JP7810346B2 - Screening method for substances that inhibit aging - Google Patents

Description

The present invention relates to a method for screening substances that inhibit aging.

AGEs (advanced glycation end products) are known to be one of the substances believed to cause aging, but there has been little research on the relationship between glycated proteins and aging. Most research on the relationship between glycated proteins and aging has focused on receptors for AGEs (RAGE) under conditions where single compounds, such as Nε-carboxymethyllysine (CML) or Nω-carboxymethylarginine (CMA), which are the final products of the glycation reaction, are purified or synthesized (see, for example, Non-Patent Document 1), and are locally accumulated. This research is unlikely to reflect realistic conditions that represent reactions in the body.

In order to reflect the reaction conditions in vivo, it is necessary to focus on how glycated proteins, which are at an intermediate stage in the glycation reaction, affect mammalian cells.

Kislinger T, Fu C, Huber B, Qu W, Taguchi A, Yan S D, Hofmann M, Yan S F, Pischetsrieder M, Stern D, Schmidt A M. J Biol Chem. (1999) Vol.274, No.44, pp.31740-31749.

The objective of the present invention is to provide a method for screening for substances that inhibit the aging phenomenon.

The present invention includes the following inventions to solve the above problems.
[1] A method for screening for a substance that inhibits aging, comprising the steps of contacting a test substance with one or more nucleic acids having a base sequence selected from the group consisting of wwcrtgraaaatgagaaatrym (SEQ ID NO: 1), tndgaaanaata (SEQ ID NO: 2), aaaantdawaawnntdgaawww (SEQ ID NO: 3), tnwkntatnnnttaarnwkw (SEQ ID NO: 4), drgvaavagarvnarrnrva (SEQ ID NO: 5), carvcchvaghnchkscann (SEQ ID NO: 6), nanavmasannshcwtnnvh (SEQ ID NO: 7), and ttsaaa; measuring the state of interaction between the nucleic acid and the test substance; and selecting a test substance that binds to the nucleic acid.
[2] The screening method according to [1], further comprising a step of confirming that the selected test substance reduces the expression level of a gene downstream of the base sequence.
[3] The screening method according to [1] or [2], wherein the aging phenomenon is an aging phenomenon selected from the group consisting of decreased motor function, decreased muscle strength, decreased physical strength, decreased bone density, decreased bone strength, decreased basal metabolic rate, decreased immunity, decreased vision, decreased hearing, decreased sense of smell, decreased sense of taste, decreased skin sensation, decreased digestion and absorption, decreased kidney function, decreased thermoregulation ability, decreased memory, skin discoloration, decreased skin elasticity, decreased skin flexibility, decreased skin moisture content, fine lines, wrinkles, rough skin surface, gray hair, hair loss, cataracts, decreased nerve cell count, decreased neurofibrillary fibers, decreased brain weight, slowing of electroencephalograms, atrophy of airway mucosa and loss of ciliary movement, atrophy of bronchial wall mucosa and the like, decreased alveolar area, decreased vital capacity and ventilation, arteriosclerosis, decreased reproductive ability, loss of ovarian function, arthritis, neurological and psychological disorders, decreased lymphatic tissue and spleen weight, increased incidence of malignant tumors, frequent urination, and decreased insulin secretion ability.
[4] The screening method according to any one of [1] to [3], wherein the step of measuring the interaction state between the nucleic acid and the test substance comprises a measurement step selected from the group consisting of gel shift assay, pull-down assay, chromatin immunoprecipitation analysis, reporter assay, Förster resonance energy transfer analysis, and ELISA.
[5] The screening method according to any one of [2] to [4], wherein the step of confirming that the gene expression level is reduced is performed by microarray analysis or quantitative PCR analysis.
[6] A pharmaceutical composition comprising, as an active ingredient, a substance selected by the screening method according to any one of [1] to [5].
[7] A cosmetic comprising a substance selected by the screening method according to any one of [1] to [5].
[8] A food or drink containing a substance selected by the screening method according to any one of [1] to [5].
[9] A feed containing a substance selected by the screening method according to any one of [1] to [5].
[10] A screening kit for substances that inhibit aging, comprising one or more nucleic acids having a base sequence selected from the group consisting of wwcrtgraaaatgagaaatrym (SEQ ID NO: 1), tndgaaanaata (SEQ ID NO: 2), aaaantdawaawnntdgaawww (SEQ ID NO: 3), tnwkntatnnnttaarnwkw (SEQ ID NO: 4), drgvaavagarvnarrnrva (SEQ ID NO: 5), carvcchvaghnchkscann (SEQ ID NO: 6), nanavmasannshcwtnnvh (SEQ ID NO: 7), and ttsaaa.

