Anti-aging products, anti-aging preparations, and methods for manufacturing anti-aging products.

Abstract

The invention relates to a product which rejuvenates aging cells or induces SASP resistance and thereby inhibits aging; and a food, cosmetic, pharmaceutical, or other similar preparation which includes such a product. The invention relates to an anti-aging product which has anti-aging activity and is produced from macrophages stimulated by lipopolysaccharide; and an anti-aging preparation such as a food, cosmetic, skin care preparation, food supplement, semi-pharmaceutical product, or pharmaceutical product which includes an anti-aging product. The invention also relates to a method for producing an anti-aging product.

Description

Anti-aging products, anti-aging products, and methods for producing the same

[0001] The present invention relates to an anti-aging product, an anti-aging product, and a method for making the same.

[0002] In 2019, the World Health Organization defined aging (excluding age-related functional decline and dementia) as a disease in the 11th edition of the International Classification of Diseases. If aging is a disease, it can be prevented or treated. The prevention or treatment (rejuvenation) of aging is collectively called anti-aging. Currently, vigorous research is being conducted into the nature of senescent cells, which are at the root of aging, as well as the causes and mechanisms of aging, and methods of anti-aging are being sought.

[0003] Cellular aging has a wide range of causes. For example, it can be induced by everyday inflammation, various stresses such as oxidative stress and lysosomal stress, telomere abnormalities, and damage to genomic DNA. Therefore, sirtuins, which are involved in genomic DNA repair, have attracted attention. Furthermore, NMN, a sirtuin coenzyme and a precursor of NAD, which decreases with aging, has been spotlighted as an anti-aging substance (Non-Patent Document 1). However, the mechanism by which NMN prevents aging has not yet been clarified, and there are still questions as to whether NMN truly has an anti-aging effect.

[0004] Furthermore, this method also has its own challenges. Namely, it is unclear whether the decrease in NAD is a result or a cause of aging, and the idea of ​​NMN as a way to prevent aging was conceived as a way to supplement what is lacking. For these reasons, even if oral administration of NMN has an anti-aging effect, it is thought that its effectiveness would be limited. In addition, at present, it is very expensive to orally ingest enough NMN to synthesize NAD.

[0005] On the other hand, if there were a way to prevent aging using the cellular abilities that living organisms naturally possess, it would be possible to create foods and medicines that would help prevent aging in a broad sense by utilizing the potential that cells naturally possess, rather than simply compensating for deficiencies.

[0006] Senescent cells have increased levels of proteins that inhibit the cell cycle, such as p16 (cyclin-dependent kinase inhibitor 2A) and p21 (cyclin-dependent kinase inhibitor 1) (Non-patent Documents 2 and 3). In other words, the cell cycle is arrested in senescent cells.

[0007] Senescent cells are known to induce the Senescence-Associated Secretory Phenotype (SASP), which causes the senescence of surrounding young cells (Non-Patent Document 4). Senescent cells continue to secrete inflammatory cytokines, chemokines, extracellular matrix degrading enzymes, cell growth factors, and fragmented DNA, encapsulated in exosomes (Non-Patent Document 5). These xenobiotics induce chronic inflammation throughout the body, leading to a further increase in senescent cells. Therefore, the concept of senolytics, which removes senescent cells, has emerged as an anti-aging method (Non-Patent Document 6).

[0008] The immune system attempts to monitor and eliminate not only foreign substances and cancer cells, but also senescent cells. However, some senescent cells express the immune checkpoint protein PD-L1, which suppresses the function of immune cells, allowing them to escape the immune system's surveillance and elimination mechanism (Non-Patent Document 7). In response, research is being conducted to improve the efficiency of the immune system's elimination of senescent cells by using anti-PD-1 antibodies (Non-Patent Document 7). However, the limitation of this method is that it assumes that the immune system is functioning normally. Since immune cells naturally age, it is unlikely that senescent immune cells will efficiently eliminate senescent cells.

