Inflammatory cytokine expression inhibitor and method for inhibiting expression of inflammatory cytokine

Abstract

[Problem] To provide an inflammatory cytokine expression inhibitor or the like that can effectively suppress the expression amount of inflammatory cytokine and that can be used safely without any risks of side effects. [Solution] This inflammatory cytokine expression inhibitor contains nicotinamide mononucleotide as an active ingredient.

Description

Inhibitor of inflammatory cytokine expression and method for inhibiting inflammatory cytokine expression

[0001] The present invention relates to an inhibitor of inflammatory cytokine expression and a method for inhibiting inflammatory cytokine expression.

[0002] Cytokines, proteins secreted primarily by immune cells, are classified into "pro-inflammatory cytokines," which cause various inflammatory symptoms in the body, and "anti-inflammatory cytokines," which suppress inflammatory symptoms. Normally, these two cytokines are balanced to prevent adverse effects. However, if this balance is disrupted and excessive pro-inflammatory cytokines are released into the bloodstream, various symptoms, such as chills, nausea, fatigue, headache, fever, tachycardia, and blood pressure fluctuations, occur. If the condition worsens, it can lead to autoimmune diseases and other conditions, resulting in a condition known as a cytokine storm.

[0003] A cytokine storm can cause serious damage to organ tissues. For example, if a cytokine storm occurs in the lungs, serous fluid and immune cells can concentrate in the airways, causing blockage and potentially leading to death. COVID-19 is known to cause interstitial pneumonia, and cytokine storms are thought to be involved in this process. Furthermore, when patients are diagnosed with cancer, chronic inflammation occurs in the body due to stimulation by cancer cells, leading to the secretion of large amounts of inflammatory cytokines from the immune cells of cancer patients. Cytokine storms are thought to be a cause of cancer cachexia.

[0004] In addition, antigen stimulation produces inflammatory cytokines, which activate neutrophils, causing neutrophil degranulation and immune complex deposition in the vascular wall, which can lead to vascular endothelial cell damage and necrotizing vasculitis.

[0005] Furthermore, TNF-α, an inflammatory cytokine, is known to have vascular damage effects, and it has been reported that blood TNF-α levels are elevated in Kawasaki disease. Sepsis is also known as an inflammatory cytokine-related disease, and it has been reported that blood interleukin-6 levels are elevated in sepsis.

[0006] As described above, when the balance between inflammatory and anti-inflammatory cytokines is disrupted and inflammatory cytokines are produced in excess, it can lead to a variety of pathological conditions. Therefore, there is a need for drugs that can reduce the expression level of inflammatory cytokines (especially when they are excessive) so that they are not produced in excess and released into the blood.

[0007] Corticosteroids are drugs that suppress the production of inflammatory cytokines. While these drugs have a strong anti-inflammatory effect, they also have side effects such as the induction and exacerbation of infections, osteoporosis and fractures, growth retardation in infants and children, arteriosclerotic lesions (myocardial infarction, cerebral infarction, aneurysms, thrombosis), gastrointestinal disorders, the induction and exacerbation of diabetes, and psychotremor disorders (mental disturbances, depression, convulsions). Furthermore, the use of corticosteroids can weaken the therapeutic effects of other drugs. Therefore, corticosteroids cannot be used safely for long periods of time.

[0008] The renin-angiotensin system (RAS) is believed to be related to cytokine storm, and it has been reported that angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs) can suppress cytokine storm. However, because ACE inhibitors and ARBs are originally used as antihypertensive drugs, they may cause excessive hypotensive symptoms such as fainting, dizziness, and falls, and are therefore contraindicated in pregnant women. Therefore, these drugs also have the disadvantage of not being able to be used safely for long periods of time.

[0009] TNF-α inhibitors (infliximab, etanercept, etc.) inhibit TNF-α and suppress the activation of immune cells, but it has not been confirmed whether they are effective in suppressing cytokine storms. They have side effects such as infections and reactions upon administration (anaphylaxis, headache, fever). Therefore, TNF-α inhibitors also have the drawback of not being able to be used safely over long periods of time.

[0010] As an inflammatory cytokine expression inhibitor capable of suppressing the expression of inflammatory cytokines such as TNF-α, an inflammatory cytokine expression inhibitor containing 4'-hydroxy-3,4,2',6'-tetramethoxychalcone or 4',6'-dihydroxy-3,4,2'-trimethoxychalcone as an active ingredient has been reported (Patent Document 1).

[0011] JP 2014-1010329 A

[0012] In view of the above-mentioned circumstances, there is a need for the development of a drug with few side effects, which can be used safely and has excellent inhibitory effect on the expression of inflammatory cytokines. Therefore, an object of the present invention is to provide an inhibitor of inflammatory cytokine expression, etc., which can effectively suppress the expression level of inflammatory cytokines and can be used safely without the risk of side effects.

[0013] As a result of intensive research to solve the above-mentioned problems, the present inventors discovered that nicotinamide mononucleotide (NMN), an intermediate metabolite involved in the biosynthesis of the coenzyme NAD (nicotinamide adenine dinucleotide), reduces the expression levels of inflammatory cytokines, leading to the completion of the present invention.

[0014] The present invention provides the following: [1] An inflammatory cytokine expression inhibitor containing nicotinamide mononucleotide as an active ingredient. [2] The inflammatory cytokine expression inhibitor according to [1] above, wherein the inflammatory cytokine is at least one of interleukin-1, interleukin-6, and TNF-α. [3] The inflammatory cytokine expression inhibitor according to [1] or [2] above, wherein the inflammatory cytokine is expressed in pulmonary microvascular endothelial cells or coronary artery endothelial cells. [4] The inflammatory cytokine expression inhibitor according to any of [1] to [3] above, wherein the production of the inflammatory cytokine is induced by dsRNA or synthetic dsRNA (poly(I:C)). [5] The inflammatory cytokine expression inhibitor according to any of [1] to [4] above, which has an effect of preventing or treating cytokine storm or vasculitis. [6] The inflammatory cytokine expression inhibitor according to any of [1] to [5] above, wherein the inflammatory cytokine expression inhibitor is a food for preventing or treating cytokine storm or vasculitis. [7] The inflammatory cytokine expression inhibitor according to any one of [1] to [5] above, wherein the inflammatory cytokine expression inhibitor is a pharmaceutical agent for preventing or treating cytokine storm or vasculitis. [8] A method for suppressing inflammatory cytokine expression in a mammal other than a human, comprising administering an effective amount of nicotinamide mononucleotide to a subject, the mammal other than a human, in need of suppression of inflammatory cytokine expression. [9] The method for suppressing inflammatory cytokine expression according to [8] above, wherein the inflammatory cytokine is expressed in pulmonary microvascular endothelial cells or coronary artery endothelial cells.

