Biomarker composition for diagnosing or inducing senescence, comprising HMGB1

Abstract

A biomarker composition which is for diagnosing or inducing senescence and comprises HMGB1, particularly reduced HMGB1, according to the present invention enables more sensitive and specific diagnosis of secondary senescence than existing typical senescence markers, and enables the provision of a candidate drug screening tool for senescence therapeutic agent development and the prediction and monitoring of senescence treatment responses.

Description

Biomarker composition for diagnosing or inducing aging comprising HMGB1

[0001] The present invention relates to a biomarker composition for diagnosing or inducing aging, comprising HMGB1.

[0002]

[0003] With the global aging trend, the accumulation of senescent cells in the body is rapidly increasing, leading to a rise in the incidence of various age-related diseases. Cellular senescence is induced by DNA damage, telomere shortening, inflammatory signaling activation, and carcinogenic stimuli, and is closely related to the pathophysiology of aging and various chronic diseases. Senescent cells exhibit impaired homeostasis and secrete a senescence-associated secretory phenotype (SASP), which includes inflammatory cytokines, chemokines, growth factors, and proteases. This alters the tissue microenvironment and induces chronic inflammation and tissue dysfunction.

[0004] Senescent cells can transmit their senescent phenotype to adjacent normal cells through paracrine mechanisms, a phenomenon known as secondary senescence or paracrine senescence. Furthermore, xenograft parabiosis or blood exchange experiments between young and old mice have demonstrated that circulating senescence-inducing factors can induce systemic aging, leading to tissue dysfunction and increased mortality. Accordingly, elucidating the pathogenesis of circulating senescence-inducing factors and the mechanisms underlying secondary senescence are emerging as key challenges in the diagnosis and treatment of age-related diseases.

[0005] As an approach to controlling these aging mechanisms, active research has recently been conducted on senolytic and senomorphic agents. Senolytic agents selectively eliminate primary senescent cells, while senomorphic agents restore function by modulating the detrimental secretory phenotype of senescent cells. These agents are attracting attention as universal tools for delaying aging and treating age-related diseases, and research is ongoing on the discovery of various candidate agents and their mechanisms of action. However, most existing studies have focused on the removal or inhibition of senescent cells themselves, and the molecular mechanisms of secondary aging transition induced by senescent cell-derived factors remain insufficiently elucidated.

[0006] In particular, musculoskeletal tissues are vulnerable to aging. As people age, muscle mass decline and osteoarthritis deteriorate rapidly, leading to an increased incidence of age-related musculoskeletal diseases such as sarcopenia and osteoarthritis. Currently, treatment for these conditions is primarily limited to steroids or anti-inflammatory drugs, and a fundamental cure remains elusive. Furthermore, numerous global pharmaceutical companies have failed to develop treatments.

[0007] Meanwhile, HMGB1 is a dual-function protein that binds to DNA within the nucleus in normal cells and performs transcriptional regulation functions. However, under stress or damage stimuli, it is released extracellularly and induces inflammatory responses and aging signals through receptors (RAGE, TLR, etc.). In particular, HMGB1 is a redox-sensitive protein, and its inflammatory or immune-related functions are known to vary depending on the oxidation state of its cysteine ​​residues. However, the role of HMGB1, especially as a factor in inducing aging metastasis and mediating the spread of systemic aging according to its redox state, has not yet been clearly elucidated.

[0008] Against this backdrop, the present inventors focused on high mobility group box 1 protein (HMGB1) as a key factor mediating aging transition (secondary aging).

[0009]

[0010] The present invention aims to solve the above-mentioned problems and other problems related thereto.

[0011] An object of the present invention is to provide a biomarker composition for diagnosing or inducing aging, comprising HMGB1.

[0012] Another object of the present invention is to provide a method for producing a method for producing a biological sample, comprising: (a) analyzing the expression level of HMGB1 protein or a gene encoding the same from a biological sample; and

[0013] (b) A method for providing information for diagnosing aging is provided, comprising a step of comparing the expression level of the HMGB1 protein or the gene encoding it analyzed in step (a) with the expression level of the HMGB1 protein or the gene encoding it in a control sample.

[0014] Another object of the present invention is to provide a method for improving or preventing aging, comprising the steps of: (i) analyzing the expression level of HMGB1 protein or a gene encoding the same in a biological sample obtained from a subject before treating the subject with a candidate drug for improving or preventing aging;

[0015] (ii) a step of analyzing the expression level of the biological HMGB1 protein or the gene encoding it obtained from the subject after treatment with the candidate drug; and

[0016] (iii) A method for selecting a candidate drug for improving or preventing aging is provided, comprising a step of screening a candidate drug by comparing the expression level of the HMGB1 protein or the gene encoding it analyzed in step (i) with the expression level of the HMGB1 protein or the gene encoding it analyzed in step (ii).

[0017] Another object of the present invention is to provide a method for predicting or monitoring an aging treatment response, comprising a step of inhibiting the expression of HMGB1 protein or a gene encoding the same.

[0018] Another object of the present invention is to provide a food composition for improving or preventing aging, comprising an HMGB1 inhibitor.

[0019] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating age-related diseases, comprising an HMGB1 inhibitor.

[0020] Another object of the present invention is to provide a method for producing an aging model, comprising a step of treating isolated cells with reduced HMGB1.

[0021] Another object of the present invention is to provide a method for diagnosing aging, comprising the steps of (a) analyzing the expression level of HMGB1 from a biological sample; and (b) comparing the expression level of HMGB1 analyzed in step (a) with the expression level of HMGB1 in a control sample.

[0022] Another object of the present invention is to provide a method for inducing senescence, comprising the step of treating a biological sample with a composition comprising HMGB1.

[0023] Another object of the present invention is to provide a method for improving or preventing aging or preventing or treating an aging-related disease, comprising administering to a subject in need thereof a composition comprising an HMGB1 inhibitor in an amount effective for improving or preventing aging or preventing or treating an aging-related disease.

[0024] Another object of the present invention is to provide a use of a composition comprising HMGB1 for diagnosing or inducing aging.

[0025] Another object of the present invention is to provide a use of a composition comprising an HMGB1 inhibitor for improving or preventing aging; or preventing or treating aging-related diseases.

[0026] Another object of the present invention is to provide a use of a composition comprising an HMGB1 inhibitor for the manufacture of a medicament for improving or preventing aging or for preventing or treating aging-related diseases.

[0027] The technical problems that the invention disclosed in this specification seeks to achieve in accordance with the technical idea are not limited to problems for solving the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0028]

[0029] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of this application. Furthermore, the scope of this application is not limited by the specific descriptions described below.

[0030] As one aspect for achieving the above purpose, the present invention provides a biomarker composition for diagnosing or inducing aging, comprising HMGB1.

[0031] In the present invention, the term “HMGB1 (High mobility group box 1)” means a multifunctional protein that interacts with nucleosomes, transcription factors, and histones in the nucleus, and binds to DNA to affect gene expression, replication, and repair processes.

[0032] In the present invention, the HMGB1 functions within the cell nucleus under normal physiological conditions, but is secreted outside the cell due to cell damage or stress, and is involved in various pathological processes such as inflammatory response, immune response, tissue regeneration, tumor microenvironment formation, and aging progression.

[0033] In the present invention, the HMGB1 is a redox-sensitive protein, and can be classified into oxidized HMGB1 or reduced HMGB1 depending on the oxidation state of the cysteine ​​residue.

[0034] In an embodiment of the present invention, it was confirmed that the expression level of reduced HMGB1 protein or a gene encoding the same increased in aging cells, but that there was no significant change in oxidized HMGB1.

[0035] In the present invention, the expression of HMGB1 can be observed in various cell types in vivo, and specifically, may be fibroblasts, epithelial cells, hepatocytes, or skeletal muscle cells, but is not limited thereto.

[0036] In the present invention, the term “senescence” may mean cellular senescence or senescence at the tissue / organ level, and senescence means a physiological process including a decline in biological function.

[0037] In the present invention, the aging may be primary aging or secondary aging.

[0038] In the present invention, the term “secondary senescence” means inducing or spreading a senescent state in surrounding cells by a senescence-associated secretory phenotype (SASP) or senescence-related marker secreted from damaged or stressed cells, and is also called paracrine senescence.

[0039] In the present invention, the reduced HMGB1 may induce senescence of surrounding cells by increasing the expression of a senescence-associated secretory phenotype (SASP) or senescence-associated marker.

[0040] In the present invention, the term “secretome” refers to the totality of biological factors such as proteins, peptides, lipids, metabolites, exosomes, and micro RNAs secreted by cells to the outside, and the composition and function of the secretome may change depending on the physiological state of the cell or environmental stimuli.

[0041] In the present invention, the secretome may include a senescence-associated secretory phenotype (SASP) or an aging-associated factor.

[0042] In the present invention, the term “senescence-associated secretory phenotype (SASP)” refers to cytokines, chemokines, growth factors, matrix metalloproteinases (MMPs) and other inflammatory factors that are abnormally secreted by cells that have entered a senescent state, and the SASP may modify the microenvironment of senescent cells and exert various biological effects on adjacent normal cells, such as inducing senescence, inducing an inflammatory response, tissue restructuring, and promoting or suppressing tumors.

[0043] In the present invention, the factors included in the SASP or SASP regulation may include at least one selected from the group consisting of IL6, IL1β, IL8, TNF-α, CXCL1, CXCL10, CCL2, SERPINE1, VCAM1, ICAM1, IGFBP5, TGFB1, PDGFRB, MMP2, MMP3, IL15, and GSK3A, and their expression may be increased at both the gene level and the protein level.

[0044] In the present invention, reduced HMGB1 released extracellularly acts as a key mediator involved in the induction and amplification of SASP, thereby inducing secondary senescence of surrounding cells and mediating paracrine senescence.

[0045] In the present invention, the term "aging-related marker" means a physiological or molecular biological indicator indicating that a cell or tissue has entered a state of aging, and refers to various types of biomarkers such as genes, proteins, enzyme activity, or fluorescent signals that can be utilized for diagnosing whether aging has been induced, determining the stage of aging progression, or evaluating the effect of inhibiting aging.

[0046] In the present invention, the aging-related marker may be one or more selected from the group consisting of p16, p15, p21, p53, and SA-β-gal, but is not limited thereto.

[0047] In the present invention, the HMGB1 may spread secondary aging by activating the PI3K-AKT / NF-KB, JAK / STAT pathway, or TNF signal pathway.

[0048] In the present invention, the HMGB1 may induce one or more symptoms selected from the group consisting of decreased muscle function, systemic inflammatory response, and decreased cell repair ability, and the symptoms may be alleviated by suppressing the expression of HMGB1 in the body.

[0049] In the present invention, the term “diagnosis” means determining or predicting the possibility of occurrence, degree of progression, or prognosis of an aging state or a disease related to aging by measuring the presence or absence, quantitative change, expression level, etc. of a specific biomarker from a biological sample.

[0050] The present invention includes diagnosing an aging state by measuring the expression level of blood HMGB1, particularly reduced HMGB1 protein, or a gene encoding the same.

[0051] In the present invention, the term “biomarker” refers to a biological substance that can be used as an indicator of a disease or physiological state, and may include a protein, nucleic acid, metabolite, or other physiological indicator.