[0009] As mentioned above, various approaches have been taken to prevent the progression of aging, primarily two methods: slowing the progression of cellular aging, such as by taking NMN, and quickly removing aging cells, such as by using anti-PD-1 antibodies. These approaches should be used comprehensively, but if aging could be controlled (prevented or rejuvenated) by utilizing the inherent cellular abilities of living organisms, it is possible to create foods and medicines that contribute to the prevention and recovery of aging in a broader sense than the two methods mentioned above. However, little progress has been made in research into safely controlling the cellular abilities of living organisms to rejuvenate aging cells or inhibiting aging by inducing resistance to SASP.

[0010] We have demonstrated that oral administration of lipopolysaccharide (LPS) can prevent and improve various intractable diseases (Non-Patent Document 8). In this study, we found that oral administration of LPS can convert the characteristics of brain microglia to neuroprotective ones (Non-Patent Document 8). LPS is not normally absorbed into the body, and the conversion of microglia characteristics is not a direct effect of LPS. Therefore, these results suggest that LPS acts on innate immune cells such as mucosal macrophages, and then transmits signals to cells throughout the body via cell migration or secreted humoral factors. We call this system the macrophage network (Non-Patent Document 9). To confirm the usefulness of this system for preventing and reversing aging, we investigated whether rejuvenation or anti-aging occurs when LPS-stimulated macrophage culture medium is added to young cells containing culture supernatant of senescent fibroblasts or senescent cells.

[0011] As a result, they discovered that the expression of p16 and p21, which are characteristic of senescent cells, decreases, rejuvenating the cells, and that the simultaneous addition of culture medium from macrophages stimulated with LPS prevents aging in young cells, which would normally undergo accelerated aging due to the influence of the SASP of senescent cells, thereby completing the present invention.

[0012] KF Mills et al., “Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice”, Cell Metabolism, 2016.12, 24(6), p.795-806J. Krishnamurthy et al., “Ink4a / Arf expression is a biomarker of aging”, The Journal of Clinical Investigation, 2004.11, 114(9), p.1299-1307X. Chen et al., “Senescence-like changes induced by expression of p21Waf1 / Cip1 in NIH3T3 cell line”, Cell Research, 2002.09, 12(3-4), p.229-233G. Nelson et al., “A senescent cell bystander effect: senescence-induced senescence”, Aging Cell, 2012.02, 11(2), pp.345-349. Chika Misawa et al., "Function of extracellular vesicles secreted by senescent cells," Drug Delivery System, June 2021, 36(2), pp.130-137Y. Zhu et al., "The Achilles' heel of senescent cells: from transcriptome to senolytic drugs," Aging Cell, March 2015, 14(4), pp.644-658TW. Wang et al., "Blocking PD-L1-PD-1 improves senescence surveillance and aging phenotypes," Nature, November 2022, 611, pp.358-364H.Mizobuchi, “Oral route lipopolysaccharide as a potential dementia preventive agent inducing neuroprotective microglia”, Frontiers in Immunology, 2023.03.09, 14https: / / doi.org / 10.3389 / fimmu.2023.1110583C. Kohchi et al., “Applications of Lipopolysaccharide Derived from Pantoea agglomerans (IP-PA1) for Health Care Based on Macrophage Network Theory”, Journal of Bioscience and Bioengineering, 2006.12, 102(6), p.485-496.

[0013] The present invention provides a product that rejuvenates aged cells or prevents aging by inducing SASP resistance, and also provides foods, cosmetics, pharmaceuticals, etc. that contain the product.

[0014] The anti-aging product of the present invention is produced from macrophages stimulated with lipopolysaccharide and has anti-aging activity. The lipopolysaccharide may be Enterobacteriaceae bacteria. The anti-aging product of the present invention contains the anti-aging product. The anti-aging product is preferably a food product, cosmetic, skin care product, supplement, quasi-drug, or pharmaceutical product. The anti-aging product is preferably in the form of a solid, liquid, gel, or aerosol. The method of producing an anti-aging product of the present invention is characterized by stimulating macrophages with lipopolysaccharide to produce an anti-aging product with anti-aging activity.

