Composition for improving brain function

Abstract

To provide a composition for promoting NGF production, a composition for promoting BDNF production, a composition for improving brain function, a composition for promoting Tollip production, and an anti-inflammatory composition.SOLUTION: Each of the compositions contains at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p as an active constituent.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition for improving brain function that has an effect of promoting the production of nerve growth factor (NGF) and / or brain-derived neurotrophic factor (BDNF), and also to a composition for promoting NGF production and a composition for promoting BDNF production. The present invention further relates to an anti-inflammatory composition having an effect of promoting Tollip production, and a composition for promoting Tollip production. [Background technology]

[0002] The decline in brain function due to stress and aging can lead to memory loss, depression, insomnia, dementia, and other conditions, significantly reducing quality of life (QOL). In Japan, where stress levels are rising and the population is rapidly aging, the increase in the number of people suffering from mental illnesses such as depression and dementia has become a social problem, and active research is being conducted to discover substances that can suppress, improve, or enhance the decline in brain function.

[0003] In recent years, nerve growth factor (NGF) (simply referred to as "NGF" in the present invention) and brain-derived neurotrophic factor (BDNF) (simply referred to as "BDNF" in the present invention) have attracted attention as factors that have functions such as maintaining the survival and growth of nerve cells and enhancing synaptic function, and contribute to maintaining and improving cognitive ability and memory.

[0004] NGF is abundant in the brain, particularly in the hippocampus and cerebral cortex, where NGF mRNA levels are also high. NGF is believed to be an essential trophic factor for the survival and function of cholinergic neurons, suggesting its potential as a therapeutic agent for Alzheimer's disease, a disease characterized by the degeneration and loss of these neurons. However, NGF is a protein with a molecular weight of approximately 13,000 and does not cross the blood-brain barrier. Therefore, if low-molecular-weight compounds that can cross the blood-brain barrier and promote NGF production in the brain can be identified, these compounds may be able to activate impaired basal forebrain cholinergic neurons and improve brain function.

[0005] BDNF is known to play an important role in synaptic plasticity and neuronal survival. It has been suggested that a decrease in BDNF is involved in the onset of neuropsychiatric disorders such as depression. Indeed, decreased BDNF expression has been confirmed in the brains of many patients with neurodegenerative diseases, psychiatric disorders, and depression (Non-Patent Documents 1 and 2). Furthermore, in experiments using depression model animals, it has been reported that intravenous administration of BDNF improves behavioral abnormalities, and that increased BDNF enhances performance such as memory and learning (Non-Patent Document 3). Therefore, increasing BDNF is expected to exert effects such as improved or improved brain function, improved cognition or memory, antidepressant or anti-anxiety effects, increased energy, and mental stabilization.

[0006] Toll-interacting protein (Tollip) (referred to simply as "Tollip" in this study) is a major inhibitory adaptor protein (TLR negative regulator) in the TLR signaling pathway. Tollip contains an N-terminal Myb1-binding domain, a conserved core domain 2, and a C-terminal portion that binds ubiquitin for endoplasmic reticulum degradation. It regulates the TLR-induced MyD88-dependent NF-κB activation pathway in two ways. First, Tollip directly interacts with IL-1R and TLR4 via the TLR-TIR domain via the intact Tollip C-terminal region, conjugating ubiquitin to the endoplasmic reticulum degradation domain, thereby suppressing TLR-induced immune responses. Second, Tollip potently suppresses the activity of IL-1R-associated kinases after TLR activation. In fact, overexpression of Tollip inhibits the activation of NF-κB in response to TLR and IL-1R ligand binding, suggesting that Tollip negatively regulates TLR-mediated signal transduction (see Non-Patent Document 4).

[0007] TLRs transmit downstream signals via MyD88 and several adaptor proteins, including MyD88. TLR signaling is essential for regulating the innate immune system, and recent studies have suggested that abnormal TLR signaling is involved in several diseases, including insulin resistance, Alzheimer's disease, tumor metastasis, chronic kidney disease, and cardiovascular disease. Furthermore, recent studies have shown that inhibition of the TLR-4 signaling pathway may be a target for suppressing chronic inflammatory diseases (see Non-Patent Document 4).

