Application of SASP inhibiting compound in preparation of anti-aging product

Abstract

The invention relates to application of a compound for inhibiting SASP in preparation of an anti-aging product, and belongs to the technical field of new application of known medicines. The compound for inhibiting the SASP is pinocembrin, and the pinocembrin is used for inhibiting replicative senescence of cells and cell stress senescence induced by etoposide, adriamycin and H2O2. The invention researches that pinocembrin realizes an anti-aging effect by inhibiting characteristic senescence-related secretion phenotype factors of senescence cells, reducing expression of senescence-related cell cycle arrest marker genes and relieving the senescence condition of the cells, and does not have an effect of eliminating and damaging the cells.

Description

Technical Field

[0001] This invention belongs to the field of novel applications of known drugs, specifically relating to the application of a compound that inhibits SASP in the preparation of anti-aging products. Background Technology

[0002] With the increasing elderly population, the incidence of age-related diseases, including Alzheimer's disease, cardiovascular and cerebrovascular diseases, chronic liver disease, and tumors, is also rising. Cellular senescence plays a crucial role in individual aging and is a major driving force behind it. Cellular senescence is a stable, terminal state characterized by permanent cell cycle arrest. The type of senescence where cells exhibit irreversible cell cycle arrest after multiple passages is called replicative senescence. In the elderly, replicative senescence can lead to various diseases. In addition, stimuli such as oxidative stress and DNA damage can also induce cellular senescence; this type of senescence is called stress-induced cellular senescence.

[0003] Cellular senescence can influence neighboring cells and their microenvironment through the secretion of senescence-associated secretory phenotype (SASP) factors. Typical SASP factors include inflammatory chemokines, cytokines, growth factors, matrix metalloproteinases, and extracellular matrix substances secreted by senescent cells, such as IL-1β, IL-6, Ccl2, and Cxcl1. SASP induces cell cycle arrest in surrounding normal cells via paracrine signals, triggering senescence in neighboring cells, leading to the accumulation of senescent cells and further promoting the occurrence and development of senescence-related damage and diseases. It is noteworthy that the SASP of senescent cells exhibits a degree of heterogeneity; it can change over time and its composition varies depending on the cell type and the mode of senescence induction. For example, the composition of SASP factors produced by replicative senescence, doxorubicin-induced senescence, H2O2-induced senescence, and etoposide-induced senescence differs. However, although the composition of SASP depends on different cell types and inducing factors, some common SASP components exist across different cell types. For instance, IL-6 and IL-8 can stimulate angiogenesis, affect cell signaling, induce innate immune responses and inflammation, and promote damage to hepatocytes, epithelial cells, and endothelial cells. During the skin aging process, in addition to the characteristic factors IL-8 and IL-6, the SASP secreted by aging skin cells also secretes matrix metalloproteinases (MMPs), which lead to the excessive degradation of collagen and elastin in the dermis, resulting in skin aging phenotypes such as wrinkles and decreased elasticity.

[0004] Currently, commonly used anti-aging strategies or therapeutic drugs are mainly divided into two categories: senolytics (serotonin-reducing agents) and senomorphics (senomorphic agents that regulate aging-related secretory phenotypes). Drugs that target and eliminate senescent cells exert their effects by directly killing senescent cells. For example, the combination of quercetin and dasatinib has a strong senescent cell-clearing function and can function as senolytics. However, the discovery of these drugs is mostly based on fibroblast screening, and they are only effective for certain types of senescent cells. Elimination of other cells with structural and functional components, such as hepatocytes, endothelial cells, and skin epithelial cells, may cause damage to tissues such as the liver, blood vessels, and skin, lacking cell selectivity. On the other hand, drugs that regulate aging-related secretory phenotypes do not kill cells and can maintain cell survival. They mainly interfere with the pro-inflammatory properties of senescent cells by affecting the production of SASPs (senescent cellular senescence enzymes) and the secretion of related factors, inhibiting cellular senescence phenotypes and the senescence of neighboring cells. Therefore, developing drugs that regulate age-related secretory phenotypes and inhibiting cellular senescence and age-related phenotypes through novel anti-aging methods and strategies has important application value and significance for delaying individual aging and improving chronic liver disease, vascular disease and skin aging caused by bodily function and cellular senescence in the elderly population.

