Prophylactic or ameliorating agent for cell senescence

Abstract

The present invention provides a prophylactic or ameliorating agent for cell senescence, which contains 2 '-fucosyllactose.

Description

Technical Field

[0001] The present invention relates to an agent for preventing or improving cellular senescence. Background Art

[0002] Human milk oligosaccharides (HMOs) are oligosaccharides found in human breast milk. HMOs serve as nutrients for beneficial bacteria such as Bifidobacterium and are expected to have various physiological applications.

[0003] For example, Patent Document 1 describes a growth inhibitor against oral pathogens containing one or more selected from lacto-oligosaccharides and lactulose. Sialyl lactose (Patent Documents 2 to 11) and fucosyllactose (Patent Documents 12 to 17) are known as HMOs.

[0004] Furthermore, in recent years, with the increasing aging of society, technologies related to anti-aging have received particular attention. The GLS1 gene, known as an anti-aging gene, is associated with glutamine metabolism and is essential for the survival of senescent cells. Therefore, to prevent or ameliorate cellular aging, for example, it is necessary to inhibit the expression of the GLS1 gene. For example, Patent Document 18 describes a GLS1 gene inhibitor characterized by containing a substance derived from Euglena as an active ingredient for inhibiting GLS1 gene expression. Furthermore, the use of endoplasmic reticulum stress ameliorators (e.g., Patent Document 19) to prevent or ameliorate cellular aging is also anticipated.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Laid-Open No. 2021-065176

[0008] Patent Document 2: Korean Patent No. 2019-0067499

[0009] Patent Document 3: International Publication No. 2012 / 053849

[0010] Patent Document 4: International Publication No. 2014 / 138578

[0011] Patent Document 5: International Publication No. 2000 / 006115

[0012] Patent Document 6: Korean Patent No. 2011-0092602

[0013] Patent Document 7: Korean Patent No. 2016-0009738

[0014] Patent Document 8: Japanese Patent Application Laid-Open No. 2001-206848

[0015] Patent Document 9: Japanese Patent Application Laid-Open No. 3-049648

[0016] Patent Document 10: Japanese Patent Application Laid-Open No. 2-207089

[0017] Patent Document 11: International Publication No. 2016 / 172183

[0018] Patent Document 12: Korean Patent No. 2018-0051688

[0019] Patent Document 13: Korean Patent No. 2019-0045079

[0020] Patent Document 14: Chinese Patent No. 109043542

[0021] Patent Document 15: European Patent No. 3127543

[0022] Patent Document 16: U.S. Patent No. 5,002,759

[0023] Patent Document 17: U.S. Patent No. 5,401,723

[0024] Patent Document 18: Japanese Patent Application Laid-Open No. 2022-112464

[0025] Patent Document 19: Japanese Patent Application Publication No. 2020-011930 Summary of the Invention

[0026] Problems to be solved by the invention

[0027] An object of the present invention is to provide a novel agent for preventing or ameliorating cellular senescence.

[0028] Methods used to solve problems

[0029] The present invention relates to, for example, the following [1] to

[51] .

[0030] [1] A preventive or improving agent for cell aging, comprising 2'-fucosyllactose.

[0031] [2] The preventive or improving agent according to [1], which is used for promoting the proliferation of normal cells.

[0032] [3] The preventive or improving agent according to [1], which is used for removing senescent cells.

[0033] [4] The preventive or ameliorating agent according to any one of [1] to [3], wherein the cellular senescence is cellular senescence caused by GLS1 expression.

[0034] [5] The preventive or ameliorating agent according to any one of [1] to [3], wherein the cellular senescence is cellular senescence caused by endoplasmic reticulum stress.

[0035] [6] The preventive or improving agent according to any one of [1] to [5], wherein the cells are skin cells.

[0036] [7] The preventive or improving agent according to any one of [1] to [6], wherein the cells are epidermal keratinocytes.

[0037] [8] The preventive or improving agent according to any one of [1] to [6], wherein the cells are dermal fibroblasts.

[0038] [9] The preventive or improving agent according to any one of [1] to [8], which is at least one selected from the group consisting of a skin wound treatment agent, a skin metabolism promoter, a pigmentation preventive and improving agent, a wrinkle preventive and improving agent, a sagging preventive and improving agent, and a rough skin preventive and improving agent.

[0039] [9a] The preventive or improving agent according to any one of [1] to [5], wherein the cells are renal cells, gastric cells, microglial cells, gingival cells, or vascular endothelial cells.

[0040] [9b] The preventive or ameliorative agent according to any one of [1] to [5] and [9a], which is at least one selected from the group consisting of a preventive or ameliorative agent for glomerulosclerosis, a preventive or ameliorative agent for decreased renal function, a preventive or ameliorative agent for decreased gastric acid secretion by gastric parietal cells, a preventive or ameliorative agent for decreased pepsinogen secretion by gastric chief cells, a preventive or ameliorative agent for decreased cognitive function, a preventive or ameliorative agent for decreased salivary secretion, a preventive or ameliorative agent for gingival inflammation, a preventive or ameliorative agent for arteriosclerosis, and a preventive or ameliorative agent for obesity, diabetes, or impaired glucose tolerance.

[0041]

[10] The preventive or ameliorating agent according to any one of [1] to [9b], which is an external preparation.

[0042]

[11] The preventive or ameliorating agent according to any one of [1] to

[10] , which is a cosmetic.

[0043] [11a] The preventive or ameliorating agent according to any one of [1] to [9b], which is a food.

[0044]

[12] A method for preventing or improving cellular senescence, comprising the step of administering 2'-fucosyllactose to a subject in need thereof.

[0045]

[13] A method for promoting normal cell proliferation, comprising the step of administering 2'-fucosyllactose to a subject in need thereof.

[0046]

[14] A method for removing senescent cells, comprising the step of administering 2'-fucosyllactose to a subject in need thereof.

[0047]

[15] The prevention or improvement method according to

[12] , wherein the cell senescence is cell senescence caused by GLS1 expression.

[0048]

[16] The prevention or improvement method according to

[12] , wherein the cell senescence is cell senescence caused by endoplasmic reticulum stress.

[0049]

[17] The method according to any one of

[12] to

[16] , wherein the cells are skin cells.

[0050]

[18] The method according to any one of

[12] to

[17] , wherein the cells are epidermal keratinocytes.

[0051]

[19] The method according to any one of

[12] to

[17] , wherein the cells are dermal fibroblasts.

[0052]

[20] A method for treating skin wounds, a method for promoting skin metabolism, a method for preventing or improving pigmentation, a method for preventing or improving wrinkles, a method for preventing or improving sagging, or a method for preventing or improving rough skin, comprising the step of administering 2'-fucosyllactose to a subject in need thereof.

[0053] [20a] The method according to any one of

[12] to

[16] , wherein the cells are kidney cells, gastric cells, microglial cells, gingival cells or vascular endothelial cells.

[0054] [20b] A method for preventing or improving glomerulosclerosis, a method for preventing or improving decreased renal function, a method for preventing or improving decreased gastric acid secretion by gastric parietal cells, a method for preventing or improving decreased pepsinogen secretion by gastric chief cells, a method for preventing or improving decreased cognitive function, a method for preventing or improving decreased salivary secretion, a method for preventing or improving gingival inflammation, a method for preventing or improving arteriosclerosis, or a method for preventing or improving obesity, diabetes, or impaired glucose tolerance, comprising the step of administering 2'-fucosyllactose to a subject in need thereof.

[0055]

[21] The method according to any one of

[12] to [20b], wherein the 2'-fucosyllactose is administered transdermally.

[0056] [21a] The method according to any one of

[12] to [20b], wherein the 2'-fucosyllactose is orally ingested.

[0057]

[22] 2'-fucosyllactose for use in therapeutic purposes, preventing or improving cell senescence.

[0058]

[23] 2'-fucosyllactose for promoting normal cell proliferation for therapeutic purposes.

[0059]

[24] 2'-fucosyllactose for use in removing senescent cells for therapeutic purposes.

[0060]

[25] The 2'-fucosyllactose for use according to

[22] , wherein the cell senescence is caused by GLS1 expression.

[0061]

[26] The 2'-fucosyllactose for use according to

[22] , wherein the cell senescence is cell senescence caused by endoplasmic reticulum stress.

[0062]

[27] The 2'-fucosyllactose for use according to any one of

[22] to

[26] , wherein the cells are skin cells.

[0063]

[28] The 2'-fucosyllactose for use according to any one of

[22] to

[27] , wherein the cells are epidermal keratinocytes.