[0008] Inflammation is typically considered an important signal that determines a patient's pathological state and is defined as the tissue response to harmful stimuli. Inflammation is associated with arthritis, asthma, cancer, diabetes, autoimmune diseases, and other conditions and is recognized as a major precursor to these diseases. Macrophages, which play a central role in the innate immune response, are involved in phagocytosis, the generation of bactericidal reactive oxygen and nitrogen intermediates, and the production of cytokines and chemokines. Proinflammatory cytokines and chemokines produced by stimulated macrophages include interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α).

[0009] Epigallocatechin-3-O-gallate (EGCG), the major polyphenol in green tea, is known to play a central role in the anti-inflammatory effects of green tea polyphenols. Recent studies have shown that upregulation of Tollip (increased expression) also plays an essential role in the anti-inflammatory effects of EGCG. This EGCG-induced increase in Tollip expression is suppressed by Src-1 inhibitors, Akt1 / 2 inhibitors, and NO synthase inhibitors, suggesting that the Src / Akt / eNOS axis acts upstream of EGCG-induced Tollip expression (see Non-Patent Document 4).

[0010] Based on these findings, it is expected that inflammation can be suppressed and improved by increasing Tollip expression. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Chen B et al, Biol Psychiatry. 50:260-265(2001). [Non-patent document 2] Phillips HS et al. Neuron. 7:695-702(1991). [Non-patent document 3] Neeper SA et al. Brain Res, 726:49-56(1996). [Non-patent document 4] Motofumi Kumaoe et al., J immunol 199(9): 3261-3269 (2017) [Non-patent document 5] van Rooij and Kauppinen, EMBO Mol Med. 2014;6(7):851-64 [Non-patent document 6] Chorn et al., RNA. 2012;18(10):1796-804 Summary of the Invention [Problem to be solved by the invention]

[0012] An object of the present invention is to provide a composition for improving brain function that has the effect of promoting the production of NGF and / or BDNF. Another object of the present invention is to provide a composition for promoting NGF production and a composition for promoting BDNF production. Another object of the present invention is to provide an anti-inflammatory composition and a composition for promoting Tollip production. [Means for solving the problem]

[0013] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that the introduction of miR-26a-5p mimic and / or miR-181a-5p mimic into human neuroblastoma significantly increases the expression of NGF and BDNF. As described above, NGF and BDNF are factors that maintain neuronal survival and growth, enhance synaptic function, and contribute to the maintenance and improvement of cognitive ability and memory. Therefore, increasing these factors in neurons can improve brain function, and can be expected to have effects such as improved cognition, improved performance in memory and learning, suppression of cognitive decline associated with stress and aging, and prevention or improvement of mental disorders such as depression and dementia.

[0014] Furthermore, the present inventors found that introducing miR-26a-5p mimic and / or miR-181a-5p mimic into human umbilical vein endothelial cells significantly increased Tollip expression and production. As described above, Tollip is known to negatively regulate TLR-mediated signaling, thereby suppressing TLR-induced inflammatory diseases. Therefore, increasing the above factors in vascular endothelial cells, for example, can be expected to have the effect of suppressing or ameliorating inflammation.

[0015] The present invention was completed through further investigation based on these findings, and includes the following embodiments. (I) Composition based on the effect of promoting NGF and / or BDNF production (I-1) A composition for promoting NGF production, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p. (I-2) A composition for promoting BDNF production, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p. (I-3) A composition for improving brain function, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p.

[0016] (II) Composition Based on Tollip Production Promoting Activity (II-1) A composition for promoting Tollip production, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p. (II-2) An anti-inflammatory composition comprising, as an active ingredient, at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p. [Effects of the Invention]

[0017] The composition for promoting NGF production of the present invention is used to increase the expression and production of NGF in nerve cells. By using the composition, the expression and production of NGF in nerve cells can be increased, thereby improving brain function in particular.

[0018] The composition for promoting BDNF production of the present invention is used to increase the expression and production of BDNF in neurons. By using the composition for promoting BDNF production of the present invention, the expression of BDNF in neurons can be increased, thereby improving brain function in particular.

[0019] The brain function-improving composition of the present invention is used to improve brain function in mammals, including humans. The brain function-improving composition of the present invention has the effect of increasing the expression and production of NGF and / or BDNF in neurons, and based on this effect, human brain function can be improved. Therefore, the brain function-improving composition of the present invention can be expected to have effects such as improving cognition, improving performance such as memory and learning, suppressing cognitive decline associated with stress and aging, and preventing or ameliorating mental disorders such as depression and dementia.