[10] The method for suppressing inflammatory cytokine expression according to [8] or [9] above, wherein production of the inflammatory cytokine is induced by dsRNA or synthetic dsRNA (poly(I:C)).

[0015] The present invention can effectively suppress the expression of inflammatory cytokines, and can be used safely with little risk of side effects.

[0016]

[0023] Figure 1 is an explanatory diagram showing the metabolic pathway involved in niacin (a collective term for nicotinamide and nicotinic acid). Figure 2 is a diagram showing the expression levels of IL6 and IL12 in HPMEC after 6 and 9 hours of treatment with control, poly(I:C) (2.5 μg / mL), and poly(I:C) (25 μg / mL). Figure 3 is a diagram showing the expression levels of IL6 and IL12 in HCAEC after 6 and 9 hours of treatment with control, poly(I:C) (2.5 μg / mL), and poly(I:C) (25 μg / mL). Figure 4 is a diagram showing PCA plots of expression profiles in HPMEC after activation with poly(I:C) (2.5 μg / mL) and under poly(I:C) non-activation conditions. Figure 5 is a diagram showing PCA plots of expression profiles in HCAEC after activation with poly(I:C) (2.5 μg / mL) and under poly(I:C) non-activation conditions. Figure 1 shows a volcano plot of differentially expressed genes (DEGs) between control and high-dose NMN treatment under poly(I:C) non-activating and poly(I:C) activating conditions in HPMEC. Figure 2 shows a volcano plot of differentially expressed genes (DEGs) between control and high-dose NMN treatment under poly(I:C) non-activating and poly(I:C) activating conditions in HCAEC. Figure 3 shows the number of genes up- or down-regulated by high-dose (10 mM) NMN under poly(I:C) activating conditions in HPMEC, as well as a list of over-represented pathways. Figure 4 shows the number of genes up- or down-regulated by high-dose (10 mM) NMN under poly(I:C) activating conditions in HCAEC, as well as a list of over-represented pathways. Figure 5 shows a color-coded dendrogram of all genes and modules for HPMEC. Figure 6 shows a color-coded dendrogram of all genes and modules for HCAEC. Figure 1 shows scatter plots of the fold change (FC) of the eigengenes of the identified modules from control to high dose (10 mM) NMN under poly(I:C) non-activation (x-axis) and poly(I:C) activation (y-axis) conditions in HPMEC. Figure 2 shows scatter plots of the fold change (FC) of the eigengenes of the identified modules from control to high dose (10 mM) NMN under poly(I:C) non-activation (x-axis) and poly(I:C) activation (y-axis) conditions in HCAEC.4A and 4B show box plots of eigengene expression and a list of overrepresented pathways in the p-green module and p-sienna3 module of HPMEC. 4B shows box plots of eigengene expression and a list of overrepresented pathways in the c-magenta module and c-purple module of HCAEC. 4C shows the expression level of IL6 in HPMEC. 4D shows the expression level of IL6 in HCAEC. 4E shows the expression level of an eigengene (SAMHD1) in the purple module of HPMEC. 4F shows the expression level of an eigengene (ANKFY1) in the yellow module of HCAEC. 4G shows predicted causal networks of WGCNA eigengenes, with FIG. 4A showing the causal relationships between all eigengenes of the WGCNA module predicted by Bayesian network analysis in HPMEC and FIG. 4B showing the causal relationships in HCAEC. 1 shows the expression level of an eigengene (TNFRSF10D) in the HPMEC p-darkred module and the results of functional enrichment analysis. 2 shows the expression level of an eigengene (HERPUD1) in the HCAEC c-black module and the results of functional enrichment analysis. 3 shows a common functional pathway between the HPMEC p-darkred module and the HCAEC c-black module. 4 shows a box plot of the expression level of a representative common gene (EIF4B) in HPMEC and HCAEC. 5 shows a schematic model of the regulatory mechanism of NMN-mediated reduction of IL6 expression under poly(I:C)-activating conditions. 6 shows a predicted causal network between the c-black, c-tan, c-yellow, and c-green modules by Bayesian network analysis of eigengenes in HCAEC transcriptome data, as well as the expression level of an eigengene (PARP14) in the c-green module. 10 is a box plot of the expression levels of both PARP9 and PARP12 contained in the p-purple module of HPMEC, and FIG. 11 is a box plot of the expression levels of both PARP9 and PARP12 contained in the c-green module of HCAEC.

[0017] The inflammatory cytokine expression inhibitor according to the present invention (hereinafter sometimes simply referred to as the "agent of the present invention") contains nicotinamide mononucleotide as an active ingredient, and exhibits the effect of suppressing inflammatory cytokine expression. In the present invention, suppression of inflammatory cytokine expression refers not only to a decrease in the amount of inflammatory cytokine expression, but also to suppression or delay of an increase in the amount of inflammatory cytokine expression. The detailed reasons why such an effect is achieved by using nicotinamide mononucleotide as an active ingredient are currently under investigation, but it is believed that administration of nicotinamide mononucleotide increases intracellular NAD ＋ This increases the histone deacetylase activity of the intracellular protein sirtuin 1. As a result, epigenetic control of genomic DNA occurs, which is thought to be one of the reasons for this, leading to a decrease in the cellular gene expression pattern, i.e., the expression level of inflammatory cytokine genes. The present invention will be described in detail below.

[0018] Nicotinamide mononucleotide (chemical formula: C １１ H １５ N ２ O ８ Nicotinamide mononucleotide (NMN) is a compound expressed by the following structural formula [Chemical Formula 1], which is produced in the bodies of many living organisms, including humans. It is commonly called NMN (nicotinamide mononucleotide) and is a coenzyme NAD ＋ Nicotinamide mononucleotide is known as an intermediate metabolite involved in the biosynthesis of . Nicotinamide mononucleotide also includes pharmacologically acceptable salts.

[0019]

[0020] The active ingredient, nicotinamide mononucleotide, is produced in vivo in the liver tissue through the NAD metabolic pathway, i.e., the pathway involved in the synthesis of nicotinamide adenine dinucleotide (NAD) from quinolinic acid via the kynurenine pathway. This point will be specifically explained with reference to Figure 1. Figure 1 shows the structure of vitamin B ３This diagram shows the metabolic pathway involved in niacin (a collective term for nicotinamide and nicotinic acid), also known as niacin. Nicotinic acid ingested through diet is taken up by the liver and converted to nicotinamide, which is then distributed throughout the body via the bloodstream. Each cell takes in nicotinamide from the blood and converts it to NAD and NADP for use. Nicotinamide can also be biosynthesized from tryptophan.

