Agent for suppressing oxidative stress

Abstract

The present invention provides an agent for suppressing oxidative stress, the agent including at least one of the following (1)-(4): (1) apoptosis inhibitor of macrophage (AIM); (2) an AIM fragment including the SRCR2 domain of the AIM; (3) a modified object of the AIM or the AIM fragment, the modified object having an amino acid sequence that has an identity of 90% or more with respect to the AIM or the AIM fragment (no substitution is present in a free cysteine residue in the SRCR2 domain); and (4) a nucleic acid encoding the AIM, the AIM fragment, or the modified object thereof.

Description

Oxidative stress inhibitors

[0001] The present invention relates to an agent for suppressing oxidative stress, which comprises an apoptosis inhibitor of macrophage (AIM).

[0002] Excessive production of reactive oxygen species (ROS) and free radicals in the body causes oxidative stress, which has various adverse effects on the body.

[0003] Examples of adverse effects of oxidative stress on living organisms include aging, lipid peroxidation, mitochondrial dysfunction, and decreased immune function.

[0004] Oxidative stress promotes the breakdown of structural proteins such as collagen and elastin in subcutaneous cells. This results in loss of skin elasticity and skin aging, such as wrinkles and sagging. Oxidative stress also promotes the oxidation of lipid membranes in cells, reducing their fluidity and permeability, thereby impairing cell viability. Excessive retinal cell dysfunction and apoptosis due to this mechanism can lead to age-related macular degeneration and cataracts. Excessive hair follicle cell dysfunction and apoptosis can lead to hair loss and poor hair growth. Damage to mitochondrial DNA due to oxidative stress reduces mitochondrial ATP production or induces mitochondrial apoptosis, resulting in cellular energy deficiency. Oxidative stress also reduces immune cell function and apoptosis, impairing immune function in the body.

[0005] As mentioned above, the adverse effects of oxidative stress in living organisms are diverse, so there is a strong need for the development of safe and effective antioxidants. Antioxidants are substances that reduce oxidative stress by directly or indirectly neutralizing ROS and free radicals. Examples of major antioxidants include vitamin C, vitamin E, polyphenols, and carotenoids.

[0006] Apoptosis inhibitor of macrophage (AIM, also known as CD5 antigen-like (CD5L)) is a protein with unique characteristics (Non-Patent Document 1). It is a circulating protein present in serum at relatively high concentrations (∼5 μg / mL in humans and ∼3 μg / mL in mice). It was initially identified as a protein that supports macrophage survival, but is now known to promote repair in many diseases (Non-Patent Document 2). The promotion of phagocytic clearance of a wide range of organic waste products, including dead cell debris and damage-associated molecular patterns (DAMPs), has been most emphasized as the primary mechanism underlying disease repair by AIM (Non-Patent Documents 3 and 4). AIM associates with waste products through charge-based interactions using a cluster of positively charged surface amino acids located at the C-terminus of AIM and disulfide bond formation using an isolated cysteine ​​residue in the second SRCR domain (Non-Patent Document 4). The binding of AIM to waste materials strongly promotes the uptake of waste materials by phagocytes, because AIM itself is highly internalized by phagocytes upon binding to scavenger receptors on the surface of phagocytes (Non-Patent Document 2). Based on this mechanism, administration of recombinant AIM (rAIM) protein to disease model animals has shown therapeutic effects in a variety of diseases and symptoms, including acute kidney injury (AKI) (Non-Patent Documents 3 and 5), peritonitis (Non-Patent Document 6), cerebral infarction (Non-Patent Document 4), obesity (Non-Patent Document 7), hepatocellular carcinoma associated with fatty liver or non-alcoholic steatohepatitis (Non-Patent Document 8), nephrolithiasis (Non-Patent Document 9), and delayed graft function in kidney transplantation (Non-Patent Document 10).

[0007] Miyazaki, T., Hirokami, Y., Matsuhashi, N., Takatsuka, H. Naito, N., Increased susceptibility of thymocytes to apoptosis in mice lacking AIM, a novel murine macrophage-derived soluble factor belonging to the scavenger receptor cysteine-rich domain superfamily, Journal of Experimental Medicine. 189(2) (1999) 413-422.Arai, S., Miyazaki, T., A scavenging system against internal pathogens promoted by the circulating protein apoptosis inhibitor of macrophage(AIM), Seminars in Immunopathology. 40(6) (2018) 567-575.Arai, S., Kitada, K., Yamazaki, T., Takai, R., Zhang, X., Tsugawa, Y., Sugisawa, R., Matsumoto, A., Mori, M., Yoshihara, Y., Doi, K., Maehara, N., Kusunoki, S.,. Takahata , A. , Noiri , E. , Suzuki , Y. , Yahagi , N. , Nishiyama , A. , Gunaratnam , L. , Takano , T. , Miyazaki , T. , Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice , Nature Medicine . 22(2) (2016) 183-193.Maehara, N., Taniguchi, K., Okuno, A., Ando, ​​H. (2016)., Hirota , A. , Li , Z. , Wang , CT , Arai , S. , Miyazaki , T. , AIM / CD5L attenuates DAMPs in the injured brain and thereby ameliorates ischemic stroke , Cell Reports . 36(11) (2021) 109693.Wang, CT, Tezuka, T., Takeda, N., Araki, K., Arai, S., Miyazaki, T., High salt exacerbates acute kidney injury by disturbing the activation of CD5L / apoptosis inhibitor of macrophage (AIM) protein, PLOS ONE. 16(11) (2021) e0260449.Tomita, T., Arai, S., Kitada, K., Mizuno, M., Suzuki, Y., Sakata, F., Nakano, D., Hiramoto, E., Takei, Y., Maruyama S., Nishiyama, A., Matsuo, S., Miyazaki, T., Ito, Y., Apoptosis inhibitor of macrophage ameliorates fungus-induced peritoneal injury model in mice, Scientific Reports. 7(1) (2017) 6450.Kurokawa, J., Arai, S., Nakashima, K., Nagano, H., Nishijima, A., Miyata, K., Ose, R., Mori, M., Kubota, N., Kadowaki, T., Oike, Y., Koga, H., Febbraio, M., Iwanaga, T., Miyazaki, T. (1999)., Macrophage-derived AIM is endocytosed into adipocytes and decreases lipid droplets via inhibition of fatty acid synthase activity, Cell Metabolism. 11(6) (2010) 479-492.Maehara, N., Arai, S., Mori, M., Iwamura, Y., Kurokawa, J., Kai, T., Kusunoki, S., Taniguchi, K., Ikeda, K., Ohara, O., Yamamura, K.I., Miyazaki, T. Circulating AIM prevents hepatocellular carcinoma through complement activation, Cell Reports. 9(1) (2014) 61-74.Matsuura, K., Maehara, N., Hirota, A., Eguchi, A., Yasuda, K., Taniguchi, K., Nishijima, A., Matsuhashi, N., Shiga, Y., Ishii, R., Iguchi Y., Tanabe, K., Arai, S., Miyazaki, T., Two independent modes of kidney stone suppression achieved by AIM / CD5L and KIM-1, Communications Biology. 5(1) (2022) 783.Lee, J.Y., Arumugarajah, S., Lian, D., Maehara, N., Haig, A.R., Suri, R.S., Miyazaki, T., Gunaratnam, L., Recombinant apoptosis inhibitor of macrophage protein reduces delayed graft function in a murine model of kidney transplantation, PLOS ONE. 16(4) (2021) e0249838.

[0008] As mentioned above, many types of antioxidants already exist and are used safely. However, some of the known antioxidants have issues with bioavailability, and there is still a strong demand for the development of new antioxidants that have more favorable effects against the various adverse effects caused by oxidative stress.