[0064]

[29] The 2'-fucosyllactose for use according to any one of

[22] to

[27] , wherein the cells are dermal fibroblasts.

[0065]

[30] 2'-fucosyllactose for use in at least one of the following: skin wound treatment and skin metabolism promotion for therapeutic purposes.

[0066] [30a] 2'-fucosyllactose for use according to any one of

[22] to

[26] , wherein the cells are renal cells, gastric cells, microglial cells, gingival cells, or vascular endothelial cells.

[0067] [30b] 2'-fucosyllactose for use in at least one of the following therapeutic purposes: prevention or improvement of glomerulosclerosis, prevention or improvement of decreased renal function, prevention or improvement of decreased gastric acid secretion by gastric parietal cells, prevention or improvement of decreased pepsinogen secretion by gastric chief cells, prevention or improvement of decreased cognitive function, prevention or improvement of decreased salivary secretion, prevention or improvement of gingival inflammation, prevention or improvement of arteriosclerosis, and prevention or improvement of obesity, diabetes, or impaired glucose tolerance.

[0068]

[31] The 2'-fucosyllactose for use according to any one of

[22] to [30b], which is for transdermal use.

[0069] [31a] The 2'-fucosyllactose for use according to any one of

[22] to [30b], which is for oral use.

[0070]

[32] Non-therapeutic application of 2'-fucosyllactose for preventing or improving cell aging.

[0071]

[33] Non-therapeutic application of 2'-fucosyllactose for promoting normal cell proliferation.

[0072]

[34] Non-therapeutic application of 2'-fucosyllactose for the removal of senescent cells.

[0073]

[35] The use according to

[32] , wherein the cell senescence is caused by GLS1 expression.

[0074]

[36] The use according to

[32] , wherein the cell senescence is cell senescence caused by endoplasmic reticulum stress.

[0075]

[37] The use according to any one of

[32] to

[36] , wherein the cells are skin cells.

[0076]

[38] The use according to any one of

[32] to

[37] , wherein the cells are epidermal keratinocytes.

[0077]

[39] The use according to any one of

[32] to

[37] , wherein the cells are dermal fibroblasts.

[0078]

[40] Use of 2'-fucosyllactose for non-therapeutic purposes, wherein the use is for at least one selected from the group consisting of prevention and improvement of pigmentation, prevention and improvement of wrinkles, prevention and improvement of sagging, and prevention and improvement of rough skin.

[0079] [40a] The use according to any one of

[32] to

[36] , wherein the cells are kidney cells, gastric cells, microglial cells, gingival cells or vascular endothelial cells.

[0080] [40b] Use of 2'-fucosyllactose for non-therapeutic purposes, for at least one of the following: prevention or improvement of glomerulosclerosis, prevention or improvement of decreased renal function, prevention or improvement of decreased gastric acid secretion by gastric parietal cells, prevention or improvement of decreased pepsinogen secretion by gastric chief cells, prevention or improvement of decreased cognitive function, prevention or improvement of decreased salivary secretion, prevention or improvement of gingival inflammation, prevention or improvement of arteriosclerosis, and prevention or improvement of obesity, diabetes, or impaired glucose tolerance.

[0081]

[41] The use according to any one of

[32] to [40b], which is transdermal application.

[0082] [41a] The use according to any one of

[32] to [40b], which is an oral application.

[0083]

[42] Application of 2'-fucosyllactose for the manufacture of a preventive or improving agent for cell aging.

[0084]

[43] Application of 2'-fucosyllactose in the manufacture of a normal cell proliferation promoter.

[0085]

[44] Application of 2'-fucosyllactose for the manufacture of an agent for removing aged skin cells.

[0086]

[45] The use according to

[42] , wherein the cell senescence is caused by GLS1 expression.

[0087]

[46] The use according to

[42] , wherein the cell senescence is cell senescence caused by endoplasmic reticulum stress.

[0088]

[47] The use according to

[42] to

[46] , wherein the cells are skin cells.

[0089]

[48] ​​The use according to any one of

[42] to

[47] , wherein the cells are epidermal keratinocytes.

[0090]

[49] The use according to any one of

[42] to

[47] , wherein the cells are dermal fibroblasts.

[0091]

[50] Use of 2'-fucosyllactose for the manufacture of at least one agent selected from the group consisting of a skin wound treatment agent, a skin metabolism promoter, a pigmentation preventive and ameliorative agent, a wrinkle preventive and ameliorative agent, a sagging preventive and ameliorative agent, and a rough skin preventive and ameliorative agent.

[0092] [50a] The use according to any one of

[42] to

[46] , wherein the cells are kidney cells, gastric cells, microglial cells, gingival cells or vascular endothelial cells.

[0093] [50b] Use of 2'-fucosyllactose for the manufacture of at least one agent selected from the group consisting of an agent for preventing or improving glomerulosclerosis, an agent for preventing or improving decreased renal function, an agent for preventing or improving decreased gastric acid secretion by gastric parietal cells, an agent for preventing or improving decreased pepsinogen secretion by gastric chief cells, an agent for preventing or improving decreased cognitive function, an agent for preventing or improving decreased salivary secretion, an agent for preventing or improving gingival inflammation, an agent for preventing or improving arteriosclerosis, and an agent for preventing or improving obesity, diabetes, or impaired glucose tolerance.

[0094]

[51] The use according to any one of

[42] to [50b], wherein the agent is an external preparation.

[0095] [51a] The use according to any one of

[42] to [50b], wherein the agent is an oral agent.

[0096] Effects of the Invention

[0097] According to the present invention, a novel agent for preventing or improving cellular senescence can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 It is a graph showing the cell proliferation rate of normal epidermal keratinocytes.

[0099] Figure 2 is a graph showing the cell proliferation rate of normal skin fibroblasts.

[0100] Figure 3 Graph showing the cell proliferation rate of senescent skin fibroblasts.

[0101] Figure 4 This is a graph showing the GLS1 expression inhibitory effect of 2'-fucosyllactose (2'-FL).

[0102] Figure 5 Graph showing the cell survival rate of epidermal keratinocytes treated with endoplasmic reticulum stress.

[0103] Figure 6 Graph showing the cell survival rate of skin fibroblasts treated with endoplasmic reticulum stress.

[0104] Figure 7 This is a graph showing the effect of 2'-FL on restoring laminin secretion in epidermal keratinocytes.

[0105] Figure 8 This figure shows the effect of 2'-FL on restoring laminin secretion in skin fibroblasts.

[0106] Figure 9 This is a graph showing the effect of 2'-FL on promoting type I collagen expression in skin fibroblasts.

[0107] Figure 10 This is a graph showing the IL-1α expression inhibitory effect of 2'-FL in skin fibroblasts.

[0108] Figure 11 This figure shows the results of verifying the GLS1 expression inhibitory effect of lactose in renal cells, gastric cells, microglial cells, gingival cells, or vascular endothelial cells.

[0109] Figure 12 The graph shows the inhibitory effect of 2'-FL on GLS1 expression in renal cells, gastric cells, microglial cells, gingival cells, or vascular endothelial cells. DETAILED DESCRIPTION

[0110] One embodiment of a preventive or ameliorative agent for cellular senescence contains 2'-fucosyllactose (2'-FL). 2'-FL is a trisaccharide composed of L-fucose, D-galactose, and D-glucose, in which the 2'-position of lactose is fucosylated. 2'-FL can be synthesized and purified in Escherichia coli, or a commercially available product can be used.

[0111] The agent for preventing or ameliorating cellular senescence according to one embodiment can inhibit GLS1 gene expression by containing 2'-FL. Specifically, the agent for preventing or ameliorating cellular senescence according to one embodiment can be a GLS1 expression inhibitor or a GLS1 gene expression inhibitor. "Inhibiting GLS1 gene expression" includes not only inhibiting expression at the transcriptional level (expression in the form of mRNA) but also inhibiting expression at the translational level (expression in the form of protein).

[0112] When GLS1 gene expression is inhibited, senescent cells are eliminated. Therefore, a GLS1 expression inhibitor according to one embodiment can be used to eliminate senescent cells and can be a senescent cell-eliminating agent containing 2'-FL. In this specification, a "senescent cell-eliminating agent" refers to an agent used to eliminate senescent cells from skin tissue. "Senescent cell elimination" means clearing senescent cells from a tissue or causing senescent cells to die within a tissue. "Senescent cell elimination" refers, for example, to inducing cell death in senescent cells in vivo or in vitro or selectively eliminating senescent cells from a cell population containing senescent cells. A GLS1 expression inhibitor and a senescent cell-eliminating agent according to one embodiment can selectively cause senescent cell death due to their ability to inhibit GLS1 gene expression. Therefore, according to one embodiment, a GLS1 expression inhibitor and a senescent cell-eliminating agent can inhibit the accumulation of senescent cells in tissues (e.g., skin tissue, kidneys, stomach, brain, spinal cord, gums, blood vessels, etc.), and can inhibit the decline in the health of tissues (e.g., skin, kidneys, stomach, brain, spinal cord, gums, blood vessels, etc.).