[0020] The composition for promoting Tollip production of the present invention is used to increase the expression and production of Tollip in the biological cells of mammals, including humans. By increasing the expression and production of Tollip in cells, the composition can control biological functions and exert an effect of suppressing or ameliorating inflammation.

[0021] The anti-inflammatory composition of the present invention is used to suppress or ameliorate inflammation. The anti-inflammatory composition of the present invention can suppress or ameliorate inflammation, particularly TLR-induced inflammation, by increasing the expression and production of Tollip in living cells. [Brief explanation of the drawings]

[0022] [Figure 1]The figures show the results of measuring the expression levels of miRNAs (Fig. 1(A): miR-26a-5p, Fig. 1(B): miR-181a-5p) in SH-SY5Y cells after introduction of miR-26a-5p mimic and / or miR-181a-5p mimic in Experimental Example 1. In the figures, the "+" and "-" symbols next to "26a mimic" and "181a mimic" on the horizontal axis indicate the presence or absence of introduction of miR-26a-5p mimic and miR-181a-5p mimic into SH-SY5Y cells (the same applies to Figs. 2 and 3 below). [Figure 2] The results show the evaluation of the effect of introduction of miR-26a-5p mimic and / or miR-181a-5p mimic on NGF mRNA expression in SH-SY5Y cells in Experimental Example 1. Mean ± SE, n = 6, Dunnett's Multiple Comparison Test, *P < 0.05, **P < 0.01, Student's t-test, ##P < 0.01 (same as in Figure 3). [Figure 3] 1 shows the results of evaluating the effect of introduction of miR-26a-5p mimic and / or miR-181a-5p mimic on BDNF mRNA expression in SH-SY5Y cells in Experimental Example 1. [Figure 4] This shows the results of measuring the amount of Tollip produced in HUVEC cells after introduction of miR-26a-5p mimic and / or miR-181a-5p mimic in Experimental Example 2. Mean ± SE, n = 4, Dunnett's Multiple Comparison Test, *P < 0.05. DETAILED DESCRIPTION OF THE INVENTION

[0023] (1) Active ingredient of the composition The present invention provides a composition for promoting NGF production, a composition for promoting BDNF production, a composition for improving brain function, a composition for promoting Tollip production, and an anti-inflammatory composition. Hereinafter, these compositions may be collectively referred to as "the present composition." Each of the compositions is characterized by containing miRNA as an active ingredient.

[0024] Examples of miRNAs used as active ingredients in compositions for promoting NGF production or for improving brain function include miRNAs that, when transfected into human neuroblastoma cells, can increase the expression level of NGF mRNA compared to cells transfected with a negative control miRNA mimic (NC miRNA mimic) that does not contain the target miRNA. As shown in Experimental Example 1 below, examples of miRNAs with such an effect include at least one selected from the group consisting of miR-26a-5p and miR-181a-5p. All of these miRNAs are human miRNAs (has-miRNAs).

[0025] Furthermore, miRNAs used as active ingredients in compositions for promoting BDNF production or for improving brain function include miRNAs that, when transfected into human neuroblastoma cells, can increase the expression level of BDNF mRNA compared to the same cells transfected with an NC miRNA mimic that does not contain the target miRNA. As shown in Experimental Example 1 below, miRNAs with such an effect can include at least one selected from the group consisting of miR-26a-5p and miR-181a-5p. All of these miRNAs are human miRNAs (has-miRNAs).

[0026] Examples of miRNAs used as active ingredients in compositions for promoting Tollip production and anti-inflammatory compositions include miRNAs that, when transfected into human umbilical vein endothelial cells, can increase Tollip production compared to the same cells transfected with an NC miRNA mimic that does not contain the target miRNA. As shown in Experimental Example 2 below, miRNAs with this effect include at least one selected from the group consisting of miR-26a-5p and miR-181a-5p. All of these miRNAs are human miRNAs (has-miRNAs).

[0027] miRBase (http: / / www.mirbase.org / ) is an online database where miRNA sequences and annotations are registered and stored. The nucleotide sequences and accession numbers of the mature miRNAs hsa-miR-26a-5p and hsa-miR-181a-5p registered in miRBase, as well as the sequence numbers (SEQ ID No.:) in the attached sequence listing, are shown in Table 1.