[0009] In analyzing the various properties of AIM, the present inventors have found that: (1) AIM has extremely strong antioxidant activity; (2) the antioxidant activity of AIM is based on two different mechanisms (direct scavenging of free radicals (Figure 7) and enhanced gene expression of antioxidant-related genes controlled by Nrf2 (Figures 8 and 9)); (3) AIM suppresses cellular oxidative stress, thereby inhibiting cellular aging (Figures 10 to 13); (4) AIM can improve the CSF clearance function (Figure 14); and (5) the improvement of CSF clearance function by AIM suppresses the accumulation of proteins that fluctuate in the CSF of Alzheimer's disease patients (Table 2). The present inventors have conducted further research based on these findings, and have thereby completed the present invention. Specifically, the present invention is as follows: [1] An agent for suppressing oxidative stress, comprising any of the following (1) to (4): (1) Apoptosis inhibitor of macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to the AIM or the AIM fragment (provided that the free cysteine ​​residue in the SRCR2 domain is not substituted), (4) a nucleic acid encoding the AIM, the AIM fragment, or a variant thereof. [2] An agent for treating, preventing, or ameliorating aging, disease, or disorder caused by oxidative stress, comprising the agent according to [1]. [3] The agent according to [2], wherein the aging caused by oxidative stress is skin aging. [4] The agent according to [2], wherein the disease caused by oxidative stress is an aging-related ophthalmic disease. [5] The agent according to [4], wherein the aging-related ophthalmic disease is any one selected from the group consisting of age-related macular degeneration and cataract. [6] The agent according to [2], wherein the disorder caused by oxidative stress is at least one selected from the group consisting of (1) to (3) below. (1) Fatigue or decreased physical strength; (2) decreased immune function; (3) hair loss or poor hair growth [7] A method for suppressing oxidative stress in a subject, comprising administering to the subject any of the following (1) to (4):(1) Apoptosis inhibitor of Macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that free cysteine ​​residues in the SRCR2 domain are not substituted), (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof [8] A method for treating, preventing or ameliorating aging, disease or disorder caused by oxidative stress in a subject, comprising administering to the subject any of the following (1) to (4): (1) Apoptosis inhibitor of Macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that free cysteine ​​residues in the SRCR2 domain are not substituted), (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof [9] The method described in [8], wherein the aging caused by oxidative stress is aging of the skin.

[0010] [8] The method described in [8], wherein the disease caused by oxidative stress is an aging-related eye disease.

[0011] The method described in

[0010] , wherein the aging-related eye disease is any one selected from the group consisting of age-related macular degeneration and cataracts.

[0012] [8] The method according to [8], wherein the disorder caused by oxidative stress is at least one selected from the group consisting of the following (1) fatigue or decreased physical strength, (2) decreased immune function, and (3) hair loss or poor hair growth.

[0013] Any of the following substances (1) to (4) for use in suppressing oxidative stress: (1) Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment containing the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted); (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof.

[0014] Any of the following substances (1) to (4) for use in the treatment, prevention, or amelioration of aging, disease, or disorder caused by oxidative stress: (1) Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment containing the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted); (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

[0015] A substance for use as described in

[0014] , wherein the aging caused by oxidative stress is aging of the skin.

[0016] A substance for use as described in

[0014] , wherein the disease caused by oxidative stress is an aging-related eye disease.

[0017] The substance for use described in

[0016] , wherein the aging-related eye disease is any one selected from the group consisting of age-related macular degeneration and cataracts.

[0018] The substance for use according to

[0014] , wherein the disorder caused by oxidative stress is at least one selected from the group consisting of the following (1) to (3): (1) fatigue or decreased physical strength, (2) decreased immune function, and (3) hair loss or poor hair growth.

[0019] Use of any of the following (1) to (4) in the manufacture of a pharmaceutical for suppressing oxidative stress: (1) Apoptosis inhibitor of macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted), or (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

[0020] Use of any of the following (1) to (4) in the manufacture of a pharmaceutical for treating, preventing, or ameliorating aging, disease, or disorder caused by oxidative stress: (1) Apoptosis inhibitor of macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted), or (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

[0021] The use described in

[0020] , wherein the aging caused by oxidative stress is skin aging.

[0022] The use described in

[0020] , wherein the disease caused by oxidative stress is an aging-related eye disease.

[0023] The use described in

[0022] , wherein the aging-related eye disease is any one selected from the group consisting of age-related macular degeneration and cataracts.

[0024] The use according to

[0020] , wherein the disorder caused by oxidative stress is at least one selected from the group consisting of the following (1) fatigue or decreased physical strength, (2) decreased immune function, and (3) hair loss or poor hair growth.

[0025] An agent for promoting cerebrospinal fluid clearance function in a subject, comprising any of the following (1) to (4): (1) an Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment comprising the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted); (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof.

[0026] The agent described in

[0025] , characterized in that it is administered intrathecally.

[0027] An agent for treating, preventing, or ameliorating brain dysfunction or neurodegenerative disease in a subject, comprising any of the following (1) to (4): (1) an Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment comprising the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that free cysteine ​​residues in the SRCR2 domain are not substituted); (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

[0028] The agent described in

[0027] , characterized in that it is administered intrathecally.

[0029] The agent according to

[0027] or

[0028] , wherein the brain dysfunction or neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Pick disease, corticobasal degeneration, progressive supranuclear paralysis, amyotrophic lateral sclerosis, spinobulbar muscular atrophy, Huntington's disease, spinocerebellar degeneration, dentatorubral-pallidoluysian atrophy, and depression.

[0030] A method for promoting cerebrospinal fluid clearance function in a subject, comprising administering to the subject any of the following (1) to (4): (1) Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment comprising the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted); or (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof.

[0031] The method described in

[0030] , characterized in that it is administered intrathecally.

[0032] A method for treating, preventing, or ameliorating brain dysfunction or neurodegenerative disease in a subject, comprising administering to the subject any of the following (1) to (4): (1) Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment comprising the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted); or (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

[0033] The method described in

[0032] , characterized in that it is administered intrathecally.

[0034] The method of

[32] or

[33] , wherein the brain dysfunction or neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Pick's disease, corticobasal degeneration, progressive supranuclear paralysis, amyotrophic lateral sclerosis, spinal-bulbar muscular atrophy, Huntington's disease, spinocerebellar degeneration, dentatorubral-pallidoluysian atrophy, and depression.

[0035] A substance selected from the following (1) to (4) for use in promoting cerebrospinal fluid clearance in a subject: (1) Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment comprising the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted); or (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof.

[0036] A substance for use as described in

[0035] , characterized in that it is administered intrathecally.

[0037] Any of the following substances (1) to (4) for use in treating, preventing, or ameliorating brain dysfunction or neurodegenerative disease in a subject: (1) Apoptosis inhibitor of Macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted), or (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

[0038] A substance for use as described in

[0037] , characterized in that it is administered intrathecally.

[0039] The substance for use according to

[37] or

[38] , wherein the brain dysfunction or neurodegenerative disease is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Pick's disease, corticobasal degeneration, progressive supranuclear paralysis, amyotrophic lateral sclerosis, spinal-bulbar muscular atrophy, Huntington's disease, spinocerebellar degeneration, dentatorubral-pallidoluysian atrophy, and depression.

[0040] Use of any of the following (1) to (4) in the manufacture of a pharmaceutical for promoting cerebrospinal fluid clearance function in a subject: (1) Apoptosis inhibitor of Macrophage (AIM), (2) an AIM fragment comprising the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted), or (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof.

[0041] The use described in

[0040] , characterized in that the medicine is administered intrathecally.

[0042] Use of any of the following (1) to (4) in the manufacture of a pharmaceutical for treating, preventing, or ameliorating brain dysfunction or neurodegenerative disease in a subject: (1) Apoptosis inhibitor of Macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted), or (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

[0043] The use described in

[0042] , characterized in that the medicine is administered intrathecally.

[0044] The use according to

[42] or

[43] , wherein the brain dysfunction or neurodegenerative disease is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Pick's disease, corticobasal degeneration, progressive supranuclear paralysis, amyotrophic lateral sclerosis, spinal-bulbar muscular atrophy, Huntington's disease, spinocerebellar degeneration, dentatorubral-pallidoluysian atrophy, and depression.

[0010] According to the present invention, oxidative stress in a living body can be suppressed extremely efficiently, and as a result, aging, diseases, or disorders caused by oxidative stress can be efficiently treated, prevented, or improved.