[0021] As shown in Figure 1, in vivo, when tryptophan is used as the starting material, tryptophan is converted to quinolinic acid (QA) via the kynurenine pathway, which is the tryptophan metabolic pathway, and then to nicotinic acid mononucleotide (NaMN). On the other hand, when nicotinic acid (Na) is used as the starting material, nicotinic acid is directly converted to NaMN. NaMN then passes through nicotinic acid adenine dinucleotide (NaAD) and is interconverted to NAD, nicotinamide (NaM), and nicotinamide mononucleotide via the NAD cycle. Nicotinamide (NaM) is converted to nicotinamide mononucleotide by nicotinamide phosphoribosyltransferase (NAMPT), and nicotinamide mononucleotide is then converted by nicotinamide mononucleotide adenyltransferase (NMNAT) to produce NAD. Nicotinamide mononucleotide can also be produced from nicotinamide riboside (NR), an NAD intermediate metabolic product.

[0022] Nicotinamide mononucleotide exists as two optical isomers, α- and β-, and the β-isomer is used in the present invention. Nicotinamide mononucleotide can be obtained, for example, by synthesizing nicotinamide riboside from nicotinamide and ribose (see Bioorg. Med. Chem. Lett., 12, 1135-1137 (2002)), followed by phosphorylating the 5-hydroxyl group of the ribose moiety (see Chem. Comm., 1999, 729-730). Specifically, for example, nicotinamide and L-ribose tetraacetate are first dissolved in anhydrous acetonitrile, and an excess amount of trimethylsilyltrifluorosulfonic acid is added under a nitrogen stream. The mixture is stirred at room temperature, and the reaction is terminated by adding methanol. The reaction solution is then loaded onto a column packed with activated carbon, washed with distilled water, and eluted with methanol to recover the product. Next, to phosphorylate the 5-hydroxyl group of the L-ribose moiety of this product, the product is dissolved in trimethoxyphosphate, and phosphorus oxychloride is added dropwise under ice cooling. The mixture is stirred under a nitrogen stream, and aqueous sodium hydroxide is added to neutralize the reaction. To the resulting reaction solution, a cold acetonitrile-ether solution is added. The lower layer (aqueous phase) is then passed through an anion exchange resin to recover the reaction product, which is then further purified with a cation exchange resin to recover highly pure nicotinamide mononucleotide. Nicotinamide mononucleotide is commercially available, and such commercially available products can be purchased and used.

[0023] The nicotinamide mononucleotide is a purified product with a low content of impurities, and its purity is preferably 90% or more, and more preferably 95% or more. If the purity is less than 90%, an unpleasant odor may be generated or the effect of nicotinamide mononucleotide may be weakened, making it difficult to fully obtain the effects of the present invention.

[0024] The agent of the present invention can be easily produced by using nicotinamide mononucleotide alone or by mixing it with other ingredients. The other ingredients are not particularly limited as long as the effects of the present invention can be obtained. Examples of other ingredients are not particularly limited, but may include, for example, supplementary ingredients commonly used in the food industry, such as various vitamins, trace elements, citric acid, malic acid, flavorings, inorganic salts, etc.

[0025] In one embodiment, the agent of the present invention is an inflammatory cytokine expression inhibitor having an effect of preventing or treating cytokine storm or vasculitis, and can be applied for the purpose of suppressing the expression of inflammatory cytokines, and thereby preventing or treating cytokine storm or vasculitis. It can also be applied to a subject already infected with cytokine storm or vasculitis to prevent the symptoms from worsening.

[0026] In one embodiment of the present invention, the inflammatory cytokine whose expression is to be inhibited is at least one of interleukin-1, interleukin-6, and TNF-α. The agent of the present invention is particularly effective in reducing the expression levels of the above-mentioned inflammatory cytokines. Furthermore, the agent of the present invention is particularly suitable for inflammatory cytokines produced in pulmonary microvascular endothelial cells or coronary artery endothelial cells. Furthermore, the agent of the present invention is particularly suitable for inflammatory cytokines induced by dsRNA or synthetic dsRNA (poly(I:C)). Poly(I:C) is a synthetic analog (synthetic dsRNA) of double-stranded RNA (dsRNA) present in some viruses, and is used to model the effects of extracellular dsRNA and mimic the inflammatory response induced by viral infection.

[0027] The method for producing the agent is not particularly limited, and a general production method used for producing the agent may be appropriately selected depending on the form. For example, if the agent is in the form of a powder, it can be produced by uniformly kneading nicotinamide mononucleotide and other ingredients that are added as needed. Note that nicotinamide mononucleotide, the active ingredient, is distributed on the market and can be commercially obtained. In particular, with regard to nicotinamide mononucleotide, a quality control system and mass production system have been established in recent years, making it possible to supply it as a food composition ingredient, and its stability as a food composition has also been confirmed.

[0028] The agent of the present invention can be used as a food or drink or a pharmaceutical. When used as a food or drink, the agent can be provided in the food or drink field as a food or drink for preventing or treating cytokine storm or vasculitis. In this case, daily intake in the form of a food or drink is particularly effective because it continuously exerts the effects of preventing and treating cytokine storm or vasculitis. The types of food and drink that are the subject of the present invention are not particularly limited, and include general food and drink, as well as functional foods, foods for specified health uses, nutritional supplements, food additives, feed, nursing care foods, dietary diets, therapeutic foods, dietary foods, and diet foods. Specific examples include confectioneries (gum, candy, cookies, gummies, biscuits, cakes, chocolates, Japanese sweets, jellies, etc.), bread, noodles, processed rice and grain products (cereals, etc.), processed meat products, processed seafood products, processed vegetables, prepared dishes, fermented foods, seasonings (sauces, dressings, ketchup, etc.), spices, dairy products (yogurt, cheese, milk, etc.), ice cream, frozen foods, retort pouch foods, beverages (carbonated drinks, soft drinks, dairy drinks, alcoholic drinks, sports drinks, fruit juice drinks, teas, nutritional drinks, concentrated drinks, etc.), powdered beverages (powdered juice, powdered soup, etc.), etc. The form of the food and beverage is not limited, and in the case of functional foods and foods for specified health uses, for example, they can be processed and provided as powders, tablets, pills, granules, hard capsules, soft capsules, jellies, liquids, pastes, etc.