[0052] As another aspect for achieving the above purpose, the present invention comprises the steps of (a) analyzing the expression level of HMGB1 protein or a gene encoding the same from a biological sample; and

[0053] (b) Provided is a method for providing information for diagnosing aging, comprising a step of comparing the expression level of the HMGB1 protein or the gene encoding it analyzed in step (a) with the expression level of the HMGB1 protein or the gene encoding it in a control sample.

[0054] The above “HMGB1”, “aging” and “diagnosis” are as described above.

[0055] In the present invention, the term “biological sample” means any material, biological fluid, tissue or cell obtained from or derived from an individual, preferably tissue, cell, blood, serum or plasma, and for example, whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, It may be a nipple aspirate, a bronchial aspirate, a synovial fluid, a joint aspirate, organ secretions, cells, a cell extract, or a cerebrospinal fluid.

[0056] In the present invention, as a result of comparing the expression level of the HMGB1 protein or the gene encoding it analyzed in step (a) with the expression level of the HMGB1 protein or the gene encoding it in a control sample, a sample having a high expression level of the HMGB1 protein or the gene encoding it may be diagnosed as aged, and the aging may be secondary aging.

[0057] In another aspect for achieving the above object, the present invention provides a method for improving or preventing aging, comprising: (i) analyzing the expression level of HMGB1 protein or a gene encoding the same in a biological sample obtained from a subject before treating the subject with a candidate drug for improving or preventing aging;

[0058] (ii) a step of analyzing the expression level of the biological HMGB1 protein or the gene encoding it obtained from the subject after treatment with the candidate drug; and

[0059] (iii) A method for selecting a candidate drug for improving or preventing aging is provided, comprising a step of screening a candidate drug by comparing the expression level of the HMGB1 protein or the gene encoding it analyzed in step (i) with the expression level of the HMGB1 protein or the gene encoding it analyzed in step (ii).

[0060] The above “HMGB1”, “biological sample” and “aging” are as described above.

[0061] In the present invention, the term "candidate drug" may be a new drug or a candidate substance for a drug, and includes any substance, molecule, element, compound, entity, or a combination thereof. Specifically, it may include proteins, polypeptides, small organic molecules, polysaccharides, polynucleotides, etc., and may also be a natural product, a synthetic compound, or a combination of two or more substances, but is not limited thereto.

[0062] In the present invention, the candidate drug may be for preventing, improving, or treating aging symptoms or aging-related diseases.

[0063] In the present invention, the term “improvement” means any act that improves or benefits aging symptoms or aging-related diseases.

[0064] In the present invention, the term “prevention” means any act of inhibiting or delaying aging or aging-related diseases.

[0065] As another aspect for achieving the above object, the present invention provides a method for predicting or monitoring an aging treatment response, comprising a step of inhibiting the expression of HMGB1 protein or a gene encoding the same.

[0066] The above “HMGB1”, “aging” and “diagnosis” are as described above.

[0067] In the present invention, the term "treatment" means any action that improves, alleviates, or benefits aging symptoms or aging-related diseases.

[0068] As another aspect for achieving the above purpose, the present invention provides a food composition for improving or preventing aging, comprising an HMGB1 inhibitor.

[0069] The above “HMGB1”, “aging”, “improvement” and “prevention” are as described above.

[0070] In the present invention, the HMGB1 inhibitor may be, but is not limited to, an HMGB1 antibody or compound.

[0071] The food composition of the present invention may contain the HMGB1 inhibitor in various amounts as long as it has the effect of improving or preventing aging.

[0072] The term "food" in the present invention includes dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, nutritional supplements, functional foods, food additives, health functional foods, and health foods, and includes all foods in the conventional sense.

[0073] The above-mentioned "health food" refers to foods that have a more active health maintenance or promotion effect than regular foods, while "health supplement food" refers to foods intended for health supplementation. In some cases, the terms "health functional food," "health food," and "health supplement food" are used interchangeably.

[0074] The term "health functional food" in the present invention is the same as food for special health use (FoSHU), and refers to a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition. Here, "function" means regulating nutrients for the structure and function of the human body or obtaining a useful effect for health purposes such as physiological action. The food of the present invention can be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. In addition, the formulation of the food can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term administration of drugs by using food as a raw material, and is highly portable, so the food of the present invention can be consumed as a supplement for preventing, suppressing, or improving vascular aging or cardiovascular disease.

[0075] Specifically, the above health functional food means a food product manufactured by adding an HMGB1 inhibitor to food materials such as beverages, teas, spices, gums, and confectionery, or in the form of pills, tablets, capsules, powders, and suspensions, and which has a specific health effect when consumed. Unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs made from food.

[0076] The food composition of the present invention is very useful because it can be consumed on a daily basis and can be expected to have an effect of improving or preventing aging.

[0077] The food composition of the present invention can be manufactured in any form, for example, beverages such as tea, juice, carbonated beverages, and sports beverages; processed dairy products such as milk and yogurt; foods such as gums, rice cakes, Korean traditional sweets, bread, confectionery, and noodles; and preparations such as tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jellies, and bars. In addition, the food composition of the present invention can have any product classification as long as it complies with the laws and regulations in effect at the time of manufacturing and distribution in terms of legal and functional classification. For example, it can be a health functional food according to the Korean 'Act on Health Functional Foods', or confectionery, tea, beverages, special-purpose foods, etc. according to each food type according to the Food Codex of the Korean 'Food Sanitation Act' (Food and Drug Safety Notice 'Standards and Specifications of Foods').

[0078] Additionally, the food composition of the present invention may include food additives in addition to its active ingredients. Food additives are generally understood as substances added to, mixed with, or infiltrated into foods during the manufacturing, processing, or preservation of foods. Since these additives are consumed daily and over long periods of time, their safety must be guaranteed. These food additives are categorized by their intended use into sweeteners, flavor enhancers, preservatives, emulsifiers, and flavoring agents.

[0079] The above sweeteners are used to impart an appropriate sweetness to foods, and may be natural or synthetic. For example, sweeteners such as neotame, lactitol, D-ribose, mannitol, D-maltitol, and sodium saccharin may be used.

[0080] The above flavor enhancers are food additives that enhance the taste and aroma of food. Both natural and synthetic flavor enhancers can be used. For example, flavor enhancers such as 5'-disodium guanylate, L-glutamic acid, glycine, and betaine can be used.

[0081] As the above preservatives, sodium metabisulfite, sulfurous anhydride, sorbic acid, benzoic acid, grapefruit seed extract, etc. can be used.

[0082] The above emulsifier is a food additive that homogeneously mixes or maintains two or more immiscible phases, such as water and oil, and sodium gluconate, glycerin fatty acid ester, lecithin, magnesium stearate, alginic acid, etc. can be used.

[0083] The above flavoring agent is a food additive that provides a unique flavor to food or reinforces the original flavor of food lost during the manufacturing process, and ethyl vanillin, ethyl octanoate, linalyl acetate, allyl caproate, paramethylacetophenone, etc. can be used.

[0084] In addition to the food additives described above, the food composition of the present invention may contain physiologically active substances or minerals known in the art and guaranteed to be safe as food additives for the purpose of supplementing and reinforcing functionality and nutrition. Examples of the physiologically active substances include catechins contained in green tea, etc., vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoylthiamine, etc., and examples of the minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferrous citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, zinc, etc.

[0085] The food composition of the present invention may contain the aforementioned food additives in an appropriate amount that can achieve the purpose depending on the product type, and with respect to other food additives that may be contained in the food composition of the present invention, reference may be made to the food codes or food additive codes of each country.

[0086] As another aspect for achieving the above purpose, the present invention provides a pharmaceutical composition for preventing or treating an aging-related disease, comprising an HMGB1 inhibitor.

[0087] The above “HMGB1”, “aging”, “prevention” and “treatment” are as described above.

[0088] In the present invention, the term “age-related disease” is not limited to anything caused by aging, but specifically may be at least one selected from the group consisting of sarcopenia, muscular dystrophy, muscle fiber regeneration deficiency, muscular dystrophy, osteoarthritis, hypotonia, muscular dystrophy, myasthenia gravis, non-alcoholic fatty liver disease, liver fibrosis, liver cirrhosis, senile liver dysfunction, cardiovascular disease, neurodegenerative disease, skin aging, idiopathic pulmonary fibrosis, pulmonary fibrosis, glaucoma, cataract, and type 2 diabetes, but is not limited thereto.

[0089] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient, or diluent according to a conventional method. Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or dosage form, and specific examples thereof may be found in the pharmacopoeias of each country, including the 'Korean Pharmacopoeia'. Carriers, excipients, and diluents that may be included in the composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, carriers, excipients, and diluents that may be included in the composition of the present invention may be non-natural carriers, but are not limited thereto.

[0090] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injection solutions according to conventional methods. Specifically, when formulating, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing the above compound with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include withepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin. Specific formulations of pharmaceutical compositions are known in the art, and reference may be made, for example, to Remington's Pharmaceutical Sciences (19th ed., 1995). The above document is considered to be a part of the present specification.

[0091] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects. The effective dosage level may be determined based on factors including the patient's health condition, type and severity of the disease, drug activity and sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, combination or concurrent drug use, and other factors well known in the medical field. The dosage and frequency of administration do not limit the scope of the present invention in any way.

[0092] The pharmaceutical composition of the present invention can be administered to mammals, including rats, dogs, cats, cows, horses, pigs, and humans, via various routes, preferably to humans. Any route of administration is conceivable, including, but not limited to, oral, intravenous, intramuscular, or subcutaneous injection.

[0093] As another aspect for achieving the above object, the present invention provides a method for producing an aging model, comprising a step of treating isolated cells with reduced HMGB1.

[0094] The above “HMGB1” and “aging” are as described above.

[0095] In the present invention, the term "model" may mean an animal model, and animal models include mice, hamsters, rats, guinea pigs, monkeys, dogs, cats, rabbits, cows, sheep, pigs, and goats, and the meaning of animal models is due to the physiological or genetic similarities between humans and animals. In particular, in disease research, biomedical disease animal models provide research materials for various causes, pathogenesis, and diagnosis of diseases, and through research on disease model animals, genes related to diseases can be identified, interactions between genes can be understood, and basic data can be obtained for determining the possibility of practical application through actual efficacy and toxicity tests of developed new drug candidates.

[0096] In the present invention, the separated cells are not particularly limited, and specifically may be at least one selected from the group consisting of skin cells, epithelial cells, epidermal cells, dermal fibroblasts, hepatocytes, kidney cells, lung cells, muscle cells, blood-brain barrier endothelial cells, cancer cells, blood cells, cervical cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells, neural stem cells, T cells, B cells, natural killer cells, macrophages, monocytes, microglia, neurons, glial cells, astrocytes, brain cells, and laryngeal cells, and more specifically, may be skin cells, epithelial cells, liver cells, kidney cells, lung cells, or muscle cells, but are not limited thereto.

[0097] As another aspect for achieving the above object, the present invention provides an aging model manufactured by the above manufacturing method.

[0098] The above “aging” etc. are as described above.

[0099] In another aspect for achieving the above object, the present invention provides a method for diagnosing aging, comprising: (a) analyzing the expression level of HMGB1 from a biological sample; and (b) comparing the expression level of HMGB1 analyzed in step (a) with the expression level of HMGB1 in a control sample.