[0011] Figure 1 shows the three-dimensional structural characteristics of recombinant AIM (hAIM and mAIM) and AIM mutants (2CSmAIM and △SRCR3mAIM) used in the examples. The three-dimensional structure of each protein was predicted using Alphafold2. Figure 2 shows the cell viability (vertical axis: RFU) when a human renal tubular epithelial cell line (HK-2) was stimulated with hydrogen peroxide (H2O2) in the presence of recombinant AIM, AIM mutant, or BSA. Figure 3 shows the staining results for ROS (upper panel) and lipid peroxides (lower panel) in HK-2 cells stimulated with H2O2 (50 μM) for 2 hours in the presence or absence of recombinant AIM. Figure 4 shows the results of quantifying the average brightness in the images shown in Figure 3. Figure 5 shows the results of immunocytochemistry (ICC) for 4-HNE (4-hydroxynonenal) or malondialdehyde (MDA) in HK-2 cells stimulated with H2O2 (50 μM) for 2 hours in the presence or absence of recombinant AIM. Figure 6 shows the results of Western blotting of the cell lysates prepared in Figure 5 using an anti-4-HNE antibody. Figure 7 shows the results of a cell-free antioxidant assay (DHHP radical scavenging assay) measuring the direct antioxidant effects of vitamin C (L-ascorbate), GSH, 2CS, and mAIM. Figure 8 shows the results of quantifying the amount of Nrf2 protein by Western blotting of nuclei fractionated from HK-2 cells cultured for 2.5 hours with recombinant AIM or 2CS (both at 100 μg / mL), 1.5 hours with HO (100 μM), or 2.5 hours without either (PBS). Lamin B1 was quantified as an internal control. Figure 9 shows the results of quantifying the expression levels of antioxidant-related genes (NQO1, GCLC, GCLM, PRDX1, PRDX2, and PRDX6) regulated by Nrf2 in HK-2 cells stimulated with HO by qPCR.Figure 10 shows the effect of AIM on the senescence of hepatic sinusoidal endothelial cells in AIM-deficient mice subjected to a high-fat diet, as confirmed by LaminB1 immunohistochemical staining (IHC). Figure 11 shows the number of LaminB1-positive cells in the liver tissue of AIM-deficient mice subjected to a high-fat diet. Figure 12 shows the time-dependent changes in cfDNA (A) and mtDNA (B) levels in the blood of wild-type or AIM-deficient mice subjected to a high-fat diet. Figure 13 shows that administration of AIM significantly reduces the amount of mtDNA (mitochondrial COX1 and mitochondrial ND1) in the blood of AIM+ / - mice subjected to a high-fat diet. Figure 14 shows the improvement of CSF clearance by AIM.

[0012] The present invention will now be described in detail.

[0013] 1. Agent for suppressing oxidative stress The present invention provides an agent for suppressing oxidative stress (hereinafter sometimes referred to as "the inhibitor of the present invention"), which comprises any of the following (1) to (4): (1) Apoptosis inhibitor of Macrophage (AIM), (2) an AIM fragment comprising the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (however, the free cysteine ​​residue in the SRCR2 domain is not substituted), (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof. (1) Apoptosis inhibitor of macrophage (AIM) The active ingredient of the inhibitor of the present invention can be an apoptosis inhibitor of macrophage (AIM). AIM (also called "CD5L" etc.) is a blood protein secreted by macrophages and is known to be conserved in various species.

[0015] The biological species of the AIM used in the present invention is not particularly limited. Examples of the biological species of the AIM include, but are not limited to, warm-blooded animals (e.g., humans, mice, cats, rats, rabbits, sheep, pigs, cattle, horses, dogs, monkeys, chimpanzees, birds, etc.). In one aspect of the present invention, it may be preferable that the biological species from which the AIM is derived matches the biological species of the subject to which the agent of the present invention is applied. For example, when the subject to which the inhibitor of the present invention is applied is humans, it is preferable to use human AIM (SEQ ID NO: 1). Furthermore, when the subject to which the agent is applied is mice or cats, it is preferable to use mouse AIM (SEQ ID NO: 3) or cat AIM (SEQ ID NO: 5), respectively.

[0016] AIM can be produced by a method known per se. AIM can be purified from the above-mentioned mammalian macrophages by a known protein purification method. Specifically, mammalian macrophages are homogenized, and cell debris is removed by low-speed centrifugation. The supernatant is then centrifuged at high speed to precipitate a cell membrane-containing fraction, and the supernatant is then subjected to chromatography such as reverse-phase chromatography, ion-exchange chromatography, or affinity chromatography, thereby preparing AIM or a salt thereof.

[0017] AIM can also be produced according to known peptide synthesis methods, such as solid-phase synthesis or liquid-phase synthesis.

[0018] When the obtained AIM is in a free form, the free form can be converted into a suitable salt by a known method. Conversely, when the AIM is obtained as a salt, the salt can be converted into the free form or another salt by a known method.

[0019] AIM can also be produced by culturing a transformant containing a nucleic acid encoding it, and isolating and purifying AIM from the resulting culture. The nucleic acid encoding AIM may be DNA or RNA, or may be a DNA / RNA chimera. DNA is preferred. The nucleic acid may be double-stranded or single-stranded. If double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. If single-stranded, it may be the sense strand (i.e., the coding strand) or the antisense strand (i.e., the non-coding strand). (2) AIM fragment containing the SRCR2 domain of AIM Furthermore, an AIM fragment containing the SRCR2 domain of AIM can be used as the active ingredient of the inhibitor of the present invention.

[0021] AIM is composed of SRCR (Scavenger-Receptor Cysteine-Rich) domains (i.e., SRCR1, SRCR2, and SRCR3). For example, in the case of human AIM (SEQ ID NO: 1), SRCR1, SRCR2, and SRCR3 correspond to the amino acid sequences of positions 24 to 125, 138 to 239, and 244 to 346 in SEQ ID NO: 1, respectively. Here, as demonstrated in the Examples below, SRCR1 and SRCR3 are not thought to be involved in the antioxidant activity of AIM. Therefore, an AIM fragment containing the SRCR2 domain of AIM can be used as the active ingredient of the inhibitor of the present invention. In a preferred embodiment, the AIM fragment used in the inhibitor of the present invention may be a fragment that contains the SRCR2 domain and has substantially the same antioxidant activity as that of wild-type AIM (i.e., either or both of the activity of scavenging reactive oxygen species (ROS) or free radicals and the activity of enhancing Nrf2 gene expression), or has an antioxidant activity that is improved over that of wild-type AIM.

[0022] AIM fragments can be produced by methods known per se. Furthermore, the antioxidant activity of AIM and AIM fragments can be confirmed by methods known per se. Briefly, the activity of scavenging reactive oxygen species (ROS) and free radicals can be confirmed, for example, by the in vitro DPPH antioxidant assay described in the Examples of the present application. Furthermore, the activity of enhancing Nrf2 gene expression can be confirmed, for example, by Western blotting of Nrf2 protein described in the Examples of the present application.

[0023] The size of the AIM fragment that can be used in the inhibitor of the present invention is not particularly limited, as long as it contains SRCR2. Therefore, the AIM fragment is a concept that includes all AIM fragments as long as they contain SRCR2, but does not include wild-type AIM itself.

[0024] (3) A modified AIM or AIM fragment containing an amino acid sequence having 90% or more identity to AIM or an AIM fragment.Furthermore, as an active ingredient of the inhibitor of the present invention, a modified AIM or AIM fragment containing an amino acid sequence having 90% or more identity to the above-mentioned AIM or AIM fragment can be used. However, as demonstrated in the Examples below, the antioxidant activity of AIM requires a free cysteine ​​in the SRCR2 domain that is not involved in intraprotein disulfide bonds (cysteine ​​at position 191 in human AIM, cysteine ​​at position 194 in mouse AIM). Therefore, a variant of AIM or an AIM fragment is characterized in that the free cysteine ​​residue in the SRCR2 domain is not substituted. In a preferred embodiment, the variant of AIM or an AIM fragment used in the inhibitor of the present invention may be a variant having substantially the same or improved antioxidant activity as that of wild-type AIM (i.e., either or both of the activity of scavenging reactive oxygen species (ROS) or free radicals and the activity of enhancing Nrf2 gene expression). The above-described methods can be used to confirm whether a variant of AIM or an AIM fragment has antioxidant activity. Furthermore, as used herein, "substantially the same" indicates that the activities are qualitatively (e.g., physiologically or pharmacologically) the same. Therefore, the activity is preferably equivalent, but the degree of activity (for example, about 0.1 to about 10 times, preferably about 0.5 to about 2 times) and quantitative factors such as the molecular weight of the protein may differ.

[0026] The variant of AIM or an AIM fragment used in the inhibitor of the present invention may have an amino acid sequence that is about 90% or more, preferably about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more or about 99% or more identical or similar to the amino acid sequence of AIM or an AIM fragment.