[0029] The intake amount of the food or beverage varies depending on the type of food or beverage, the age, sex, weight, expected effects, symptoms, etc. of the person taking it, but the daily intake amount of nicotinamide mononucleotide contained in the food or beverage is typically 1 mg to 500 mg, preferably 5 mg to 350 mg, and more preferably 50 mg to 300 mg per adult. If the amount is less than 1 mg, the effects of the present invention may not be obtained, while if the amount is more than 500 mg, the obtained effects will not change significantly and it will be economically disadvantageous. The blending ratio of nicotinamide mononucleotide in the food or beverage can be appropriately set within a range of 100% or less of the total weight of the food or beverage.

[0030] The food and drink products are safe and have no particular side effects, and therefore can be taken over a long period of time for the purpose of preventing or treating cytokine storm or vasculitis. Furthermore, they can be applied not only to subjects in whom prevention of cytokine storm or vasculitis is desired, but also to subjects suffering from the disease, as well as to healthy subjects, so as to prevent the cytokine storm or vasculitis from becoming severe.

[0031] Meanwhile, in the pharmaceutical field, the agent of the present invention can be administered orally or parenterally as a pharmaceutical (including quasi-drugs) for the prevention or treatment of cytokine storm or vasculitis. The dosage form of the pharmaceutical is not particularly limited, and examples thereof include oral administration preparations such as powders, tablets, sustained-release tablets, chewable tablets, effervescent tablets, troches, buccal tablets, sublingual tablets, capsules, fine granules, granules, pills, dry syrups, liquids, suspensions, syrups, and elixirs, as well as injections, infusions, and topical preparations. Among these, oral administration preparations such as powders, tablets, and capsules, and topical preparations are preferred in terms of ease of administration and stability of the active ingredient.

[0032] The pharmaceutical may be appropriately blended with known pharmaceutically acceptable additives suitable for the dosage form, taking into consideration physicochemical properties, biological properties, etc. Examples of such additives include excipients (lactose, starch, crystalline cellulose, sodium phosphate, etc.), solvents (water, soybean oil, saline, non-aqueous solvents for injection, etc.), binders (starch, gelatin, gum arabic, sodium alginate, carmellose sodium, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, etc.), disintegrants (starch, carmellose sodium, etc.), lubricants (talc, magnesium stearate, calcium stearate, macrogol, sucrose fatty acid esters, etc.), coating agents (sucrose, HPC, shellac, gelatin, glycerin, hydroxypropylmethylcellulose, hydroxypropylmethylcellulose phthalate, cellulose acetate phthalate, etc.), stabilizers (sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxybenzoate, etc.), and the like. Examples of the additives include benzoyl perfluorooctyl alcohol (e.g., benzoyl perfluorooctyl alcohol), preservatives (e.g., methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, benzyl alcohol, phenol, chlorobutanol, benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, thimerosal), thickeners (e.g., methylcellulose, carmellose sodium, chondroitin sulfate, sodium alginate), suspending agents (various nonionic surfactants, methylcellulose, carmellose sodium), emulsifiers (e.g., gum arabic, cholesterol, sorbitan sesquioleate, polysorbate 80, sodium lauryl sulfate), buffers (citric acid, acetic acid, sodium phosphate, boric acid), surfactants (e.g., hydrogenated castor oil, polysorbate 80), colorants (e.g., water-soluble food dyes, lake dyes), flavoring agents (e.g., lactose, sucrose, glucose, mannitol), odorants (e.g., aromatic essential oils), and plasticizers (e.g., phthalate esters, vegetable oils, polyethylene glycol).

[0033] The dosage of the pharmaceutical varies depending on the age, weight, symptoms, frequency of administration, purpose, etc. of the recipient and cannot be uniformly defined. However, the dosage of the pharmaceutical is typically 1 mg to 500 mg, preferably 5 mg to 350 mg, and more preferably 50 mg to 300 mg, of nicotinamide mononucleotide administered per day to an adult. If the dosage is less than 1 mg, the effects of the present invention may not be achieved. On the other hand, if the dosage is more than 500 mg, the effects obtained will not change significantly and it will be economically disadvantageous. The proportion of nicotinamide mononucleotide in the pharmaceutical can be appropriately determined depending on the dosage form, dosage, etc. of the pharmaceutical.

[0034] The number of times the pharmaceutical is administered can be appropriately determined depending on the age, weight, symptoms, and dosage of the pharmaceutical per administration of the subject, etc. One example of the number of times the pharmaceutical is administered per day is 1 to 3 times.

[0035] Another embodiment of the present invention is a method for suppressing inflammatory cytokine expression, comprising administering an effective amount of nicotinamide mononucleotide to a subject in need of suppression of inflammatory cytokine expression. The subject is preferably a mammal such as a human, mouse, rat, rabbit, dog, cat, cow, horse, pig, or monkey. The method is also effective for mammals other than humans, but humans are particularly preferred. In the method, the intake amount of nicotinamide mononucleotide, the number of times of intake per day, and the like are as described for the agent. Furthermore, nicotinamide mononucleotide can be administered at any time and in any circumstances, and can be administered to a subject on a daily basis for a long period of time.

[0036] Yet another embodiment of the present invention is a method for preventing or treating cytokine storm or vasculitis, comprising administering an effective amount of nicotinamide mononucleotide to a subject in need of such prevention or treatment. The subject is preferably a mammal such as a human, mouse, rat, rabbit, dog, cat, cow, horse, pig, or monkey. The method is also effective for mammals other than humans, but humans are particularly preferred. In the method, the intake amount of nicotinamide mononucleotide, the number of times per day to be taken, and the like are the same as those described for the agent. Furthermore, nicotinamide mononucleotide can be taken at any time and in any situation, and can be taken by a subject on a daily basis for a long period of time.