[0113] The preventive or ameliorative agent for cell senescence of one embodiment can promote the proliferation of normal cells by containing 2'-FL. That is, the preventive or ameliorative agent for cell senescence of one embodiment can be a normal cell proliferation promoter containing 2'-FL. The preventive or ameliorative agent for cell senescence of one embodiment can also promote the proliferation of normal cells at the site of wound in tissues (e.g., skin, kidney, stomach, brain, spinal cord, gums, blood vessels, etc.), thereby promoting wound healing. Therefore, the preventive or ameliorative agent for cell senescence of one embodiment can be used for the treatment of tissue wounds and can be a tissue wound treatment agent. The preventive or ameliorative agent for cell senescence of one embodiment can be used for the treatment of skin wounds and can be a skin wound treatment agent.

[0114] The agent for preventing or ameliorating cellular senescence according to one embodiment contains 2'-FL, thereby promoting normal cell proliferation and removing senescent cells.

[0115] The agent for preventing or ameliorating cellular senescence according to one embodiment can be used for at least one application selected from the group consisting of tissue wound treatment and cell metabolism promotion. That is, the agent for preventing or ameliorating cellular senescence according to one embodiment can be at least one selected from the group consisting of tissue wound treatment agents and cell metabolism promoters containing 2'-FL. The agent for preventing or ameliorating cellular senescence according to one embodiment can be used for at least one application selected from the group consisting of skin wound treatment, skin metabolism promotion, prevention and improvement of pigmentation, prevention and improvement of wrinkles, prevention and improvement of sagging, and prevention and improvement of rough skin. That is, the agent for preventing or ameliorating cellular senescence according to one embodiment can be at least one selected from the group consisting of skin wound treatment agents, skin metabolism promoters, prevention and improvement of pigmentation, prevention and improvement of wrinkles, prevention and improvement of sagging, and prevention and improvement of rough skin containing 2'-FL.

[0116] The agent for preventing or ameliorating cellular aging according to one embodiment, by containing 2'-FL, can promote the expression of type I collagen in cells, more effectively promote type I collagen expression in skin cells, and further effectively promote type I collagen expression in dermal fibroblasts. Type I collagen present in the skin, particularly the dermis, imparts elasticity and strength to the skin. Therefore, the agent for preventing or ameliorating cellular aging according to one embodiment can increase type I collagen in the skin, particularly the dermis, and can therefore also be used as an agent for preventing or ameliorating wrinkles and / or sagging.

[0117] The preventive or ameliorative agent for cell aging according to one embodiment can inhibit the expression of IL-1α caused by an inflammatory response in cells by containing 2'-FL, can more effectively inhibit the expression of IL-1α in skin cells, and can further effectively inhibit the expression of IL-1α in dermal fibroblasts. Inhibiting IL-1α caused by an inflammatory response in the epidermis or dermis can have the effects of maintaining skin whitening, inhibiting sunburn, and improving atopic symptoms. Therefore, the preventive or ameliorative agent for cell aging according to one embodiment can inhibit the expression of IL-1α caused by an inflammatory response in skin cells, and therefore can also be used as a preventive and ameliorative agent for pigmentation and / or a preventive and ameliorative agent for rough skin. As an inflammatory response, it can be an inflammatory response caused by active oxygen species such as hydrogen peroxide.

[0118] The target cells of the preventive or ameliorative agent for cell aging according to one embodiment can be animal cells, for example, cells of tissues or organs such as skin, muscle, bone, joints, fat, brain, spinal cord, digestive organs, reproductive organs, endocrine organs, respiratory organs, circulatory system, immune system, bone, joints and adipose tissue. GLS1 can be expressed in any of these tissues and organs, and endoplasmic reticulum stress can also be generated. The target cells of the preventive or ameliorative agent for cell aging according to one embodiment can be skin cells. Examples of skin cells include cells constituting the epidermis and dermis. Examples of cells constituting the epidermis include epidermal keratinocytes (keratinocytes), pigment cells (melanocytes), Langerhans cells, and Merkel cells, with epidermal keratinocytes being preferred. Cells constituting the epidermis are preferably cells present in the basal layer, spinous layer, granular layer or stratum corneum of the epidermis. Examples of cells constituting the dermis include fibroblasts, macrophages, mast cells, plasma cells, and dermal dendritic cells, with dermal fibroblasts being preferred. The cells constituting the dermis are preferably cells present in the papillary layer or the reticular layer of the dermis.

[0119] The target cells for the agent for preventing or ameliorating cell senescence according to one embodiment are also preferably renal cells. Examples of renal cells include glomerular cells, renal tubular cells, and interstitial cells. Inhibiting the expression of the GLS1 gene in renal cells can prevent or ameliorate glomerulosclerosis or prevent or ameliorate decreased renal function. Glomerulosclerosis and decreased renal function can be caused by renal cell senescence. For example, Johmura, Yoshikazu, et al. "Senolysis by glutaminolysis inhibition ameliorates various age-associated disorders." Science 371.6526(2021): 265-270. states that inhibiting GLS1 in 80-week-old male mice improves glomerulosclerosis, macrophage infiltration into the kidneys, and renal dysfunction associated with aging. Therefore, the agent for preventing or ameliorating cell senescence according to one embodiment can be used as an agent for preventing or ameliorating glomerulosclerosis or an agent for preventing or ameliorating decreased renal function.

[0120] The target cells for the agent for preventing or ameliorating cell aging according to one embodiment are also preferably gastric cells. Examples of gastric cells include principal cells, accessory cells, and parietal cells. Inhibiting GLS1 gene expression in principal cells can prevent or ameliorate the decrease in pepsinogen secretion by these principal cells. This decrease in pepsinogen secretion can be caused by principal cell aging. For example, https: / / www.tyojyu.or.jp / net / kenkou-tyoju / rouka / saibou-kotai-rouka.html (accessed on February 6, 2024) states that pepsinogen secretion by principal cells decreases during gastric aging. Therefore, the agent for preventing or ameliorating cell aging according to one embodiment can be used as an agent for preventing or ameliorating decreased renal function.

[0121] In addition, inhibiting the expression of the GLS1 gene in gastric parietal cells can prevent or improve the reduction in gastric acid secretion by the above-mentioned gastric parietal cells. The above-mentioned reduction in gastric acid secretion can be caused by aging of gastric parietal cells. For example, https: / / www.tyojyu.or.jp / net / kenkou-tyoju / rouka / saibou-kotai-rouka.html (accessed on February 6, 2024) states that gastric acid secretion by gastric parietal cells decreases during gastric aging. Therefore, the preventive or ameliorative agent for cell aging of one embodiment can be used as a preventive or ameliorative agent for reduced gastric acid secretion.

[0122] The target cells of the agent for preventing or improving cell aging in one embodiment are also preferably microglia. When the expression of the GLS1 gene is inhibited in microglia, cognitive impairment can be prevented or improved. The above-mentioned cognitive impairment can be caused by microglial aging. For example, Shohei Kawanishi's "Is the removal of aging glial cells the key to preventing cognitive impairment?" Phallus 55.9 (2019): 889-889. states that in mice in which aging microglia were removed, the amount of phosphorylated tau protein in the brain was significantly reduced, brain atrophy was alleviated, the decrease in the area of ​​the dentate gyrus of the hippocampus was suppressed, and cognitive function was improved. Therefore, the agent for preventing or improving cell aging in one embodiment can be used as an agent for preventing or improving cognitive impairment.

[0123] The target cells of the agent for preventing or improving cell aging in one embodiment are also preferably gingival cells. When the expression of the GLS1 gene is inhibited in gingival cells, gingival inflammation can be prevented or improved. The above-mentioned gingival inflammation can be caused by gingival cell aging. For example, https: / / www.ncgg.go.jp / ncgg-kenkyu / documents / 2021 / 21xx_6.pdf (accessed on February 6, 2024) records that a significant increase in the expression of IL-1β and TNFα was observed in the gums of old mice compared to young mice. Therefore, the agent for preventing or improving cell aging in one embodiment can be used as an agent for preventing or improving gingival inflammation.