[0027] As used herein, "identity" refers to the percentage (%) of identical and similar amino acid residues relative to the total number of overlapping amino acid residues in an optimal alignment (preferably, the algorithm can consider the introduction of gaps into one or both of the sequences for optimal alignment) when two amino acid sequences are aligned using a mathematical algorithm known in the art. Furthermore, "similarity" refers to the percentage (%) of the number of positions at which identical or similar amino acid residues exist in both aligned amino acid sequences relative to the total number of amino acid residues. "Similar amino acids" refer to amino acids that are similar in physicochemical properties, and include, for example, amino acids classified in the same group, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), and amino acids with small side chains (Gly, Ala, Ser, Thr, Met). Substitution with such similar amino acids is expected to have no effect on the phenotype of the protein (i.e., it is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and have been described in various publications (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).

[0028] The identity or similarity of amino acid sequences in this specification can be calculated using the identity or similarity calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expectation value = 10; gaps allowed; matrix = BLOSUM62; filtering = OFF). Other algorithms for determining identity or similarity of amino acid sequences include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993) [this algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) (Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997))], the algorithm described in Needleman et al., J. Mol. Biol., 48:444-453 (1970) [this algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4:11-17 (1988) [this algorithm is incorporated into the ALIGN program (version 2.0) which is part of the CGC sequence alignment software package], and the algorithm described in Pearson et al., Proc. Natl. Acad. Sci. USA, 85:2444-2448 (1988) [this algorithm is incorporated into the FASTA program in the GCG software package], and the like, which can also be preferably used.

[0029] The AIM or AIM fragment used in the present invention may include, for example, (1) an amino acid sequence in which one or two or more amino acids (preferably about 1 to 34, preferably about 1 to 30, more preferably about 1 to 20, particularly preferably one to several (2, 3, 4, or 5)) have been deleted from the amino acid sequence of AIM or an AIM fragment, (2) an amino acid sequence in which one or two or more amino acids (preferably about 1 to 34, preferably about 1 to 30, more preferably about 1 to 20, particularly preferably one to several (2, 3, 4, or 5)) have been added from the amino acid sequence of AIM or an AIM fragment, or (3) an AIM or (4) an amino acid sequence in which one or two or more amino acids (preferably about 1 to 34, preferably about 1 to 30, more preferably about 1 to 20, and particularly preferably one to several (2, 3, 4, or 5)) have been inserted into the amino acid sequence of an AIM fragment; (5) an amino acid sequence in which one or two or more amino acids (preferably about 1 to 34, preferably about 1 to 30, more preferably about 1 to 20, and particularly preferably one to several (2, 3, 4, or 5)) have been substituted with other amino acids in the amino acid sequence of AIM or an AIM fragment; or (6) a polypeptide containing an amino acid sequence that is a combination thereof. When an amino acid sequence is inserted, deleted, or substituted as described above, the position of the insertion, deletion, or substitution is not particularly limited, as long as the desired biological activity of the protein (e.g., one or both of the activity of scavenging reactive oxygen species (ROS) or free radicals and the activity of enhancing Nrf2 gene expression) is maintained.

[0030] When human AIM is used, preferred examples of variants include, but are not limited to, the following: (1) an amino acid sequence in which the cysteine ​​at amino acid number 300 in the amino acid sequence represented by SEQ ID NO: 1 is substituted with serine; (2) a variant that has about 90% or more (preferably, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, or about 99% or more) identity with the amino acid sequence of (1), and in which the cysteine ​​and the substituted serine present in the amino acid sequence of (1) are retained, and that has substantially the same or improved antioxidant activity as that of wild-type AIM. (3) A variant that further comprises deletion, addition, insertion or substitution of one to several (2, 3, 4 or 5) amino acids, or a combination thereof, at a position other than the cysteine ​​and the substituted serine present in the amino acid sequence of (1), and that has substantially the same or improved antioxidant activity as that of wild-type AIM.

[0031] In this specification, AIM, an AIM fragment, or a variant thereof is described in accordance with the convention for peptide notation, with the left end being the N-terminus (amino terminus) and the right end being the C-terminus (carboxyl terminus).

[0032] In the present invention, the C-terminus of AIM, AIM fragment, or modified form thereof used in the present invention may be any of a carboxyl group (-COOH), carboxylate (-COO-), amide (-CONH2) or ester (-COOR).

[0033] Here, R in the ester is, for example, C such as methyl, ethyl, n-propyl, isopropyl, n-butyl, etc. 1-6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3-8 Cycloalkyl groups such as phenyl, α-naphthyl, etc. 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl group: α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0034] When the AIM, AIM fragment, or modified form thereof used in the present invention has a carboxyl group (or carboxylate) at a position other than the C-terminus, the AIM, AIM fragment, or modified form thereof used in the present invention may also include those in which the carboxyl group is amidated or esterified. In this case, the ester may be, for example, the C-terminal ester described above.

[0035] Furthermore, in the AIM, AIM fragment, or modified form thereof used in the present invention, the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1-6 C such as alkanoyl 1-6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1-6 C such as alkanoyl group 1-6 These may include those protected with an acyl group or those bound to a sugar chain.

[0036] The AIM, AIM fragment, or modified form thereof used in the present invention may be in the form of a salt. For example, a salt with a physiologically acceptable acid (e.g., inorganic acid, organic acid) or base (e.g., alkali metal salt) is used, with physiologically acceptable acid addition salts being particularly preferred. Examples of such salts include salts with inorganic acids (e.g., hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) and salts with organic acids (e.g., acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid). (4) Nucleic acids encoding AIM, AIM fragments or variants thereof Furthermore, nucleic acids encoding the above-mentioned AIM, AIM fragments or variants thereof can be used as the active ingredient of the inhibitor of the present invention.

[0038] In one embodiment, examples of nucleic acids encoding AIM include, but are not limited to, human AIM cDNA (SEQ ID NO:2), mouse AIM cDNA (SEQ ID NO:4), and feline AIM cDNA (SEQ ID NO:6). In another embodiment, examples of nucleic acids encoding AIM fragments include, but are not limited to, nucleic acids encoding AIM fragments containing the SRCR2 domain of human AIM (amino acid sequence consisting of amino acid numbers 138 to 239 of SEQ ID NO:1). In another embodiment, examples of nucleic acids encoding variants of human AIM include, but are not limited to, nucleic acids encoding variants of AIM containing an amino acid sequence in which the cysteine ​​at amino acid number 300 of the amino acid sequence represented by SEQ ID NO:1 is substituted with serine.

[0039] In one embodiment, a nucleic acid encoding AIM, an AIM fragment, or a variant thereof may be codon-optimized for the purpose of increasing expression efficiency in an organism to which the nucleic acid is to be applied. Codon optimization may be performed using a method known per se.

[0040] Nucleic acids encoding AIM, AIM fragments or variants thereof can be prepared according to methods known per se (for example, the methods described in Molecular Cloning, 2nd Edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989)).

[0041] In one aspect, the nucleic acid encoding AIM, an AIM fragment or a variant thereof may be added with known sequences such as a promoter, a Poly A addition signal, a Kozak consensus sequence, a tag sequence, a linker sequence, and an NLS.

[0042] In a preferred embodiment, a nucleic acid encoding AIM, an AIM fragment, or a variant thereof may be carried by a viral vector. Suitable viral vectors include, but are not limited to, adeno-associated viruses, adenoviruses, lentiviruses, and Sendai viruses. Considering use in gene therapy, adeno-associated viruses are preferred because they allow long-term expression of transgenes and are derived from non-pathogenic viruses, making them highly safe. The serotype of the adeno-associated virus is not particularly limited as long as the desired effects of the present invention are achieved; any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 may be used. The viral vectors used in the present invention also include their derivatives. Derivatives of viral vectors, such as those with modified capsids, are well known. In particular, AAV derivatives include, but are not limited to, those disclosed in WO2012 / 057363.

[0043] A viral vector containing a nucleic acid encoding AIM, an AIM fragment, or a variant thereof can be prepared by a known method. Briefly, a viral expression plasmid vector is prepared by inserting a nucleic acid encoding AIM, an AIM fragment, or a variant thereof, and, if necessary, a nucleic acid having a desired function (e.g., a promoter, etc.), and this is transfected into an appropriate host cell to transiently produce a viral vector containing a nucleic acid encoding AIM, an AIM fragment, or a variant thereof, and then this can be recovered. For example, when preparing an AAV vector, a vector plasmid is first prepared by retaining the ITRs at both ends of the wild-type AAV genome sequence and inserting a nucleic acid encoding AIM or an AIM fragment in place of the DNA encoding the remaining Rep and capsid proteins. Meanwhile, the DNA encoding the Rep and capsid proteins required for viral particle formation is inserted into a separate plasmid. Furthermore, a plasmid containing genes (E1A, E1B, E2A, VA, and E4orf6) responsible for the adenovirus helper function required for AAV propagation is prepared as an adenovirus helper plasmid. By cotransfecting these three plasmids into host cells, recombinant AAV (i.e., an AAV vector) is produced in the cells. It is preferable to use host cells (e.g., 293 cells) capable of supplying some of the gene products (proteins) of the genes responsible for the helper function. When such cells are used, it is not necessary to incorporate genes encoding proteins that can be supplied by the host cells into the adenovirus helper plasmid. Since the produced AAV vector is present in the nucleus, the host cells are frozen and thawed to recover the vector, and the desired AAV vector is prepared by separating and purifying it using cesium chloride density gradient ultracentrifugation or column methods.