[0015] Previously, methods for slowing the progression of cellular senescence and methods for quickly removing senescent cells have been reported as anti-aging measures. However, in this invention, by using factors produced by macrophages stimulated with LPS, it is possible to rejuvenate senescent cells and prevent the aging that senescent cells cause to younger cells.

[0016] Gene expression in each NB1RGB cell. p16 (A), p21 (B), Ki-67 (C). Gene expression in each NB1RGB cell. p16 (A), p21 (B), Ki-67 (C).

[0017] Examples of the present invention will be described below, but the present invention is not limited to the following examples.

[0018] [Experiment in which culture medium of macrophages stimulated with LPS was added to aged fibroblasts] (1) Materials and Methods 1) Cell Culture

[0019] Human skin fibroblast cell lines NB1RGB PDL14.9 (hereafter referred to as "young cells") and PDL53.9 (hereafter referred to as "old cells") (RIKEN BRC Cell Bank) were maintained in Minimum Essential Medium α (MEMα) containing 10% fetal bovine serum (FBS). Three days before the start of the test, the medium was changed to Eagle's Minimum Essential Medium (EMEM) containing 10% FBS.

[0020] 2) Preparation of conditioned medium Human macrophage cell line THP-1 (ATCC) was cultured in RPMI-1640 containing 10% FBS. THP-1 was cultured at a concentration of 1 x 10 6 THP-1 cells were seeded at 1000 cells / mL, and LPS (LPSp) derived from Pantoea agglomerans was added to a final concentration of 100 ng / mL. The culture supernatant was collected after 24 hours. This LPSp-containing culture supernatant is designated as THP / LPS-CM. As a control, THP-1 culture supernatant without LPSp was also collected. This culture supernatant is designated as THP-CM.

[0021] 3) RNA extraction and real-time quantitative PCR (RT-qPCR) Young cells (n=3) and old cells (n=9) were diluted to 3 x 10 5 Cells were seeded into 6-well plates at 3 mL / well. After 24 hours of pre-culture, the medium was replaced as follows: ・Young cell group: Three wells of young cells were replaced with fresh EMEM. ・Old cell group: Three wells of old cells were replaced with fresh EMEM. ・Old + THP-CM group: Another three wells of old cells were replaced with a 1:1 mixture of EMEM and THP-CM. ・Old + THP / LPS-CM group: Another three wells of old cells were replaced with a 1:1 mixture of EMEM and THP / LPS-CM. All groups were then cultured for 24 hours.

[0022] Total RNA from young and old cells was extracted using the Fast Gene™ RNA Basic Kit (Nihon Genetics) according to the manufacturer's protocol. RNA was quantified by absorbance at 260 nm, and cDNA was synthesized using the TOYOBO ReverTra Ace™ qPCR RT Master Mix and rDNA Remover (Toyobo Co., Ltd.). Primers for p16, p21, Ki-67, and GAPDH used in the experiment were prepared by FASMAC Co., Ltd. Ki-67 is a marker for proliferating cells.

[0023] RT-qPCR was performed using THUNDERBIRD™ SYBR™ qPCR Mix (Toyobo Co., Ltd.). PCR conditions were 95°C for 1 minute, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute, followed by 95°C for 1 minute, 55°C for 30 seconds, and 95°C for 30 seconds. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard, and results were expressed as fold changes relative to young cells.

[0024] 4) Statistical analysis: Statistical analysis of RT-qPCR results was performed using BellCurve for Excel (ver. 4.04, Social Survey Research Information) using ANOVA followed by Tukey-Kramer multiple comparison test. p < 0.05 was considered significant.

[0025] (2) Results p16 and p21 are cyclin-dependent kinase inhibitors, involved in cell cycle progression, and are each considered to be senescence markers. Increases in either of these factors cause cell cycle arrest. Ki-67 is a marker for proliferating cells, and its increase leads to cell rejuvenation. Therefore, we first examined the expression of three genes (p21, p16, and Ki-67) in the young cell group, old cell group, old + THP-CM group, and old + THP / LPS-CM group.