[0028] [Table 1]

[0029] In the present invention, the term "miRNA" refers to microRNA. miRNAs are short non-coding RNAs consisting of 20 to 25 nucleotides and include those that bind to target mRNAs and inhibit their translation (mature miRNAs). miRNAs may also be precursors that generate the mature miRNAs in vivo. In other words, the miRNAs targeted by the present invention may be single-stranded (mature, capable of immediately binding to target mRNAs) or may be double-stranded (pri-miRNA, pre-miRNA, double-stranded miRNA) that correspond to the precursors.

[0030] The typical process for generating mature miRNA is as follows. RNA polymerase II generates single-stranded primary transcripts (primary miRNAs: pri-miRNAs) from miRNA genes, each of which has one or more hairpin structures. The pri-miRNAs are then cleaved by the RNase III enzyme Drosha to generate precursor miRNAs (pre-miRNAs), which are intermediate precursors with hairpin structures. The pre-miRNAs are then cleaved in the cytoplasm by the RNase III enzyme Dicer to generate double-stranded miRNAs consisting of a mature miRNA (guide strand) and its antisense miRNA* (passenger strand). The double-stranded miRNAs are incorporated into the RNA-induced silencing complex (RISC). The double-stranded miRNAs incorporated into the RISC are unwound in the RISC, resulting in two single-stranded miRNAs. One of the unstable single strands is degraded, while the other stable single strand functions as a mature miRNA.

[0031] The miRNAs targeted by the present invention may be those produced within an organism, those produced in cells or cell lines extracted from an organism, or those artificially synthesized. They may also be commercially available. Furthermore, the miRNAs targeted by the present invention are not limited to endogenous miRNAs derived from an organism or those having the same structure as endogenous miRNAs, but may also be nucleic acid molecules that mimic the function of endogenous miRNAs (miRNA mimics). MiRNA mimics are compounds well known in the art as nucleic acid molecules that mimic endogenous miRNAs (see, for example, Non-Patent Documents 5 and 6). In this specification, when a miRNA is specifically a miRNA mimic, it is clearly distinguished by adding the term "mimic" to the end of the term.

[0032] A miRNA mimic can have a base sequence (similar sequence) in which one or more nucleotides are deleted, substituted, or added to the base sequence of an endogenous miRNA, as long as it mimics the function of the endogenous miRNA. In this case, the identity with the base sequence of the target endogenous miRNA (e.g., mature miRNA) is preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and particularly preferably 95% or more. The "percent identity" between the base sequences of two miRNAs refers to the percentage of identical nucleotides between the two sequences compared after best alignment (optimal alignment). This percent identity can be calculated using any sequence analysis method well known to those skilled in the art. Furthermore, miRNA mimics may be double-stranded or single-stranded, as long as they mimic the function of endogenous miRNAs. If they are double-stranded, they may contain one or more single-stranded portions. Furthermore, miRNA mimics may contain chemical modifications, as long as they mimic the function of endogenous miRNAs. Examples of chemical modifications include, but are not limited to, 2'-fluoro modifications, 2'-O-methyl modifications, 2'-O-methoxyethyl modifications, LNA, phosphorothioate bonds, morpholinos, and PNAs. Chemical modifications include those for increasing stability against RNases and RISCs.

[0033] For example, the mimics of the aforementioned hsa-miR-26a-5p (mature miRNA) and hsa-miR-181a-5p (mature miRNA) are synthetic RNA molecules that have sequences similar to those of hsa-miR-26a-5p (mature miRNA) and hsa-miR-181a-5p (mature miRNA), respectively, and are capable of upregulating the expression of target genes (NGF gene, BDNF gene, or Tollip gene). These are commercially available.

[0034] The miRNA, the active ingredient of the present composition, may be used in its naked state. However, it is preferable to use it in combination with any known delivery vehicle that has the relevant function to increase its stability and to support, promote, or facilitate delivery to the site of action (target cells). Delivery vehicles can be viral or non-viral vectors. Viral vectors include, but are not limited to, vectors based on adenovirus, adeno-associated virus (AAV), retrovirus, vaccinia virus, poxvirus, lentivirus, herpes virus, etc. Non-viral vectors include, but are not limited to, particulate carriers such as polymer particles, lipid particles, and inorganic particles, as well as bacterial vectors. Particulate carriers can be nanoparticles with nanometer-sized particles. Polymer particles include, but are not limited to, polymers such as cationic polymers, polyamidoamine (PAMAM), chitosan, cyclodextrin, poly(lactic-co-glycolic acid) (PLGA), poly(lactic-co-caprolactonic acid) (PLCA), poly(β-amino ester), and atelocollagen. Lipid particles include liposomes and non-liposomal lipid particles. Liposomes are vesicles with an internal lumen surrounded by a lipid bilayer membrane, while non-liposomal lipid particles are lipid particles that do not have such a structure. Examples of inorganic particles include gold nanoparticles, quantum dots, silica nanoparticles, iron oxide nanoparticles (e.g., superparamagnetic iron oxide nanoparticles (SPIONs)), nanotubes (e.g., carbon nanotubes (CNTs)), nanodiamonds, and fullerenes. Examples of bacterial vectors include, but are not limited to, those based on Listeria monocytogenes, Bifidobacteria, and Salmonella typhimurium.