[0045] The inhibitor of the present invention may be applied to any living species that may be subject to oxidative stress, including, but not limited to, humans, mice, cats, rats, rabbits, sheep, pigs, cows, horses, dogs, monkeys, chimpanzees, and birds.

[0046] The amount of the active ingredient contained in the inhibitor of the present invention is not particularly limited as long as the desired effect is obtained. The amount of the active ingredient contained in the inhibitor of the present invention may be optimized appropriately depending on the type of active ingredient, the type and form of the active ingredient in the subject, the sex, age, weight, administration schedule, administration route, etc. of the subject.

[0047] The route of administration of the inhibitor of the present invention is not particularly limited, as long as the active ingredient (i.e., AIM, an AIM fragment, or a modified form thereof) is delivered to the site where oxidative stress is occurring. Since AIM is a protein that circulates in the blood, the inhibitor of the present invention does not need to be administered directly to the site where oxidative stress is occurring; any administration route that achieves the desired effect may be used. In one embodiment, the route of administration of the inhibitor of the present invention may be oral or parenteral. Parenteral administration includes, but is not limited to, intravenous administration, intraarterial administration, subcutaneous administration, and intraperitoneal administration.

[0048] When the inhibitor of the present invention is formulated for parenteral administration, it can be formulated, for example, as an injection, suppository, etc. Injections may include dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip infusion injections. Such injections can be prepared according to known methods. For example, injections can be prepared by dissolving, suspending, or emulsifying components such as AIM, nucleic acids encoding AIM or variants thereof, and / or viruses carrying nucleic acids encoding AIM, AIM fragments, or variants thereof in a sterile aqueous or oily liquid typically used for injections. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants, and the like, which may be used in combination with appropriate solubilizing agents, such as alcohols (e.g., ethanol), polyalcohols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)). As the oily liquid, for example, sesame oil, soybean oil, etc. can be used, and a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. can be used in combination. The prepared injection solution is preferably filled into a suitable ampule.

[0049] The dosage of the inhibitor of the present invention to a subject is not particularly limited as long as the desired effect is obtained, and may be optimized appropriately depending on the type and form of the active ingredient, the sex, age, weight, administration schedule, administration route, etc. of the subject.

[0050] The inhibitor of the present invention can also be used in combination with other antioxidants, such as, but not limited to, glutathione, N-acetylcysteine, ascorbic acid, α-tocopherol, butylhydroxyanisole, catechin, quercetin, uric acid, bilirubin, glucose, flavonoids, ceruloplasmin, albumin, ferritin, metallothionein, superoxide dismutase, glutathione peroxidase, glutathione transferase, catalase, or thioredoxin.

[0051] When used in combination with other antioxidants, the inhibitor of the present invention may be formulated by combining the active ingredient of the present invention with the active ingredient of the other antioxidant, or the separately formulated drugs may be administered to a subject separately.

[0052] In one embodiment, the inhibitor of the present invention may be a food composition.

[0053] When the inhibitor of the present invention is a food composition, the food composition may contain ingredients other than the active ingredient (i.e., AIM, AIM fragment, variants thereof, or nucleic acids encoding them). Such other ingredients may include, but are not limited to, various food additives, such as antioxidants, flavorings, various esters, organic acids, organic acid salts, inorganic acids, inorganic acid salts, inorganic salts, colorants, emulsifiers, preservatives, seasonings, sweeteners, acidulants, fruit juice extracts, vegetable extracts, nectar extracts, pH adjusters, and quality stabilizers.

[0054] When the inhibitor of the present invention is a food composition, the food composition may be a general food, or may be a health food, functional food, food for the sick, or food for specified health uses.

[0055] When the inhibitor of the present invention is a food composition, the food composition may be in any form, such as a solid, semi-solid, liquid, or slurry. In one embodiment, the food composition may be prepared in the form of a capsule.

[0056] When the inhibitor of the present invention is a food composition, the food composition may be livestock feed, pet food, or the like.

[0057] When the inhibitor of the present invention is a food composition, the timing of ingestion of the food composition by a subject is not particularly limited as long as the desired effect can be obtained. Examples of timing of ingestion of the food composition of the present invention include, but are not limited to, before the onset of oxidative stress, in the early stage of the onset of oxidative stress, and when the oxidative stress level is increasing.

[0058] 2. Agent for treating, preventing, or ameliorating aging, disease, or disorder caused by oxidative stress The present invention also provides an agent for treating, preventing, or ameliorating aging, disease, or disorder caused by oxidative stress (hereinafter, sometimes referred to as the "therapeutic agent of the present invention"), which comprises the inhibitor of the present invention.

[0059] The active ingredient of the therapeutic agent of the present invention is the inhibitor of the present invention described above, and the manufacturing method, target of application, administration route, dosage form, dosage amount, method of use, etc. of the therapeutic agent of the present invention may be the same as those of the inhibitor of the present invention.

[0060] The amount of the inhibitor of the present invention contained in the therapeutic agent of the present invention is not particularly limited, and may be optimized appropriately depending on the type and form of the active ingredient, the sex, age, weight, administration schedule, administration route, etc. Furthermore, the therapeutic agent of the present invention may contain ingredients other than the inhibitor of the present invention, as long as the desired effect is obtained.

[0061] Oxidative stress promotes the degradation of structural proteins such as collagen and elastin in subcutaneous cells, resulting in loss of skin elasticity and skin aging such as wrinkles and sagging. Therefore, in one aspect, aging caused by oxidative stress can be skin aging.

[0062] Oxidative stress also promotes the oxidation of lipid membranes in cells, reducing the fluidity and permeability of cell membranes and thereby reducing cell viability. Excessive retinal cell dysfunction and apoptosis due to this mechanism can lead to eye diseases (e.g., age-related macular degeneration and cataracts). Therefore, in one embodiment, the disease caused by oxidative stress is an aging-related eye disease (e.g., age-related macular degeneration and cataracts).

[0063] Furthermore, if oxidative stress causes excessive decline in hair follicle cell function or apoptosis, it can lead to hair loss or poor hair growth. Furthermore, if mitochondrial DNA is damaged by oxidative stress, the amount of ATP produced by mitochondria decreases or mitochondrial apoptosis is induced, resulting in cellular energy deficiency. Furthermore, if oxidative stress causes decline in immune cell function or apoptosis, the immune function of the living body declines. Therefore, in one aspect, the disorder caused by oxidative stress is at least one selected from the group consisting of the following (1) to (3): (1) fatigue or decline in physical strength; (2) decline in immune function; and (3) hair loss or poor hair growth.

[0064] 3. Method for Suppressing Oxidative Stress The present invention also provides a method for suppressing oxidative stress in a subject (hereinafter, may be referred to as the "suppression method of the present invention"), which comprises administering to the subject any of the following (1) to (4): (1) Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment comprising the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted); (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof.

[0065] The suppression method of the present invention is characterized by administering the suppressant of the present invention to the subject, and the active ingredients thereof are all the same as those of the suppressant of the present invention.

[0066] 4. Method for treating, preventing, or ameliorating aging, disease, or disorder caused by oxidative stress The present invention also provides a method for treating, preventing, or ameliorating aging, disease, or disorder caused by oxidative stress in a subject (hereinafter, sometimes referred to as the "treatment method of the present invention"), which comprises administering to the subject any of the following (1) to (4): (1) Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment comprising the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted); (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

[0067] The therapeutic method of the present invention is characterized by administering the therapeutic agent of the present invention to a subject, and the active ingredients thereof are all the same as those of the therapeutic agent of the present invention.

[0068] 5. Agent for Promoting Cerebrospinal Fluid (CSF) Clearance Function The present invention also provides an agent for promoting CSF clearance function in a subject (hereinafter, sometimes referred to as "the promoter of the present invention"), which comprises administering to the subject any of the following (1) to (4): (1) Apoptosis inhibitor of Macrophage (AIM); (2) an AIM fragment comprising the SRCR2 domain of AIM; (3) a variant of AIM or an AIM fragment comprising an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted); (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof.