[0026] The results are shown in Figure 1. p16 was significantly increased in the old cell group and the old + THP-CM group compared with the young cell group (p = 0.001, p < 0.001, respectively). The old + THP / LPS-CM group was significantly decreased compared with both the old cell group and the old + THP-CM group (p = 0.023, p = 0.003, respectively). No significant difference was observed between the young cell group and the old + THP / LPS-CM group. There was also no significant difference between the old cell group and the old + THP-CM group (Figure 1A).

[0027] p21 was significantly increased in the old cell group and the old + THP-CM group compared with the young cell group (both p<0.001). The old + THP / LPS-CM group was significantly decreased compared with both the old cell group and the old + THP-CM group (both p<0.001). There was no significant difference between the young cell group and the old + THP / LPS-CM group. There was also no significant difference between the old cell group and the old + THP-CM group (Figure 1B).

[0028] Ki-67 was significantly decreased in the old cell group and the old + THP-CM group compared with the young cell group (both p<0.001). The old + THP / LPS-CM group had lower levels than the young cell group, but was significantly increased compared with both the old cell group and the old + THP-CM group (both p<0.001). No significant difference was observed between the old cell group and the old + THP-CM group (Fig. 1C).

[0029] These results indicate that the old cell group and old + THP-CM group showed more advanced senescence at the gene expression level than the young cell group. Addition of THP / LPS-CM decreased p16 and p21 in the old cells, while increasing Ki-67, bringing them closer to the state of young cells. Furthermore, 24 hours after addition of LPSp (final concentration 100 ng / mL) to the old cell group, there was no significant difference in the gene expression patterns of p16, p21, and Ki-67 compared to the untreated group. These findings suggest that factors secreted by LPS-stimulated macrophages, rather than LPS itself, are responsible for cell rejuvenation.

[0030] [Experiment in which LPS-stimulated macrophage culture medium was added to young cells supplemented with culture supernatant from senescent fibroblasts] (1) Method 1) Cell culture and preparation of culture supernatant for senescence induction

[0031] Human dermal fibroblast cell lines NB1RGB PDL14.9 (hereafter referred to as "young cells") and PDL61.9 (hereafter referred to as "old cells") (RIKEN BRC Cell Bank) were maintained and passaged in Minimum Essential Medium α (MEMα) containing 10% FBS. The medium for young cells was replaced with EMEM containing 10% FBS 3 days before the start of the experiment. The medium for old cells was replaced with EMEM containing 10% FBS, and the culture supernatant was collected 7 days later. The culture supernatant was filtered through a 0.2 μm filter, and the filtrate (hereafter referred to as "old sup") was used to induce senescence in young cells.

[0032] 2) Preparation of conditioned medium: Same as in Example 1 (1) 2).

[0033] 3) RNA extraction and real-time quantitative PCR (RT-qPCR) Young cells (n=21) were diluted to 3 x 10 5 Cells were seeded into 6-well plates at 3 mL / well. After 24 hours of pre-culture, the medium was replaced as follows: ・Old sup group: The medium in 3 wells was replaced with old sup. ・Old sup+THP-CM group: Three wells were replaced with a 1:1 mixture of old sup and THP-CM (Ki-67 was not tested). ・1, 10, 100, 1000 ng / mL groups: Three wells were replaced with a 1:1 mixture of old sup and THP / LPS-CM at 1, 10, 100, or 1000 ng / mL (Ki-67 was only 100 ng / mL). ・Young cell group: The remaining 3 wells were replaced with EMEM as a control.

[0034] All were cultured for 24 hours. The primers used for total RNA and RT-qPCR were the same as those used in Example 1(1)3). However, RT-qPCR of Ki-67 was not performed.

[0035] 5) Statistical analysis: Same as in Example 1(1)4).