[0035] (2) Form of the composition The composition can be a parenteral or an oral composition. Parenteral compositions include pharmaceuticals and quasi-drugs that are administered intravenously, intramuscularly, intradermally, subcutaneously, intraperitoneally, or intraintestinally. For example, they may have forms known in the art, such as injections, drip infusions, enteral preparations, suppositories, and topical preparations. Oral compositions include foods and beverages, quasi-drugs, and pharmaceuticals, and may be prepared in forms known in the art. These forms include tablets, capsules, powders, granules, pills, suspensions, emulsions, liquids, drinks, syrups, and other formulations, as well as common food and beverage forms. Furthermore, the foods and drinks covered by the present invention include health foods, functional foods, nutritional supplements, foods with functional claims, foods for specified health uses, and foods for the sick.

[0036] Therefore, in addition to an effective amount of miRNA (at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p), the present composition can contain pharmaceutically acceptable or edible ingredients (food and beverage ingredients, food additives) to the extent that the effects of the present invention are not impaired. Pharmaceutically acceptable ingredients can be selected from ingredients known in the art depending on the intended use and formulation, and examples include excipients, diluents, dispersants, preservatives, stabilizers, lubricants, buffers, and pH adjusters. Edible ingredients include proteins, carbohydrates, lipids, minerals, and vitamins as nutrient sources; sugars, sugar alcohols, sweeteners, acidulants, fruit juices, flavorings, flavorings, and other ingredients for imparting palatability; and thickening polysaccharides and emulsifiers.

[0037] The optimal mode of administration, dosage and formulation of the present composition can be determined depending on the age or weight of the subject to be administered, the condition (pathological condition) of the subject, and the purpose (application) of administration.

[0038] (3) Use of the composition The present composition can be used for the following applications, which are not limited to therapeutic applications but also include non-therapeutic applications.

[0039] (a) Composition for promoting NGF production The composition can be a composition for promoting NGF production. The composition for promoting NGF production is a parenteral or oral composition for mammals (humans and non-human animals), including humans (hereinafter referred to as "humans"), used to promote NGF production in living cells. Here, "promoting NGF production" also means promoting "NGF mRNA expression," which is a prerequisite for NGF production. "Biological cells" also include human nerve cells. The miRNA to be incorporated into the composition for promoting NGF production may be at least one selected from the group consisting of miR-26a-5p and miR-181a-5p, and may also be incorporated in combination with both, preferably a combination of both (see Figure 2). The amount of miRNA in the composition for promoting NGF production may be determined as long as the amount of NGF production (including the amount of NGF mRNA expression) in living cells increases by a factor of 1 to 100% by mass when the composition is administered to humans, compared to when the composition is not administered. For example, whether or not the amount of NGF mRNA expression in living cells increases can be evaluated according to the method described in Experimental Example 1 below.

[0040] (b) Composition for promoting BDNF production The composition may be a composition for promoting BDNF production. The composition for promoting BDNF production is a parenteral or oral composition for humans that is used to promote BDNF production in living cells. Here, "promoting BDNF production" also means promoting "BDNF mRNA expression," which is a prerequisite for BDNF production. "Biological cells" also include human nerve cells. The miRNA to be incorporated into the composition for promoting BDNF production may be at least one selected from the group consisting of miR-26a-5p and miR-181a-5p, or a combination of both. Preferably, miR-181a-5p is used due to its high BDNF production-promoting effect (see Figure 2). The amount of miRNA in the composition for promoting BDNF production may be set appropriately within the range of 1 to 100% by mass, as long as the amount of BDNF production (including the amount of BDNF mRNA expression) in biological cells increases when the composition is administered to humans compared to when the composition is not administered. For example, whether or not the amount of BDNF mRNA expression in biological cells increases can be evaluated according to the method described in Experimental Example 1 below.

