Application of SRT1720 in preparation of skin antioxidant repair agent

Abstract

The invention belongs to the technical field of skin care products, and discloses application of SRT1720 in preparation of a skin antioxidant repair agent. The repairing agent can effectively activate SIRT1 and promote expression of antioxidant protein Nrf-2, so that the anti-oxidative stress response of skin fibroblasts is adjusted, and skin aging caused by oxidative damage is resisted.

Description

Technical Field

[0001] The invention belongs to the technical field of skin care products, and particularly relates to the application of SRT1720 in preparing a skin antioxidant repairing agent. Background Art

[0002] The skin is the largest organ in the human body. Long-term exposure to external factors such as environmental pollution, ultraviolet rays, and tobacco smoke makes it susceptible to oxidative stress. [1,2] Oxidative stress is cell damage caused by free radicals, which leads to impaired cell function and accelerates skin aging. In recent years, many studies have shown that antioxidants can slow down skin damage caused by oxidative stress and help improve the skin aging process by regulating the redox state of skin cells. [3] . SIRT1, a member of the histone deacetylase (Sirtuins) family, was found to have significant antioxidant effects [4] , and has potential in skin repair and anti-aging [5] SRT1720 is a selective agonist of SIRT1 and has been shown to activate SIRT1, thereby promoting the antioxidant response of cells and enhancing the repair capacity of cells. [6] However, there is insufficient research on how SRT1720 specifically improves skin repair through antioxidant mechanisms, especially in skin fibroblasts. Summary of the invention

[0003] In order to overcome the deficiencies of the prior art, the present invention provides the use of SRT1720 in the preparation of a skin antioxidant repair agent, which can effectively activate SIRT1 and promote the expression of the antioxidant protein Nrf-2, thereby regulating the antioxidant stress response of skin fibroblasts and resisting skin aging caused by oxidative damage. SRT1720 can restore the healthy state of the skin and improve the repair ability and vitality of the skin. The invention has broad application prospects in repairing skin cell damage as an antioxidant skin care product.

[0004] The above object of the present invention is achieved by the following technical solution: application of SRT1720 in the preparation of skin antioxidant repair agent, wherein the chemical structural formula of SRT1720 is:

[0005]

[0006] Furthermore, the skin antioxidant repair agent ingredients include SRT1720 and auxiliary materials.

[0007] Furthermore, the auxiliary materials include one or more of purified water, oil, lipids, esters, waxes, surfactants, moisturizers, thickeners, chelating agents, pigments, fragrances, antioxidants, preservatives, and pH regulators.

[0008] Furthermore, the dosage form of the skin antioxidant repair agent includes cream, lotion, solution, spray, mask, essence water, gel or powder.

[0009] The skin antioxidant repair agent can improve skin condition, repair oxidative damage, and resist skin aging.

[0010] The beneficial effects of the present invention compared with the prior art are: the repair agent can effectively activate SIRT1 and promote the expression of the antioxidant protein Nrf-2, thereby regulating the antioxidant stress response of skin fibroblasts and resisting skin aging caused by oxidative damage. SRT1720 can restore the healthy state of the skin and improve the repair ability and vitality of the skin. The invention has a wide range of application prospects in repairing skin cell damage as an antioxidant skin care product. The repair agent is suitable for daily skin care, can improve skin condition, repair oxidative damage, and resist skin aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0012] Figure 1 is the ROS fluorescence staining images and quantitative analysis of ROS fluorescence intensity in different experimental groups (B);

[0013] Figure 2 Western blot was used to detect the protein expression levels of SIRT1 (A) and antioxidant protein Nrf-2 (B) in different experimental groups;

[0014] Figure 3 Figure 2 shows pictures of different experimental groups (A) and quantitative analysis of β-galactosidase-positive cells (B). DETAILED DESCRIPTION

[0015] The present invention is described in detail below by specific examples, but the protection scope of the present invention is not limited. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can be obtained from commercial channels.

[0016] Example 1

[0017] Human dermal fibroblasts were used for the experiment:

[0018] SRT1720 was dissolved in DMSO at the required concentration, filtered using a 0.22 μm sterile filter membrane, and the prepared stock solution was dispensed into small tubes and stored at -20°C (short-term storage).

[0019] 1. After plating, the cells were divided into 4 groups: control group, SRT1720 group, H2O2 group, and H2O2+SRT1720 group. (1) The control group was treated with DMSO solvent for 4 hours; (2)

[0020] The SRT1720 group was treated with 4 μmol / L SRT1720 for 4 h; (3) the H2O2 group was treated with DMSO solvent for 2 h, and then with 200 μmol / L H2O2 for 2 h; (4) the H2O2+SRT1720 group was pretreated with 4 μmol / L SRT1720 for 2 h, and then with 200 μmol / L H2O2 for 2 h. H2O2 was used to induce cellular oxidative stress response and establish a cell oxidative damage model.

[0021] 2. Reactive oxygen species (ROS) kit was used to detect the level of reactive oxygen species in each experimental group. (1) Human dermal fibroblasts were cultured at a rate of 1*10 5 The cells were seeded at a density of 1000 in 96-well plates and cultured to the logarithmic growth phase.

[0022] (2) Culture according to the experimental grouping and treatment scheme in Example 1. (3) Add the ROS probe (DCF-DA) to the cell culture medium. The concentration range is 10-20 μM (according to the kit instructions), and incubate in a 37°C incubator for 1 hour to allow the probe to enter the cells and react with ROS. (4) After the incubation, gently wash the cells with PBS buffer or culture medium to remove unpenetrated or unreacted dye and reduce background noise. (5) Use a fluorescence microscope to observe the stained cells, record the fluorescence image, and quantify the fluorescence intensity using an enzyme marker.