[0036] (2) Results Figure 2 shows the gene expression of p16 and p21, which are known as senescence markers, and Ki-67, a marker for proliferating cells.

[0037] p16 was significantly increased in the old sup and old sup + THP-CM groups compared with the young cell group (p = 0.003 and p < 0.001, respectively). At all concentrations, the old sup + THP / LPS-CM group significantly decreased compared with both the old sup and old sup + THP-CM groups (both p < 0.05). No significant differences were observed between the young cell group and the old sup + THP / LPS-CM group. There were also no significant differences between the old cell group and the old sup + THP-CM group. When comparing the old sup + THP / LPS-CM groups, significant differences were observed between the 1 ng / mL group and the 100 ng / mL and 1000 ng / mL groups (all p < 0.05), and p16 decreased in the 100 ng / mL and 1000 ng / mL groups. No significant differences were observed when concentrations of 10 ng / mL or higher were added (Figure 2A).

[0038] p21 was significantly increased in the old sup and old sup + THP-CM groups compared with the young cell group (both p<0.001). At all concentrations, the old sup + THP / LPS-CM group was significantly decreased compared with both the old sup and old sup + THP-CM groups (both p<0.001). Significant differences were observed between the young cell group and the old sup + THP / LPS-CM group at 10, 100, and 1000 ng / mL (p=0.002, p=0.002, and p=0.001, respectively), and the old sup + THP / LPS-CM group was decreased. There was no significant difference between the old cell group and the old sup + THP-CM group. There was also no significant difference between the old sup + THP / LPS-CM groups (Figure 2B).

[0039] Ki-67 was significantly decreased in the old sup cell group compared with the young cell group (p<0.001). It was significantly increased in the old sup + THP / LPS-CM group compared with the old sup cell group (p<0.001). No significant difference was observed between the young cell group and the old sup + THP / LPS-CM group (Fig. 2C).

[0040] These results suggest that the old sup and old sup + THP-CM groups exhibited increased p16 and p21 gene expression levels compared with the young cell group, indicating the progression of senescence. However, the addition of THP / LPS-CM to young cells simultaneously with old cell conditioned media resulted in decreased p16 and p21 expression and increased Ki-67 expression. This suggests that THP / LPS-CM suppresses the senescence-induced progression of young cells in response to old cell conditioned media. Furthermore, the gene expression patterns of p16, p21, and Ki-67 in young cells 24 hours after the addition of old sup and LPSp (final concentration 100 ng / mL) were not significantly different from those in the old sup cell group. These findings suggest that factors secreted by LPS-stimulated macrophages, rather than LPS itself, are responsible for the suppression of cellular senescence.

[0041] [Application Examples] From the above examples, it can be seen that macrophages can be stimulated with lipopolysaccharide to produce anti-aging products with anti-aging effects.

[0042] The effect of lipopolysaccharides on macrophages is known for many lipopolysaccharides, and the lipopolysaccharides are not limited to those derived from Pantoea agglomerans, but may be broadly derived from bacteria of the Enterobacteriaceae family. The anti-aging product may be a food, cosmetic, skin care product, supplement, quasi-drug, or pharmaceutical. The anti-aging product may be in the form of a solid, liquid, gel, or aerosol.

[0043] All publications cited herein are incorporated herein by reference in their entirety.

Claims

1. An anti-aging product that is produced by macrophages stimulated with lipopolysaccharide and is characterized by its anti-aging effects.

2. The anti-aging product according to claim 1, characterized in that the lipopolysaccharide is a bacterium of the Enterobacteriaceae family.

3. An anti-aging product comprising the anti-aging product according to claim 1.

4. The anti-aging product according to claim 3, which is a food product, a cosmetic product, a skin care product, a supplement, a quasi-drug, or a pharmaceutical product.

5. The anti-aging product according to claim 3, characterized in that the anti-aging product is in the form of a solid, liquid, gel, or aerosol.

6. A method for producing an anti-aging product, comprising stimulating macrophages with lipopolysaccharide to produce an anti-aging product having an anti-aging effect.

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