[0005] Pinocene ester (PCE) is a natural flavonoid compound, a 5,7-dihydroxyflavanone, found in relatively high amounts in some plants (such as Swiss white pine and eucalyptus leaves). Besides natural extraction from plants, it can also be obtained through biosynthesis and chemical synthesis. In preclinical studies, PCE has demonstrated good antibacterial, antiviral, and neuroprotective effects.

[0006] Relevant patent documents retrieved: The patent, published in China (CN116687911) on September 5, 2023, discloses a novel use of geraniol in the preparation of drugs to protect against heat injury. It verifies that geraniol can alleviate heat injury by regulating body temperature through cytokines, thereby reducing cellular and systemic heat stress responses and enhancing the body's antioxidant capacity.

[0007] Relevant non-patent literature retrieved: The journal or book title is "Food Industry Technology", the article title is "Study on the Optimization of the Formula of Lycium barbarum Leaf Flavonoid Oral Liquid and its Antioxidant Effect on Aging Mice", the publication date is February 8, 2025. This article discloses that Lycium barbarum leaf flavonoid oral liquid can significantly increase the content of SOD, CAT and MDA in the serum, liver and brain tissue of aging model mice, and has good antioxidant capacity.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: Patent CN116687911 did not study the anti-aging mechanism of jugalosin. The relevant evidence is that the beneficial effects only mention that jugalosin can reduce cellular and systemic heat stress response by adjusting cytokines to regulate body temperature and enhance the body's antioxidant capacity. It does not mention the anti-aging effect of jugalosin in regulating SASP cytokine expression, nor has it been verified by relevant experiments. However, it is known to those skilled in the art that the mechanisms of anti-aging and body temperature regulation are different.

[0009] The literature "Optimization of the formulation of Lycium barbarum leaf flavonoid oral liquid and its antioxidant effect on aging mice" also did not study the anti-aging mechanism of ginsenoside. The relevant evidence is that the abstract mentions that the oral liquid can significantly increase the content of SOD, CAT, GSH and MDA in the serum, liver and brain tissue of aging mouse models, showing good antioxidant capacity. However, the expression of aging-related factors was not verified in the animal experiments, and its mechanism of action was not studied. Summary of the Invention

[0010] The purpose of this invention is to provide: The application of a compound that inhibits SASP in the preparation of anti-aging products, and related technologies, to solve technical problems such as inhibiting the expression of SASP and related factors in senescent cells, downregulating the expression of cell cycle arrest marker genes, regulating the senescence phenotype and SASP factor expression in liver cells of senescent mice, and alleviating aging-related cell and tissue organ damage, or combinations thereof.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] The definitions of standard chemical terms can be found in the reference "Pharmacopoeia of the People's Republic of China (2020 Edition): China Medical Science and Technology Press: May 2020: 1st Edition."

[0014] Unless otherwise stated, conventional methods within the scope of the art, such as cell experiments, shall be used.

[0015] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0016] The term "PCE" used in this article refers to: pine oleoresin.

[0017] The term "MEF cell" used in this article refers to mouse embryonic fibroblasts.

[0018] The term "DOX" used in this article refers to doxorubicin.

[0019] The term "ETO" used in this article refers to etoposide.

[0020] The term "NAR" used in this article refers to naringenin.

[0021] In a first aspect, the present invention provides the application of a compound that inhibits SASP in the preparation of anti-aging products.

[0022] This includes technical features such as: SASP inhibition, compounds, anti-aging, products, and applications.

[0023] Among them, the preferred technical feature for inhibiting SASP is: inhibiting the expression of SASP-related factors.

[0024] The preferred technical feature compound is arbutin, whose structure is shown in formula (1):

[0025] Equation (1).

[0026] The technically characteristic compound is further preferably: arbutin is used to inhibit cellular replicative senescence, as well as cellular stress-induced senescence induced by etoposide, doxorubicin, and H2O2; arbutin is used to alleviate and / or treat liver function damage caused by aging, by inhibiting hepatocyte senescence and SASP-related factor expression, reducing the leakage of liver injury marker enzymes, lowering their content in serum, and improving liver function; the cells are selected from at least one of fibroblasts, endothelial cells, epithelial cells, skin keratinocytes, and hepatocytes.