[0100] The above “biological sample”, “HMGB1”, “aging” and “diagnosis” are as described above.

[0101] As another aspect for achieving the above object, the present invention provides a method for inducing aging, comprising the step of treating a biological sample with a composition containing HMGB1.

[0102] The above “biological sample”, “HMGB1” and “aging” are as described above.

[0103] As another aspect for achieving the above object, the present invention provides a method for improving or preventing aging or preventing or treating an aging-related disease, which comprises administering a composition comprising an HMGB1 inhibitor to a subject in need thereof in an amount effective for improving or preventing aging or preventing or treating an aging-related disease.

[0104] The above “biological sample”, “HMGB1”, “aging”, “improvement”, “prevention”, “treatment” and “aging-related disease” are as described above.

[0105] In the present invention, “effective amount” means an amount sufficient to achieve the effect of improving or preventing aging, or preventing or treating aging-related diseases, of the composition of the present invention described above.

[0106] The term "subject" as used herein includes, but is not limited to, a human, monkey, cow, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit or guinea pig, and preferably may be a mammal.

[0107] As used herein, the term “administration” means providing a given substance to a subject by any suitable method.

[0108] The composition of the present invention may be administered orally or parenterally via any conventional route capable of reaching the target tissue. Furthermore, the composition of the present invention may be administered using any device capable of delivering the active ingredient to target cells, tissues, or organs.

[0109] As another aspect for achieving the above object, the present invention provides the use of a composition containing HMGB1 for diagnosing or inducing aging.

[0110] The above “aging,” “diagnosis,” and “HMGB1” are as described above.

[0111] As another aspect for achieving the above object, the present invention provides a use of a composition comprising an HMGB1 inhibitor for improving or preventing aging, or preventing or treating aging-related diseases.

[0112] The above “aging”, “improvement”, “prevention”, “treatment” and “aging-related diseases” and “HMGB1”, “etc. are as described above.

[0113] As another aspect for achieving the above object, the present invention provides a use of a composition comprising an HMGB1 inhibitor for the manufacture of a drug for improving or preventing aging, or for preventing or treating aging-related diseases.

[0114] The above “aging”, “improvement”, “prevention”, “treatment” and “aging-related diseases” and “HMGB1”, “etc. are as described above.

[0115] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0116]

[0117] The biomarker composition for diagnosing or inducing aging, comprising HMGB1, particularly reduced HMGB1, of the present invention enables the diagnosis of secondary senescence that is more sensitive and specific than conventional general aging markers, and provides a tool for screening candidates for aging therapeutic agents and predicting and monitoring aging treatment responses. Furthermore, a food composition or pharmaceutical composition comprising an HMGB1 inhibitor as an active ingredient has the advantage of being effectively utilized for the prevention or treatment of aging-related diseases such as sarcopenia and systemic inflammation.

[0118]

[0119] Figure 1 shows the process of producing reduced HMGB1 (A: Western blotting and SDS-PAGE of reduced HMGB1 protein (ReHMGB1), B: Endotoxin Assay analysis).

[0120] Figure 2 confirms the effect of reducing the senescence phenotype according to HMGB1 antibody treatment in mouse fibroblasts (A: analysis of luminescent signals derived from p16-expressing senescent cells, B: analysis of p16^INK4a, p21, IL6, and Mmp3 gene expression).

[0121] Figure 3 confirms the paracrine aging-inducing effect of HMGB1 (A: schematic diagram of experimental design, B and C: evaluation of cell proliferation rate using WST-1 assay, D: analysis of p16 gene expression level).

[0122] Figure 4 confirms the inhibitory effect of paracrine aging according to HMGB1 antibody treatment (Figure 4a: schematic diagram of experimental design, Figure 4b: immunofluorescence staining results, Figure 4c: SA-β-gal positivity ratio, Figure 4d: EdU positivity ratio, Figure 4e: HMGB1 fluorescence intensity in the nucleus, Figures 4f and 4g: ELISA quantification of IL6 and TNF-α concentrations, Figure 4h: RT-qPCR analysis of changes in gene expression levels in fibroblasts according to HMGB1 antibody treatment, Figure 4i: Western blot analysis results).

[0123] Figure 5 shows the induction of senescence phenotype in fibroblasts according to the redox status of HMGB1 (Figure 5a: schematic diagram of experimental design, Figure 5b: results of WST-1 analysis, Figure 5c: results of SA-β-gal activity, EdU activity, and p21 immunofluorescence staining, Figures 5d to 5f: graphs showing the quantification of the results of Figure 5c, Figure 5g: analysis of RNA expression levels in fibroblasts according to treatment with HMGB1 in a redox status).

[0124] Figure 6 shows the results of confirming the aging-inducing effect of ReHMGB1 according to cell type (A: cell coverage analysis of BJ foreskin fibroblasts, renal epithelial cells, and human skeletal muscle cells, B and C: SA-β-gal and EdU staining).

[0125] Figure 7 shows the transcriptome changes in fibroblasts according to the redox status of HMGB1 (Figure 7a: PCA plot results, Figure 7b: RNA sequencing results after treating fibroblasts with PBS (vehicle) or HMGB1 in redox state, Figure 7c: Heatmap of gene signatures related to senescence and cell cycle arrest that were significantly changed in RNA-seq data, Figure 7d: Venn diagram of up- and down-regulated genes in ReHMGB1 and OxHMGB1 treatment groups, Figure 7e: Representative heatmap showing top 10 GO terms significantly enriched in GO-Biological Process and GO-Molecular Function, Figure 7f: Representative heatmap showing significantly enriched KEGG pathways).

[0126] Figure 8 shows the protein expression levels according to the treatment with redox HMGB1 (A to C: Measurement of expression of senescence markers and paracrine senescence-related factors by Western blotting, D: Schematic of paracrine factor validation experiment using a co-culture system, E: RT-qPCR in co-cultured fibroblasts).

[0127] Figure 9 shows the analysis of the effect of ReHMGB1 treatment on the induction of senescent cell secretome (A: Venn diagram visualizing the overlapping results comparing DEGs, B: heatmap showing the expression pattern of cell senescence-related signatures, C: GO and KEGG pathway analysis results).

[0128] Figure 10 shows the regulatory network of ReHMGB1-induced senescent fibroblasts (Figure 10a: interaction network for up-regulated DEGs based on STRING database, Figure 10b: interaction network of 380 DEGs down-regulated in ReHMGB1 treatment group based on STRING, Figure 10c: representative graph calculated by GSEA (gene set enrichment analysis), Figure 10d: heatmap showing DESeq2 normalized counts (row Z-score) of selected transcription factors (TFs) in color, Figures 10e-f: Western blot analysis results).

[0129] Figure 11 confirms inhibition of ReHMGB1-induced senescence by RAGE / JAK2 inhibition (A: Cell density measured before ReHMGB1 treatment, B: Cell density measured under JAK inhibitor (JAKi) or DMSO pretreatment conditions, C: SA-β-gal activity (blue cytoplasmic staining), EdU activity (green), DAPI (white) staining according to treatment with RAGEa (100 nM) or JAKi (0.3 μM), D: Quantification of the ratio of SA-β-gal positive cells among total cells stained with DAPI, E: Quantification of the EdU incorporation ratio relative to the total number of cells).

[0130] Figure 12 shows age-dependent changes in HMGB1 in mouse serum (A: Immunoblotting of HMGB1 expression, B: Schematic of experimental design analyzing reduced HMGB1 status in serum, C: Pull-down assay performed and loading uniformity confirmed by Ponceau S staining).

[0131] Figure 13 shows age-dependent changes in HMGB1 in human serum (A: immunoblotting of HMGB1 expression, B: reduced HMGB1 pull-down assay).

[0132] Figure 14 shows the results of analyzing the promotion of aging in various tissues according to ReHMGB1 injection (Figure 14a: experimental design, Figure 14b: quantification of IL6, IL1β, and TNF-α concentrations in plasma and serum using a multiplex ELISA kit, Figure 14c: measurement of p21(Cdkn1a) expression by RT-qPCR, Figure 14d: analysis by RT-qPCR, Figure 14e: images of TA muscle in paraffin sections stained with laminin, analysis of HMGB1 loss in the nucleus, and p16^INK4a / p21 expression, Figure 14f: ReHMGB1 pull-down assay in serum after intravenous injection of ReHMGB1).

[0133] Figure 15 confirms that HMGB1 promotes cell aging and functional decline during muscle damage (Figure 15a: schematic diagram of the in vivo experiment, Figure 15b: morphological images of injured TA muscle and uninjured muscle (control), Figure 15c: HMGB1 concentration quantified by ELISA, Figure 15d: images from TA muscle sections, Figure 15e: quantification of SA-β-gal activity, Figure 15f: analysis of muscle cross-sectional area (CSA) of mice before and after injury, Figure 15g: measurement of distribution of regenerated muscle fibers in TA muscle 5 days after injury, Figure 15h: quantification of expression of myogenic marker MyoD, Figure 15i: quantification of number of p21-stained cells, Figure 15j: measurement of physical function).

[0134] Figure 16 shows the age-related HMGB1 gene expression synchrony across human tissues (A: HMGB1 gene expression change rate, B is a graph of HMGB1 expression change according to age).

[0135]

[0136] Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0137]

[0138] [Experimental Methods and Materials]

[0139] 1. Production of reduced HMGB1

[0140] Reduced HMGB1, a major aging-inducing factor of the present invention, was produced. After HMGB1 extraction, an experiment was conducted to confirm the size of the reduced HMGB1. First, recombinant human HMGB1 with six His-tags attached to the N-terminus was produced using SoluBL21™ E. coli (AMSBIO, Abingdon, UK). This protein was sequentially processed and purified through Ni++-NTA agarose (Qiagen, Hilden, Germany) and heparin-Sepharose (Cytiva, Marlborough, MA, USA) chromatography. The purity of the protein was confirmed through Western blotting and Coomassie blue staining (Fig. 1A).

[0141] Subsequently, safety experiments were conducted using an endotoxin test. Endotoxin was removed from purified HMGB1 through Triton X-114 phase separation, and endotoxin levels were confirmed using a biological endotoxin test method (Lonza, Basel, Switzerland). HMGB1 was used when the endotoxin level was confirmed to be 1.0 EU / μg of protein or less (Fig. 1B).

[0142]

[0143] 2. Cell lines and culture systems

[0144] Human fetal lung fibroblasts (WI-38; ATCC® CCL-75™), human foreskin fibroblasts (BJ; ATCC® CRL-2522™, Manassas, VA, USA), and human kidney epithelial cells (ATCC® PCS-400-011™) were obtained from the American Type Culture Collection (ATCC®), and human skeletal muscle cells (SkMC) were supplied by Lonza Bioscience (Basel, Switzerland).

[0145] WI-38 and BJ cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 4.5 g / L glucose (D-glucose, without sodium pyruvate), 100 U / mL penicillin, and 0.1 mg / mL streptomycin in a cell incubator at 37°C and 3% oxygen.

[0146] Renal epithelial cells were cultured in Renal Epithelial Cell Basal Medium (ATCC® PCS-400-030™) and SkMC were cultured in StemLife SK Medium complete kit (LifeLine Cell Technology, LL-0069, California, USA) at 37°C and 5% CO2, respectively.

[0147] After stabilization for 24 hours, cells were treated with ReHMGB1 (20 μg / mL) for 3 days, and senescence indices were analyzed.