[0124] The target cells for the agent for preventing or ameliorating cellular senescence according to one embodiment are also preferably vascular endothelial cells. Inhibiting GLS1 gene expression in vascular endothelial cells can prevent or ameliorate arteriosclerosis. Arteriosclerosis can be caused by vascular endothelial cell senescence. For example, Johmura, Yoshikazu, et al. "Senolysis by glutaminolysis inhibition ameliorates various age-associated disorders." Science 371.6526 (2021): 265-270. reported that inhibiting GLS1 in mice with induced thoracic aorta arteriosclerosis alleviated the arteriosclerosis. Therefore, the agent for preventing or ameliorating cellular senescence according to one embodiment can be used as an agent for preventing or ameliorating arteriosclerosis.

[0125] Furthermore, inhibiting GLS1 gene expression in vascular endothelial cells can prevent or improve obesity, diabetes, or impaired glucose tolerance. These obesity, diabetes, or impaired glucose tolerance can be caused by vascular endothelial cell senescence. For example, Masayoshi Suda, Itsuki Shimizu, and Toru Minamino, "On Vascular Endothelial Cell Senescence," Journal of the Japanese Society of Thrombosis and Haemostasis 30.3 (2019): 521-528, describe that diabetes promotes vascular cell senescence; mice with endothelial-specific p53 deficiency, which suppresses endothelial senescence, exhibit reduced obesity compared to wild-type mice even after being fed a high-fat diet, and exhibit mild systemic impaired glucose tolerance. On the other hand, mice with endothelial cell senescence that overexpress p53 in endothelial cells by endothelial-specific deficiency of Mdm2 / Mdm4, which degrades p53, exhibit exacerbated impaired glucose tolerance and increased obesity when fed a high-fat diet. Therefore, the agent for preventing or ameliorating cellular senescence according to one embodiment can be used as an agent for preventing or ameliorating obesity, diabetes, or impaired glucose tolerance.

[0126] In the present invention, "normal cells" refer to cells that are capable of sustained cell proliferation and cell cycle progression. Cells are considered normal if the expression level of a senescence marker is comparable to the expression level of common senescence markers in cells capable of sustained cell proliferation. Examples of senescence markers include acid β-galactosidase, P53, P16INK4a, and P21CIP1.

[0127] In the present invention, "senescent cells" refer to cells in which cell proliferation or cell cycle arrest has irreversibly occurred. Cells are considered senescent if the expression of senescence markers is significantly increased compared to the expression of common senescence markers in normal cells.

[0128] One embodiment of a preventive or ameliorative agent for cellular senescence can ameliorate endoplasmic reticulum stress in cells by containing 2'-FL. That is, one embodiment of a preventive or ameliorative agent for cellular senescence can be an agent that ameliorate endoplasmic reticulum stress in cells. Endoplasmic reticulum stress refers to the accumulation of proteins that fail to fold into their normal higher-order structures (denatured proteins) in the endoplasmic reticulum due to gene mutations, viral infections, inflammation, harmful chemicals, and the like, which adversely affects cells. Cells experiencing endoplasmic reticulum stress tend to lose their normal functions and sometimes undergo apoptosis. It is speculated that reduced cellular function and apoptosis can lead to decreased metabolism or the development of spots, wrinkles, sagging, rough skin, and the like. It is believed that ameliorating endoplasmic reticulum stress in cells can prevent or ameliorate the aging of the tissue to which the cells belong. Furthermore, one embodiment of a preventive or ameliorative agent for cellular senescence can also ameliorate endoplasmic reticulum stress caused by inflammation, etc., resulting in tissue trauma, and thus can also be used to treat tissue trauma.

[0129] When endoplasmic reticulum stress is induced in cells, the amount of laminin secreted by the above-mentioned cells will decrease. The preventive or ameliorative agent for cell aging in one embodiment can restore the reduced amount of laminin secretion caused by endoplasmic reticulum stress in cells by containing 2'-FL, can more effectively restore the reduced amount of laminin secretion caused by endoplasmic reticulum stress in skin cells, and can further effectively restore the reduced amount of laminin secretion caused by endoplasmic reticulum stress in epidermal keratinocytes and dermal fibroblasts. Therefore, the preventive or ameliorative agent for cell aging in one embodiment can restore the reduced amount of laminin secretion when endoplasmic reticulum stress occurs in cells, and can therefore also be used as an endoplasmic reticulum stress ameliorative agent in cells.

[0130] Furthermore, laminin is one of the proteins that constitutes the basement membrane between the epidermis and dermis, and promotes cell adhesion, cell migration, and cell proliferation of skin cells and other cells. Therefore, since it can restore the reduced amount of laminin secretion caused by endoplasmic reticulum stress in skin cells and promote cell adhesion, cell migration, and cell proliferation of skin cells, it can also be used for at least one purpose selected from the group consisting of skin wound treatment, skin metabolism promotion, prevention and improvement of pigmentation, prevention and improvement of wrinkles, prevention and improvement of sagging, and prevention and improvement of rough skin.

[0131] Subjects to which the preventive or ameliorating agent for cellular senescence according to one embodiment can be applied include humans and non-human mammals, preferably humans. Examples of non-human mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, and monkeys.

[0132] When the agent for preventing or ameliorating cellular senescence according to one embodiment is applied to the skin, examples of application sites include the scalp, face (forehead, cheeks, lips, nose, ears, etc.), head, shoulders, back, chest, abdomen, sexual organs, arms, hands, lower limbs, feet, nails, and hair, and other areas of the body. The agent for preventing or ameliorating cellular senescence according to one embodiment can be applied to the liver, kidneys, spleen, large intestine, small intestine, stomach, and bladder, among others.

[0133] The agent for preventing or ameliorating cellular senescence according to one embodiment preferably contains 0.001% by mass to 5% by mass of 2'-FL, and more preferably 0.01% by mass to 0.5% by mass, based on the total amount of the agent.

[0134] For a subject weighing 60 kg, the dosage of the preventive or ameliorating agent for cellular senescence according to one embodiment can be set to 1 mg to 60 g, 0.01 mg to 1 g, 6 mg to 30 g, or 60 mg to 15 g per day.

[0135] The frequency of administration of the agent for preventing or ameliorating cellular senescence according to one embodiment may be once a day or twice a day.

[0136] The method of administration of the preventive or ameliorative agent for cellular senescence according to one embodiment may be transdermal administration, oral administration, intravenous administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, or eye drop administration, preferably transdermal administration. That is, the preventive or ameliorative agent for cellular senescence according to one embodiment may be an external preparation. Examples of transdermal administration methods include methods of applying the agent to a body surface such as the skin or methods of applying the agent to the skin.

[0137] The agent for preventing or ameliorating cellular senescence according to one embodiment may have the following forms, for example: solid; liquid (solution or suspension); emulsion such as lotion or cream; paste; gel; or mousse.

[0138] The preventive or improving agent for cell aging of one embodiment can be used for any of therapeutic purposes and non-therapeutic purposes (such as applications for cosmetic purposes). Specifically, for example, the preventive or improving agent for cell aging of one embodiment can be a drug, a quasi-drug, a cosmetic or a food, preferably a cosmetic. The drugs, quasi-drugs, cosmetics and foods containing the preventive or improving agent for cell aging of one embodiment can be manufactured according to conventional methods. The content of the preventive or improving agent for cell aging or 2'-FL of one embodiment in the drug, quasi-drug, cosmetic or food is not particularly limited and can be freely set according to the purpose. The preventive or improving agent for cell aging of one embodiment can be used, ingested or administered preventively in order to proliferate normal cells and / or to remove senescent cells. When the preventive or improving agent for cell aging of one embodiment is a food, the ingestion can be oral ingestion.

[0139] The agent for preventing or ameliorating cellular senescence according to one embodiment may contain ingredients other than 2'-FL, as long as the effectiveness of the agent for preventing or ameliorating cellular senescence is not impaired. Specific examples of ingredients other than 2'-FL include moisturizing ingredients, anti-inflammatory ingredients, antibacterial ingredients, cell-activating ingredients, anti-aging ingredients, blood circulation promoting ingredients, UV protection ingredients, whitening ingredients, vitamins, proteins, peptides, amino acids, alcohols, and the like. These active ingredients may be used alone or in combination of two or more, as needed.