[0027] Among them, the preferred anti-aging technology feature is achieved by inhibiting the secretion of characteristic aging-related phenotypic factors of senescent cells, reducing the expression of aging-related marker cell cycle arrest genes, or alleviating cellular senescence through boraxone.

[0028] Among them, the anti-aging technical features are further optimized as follows: the activity of galactosidase, a marker of cellular aging, is reduced; the expression of cell cycle arrest marker genes p16 and p21 is inhibited; and the expression of aging-related SASP factors is inhibited.

[0029] The anti-aging technical features are further optimized as follows: inhibiting the replicative senescence of MEF cells, downregulating the expression levels of cell cycle arrest-related senescence marker genes, and significantly inhibiting the expression of SASP-related factors; inhibiting etoposide (ETO)-induced senescence of NIH3T3 cells, downregulating the expression levels of cell cycle arrest-related senescence marker genes, and significantly inhibiting the expression of SASP-related factors Cxcl1, Ccl2, and IL-8, rather than by targeting and clearing senescent fibroblasts; inhibiting DOX-induced senescence of endothelial cells EA.hy926, downregulating the expression levels of cell cycle arrest-related senescence marker genes, and significantly inhibiting SAS Instead of targeting and eliminating senescent endothelial cells, this study downregulated the expression of H2O2-induced cell cycle arrest-related aging marker genes without affecting the survival of HaCaT skin keratinocytes, and significantly inhibited the expression of SASP-related factors Ccl2, IL-8, and MMP8. It also reduced the release of damage markers such as AST and ALT in the liver of senescent mice, decreased the number of β-gal-positive cells in the liver of senescent mice, inhibited the expression of aging-related marker genes p16 and p21 in senescent mice, and inhibited the expression of SASP-related factors IL-6 and TNF-α in the liver of senescent mice.

[0030] The preferred technical feature of the product is that it contains a combination of arbutin or its pharmaceutically acceptable salt and pharmaceutically acceptable additives.

[0031] The technical feature additive is further preferably selected from one or more of the following: nutritional supplements, dietary supplements, excipients, disintegrants, and lubricants.

[0032] Among them, the technical features of the product are further preferably: oral dosage form, external dosage form or injectable dosage form.

[0033] Among them, the preferred oral dosage form is: tablets, capsules, oral liquids, enteric-coated tablets, granules, syrups, pills, water-honey pills, powders, mixtures, decoctions, medicated wines, syrups, tinctures, or sublingual tablets.

[0034] The preferred external dosage form is further specified as: ointment, suppository, hard ointment, liniment, eye drops, oil, cream, aerosol or spray, or external dosage form as toner, moisturizing lotion, face mask, face cream, powder, shower gel, soap, liquid foundation, foundation cream, serum, hand cream, body lotion, hand soap, shampoo, conditioner, facial cleanser, facial wash, sunscreen, moisturizing cream, snow cream, nutrient solution or perfume.

[0035] Among them, the preferred technical characteristic of aging is: age-related decline in organ function and chronic liver disease, vascular disease and / or skin aging.

[0036] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: 1. Compared with existing technologies, this invention inhibits the replicative senescence of MEF cells, downregulates the expression levels of cell cycle arrest-related senescence marker genes, and inhibits the expression of SASP-related factors; inhibits etoposide (ETO)-induced senescence of NIH3T3 cells, downregulates the expression levels of cell cycle arrest-related senescence marker genes, and inhibits the expression of SASP-related factors Cxcl1, Ccl2, and IL-8, rather than by targeting and eliminating senescent fibroblasts; inhibits DOX-induced senescence of endothelial cells EA.hy926, downregulates the expression levels of cell cycle arrest-related senescence marker genes, and inhibits the expression of SASP-related factors Cxcl1, Ccl2, and IL-8. It achieves better technical effects by downregulating the expression of H2O2-induced cell cycle arrest-related aging marker genes, inhibiting the expression of SASP-related factors Ccl2, IL-8, and MMP8, without affecting the survival of HaCaT cells in skin keratinocytes; reducing the release of damage markers such as AST and ALT in the liver of aging mice, reducing the number of β-gal positive cells in the liver of aging mice, inhibiting the expression of aging-related marker genes p16 and p21 in aging mice, and inhibiting the expression of SASP-related factors IL-6 and TNF-α in the liver of aging mice.