[0148] For conditioned media (CM) experiments, WI-38 fibroblasts were seeded in T-175 flasks at 2 Х 10 6After seeding at a cell density of 10, the cells were cultured for 3 days in DMEM medium containing 0.2% FBS to induce a quiescent state, and then irradiated with 20 Gy of X-rays and cultured for 10 days to induce a senescent state. After culturing for 96 hours in fresh medium, the CM was collected, centrifuged at 2000 X g for 10 minutes, and filtered through a 0.22 μm filter. The obtained CM was mixed with DMEM containing 40% FBS at a ratio of 3:1 to prepare a final CM containing 10% FBS.

[0149] WI-38 cells were treated with ReHMGB1 and JAK inhibitor for 3 days, and RAGE antagonist (FPS-ZM1) was pretreated 30 min before ReHMGB1 treatment.

[0150] Momelotinib (CYT387), a JAK inhibitor, was purchased from MedChemExpress (HY-10961), and FPS-ZM1 was purchased from Sigma-Aldrich (St. Louis, MO, USA; 553030).

[0151]

[0152] 3. Mice and Experimental Procedures

[0153] Male C57BL / 6J mice, aged 3 months (young), 15 months (middle-aged), and 24 months (old), were provided by the Jackson Laboratory (Bar Harbor, ME, USA) and the Aging Research Center of the Korea Basic Science Institute (Daejeon, Republic of Korea). Mice were housed in a pathogen-free environment, with free access to food and water. They were maintained under a 12-h light-dark cycle (photoperiod 6:00–18:00) and a temperature of 20–22°C.

[0154] All experiments were conducted with approval from the Institutional Animal Care and Use Committee of Korea University College of Medicine (KOREA-2020-0168 and KOREA-2023-0171). Mice were randomly assigned to control or treatment groups.

[0155] To analyze systemic changes in the aging phenotype following ReHMGB1 administration, 3-month-old male mice were intravenously injected with ReHMGB1 (5 mg / kg, supplied by Yonsei University) or vehicle once at weekly intervals.

[0156] Furthermore, to confirm the in vivo HMGB1 inhibitory effect, purified anti-HMGB1 antibody (3E8 clone mAb, BioLegend; 651402) was intravenously injected (iv, 0.1 mg / kg) into 15-month-old middle-aged male mice. After 24 h, 50 μL of 1.2% barium chloride (BaCl2) solution was injected into the tibialis anterior (TA) muscle of anesthetized mice to induce unilateral muscle damage (Sigma-Aldrich; 202738).

[0157]

[0158] 4. Isolation of mouse tissue and serum

[0159] Tissues were dissected postmortem, and muscle tissues were either flash frozen in dry ice-cooled isopentane or fixed overnight in paraformaldehyde. Samples were embedded in OCT compound (Sakura Finetek; 4583) or paraffin.

[0160] Tissues for RNA extraction were immediately stored in liquid nitrogen and then stored at -80°C. Serum was isolated by collecting blood in 1.5 mL Eppendorf tubes, allowing it to coagulate at room temperature, and centrifuging at 2,000 Х g for 15-30 minutes.

[0161]

[0162] 5. Preparation of HMGB1 redox form

[0163] Recombinant human HMGB1 protein with an N-terminal His tag was expressed in SoluBL21™ transformed E. coli (AMSBIO, Abingdon, UK) and prepared according to a previous study. The protein was purified by chromatography on Ni++-NTA agarose (Qiagen, Germany) and heparin-Sepharose columns (Cytiva, USA). The purity of the purified protein was confirmed by Western blotting and Coomassie blue staining.

[0164] Endotoxin was removed using Triton X-114 phase separation, and endotoxin levels were measured using the Limulus amebocyte lysate (LAL) assay (Lonza). HMGB1 was used when the endotoxin level was 1.0 EU / μg or less.

[0165] For in vitro analysis, fully oxidized HMGB1 (OxHMGB1, HMGBiotech, Italy; HM060) without LPS was used.

[0166]

[0167] 6. SA-β-gal and EdU staining

[0168] SA-β-gal staining was performed using the Senescence β-Galactosidase Staining kit (Cell Signaling Technology, USA; #9860) according to the manufacturer's instructions. Tissue sections were counterstained with nuclear fast red for nuclear staining.

[0169] After SA-β-gal staining, EdU detection was performed using the Click-iT EdU Alexa Fluor 488 imaging kit (Invitrogen; #C10337) according to the manufacturer's protocol.

[0170]

[0171] 7. Immunofluorescence staining

[0172] 3-8 Х 10 on a chamber slide (Thermo Scientific, USA) 4 Cells cultured at a density of 10 cells / mL and paraffin-embedded tissue sections were fixed with 4% paraformaldehyde (Biosesang; P2031) and permeabilized with 0.5% Triton X-100 (Biosesang; PR2294-100-74) for 20 minutes at room temperature.

[0173] Tissue sections were subjected to antigen retrieval with pH 6.0 sodium citrate buffer containing Tween-20 (Biosesang; SR2189-050-60) and pretreated with goat anti-mouse Fab fragment antibody (Jackson Immunoresearch; 115-007-003) to block endogenous mouse IgG.

[0174] Samples were blocked with 0.5% Triton X-100 containing 10% normal donkey serum for 1 hour and reacted with primary antibody overnight at 4°C.

[0175] Donkey anti-Rabbit IgG (Invitrogen) conjugated with Alexa Fluor 488 and 594 was used as the secondary antibody, and nuclei were stained with DAPI (EMD Millipore; 90229).

[0176] The stained slides were photographed with a fluorescence microscope (Olympus BX53, Japan), and the primary antibodies for immunofluorescence staining are shown in Table 1 below.

[0177] AntibodiesHost speciesSourceIdentifierDilution factorAnti-HMGB1 antibodyRabbitAbcamCat# ab182561:250p16-INK4A Polyclonal antibodyRabbitProteintechCat# 10883-1-AP1:400p21 Waf1 / Cip1(12D1) AntibodyRabbitCell signalingCat# 2947S1:500p21 Polyclonal AntibodyRabbitInvitrogenCat# PA1-303991:200Laminin α-2 AntibodyRatSanta Cruz BiotechnologyCat# sc-598541:200PCRP-MYOD1-2A5MouseDSHBCat# PCRP-MYOD1-2A51:100Donkey anti-Rabbit IgG(H+L) Highly Cross-Adsorbed Secondary AntibodyDonkeyInvitrogenCat# A-21206Cat# A-212071:500Donkey anti-Mouse IgG(H+L) Highly Cross-Adsorbed Secondary AntibodyDonkeyInvitrogenCat# A-21202Cat# A-212031:500Donkey anti-Rat IgG(H+L) Highly Cross-Adsorbed Secondary AntibodyDonkeyInvitrogenCat# A-212081:200

[0178]

[0179] 8. Real-time quantitative PCR (RT-qPCR)

[0180] Total RNA was isolated using the Direct-zol RNA Miniprep Kit (Zymo Research; R2050) with TRIzol (Ambion; 1596026), and RNA quality was assessed using NanoDrop ND-2000c (Thermo).

[0181] cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems; 4368814), and relative mRNA expression levels were analyzed using a QuantStudio 3 Real-Time PCR System with Power SYBR Green PCR Master Mix (Applied Biosystems; 4368706).

[0182] The β-actin gene was used as an endogenous control (housekeeping gene). Primer sequences for qRT-PCR analysis of human and mouse genes are shown in Table 2.

[0183] OligonucleotidesSequenceSpecies서열번호ACTB-F5′-GCT CCT GAG CGC AAG TAC-3′H1ACTB-R5′-GGA CTC GTC ATA CTC CTG CTT GC-3′H2CDKN1A(p21)-F5′-TGT CCG TCA GAA CCC ATG C-3′H3CDKN1A(p21)-R5′-AAA GTC GAA GTT CCA TCG CTC-3′H4CDKN2A(p16INK4a)-F5′-CCA ACG CAC CGA ATA GTT ACG-3′H5CDKN2A(p16INK4a)-R5′-GCG CTG CCC ATC ATC ATG-3′H6C-C motif chemokine ligand 2(CCL2)-F5’-ATC AAT GCC CCA GTC ACC TG-3’H7C-C motif chemokine ligand 2(CCL2)-R5’-TCT CCT TGG CCA CAA TGG TC-3’H8CXC motif chemokine ligand 1(CXCL1)-F5’-GCT GAA CAG TGA CAA ATC CAA C-3’H9CXC motif chemokine ligand 1(CXCL1)-R5’-CTT CAG GAA CAG CCA CCA GT-3’H10Glycogen synthase kinase 3 alpha(GSK3A)-F5’-GGC TCA TTT GGG GTC GTG TA-3’H11Glycogen synthase kinase 3 alpha(GSK3A)-R5’-CAG TGG TCC AGC TTA CGC AT-3’H12Insulin-like growth factor 1 receptor(IGF1R)-F5’-GTG GGG GCT CGT GTT TCT C-3’H13Insulin-like growth factor 1 receptor(IGF1R)-R5’-ACC TGA GAT GAG ACA GGA GTC-3’H14Interleukin 6(IL6)-F5′-CCC CTG ACC CAA CCA CAA AT-3′H15Interleukin 6(IL6)-R5′-ATT TGCCGA AGA GCC CTC AG-3′H16Interleukin 8(IL8)-F5’-ACT GAG AGT GAT TGA GAG TGG AC-3’H17Interleukin 8(IL8)-R5’-AAC CCT CTG CAC CCA GTT TTC-3’H18Interleukin 15(IL15)-F5’-GCT ATG CTG GTA GGC TCC TG-3’H19Interleukin 15(IL15)-R5’-CCG TGA TTG GAC AAG CTT GC-3’H20Matrix Metallopeptidase 2(MMP13)-F5’-CGA TGG ATA CCC CTT TGA CGG-3’H21Matrix Metallopeptidase 2(MMP13)-R5’-CCA TAC TTC ACA CGG ACC ACT TG-3’H22Matrix Metallopeptidase 13(MMP13)-F5’-TGG TCC AGG AGA TGA AGA CC-3’H23Matrix Metallopeptidase 13(MMP13)-R5’-TCC TGG GAG ACT GGT AAT GG-3’H24Plasminogen activator inhibitor 1(PAI1; Serpine1)-F5’-AAG GCA CCT CTG AGA ACT TCA-3’H25Plasminogen activator inhibitor 1(PAI1; Serpine1)-R5'-GTG GAC GGA TCA GGA CCC-3'H26Platelet derived growth factor receptor beta(PDGFRB)-F5’-GTT CAA AGA CAA CCG CAC CC-3’H27Platelet derived growth factor receptor beta(PDGFRB)-R5’-GTC TGT TCC CCA CTG TCA GG-3’H28Transforming growth factor beta 1(TGFB1)-F5’-CCA CCA TCA CCA ACA TCC AG-3’H29Transforming growth factor beta1(TGFB1)-R5’-GCC GTT ACC TTC AAG CAT CGT G-3’H30Tumor necrosis factor alpha(TNF-α)-F5’-CCT CTC TCC CCT GGA AAG GA-3’H31Tumor necrosis factor alpha(TNF-α)-R5’-CA CGA ACA AGG AGT CGG AGA-3’H32TP53(p53)-F5’-GTA GCT GGG ACC ACA GGT TC-3’H33TP53(p53)-R5’-CC AGA GTT TGA GGA CCC GAG-3’H34Vascular cell adhesion protein 1(Vcam1)-F5’-GGG AAG CCG ATC ACA GTC AA-3’H35Vascular cell adhesion protein 1(Vcam1)-R5’-CC TAC GTC TGT CCT TCA GGG-3’H36Actin-F5’-CTA AGG CCA ACC GTG AAA AG-3’M37Actin-R5’-AC AGG GAC ATA CGG AGA CCA-3’M38Cdkn1a(p21)-F5’-TCC ACA GCG ATA TCC AGA CA-3’M39Cdkn1a(p21)-R5’-CA GGT TAG GAC CAC TAC AGG-3’M40Cdkn2a(p16INK4a)-F5’-TCC TCG CAG TTC GAA TCT G-3’M41Cdkn2a(p16INK4a)-R5’-C CCA TGC TGG CTT TCT CAA-3’M42Cdkn2b(p15INK4b)-F5’-AGA TCC CAA CGC CCT GAA-3’M43Cdkn2b(p15INK4b)-R5’-GAA GGA CCT GTG CGA ACA-3’M44C-X-C motif chemokine ligand 10(Cxcl10)-F5’-GGT CTG AGT GGG ACT CAA GGG ATC-3’M45C-X-C motif chemokine ligand 10(Cxcl10)-R5’-CAC AAC TCT AGT AAC GGT GCT ACT-3’M46MatrixMetallopeptidase 13(Mmp13)-F5'-GGA GCC CTG ATG TTT CCC AT-3'M47Matrix Metallopeptidase 13(Mmp13)-R5'-ATA CCA GGT CCG CTA CTT CTG-3'M48TIMP Metallopeptidase inhibitor 1(Timp1)-F5'-CAC ACC AGA GCA GAT ACC ATG A-3'M49TIMP Metallopeptidase inhibitor 1(Timp1)-R5'-CCG ATT TAA GTA CCC AAG GGG-3'M50Tumor necrosis factor alpha(Tnf-α)-F5'-GTC CAT TCC TGA GTT CTG-3'M51Tumor necrosis factor alpha(Tnf-α)-R5'-GGA TGG AAG TCT GGA AAG-3'M52