[0041] (c) Composition for improving brain function The composition may be a composition for improving brain function. The composition for improving brain function is a parenteral or oral composition used to improve brain function in humans. Here, "improving function" includes the meanings of suppressing functional decline, maintaining function (maintaining the current state by suppressing functional decline), and improving function. The composition for improving brain function has the effect of promoting NGF production and the effect of promoting BDNF production based on the respective effects of miR-26a-5p and / or miR-181a-5p described above. As mentioned above, NGF is abundant in the brain, particularly in the hippocampus and cerebral cortex, and is a trophic factor essential for the survival and function of cholinergic neurons. BDNF is also a neurotrophic factor involved in the development, growth, maintenance, and regeneration of neurons, and is known to enhance memory and learning performance (Non-Patent Document 3). Therefore, the present composition can be ingested or administered to individuals seeking to improve memory, learning ability, or learning efficiency (e.g., students preparing for exams, individuals concerned about declining memory, learning ability, or learning efficiency) as a composition to aid in the improvement of memory or learning performance. Furthermore, it is known that BDNF expression levels in the brain are reduced in patients with depression or Alzheimer's dementia (Non-Patent Documents 1 and 2). Therefore, the present composition can be ingested or administered to patients with dementia, particularly Alzheimer's dementia, or those at risk of developing the condition, as a composition for improving cognitive function, preventing cognitive decline, or enhancing cognitive function. Here, "cognitive function" refers to the ability to correctly understand and appropriately perform tasks, and includes memory, language ability, judgment (including orientation and attention), calculation ability, and / or executive function. Pre-dementia also includes mild cognitive impairment. Since it is estimated that one in four people aged 65 or older has dementia or mild cognitive impairment, elderly people aged 65 or older may also be considered pre-dementia in the present invention. Furthermore, the present composition can be administered or ingested as a depression-improving composition to patients with depression (including new-type depression), or to those in a depressed state or prone to depression. Furthermore, the brain function-improving composition can also be effectively used as an agent for preventing and ameliorating the progression of degenerative diseases of the central nervous system. In addition to Alzheimer's disease, such diseases include diabetes, familial autonomic neuropathy, neurofibroma, neuroblastoma, and pheochromocytoma.

[0042] (d) Composition for promoting Tollip production The composition may be a composition for promoting Tollip production. The composition for promoting Tollip production is a parenteral or oral composition for humans that is used to promote Tollip production in living cells. Here, "promoting Tollip production" also means promoting "Tollip mRNA expression," which is a prerequisite for Tollip production. "Biological cells" also include human vascular endothelial cells. The miRNA to be incorporated into the composition for promoting Tollip production may be at least one selected from the group consisting of miR-26a-5p and miR-181a-5p, or a combination of both. Preferably, miR-181a-5p is used because of its high Tollip production-promoting effect (see Figure 3). The amount of miRNA in a composition for promoting Tollip production may be any amount that increases the amount of Tollip production (including the amount of Tollip mRNA expression) in biological cells when the composition is administered to humans compared to when the composition is not administered, and can be appropriately set within the range of 1 to 100% by mass. For example, whether or not there is an increase in the amount of Tollip production in biological cells can be evaluated according to the method described in Experimental Example 2 below.

[0043] (e) Anti-inflammatory composition The composition may be an anti-inflammatory composition. The anti-inflammatory composition is a parenteral or oral composition used to suppress or ameliorate inflammation (inflammatory diseases) occurring in the human body. The anti-inflammatory composition has a Tollip production-promoting effect based on the effects of the aforementioned miR-26a-5p and / or miR-181a-5p, respectively. As mentioned above, TLR signaling is essential for controlling the innate immune system. Recent studies have reported that abnormal TLR signaling is involved in chronic inflammatory diseases, that Tollip negatively regulates TLR signaling, and that upregulation of Tollip (increased expression) plays an essential role in the anti-inflammatory effects of EGCG (see Non-Patent Document 4). For this reason, the inflammation targeted by the present composition is preferably TLR-induced inflammation, including non-infectious inflammation, autoimmune inflammation, and the like. The present composition can be used as a composition for suppressing or ameliorating such TLR-induced inflammation, and can be preferably ingested or administered to patients with inflammation induced by enhanced TLR signaling or to those at risk of developing inflammation.