[0037] 2. Compared with existing technologies, this invention provides a technical solution with a different technical concept, and its technical effect is slightly improved compared with existing technologies. The difference in technical concept between this invention and existing technologies includes, but is not limited to, the discovery of a new application of pine resin in this invention, namely: proposing and verifying the application of pine resin in the preparation of anti-aging products from the perspective of regulating aging-related secretory phenotypes, i.e., inhibiting SASP anti-cellular aging. Attached Figure Description

[0038] Figure 1 The effect of PCE on the expression levels of p16 and p21 in senescent MEF cells (compared with the senescent group, **p<0.01, ***p<0.001).

[0039] Figure 2 The effect of PCE on the expression of SASP-related factors in senescent MEF cells (***p<0.001 compared with the senescent group).

[0040] Figure 3 The effect of PCE on the survival rate of ETO-induced senescent NIH3T3 cells.

[0041] Figure 4 The effect of NAR on the survival rate of ETO-induced senescent NIH3T3 cells.

[0042] Figure 5 Effects of PCE and NAR on the expression levels of p16 and p21 in ETO-induced senescent NIH3T3 cells (compared with the senescent group, **p<0.01, ***p<0.001).

[0043] Figure 6 The effect of PCE on SASP factor expression in ETO-induced senescent NIH3T3 cells (compared with the senescent group, *p<0.05, ***p<0.001).

[0044] Figure 7 The effect of PCE on the survival rate of DOX-induced senescent EA.hy926 cells.

[0045] Figure 8 The effect of PCE on the expression levels of p16 and p21 in DOX-induced senescent EA.hy926 cells (***p<0.001 compared with the senescent group).

[0046] Figure 9 The effect of PCE on SASP factor expression in DOX-induced senescent EA.hy926 cells (***p<0.001 compared with the senescent group).

[0047] Figure 10 The effect of PCE on the survival rate of H2O2-induced senescent HaCaT cells.

[0048] Figure 11 Effects of PCE on p16 and p21 expression levels in H2O2-induced senescent HaCaT cells (*p<0.05, **p<0.01, ***p<0.001 compared with the senescent group).

[0049] Figure 12 The effect of PCE on SASP factor expression in H2O2-induced senescent HaCaT cells (***p<0.001 compared with the senescent group). Detailed Implementation

[0050] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0051] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0052] Experimental materials PCE was purchased from Beijing Yuanye Biotechnology Co., Ltd. H2O2, etoposide (ETO), and doxorubicin (DOX) were purchased from Beijing Solarbio Technology Co., Ltd.

[0053] C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). MEF cells were extracted from pregnant C57BL / 6 mice at gestation days 13-14. MEF cell culture medium consisted of 89% DMEM, 10% fetal bovine serum, and 1% NEAA. Mouse embryonic fibroblast NIH / 3T3 cell line, human umbilical vein endothelial cell line EA.hy926 cell line, and human immortalized keratinocyte HaCat cell line were all purchased from Wuhan Pronosei Biotechnology Co., Ltd.

[0054] DMEM medium, 100U penicillin and streptomycin antibiotics, fetal bovine serum, and trypsin used for cell culture were all purchased from Gibco, USA. TransZol Up reagent was purchased from TransGen Biotech, Beijing. The HiFiScript cDNA Synthesis Kit and SYBR Green PCR Master Mix were purchased from Jiangsu Kangwei Century Biotechnology Co., Ltd. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. Cell senescence β-galactosidase staining kit and MTT (thiazolyl blue) powder were purchased from Beyotime Biotechnology Co., Ltd.

[0055] Experimental instruments The BioTek Cytation 5 multi-functional cell imaging system was purchased from BioTek Inc., USA. The upright fluorescence microscope was purchased from Carl Zeiss Optics Co., Ltd. Medical refrigeration, low-temperature, and ultra-low-temperature storage boxes were purchased from Haier Co., Ltd. The LightCycler® 96 real-time quantitative PCR instrument was purchased from Roche Pharmaceuticals Ltd., Shanghai. The SORVALLST16R low-temperature high-speed centrifuge was purchased from Thermo Fisher Scientific, USA.