[0184]

[0185] 9. Cell proliferation assay

[0186] Cell proliferation was performed using the WST-1 assay (TAKARA, Japan; MK400) according to the manufacturer's instructions. Absorbance was measured at 450 nm using a SpectraMax i3x multimode microplate reader (Molecular Devices, USA).

[0187] Additionally, cell density (confluence) was analyzed using the Incucyte® SX1 Live-Cell Imaging System.

[0188]

[0189] 10. RNA sequencing and differentially expressed gene analysis

[0190] mRNA was extracted using the Poly(A) RNA Selection Kit (Lexogen, Austria), and cDNA was synthesized and sheared according to the manufacturer's protocol.

[0191] The library was indexed with Illumina index 1-12, and quality was checked with TapeStation HS D1000 Screen Tape (Agilent Technologies, Netherlands).

[0192] Quantification was performed using a library quantification kit using the StepOne Real-Time PCR System (Life Technologies, USA). Sequencing was performed in a 100-bp paired-end manner on the NovaSeq 6000 (Illumina) platform. Quality control was performed using FastQC, and low-quality reads and adapters were removed using Cutadapt (v3.5).

[0193] The data were aligned to GRCh38 using STAR (v2.7.8a), and gene counts were extracted using HTSeq (v2.0.2). Differentially expressed genes (DEGs) were analyzed using DESeq2 (v1.38.3), and genes with adjusted p-value < 0.05 and |log2-fold change| > 1 were considered significant.

[0194] Gene symbols were matched to Ensembl IDs, and aging-related DEGs were identified using the CellAge database and GSEA resources. Normalized expression values ​​were visualized using the pheatmap function in R.

[0195]

[0196] 11. Functional pathway enrichment analysis

[0197] Significant DEGs were analyzed using DAVID and clusterProfiler (v4.0.5, R package) for Gene Ontology (GO) and KEGG pathway enrichment analysis.

[0198] The significance of the path was set to p-adj < 0.05 using the Benjamini-Hochberg method.

[0199] Molecular network analysis was performed using the STRING database (v11.5), with a confidence score set to >0.7. Visualization was performed using Cytoscape (v3.6.1), and the top 20 core hub genes were identified using cytoHubba.

[0200] Additionally, gene clusters with active interactions were detected using the MCODE algorithm (Node score cutoff = 0.2, K-core, degree cutoff = 2), and transcription factor prediction was performed using the TRRUST database (https: / www.grnpedia.org / trrust / ).

[0201]

[0202] 12. Western blotting

[0203] Cells were lysed in RIPA buffer (Pierce; 89901) supplemented with a protease / phosphatase inhibitor cocktail (Thermo; 78442). Protein concentration was measured using a BCA assay (Pierce; A55861) at 562 nm with a SpectraMax i3x reader.

[0204] Samples were prepared with 4X Bolt LDS buffer (Invitrogen; B0007), heated at 80°C for 5 minutes, centrifuged, loaded onto Bolt 4-12% Bis-Tris gel (Invitrogen), and transferred to PVDF membrane. The membrane was stained with Ponceau S, blocked with 5% skimmed milk or TBS-T containing BSA, and reacted with primary antibodies overnight at 4°C.

[0205] Subsequently, the cells were reacted with HRP-conjugated secondary antibodies (Cell Signaling Technology; 7074S, 7076S), and signals were detected with SuperSignal™ West Femto substrate (Thermo; 34094). Images were visualized using the Fusion Solo S system (Vilber, France), and band quantification was performed using ImageJ. The list of primary antibodies used for Western blot analysis is shown in Table 3 below.

[0206] AntibodiesHost SpeciesSourceIdentifierDilution factorCDKN2BMouseSanta CruzBiotechnologyCat# sc-2717911:1000E2F8(E8I3L)RabbitCell SignalingCat# 34661T1:1000GAPDH Antibody(0411)MouseSanta Cruz BiotechnologyCat# sc-477241:1000RAGE Antibody(A-9)MouseSanta Cruz BiotechnologyCat# sc-3651541:1000Anti-p21 Waf1 / Cip1RabbitCell SignalingCat# 2947S1:1000p38 MAPK AntibodyRabbitCell SignalingCat# 9211S1:1000p-p38 MAPK Antibody(E-1)MouseSanta Cruz BiotechnologyCat# sc-1661821:1000p53 Antibody(DO-1)MouseSanta Cruz BiotechnologyCat# sc-1261:1000AKT 1 / 2 / 3 Antibody(5C10)MouseSanta Cruz BiotechnologyCat# sc-814341:1000ICAM / CD54(E3Q9N)RabbitCell SignalingCat# 67836T1:1000Phospho-AKT(Ser473)(D9E) AntibodyRabbitCell SignalingCat# 4060T1:1000JAK2(D2E12) XP® AntibodyRabbitCell SignalingCat# 3230S1:1000Phospho-Jak2(Tyr1007 / 1008) AntibodyRabbitCell SignalingCat# 3771S1:1000RRM2(E7Y9L)RabbitCell signalingCat# 65939T1:1000Stat1 Antibody(C-136)MouseSanta Cruz BiotechnologyCat# sc-4641:1000p-Stat1Antibody(A-2)MouseSanta Cruz BiotechnologyCat# sc-83941:1000Stat3 Antibody(F-2)MouseSanta Cruz BiotechnologyCat# sc-80191:1000p-Stat3 Antibody(B-7)MouseSanta Cruz BiotechnologyCat# sc-80591:1000RELA / NFkB p65 Antibody(F-6)MouseSanta Cruz BiotechnologyCat# sc-80081:1000p-RELA / NFkB p65 Antibody(27. Ser 536)MouseSanta Cruz BiotechnologyCat# sc-1365481:1000VCAM1 AntibodyMouseSanta CruzBiotechnologyCat# sc-131601:1000

[0207]

[0208] 13. Enzyme-linked immunosorbent assay (ELISA)

[0209] Cytokine levels were detected using bead-based ELISA kits (R&D systems, Minneapolis, MN, USA; HS600C & HSTA00E, Arigo Biolaboratories, Hsinchu City, Taiwan; ARG82842, Chondrex, Woodinville, WA, USA; 6010) according to the manufacturer's protocol. Cells were trypsinized and counted for normalization. ELISA plates were measured at 450 nm using a SpectraMax i3x reader. The obtained data were used to calculate the cytokine concentrations of the samples using the values ​​obtained from the standards.

[0210]

[0211] 14. Cytokine antibody array assay

[0212] Cell supernatants were collected with lysis beads and buffer (Full Moon Biosystems, Sunnyvale, CA, USA) containing protease / phosphatase inhibitors (Sigma-Aldrich) for protein extraction. Proteins were purified through a gel matrix column, mixed, hydrated, and centrifuged at 750 g for 2 min.

[0213] The purified protein was reacted with a 10 μg / μL biotin / DMF solution, and the antibody microarray slide (Fullmoon Biosystems) was blocked and incubated at room temperature for 2 h at 60 rpm. The protein was then reacted with Cy3-streptavidin (GE Healthcare) and detected at 10 μm resolution using a GenePix 4100A scanner (Axon, Union City, CA, USA). Total protein amount was calculated based on fold change and normalized to cell number.

[0214]

[0215] 15. Pull-down assay

[0216] Mouse serum was incubated with Biotin Polyethylene Oxide Iodoacetamide (biotin-IAM, Sigma; B2059) at 4°C for 18 h, and then incubated with streptavidin agarose beads (Pierce; 20349) for 1 h at 4°C with rotation. After the reaction, the pellet containing reduced HMGB1 was collected by centrifugation.

[0217]

[0218] 16. Behavioral experiments

[0219] Grip strength was measured using a force meter (Bioseb, Pinellas Park, FL, USA), and the average value after 5 repetitions was normalized to body weight.

[0220] The Rotarod test (Ugo Basile, Gemonio, Italy) was performed three times in 300-second, 0-40 rpm acceleration scenarios after 1-hour training. Latency to fall was recorded. Trials exceeding 300 seconds were excluded.

[0221] Treadmill work was performed after two days of training, with subjects running for 5 minutes at a 1% incline, increasing the speed from 5 to 25 m / min. Running was induced using weak electrical stimulation (2 Hz, intensity 0.5), and the point at which the response ceased was recorded as the time to exhaustion.

[0222]

[0223] 17. Quantification and statistical analysis

[0224] All experiments were performed independently at least three times, and the results are presented as the mean ± standard error of the mean (SEM).

[0225] A statistically significant difference was considered when the p value was < 0.05, and the significance level was expressed as follows (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0226] Pairwise comparisons were performed using a two-tailed unpaired t-test with Welch correction, taking into account differences in variance between groups.

[0227] One-way analysis of variance (ANOVA) was used for multiple comparisons, and Dunnett's or Tukey's tests were applied as post hoc analyses. If normality was not satisfied, the Mann-Whitney U test was performed. The Benjamini-Hochberg correction was applied for multiple comparisons.

[0228] Statistical analysis was performed using GraphPad Prism (v10.1.1) and R software (v4.3.3).