[0037] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0038] [Methods] The specific details of the various methods performed in the test examples described below are summarized below. (Human primary cell culture) Human pulmonary microvascular endothelial cells (HPMEC) and human coronary artery endothelial cells (HCAEC) were obtained from ScienCell Research Laboratories and cultured in endothelial cell medium (ScienCell Research Laboratories, Carlsbad, USA) at 37°C and 5% CO ２ The culture dishes and plates were kept in a humidified atmosphere at 2 μg / cm ２The cells were pre-coated with fibronectin at a concentration of 0.01%. After thawing the frozen vial in a 37°C water bath, the entire sample was seeded onto a single 100 mm dish and cultured with medium changes every 2-3 days until a sufficient cell number was obtained. Cells were detached with 0.05% trypsin-EDTA and subcultured. Subculture expansion continued until the required cell number for testing was reached. Both cell lines were passaged twice after thawing, spread onto 12-well tissue culture-treated plates, and cultured for 2 days. For RT-PCR, cells were treated with polyinosinic acid-polycytidylic acid (poly(I:C); Enzo Life Sciences) at final concentrations of 0, 2.5, and 25 μg / ml for 6 or 9 hours. Total RNA was prepared using the RNeasy Plus Mini Kit (QIAGEN) and reverse transcribed using the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific) to obtain cDNA. Quantitative PCR was performed using TaqMan Gene Expression Master Mix, PowerUp SYBR Green Master Mix, and TaqMan Gene Expression Assays (Thermo Fisher Scientific). For transcriptome analysis, CO ２ The cells were treated with or without 25 μg / ml poly(I:C) for 6 hours in an incubator, and then cultured in fresh medium containing 0, 0.1 (low), 1.0 (medium), or 10.0 (high) mM NMN for 48 hours. NMN was provided by Mirai Lab Biosciences, Inc. (Tokyo, Japan). In the poly(I:C)-induced group, the supernatant from the 6-well plate in which the cells were seeded was removed and replaced with poly(I:C)-containing medium. In the non-poly(I:C)-induced group, the medium contained water instead of poly(I:C). ２ After 6 hours of poly(I:C) induction in an incubator, the supernatant was removed, the medium was replaced with fresh medium containing 0, 0.1 (low), 1.0 (medium), or 10.0 (high) mM NMN, and the cells were further incubated in CO ２The cells were cultured in an incubator for 48 hours. Total RNA was prepared from cells from eight different cultures (the above conditions with or without poly(I:C) induction and four different NMN doses) in triplicate, using TRI reagent (Molecular Research Center). Cells were approximately 40-50% confluent at the time of poly(I:C) induction and 100% confluent 48 hours after NMN addition.

[0039] (RNA-seq and RNA-seq Data Analysis) Library construction for whole transcriptome sequencing was performed using the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina, San Diego, CA) according to the manufacturer's instructions. Transcriptome library sequencing was performed using an Illumina NovaSeq 6000 in 101-bp paired-end mode. Data integrity of raw sequences was assessed by FastQC v0.11.7 (http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ), and sequences were trimmed with Trimmomatic 0.38 (http: / / www.usadellab.org / cms / ?page=trimmomatic) to exclude reads shorter than 36 bp in length. Trimmed reads were mapped to the reference genome GRCh38 using HISAT2 version 2.1.0 (https: / / ccb.jhu.edu / software / hisat2 / index.shtml) and Bowtie2 2.3.4.1 aligner. For each sample, gene / transcript abundance was calculated from the number of reads and normalized to FPKM (Fragments Per Kilobase of transcript per Million mapped reads) (see data availability below). Sample preparation and RNA-seq analysis were performed by Cosmobio Co., Ltd. (Sapporo, Japan).

[0040] Principal Component Analysis: Principal component analysis (PCA) of the transcriptome data was performed using the PCA function implemented as part of Python's sklearn.decomposition. Potential outliers were excluded from further analysis.

[0041] (Analysis of differentially expressed genes) Differentially expressed genes (DEGs) between the control group and the high-dose (10 mM) NMN group were identified using R version 4.1.0 (2021-05-18). Adjusted p-values ​​were calculated based on the false discovery rate (FDR) using the fdrtool library. The thresholds for log2 fold change (FC), p-value, and local FDR were set to 1.0 (2-fold), 0.05, and 0.1, respectively.

[0042] (Weighted Gene Correlation Network Analysis) Using the WGCNA package (v1.70) in R (4.1.0, 2021-05-18), all genes from the transcriptome data were clustered according to their expression levels across different NMN doses under poly(I:C)-inactive or poly(I:C)-active conditions. The minimum module size was set to 30, and the soft power of the scale-free topology model fit was set to 0.9. The eigengenes (eigengenes) of the identified modules were identified using the moduleEigengenes() function, which calculates the first principal component of each module. The correlation between the calculated module eigengenes and gene expression was calculated to quantify the similarity of all genes contained in the module.

[0043] Pathway Enrichment Analysis Pathway definitions were obtained from Gene Ontology (GO) (searched October 17, 2020). For each module identified by WGCNA, the overrepresentation of genes within the pathway was tested based on the pathway definition using the Benjamini and Hochberg method for FDR, and adjusted p-values ​​were calculated.

[0044] (Bayesian Network Analysis) Causal network inference was performed using a general Bayesian network in which each node models the expression level of an eigengene. A directed acyclic graph (DAG) of the Bayesian network structure was fitted to the expression levels of eigengenes using the bn.boot() function in the bnlearn package (version 4.6.2). To obtain a consensus network, network training was performed 500 times using three different hybrid structure learning algorithms: Max-Min Hill Climbing (MMHC), Hybrid HPC (H2PC), and the more general 2-phase Restricted Maximization (RSMAX2). The strength and directionality of each edge in each algorithm were calculated using boot.boot(). The strength() function was used to calculate the probability of observing an edge regardless of its direction in bootstrap replicates (ranging from 0.0 to 1.0) and the probability of a particular direction in bootstrap replicates given the presence of the edge (ranging from 0.0 to 1.0). Edges with strength > 0.1 and directionality > 0.5 were considered valid directed edges. The consensus network shown in Figure 5 was constructed using the sum of the strengths calculated by the three algorithms, so the strengths range from 0.0 to 3.0.

[0045] (Test Example 1) As described above for human primary cell culture, HPMEC and HCAEC were treated with poly(I:C). RT-PCR confirmed that the mRNA expression levels of IL6 and IL12 were significantly increased in both HPMEC (Figure 2A) and HCAEC (Figure 2B) after poly(I:C) activation. IL6 is a major inflammatory cytokine that is significantly and strongly upregulated during viral infections, such as SARS-CoV-2, influenza, and RSV infections. The increase in mRNA expression levels of IL6 and IL12 indicates that the addition of poly(I:C) to the culture medium successfully induced an intracellular inflammatory response.

[0046] Next, to examine the effect of NMN treatment on poly(I:C)-induced inflammatory responses, transcriptome data were obtained from three samples of HPMECs and HCAECs for each of eight different conditions, using three different NMN dose levels (0.1 mM, 1.0 mM, and 10.0 mM) and a control (0 mM) under poly(I:C) non-activation or poly(I:C) activation conditions. The results indicated that samples treated with a high NMN dose were clustered into small clusters in both HPMECs and HCAECs, regardless of poly(I:C) activation, suggesting that high NMN doses result in gene expression profiles that are more similar to each other than low and medium doses of NMN.