[0069] The active ingredient of the promoter of the present invention is the same as that of the inhibitor of the present invention described above, and the manufacturing method and target of the promoter of the present invention are the same as those of the inhibitor of the present invention, but the promoter of the present invention is administered so that the active ingredient reaches the CSF of the subject. The administration route of the promoter of the present invention is not particularly limited as long as the active ingredient of the promoter of the present invention can reach the CSF of the subject, but is preferably intrathecal administration.

[0070] The amount of the enhancer of the present invention to be administered to a subject is not particularly limited as long as the desired effect is obtained, and may be optimized appropriately depending on the type and form of the active ingredient, the sex, age, weight, administration schedule, administration route, etc. of the subject.

[0071] 6. Agent for treating, preventing, or ameliorating brain dysfunction or neurodegenerative disease The present invention also provides an agent for treating, preventing, or ameliorating brain dysfunction or neurodegenerative disease in a subject, comprising the promoter of the present invention (hereinafter, sometimes referred to as the "agent for treating brain dysfunction or neurodegenerative disease of the present invention").

[0072] The active ingredient of the therapeutic agent for cerebral dysfunction or neurodegenerative disease of the present invention is the promoter of the present invention described above. The therapeutic agent for cerebral dysfunction or neurodegenerative disease of the present invention can treat, prevent, or ameliorate cerebral dysfunction or neurodegenerative disease whose symptoms can be improved by improving CSF clearance function. Examples of such cerebral dysfunction or neurodegenerative disease include, but are not limited to, the following:

[0073] Alzheimer's disease, Parkinson's disease, Lewy body dementia, multiple system atrophy, Pick's disease, corticobasal degeneration, progressive supranuclear paralysis, amyotrophic lateral sclerosis, spinobulbar muscular atrophy, Huntington's disease, spinocerebellar degeneration, dentatorubral-pallidoluysian atrophy

[0074] The above-mentioned diseases involve amyloid accumulation during onset and progression, and improving CSF clearance is expected to treat, prevent, or improve symptoms. Additionally, impaired CSF clearance has been reported to be involved in the onset and exacerbation of psychiatric disorders (especially depression) (1748482131104_0 et al., Mol Psychiatry. 2021 Dec;26(12):7072-7073; Thea Overgaard Wichmann et al., Front Hum Neurosci. 2022 Jan 21:15:737217; Ina Viktoria Mousten et al., JAMA Psychiatry. 2022 Jun 1;79(6):571-581). Therefore, improving clearance function with AIM is expected to treat, prevent, or improve psychiatric disorders (especially depression) caused by impaired CSF clearance.

[0075] The therapeutic agent for cerebral dysfunction or neurodegenerative disease of the present invention has a main mechanism of action of improving the clearance function of CSF, and is therefore administered so that the active ingredient reaches the CSF of the subject. The route of administration of the therapeutic agent for cerebral dysfunction or neurodegenerative disease of the present invention is not particularly limited as long as the active ingredient of the therapeutic agent for cerebral dysfunction or neurodegenerative disease of the present invention can reach the CSF of the subject, but is preferably intrathecal administration.

[0076] 7. Method for Promoting CSF Clearance Function The present invention also provides a method for promoting the CSF clearance function in a subject (hereinafter sometimes referred to as the "promoting method of the present invention"), which comprises administering a promoter of the present invention to the subject.

[0077] The promoting method of the present invention is characterized by administering (particularly, intrathecally administering) the promoting agent of the present invention to the subject, and the active ingredients thereof are all the same as those of the promoting agent of the present invention.

[0078] 8. Method for treating, preventing, or ameliorating cerebral dysfunction or neurodegenerative disease The present invention also provides a method for treating, preventing, or ameliorating cerebral dysfunction or neurodegenerative disease in a subject, which comprises administering (particularly, intrathecally administering) the therapeutic agent for cerebral dysfunction or neurodegenerative disease of the present invention to the subject (hereinafter, sometimes referred to as the "method for treating cerebral dysfunction or neurodegenerative disease of the present invention").

[0079] The method for treating brain dysfunction or neurodegenerative disease of the present invention is characterized by administering (particularly, intrathecally administering) the therapeutic agent for brain dysfunction or neurodegenerative disease of the present invention to a subject to which it is to be applied, and the active ingredients thereof are all the same as those of the therapeutic agent for brain dysfunction or neurodegenerative disease of the present invention.

[0080] In this specification, "treatment" of aging or disease includes not only the cure of aging or disease, but also the remission of aging or disease, improvement of the severity of aging or disease, suppression of the worsening of aging or disease, slowing the rate of worsening of aging or disease, and alleviation of some of the multiple symptoms of aging or disease.

[0081] Furthermore, "prevention" of aging or disease in this specification includes not only preventing the onset of aging or disease, but also delaying the onset of aging or disease, etc. In addition, "prevention" of aging or disease in this specification can also include preventing the recurrence of the aging or disease after treatment, delaying the recurrence of the aging or disease after treatment, preventing re-exacerbation after remission of the aging or disease, delaying the re-exacerbation after remission of the aging or disease, preventing re-exacerbation after alleviation of symptoms of the aging or disease, delaying the re-exacerbation after alleviation of symptoms of the aging or disease, etc.

[0082] As used herein, "illness" refers to a state in which a subject's health condition has temporarily deteriorated or a state in which a biological function has temporarily declined. Furthermore, "improvement of illness" not only refers to restoring a temporarily deteriorated health condition and / or a temporarily declined biological function to its original state, but also includes suppressing or delaying further deterioration or decline of a temporarily deteriorated health condition and / or a temporarily declined biological function.

[0083] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[0084] 1. Prediction of the three-dimensional structural features of recombinant AIM and AIM mutants The three-dimensional structural features of the recombinant AIM (hAIM and mAIM) and AIM mutants (2CS mAIM (SEQ ID NO: 19) and △SRCR3 mAIM (SEQ ID NO: 20)) used in this example were predicted using Alphafold2 in the AlphaFold Protein Structure Database. The prediction results are shown in Figure 1.

[0085] 2. Viability Assay of Human Renal Tubular Epithelial Cell Line (HK-2) with H2O2. HK-2 cells were cultured in DMEM, high glucose, and GlutaMAX. TM(GIBCO, 10569-044), supplemented with 10% (vol / vol) fetal bovine serum (FBS; Thermo Fisher Scientific), 20 μg / mL gentamicin (Thermo Fisher Scientific 15710072), maintained at 37°C and 5% CO2. Cells were harvested from confluent 10-cm dishes with 0.25% Trypsin-EDTA (GIBCO, 25200-072) and plated at 2 × 10 cells per well in a 96-well plate. 5 Cells were plated at 1 / well. After cells adhered to the plate and reached 80% confluence, the culture medium was replaced with glutamine-deficient assay medium (D-MEM, High Glucose (Fujifilm, 4530285), supplemented with 10% (vol / vol) FBS, 20 μg / mL gentamicin) the day before the assay and incubated overnight. H2O2 (Wako, 081-04215) was then added to the medium at 25–100 μM, and bovine serum albmin (BSA; Sigma-Aldrich, A8806), 2CS mAIM, △SRCR3 mAIM, mAIM, and hAIM were added at 50 μg / mL each, and the cells were incubated for a total of 3 hours. The supernatant was then removed and replaced with 10% Prestoblue (Invitrogen A13261) assay medium. The cells were incubated at 37°C, 5% CO for 1 hour, and the fluorescence of the supernatant was measured (Ex: 560 / Em: 590 nm). The results are shown in Figure 2.

[0086] As shown in FIG. 2, the addition of ΔSRCR3 mAIM, mAIM, or hAIM improved cell viability.