[0229]

[0230] Example 1. Confirmation of secondary senescence induction by HMGB1

[0231] 1.1 Confirmation of secondary aging induction in vivo

[0232] To determine whether HMGB1 derived from senescent cells has the ability to induce senescence in surrounding cells (secondary senescence), mouse dermal fibroblasts (MDFs) were treated with sera from young (young serum: 2–4 months old) and aged (old) mice. These sera were pretreated with control IgG or anti-HMGB1 antibody (HMGB1 ab), respectively. The luminescence of p16-3MR-expressing cells and the expression of p16, p21, IL6, and Mmp3 were analyzed.

[0233] As shown in Figure 2A, anti-HMGB1 antibody treatment of MDFs treated with serum from aged mice resulted in a decrease in the luminescence of 3MR-expressing cells. Furthermore, as shown in Figure 2B, significant decreases in the expression of p16, IL6, and Mmp3 were observed. This confirms that HMGB1 contributes to the spread of systemic aging.

[0234]

[0235] 1.2. Confirmation of secondary aging induction in vitro

[0236] To determine whether HMGB1-induced secondary senescence also functions in vitro, WI-38 human lung fibroblasts were treated with conditioned medium (IR-CM) from cells induced by primary senescence by exposure to ionizing radiation. Conditioned medium (Qui-CM) from quiescent cells induced by low serum (0.2% FBS) treatment served as a negative control (Fig. 3A). IR-CM was treated with control IgG (IgG-CM) or anti-HMGB1 antibody (aHM-CM) (Fig. 4A).

[0237] As a result, as shown in Figures 4b to 4d, a strong senescence response was induced in WI-38 cells by IR-CM, with an increase in SA-β-gal activity and a decrease in EdU incorporation. Treatment with aHM-CM also resulted in a decrease in SA-β-gal activity and an increase in EdU incorporation. From this, it was confirmed that the senescence-inducing effect was significantly alleviated by treatment with an anti-HMGB1 antibody.

[0238] In addition, as shown in Figures 3B to 3D, it was confirmed that the proliferation of fibroblasts increased and the expression of p16 decreased with aHM-CM treatment, which was almost at a similar level to Qui-CM.

[0239] In addition, as shown in Fig. 4b (arrows indicate locations where expression was reduced due to secretion of HMGB1 into the nucleus) and Fig. 4e, the localization of HMGB1 into the nucleus was increased in cells treated with aHM-CM, confirming that the phenomenon of HMGB1 loss into the nucleus was suppressed in senescent cells.

[0240] Furthermore, as shown in Figures 4f and 4g, the levels of IL6 and TNF-α in the medium of aHM-CM-treated cells were significantly reduced compared to IgG-CM.

[0241] Additionally, as shown in Figures 4h and 3D, the expression of p16, p21, p53, IL6, and CCL2 decreased with aHM-CM treatment. In particular, it was confirmed that the levels of p21 and p53 proteins were also significantly reduced by HMGB1 inhibition (Figure 4i).

[0242] From this, we confirmed that extracellular HMGB1 plays a key role in the paracrine propagation of the aging phenotype, and that SASP-based aging propagation can be effectively suppressed through inhibition of extracellular HMGB1.

[0243]

[0244] Example 2. Confirmation of the aging-inducing effect of HMGB1 according to its redox state.

[0245] 2.1. Effect of oxidation-reduction status on senescence induction in WI-38 fibroblasts

[0246] To evaluate the senescence-inducing effects of various redox states of HMGB1 in vivo, WI-38 fibroblasts were treated with ReHMGB1 (reduced form) or OxHMGB1 (fully oxidized form) and compared with primary senescent cells induced by ionizing radiation (IR) (Fig. 5a).

[0247] WST-1 analysis results 4 days after treatment showed that the proliferation rate of ReHMGB1-treated cells was significantly reduced, similar to that of IR-treated cells, but there was no significant change in OxHMGB1- or PBS-treated cells (Fig. 5b).

[0248] In addition, as shown in Figures 5c to 4e, ReHMGB1-treated WI-38 fibroblasts showed increased SA-β-gal activity and decreased EdU incorporation. As shown in Figures 5c and 5f, p21 expression also significantly increased in the ReHMGB1-treated group, and as shown in Figure 5g, all of the major aging-regulating factors and major SASP factors, p16, p21, p53, IL6, IL8, CXCL1, SERPINE1, TNF-α, CCL2, VCAM1, and MMP13, were increased, confirming that ReHMGB1-treated cells were in a state of senescent cells.

[0249]

[0250] 2.2. Anti-aging effects according to cell type

[0251] Additionally, to determine whether the effects of ReHMGB1 are limited to specific cell types, experiments were conducted in human foreskin fibroblasts (BJ foreskin fibroblasts), renal epithelial cells, and human skeletal muscle cells.

[0252] As a result, as shown in Figures 6A to 6C, increased SA-β-gal activity and decreased EdU incorporation were observed in all cell types. This confirms that ReHMGB1 extensively induces senescence.

[0253] In summary, we confirmed that extracellular ReHMGB1 induced secondary senescence in various cells, including WI-38, but OxHMGB1 had no effect.

[0254]

[0255] Example 3. Analysis of transcriptome changes and aging-induced characteristics following HMGB1 treatment.

[0256] 3.1. Analysis of aging gene signatures at the transcriptome level

[0257] To identify aging-related transcriptome changes in fibroblasts in response to redox state extracellular HMGB1, RNA sequencing analysis (RNA-seq) was performed on cells treated with ReHMGB1 and OxHMGB1.

[0258] First, the PCA (Principal component analysis) analysis results confirmed that the ReHMGB1, OxHMGB1, and PBS treatment groups formed distinct clusters (Fig. 7a).

[0259] Subsequently, RNA sequencing was performed after treating WI-38 fibroblasts with PBS (vehicle) or HMGB1 at different redox states. The volcano plot shows differentially expressed genes (DEGs) according to HMGB1 redox treatment. Red dots represent upregulated genes, and blue dots represent downregulated genes. Based on |log2FC| ≥ 1 and p-adj < 0.05, a total of 1,087 differentially expressed genes (DEGs) were compared in the ReHMGB1-treated group and 613 DEGs in the OxHMGB1-treated group compared to the PBS control group. Among these, 55 DEGs related to cell senescence and 35 DEGs related to cell cycle regulation were selected to create a heatmap of gene signatures related to senescence and cell cycle regulation.

[0260] As a result, as shown in Fig. 7b and Fig. 7c, the ReHMGB1-treated group (Re-H) showed a strong aging gene signature compared to the OxHMGB1-treated group (Ox-H), and showed an association with inflammatory pathways such as VCAM1, ICAM1, CCL2, CXCL10, ATF3, IGFBP, and RELB, and a decrease in the expression of oxidative stress-related genes RRM2, MYBL2, and FOXM1. In addition, ReHMGB1 significantly regulated cell cycle regulators such as p15^INK4b (CDKN2B) and the regulatory marker E2F8, which is consistent with the observed decrease in proliferation.

[0261] As a result of showing the heatmap of gene signatures related to aging and cell cycle arrest that were significantly changed in the RNA-seq data, as shown in Fig. 7d, 601 genes were upregulated and 486 were downregulated in the ReHMGB1 treatment group, and 392 were upregulated and 221 were downregulated in the OxHMGB1 treatment group. Among these, 295 DEGs overlapped between ReHMGB1 and PBS, and 154 DEGs overlapped between OxHMGB1 and PBS.

[0262] GO (Gene Ontology) and KEGG pathway analyses of these DEGs revealed that the ReHMGB1-treated group was enriched in biological processes related to fibrosis and ossification (GO-BP), such as cell migration, extracellular matrix (ECM) composition, and signal transduction. Molecular function (GO-MF) analysis confirmed a tendency toward inhibition of ATP consumption (Fig. 7e), and KEGG analysis highlighted cytokine-receptor interactions, including the PI3K-AKT, JAK / STAT, and TNF signaling pathways (Fig. 7f).

[0263] In contrast, in OxHMGB1-treated cells, the GO-BP pathway related to interferon activation was mainly activated, and inflammation and aging pathways, as with ReHMGB1, were not activated.

[0264] Additionally, it was confirmed that the TGF-β signaling pathway, which is involved in aging and tissue immune regulation, as well as paracrine aging, was suppressed in the OxHMGB1-treated group.

[0265]

[0266] 3.2. Analysis of aging markers and paracrine aging-related factors at the protein level

[0267] The protein expression levels of aging markers and paracrine aging-related factors were analyzed according to treatment with redox-conjugated HMGB1.

[0268] As a result, changes in the expression of major aging markers were confirmed, as shown in Figures 8A to 8C (Figures 8A to 8C).

[0269] Furthermore, when WI-38 cells were co-cultured, and the results of gene expression analysis (RT-qPCR) as shown in Figures 8D and 8E, SASP factors of IGFBP5, IL15, TGFB1, MMP2, GSK3A, IGF1R, and PDGFRB were significantly increased in recipient cells co-cultured with IR-induced senescent cells, confirming that ReHMGB1 mediates paracrine senescence.

[0270]

[0271] 3.3. Induction of the Secretome in Senescent Cells by ReHMGB1 Treatment

[0272] First, we compared the DEG signatures of ReHMGB1-induced senescence with those of IR-induced senescence (WI-38 fibroblasts, GSE13027).

[0273] As a result, it was confirmed that ReHMGB1-induced senescence showed transcriptomic characteristics very similar to IR-induced senescence, and the OxHMGB1 treatment group showed a large difference (Fig. 9A).

[0274] Additionally, to further investigate the contribution of ReHMGB1 to SASP proliferation, conditioned medium (CM) proteins from ReHMGB1, OxHMGB1, and IR-treated cells were analyzed.

[0275] As a result, the ReHMGB1-treated group was enriched in SASP factors such as IGFBP5, IL15, TGFB1, MMP2, GSK3A, IGF1R, and PDGFRB, which is similar to the secretome of IR-induced senescent cells. In contrast, the secretome of the OxHMGB1-treated group was similar to the PBS control group and was not enriched in SASP (Fig. 9B). In particular, we confirmed that SASP factors commonly present in ReHMGB1- and IR-induced CMs are associated with aging-related biological processes (Fig. 9C).

[0276]

[0277] Example 4. Elucidation of the molecular mechanism of aging induction by ReHMGB1.

[0278] To elucidate the molecular mechanisms of secondary senescence induced by extracellular ReHMGB1, we constructed a protein-protein interaction (PPI) network based on RNA-seq data obtained after treating WI-38 fibroblasts with ReHMGB1 or OxHMGB1.

[0279] We analyzed the interaction network of 496 DEGs upregulated in the ReHMGB1 treatment group based on the STRING database. The network score cutoff value was 0.7, and node color indicates expression change (fold change), and node size indicates the number of interactions (links). In addition, significant network clusters were indicated with colored circles according to the cluster score (5 or higher) calculated using the MCODE algorithm.

[0280] As a result, it was confirmed that ReHMGB1-treated cells formed a strongly connected network of pathways related to cytokine-mediated signaling, inflammatory response, interferon signaling, and extracellular matrix (ECM) organization (Fig. 10a). Key factors in this pathway were identified as IL6, MX1, and ADAMST8.