[0044] As described above, in this specification, the terms "comprise" and "contain" encompass the meanings of "consist of" and "consist essentially of." [Example]

[0045] The present invention will be explained below using experimental examples to aid in understanding the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. Unless otherwise specified, the following experiments were carried out at room temperature (25±5°C) and atmospheric pressure. Unless otherwise specified, "%" and "parts" in the following descriptions mean "% by mass" and "parts by mass," respectively.

[0046] The materials and equipment used in the following experimental examples are as follows: SH-SY5Y cells: human neuroblastoma (obtained from ATCC). HUVEC cells: normal human umbilical vein endothelial cells (obtained from Lonza: lot C2519A). 10% FBS-EMEM / F12 medium: A medium for culturing SH-SY5Y cells prepared by adding FBS (fetal bovine serum) to EMEM / F12 medium (a 1:1 mixture of EMEM (Eagle's Minimum Essential Medium) and Ham's F12 medium) to a concentration of 10% by mass. EGM2 medium: EBM2 medium (manufactured by Lonza) TM -2 BulletKit TM (manufactured by Lonza) and added the accompanying reagents (except FBS). 2% FBS-EGM2 medium: a medium for culturing HUVEC cells prepared by adding FBS to the EGM2 medium to a concentration of 5% by mass. The FBS used here was the FBS included with the EBM2 medium (manufactured by Lonza). miR-26a-5p mimic: A small double-stranded RNA molecule (chemically synthesized) designed to mimic the endogenous mature miRNA (hsa-miR-26a-5p: SEQ ID NO: 1) molecule when introduced into cells (obtained from Thermo Fisher Scientific). miR-181a-5p mimic: A small double-stranded RNA molecule (chemically synthesized) designed to mimic the endogenous mature miRNA (hsa-miR-181a-5p: SEQ ID NO: 2) molecule when introduced into cells (obtained from Thermo Fisher Scientific). NC mimic: Negative Control mimic: miRNA mimic molecule with a unique sequence that does not target any human, mouse, or rat gene (obtained from Thermo Fisher Scientific). mRNA delivery reagent: Lipofectamine TM RNAiMAX Transfection Reagent (Thermo Fisher Scientific) Anti-Tollip antibody: obtained from Proteintech.

[0047] Experimental Example 1: Evaluation of NGF and BDNF expression by miRNA using human neuroblastoma (1) Experimental method SH-SY5Y cells were cultured at 1.0 × 10 in 10% FBS-EMEM / F12 medium. 5 The cells were seeded at 1000 cells / mL and pre-cultured at 37°C for 24 hours. Then, using an mRNA transfection reagent, SH-SY5Y cells were transfected with miR-26a-5p mimic and / or miR-181a-5p mimic (both at a final concentration of 10 nM). The control group was transfected with NC-mimic (final concentration of 10 nM) instead of the miR mimic. After transfection, the cells were cultured in the same medium at 37°C for 24 hours, and then the expression levels of miRNAs (miR-26a-5p, miR-181a-5p) and mRNAs encoding NGF and BDNF (NGF mRNA, BDNF mRNA) in the SH-SY5Y cells were measured. The expression levels of each miRNA (miR-26a-5p, miR-181a-5p) in SH-SY5Y cells were measured by quantitative real-time PCR according to standard methods. The expression levels of NGF mRNA and BDNF mRNA in SH-SY5Y cells were also measured using quantitative real-time PCR. Specifically, SH-SY5Y cells were recovered from the culture and lysed with total RNA extraction reagent to extract RNA, and cDNA was synthesized. Using the resulting cDNA as a template, quantitative real-time PCR was performed according to standard methods using primers for the NGF and BDNF genes to measure the mRNA expression levels of NGF and BDNF. As an endogenous control, the expression level of ACTB (actin β) mRNA was measured using primers for the ACTB gene.

[0048] (2) Experimental results Figures 1(A) and (B) show the results of measuring the expression levels of miRNAs (miR-26a-5p, miR-181a-5p) in SH-SY5Y cells after introduction of miR-26a-5p mimic and / or miR-181a-5p mimic. Figures 2 and 3 show the effects of introduction of miR-26a-5p mimic and / or miR-181a-5p mimic on NGF mRNA expression and BDNF mRNA expression in SH-SY5Y cells. In each figure, the "+" and "-" symbols next to "26a mimic" and "181a mimic" on the horizontal axis indicate the presence or absence of introduction of miR-26a-5p mimic and miR-181a-5p mimic into SH-SY5Y cells. For example, if both "26 a mimic" and "181 a mimic" are "-", it means that neither of the two miRNA mimics has been introduced into SH-SY5Y cells, and if both are "+", it means that both of the two miRNA mimics have been introduced into SH-SY5Y cells.