The present invention provides a method for screening for substances that inhibit aging.

The inventors contacted mammalian cells with glycated proteins, which are present in the middle stage of the glycation reaction, and comprehensively analyzed changes in gene expression levels. Furthermore, they analyzed the base sequences near the transcription start sites of genes whose expression levels increase upon contact with glycated proteins, and identified eight consensus sequences that are highly conserved in mammals (see Table 1 in the Examples). Because glycated proteins are involved in the aging phenomenon, substances that specifically bind to these consensus sequences and suppress the transcription of downstream genes are thought to be substances that can suppress the aging phenomenon.

The present invention provides a method for screening for substances that inhibit aging (hereinafter referred to as the "screening method of the present invention"). The screening method of the present invention may include the following steps (1) to (3):
(1) contacting a test substance with one or more nucleic acids consisting of a base sequence selected from the group consisting of wwcrtgraaaatgagaaatrym (SEQ ID NO: 1), tndgaaanaata (SEQ ID NO: 2), aaaantdawaawnntdgaawww (SEQ ID NO: 3), tnwkntatnnnttaarnwkw (SEQ ID NO: 4), drgvaavagarvnarrnrva (SEQ ID NO: 5), carvcchvaghnchkscann (SEQ ID NO: 6), nanavmasannshcwtnnvh (SEQ ID NO: 7) and ttsaaa; (2) measuring the interaction state between the nucleic acid and the test substance; (3) selecting a test substance that binds to the nucleic acid.

Aging refers to various phenomena that occur irreversibly with age. Examples include decline in motor function, balance, muscle strength, physical fitness, bone density and strength, basal metabolic rate, immune system, decreased five senses (sight, hearing, smell, taste, and skin), digestive and absorptive capacity, renal function, decreased thermoregulation, memory loss, skin discoloration, decreased skin elasticity, decreased skin flexibility, decreased skin moisture content, fine lines, wrinkles, rough skin surface, gray hair, hair loss, cataracts, decreased nerve cell count, decreased neurofibrillary activity, decreased brain weight, slowed EEG, atrophy of airway mucosa and loss of ciliary movement, atrophy of bronchial wall mucosa, decreased alveolar area, decreased vital capacity and ventilation, arteriosclerosis, decreased reproductive ability, loss of ovarian function, arthritis, neurological and psychological disorders, decreased lymphatic tissue and spleen weight, increased incidence of malignant tumors, frequent urination, and decreased insulin secretion.

The test substances used in the screening methods of the present invention are not particularly limited and may include, for example, single substances such as natural compounds, organic compounds, inorganic compounds, nucleic acid oligos, proteins, and peptides, as well as expression products of compound libraries, nucleic acid oligo libraries, peptide libraries, gene libraries, cell extracts, cell culture supernatants, fermentation microbial products, marine organism extracts, plant extracts, prokaryotic cell extracts, eukaryotic single-cell extracts, and animal cell extracts. Test substances may be novel or known substances. These test substances may form salts. Salts of test substances are preferably salts with physiologically acceptable acids or bases. These test substances may also be modified with a labeling substance. Labeling substances are not particularly limited as long as they are detectable, and examples include radioisotope-containing compounds, stable isotope-containing compounds, biotin, lipids, fluorescent dye molecules, non-fluorescent dye molecules, enzymes, enzyme substrates, and affinity tags.

In step (1), a test substance is contacted with one or more nucleic acids comprising the aforementioned base sequence. The nucleic acid comprising the aforementioned base sequence may be an artificially synthesized oligonucleotide, or may be extracted, isolated, purified, and/or amplified from any mammalian cell by known techniques. Oligonucleotides can be synthesized by standard methods, for example, easily synthesized using a commercially available DNA synthesizer. These nucleic acids may also be modified with a labeling substance. The labeling substance is not particularly limited as long as it is detectable, and examples include radioisotope-containing compounds, stable isotope-containing compounds, nucleic acid analogs, digoxigenin, biotin, fluorescent dye molecules, non-fluorescent dye molecules, enzymes, etc.

The method for contacting the nucleic acid consisting of the above-mentioned base sequence with the test substance is not particularly limited. For example, the nucleic acid may be added to a solution containing the test substance, the test substance may be added to a solution containing the nucleic acid, a solution containing the nucleic acid and a solution containing the test substance may be mixed, or the nucleic acid and the test substance may be mixed in a medium such as water or a buffer solution. The conditions for contacting the nucleic acid with the test substance, such as temperature and time, are not particularly limited and can be set appropriately based on the technical common sense of those skilled in the art.