[0281] Analysis of the interaction network of 380 DEGs downregulated in the STRING-based ReHMGB1 treatment group revealed that, conversely, the PPI network related to cell cycle and DNA replication was significantly reduced in the ReHMGB1 treatment group (Fig. 10b), and the central hub gene of the pathway was identified as Inhibitor of DNA binding 1 (ID1), which has a cell cycle inhibitory function.

[0282] In addition, the results of showing the gene sets significantly enriched in the ReHMGB1 treatment group (based on GSEA score and p value) among the KEGG pathways as a graph calculated through GSEA (Gene Set Enrichment Analysis) showed that the cytokine-receptor interaction and JAK / STAT signaling pathways had the highest NES scores (normalized enrichment scores), while the DNA replication and cell cycle pathways had the lowest NES scores (Fig. 10c).

[0283] From this, we confirmed that ReHMGB1 induces inflammatory cytokines in normal cells, activates SASP-related signaling pathways, and simultaneously induces senescence by suppressing cell cycle positive regulatory factors.

[0284] Additionally, to identify key molecular factors of the aging progression induced by ReHMGB1, core genes within the network were identified using three centrality indices (degree centrality, betweenness centrality, and closeness centrality) based on the DEGs expressed in ReHMGB1-treated cells (Tables 4 and 5).

[0285] DEGsKey TFDescriptionP valueQ valueUp-regulatedRELAv-rel reticuloendotheliosis viral oncogene homolog A(avian)9.28E-077.14E-06NFKB1nuclear factor of kappa light polypeptide gene enhancer in B-cells 19.53E-077.14E-06STAT1signal transducer and activator of transcription 1, 91 kDa1.69E-068.43E-06ZFP36ZFP36 ring finger protein2.36E-068.84E-06STAT3signal transducer and activator of transcription 3(acute-phase response factor)8.21E-062.46E-05XBP1X-box binding protein 11.44E-053.59E-05RELv-rel reticuloendotheliosis viral oncogene homolog(avian)1.94E-054.16E-05ATF4activating transcription factor 4(tax-responsive enhancer element B67)4.99E-059.36E-05KLF4Kruppel-like factor 4(gut)6.54E-050.000109CEBPACCAAT / enhancer binding protein(C / EBP), alpha0.0001070.00016EP300E1A binding protein p3000.0001290.000176SP1Sp1 transcription factor0.0002940.000367EGR1early growth response 10.0003190.000368MYCv-myc myelocytomatosis viral oncogene homolog(avian)0.0004110.000441JUNjun proto-oncogene0.000910.00091Down-regulatedE2F3E2F transcription factor 32.55E-091.79E-08E2F4E2F transcription factor 4, p107 / p130-binding1.58E-085.53E-08E2F1E2F transcription factor 13.46E-068.08E-06PTTG1pituitary tumor-transforming 15.89E-068.91E-06TP53tumor protein p536.36E-068.91E-06IRF1interferon regulatory factor 17.13E-058.31E-05MYCv-myc myelocytomatosis viral oncogene homolog(avian)0.0002750.000275.

[0286]

[0287]

[0288]

[0289] RankUp-regulatedDown-regulatedTermsDC(k)TermsBCTermsCCTermsDC(k)TermsBCTermsCC1IL618VEGFA7511.3IL60.0026539CDK1113UBE2C4462.4CDK10.00494192MX115BMP46186.0VEGFA0.0026536CCNA2109CXCL84439.4CCNA20.00494173VEGFA14PTCH5518.0CXCL100.0026535TOP2A106EGF3999.7TOP2A0.00494124MX211GPC65074.0MX10.0026532BUB1106FOS3664.5BUB10.00494125OAS111GPC44820.0LIF0.0026532CCNB1102BIRC52810.8CCNB10.00494116CXCL1011FZD54735.7ICAM10.0026531AURKB100HIST2H2BE1709.7BUB1B0.00494117PDGFRA11CXCL103601.0PDGFRA0.0026531BUB1B100MMP31564.0AURKB0.00494088IFI44L10WNT23358.0CCL20.0026527KIF2C99CDK11477.9KIF2C0.00494069IFITM110MUSK3080.0OAS10.0026527NCAPG98EDN11410.2NCAPG0.004940610IFI3510PAK32553.8KIT0.0026527KIF1197ITGA61050.0KIF110.004940511LIF9IL62398.0IFI44L0.0026527TTK96ELN1050.0CCNB20.004940412XAF19PARP92385.1STAT5A0.0026527CCNB296RRM2834.4CDC200.004940413KIT9MX12200.0VCAM10.0026527CDC2096CCNA2814.2TTK0.004940414GBP29PARP141998.0JAK30.0026527CDCA894TACC3803.7CDCA80.004940415STAT5A9HELZ21822.0NGF0.0026526ASPM93CCNB1777.0ASPM0.004940316SCN5A9TRIB31739.6IL12A0.0026525KIF20A93TTK775.6KIF20A0.004940017S CN4B8IFI44L1398.0TLR30.0026525AURKA92CDC45743.7AURKA0.004940018ANK28MUC11389.6CSF1 0.0026525NDC8091VCAN706.0NDC800.004940019ICAM18ICAM11381.9SHC20.0026525DLGAP590COL 17A1704.0UBE2C0.004940020IFITM38NGF947.5IL6R0.0026524TPX289EGR1704.0TPX20.0049400.

[0290]

[0291] Although the ranking of genes by criteria showed some differences, IL6, MX1, VEGFA, CXCL10, IFI44L, and ICAM1 were all centrally located in the DEG network, while CDK1, CCNA2, CCNB1, and TTK appeared as the intersection of the repressed gene group.

[0292] Integrated transcription factor prediction for key DEGs was performed using the TRRUST database.

[0293] As a result, as shown in Table 4, from these core genes, RELA, NFKB1, and STAT1 were predicted to be major transcription factors (TFs) in the activated network, and the E2F family (E2F1, E2F3, and E2F4) was predicted to be a factor regulating the repressed gene group. This is consistent with the effect of ReHMGB1 in promoting SASP and arresting the cell cycle.

[0294] In fact, even when referring to the results of analyzing the heatmap in which the DESeq2 normalized counts (row Z-scores) of the selected transcription factors (TFs) are color-coded, the selected transcription factors were significantly increased at the mRNA level in the ReHMGB1 treatment group (Fig. 10d), confirming that the predicted results are consistent with the experimental results.

[0295] That is, ReHMGB1 binds to RAGE and activates the PI3K-AKT / NF-κB and JAK / STAT pathways, and we confirmed whether these sub-pathways were activated.

[0296] Specifically, the activities of RAGE, AKT (p-AKT), p65 (p-p65), JAK2 (p-JAK2), and STAT1 (p-STAT1) were measured using Western blot on proteins extracted from ReHMGB1, OxHMGB1-treated WI-38 fibroblasts, and vehicle control. GAPDH was used as a loading control.

[0297] As a result, as shown in Figures 10e and 10f, in ReHMGB1-treated cells, RAGE expression increased, phosphorylation of AKT and NF-ΚB (p65) increased, and activation of JAK2 and STAT1 was more pronounced than in the OxHMGB1 or PBS-treated groups.

[0298] Additionally, WI-38 fibroblasts were treated with a RAGE antagonist (FPS-ZM1, 100 nM) and a JAK2 inhibitor (Momelotinib, 0.3 μM). Automated cell density (confluence) was calculated using an Incucyte SX1 instrument, and images were captured in real time at 2-hour intervals for 3 days under a 10x magnification lens condition. First, cell density was measured under conditions pretreated with a RAGE antagonist (RAGEa) or DMSO (control) before ReHMGB1 treatment, and in the same way, cell density was measured under conditions pretreated with a JAK inhibitor (JAKi) or DMSO.

[0299] As a result, as shown in Figures 11A and 11B, it was confirmed that both drugs overcame the initial cell density difference and restored continuous cell proliferation.

[0300] Additionally, representative images of SA-β-gal activity (blue cytoplasmic staining), EdU activity (green), and DAPI (white) staining were analyzed in WI-38 senescent cells induced by ReHMGB1, depending on whether RAGEa (100 nM) or JAKi (0.3 μM) was treated.

[0301] As a result, as shown in Figures 11C to 11E, it was confirmed that SA-β-gal activity was significantly reduced.

[0302]

[0303] Example 5. Confirmation of the age- and tissue-dependent aging-inducing effects of ReHMGB1.

[0304] 5.1. Confirmation of age-related aging induction

[0305] First, to confirm age-dependent changes in HMGB1, total HMGB1 expression in the serum of young and old mice was analyzed by immunoblotting. Figure 12B schematically illustrates the experimental design for analyzing reduced HMGB1 status in serum. Serial pull-down assays were performed on the serum of young and old mice, and loading uniformity was confirmed by Ponceau S staining.

[0306] As a result, as shown in Figures 12A and 12C, in C57BL / 6 mice, the total HMGB1 level in 24-month-old (old) mice was significantly increased compared to 3-month-old (young) mice, and the circulating level of ReHMGB1 was also markedly increased.

[0307] Similarly, the results showing age-dependent changes in HMGB1 in human serum showed that the concentration of ReHMGB1 in the elderly group of 70-80 years old was significantly higher than that in the young group of 40 years old in human serum samples, and the total HMGB1 was confirmed to increase, although not significantly (Fig. 13A and Fig. 13B).

[0308]

[0309] 5.2. Confirmation of aging induction by tissue

[0310] To determine whether systemic ReHMGB1 injection promotes aging in various tissues, 3-month-old C57BL / 6J mice were intravenously administered ReHMGB1 or PBS (vehicle) (iv, 5 mg / kg) (Fig. 14a), and tissue and serum analyses were performed 7 days later.

[0311] First, the concentrations of IL6, IL1β, and TNF-α in plasma and serum were quantified using a multiplex ELISA kit. The groups were ReHMGB1 injection group (n=6) and PBS (vehicle) control group (n=8).

[0312] As a result, as shown in Figure 14b, the levels of inflammatory cytokines such as IL-1β and IL-6 significantly increased in the ReHMGB1 treatment group.

[0313] Afterwards, p21 (Cdkn1a) expression was measured by RT-qPCR using RNA isolated from skeletal muscle (TA, GA), heart, liver, and kidney, and expressed as a value normalized to Actin.

[0314] As a result, it was confirmed that p21 (Cdkn1a) mRNA expression was significantly increased in skeletal muscle (tibialis anterior, TA and gastrocnemius, GA) and liver (Fig. 14c).

[0315] Additionally, p15INK4b (Cdkn2b), Cxcl10, Mmp13, TNF-α, and Timp1 mRNAs were analyzed by RT-qPCR using RNA isolated from skeletal muscle (TA, GA) and liver. All values ​​were normalized to actin. Mean ± SEM was expressed.

[0316] As a result, as shown in Fig. 14d, it was confirmed that Cxcl10 and Timp1 in TA muscle, and Tnf-α, Mmp13, Timp1, and p15^INK4b (Cdkn2b) in liver were also significantly increased, especially in skeletal muscle.

[0317] Paraffin-embedded TA muscles were analyzed for laminin staining, nuclear HMGB1 loss, and p16^INK4a / p21 expression. Scale bar = 20 μm. Quantitative analysis of cross-sectional area (CSA), nuclear HMGB1+, and p16^INK4a+ / p21+ myofibers was performed using ImageJ. Mean ± SEM was expressed, and a two-tailed t-test with Welch's correction was used for statistical analysis.