[0087] 3. Total ROS / Lipid Peroxidation Detection. HK-2 cells were pre-cultured under the same conditions as in the Viability Assay described above, then stimulated with 50 μM H2O2 for 2 hours. The medium was then replaced to remove H2O2. 2CS mAIM, △SRCR3 mAIM, mAIM, or hAIM was added at 50 μg / mL and incubated for 3 hours. Subsequently, cells were stained with 10 μM ROS Assay Kit - Photo-oxidation Resistant DCFH-DA (Dojindo, R253) at 37°C, 5% CO2 for 30 minutes, and then washed with HBSS (+) (NACALAI TESQUE, 09735-75). The cells were then observed and photographed using an LSM980 system (Carl Zeiss) under reduced laser power (Ex 493 nm; Em 513 nm; laser power, 0.05%; averaging, 2; Gain, 900V; LSM plus). Separately, HK-2 cells stimulated under the same culture and stimulation conditions were stained for lipid peroxides with Liperfluo (Dojindo, L248). The staining conditions were 20 μM, 15 min, 37°C, and 5% CO2. Images were taken using the LSM980 system (Lambda mode; Ex 488 nm; averaging, 4). Figure 3 shows confocal images of cells stimulated with H2O2 under each condition. Figure 4 also shows the results of quantifying the average intensity of the confocal images under each condition using ZEN software (the region of interest (ROI) was set to include at least 20 cells per image).

[0088] As shown in Figures 3 and 4, when △SRCR3 mAIM, mAIM, and hAIM were added, the amounts of intracellular ROS and lipid peroxides were maintained at low levels even after stimulation with H2O2.

[0089] 4. Immunocytochemistry (ICC) of Lipid Peroxidation Metabolites (4-HNE and MDA). HK-2 cells were cultured under the same conditions as in 3. Total ROS / Lipid Peroxidation Detection and then stimulated with H2O2. After stimulation, mAIM protein was added and incubated. After incubation, cells were fixed with 4% PFA for 10 min at room temperature and permeabilized with 0.1% Triton-X for 10 min. Cells were then blocked with Blocking Solution (G-Block; Genostaff, GB-01) for 20 min at room temperature. Primary antibodies used were anti-4-HNE rabbit polyclonal antibody (1:100 dilution; Bioss, bs-6313) and anti-MDA rabbit polyclonal antibody (1:50 dilution; MyBioSource, MBS2032894), and incubated for 1.5 h at room temperature. The secondary antibody used was Chicken anti-Rabbit IgG (H+L) (1:1000 dilution; Invitrogen, A-21441). After adding the secondary antibody, the cells were incubated for 30 minutes at room temperature in the dark, and then observed and photographed using the LSM980 system. The results are shown in Figure 5.

[0090] As shown in Figure 5, the addition of mAIM reduced the amounts of 4-HNE and MDA in HK-2 cells stimulated with H2O2.

[0091] 5. Western analysis of H2O2- and AIM-treated cell lysates HK-2 cells were cultured under the same conditions as those used in 3. Total ROS / Lipid peroxidation detection and then stimulated with H2O2. After stimulation, each AIM protein (mAIM or hAIM) was added, and after incubation, the cells were gently washed twice with cold PBS to avoid cell detachment. Next, cells were lysed using Mammalian Cell Lysis Buffer (Abcam, ab179835) according to the manufacturer's protocol, and then lysed with a protease inhibitor cocktail (cComplete). TM, Mini, Roche, 11836153001) was added and temporarily stored at -80°C. Lysate samples were quantified using a BCA assay (Thermo Fisher Scientific, 23225) and then diluted with 0.1X Sample Buffer (Simple Western, 042-195) to standardize the protein content. The obtained samples were analyzed by ProteinSimple Western protein analysis (U. Nguyen, N. Squaglia, A. Boge, and PA Fung, "The Simple Western"). TM 4-HNE was quantified using a Western blotting technique (Nature Methods, Vol. 8, No. 11, pp. v-vi, 2011 / 11 / 01 2011, doi: 10.1038 / nmeth.f.353). Anti-4-HNE rabbit polyclonal antibody was used at a 1:50 dilution, and anti-human β-Actin antibody (rabbit monoclonal antibody; clone 13E5; Cell Signaling, 4970S) was used at a 1:100 dilution. The results are shown in Figure 6.

[0092] As shown in FIG. 6, the reduction of oxidative stress by the addition of AIM was also confirmed by Western analysis.

[0093] 6. DPPH Antioxidant Assay. The antioxidant activities of L-ascorbate (Vitamin C), reduced GSH (both of which are representative organic compounds with reducing activity), 2CS-mAIM, and mAIM were analyzed using a DPPH antioxidant assay kit (Dojindo, D678) according to the manufacturer's protocol. Briefly, 20 μL of sample (250 μg / mL each) was added to each well of a 96-well plate, followed by 80 μL of assay buffer and 100 μL of DPPH working solution. The plate was then incubated at 25°C for 30 minutes in the dark and measured at 517 nm using a microplate reader. The radical scavenging rate (%) was calculated as the antioxidant activity of the sample. This value was divided by the molar concentration of each molecule per 250 μg / mL and plotted relative to Vitamin C. The results are shown in Figure 7.

[0094] As shown in Figure 7, mAIM exhibited very strong antioxidant activity in a cell-free system.

[0095] 7. Western analysis of nuclear fraction lysate from H2O2- and AIM-treated cells. HK-2 cells were cultured under the same conditions as those used in 2. Viability assay of human renal tubular epithelial cell line (HK-2) with H2O2. Then, 8 × 10 cells were plated on a 6-cm dish. 4Cells were seeded at 1000 x g for 1 hour. The medium was replaced with assay medium 4 hours before the start of the assay. Cells were incubated for 1.5 hours with 50 μM H2O2, or for 2.5 hours with mAIM or 2CS-mAIM at 50 μg / mL, or without any of the three. After incubation, cells were gently washed twice with PBS to avoid detachment. Cells were then detached with a scraper and collected in a 1.5 mL tube. The collected cells were pelleted by centrifugation at 12,000 x g for 5 minutes at 4°C. Nuclear fractions were extracted from the pellets using the LysoPure™ Nuclear and Cytoplasmic Extractor Kit according to the manufacturer's protocol. Each fraction was stored at -80°C with the addition of a protease inhibitor cocktail. Protein content was quantified by BCA assay immediately before Western assay. Protein amounts were standardized by dilution with 0.1x sample buffer. The rest of the protocol was performed according to the user's guide. Anti-human NRF2 antibody (rabbit polyclonal; Proteintech, 16396-1-AP) was used at a dilution of 1:50, and anti-Lamin B1 antibody (rabbit polyclonal; Abcam, ab16048) was used at a dilution of 1:100. The results are shown in Figure 8.

[0096] It is known that H2O2 promotes the nuclear translocation of Nrf2 and increases Nrf2 in the nuclear fraction. As shown in Figure 8, the addition of AIM also increased Nrf2 in the nuclear fraction.

[0097] 8. Analysis of transcription levels of antioxidant-related genes regulated by Nrf2 by quantitative PCR (qPCR) HK-2 cells were cultured under the same conditions as those used in 2. Viability assay of human renal tubular epithelial cell line (HK-2) with H2O2 addition, and then plated at 5x10 cells in a 12-well plate. 5Cells were seeded at 1 / well. 24 hours before the start of the assay, the medium was replaced with assay medium and incubated. H2O2 stimulation was performed at 50 μM for 2 hours. After the medium was replaced, 2CS-mAIM, △SRCR3-mAIM, mAIM, and hAIM were added at 50 μg / mL each and incubated for 3 hours. After incubation, the cells were gently washed twice with PBS to avoid detaching. Cells were harvested and RNA was extracted from the cells using ReliaPrep RNA Miniprep Systems (Promega). cDNA was prepared using SuperScript IV VILO Master Mix (Thermo Fisher Scientific) with the extracted RNA as a template. Quantitative evaluation of mRNA was performed using the △△CT method using the QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific) and Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). The transcription levels of antioxidant-related genes NQO1, hGCLC, hGCLM, hPRDX1, hPRDX2, and hPRDX6 were confirmed. The sequences of the oligonucleotides used in PCR are shown below, and the results are shown in Figure 9.

[0098]

[0099] As shown in FIG. 9, when ΔSRCR3 mAIM, mAIM, or hAIM was added, the transcription levels of antioxidant-related genes regulated by Nrf2 tended to increase.

[0100] 9. Effect of AIM on High-Fat Diet-Induced Senescence of Liver Sinusoidal Endothelial Cells Wild-type (WT) and AIM-deficient (AIM KO) mice (C57BL / 6J background, 8 weeks old) were fed a high-fat diet (HFD) for 8 weeks. During the challenge, mice were intraperitoneally injected with rAIM (300 μg / mouse) once a week. As a control, the same volume of PBS (250 μL / mouse) was intraperitoneally injected once a week. Mice were sacrificed at 16 weeks of age, and liver specimens were fixed with formaldehyde and immunostained for LaminB1, a representative senescence marker that decreases with cellular senescence. The immunostaining results are shown in Figure 10. 2 The graph of the number of LaminB1-positive cells per 1000 cells is shown in FIG.