In step (2), the state of interaction between the nucleic acid and the test substance is measured. The method for measuring the interaction is not limited, as long as it allows detection of the binding between the nucleic acid and the test substance. For example, luminescence methods such as gel shift assay, pull-down assay, chromatin immunoprecipitation (ChIP) analysis, and reporter assay, fluorescence methods such as Förster resonance energy transfer (FRET) analysis, and colorimetric methods such as enzyme-linked immunosorbent assay (ELISA) may be used, or commercially available transcription factor assay kits may be used.

In step (3), a test substance that binds to the nucleic acid is selected. Binding between the nucleic acid and the test substance can be determined by a standard method using the measurement method used in step (2), and substances that are determined to bind to the nucleic acid can be selected.

When a gel shift assay (EMSA: Electrophoretic Mobility Shift Assay) is used in step (2), the state of interaction between the nucleic acid comprising the above-mentioned base sequence and the test substance can be measured from gel mobility using standard methods. For example, the test substance can be contacted with a nucleic acid comprising the above-mentioned base sequence labeled with any labeling substance (step (1)), and after gel electrophoresis, the labeled nucleic acid can be visualized using standard methods and the gel mobility compared (step (2)). In this case, in step (3), the gel mobility of the nucleic acid before and after contact with the test substance can be compared, and the test substance contacted with the nucleic acid that has a shorter migration distance can be selected.

When a pull-down assay is used in step (2), the state of interaction between the nucleic acid comprising the above base sequence and the test substance can be determined based on the presence or absence of complex formation using standard methods. In this case, in step (1), a nucleic acid comprising the above base sequence modified with an arbitrary labeling substance may be immobilized as bait and contacted with the test substance as prey. Alternatively, a test substance modified with an arbitrary labeling substance may be immobilized as bait and contacted with a nucleic acid comprising the above base sequence as prey. In step (2), the bait/prey complex can be eluted using standard methods. The eluted complex may be isolated and/or digested using known techniques and identified by mass spectrometry. Alternatively, the eluted complex may be visualized by electrophoresis gel staining, Western blotting, radioisotope detection, or the like, and the bound test substance may be confirmed. Alternatively, the eluted complex may be isolated using known techniques, and the bound nucleic acid may be detected by Southern blotting, PCR analysis, or the like. In this case, in step (3), a test substance confirmed to have formed a complex with the nucleic acid comprising the above base sequence in step (2) may be selected.

If ChIP analysis is used in step (2), the state of interaction between the nucleic acid comprising the above base sequence and the test substance can be measured based on the presence or absence of complex formation using standard methods. In this case, in step (1), the test substance modified with any labeling substance may be contacted with the nucleic acid comprising the above base sequence. The complex of the test substance and the nucleic acid comprising the above base sequence may be fixed by crosslinking using a solution containing a chemical fixative such as formaldehyde. In step (2), the complex of the test substance and the nucleic acid comprising the above base sequence may be recovered and concentrated by contacting it with beads or a column to which a specific antibody against the labeling substance is bound. The concentrated complex can be eluted using known techniques and detected using known techniques such as quantitative PCR or Western blotting. In this case, in step (3), the test substance detected from the complex recovered in step (2) can be selected.

When a reporter assay is used in step (2), the state of interaction between the nucleic acid comprising the above base sequence and the test substance can be measured using the expression of the reporter gene protein as an indicator according to standard methods. In this case, an expression vector constructed to express any luminescent protein gene or fluorescent protein gene downstream of any base sequence selected from the above base sequences can be introduced into any mammalian cell, and the transfected cell can be contacted with the test substance (step (1)). The presence or absence of binding between the nucleic acid comprising the above base sequence and the test substance can be detected by measuring the luminescence, absorbance, or fluorescence of the transfected cell (step (2)). In this case, in step (3), a test substance for which a change in luminescence, fluorescence, or absorbance was detected in step (2) can be selected.

When FRET is used in step (2), the state of interaction between the nucleic acid consisting of the above base sequence and the test substance can be measured based on changes in fluorescence wavelength using standard methods. For example, in step (1), a nucleic acid containing the above base sequence labeled with any fluorescent molecule is contacted with a test substance labeled with a different fluorescent molecule, and in step (2), the original fluorescence wavelength is compared with the fluorescence wavelength after contact. If the contacted nucleic acid and the test substance show a strong interaction, different fluorescence wavelengths can be detected before and after contact. In this case, in step (3), a test substance for which different fluorescence wavelengths were detected before and after contact with the nucleic acid in step (2) can be selected.