[0318] As a result, as shown in Fig. 14e, there was no significant difference in the average muscle fiber cross-sectional area (CSA), but the expression of p16^INK4a and p21 was increased in the TA muscles of ReHMGB1-treated mice, which was accompanied by a decrease in HMGB1 staining in the nucleus.

[0319] ReHMGB1 pull-down assay was performed on serum after intravenous injection of ReHMGB1. Measurements were taken from serum of three C57BL / 6J mice (n=3) at 0, 3, 6, and 24 h, and all data are expressed as mean ± SEM. Statistical analysis was performed using a two-tailed t-test with Welch's correction.

[0320] As a result, as shown in Fig. 14f, the concentration of ReHMGB1 reached a peak at the 6-hour point and was detectable even after 24 hours, confirming that it was expressed stably enough to function in vivo despite its redox sensitivity.

[0321] In summary, we demonstrate that circulating ReHMGB1 induces a systemic aging-like phenotype, upregulates SASP factors, and contributes to tissue dysfunction in vivo, thereby acting as a senescence accelerator and systemic mediator of senescence propagation.

[0322]

[0323] Example 6. Muscle regeneration therapeutic effect according to in vivo HMGB1 inhibition

[0324] Based on the skeletal muscle aging-inducing effect of ReHMGB1 confirmed in Example 5, an experiment was conducted to determine whether a therapeutic strategy targeting HMGB1 could improve damaged muscle regeneration.

[0325] First, purified anti-HMGB1 antibody (aHMGB1) was administered intravenously (iv, 0.1 mg / kg) in a BaCl2-induced muscle injury model in 15-month-old mice, and acute muscle injury was induced by injecting 50 μL of 1.2% barium chloride (BaCl2) dissolved in 0.9% saline into the tibialis anterior (TA) muscle (Fig. 15a).

[0326] Afterwards, images of the injured TA muscle and the uninjured muscle (control group) after BaCl2 injection were measured, and TA muscle mass was measured in the IgG-injured group (IgG-injured, n = 6), the anti-HMGB1 antibody-injured group (aHMGB1-injured, n = 6), and the uninjured group (n = 6). Muscle mass was normalized to body weight, and the bar graph was expressed as the mean ± standard error of the mean (SEM). Muscle damage was observed histomorphologically, and a decrease in muscle mass was confirmed (Fig. 15b). The circulating HMGB1 concentration in the serum of each group (uninjured TA, IgG-injured TA, and anti-HMGB1 antibody-injured TA) was quantified by ELISA.

[0327] As a result, as shown in Figure 15c, the serum HMGB1 level in the aHMGB1 (antibody)-administered group was significantly reduced compared to the IgG group, demonstrating the efficacy of HMGB1 blockade.

[0328] Subsequently, to assess tissue inflammation and regeneration, SA-β-gal activity (blue cytoplasmic staining), myofibers stained with laminin, regenerative muscle progenitors labeled with MyoD, and senescent cells identified with p21 were stained and quantified in TA muscle sections.

[0329] As a result, as shown in Figures 15d to 15g, SA-β-gal activity in the TA muscles of the IgG-damaged group significantly increased compared to the control group, but in the aHMGB1-damaged group, the activity was significantly reduced and similar to the level of the undamaged group. In addition, it was confirmed that the muscle fiber cross-sectional area (CSA) that decreased due to damage in the IgG-damaged group was partially recovered in the aHMGB1-administered group.

[0330] Additionally, to evaluate changes in regenerative and senescent cells in the muscle, immunofluorescence staining for MyoD (a muscle regeneration marker) and p21 (a senescence marker) was performed.

[0331] As a result, MyoD+ regenerative cells increased and p21+ senescent cells decreased in aHMGB1-treated muscles (Figs. 15h and 15i). This confirmed that the muscle regeneration promotion effect was exerted through inhibition of aging.

[0332] Finally, total physical function was measured using grip strength, rotarod endurance, and treadmill distance. Data were normalized to body weight at 4 days post-injury (dpt) and compared. Bar graphs are presented as the mean ± SEM of six independent experiments, and statistical analysis was performed using a two-tailed t-test with Welch's correction.

[0333] As a result, as shown in Fig. 15j, it was confirmed that aHMGB1-administered mice had superior muscle function in grip strength, rotarod endurance (latency), and treadmill work than the IgG-damaged group.

[0334] In summary, it was confirmed that blocking HMGB1 in vivo not only inhibits cell aging but also has a muscle regeneration therapeutic effect.

[0335]

[0336] Example 7. Confirmation of changes in HMGB1 gene expression across human tissues and with age.

[0337] To confirm the synchronous expression of HMGB1 gene in various organs throughout the human body, the rate of change in HMGB1 gene and the change in HMGB1 expression in muscle according to age were calculated using the TPM method and presented in a graph.

[0338] First, using GTEx open data, Figure 16A shows the proportion of tissues with a >75% decrease in HMGB1 expression in normal adults of various age groups by tissue. Tissues with high proportions can be interpreted as the result of decreased intracellular expression and increased extracellular release due to aging, tissue damage, or other physiological causes. At the same time, the pattern was very different for each tissue, confirming that it is a sensitive protein that can act as a physiological signal for inflammatory responses, damage, and stress in tissues and cells. In other words, increased extracellular release means that it is produced in large quantities under specific environments and conditions. Therefore, as shown in Figure 16B with the variable of age, it was found to increase in muscle tissue with age, confirming that extracellular release of circulating hmgb1 (hmgb1) or circulating Hmgb1 has a systemic effect in humans, and has the potential to act as an aging metastasis factor.

[0339] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. A biomarker composition for diagnosing or inducing aging, comprising HMGB1.

2. In paragraph 1, A biomarker composition for diagnosing or inducing aging, wherein the above HMGB1 is reduced HMGB1.

3. In paragraph 1, A biomarker composition for diagnosing or inducing aging, wherein the above aging is secondary senescence.

4. In paragraph 1, The above HMGB1 is a biomarker composition for diagnosing or inducing aging, which induces aging of surrounding cells.

5. In paragraph 1, A biomarker composition for diagnosing or inducing aging, wherein the above HMGB1 induces one or more symptoms selected from the group consisting of decreased muscle function, systemic inflammatory response, and decreased cell regenerative ability.

6. In paragraph 1, A biomarker composition for diagnosing or inducing aging, wherein the above HMGB1 increases the expression of a senescence-associated secretory phenotype (SASP) or aging-related marker.

7. In paragraph 6, A biomarker composition for diagnosing or inducing aging, wherein the aging-related secretory phenotype is at least one selected from the group consisting of IL6, IL1β, IL8, TNF-α, CXCL1, CXCL10, CCL2, SERPINE1, VCAM1, ICAM1, IGFBP5, TGFB1, PDGFRB, MMP2, MMP3, IL15, and GSK3A.

8. In paragraph 6, A biomarker composition for diagnosing or inducing aging, wherein the aging-related marker is at least one selected from the group consisting of p16, p15, p21, p53, and SA-β-gal.

9. In paragraph 1, A biomarker composition for diagnosing or inducing aging, wherein the above HMGB1 activates the PI3K-AKT / NF-KB, JAK / STAT pathway or TNF signal pathway. 10.(a) a step of analyzing the expression level of HMGB1 protein or a gene encoding it from a biological sample; and (b) A method for providing information for diagnosing aging, comprising a step of comparing the expression level of the HMGB1 protein or the gene encoding it analyzed in step (a) with the expression level of the HMGB1 protein or the gene encoding it in a control sample.

11. In paragraph 10, A method for providing information for diagnosing aging, wherein the above HMGB1 is reduced HMGB1.

12. In paragraph 10, A method for providing information for diagnosing aging, wherein the above aging is secondary senescence.

13. In paragraph 10, A method for providing information for diagnosing aging, wherein the biological sample is tissue, cell, blood, serum or plasma. 14.(i) A step of analyzing the expression level of HMGB1 protein or a gene encoding it in a biological sample obtained from a subject before treatment with a candidate drug for improving or preventing aging; (ii) a step of analyzing the expression level of the biological HMGB1 protein or the gene encoding it obtained from the subject after treatment with the candidate drug; and (iii) A method for selecting a candidate drug for improving or preventing aging, comprising a step of screening a candidate drug by comparing the expression level of the HMGB1 protein or the gene encoding it analyzed in step (i) with the expression level of the HMGB1 protein or the gene encoding it analyzed in step (ii).

15. In paragraph 14, A method for selecting a candidate drug for improving or preventing aging, wherein the above HMGB1 is reduced HMGB1.

16. In paragraph 14, A method for selecting a candidate drug for improving or preventing aging, wherein the above aging is secondary senescence.

17. A method for predicting or monitoring an aging improvement or prevention response, comprising a step of inhibiting the expression of HMGB1 protein or a gene encoding the same.

18. A food composition for improving or preventing aging, comprising an HMGB1 inhibitor.

19. A pharmaceutical composition for preventing or treating age-related diseases, comprising an HMGB1 inhibitor.

20. In paragraph 19, A pharmaceutical composition for preventing or treating an aging-related disease, wherein the aging-related disease is at least one selected from the group consisting of sarcopenia, muscular dystrophy, muscle fiber regeneration deficiency, muscular dystrophy, osteoarthritis, hypotonia, muscular dystrophy, myasthenia gravis, non-alcoholic fatty liver disease, liver fibrosis, liver cirrhosis, senile liver dysfunction, cardiovascular disease, neurodegenerative disease, skin aging, idiopathic pulmonary fibrosis, pulmonary fibrosis, glaucoma, cataract, and type 2 diabetes.

21. A method for producing an aging model, comprising a step of treating isolated cells with reduced HMGB1.

22. In paragraph 21, A method for manufacturing an aging model, wherein the above aging is secondary senescence.

23. In paragraph 21, A method for producing an aging model, wherein the separated cells are skin cells, epithelial cells, epidermal cells, dermal fibroblasts, hepatocytes, kidney cells, lung cells, muscle cells, blood-brain barrier endothelial cells, cancer cells, blood cells, cervical cells, lymphocytes, immune cells, stem cells, induced pluripotent stem cells, neural stem cells, T cells, B cells, natural killer cells, macrophages, monocytes, microglia, neurons, glial cells, astrocytes, brain cells, or laryngeal cells. 24.(a) A step of analyzing the expression level of HMGB1 from a biological sample; and (b) A method for diagnosing aging, comprising a step of comparing the expression level of HMGB1 analyzed in step (a) with the expression level of HMGB1 in a control sample.

25. A method for inducing senescence, comprising the step of treating a biological sample with a composition containing HMGB1.

26. A method for improving or preventing aging, or preventing or treating an aging-related disease, comprising administering to a subject in need thereof a composition comprising an HMGB1 inhibitor in an amount effective for improving or preventing aging, or preventing or treating an aging-related disease.

27. Use of a composition according to any one of claims 1 to 9 for diagnosing or inducing aging.

28. Use of a composition comprising an HMGB1 inhibitor for improving or preventing aging; or preventing or treating aging-related diseases.

29. Use of a composition comprising an HMGB1 inhibitor for the manufacture of a medicament for improving or preventing aging; or for preventing or treating aging-related diseases.

Images