 minutes each, the sections were incubated with anti-MHC IIa antibody (6 μg / mL, diluted in BSA solution) in a dark humidified chamber at room temperature for 3 hours, rinsed three times with PBS buffer at room temperature for 5 minutes each, and then incubated with Alexa Fluor 647-conjugated goat anti-mouse IgG1 antibody (1:200 diluted in BSA solution) at room temperature for another 1 hour. The sections were then washed three times with PBS buffer at room temperature for 5 minutes each. The tissue sections were mounted with 90% glycerol and then imaged with fluorescence under a confocal microscope.

[0180] Measurement of mouse muscle NAD using CE-MS mass spectrometry + Mouse skeletal muscle was clamped with liquid nitrogen and rapidly sheared, snap-frozen in liquid nitrogen, and ground. The muscle was then vortex-lyzed with 1 mL of pre-chilled methanol containing an internal standard. 1 mL of chloroform and 400 μL of ultrapure water were added and vortexed. The muscle was then centrifuged at 15,000 g for 15 minutes, and the upper aqueous phase was collected, lyophilized, reconstituted, and analyzed by mass spectrometry.

[0181] The mouse muscle mitochondrial function assay involves starving and sacrificing mice. The gastrocnemius muscle from the hind leg is then digested in 4 mL of dissociation medium containing collagenase for 1.5 hours. The muscle is then rinsed with 4 mL of preheated dissociation medium without collagenase and incubated in 0.5 mL of dissociation medium without collagenase. The muscle is then dispersed six times using a 20G needle to obtain a dispersed muscle homogenate. Twenty microliters of muscle homogenate is inoculated onto a Seahorse XF24Islet Capture Microplate and incubated overnight at 37°C in the absence of carbon dioxide using aCSF. Oligomycin is used as a respiratory chain inhibitor at a final concentration of 10 μM during the assay. Data are collected using Wave 2.6.3 Desktop software (Agilent Technologies).

[0182] The experimental method for detecting the maximum running ability of mice is to train the mice in a normal light-dark cycle for 3 days in a Rodent Treadmill NG (UGO Basile, cat.47300) and test them during the dark period. Before the experiment, the mice were fasted for 2 hours. The treadmill was set to a 5° incline, and the speed of the treadmill was set to increase linearly (starting at a speed of 5 m / min and then increasing to a final speed of 25 m / min within 120 minutes). After accumulating 5 or more electric shocks (0.1 mA) per minute for two consecutive minutes, the mice were considered exhausted and removed from the treadmill. The distance walked was recorded as running ability.

[0183] Mouse grip strength is measured using a handgrip dynamometer (Ugo Basile, cat. 47200). The mouse is clamped by its tail and lowered ("dropped") until its forelimbs or all four limbs grasp a T-bar connected to a digital dynamometer. The mouse is lowered further until its body is level with the instrument and then slowly and steadily pulled away from the T-bar until its forelimbs or all four limbs are free of the bar, generating a peak force measured in grams. This is repeated five times per mouse, with a 5-minute interval between measurements.

[0184] Experimental methods for the insulin tolerance test (ITT) and glucose tolerance test (GTT) in mice. Before each experiment, mice were housed in a single cage for one week. For the GTT, mice were fasted for 16 hours (5:00 PM to 9:00 AM) and then injected with 2 g / kg of glucose (intraperitoneal injection). For the ITT, mice were fasted for 6 hours (8:00 AM to 2:00 PM) and then injected with 0.5 U / kg of insulin (intraperitoneal injection). Conventional blood glucose concentrations were measured 15, 30, 60, and 120 minutes after glucose or insulin injection.

[0185] Figure 4a shows the changes in muscle fiber types in aged mice. After feeding LCA, the number of oxidative muscle fibers in the mouse muscles increased significantly (determined by the expression levels of oxidative muscle fiber markers MHCI and MHCIIa), and the number of glycolytic muscle fibers decreased (determined by the expression level of MHCIIb).

[0186] Figure 4(b) shows the changes in muscle atrophy in aged mice. It can be seen that after feeding LCA, the mRNA levels of Trim63 and Fbxo3, marker genes for muscle atrophy in aged mice, decreased significantly.

[0187] Figure 4(c) shows the body weight and muscle weight of elderly mice, which shows that the mice did not lose muscle mass after being fed LCA.

[0188] Figure 4d shows that LCA feeding improved the muscle damage repair ability of aged mice.

[0189] Figure 4e shows the NAD in the muscles of LCA-fed aged mice + The content increased.

[0190] Figures 4(f) and 4(g) show an increase in mitochondrial content in the muscle of LCA-fed aged mice. Specifically, electron microscopy revealed a significant increase in the area of ​​mitochondria in muscle fibers of mice fed LCA. Western blot analysis of muscle tissue also revealed an increase in the levels of oxidative phosphorylation-related proteins (ATP5A, UQCRC2, MTCO1, SDHB, and NDUFB8), as well as an increase in the mtDNA / nDNA ratio.

[0191] Figure 4h shows that mitochondrial function was increased in muscles of LCA-fed aged mice.

[0192] FIG4i shows that energy expenditure of LCA-fed aged mice was also significantly increased.

[0193] Figure 4j-l shows that the running time and running distance, as well as the gripping strength of the forelimbs and limbs of the old mice fed with LCA were significantly increased.

[0194] m~o in Figure 4 show that LCA feeding not only improves muscle function, but also improves age-related glucose intolerance and insulin resistance in mice.