[0056] The experimental methods used in the following examples are as follows: SA-β-gal activity detection experiment Cell lines were seeded into 24-well plates and incubated overnight. After the appropriate drug treatment, the cells were fixed in PBS containing 2% glutaraldehyde for 20 minutes. Then, the cells were incubated overnight at 37°C with a solution containing X-gal. After staining, the cells were observed and photographed under an inverted microscope; cells showing blue granules were identified as SA-β-gal positive cells. Five fields of view were randomly analyzed using ImageJ software, and the staining positivity rate was calculated as the percentage of positive cells out of the total number of cells: SA-β-gal positivity rate (%) = number of positive cells / total number of cells × 100%.

[0057] MTT Experiment After digestion of normal or senescent cells, the cells were counted and seeded at an appropriate density into 96-well plates. Once the cells adhered, different concentrations of drugs were added to treat the cells for 24 hours. The 96-well plates were then removed, and 20 μL of MTT solution was added to each well. After incubation at 37°C for 4 h, the supernatant was carefully aspirated from the wells, and 150 μL of DMSO was added. The plates were shaken for 10 min to fully dissolve the precipitate. The absorbance was measured at 570 nm using a microplate reader, and the results were recorded.

[0058] PCR experiment After cell modeling and drug treatment, 1 ml of TransZol Up reagent was added and thoroughly mixed by inverting. 200 μL of chloroform was added, and the mixture was inverted 10 times to mix thoroughly. The mixture was centrifuged at 12000 rpm, 4°C for 10 min. The supernatant was carefully transferred to a new sterile 1.5 ml EP tube, and an equal volume of isopropanol was added, followed by inverting to mix. The tube was centrifuged at 12000 rpm, 4°C for 20 min. The supernatant was discarded, and the precipitate was washed with 1 ml of 75% ethanol and anhydrous ethanol, respectively. The precipitate was air-dried in a laminar flow hood, and the RNA was dissolved in DEPC water. The RNA concentration was measured as a percentage of OD260 / 280 to determine the purity and content of the extracted RNA. All operations were performed on ice. Reverse transcription was performed according to the instructions of the Kangwei Century High-Capacity cDNA Reverse Transcription Kit to synthesize cDNA. PCR amplification was performed according to the SYBR Green PCR Master Mix kit instructions, using β-actin as an internal control, to detect the relative expression levels of each gene in each group of cells. Two [analytes / representations] were used. -ΔΔCt The method calculates the relative abundance of each gene, and each experiment is repeated three times.

[0059] Statistical processing Data analysis was performed using GraphPad Prism 9.0 software, employing one-way ANOVA. Results are expressed as Mean ± SEM.

[0060] Example 1: Experiment on the inhibition of replication and senescence in MEF cells Primary mouse embryonic fibroblasts (MEFs) were extracted from C57BL / 6 mouse embryos. The 10th generation (P10) MEFs were used as the replicative senescence (RS) group, and the 3rd generation (P3) MEFs were used as the normal control group. When the P9 generation cells reached 70% confluence, the treatment groups were treated with 20 μM and 40 μM PCE for 48 hours, respectively. The normal control group (Normol) and the replicative senescence group were treated with the corresponding solvent DMSO for 48 hours.

[0061] The results showed that PCE treatment significantly inhibited the replicative senescence of MEF cells, specifically manifested as a decrease in the activity of the cellular senescence marker galactosidase (Table 1) and a significant inhibition of the expression of cell cycle arrest marker genes p16 and p21. Furthermore, we examined the expression levels of some SASP factors secreted by senescent cells, and the results showed that PCE treatment significantly inhibited the expression levels of IL-6, IL-1β, and MMP3 in senescent MEF cells. Figures 1-2 ).

[0062] Table 1. β-gal staining of senescent MEF cells

[0063] Note: Compared with the aging group, **p<0.01, ***p<0.001.