[0101] LaminB1 is expressed mainly on the nuclear membrane of sinusoidal endothelial cells and Kupffer macrophage cells in the liver, and its expression is known to be attenuated or disappear as cellular senescence progresses. As shown in Figures 10 and 11, cellular senescence was significantly accelerated in AIM-deficient mice (KO) after 8 weeks of HFD. On the other hand, administration of AIM abolished cellular senescence in AIM-deficient mice (KO).

[0102] 10. AIM Suppresses Age-Related Accumulation of Circulating Cell-Free DNA (cfDNA) and Mitochondrial DNA (mtDNA). Extracellularly released cell-free DNA (cfDNA) appears in the blood as a result of cell death (apoptosis and necrosis) and immune responses such as NETosis. Mitochondrial DNA (mtDNA) in particular, due to its structural characteristics (CpG unmethylation and bacterial-like circular structure), has the potential to induce strong inflammatory responses as a damage-associated molecular pattern (DAMP). It is known that aging and aging are accompanied by abnormal cell turnover and mitochondrial dysfunction, leading to increased levels of cfDNA and mtDNA in the blood. For example, it has been reported that 12-month-old mice have significantly higher levels of cfDNA and mtDNA in the blood compared to 3-month-old mice (Pinti et al., Eur J Immunol. 2014 May;44(5):1552-62).

[0103] Three wild-type (WT) and three AIM-deficient (AIM KO) mice were fed either a normal diet or a high-fat diet (HFD). After 6 weeks (measured only in the HFD group) and 12 weeks, blood cfDNA concentrations and mtDNA (mt-COX1 DNA) copy numbers were measured. cfDNA was also measured one year later. The layout of this study is shown in Table 1 below. The results are shown in Figure 12.

[0104]

[0105] Regarding cfDNA, as shown in Figure 12A, after one year of normal diet loading, only AIM KO mice showed an increase in cfDNA blood levels. Furthermore, after HFD loading, WT mice showed an increase in cfDNA after one year, but AIM KO mice showed an increase after 12 weeks, and the increase after one year was even more pronounced than in WT mice. Regarding mtDNA, as shown in Figure 12B, no increase in mtDNA levels was observed in the blood after normal diet loading, but after 12 weeks of HFD loading, an increase in mtDNA levels was observed. The increase in mtDNA levels was even more pronounced in AIM KO mice.

[0106] 10. Suppression of Age-Related Accumulation of Blood mtDNA by AIM. AIM+ / - mice (essentially identical to WT mice) were fed an HFD and intraperitoneally injected once a week with recombinant mouse AIM (rAIM; n=2) or the same volume of PBS (n=1). Six months later, the blood levels (copy numbers) of two types of mtDNA (mitochondrial COX1 and mitochondrial ND1) were measured. The results are shown in Figure 13.

[0107] As shown in FIG. 13, a significant decrease in the blood mtDNA levels of both COX1 and ND1 was observed in rAIM-administered mice.

[0108] 11. AIM Improves Cerebrospinal Fluid (CSF) Clearance and Suppresses Age-Related Protein Accumulation The brain is surrounded by cerebrospinal fluid (CSF), which is restricted from direct contact with blood components by the blood-brain barrier. CSF is produced at a volume of over 500 mL per day, providing nutrients to neurons and removing waste products, thereby maintaining brain homeostasis. Recently, a "glymphatic system" has been proposed, in which CSF flows into the brain parenchyma driven by arterial pulsation and is exchanged with interstitial fluid (ISF) via aquaporin 4 (AQP4) in astrocytes. This system is increasingly recognized as playing an important role in the clearance of amyloid beta and tau (Jeffrey J Iliff et al., Sci Transl Med. 2012 Aug 15;4(147):147ra111.). This function declines with aging and neurodegenerative diseases, and impaired CSF clearance has been reported in Alzheimer's disease and Parkinson's disease. Therefore, we examined the amount of CSF clearance in WT and AIM KO mice.

[0109] A catheter with a 27G needle was inserted into the cisterna magna of stereotaxically fixed WT or AIM KO mice (n = 3 each). 5 μL of CSF was removed, and then 65 μg of FITC-labeled Dextran 70kDa (FD70) ± 4 μg of rAIM diluted with artificial CSF was infused into the cisterna magna at a rate of 1 μg / min.

[0110] 5 μL of CSF was collected from the catheter after 20 minutes or 1 hour, and the fluorescence intensity (Ex: 490, Em: 520) of the collected sample was measured using a dedicated low-volume microplate to analyze the amount of residual FD70.

[0111] In addition, WT mice (n=3) that had been subjected to treadmill exercise (20 m / min, 30 min) before FD70 administration were also analyzed in the same manner. The results are shown in Figure 14.

[0112] As shown in Figure 14, in WT mice, as previously reported, exercise improved FD70 clearance, but administration of rAIM into the CSF also improved clearance to a similar or greater extent. In contrast, in AIM KO mice, FD70 clearance was significantly reduced compared to WT mice. These results demonstrate that AIM improves CSF clearance.

[0113] 12. Proteomic Analysis of CSF in WT and AIM KO Mice Proteomic analysis was performed using LC-MS on CSF (a mix of three mice) from 4-month-old (4M) and 18-month-old (18M) WT and AIM KO mice (n=3 each). The protein levels in the CSF of each mouse were compared for proteins known to vary in CSF in Alzheimer's disease (AD). The results are shown in Table 2 below. The protein levels in the table are relative values, with the protein concentration in the CSF of 4-month-old WT mice set at 1.

[0114]

[0115] As shown in Table 2, significant accumulation of most proteins was observed in AIM KO mice of the same age compared to 18-month-old WT mice. This result strongly suggests that AIM may suppress age-related brain dysfunction through CSF clearance. In other words, administering AIM so that it reaches the CSF of a subject may enable the treatment or prevention of age-related brain dysfunction and neurodegenerative diseases such as Alzheimer's disease.

[0116] According to the present invention, oxidative stress in a living body can be suppressed extremely efficiently. As a result, aging, diseases, or disorders caused by oxidative stress can be efficiently treated, prevented, or improved. Therefore, the present invention is extremely useful in the medical field.

[0117] This application is based on patent application No. 2024-088911 filed in Japan (filing date: May 31, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. An agent for suppressing oxidative stress, comprising any one of the following (1) to (4): (1) Apoptosis inhibitor of Macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted), or (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

2. An agent for treating, preventing or ameliorating aging, disease or disorders caused by oxidative stress, comprising the agent according to claim 1.

3. The agent according to claim 2, wherein the aging caused by oxidative stress is aging of the skin.

4. The agent according to claim 2, wherein the disease caused by oxidative stress is an aging-related eye disease.

5. The agent according to claim 4, wherein the aging-related eye disease is any one selected from the group consisting of age-related macular degeneration and cataracts.

6. The agent according to claim 2, wherein the disorder caused by oxidative stress is at least one selected from the group consisting of the following (1) to (3): (1) fatigue or decreased physical strength, (2) decreased immune function, and (3) hair loss or poor hair growth.

7. A method for suppressing oxidative stress in a subject, comprising administering to the subject any of the following (1) to (4): (1) Apoptosis inhibitor of macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted), or (4) a nucleic acid encoding said AIM, said AIM fragment or a variant thereof.

8. A method for treating, preventing, or ameliorating aging, disease, or disorder caused by oxidative stress in a subject, comprising administering to the subject any of the following (1) to (4): (1) Apoptosis inhibitor of macrophage (AIM), (2) an AIM fragment containing the SRCR2 domain of AIM, (3) a variant of AIM or an AIM fragment containing an amino acid sequence having 90% or more identity to said AIM or said AIM fragment (with the proviso that the free cysteine ​​residue in the SRCR2 domain is not substituted), or (4) a nucleic acid encoding said AIM, said AIM fragment, or a variant thereof.

9. The method according to claim 8, wherein the aging caused by oxidative stress is aging of the skin.

10. The method according to claim 8, wherein the disease caused by oxidative stress is an aging-related eye disease.

11. The method of claim 10, wherein the aging-related eye disease is any one selected from the group consisting of age-related macular degeneration and cataracts.

12. The method according to claim 8, wherein the disorder caused by oxidative stress is at least one selected from the group consisting of the following (1) to (3): (1) fatigue or decreased physical strength, (2) decreased immune function, and (3) hair loss or poor hair growth.

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