When ELISA is used in step (2), the state of interaction between the nucleic acid comprising the above base sequence and the test substance can be determined based on the presence or absence of a color reaction, using standard methods. For example, nucleic acid comprising the above base sequence can be immobilized on a plate, contacted with a test substance labeled with an enzyme such as horseradish peroxidase (HRP) (step (1)a), and then reacted with a color reagent containing a substrate for the labeling enzyme such as 3,3',5,5'-tetramethylbenzidine (TMB) to detect color development (step (2)a). Alternatively, the test substance can be immobilized on a plate, contacted with an enzyme-labeled nucleic acid comprising the above base sequence (step (1)b), and then reacted with a color reagent containing a substrate for the labeling enzyme to detect color development (step (2)b). If an antibody specific to the test substance is obtained, a nucleic acid containing the above-mentioned base sequence may be immobilized on a plate and contacted with the test substance (step (1)c), after which the antibody specific to the test substance and an enzyme-labeled secondary antibody may be treated and reacted with a color-developing reagent containing a substrate for the labeling enzyme to detect color development (step (2)c). In this case, in step (3), the test substance for which a color development reaction was detected in step (2) may be selected.

In one embodiment, a commercially available transcription factor assay kit can be used. Various commercially available transcription assay kits may include a combination or modification of the above-mentioned assay methods. The contact of the nucleic acid containing the above-mentioned base sequence with the test substance (step (1)) and the measurement of the interaction between the above-mentioned base sequence and the test substance (step (2)) can be carried out according to the kit manufacturer's protocol. In this case, in step (3), the test substance that binds to the above-mentioned base sequence can be selected according to the evaluation method of the kit used.

The screening method of the present invention may further include a step (step (4)) of confirming that the test substance selected in step (3) reduces the expression level of a gene downstream of the base sequence. The confirmation step is not limited as long as it allows for measuring the expression level of a gene downstream of the base sequence. For example, the selected test substance may be contacted with any mammalian cell, and then nucleic acid may be extracted using a known method to confirm the expression level of the target gene. The target gene is not particularly limited; any one downstream gene of the selected consensus sequence may be selected and confirmed. The method of contacting the cells with the selected test substance is not particularly limited; for example, the test substance may be added to a culture medium containing the cells. The expression level of the gene may be confirmed by measuring the amount of mRNA or the amount of translated protein. In one embodiment, the confirmation step is performed by microarray analysis. In another embodiment, the confirmation step is performed by quantitative PCR (Polymerase Chain Reaction) analysis.

When microarray analysis is used in step (4), gene expression levels can be confirmed using standard methods. For example, sample DNA and/or RNA can be extracted from mammalian cells treated with a selected test substance using known techniques, hybridized on a microarray in which oligonucleotides or cDNA are aligned and immobilized on a glass or other substrate, and compared with a control to comprehensively examine quantitative and qualitative changes, such as gene expression levels and mutations. The method for manufacturing the microarray is not particularly limited. DNA synthesized using standard methods can be labeled with fluorescent dyes such as Cy3 or Cy5 to create DNA probes and immobilized on a substrate using known techniques. DNA can also be synthesized on a substrate, DNA can be synthesized using inkjet printing technology, or commercially available microarrays can be used. Samples can be labeled with a single fluorescent dye and hybridized separately for each prepared labeled sample. Hybridization can also be performed by mixing two or more samples labeled with different fluorescent dyes. Quantitative and qualitative changes in genes can be detected by analyzing the fluorescent signal intensity obtained from each microarray using image data analysis of the microarray. Any analysis software may be used for image data analysis, including free software such as open source and free software. A decrease in gene expression level can be determined based on a decrease in the fluorescent signal intensity for each probe. The criteria for determination are not particularly limited, but for example, a decrease of 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less compared to the fluorescent signal intensity of a control not treated with the test substance may be determined to be a decrease in gene expression level due to contact with the test substance.

When quantitative PCR is used in step (4), gene expression levels can be confirmed using standard methods. For example, sample DNA and/or RNA can be extracted from mammalian cells treated with a selected test substance using known techniques, PCR-amplified, and then subjected to agarose gel electrophoresis to compare relative gene expression levels. Alternatively, the PCR amplification level of the sample can be monitored in real time using real-time PCR, and the expression level can be measured in comparison with that of a control. Primers used for PCR amplification may be oligo-DNA that complementarily binds to sequences adjacent to the downstream gene region of the selected consensus sequence, and can be synthesized using known techniques. A decrease in gene expression level can be determined by a decrease in the amount of PCR amplification product. The criteria for determination are not particularly limited; for example, a decrease of 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less compared to a control not treated with the test substance may be determined to be a decrease in gene expression level due to contact with the test substance.