[0064] Example 2: Etoposide (ETO)-induced inhibition of cellular stress-induced senescence. Low doses of etoposide can induce cellular senescence. NIH3T3 cells were first treated with a low dose of ETO (10 μM) for 24 hours to induce senescence, then co-cultured in normal medium with PCE or naringenin (NAR) for 48 hours. Cell viability was assessed by MTT assay. Results showed that different concentrations of PCE did not significantly kill senescent cells compared to normal cells. Figure 3 ), while treatment with similar compounds (NAR) can significantly kill senescent cells ( Figure 4Meanwhile, naringenin (NAR), a compound of the same class, was selected as a pharmacodynamic control. Cell morphology was further observed and aging-related indicators were detected to verify the anti-aging activity of PCE. Experimental results showed that PCE treatment significantly inhibited ETO-induced senescence in NIH3T3 cells, and the anti-aging activity of PCE was significantly superior to that of NAR (Table 2). PCE treatment not only reduced cellular galactosidase activity but also downregulated the expression of cell cycle arrest marker genes p16 and p21. Furthermore, at the same dose, PCE had a stronger effect on reducing aging-related genes than NAR (…). Figure 5 Furthermore, the expression of SASP factors such as Cxcl1, Ccl2, and IL-8 in senescent cells was significantly inhibited by PCE. Figure 6 This further indicates that PCE exerts its anti-cellular aging effect as a drug that regulates age-related secretory phenotypes.

[0065] Table 2. β-gal staining of senescent NIH3T3 cells

[0066] Note: Compared with the aging group, ***p<0.001.

[0067] Example 3: Doxorubicin (DOX)-induced inhibition of cellular stress-induced senescence Doxorubicin (DOX) induces cellular senescence and increases the secretion of SASPs in endothelial cells, leading to DOX-induced senescence-related inflammation. After inducing EA.hy926 cells with 500 nM DOX for 24 hours, they were co-cultured in normal medium or drug-containing medium with the corresponding concentration of PCE for 48 hours. Cell viability was assessed by MTT assay. The results showed that different concentrations of PCE did not significantly kill senescent cells compared to normal cells. However, the expression of cellular senescence marker genes p16 and p21, as well as senescence-related SASP factors Ccl2, IL-8, and MMP8, were significantly inhibited, indicating that PCE significantly inhibits DOX-induced cellular senescence by suppressing SASPs rather than by targeting and eliminating senescent cells. Figures 7-9 ).

[0068] Example 4: Experiment on inhibition of cellular stress-induced senescence induced by hydrogen peroxide (H2O2) Hydrogen peroxide (H2O2)-induced cellular senescence models are mainly constructed by exposing cells to the peroxidative environment created by H2O2. Following common methods for hydrogen peroxide-induced cellular senescence models, HaCaT cells were stimulated with 200 μM H2O2 for 4 hours, followed by 24 hours of culture to verify the ameliorative effect of PCE on skin aging. Experimental results showed that PCE significantly downregulated the expression levels of H2O2-induced cell cycle arrest-related senescence marker genes without affecting HaCaT cell survival, and significantly inhibited the expression of SASP-related factors Ccl2, IL-8, and MMP8. Figure 10-12 ).

[0069] Example 5: Protective effect of PCE on the liver of aging mice Thirty-two 6-week-old male C57 mice were given free access to food and water and acclimatized to the laboratory environment. All mice were randomly divided into four groups (n=8): control group, model group, PCE-L group, and PCE-H group. For the first four weeks, the control and model groups were administered an equal volume of saline by gavage. Mice in the PCE-L group were administered PCE (20 mg / kg) by gavage daily, and mice in the PCE-H group were administered PCE (40 mg / kg) by gavage daily. After four weeks, except for the control group, all groups were subcutaneously injected with 200 mg / kg D-galactose daily to establish an aging mouse model. Simultaneously, the treatment groups continued to be administered PCE by gavage daily, while the control and model groups were administered saline by gavage. Mice were sacrificed after six weeks. Serum and liver tissues were isolated from the mice for subsequent analysis.

[0070] The levels of AST and ALT in mouse serum were detected using a Hitachi 7020 biochemical analyzer according to the reagent kit method. The experimental results (Table 3) showed that, compared with control mice, the levels of AST and ALT, indicators of liver damage, were significantly increased in the serum of aged mice, while PCE treatment decreased the levels of AST and ALT in mouse serum in a dose-dependent manner.