[0195] Through the above data, we can find that LCA improves the muscle content and structure of mice and improves the exercise capacity of elderly mice.

[0196] Example 4: The life-extending effect of LCA on aged mice depends on AMPK

[0197] In this example, based on the previous work, LCA was fed to elderly mice with AMPKα1 / 2 muscle knockout for one month to test whether the beneficial effects could continue to be achieved in the absence of AMPK.

[0198] Figure 5(a) shows the muscle weight and body weight of AMPK muscle knockout aged mice after feeding LCA.

[0199] Figure 5b shows that after AMPK muscle knockout, LCA feeding no longer increases NAD in muscle + content.

[0200] Figure 5(c) shows that the mitochondrial content in the muscles of LCA-fed aged mice increased, whereas the increase in mitochondrial content was no longer observed in AMPK knockout mice.

[0201] Figure 5(d) shows that LCA-fed aged mice increased mitochondrial function in muscle, while there was no significant difference in mitochondrial function after AMPK knockout.

[0202] Figure 5e shows that energy expenditure was significantly increased in LCA-fed aged mice, but there was no significant difference after AMPK knockout.

[0203] Figures 5f to 5h show that the running time and running distance, as well as the gripping strength of the forelimbs and limbs of LCA-fed elderly mice were significantly increased, while LCA had no effect in AMPK knockout mice.

[0204] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. Use of lithocholic acid or a soluble salt thereof as an AMPK activator, or use thereof in the preparation of an AMPK activator.

2. The use according to claim 1, wherein The AMPK activator activates AMPK in the liver, kidney or muscle tissue of a mammal.

3. Use of lithocholic acid or its soluble salt in the preparation of any of the following medicines: (a) Drugs that inhibit cholesterol synthesis; (b) drugs that reduce fatty acid synthesis; (c) drugs for the prevention and / or treatment of diabetes; (d) drugs for preventing and / or treating tumors; (e) Drugs for improving the motor performance of mammals; (f) Drugs for extending lifespan in mammals.

4. The use according to claim 3, wherein The content of lithocholic acid or its soluble salt in the drug is 0.00001 wt% to 1 wt%, preferably 0.001 wt% to 1 wt%, and more preferably 0.01 wt% to 0.1 wt%.

5. The use according to claim 3 or 4, wherein The drug also contains at least one other AMPK activator.

6. The use according to any one of claims 3 to 5, wherein The medicine also contains pharmaceutically acceptable carriers or excipients.

7. The use according to any one of claims 3 to 6, wherein The dosage form of the drug includes one or more of injection, oral liquid, suspension, emulsion, extract, powder, granule, suppository, aerosol, granule, gel, transdermal patch, sustained release agent, enteric solvent, osmotic pump, tablet, capsule, microcapsule, microsphere, nanoparticle, liposome or capsule.

8. AMPK activators, wherein: The AMPK activator contains lithocholic acid or a soluble salt thereof.

9. A pharmaceutical composition comprising an effective dose of lithocholic acid or a soluble salt thereof, or the AMPK activator according to claim 8, and a pharmaceutically acceptable excipient; The pharmaceutical composition has at least one of the following effects: (a) Drugs that inhibit cholesterol synthesis; (b) drugs that reduce fatty acid synthesis; (c) drugs for the prevention and / or treatment of diabetes; (d) drugs for preventing and / or treating tumors; (e) Drugs for improving the motor performance of mammals; (f) Drugs for extending lifespan in mammals.

10. The pharmaceutical composition according to claim 9, wherein The content of lithocholic acid or its soluble salt is 0.00001 wt% to 1 wt%, preferably 0.001 wt% to 1 wt%, and more preferably 0.01 wt% to 0.1 wt%.

11. The pharmaceutical composition according to claim 9 or 10, wherein The pharmaceutical composition also contains at least one other AMPK activator.

12. The pharmaceutical composition according to any one of claims 9 to 11, wherein The dosage form of the pharmaceutical composition includes one or more of injection, oral liquid, suspension, emulsion, extract, powder, granule, suppository, aerosol, granule, tablet, capsule, microcapsule, microsphere, nanoparticle, liposome or capsule.

13. The pharmaceutical composition according to any one of claims 9 to 12, wherein The auxiliary material includes a cosolvent, and the cosolvent is preferably sodium carboxymethylcellulose (CMC), dimethyl sulfoxide (DMSO), 2-hydroxypropyl-β-cyclodextrin (HP-CD) or corn oil.

14. Lithocholic acid or a soluble salt thereof, the AMPK activator according to claim 8, or the pharmaceutical composition according to any one of claims 9 to 13, for use in: activating AMPK, preferably, activating AMPK in the liver, kidney or muscle tissue of a mammal; Inhibit cholesterol synthesis; Reduce fatty acid synthesis; Prevent and / or treat diabetes; Prevention and / or treatment of tumors; Improving the motor performance of a mammal; and / or Extending mammalian lifespan.

15. A method for activating AMPK, inhibiting cholesterol synthesis, reducing fatty acid synthesis, preventing and / or treating diabetes, preventing and / or treating tumors, improving the motor ability of mammals and / or extending the life span of mammals, comprising administering to cells or individuals a therapeutically effective amount of lithocholic acid or a soluble salt thereof, the AMPK activator of claim 8, or the pharmaceutical composition of any one of claims 9 to 13; Preferably, the activating AMPK is activating AMPK in the liver, kidney or muscle tissue of a mammal.

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