The mammalian cells used in the confirmation process of the present invention may be cells derived from any mammal, and are not particularly limited. Examples of mammals include humans, monkeys, rats, mice, guinea pigs, dogs, cats, rabbits, goats, sheep, horses, pigs, and cows. Mammalian-derived cells may be primary culture cells, established cell lines, cancerous cells, or cells isolated from tissue collected as a biopsy sample. Examples include fibroblasts, smooth muscle cells, melanoma cells, chondrocytes, hepatocytes, peripheral blood cells, cells in lymph and tissue fluid, epithelial cells, neurons, and ovarian cells. In one embodiment, the cells are mouse-derived melanoma cells.

The present invention provides a composition for inhibiting aging phenomena (hereinafter referred to as the "composition of the present invention"). The composition of the present invention may contain, as an active ingredient, a substance selected by the screening method of the present invention, and any selected substance may be used as the active ingredient, or one or more selected substances may be used in any combination. The substance selected by the screening method of the present invention may also be concentrated by freeze-drying, spray-drying, etc. before use.

The compositions of the present invention can be implemented as pharmaceutical compositions for inhibiting aging. When implemented as pharmaceutical compositions, the compositions of the present invention can be formulated for delivery to cells and/or subjects via any administration route by appropriately blending with pharmaceutically acceptable carriers or additives. Modes of administration to a subject may include injection, infusion, inhalation, intranasal, intraocular, topical, cannula-based, or ingestion. Specifically, oral preparations may include tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions; and parenteral preparations may include injections, infusions, suppositories, ointments, patches, and enteral nutritional supplements. Injections include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injections and infusions. In some embodiments, administration includes aerosol inhalation, for example, using a nebulizer. The proportion of pharmaceutically acceptable carriers or additives may be appropriately determined based on the range commonly used in the pharmaceutical field. There are no particular limitations on the carriers or additives that can be added, and examples include water, saline, and other aqueous solvents; various carriers such as aqueous or oily bases; and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and fragrances. Pharmaceutically acceptable carriers or additives are generally safe, non-toxic, and include carriers or additives that are useful in preparing the desired pharmaceutical composition. Such carriers or additives may be solid, liquid, semi-solid, or, in the case of an aerosol composition, gaseous.

The pharmaceutical compositions described herein can also be tableted for oral administration or prepared in emulsions or syrups. Examples of additives that can be incorporated into tablets, capsules, etc. include binders such as gelatin, cornstarch, tragacanth, and gum arabic; fillers such as crystalline cellulose; bulking agents such as cornstarch, gelatin, and alginic acid; lubricants such as magnesium stearate; sweeteners such as sucrose, lactose, or saccharin; and flavoring agents such as peppermint, sucrose, or cherry. When the dosage unit is a capsule, the above-mentioned materials can further contain a liquid carrier such as an oil or fat. Sterile compositions for injection can be prepared according to conventional pharmaceutical procedures (e.g., by dissolving or suspending the active ingredient in a solvent such as water for injection or natural vegetable oil). Aqueous solutions for injection include, for example, saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), and may be used in combination with appropriate solubilizers such as alcohol (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80™, HCO-50). Oily solutions include, for example, sesame oil and soybean oil, and may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Additionally, the solution may be formulated with buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives (e.g., benzyl alcohol, phenol, etc.), antioxidants, etc.

The compositions of the present invention can be formulated as topical compositions, particularly dermatologically acceptable cosmetics. When formulated as cosmetics, the compositions of the present invention may contain excipients such as at least one compound selected from the group consisting of preservatives, emollients, emulsifiers, surfactants, moisturizers, thickeners, conditioners, matting agents, stabilizers, antioxidants, texture modifiers, bleaching agents, film formers, solubilizers, pigments, dyes, fragrances, and sunscreens. The compositions may also contain various cosmetic ingredients suitable for topical use and commonly used in the cosmetics industry. Examples of these ingredients include abrasives, absorbents, essential oils, astringents, anti-acne agents, anti-clumping agents, anti-foaming agents, antibacterial agents, binders, biological additives, buffers, swelling agents, chelating agents, additives, biocides, denaturants, topical analgesics, film-forming agents, polymers, opacifiers, pH adjusters, reducing agents, depigmenting or whitening agents, emollients and/or wound-healing agents, thickeners, and vitamins, and derivatives or equivalents thereof. These dermatologically acceptable cosmetic products can be prepared in a form selected from the group consisting of aqueous or oily solutions in pots or tubes, creams or aqueous or oily gels, milks, emulsions, particularly of the oil-in-water, water-in-oil, or complex or silicone-based types, microemulsions, or nanoemulsions, globules, liquid soaps, makeup removers, ointments, foams, anhydrous products, liquid, paste, or solid products, such as sticks.