[0140] When the preventive or improving agent for cell aging of one embodiment is a drug, quasi-drug, cosmetic or food, the drug, quasi-drug, cosmetic or food may contain ingredients that are commonly used for drugs, quasi-drugs, cosmetics or foods in addition to the preventive or improving agent for cell aging of one embodiment. The drug, quasi-drug, cosmetic or food of one embodiment may contain bases, carriers, additives, etc. that are commonly used for drugs, quasi-drugs, cosmetics or foods. As additives, for example, excipients, oils, powders, buffers, dissolution aids, antioxidants, surfactants, thickeners, preservatives, pH regulators, chelating agents, stabilizers, irritation reducing agents, preservatives, pigments, colorants, fragrances, gloss imparting agents, gelling agents, alcohols, water-soluble polymers, film forming agents, resins, keratin dissolving agents, etc. can be listed. The base, carrier and the above-mentioned various additives can be used alone or in combination of two or more as needed.

[0141] When the preventive or improving agent for cell aging of one embodiment is a medicine, the dosage form of the medicine can be, for example, an aerosol, a liquid, a suspension, an emulsion, a cream, an ointment, a gel, a liniment, a lotion, a cataplasm, a patch, an eye drop, a nose drop, an ear drop, a suppository, an elixir, a capsule, a granule, a pill, a powder, a tablet, a syrup, an injection, a lozenge, etc. When the preventive or improving agent for cell aging of one embodiment is used as a skin medicine, it is preferably an external preparation. By making an external preparation, 2'-FL can act directly on skin cells and exert a stronger effect. As an external preparation, it is preferably provided in the form of an aerosol, a liquid, a suspension, an emulsion, a cream, an ointment, a gel, a liniment, a lotion, a cataplasm, a patch, etc.

[0142] When the preventive or ameliorating agent for cellular aging according to one embodiment is a quasi-drug or cosmetic, the form of the quasi-drug or cosmetic can be, for example, a lotion, emulsion, cream, gel, beauty serum, sunscreen cosmetics, facial mask, hand cream, foot cream, body lotion, body milk and other basic cosmetics; cleansing cosmetics such as facial cleanser, makeup remover, soap, body wash, shampoo, conditioner, hair styling agent, nail polish remover; makeup cosmetics such as foundation, base makeup, lipstick, lipstick, blush, eye shadow, eyebrow pencil, nail polish, hair dye; antiperspirant; bath preparation; perfume; food and beverages such as nutritional functional foods, functional labeled foods, specific health foods, special purpose foods, etc. When used as a quasi-drug or cosmetic for skin use, it is preferably an external preparation as a pharmaceutical. By making it into an external preparation, for example, 2'-FL acts directly on skin cells, it can exert a stronger effect. Quasi-drugs or cosmetics as external preparations are preferably quasi-drugs and cosmetics in forms other than food and beverages.

[0143] When the agent for preventing or ameliorating cellular senescence according to one embodiment is a food, the food may be a general food as well as a food for specified health purposes, a food with nutritional functions, a food with functional claims, a food for hospital patients, a supplement, or the like. Furthermore, the agent for preventing or ameliorating cellular senescence according to one embodiment may also be used as a food additive.

[0144] Examples of the food composition include seasonings, processed meat products, processed agricultural products, beverages (such as lactic acid bacteria beverages, soft drinks, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, and nutritional beverages), powdered beverages (such as powdered fruit juice and powdered soup), concentrated beverages, snacks (such as candies (lozenges), cookies, biscuits, chewing gum, soft candies, chewable tablets, tablets, and chocolate), bread, and cereals. Furthermore, in the case of foods for specific health purposes, foods with nutritional functional properties, and foods with functional claims, the food composition may be in the form of capsules, tablets, syrups, granules, or powders.

[0145] Here, foods for specified health uses are foods that contain health-promoting ingredients that affect physiological functions and are approved by the Commissioner of the Consumer Affairs Agency of Japan to be labeled as suitable for specified health uses. In the present invention, foods may be marketed with a label indicating specific health uses related to inhibiting GLS1, improving endoplasmic reticulum stress, removing senescent cells, or promoting normal cell proliferation.

[0146] Furthermore, functional foods are foods that supplement nutrients (vitamins, minerals) and are labeled to indicate the function of these nutrients. To be marketed as functional foods, the nutrients contained in the standard daily intake must fall within specified upper and lower limits, and the labeling must include not only the nutritional function but also precautions.

[0147] In addition, functional food refers to food that the business operator is responsible for labeling the functionality based on scientific evidence. Before selling, the business operator must report relevant information on safety and functionality to the Director-General of the Consumer Affairs Agency of Japan.

[0148] Example

[0149] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0150] <Example 1>

[0151] For 2'-fucosyllactose, epidermal cell proliferation activity was evaluated.

[0152] <Example 1-1: Absorbance Measurement>

[0153] Human normal epidermal keratinocytes (HaCaT) were cultured in DMEM containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cells were plated at 5.0×10 3 The cells were seeded in a 96-well plate at a concentration of 50 μg / mL, 100 μg / mL, or 200 μg / mL in a culture medium for the proliferation of epidermal keratinocytes (without FBS) to which the test sample (sucrose or 2'-fucosyllactose) was added at a concentration of 50 μg / mL, 100 μg / mL, or 200 μg / mL. The cells were cultured at 37°C for 2 days. The culture medium was replaced with DMEM medium without FBS, and 10 μL of 5 mg / mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added to each well and cultured at 37°C for 3 hours. After the culture was completed, 100 μL of dimethyl sulfoxide (DMSO) was used to extract the formazan produced in the cells. After extraction, the absorbance of the formazan was measured using a microplate reader (As, 492 nm). The reference wavelength was set to 620 nm.

[0154] <Example 1-2: Reference absorbance measurement and calculation of cell proliferation rate>

[0155] Human normal epidermal keratinocytes were cultured in the same manner as in Example 1-1 except that the test sample was not added, and the absorbance of formazan was measured. The obtained absorbance was set as Ab, and the cell proliferation rate represented by the following formula (1) was calculated. The results are shown in Figure 1 .

[0156] Cell proliferation rate (%) = 100 × As / Ab …(1)

[0157] like Figure 1 As shown, 2'-FL increased the proliferation activity of normal epidermal cells. At least when the 2'-FL content in the culture medium was within the range of 50 μg / mL to 200 μg / mL, the proliferation activity of epidermal cells increased with increasing 2'-FL content.

[0158] <Example 2>

[0159] For 2'-fucosyllactose, fibroblast proliferation activity was evaluated.

[0160] <Example 2-1: Absorbance Measurement>

[0161] Human normal skin fibroblasts (NB1RGB) with a PDL value below 30 and human senescent skin fibroblasts (NB1RGB) with a PDL value above 50 were cultured in DMEM supplemented with 10% FBS. The cultured cells were harvested by trypsinization. The harvested cells were plated at 5.0 × 10 cells per well. 3 The cells were seeded in a 96-well plate at a concentration of 50 μg / mL, 100 μg / mL, or 200 μg / mL in a fibroblast proliferation medium (without FBS) supplemented with the test sample (sucrose or 2'-fucosyllactose) at a concentration of 50 μg / mL, 100 μg / mL, or 200 μg / mL, and cultured at 37°C for 2 days. The culture medium was replaced with DMEM medium without FBS, and 10 μL of 5 mg / mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was added to each well, and the cells were cultured at 37°C for 3 hours. After the culture was completed, the formazan produced in the cells was extracted with 100 μL of dimethyl sulfoxide (DMSO). After extraction, the absorbance of the formazan was measured using a microplate reader (As, 492 nm). The reference wavelength was set to 620 nm.

[0162] <Example 2-2: Reference absorbance measurement and calculation of cell proliferation rate>

[0163] In the same manner as in Example 2-1, except that no test sample was added, normal human skin fibroblasts with a PDL of 30 or less and senescent human fibroblasts with a PDL of 50 or more were cultured, and the absorbance of formazan was measured. The obtained absorbance was designated as Ab, and the cell proliferation rate expressed in the above formula (1) was calculated for the cases of PDL 30 or less and PDL 50 or more. The results for the cases of PDL 30 or less are shown in FIG. Figure 2 The results above PDL50 are shown in Figure 3 .

[0164] like Figure 2 As shown, 2'-FL increased the proliferation activity of normal fibroblasts (PDL30 or less). At least when the 2'-FL content in the culture medium was within the range of 50 μg / mL to 200 μg / mL, the proliferation activity of normal fibroblasts increased with increasing 2'-FL content.

[0165] like Figure 3 As shown, 2'-FL inhibited the proliferation of senescent fibroblasts (PDL50 or higher). At least when the 2'-FL content in the culture medium was in the range of 50 μg / mL to 200 μg / mL, the senescent fibroblast proliferation inhibitory activity increased with increasing 2'-FL content.

[0166] <Example 3>

[0167] To evaluate the effect of 2-fucosyllactose on the longevity-related gene (GLS1).