[0049] As shown in Figure 1(A) and (B), the introduction of miR-26a-5p mimic and / or miR-181a-5p mimic into SH-SY5Y cells increased the expression of miRNAs (hsa-miR-26a-5p, hsa-miR-181a-5p) in SH-SY5Y cells depending on the miRNA mimic introduced. This indicates that miR-26a-5p mimic and miR-181a-5p mimic act by mimicking the corresponding mature miRNAs (hsa-miR-26a-5p, hsa-miR-181a-5p) in SH-SY5Y cells.

[0050] As shown in Figure 2, we confirmed that the introduction of miR-26a-5p mimic or miR-181a-5p mimic into SH-SY5Y cells significantly increased the expression level of NGF mRNA in SH-SY5Y cells. Furthermore, we confirmed that the introduction of both miR-26a-5p mimic and miR-181a-5p mimic further increased the expression level of NGF mRNA. From this, it is thought that both miR-26a-5p and miR-181a-5p exert an effect of promoting the expression and production of NGF in cells, particularly in nerve cells.

[0051] As shown in Figure 3, it was confirmed that the introduction of miR-26a-5p mimic or miR-181a-5p mimic into SH-SY5Y cells significantly increased the BDNF mRNA expression level in SH-SY5Y cells. From this, it is thought that both miR-26a-5p and miR-181a-5p exert an effect of promoting the expression and production of BDNF in cells, particularly in nerve cells.

[0052] As mentioned above, NGF is a trophic factor essential for the survival and function of neurons in the brain, and BDNF is one of the neurotrophic factors involved in the development, growth, maintenance, and regeneration of neurons. Therefore, introducing miR-26a-5p and / or miR-181a-5p into the body is expected to promote the expression and production of NGF and BDNF in brain cells, particularly neurons, thereby suppressing or improving brain function.

[0053] Experimental Example 2: Evaluation of Tollip expression by miRNA using human umbilical vein endothelial cells (1) Experimental method The HUVEC cells used were cultured in 2% FBS-EGM2 medium at 37°C under water vapor-saturated 5% CO2 conditions, and then passaged. HUVEC cells were cultured at 2.5 x 10 4 HUVEC cells were seeded in 2% FBS-EGM2 medium at a concentration of 10 nM cells / mL and precultured for 24 hours. Then, miR-26a-5p mimic and / or miR-181a-5p mimic (both final concentrations of 10 nM) were transfected into the cells using an mRNA transfection reagent. The control group was transfected with NC-mimic (final concentration of 10 nM) instead of the miR mimic. After transfection, the cells were cultured in the same medium at 37°C for 24 hours, and then the amount of Tollip produced in the HUVEC cells was measured by Western blotting using an anti-Tollip antibody. As an endogenous control, GAPDH production was also measured using an antibody against GAPDH.

[0054] (2) Experimental results Figure 4 shows the results of measuring the amount of Tollip produced in HUVEC cells after introduction of miR-26a-5p mimic and / or miR-181a-5p mimic. As shown in Figure 4, it was confirmed that introduction of miR-181a-5p mimic into HUVEC cells significantly increased the amount of Tollip produced in HUVEC cells. On the other hand, no significant difference was observed in the miR-26a-5p mimic compared to the control group, but introduction of miR-26a-5p mimic tended to increase the amount of Tollip produced. These findings suggest that both miR-26a-5p and miR-181a-5p promote the expression and production of Tollip in cells, particularly endothelial cells, and that this action contributes to their anti-inflammatory effects. [Sequence List Free Text]

[0055] SEQ ID NO: 1 shows the nucleotide sequence of the mature miRNA hsa-miR-26a-5p registered in miRBase. SEQ ID NO: 2 shows the nucleotide sequence of the mature miRNA hsa-miR-181a-5p.

Claims

1. A composition for promoting NGF production, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p.

2. A composition for promoting BDNF production, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p.

3. A composition for improving brain function, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p.

4. A composition for promoting Tollip production, comprising as an active ingredient at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p.

5. An anti-inflammatory composition comprising, as an active ingredient, at least one miRNA selected from the group consisting of miR-26a-5p and miR-181a-5p.

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