As used herein, the term "dermatologically acceptable" means that the composition or ingredients described thereafter are suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic reaction, or the like.

The content of the active ingredient in the pharmaceutical composition or cosmetic of the present invention is not particularly limited, and may be 0.01 to 99% (w/w), or 0.1 to 95% (w/w).

The composition of the present invention can be implemented as a food or beverage for inhibiting the aging phenomenon. Food or beverage products for inhibiting the aging phenomenon include health foods, functional foods, foods for specified health uses, foods for the sick, nutritionally fortified foods, supplements, etc. The form of the food or beverage product is not particularly limited. For example, examples include tablets, granules, powders, and energy drinks; beverages such as tea drinks, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, milk, and lactic acid drinks; sweets and confectionery foods such as candy, gum, candy tablets, gummy jelly, chocolate, snacks, biscuits, jelly, jam, yokan, rice crackers, arare, manju, and pancake mix; processed seafood foods such as kamaboko and chikuwa; processed livestock foods such as ham and sausage; dairy products such as processed milk and fermented milk; oils and fats and oil-processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressing; seasonings such as sauces, spices, and sauces; retort pouch foods such as curry, stew, rice porridge, and rice porridge; side dishes; breads; and frozen desserts such as ice cream, sherbet, and shaved ice. There are no particular limitations on the manufacturing method for these foods and beverages, as long as it does not impair the effects of the present invention, and methods used by those skilled in the art for each application may be used.

The content of the active ingredient in the food and beverage product for inhibiting aging phenomena of the present invention is not particularly limited, and may be 0.01 to 99% (w/w), or 0.1 to 95% (w/w).

The composition of the present invention can be used as feed. For example, the composition of the present invention may be added in granular, powdered, liquid, or other form to livestock feed or various pet foods such as dog food and cat food to prepare various types of feed. The method for producing these feeds is not particularly limited as long as it does not impair the effects of the present invention, and may follow methods used by those skilled in the art for each application. Furthermore, as long as it does not impair the effects of the present invention, any ingredients commonly used in feeds may be included. For example, grains such as corn, milo, barley, wheat, rye, and rice; bran and rice bran; vegetable oil cakes such as soybean meal and rapeseed meal; animal proteins such as fish meal and meat-and-bone meal; oligosaccharides; oils and fats such as animal fats and oils; various vitamins such as vitamin B1 and vitamin E; enzymes; minerals such as salt, silicic acid, calcium carbonate, and tricalcium phosphate; amino acids; organic acids, etc. The feed of the present invention can be in any suitable form, such as dry, semi-moist, or moist, and can be made into a product that is stable at refrigerated or room temperature.

The content of the active ingredient in the feed of the present invention is not particularly limited, and may be 0.01 to 99% (w/w), or 0.1 to 95% (w/w).

The present invention provides a screening kit for substances that inhibit aging (hereinafter referred to as the "kit of the present invention"). The kit of the present invention may include one or more nucleic acids having a base sequence selected from the group consisting of wwcrtgraaaatgagaaatrym (SEQ ID NO: 1), tndgaaanaata (SEQ ID NO: 2), aaaantdawaawnntdgaawww (SEQ ID NO: 3), tnwkntatnnnttaarnwkw (SEQ ID NO: 4), drgvaavagarvnarrnrva (SEQ ID NO: 5), carvcchvaghnchkscann (SEQ ID NO: 6), nanavmasannshcwtnnvh (SEQ ID NO: 7), and ttsaaa. The kit may also include a container (e.g., a tube) for holding a test sample. The kit may also include reagents necessary for testing, and may include a medium such as a buffer solution. In this case, the nucleic acid having the base sequence may be packed in the container for holding the test sample, or may be contained in a container separate from the container for holding the test sample. Furthermore, the reagents and/or media required for the test may be filled in the container that holds the specimen sample, or may be contained in a container separate from the container that holds the specimen sample. The kit of the present invention may be provided in a single packaging box, or may be a kit supplied in separate packages that are used as a set when in use. It may also optionally include other items than those exemplified above, such as an instruction manual.

The present invention will be explained in detail below using examples, but the present invention is not limited to these examples.

Example 1: Analysis of gene changes and transcription start site specificity caused by glycated proteins
To examine the effects of glycated proteins on gene expression levels and related intracellular parameters in melanoma cells, glycated proteins were prepared from glucose and BSA.