[0168] <Example 3-1: Cell Treatment>

[0169] Human normal skin fibroblasts (NB1RGB, PDL30 or less) were cultured in DMEM containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cell-containing liquid was diluted with DMEM containing 10% FBS to a cell count of 5.0 × 10 5 The cells were seeded in a 10 cm dish at a concentration of 100 μM and cultured until they reached 60% confluence. The cells were then exposed to a medium containing 50 μM hydrogen peroxide (without FBS) for 2 hours, once a day for 3 days. The medium was then replaced with DMEM containing 5% FBS, and the medium was recovered after 2 days (medium A). Skin fibroblasts not exposed to hydrogen peroxide were diluted to 5.0 × 10 5 The cells were seeded in a 10 cm culture dish and cultured for 24 hours. Then, half of the total volume of medium A used for culturing hydrogen peroxide-treated skin fibroblasts was added and cultured until 90% confluence was achieved.

[0170] <Example 3-2: Sample Preparation for GLS1 Expression Analysis>

[0171] The skin fibroblasts prepared in Example 3-1 were diluted to 8.0×10 4 The cells were seeded in 6-well plates at a concentration of 100 μg / mL and cultured until 80% confluence was achieved. The culture medium was then replaced with a fibroblast growth medium (containing neither DMEM nor FBS) supplemented with the test sample (lactose or 2'-fucosyllactose) at a concentration of 50 μg / mL or 200 μg / mL. After 24 hours of culture, the supernatant was removed. The cells were washed twice with cold PBS, and proteins were extracted using RIPA buffer (50 mmol / L Tris-HCl buffer (pH 7.9), 150 mmol / L NaCl, 1 (w / v)% Nonidet P40 substitute, 0.5 (w / v)% sodium deoxycholate, and 0.1 (w / v)% SDS). The extracted proteins were quantified using the Bradford method.

[0172] <Example 3-3: Reference Cell Treatment>

[0173] The skin fibroblasts prepared in <Example 3-1> and the skin fibroblasts cultured in DMEM containing 10% FBS were cultured in the same manner as in <Example 3-2>, except that the test sample was not added, and protein was extracted. The extracted protein was quantified by the Bradford method.

[0174] <Example 3-4: GLS1 expression analysis>

[0175] Western blot analysis was performed using antibodies against GLS1 and β-actin to measure protein levels at 24 hours. Equal amounts of protein were added to a 10% acrylamide gel. Antibodies against GLS1 (manufactured by Cell Signaling Technology) and antibodies against β-actin (HRP-labeled) (manufactured by Proteintech) were used according to the manufacturer's instructions. In the case of GLS1 alone, HRP (manufactured by Proteintech) was used as the secondary antibody, and 3,3'-diaminobenzidine (DBA) was used for color development. The depth of color development was quantified using the open source software "image J" and is shown in Figure 4 .

[0176] like Figure 4 As shown, 2'-FL inhibited the expression of GLS1. At least when the 2'-FL content in the culture medium was in the range of 50 μg / mL to 200 μg / mL, the inhibitory effect on GLS1 expression increased with increasing 2'-FL content.

[0177] <Example 4>

[0178] The survival rate of epidermal keratinocytes treated with tunicamycin was evaluated.

[0179] <Example 4-1: Absorbance Measurement>

[0180] Normal human epidermal keratinocytes (HaCaT) were cultured using DMEM containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cells were seeded in 96-well plates, and 0.1 μg / mL of tunicamycin, a drug for inducing endoplasmic reticulum stress, was added at the moment of reaching 100% confluence and cultured for 24 hours. A group was also prepared in which a DMEM culture medium containing 50 μg / mL, 100 μg / mL or 200 μg / mL of a test sample (2'-fucosyllactose) without FBS was added and cultured for 24 hours with tunicamycin. After the culture was completed, the survival rate of normal human epidermal keratinocytes was evaluated using Cell Counting Kit-8 (manufactured by Tongren Chemical Research Institute). The measurement wavelength (As) was set to 450 nm, and the reference wavelength was set to 620 nm.

[0181] <Example 4-2: Reference absorbance measurement and calculation of cell proliferation rate>

[0182] Human normal epidermal keratinocytes were cultured in the same manner as in Example 4-1 except that tunicamycin and the test sample were not added, and the absorbance at a wavelength of 450 nm was measured. The obtained absorbance was set as Ab, and the cell survival rate represented by the above formula (1) was calculated. The results are shown in Figure 5 .

[0183] like Figure 5 As shown, 2'-FL improves ER stress in normal human epidermal keratinocytes. At least when the 2'-FL content in the culture medium is within the range of 50 μg / mL to 200 μg / mL, the effect of improving ER stress in normal human epidermal keratinocytes increases with increasing 2'-FL content.

[0184] <Example 5>

[0185] The survival rate of tunicamycin-treated skin fibroblasts was evaluated.

[0186] <Example 5-1: Absorbance Measurement>

[0187] Normal human skin fibroblasts (NB1RGB) with a PDL of less than 30 were cultured in DMEM containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cells were seeded in a 96-well plate, and 0.3 μg / mL of tunicamycin was added when 100% confluence was reached, and cultured for 24 hours. A group was also prepared in which a DMEM medium without FBS containing 50 μg / mL, 100 μg / mL or 200 μg / mL of the test sample (2'-fucosyllactose) was added simultaneously with tunicamycin and cultured for 24 hours. After the culture was completed, the survival rate of normal human skin fibroblasts was evaluated using Cell Counting Kit-8 (manufactured by Dojindo Chemical Research Institute). The measurement wavelength (As) was set to 450 nm and the reference wavelength was set to 620 nm.

[0188] <Example 5-2: Reference absorbance measurement and calculation of cell proliferation rate>

[0189] Human normal skin fibroblasts with a PDL of 30 or less were cultured in the same manner as in Example 5-1, except that tunicamycin and the test sample were not added, and the absorbance at a measurement wavelength of 450 nm was measured. The obtained absorbance was set as Ab, and the cell survival rate represented by the above formula (1) was calculated. The results are shown in Figure 6 .

[0190] like Figure 6 As shown, 2'-FL improves ER stress in human normal skin fibroblasts with a PDL of 30 or less. At least when the 2'-FL content in the culture medium is within the range of 50 μg / mL to 200 μg / mL, the effect of improving ER stress in human normal skin fibroblasts with a PDL of 30 or less increases with increasing 2'-FL content.

[0191] <Example 6>

[0192] Evaluation of laminin secretion by tunicamycin-treated epidermal keratinocytes.

[0193] <Example 6-1: Preparation of samples for laminin analysis>

[0194] Normal human epidermal keratinocytes (HaCaT) were cultured in DMEM containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cells were inoculated into a 24-well plate, and 0.1 μg / mL of tunicamycin was added when 100% confluence was reached, and the culture supernatant was recovered after 24 hours. A group in which a DMEM culture medium containing no FBS and a test sample (2'-fucosyllactose) of 50 μg / mL, 100 μg / mL or 200 μg / mL was added simultaneously with tunicamycin and the culture supernatant was recovered after 24 hours was also prepared. The culture supernatant was centrifuged and the supernatant was used as a specimen. In addition, the cells after removing the culture supernatant were washed twice with PBS, 0.20 mL of RIPA buffer was added, and whole-cell protein was extracted. The whole-cell protein concentration was determined using the Bradford method.

[0195] <Example 6-2: Reference Cell Treatment>

[0196] Normal human epidermal keratinocytes were cultured in the same manner as in Example 6-1, except that tunicamycin and the test sample were not added. The culture supernatant was recovered and centrifuged, and the supernatant was used as the sample. Furthermore, after removal of the culture supernatant, the cells were washed twice with PBS, and 0.20 mL of RIPA buffer was added to extract whole-cell protein. The whole-cell protein concentration was measured using the Bradford method.

[0197] <Example 6-3: Laminin expression analysis>

[0198] The amount of laminin in the specimen was measured by enzyme-linked immunosorbent assay (ELISA) using antibodies against laminin. Anti-laminin antibody (manufactured by our company, product number F-54, Clone No. HL-4H3) was used as described on the data sheet. HRP (manufactured by SeraCare Life Sciences) was used as the secondary antibody, and after color development with 3,3',5,5'-tetramethylbenzidine (TMB), the reaction was stopped with 1N sulfuric acid. After the reaction was stopped, the absorbance at a measurement wavelength of 450nm was measured using a microplate reader. The reference wavelength was set to 620nm. The amount of laminin measured by ELISA was divided by the amount of whole-cell protein for standardization, and the resulting value was used as the amount of laminin secretion. The results are shown in Figure 7 .