[0047] (Test Example 2) Principal component analysis (PCA) was performed to obtain an overview of the transcriptome data for HPMEC and HCAEC. For HPMEC, one replicate with low NMN (0.1 mM) in poly(I:C)-nonactivating conditions and one replicate with high NMN (10.0 mM) in poly(I:C)-nonactivating conditions were clear outliers. Similarly, for HCAEC, one replicate with low NMN (0.1 mM) in poly(I:C)-activating conditions was the exception. These clear outliers were excluded from further analysis. Figures 2C and 2D show the PCA results excluding these outliers. For both HPMEC and HCAEC, principal component 1 (PC1) distinguishes high NMN samples from other samples, and PC2 distinguishes poly(I:C)-nonactivating conditions from poly(I:C)-activating conditions. Furthermore, a tight clustering of high NMN samples was observed in the PCA plots in both conditions with and without poly(I:C) activation, suggesting distinct and unique expression changes in cell cultures at high NMN concentrations.

[0048] (Test Example 3) To determine the unique characteristics of samples treated with high-dose NMN, differentially expressed genes (DEGs) were identified between control (0 mM) and high-NMN (10 mM) samples in HPMEC (Figure 2E) and HCAEC (Figure 2F) under both poly(I:C)-inactivated and poly(I:C)-activated conditions. Figure 2E shows a volcano plot of differentially expressed genes (DEGs) between control and high-dose NMN treatment in HPMEC under poly(I:C)-inactivated and poly(I:C)-activated conditions, and Figure 2F shows a volcano plot of differentially expressed genes (DEGs) between control and high-dose NMN treatment in HCAEC under poly(I:C)-inactivated and poly(I:C)-activated conditions. DEG analysis revealed that in HPMEC under poly(I:C)-inactivated conditions, the number of DEGs was low between the control and high-NMN treatment groups, with 59 up-regulated genes and 76 down-regulated genes. In contrast, the number of up-regulated genes was higher under poly(I:C)-activating conditions, with 302 up-regulated genes and 308 down-regulated genes (Figure 2G). This result indicates that high-dose NMN application is more effective under poly(I:C)-activating conditions than under non-poly(I:C)-activating conditions in HPMECs. Meanwhile, the number of DEGs in HCAECs was relatively high, regardless of poly(I:C) activation (Figure 2H). Furthermore, pathway enrichment analysis based on DEGs revealed that pathways related to the extracellular matrix and cell population proliferation were overrepresented among genes up-regulated by high NMN under poly(I:C)-activating conditions in both HPMECs and HCAECs. Furthermore, pathways related to the mitotic cell cycle, cell division, and chromosome segregation / organization were overrepresented among genes down-regulated by high NMN under poly(I:C)-activating conditions.

[0049] (Example 4) Given the above common observations from DEG-based pathway enrichment analysis in HPMEC and HCAEC, further analysis was conducted to identify common regulatory mechanisms between the two cell types that may underlie NMN-induced gene expression changes during poly(I:C) induction. Weighted gene correlation network analysis (WGCNA) was performed to evaluate regulatory transcriptome modules during poly(I:C) activation and to investigate NMN dose effects. WGCNA clustered the HPMEC transcriptome data into 41 clusters and the HCAEC transcriptome data into 15 clusters, as shown in the cluster dendrograms in Figures 3A and 3B. Module names were based on a code indicating the cell line in which the module was defined (p:HPMEC, c:HCAEC), followed by a color name. The latter was an arbitrarily assigned term to allow easy-to-remember code reference for the modules. The expression trend of each module was statistically evaluated by correlation analysis against the signatures defined by "NMN dose level (control, low, medium, and high) and poly(I:C) activation." Five out of 41 HPMEC modules and seven out of 15 HCAEC modules were identified as affected modules. These showed dramatic expression changes (p-value <1E-5) as a result of poly(I:C) induction. Meanwhile, NMN dose signature analysis identified two HPMEC modules and one HCAEC module that showed significant expression changes.

[0050] (Test Example 5) To evaluate the effects of high-dose NMN on poly(I:C)-unactivated and poly(I:C)-activated samples, the fold change (FC) in the expression levels of eigengenes for all modules was determined between the control and high-dose NMN, as shown in Figures 3C and 3D. The x-axis represents the FC for the poly(I:C)-unactivated sample, and the y-axis represents the FC for the poly(I:C)-activated sample. Scatter plots enabled us to extract modules that differed in the effect of high NMN doses between poly(I:C)-unactivated and poly(I:C)-activated conditions. In HPMECs, the eigengenes of the p-green and p-sienna3 modules had much higher fold increases under poly(I:C)-unactivated conditions than under poly(I:C)-activated conditions (Figure 3C). Similarly, in HCAECs, the eigengenes of the c-magenta and c-purple modules were more upregulated (having a larger positive FC) under poly(I:C)-inactivated conditions than under poly(I:C)-activated conditions in HCAECs (Figure 3D). In the transcriptome data from HPMECs, APLN, a eigengene of the p-green module, was upregulated by NMN approximately twofold under poly(I:C)-inactivated conditions compared with poly(I:C)-activated conditions. CTSS, a eigengene of the p-sienna3 module in HPMECs, was upregulated by NMN more than twofold under poly(I:C)-inactivated conditions compared with poly(I:C)-activated conditions. Pathway enrichment analysis of gene members of the p-green and p-sienna3 modules in HPMEC revealed that pathways related to mitochondrial oxidative phosphorylation were overrepresented in the p-green module, and inflammatory pathways were overrepresented in the p-sienna3 module (Figure 3E). Pathways similar to those in the p-sienna3 module in HPMEC were found in the c-magenta and c-purple modules in HCAEC, respectively (Figure 3F).

[0051] (Test Example 6) Functional enrichment analysis of various doses of NMN was performed with or without poly(I:C) activation. Figures 4A and 4B show the changes in IL6 expression levels in HPMEC and HCAEC, respectively. In HPMEC, IL6 expression significantly increased after poly(I:C) activation, and the level was reduced by high NMN treatment in poly(I:C)-activated cell cultures (Figure 4A). IL6 expression was slightly increased after NMN treatment in poly(I:C)-unactivated cell cultures. On the other hand, in HCAEC, IL6 expression was reduced by high NMN treatment under both poly(I:C)-unactivated and poly(I:C)-activated conditions (Figure 4B). Interestingly, IL6 was co-expressed with members of the p-purple module in HPMEC and the c-yellow module in HCAEC, and these were identified as modules whose expression was reduced by NMN treatment under poly(I:C)-activated conditions compared to non-activated conditions. Figures 4C and 4D show the changes in expression levels of these targeted modules. The intrinsic genes of the p-purple module in HPMEC and the c-yellow module in HCAEC, respectively, were SAMHD1 and ANKFY1, whose expression was significantly increased upon poly(I:C) activation, and this increase was attenuated by high-dose NMN.