Glycated protein was prepared as follows: 0.1 g of bovine serum albumin (F-V, pH 5.2, Nacalai Tesque), 0.1 M (final concentration) D-glucose, and 0.005 M (final concentration) diethylenetriamine-N,N,N',N",N"-pentaacetic acid (DTPA, Dojindo Laboratories) were mixed into 4.0 mL of 0.2 M phosphate buffer (pH 7.4), and the mixture was allowed to react in a sealed container at 37°C for 14 days. After the reaction, the protein was purified by column filtration using a desalting column (PD-10, Cytiva).

Since glycated proteins are known to inhibit the neurite outgrowth of neuron-like cells (Souichi Nakajima et al., Fragrance Journal (2021) 49(11):46-48), the production of glycated proteins was confirmed by measuring their effect on neurite outgrowth of neuron-like cells. Specifically, human neuroblastoma SH-SY5Y cells were seeded onto a collagen-coated 24-well plate (IWAKI, product number 4820-010) at 1.2 x ¹⁰⁴ cells/400 μL per well and cultured at 37°C in a 5% _CO2 atmosphere for 24 hours. The purified protein was then added and allowed to contact the cells for 48 hours. The percentage of cells that extended neurites was calculated by microscopic observation. When the purified protein was added to a final concentration of 100 μg/mL, neurite outgrowth was inhibited in approximately 80% of the cells, confirming that the protein glycation reaction had progressed.

<Extraction of melanoma cell RNA>
To investigate the effects of glycated proteins on gene expression levels and related intracellular parameters in melanoma cells, the glycated proteins prepared as described above were contacted with melanoma cells. Total RNA was extracted from the melanoma cells by a standard method, and mRNA expression levels were compared relative to a control group.

First, B16 melanoma 4A5 cells were precultured. B16 melanoma 4A5 cells were seeded at 1.0 x ¹⁰ cells/2000 μL per well in a 6-well multiplate (Greiner, product number 657185) and cultured at 37°C for 4 hours in a 5% _CO2 atmosphere. DMEM (Merck) was used as the medium. The glycated proteins prepared as described above were added to the medium at a final concentration of 100 μg/mL and cultured at 37°C for 24 hours in a 5% _CO2 atmosphere. The medium was not changed. Cultured cells were washed with PBS, and total RNA was extracted according to the protocol of an RNA extraction kit (PureLink RNA Mini Kit, Thermo Fisher Scientific). Total RNA quality was assessed using a microchip electrophoresis system (Bioanalyzer, Agilent) to confirm that the RNA sample resolution (RIN: RNA Integrity Number) exceeded 7.0.

<Construction of double-stranded cDNA library>
cDNA was synthesized from total RNA using random primers by standard methods. Next, the ribose diol in the 5' cap structure of the RNA was oxidized and biotinylated by standard methods. Biotinylated RNA/cDNA hybrid strands were selectively recovered by cap trapping using streptavidin beads. RNA was degraded using RNase H (Promega), and adapters were ligated to both ends of the cDNA obtained by standard methods to construct a double-stranded cDNA library (CAGE (Cap Analysis of Gene Expression) library).

<Analysis using next-generation sequencers>
Gene sequencing of the CAGE library was performed using a next-generation sequencer (NextSeq 500, Illumina) by single-end reads targeting the 5'-end of the CAGE library. The resulting reads (CAGE tags) were mapped to the reference genome sequence (human hg38 genome) using an alignment tool (BWA: Burrows-Wheeler Aligner 0.5.9, Li, H. and Durbin, R. Bioinformatics (2009) 25:1754-1760). Reads that were not mapped by BWA were mapped using another alignment tool (HISAT2 (2.0.5), Kim, D et al., Nat Biotechnol (2019) 37:907-915). CAGE tag count data were clustered using CAGEr (open source, Haberle, V. et al., Nucleic Acids Research (2015) 43:e51) with the Paraculu algorithm (Parametric clustering algorithm, Frith, MC et al., Genome Res. (2008) 18:1-12) with the following parameters: Total reads for all samples were normalized to 1 million, and clusters with counts per million (CPM) < 0.2 were discarded.