[0199] like Figure 7 As shown, 2'-FL restored laminin secretion in normal human epidermal keratinocytes. At least when the 2'-FL content in the culture medium was within the range of 50 μg / mL to 200 μg / mL, the effect of restoring laminin secretion in normal human epidermal keratinocytes increased with increasing 2'-FL content.

[0200] <Example 7>

[0201] Evaluation of laminin secretion by tunicamycin-treated skin fibroblasts.

[0202] <Example 7-1: Preparation of samples for laminin analysis>

[0203] Normal human skin fibroblasts (NB1RGB) with a PDL of less than 30 were cultured using DMEM containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cells were seeded in a 24-well plate, and 0.3 μg / mL of tunicamycin was added when 100% confluence was reached, and the culture supernatant was recovered after 24 hours. A group was also prepared in which a DMEM culture medium without FBS containing 50 μg / mL, 100 μg / mL or 200 μg / mL of the test sample (2'-fucosyllactose) was added simultaneously with tunicamycin and the culture supernatant was recovered after 24 hours. The culture supernatant was centrifuged and the supernatant was used as the specimen. In addition, the cells after removing the culture supernatant were washed twice with PBS, 0.20 mL of RIPA buffer was added, and the whole-cell protein was extracted. The whole-cell protein concentration was determined by the Bradford method.

[0204] <Example 7-2: Reference Cell Treatment>

[0205] Human normal skin fibroblasts with a PDL of 30 or less were cultured in the same manner as in Example 7-1, except that tunicamycin and the test sample were not added. The culture supernatant was recovered and centrifuged, and the supernatant was used as the sample. Furthermore, after the supernatant was removed, the cells were washed twice with PBS, and 0.20 mL of RIPA buffer was added to extract whole-cell protein. The whole-cell protein concentration was measured using the Bradford method.

[0206] <Example 7-3: Laminin expression analysis>

[0207] The amount of laminin in the specimen was measured by enzyme-linked immunosorbent assay (ELISA) using antibodies against laminin. Anti-laminin antibodies (manufactured by our company, product number F-54, Clone No. HL-4H3) were used as described on the data sheet. HRP (manufactured by SeraCare Life Sciences) was used as the secondary antibody, and after color development with 3,3',5,5'-tetramethylbenzidine (TMB), the reaction was stopped with 1N sulfuric acid. After the reaction was stopped, the absorbance at a measurement wavelength of 450nm was measured using a microplate reader. The reference wavelength was set to 620nm. The amount of laminin measured by ELISA was divided by the amount of whole-cell protein for standardization, and the resulting value was used as the amount of laminin secretion. The results are shown in Figure 8 .

[0208] like Figure 8 As shown, 2'-FL restored laminin secretion in human normal skin fibroblasts with a PDL of 30 or less. At least when the 2'-FL content in the culture medium was within the range of 50 μg / mL to 200 μg / mL, the effect of restoring laminin secretion in human normal skin fibroblasts with a PDL of 30 or less increased with increasing 2'-FL content.

[0209] <Example 8>

[0210] The effect of 2-'FL on the expression of type I collagen was evaluated.

[0211] <Example 8-1: Sample Preparation for Type I Collagen Expression Analysis>

[0212] Human normal skin fibroblasts (NB1RGB) with a PDL of less than 30 were cultured in DMEM (Dulbecco's Modified Eagle Medium, manufactured by Nissin) containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cells were seeded into a 96-well plate, and when they reached 100% confluence, DMEM without FBS containing 50 μg / mL, 100 μg / mL, or 200 μg / mL of the test sample (2'-FL and sucrose) was added. After 48 hours of incubation at 37°C and 5% CO2, the culture supernatant was recovered. The culture supernatant was centrifuged and used as the specimen.

[0213] <Example 8-2: Preparation of control sample>

[0214] The culture supernatant was collected as a sample in the same manner as in Example 8-1, except that FBS-free DMEM containing no test sample was used instead of FBS-free DMEM containing the test sample.

[0215] <Example 8-3: Analysis of Type I Collagen Expression>

[0216] The amount of type I collagen in the specimen was determined by enzyme-linked immunosorbent assay (ELISA) using antibodies against type I collagen. Two anti-type I collagen antibodies (manufactured by our company [product number F-56, clone number I-8H5] and Proteintech) were used as described on the data sheet. An HRP-labeled antibody (manufactured by SeraCare Life Sciences) was used as the secondary antibody, and after color development with 3,3',5,5'-tetramethylbenzidine (TMB), the reaction was stopped with 1N sulfuric acid. After the reaction was stopped, the absorbance at a measurement wavelength of 450nm was measured using a microplate reader. The reference wavelength was set to 620nm. The relative value of the amount of type I collagen when each test sample was added was calculated, with the amount of type I collagen in the control set to 1. In addition, the determination of type I collagen was carried out 3 times, Figure 9 The relative expression level of type I collagen relative to the control is shown as mean ± standard deviation.

[0217] like Figure 9 As shown, 2'-FL at a concentration of at least 100 μg / mL increased the expression of type I collagen in normal human epidermal keratinocytes. When the 2'-FL concentration in the culture medium was at least between 100 μg / mL and 200 μg / mL, the effect of increasing type I collagen expression increased with increasing 2'-FL concentration.

[0218] <Example 9>

[0219] The effect of 2'-FL on the expression of IL-1α was evaluated.

[0220] <Example 9-1: Sample Preparation for IL-1α Expression Analysis>

[0221] Human normal epidermal keratinocytes (HaCaT) were cultured in DMEM containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cells were plated at 6×10 4 Cells were seeded at a density of 100 cells / well in a 48-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours. After culture, cells were pre-cultured for 2 hours in DMEM without FBS containing 50 μg / mL, 100 μg / mL, or 200 μg / mL of the test sample (2'-FL and sucrose), or in DMEM without FBS without the test sample. The medium was then exchanged for DMEM without FBS containing 50 μg / mL, 100 μg / mL, or 200 μg / mL of the test sample and 20 μM H2O2, or in DMEM without FBS without the test sample and 20 μM H2O2. After additional culture for 24 hours in a 37°C, 5% CO2 incubator, the culture supernatant was recovered. The culture supernatant was centrifuged and used as the specimen.

[0222] <Example 9-2: Preparation of control sample>

[0223] The culture supernatant was collected as a sample in the same manner as in Example 9-1, except that FBS-free DMEM containing H 2 O 2 and the test sample was used instead of FBS-free DMEM containing H 2 O 2 and the test sample.

[0224] <Example 9-3: Analysis of IL-1α Expression>

[0225] Most of IL-1α is released outside the cells, so the IL-1α in the supernatant culture medium was measured using ELISA (AuthentiKineTM IL-1 alpha ELISA Kit, manufactured by Proteintech) to evaluate the IL-1α expression level. The measurement wavelength was set to 450 nm, and the reference wavelength was set to 630 nm. The relative value of the IL-1α amount when each test sample was added was calculated, with the control IL-1α amount set to 1. In addition, the IL-1α measurement was carried out 3 times, Figure 10 The relative expression levels of IL-1α relative to the control are shown in the form of mean ± standard deviation.

[0226] like Figure 10 As shown, 2'-FL inhibited the H2O2-induced IL-1α expression in normal human epidermal keratinocytes. The inhibitory effect on IL-1α expression increased with increasing 2'-FL levels, at least when the 2'-FL content in the culture medium was between 50 μg / mL and 200 μg / mL.

[0227] <Example 10>

[0228] To evaluate the effect of 2'-FL on the longevity-related gene (GLS1).

[0229] <Example 10-1: Sample Preparation for GLS1 Expression Analysis>

[0230] A. Human fetal kidney cells (HEK293)

[0231] Human fetal kidney cells (HEK293) were cultured in DMEM containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cells were seeded in a 6-well plate, and DMEM containing 50 μg / mL, 200 μg / mL, or 1000 μg / mL of 2'-FL as a test sample and 50 μg / mL or 200 μg / mL of lactose as a test sample were added when 100% confluence was reached, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture supernatant was removed, and the cells were washed twice with phosphate-buffered saline (PBS), and 0.20 mL of RIPA buffer (manufactured by Nakalai Tesku Co., Ltd.) was added to extract the total protein. The protein in each protein extract was quantified by the Bradford method.