[0052] (Test Example 7) Next, the characteristics of gene members in the p-purple module of HPMEC and the c-yellow module of HCAEC were evaluated by pathway enrichment analysis (Figures 4C and 4D). In HPMEC, pathways overrepresented in the p-purple module were particularly related to inflammatory responses, particularly the type I interferon signaling pathway. Interestingly, the top five pathways overrepresented in the c-yellow module of HCAEC were completely different from those overrepresented in the p-purple module of HPMEC, including vitamin D and vascular patterning pathways, which appear to be unrelated to inflammation. Thus, evaluation of the causal network between modules revealed potential regulatory mechanisms underlying NMN-induced changes in poly(I:C)-induced inflammatory responses.

[0053] (Test Example 8) Based on the results of Test Example 7, we applied Bayesian network analysis to infer potential regulatory mechanisms underlying the effect of NMN treatment on poly(I:C)-induced inflammatory responses. A general Bayesian network, in which each node models the expression level of an eigengene, was used to predict a causal network. Figures 5A and 5B show the causal relationships between the eigengenes of all WGCNA modules predicted by Bayesian network analysis. The arrows represent causal regulation, and the values ​​on the arrows are the sum of the "strengths" calculated by three different algorithms of the hybrid method in the bnlearn package for HPMECs and HCAECs, respectively. In the predicted causal network, the modules with the strongest "strengths" include p-green, c-magenta, and c-purple, as well as p-lightgreen and c-brown, which are first shown in Figures 3C-3F. The p-purple module in HPMEC is predicted to be regulated by the p-darkred module (Fig. 5A), and the c-yellow module in HCAEC is predicted to be regulated by the c-black module via the c-tan module (Fig. 5B).

[0054] CD9, an intrinsic gene of the HPMEC p-lightgreen module, was upregulated by NMN treatment independent of poly(I:C) activation, and APLN, an intrinsic gene of the HPMEC p-lightgreen module, had an expression pattern identical to that of the HPMEC p-lightgreen module. Pathway enrichment analysis of gene members of the HPMEC p-lightgreen and p-green modules revealed that pathways related to mitochondrial oxidative phosphorylation were overrepresented in the p-green module, and antigen presentation-related pathways were overrepresented in the p-lightgreen module. The HCAEC c-brown and c-purple (Figure 3F) modules were predicted to regulate the c-magenta module (Figure 5B). Pathways related to antigen processing and presentation were overrepresented in gene members of the HCAEC c-purple module. Various pathways, including the apoptosis pathway, alpha signaling pathway, and NF-kappa B signaling pathway, were overrepresented in the c-magenta module of HCAEC. In the predicted network, common pathways, such as antigen processing and mitochondrial oxidative phosphorylation, were present between HPMEC and HCAEC. Genes related to apoptosis, alpha signaling pathway, and NF-kappa B signaling were frequently found in the c-magenta module of HCAEC; these factors were specific to HCAEC and were not included as overrepresented pathways in the p-lightgreen and p-green modules of HPMEC.

[0055] Regarding the regulatory mechanisms underlying the attenuation of poly(I:C)-induced inflammatory responses by NMN treatment, it is noteworthy that the causal relationship between the HPMEC p-darkred module and the p-purple module, in which IL6 is present, was relatively strong (Figure 5A). The intrinsic gene of the HPMEC p-darkred module is TNFRSF10D, a member of the TNF receptor superfamily. TNFRSF10D expression levels were decreased by poly(I:C) activation and increased by NMN under poly(I:C) activation conditions (Figure 6A). Furthermore, translation and viral infection / transcription pathways were overrepresented in the HPMEC p-darkred module, suggesting that NMN application plays an important role in restoring the expression levels of inflammation-related genes that were decreased by poly(I:C) activation. Bayesian network analysis demonstrated that NMN treatment induced a direct causal relationship between translation and viral infection / transcription pathways and the reduced expression of genes including IL6 in HPMECs.

[0056] On the other hand, the c-yellow module of HCAECs was included in a predicted regulatory network consisting of four modules: c-black, c-tan, c-yellow, and c-green (Figure 5B). The eigengene of the c-black module of HCAECs was HERPUD1, which plays an important role in the unfolded protein response (UPR) and ER-associated protein degradation (ERAD) pathways. HERPUD1 expression was increased by NMN under poly(I:C)-activating conditions (Figure 6B). Pathways related to platelet-derived growth factor binding, actin binding, cortical actin cytoskeleton organization, extracellular matrix structural components, and axon guidance were overrepresented in the c-black module of HCAECs. In particular, the platelet-derived growth factor binding pathway was overexpressed in HCAECs but not in HPMECs. This pathway may represent a response specifically observed in cardiac cells as a regulatory mechanism for the alleviation of inflammation by NMN treatment under poly(I:C)-activating conditions. This suggests that NMN treatment may increase the expression of genes associated with the platelet-derived growth factor binding pathway under poly(I:C)-activating conditions, thereby alleviating the inflammatory response in HCAECs.

[0057] Although the top five pathways overrepresented in the putative regulatory gene clusters were different between HPMEC and HCAEC (Figures 6A and 6B), several pathways and genes were shared (Figure 6C). The number of pathways enriched in the HPMEC p-darkred and HCAEC c-black modules, as well as the number of pathways shared by the two modules, are shown in Figure 6C. Seven shared pathways are listed with their p-values ​​in the HPMEC p-darkred and HCAEC c-black modules in the panels of Figure 6C. These pathways include "cellular protein metabolic process," "cytoplasmic ribosomal large subunit," and "extracellular matrix." EIF4B (eukaryotic translation initiation factor) was one of the shared genes between the HPMEC p-darkred and HCAEC c-black modules, and is involved in the shared pathway (translation initiation) shown in Figure 6C. Other common genes were RPS3A, RPL4, RPL7A, and RPL17. NMN treatment generally increased the expression level of EIF4B in both HPMEC and HCAEC (Fig. 6D).

[0058] EIF4B is known to be a cofactor of the RNA helicase EIF4A, which unwinds local secondary structures to generate ribosome landing pads on mRNA. Although the relationship between poly(I:C)-induced inflammatory responses, NMN treatment, and EIF4B expression levels remains unclear, it is noteworthy that EIF4B, along with EIF4A, plays an important role in translation initiation. Furthermore, EIF4B is regulated by two major oncogenic signaling pathways, Ras-MAPK and PI3-K / mTOR, making them important targets for cancer therapy.