<Gene expression variation and motif analysis>
The DESeq2 package (1.20.0) in CAGEr was used to detect differentially expressed genes. For motif analysis, the genomic DNA sequence of the region from 200 bp upstream to 50 bp downstream of the differentially expressed CAGE peak was analyzed using the following de novo motif discovery software: AMD (Automated Motif Discovery; Shi, J. et al., PLoS ONE (2011) 6(9):e24576), GLAM2 (Gapped Local Alignment of Motifs; Frith, MC et al., PLoS Computational Biology (2008) 26(7):860-866), DREME (Discriminative Regular Expression Motif) Elicitation; Bailey, TL, Bioinformatics (2011) 27(12):1653-1659) and Weeder (Pavesi, G. et al., Nucleic Acids Res. (2004) 32:W199-W203). Motif expression was investigated using FIMO (Find Individual Motif Occurrences; Grant, C. E. et al., Bioinformatics (2011) 27(7):1017-1018). Similarity between the candidate motif sequences and those registered in the JASPAR database (JASPAR CORE 2016 vertebrates, open source; Mathelier, A. et al., Nucleic Acids Res. (2016) 44:D110-D115) was compared using TOMTOM (Gupta, S. et al., Genome Biology (2007) 8(2):R24). Genes with a CPM greater than 1 detected by CAGEr and a fold change in expression greater than 4 were analyzed using the clusterProfiler package in CAGEr.

<Results>
Gene clusters with similar expression patterns were grouped, and the changes in expression levels and the similarity of the transcription start site sequences were examined. A search for specific sequences (motifs) common to genes whose expression levels increased after glycated protein treatment was conducted to identify sequences highly conserved in mammals. The binding motifs shown in Table 1 were found to have a statistically significant effect on changes in gene expression. Furthermore, a review of the sequence data registered with JASPAR suggested that known transcription factors may be involved in some of the sequences.

The present invention is not limited to the above-described embodiments and examples; various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims (5)

A method for screening for an anti-aging substance, comprising:
The method includes the steps of contacting a test substance with one or more nucleic acids having a base sequence selected from the group consisting of wwcrtgraaaatgagaaatrym (SEQ ID NO: 1), aaaantdawaawnntdgaawww (SEQ ID NO: 3), tnwkntatnnnttaarnwkw (SEQ ID NO: 4), drgvaavagarvnarrnrva (SEQ ID NO: 5), carvcchvaghnchkscann (SEQ ID NO: 6), and nanavmasannshcwtnnvh (SEQ ID NO: 7), which are consensus sequences highly conserved in genes whose expression levels in mammalian cells increase upon contact of a glycosylated protein with the mammalian cells; measuring the state of interaction between the nucleic acid and the test substance; and selecting a test substance that binds to the nucleic acid,
The screening method further comprises a step of confirming that the selected test substance reduces the expression level of a gene downstream of the base sequence. 2. The method of claim 1, wherein the aging phenomenon is an aging phenomenon selected from the group consisting of decreased motor function, decreased muscle strength, decreased physical strength, decreased bone density, decreased bone strength, decreased basal metabolic rate, decreased immunity, decreased vision, decreased hearing, decreased sense of smell, decreased sense of taste, decreased skin sensation, decreased digestion and absorption, decreased kidney function, decreased thermoregulation ability, decreased memory, skin discoloration, decreased skin elasticity, decreased skin flexibility, decreased skin moisture content, fine lines, wrinkles, rough skin surface, gray hair, hair loss, cataracts, decreased nerve cell number, decreased neurofibrillary fibers, decreased brain weight, slowing of electroencephalograms, atrophy of airway mucosa and loss of ciliary movement, atrophy of bronchial wall mucosa, decreased alveolar area, decreased vital capacity and ventilation, arteriosclerosis, decreased reproductive ability, loss of ovarian function, arthritis, neurological and psychological disorders, decreased lymphatic tissue and spleen weight, increased incidence of malignant tumors, frequent urination, and decreased insulin secretion ability. The screening method according to claim 1 or 2, wherein the step of measuring the interaction state between the nucleic acid and the test substance includes a measurement step selected from the group consisting of gel shift assay, pull-down assay, chromatin immunoprecipitation analysis, reporter assay, Förster resonance energy transfer analysis, and ELISA. The screening method according to any one of claims 1 to 3, wherein the step of confirming a decrease in gene expression level is performed by microarray analysis or quantitative PCR analysis. A screening kit for use in the screening method of any one of claims 1 to 4, which is a screening kit for substances that inhibit aging, comprising one or more nucleic acids having a base sequence selected from the group consisting of wwcrtgraaaatgagaaatrym (SEQ ID NO: 1), aaaantdawaawnntdgaawww (SEQ ID NO: 3), tnwkntatnnnttaarnwkw (SEQ ID NO: 4), drgvaavagarvnarrnrva (SEQ ID NO: 5), carvcchvaghnchkscann (SEQ ID NO: 6), and nanavmasannshcwtnnvh (SEQ ID NO: 7), which are consensus sequences highly conserved in genes whose expression levels increase in mammalian cells upon contact of a glycated protein with the mammalian cells.