[0232] B. Human undifferentiated gastric carcinoma cells (KKLS)

[0233] Human gastric undifferentiated carcinoma cells (KKLS) were cultured with RPMI1640 (manufactured by Nisui Co., Ltd.) containing 10% FBS. The cultured cells were recovered by trypsin treatment. The recovered cells were inoculated into 6-well plates, and RPMI1640 containing 50 μg / mL, 200 μg / mL or 1000 μg / mL of the test sample (2'-FL and lactose) was added at the time of reaching 100% confluence, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture supernatant was removed, washed twice with PBS, 0.20 mL of RIPA buffer was added, and the whole protein was extracted. The protein in each protein extract was quantified by the Bradford method.

[0234] C. Mouse microglia (BV2)

[0235] Mouse microglial cells (BV2) were cultured in DMEM containing glutamine. The cultured cells were recovered by trypsin treatment. The recovered cells were plated at 4×10 5 Cells were seeded at a density of 100 cells / well in a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 24 hours. Following culture, glutamine-free DMEM containing 50 μg / mL, 200 μg / mL, or 1000 μg / mL of the test sample (2'-FL and lactose) was added and cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture supernatant was removed, the cells were washed twice with PBS, and 0.20 mL of RIPA buffer was added to extract total protein. The protein content of each protein extract was quantified using the Bradford method.

[0236] D. Human gingival fibroblasts (Gin-1)

[0237] Human gingival fibroblasts (Gin-1) were cultured in DMEM containing 10% FBS. The cultured cells were recovered by trypsin treatment. Cells expressing GLS1 without the addition of the test sample were used in the experiment. The cells recovered by trypsin treatment were inoculated into a 6-well plate, and DMEM containing 50μg / mL, 200μg / mL or 1000μg / mL of the test sample (2'-FL and lactose) was added when 80% confluence was reached, and cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture supernatant was removed, washed twice with PBS, and 0.20mL of RIPA buffer was added to extract the total protein. The protein in each protein extract was quantified by the Bradford method.

[0238] E. Human umbilical vein endothelial cells (HUVEC)

[0239] Human umbilical vein endothelial cells (HUVEC) were cultured with DMEM+10% FBS+5mM glucose (conventional culture medium). The cultured cells were recovered by trypsin treatment. The recovered cells were inoculated into a 10 cm culture dish and cultured in a 37 ° C, 5% CO2 incubator for 24 hours. After that, the cells were exposed to DMEM+10% FBS+30mM glucose (high glucose medium) for 10 hours and to conventional culture medium for 14 hours for 7 days. After 7 days, conventional culture medium containing 50μg / mL, 200μg / mL or 1000μg / mL of the test sample (2'-FL and lactose) was added and cultured in a 37 ° C, 5% CO2 incubator for 24 hours. The culture supernatant was removed, washed twice with PBS, 0.20mL of RIPA buffer was added, and the whole protein was extracted. The protein in each protein extract was quantified by the Bradford method.

[0240] <Example 10-2: Preparation of control sample>

[0241] A. Human fetal kidney cells (HEK293)

[0242] Protein extracts were collected in the same manner as in Example 10-1A. Human fetal kidney cells (HEK293) except that DMEM containing no test sample was used instead of DMEM containing the test sample. The amount of protein in each protein extract was quantified by the Bradford method.

[0243] B-E. Human undifferentiated gastric carcinoma cells (KKLS), mouse microglial cells (BV2), human gingival fibroblasts (Gin-1), and human umbilical vein endothelial cells (HUVEC)

[0244] Protein extracts were recovered in the same manner as in <Example 10-1 B. Human gastric undifferentiated carcinoma cells (KKLS)>, <Example 10-1 C. Mouse microglial cells (BV2)>, <Example 10-1 D. Human gingival fibroblasts (Gin-1)>, and <Example 10-1 E. Human umbilical vein endothelial cells (HUVEC)>, except that the culture medium containing the test sample was used instead of the culture medium containing the test sample. The protein content in each protein extract was quantified by the Bradford method.

[0245] <Example 10-3. GLS1 expression analysis>

[0246] A. Human fetal kidney cells (HEK293)

[0247] For each protein extract, Western blotting analysis was performed using anti-GLS1 antibody and anti-β-actin antibody. Specifically, each protein extract was added to a 10% acrylamide gel in an equal amount of protein. Anti-GLS1 antibody (manufactured by Cell Signaling) and HRP-labeled anti-β-actin antibody (manufactured by Proteintech) were used as instructed by the manufacturer. For GLS1, HRP-labeled antibody (manufactured by Proteintech) was used as a secondary antibody. HRP was developed with 3,3'-diaminobenzidine (DBA). The depth of color development was quantified using the open source software "image J". The amount of GLS1 relative to the amount of β-actin in the control was set to 1, and the relative value of the amount of GLS1 relative to the amount of β-actin when each test sample was added was calculated, and the above relative values ​​when lactose was used are shown in FIG. Figure 11 The above difference values ​​when using 2'-FL are shown in Figure 12 .

[0248] B-E. Human undifferentiated gastric carcinoma cells (KKLS), mouse microglial cells (BV2), human gingival fibroblasts (Gin-1), and human umbilical vein endothelial cells (HUVEC)

[0249] The amount of GLS1 in the specimen was determined by ELISA using an antibody against GLS1. Two anti-GLS1 antibodies (manufactured by Cell Signaling and Proteintech) were used as described on the data sheet. An HRP-labeled antibody (manufactured by SeraCare Life Sciences) was used as the secondary antibody, and after color development with 3,3',5,5'-tetramethylbenzidine (TMB), the reaction was stopped with 1N sulfuric acid. After the reaction was stopped, the absorbance at a measurement wavelength of 450nm was measured using a microplate reader. The reference wavelength was set to 620nm. The amount of GLS1 relative to the amount of β-actin in the control was set to 1, and the relative value of the amount of GLS1 relative to the amount of β-actin when each test sample was added was calculated, and the above relative values ​​when lactose was used are shown in FIG. Figure 11 The above difference values ​​when using 2'-FL are shown in Figure 12 .

[0250] like Figure 11 and Figure 12 As shown, 2'-FL inhibited GLS1 expression in human umbilical vein endothelial cells and human gingival fibroblasts, and at least 200 μg / mL of 2'-FL inhibited GLS1 expression in human fetal kidney cells, human undifferentiated gastric cancer cells, and mouse microglia. Furthermore, when the 2'-FL concentration in the culture medium was at least between 50 μg / mL and 1000 μg / mL, the inhibitory effect on GLS1 expression in human umbilical vein endothelial cells and human gingival fibroblasts increased with increasing 2'-FL concentration. Furthermore, when the 2'-FL concentration in the culture medium was at least between 200 and 1000 μg / mL, the inhibitory effect on GLS1 expression in human fetal kidney cells, human undifferentiated gastric cancer cells, and mouse microglia increased with increasing 2'-FL concentration.

Claims

A preventive or improving agent for cell senescence, comprising 2'-fucosyllactose. The preventive or improving agent according to claim 1, which is used for promoting the proliferation of normal cells. The preventive or improving agent according to claim 1 , which is used for removing senescent cells.

4. The preventive or ameliorating agent according to claim 1, wherein The cell senescence is cell senescence caused by GLS1 expression.

5. The preventive or ameliorating agent according to claim 1, wherein The cell senescence is cell senescence caused by endoplasmic reticulum stress.

6. The preventive or ameliorating agent according to claim 1, wherein The cells are skin cells.

7. The preventive or ameliorating agent according to claim 1, wherein The cells are epidermal keratinocytes.

8. The preventive or ameliorating agent according to claim 1, wherein The cells are dermal fibroblasts.

9. The preventive or improving agent according to claim 1, which is at least one selected from the group consisting of a skin wound treatment agent, a skin metabolism promoter, a pigmentation preventive and improving agent, a wrinkle preventive and improving agent, a sagging preventive and improving agent, and a rough skin preventive and improving agent.

10. The preventive or ameliorating agent according to claim 1, wherein The cells are kidney cells, gastric cells, microglial cells, gingival cells or vascular endothelial cells.

11. The preventive or improving agent according to claim 1, which is at least one selected from the group consisting of a preventive or improving agent for glomerulosclerosis, a preventive or improving agent for decreased renal function, a preventive or improving agent for decreased gastric acid secretion by gastric parietal cells, a preventive or improving agent for decreased pepsinogen secretion by gastric chief cells, a preventive or improving agent for decreased cognitive function, a preventive or improving agent for gingival inflammation, a preventive or improving agent for arteriosclerosis, and a preventive or improving agent for obesity, diabetes, or impaired glucose tolerance. The preventive or ameliorating agent according to claim 1 , which is an external preparation.

13. The preventive or ameliorating agent according to any one of claims 1 to 12, which is a cosmetic. The preventive or ameliorating agent according to any one of claims 1 to 11, which is a food.

Images