[0071] Table 3 Serum AST and ALT levels in mice

[0072] β-gal staining was performed on frozen sections of mouse liver to detect the senescence of mouse liver cells, and the results were expressed as the β-gal cell positivity rate. The experimental results (Table 4) showed that, compared with the control group, the proportion of β-gal positive cells in the liver of the model group mice was significantly increased, and PCE treatment significantly reduced the number of β-gal positive cells, suggesting that PCE has a significant anti-cellular senescence effect.

[0073] Table 4. β-gal staining of mouse liver tissue

[0074] The expression levels of mouse liver aging-related marker genes (p16, p21) were detected using real-time quantitative PCR according to the SYBR Green PCR MasterMix kit instructions, with β-actin as an internal control. The relative expression levels of p16, p21, IL-6, and TNF-α in each group of cells were measured (with the model group as 1). A 2-1 -ΔΔCt The relative abundance of each gene was calculated, and each experiment was repeated three times. The results are shown in Tables 5 and 6.

[0075] Compared with the control group mice, the liver aging-related genes p16 and p21 were significantly upregulated in the model group mice, and PCE treatment could significantly inhibit the expression of p16 and p21.

[0076] Table 5. Detection of relative expression levels of aging marker genes in mouse liver tissue.

[0077] Compared with the control group, the expression levels of aging-related phenotypic factors IL-6 and TNF-α in the liver of the model group mice were significantly upregulated, and PCE treatment significantly inhibited the expression of IL-6 and TNF-α. This indicates that PCE can effectively regulate the expression of SASP factor in the liver cells of aging mice.

[0078] Table 6. Detection of SASP-related gene expression levels in mouse liver tissue.

[0079] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of a compound that inhibits SASP in the preparation of anti-aging products, characterized in that, The compound that inhibits SASP is terpineol, and its structure is shown in formula (1). Equation (1).

2. The application according to claim 1, characterized in that, The aforementioned chorionic acid is used to inhibit cellular replicative senescence, as well as cellular stress-induced senescence induced by etoposide, doxorubicin, and H2O2.

3. The application according to claim 2, characterized in that, The cells are selected from at least one of fibroblasts, endothelial cells, epithelial cells, skin keratinocytes, and hepatocytes.

4. The application according to claim 1, characterized in that, The anti-aging effect is achieved by borsoni to inhibit the secretion of characteristic senescence-related phenotypic factors in senescent cells, reduce the expression of senescence-related cell cycle arrest marker genes, and / or alleviate cellular senescence.

5. The application according to claim 1, characterized in that, The aforementioned terpinene is used to alleviate and / or treat liver function damage caused by aging. By inhibiting liver cell aging and the expression of SASP-related factors, terpinene reduces the leakage of liver injury marker enzymes, lowers their content in serum, and improves liver function.

6. The application according to claim 1, characterized in that, The product contains geraniol or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable additive.

7. The application according to claim 6, characterized in that, The additives are selected from one or more of the following: nutritional supplements, dietary supplements, excipients, disintegrants, and lubricants.

8. The application according to claim 1, characterized in that, The product can be in the form of an oral dosage form, a topical dosage form, or an injectable dosage form.

9. The application according to claim 8, characterized in that, The oral dosage forms are tablets, capsules, oral liquids, enteric-coated tablets, granules, syrups, drop pills, water-honey pills, powders, mixtures, decoctions, tinctures, wines, lotions, tinctures, or sublingual tablets; the external dosage forms are ointments, suppositories, hard ointments, liniments, eye drops, oils, creams, aerosols, or sprays; the external dosage forms are toners, moisturizing lotions, face masks, face creams, pressed powders, shower gels, soaps, liquid foundations, foundation creams, serums, hand creams, body lotions, hand soaps, shampoos, conditioners, facial cleansers, facial washes, sunscreens, moisturizing creams, snow creams, nutrient solutions, or perfumes.

10. The application according to claim 1, characterized in that, The aging referred to here is age-related decline in organ function and chronic liver disease, vascular disease and / or skin aging.

Images