[0059] The PARP family includes major NAD(+)-consuming enzymes that mediate host defense responses via ADP-ribosylation. Poly(I:C) activation increased the expression of genes in the same module as IL6. Furthermore, NMN treatment decreased the expression of poly(ADP-ribose) polymerase (PARP) family genes. Based on these results, we hypothesized an NMN-driven mechanism for attenuating poly(I:C)-induced inflammatory responses (Figure 6E). The hypothesis is as follows: NMN treatment increases intracellular NAD(+), and the PARP family consumes NAD(+) to mediate host defense responses against viruses (poly(I:C) activation) via ADP-ribosylation. As a result, ADP-ribosylation attenuates the inflammatory response triggered by poly(I:C) activation. The PARP family includes NAD(+)-consuming enzymes that transfer a single ADP-ribose (ADPR) unit from NAD(+) to a polypeptide side chain to modify protein function. Because several PARPs are known to be induced by interferon, ADP-ribosylation is thought to be a host defense response. In Figure 6E, this defense response is indicated by a repression arrow from "ADPR" to "virus."

[0060] In HCAECs, IL6 was contained in the c-yellow module, and STAT1 was contained in the c-green module (Figure 6F). The intrinsic gene of the c-green module in HCAECs was PARP14, and NMN treatment reduced PARP14 expression in a concentration-dependent manner under poly(I:C)-activated conditions. In both HPMECs and HCAECs, NMN treatment reduced PARP9 and PARP12 expression only under poly(I:C)-activated conditions (Figures 6G and 6H). These results further support our hypothesis that PARP-dependent ADP-ribosylation may suppress the inflammatory response induced by poly(I:C) activation (Figure 6E). Poly(I:C) activation significantly increased the expression of PARP9 and PARP12, and NMN treatment attenuated this increase in expression. This indicates that NMN treatment has a significant effect on poly(I:C)-induced inflammatory responses.

[0061] (Discussion) The main discovery that led to this invention was that expression profiling revealed the anti-inflammatory activity of NMN and suggested its underlying pathway. Poly(I:C) mimics viral infection and induces inflammatory responses. IL6 is an important marker of inflammation and is upregulated in many diseases, including cardiovascular and cardiocerebrovascular diseases. In HPMECs, RT-PCR and RNA-seq revealed an increase in IL6 expression after poly(I:C) intervention, and a significant decrease in IL6 expression was observed after subsequent NMN treatment. In HCAECs, dysregulation of IL6 expression was not significant. WGCNA was applied because gene expression changes are time-dependent, RNA-seq is vulnerable to noise, and gene clusters are more robust than differential expression profiling of single genes. Because the IL6-containing gene cluster was highly enriched in the GO pathway in HCAECs and had no clear association with inflammation, this clustering approach did not immediately support the existence of NMN-mediated reduction in poly(I:C)-induced inflammation. Therefore, we next performed Bayesian network analysis to reveal regulatory control of the c-black module in HCAECs through the c-tan module and the c-yellow module, which includes IL6. Interestingly, the platelet-derived growth factor (PDGF)-binding pathway was a highly overrepresented pathway in the c-black module. PDGF is closely related to inflammation. The most representative member of the c-black module in HCAECs, its intrinsic gene HERPUD1, was significantly upregulated by poly(I:C). Therefore, a systems biology approach supported the existence of a causal relationship between NMN treatment and PDGF-mediated reduction in inflammation in both HPMECs and HCAECs.

[0062] The PARP family comprises multifaceted proteins with diverse roles, including roles in DNA damage response, and plays a functional role in responding to viral infection and inflammation. In the present invention, it has been revealed that specific members of the PARP family are dysregulated by poly(I:C) intervention and NMN treatment, as discussed in the following paragraphs.

[0063] The differential expression of PARP9, 12, and 14 is intriguing because these proteins play a central role in the concept of the "NAD+ battlefield." These three PARPs consume NAD+ to define post-translational modifications, while some viruses encode functions to reverse these modifications. In both cell lines, poly(I:C) significantly upregulated PARP9, 12, and 14. NMN treatment significantly downregulated PARP9 and 12 in HCAECs and PARP9 in HPMECs (Figures 6G and 6H). One possible explanation is that as NAD+ is increased by the addition of NMN, the PARP pool becomes more effective at inducing post-translational modifications. Utilizing abundant NAD+ may impose a lower overall energy burden on the cell than attempting to counter viral activity by upregulating PARPs, even in the absence of NAD+-boosting NMN.

[0064] The raw and processed RNA-seq data generated and analyzed in the examples are available at NCBI GEO under accession number GSE233679 (https: / / www.ncbi.nlm.nih.gov / geo / query / acc.cgi?acc=GSE233679).

Claims

1. An agent for suppressing inflammatory cytokine expression, containing nicotinamide mononucleotide as an active ingredient.

2. The inflammatory cytokine expression inhibitor according to claim 1, wherein the inflammatory cytokine is at least one of interleukin 1, interleukin 6, and TNF-α.

3. The inflammatory cytokine expression inhibitor according to claim 1, wherein the inflammatory cytokine is expressed in pulmonary microvascular endothelial cells or coronary artery endothelial cells.

4. The inflammatory cytokine expression inhibitor according to claim 1, wherein the production of inflammatory cytokines is induced by dsRNA or synthetic dsRNA (poly(I:C)).

5. The inflammatory cytokine expression inhibitor according to claim 1, which has a preventive or therapeutic effect against cytokine storm or vasculitis.

6. The inflammatory cytokine expression inhibitor according to claim 1, which is a food for preventing or treating cytokine storm or vasculitis.

7. The inflammatory cytokine expression inhibitor described in claim 1, which is a pharmaceutical for preventing or treating cytokine storm or vasculitis.

8. A method for inhibiting inflammatory cytokine expression in a mammal, other than a human, comprising administering an effective amount of nicotinamide mononucleotide to a subject, other than a human, mammal, in need of inhibition of inflammatory cytokine expression.

9. The method for inhibiting inflammatory cytokine expression according to claim 8, wherein the inflammatory cytokine is expressed in pulmonary microvascular endothelial cells or coronary artery endothelial cells.

10. A method for suppressing inflammatory cytokine expression according to claim 8 or 9, wherein the production of inflammatory cytokines is induced by dsRNA or synthetic dsRNA (poly(I:C)).

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