[0023] The ROS levels in cells were tested according to the above experimental groups ( Figure 1 ), compared with the control group, H2O2 treatment led to a significant increase in ROS levels in cells ( Figure 1 A, B), indicating that the H2O2-induced cell oxidative damage model was successfully established. The results showed that SRT1720 pretreatment could significantly inhibit the generation of ROS in both normal cells and H2O2-induced oxidative damage cells ( Figure 1 A, B).

[0024] 3. Western blot detection of protein expression (1) Collect cells from the control group, SRT1720 group, H2O2 group, and H2O2+SRT1720 group. (2) Add cell lysis buffer to extract cells, where RIPA:PMSF=10:1. (3) Protein quantification: Use BCA protein quantification method to determine the concentration of extracted protein. (4) SDS-PAGE electrophoresis: Use polyacrylamide gel electrophoresis (SDS-PAGE) to separate protein samples according to molecular weight. (5) Transfer: Assemble the gel, membrane, and transfer buffer in a transfer device, and use a wet transfer device to transfer the separated proteins from the gel to the PVDF membrane. (6) Blocking: Place the membrane in a shaker containing 5% skim milk powder and incubate for 2 hours. (7) Primary antibody incubation: The primary antibody of the target protein is fully bound to the target protein in the membrane and incubated overnight. (8) Washing: Wash the membrane 3 times with TBST buffer for 5 minutes each time to remove unbound primary antibody. (9) Secondary antibody incubation: Incubate the secondary antibody corresponding to the source of the primary antibody (such as goat anti-mouse IgG) for 2 hours to allow it to bind to the primary antibody. (10) Washing: Wash the membrane three times with TBST buffer for 5 minutes each time to remove unbound secondary antibody. (11) Signal detection: Use the substrate chemiluminescence ECL method to detect the protein labeled with the secondary antibody, and use an imaging system to image and save the target protein. (12) Result analysis: Use ImageJ to quantitatively analyze the target protein, and use GraphPad Prism 8.4 analysis software for statistical analysis.

[0025] Western blot was used to detect the protein expression of SIRT1 and Nrf-2 in the cells of the above experimental groups ( Figure 2 The results showed that the protein expression of SIRT1 was significantly decreased in the H2O2 group compared with the control group, but in the H2O2+SRT1720 group, the expression of SIRT1 was reversed ( Figure 2 A). Similarly, after adding SRT1720, the expression level of antioxidant protein Nrf-2 increased, suggesting that SRT1720 can enhance the antioxidant capacity of cells ( Figure 2 B).

[0026] 4. β-galactosidase staining to detect the aging of cells in each experimental group (1) Culture according to the experimental grouping and treatment scheme in Example 1. (2) Fix the cells with 4% paraformaldehyde and incubate at room temperature for 15 minutes. (3) Gently wash the cells three times with PBS buffer to remove the fixative. (4) According to the instructions of the kit, prepare the β-galactosidase staining reagent solution and add the staining solution to the cell culture dish to ensure that the cells are completely immersed. (5) Incubate the cells at 37°C (without CO2) for 2 hours, and senescent cells will show obvious blue staining. (6) After staining, gently wash the cells with PBS buffer to remove unreacted staining solution. (7) Observe the cells under an inverted microscope and take images for further analysis.

[0027] The cells in the above experimental groups were stained with β-galactosidase to observe the senescence state of each group of cells ( Figure 3 ). Compared with the control group, the number of blue-stained senescent cells increased in the H2O2 group, but decreased in the H2O2+SRT1720 group ( Figure 3 A, B). The above suggests that SRT1720 can improve cell senescence through anti-oxidation.

[0028] The above-described embodiments are only preferred embodiments of the present invention, but not all feasible embodiments of the present invention. For those skilled in the art, any obvious changes made thereto without departing from the principles and spirit of the present invention should be considered to be included in the scope of protection of the claims of the present invention.

[0029] References:

[0030] [1].Mark, R., et al., Oxidative stress in aging human skin. Biomolecules, 2015.5(2).

[0031] [2]. Erika, C. and R. Lucia, Skin Aging, Cellular Senescence and Natural Polyphenols. Int J Mol Sci, 2021.22(23).

[0032] [3].Monika,M.,Plant-Derived Antioxidants:Significance in Skin Health and the Aging Process.Int J Mol Sci, 2022.23(2).

[0033] [4].Qi-Jun,W.,et al.,The sirtuin family in health and disease.SignalTransduct Target Ther,2022.7(1).

[0034] [5].Angelika,B.-B.,et al.,Sirtuin 1and Skin:Implications in Intrinsicand Extrinsic Aging-A Systematic Review.Cells,2021.10(4).

[0035] [6]Chih-Chang,C.,et al.,SRT1720 as an SIRT1 activator for alleviatingparaquat-induced models ofParkinson's disease.Redox Biol,2022.58(0).

Claims

1. The application of SRT1720 in the preparation of skin antioxidant repair agent, characterized in that: The chemical structural formula of SRT1720 is:

2. The use of SRT1720 according to claim 1 in preparing a skin antioxidant repair agent, characterized in that: The skin antioxidant repair agent comprises SRT1720 and auxiliary materials.

3. The use of SRT1720 according to claim 2 in the preparation of a skin antioxidant repair agent, characterized in that: The auxiliary materials include one or more of purified water, oil, lipids, esters, waxes, surfactants, moisturizers, thickeners, chelating agents, pigments, fragrances, antioxidants, preservatives, and pH regulators.

4. The use of SRT1720 according to claim 1 in preparing a skin antioxidant repair agent, characterized in that: The dosage forms of the skin antioxidant repair agent include creams, lotions, solutions, sprays, facial masks, essence water, gels or powders.

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