A sirtuin protein promoter and uses thereof

Abstract

The application discloses a SIRT protein promoter and application thereof, and belongs to the technical field of cosmetics, and particularly relates to a SIRT protein promoter; the active ingredient of the SIRT protein promoter is oligopeptide-6; the oligopeptide-6 can not only activate an AMPK signal path, up-regulate NAMPT gene expression, and improve intracellular NAD + level, thereby activating SIRT protein in dependence on NAD + ; and can effectively promote collagen production, and realize the effects of anti-wrinkle and skin tightening.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, specifically to a SIRT protein promoter and its application. Background Technology

[0002] The Sirtuins (SIRTs) protein family is a class of proteins that depend on nicotinamide adenine dinucleotide (NAD). + SIRT proteins, including deacetylases, play a central role in key biological processes such as cellular stress response, energy metabolism, genome stability, and aging regulation. Among them, SIRT proteins located in the cell nucleus (such as SIRT1 and SIRT6) are known as "longevity proteins" due to their significant functions in maintaining cellular homeostasis, promoting DNA repair, and delaying cellular aging, and have become important targets in the development of anti-aging drugs and cosmetics. Studies have shown that increasing the activity or expression level of SIRT proteins may delay skin aging and improve skin function.

[0003] AMP-activated protein kinase (AMPK) is a key regulator of cellular energy metabolism, and its activation via phosphorylation regulates multiple downstream metabolic pathways. Studies suggest that AMPK activation may be related to intracellular NAD+. + Elevated levels are associated with NAD, while... + It is an essential cofactor for the deacetylation activity of the SIRT protein family. Nicotinamide phosphoribosyltransferase (NAMPT) is an NAD+ protein. + The rate-limiting enzyme in the synthetic salvage pathway has an expression level that directly affects intracellular NAD. + The concentration of AMPK-NAMPT-NAD. + This signaling axis may serve as a potential upstream regulatory pathway, playing a crucial role in activating the activity of SIRT proteins.

[0004] Oligopeptide-6 is an active peptide already used in cosmetics, but its specific mechanism of action, particularly its interaction with key intracellular anti-aging signaling pathways, remains poorly understood. Currently, while some SIRT protein agonists have been reported, their mechanisms of action are mostly broadly attributed to direct activation or involving only a single step. For bioactive peptides like oligopeptide-6, it remains to be seen whether and how they can influence NAD through the regulation of upstream signaling pathways (such as AMPK phosphorylation). + The level of SIRT protein expression, and its subsequent activation, remains a research gap, hindering a deeper understanding of the mechanism of action of oligopeptide-6 and its value in precision anti-aging applications. Therefore, there is an urgent need to develop a formulation that can effectively promote SIRT protein expression and to further explore the mechanism of action of its active ingredients. Summary of the Invention

[0005] The purpose of this invention is to provide a SIRT protein promoter and its application, which can not only activate the AMPK signaling pathway, upregulate NAMPT gene expression, and increase intracellular NAD+, but also... + Level, thus dependent on NAD + It activates SIRT protein and can also effectively promote collagen production, achieving anti-wrinkle and skin-firming effects.

[0006] The technical solution adopted by the present invention to achieve the above objectives is as follows: A SIRT protein promoter, characterized in that it comprises: oligopeptide-6; the amino acid sequence of said oligopeptide-6 is shown in SEQ ID No. 1.

[0007] This invention discloses a novel use of oligopeptide-6 as a SIRT protein promoter. Oligopeptide-6 can promote intracellular NAD+ expression by activating the AMPK signaling pathway and upregulating NAMPT gene expression. + Biosynthesis, and then through NAD + SIRT protein is activated through a dependency mechanism. Simultaneously, oligopeptide-6 can also promote collagen synthesis, thereby exerting anti-wrinkle and firming effects, showing promising application prospects in the cosmetics field.

[0008] Preferably, the SIRT protein includes at least one of SIRT1 protein, SIRT2 protein, SIRT3 protein and SIRT6 protein.

[0009] Preferably, the concentration of oligopeptide-6 used is 1-100 μg / mL.

[0010] The present invention also discloses the application of the above-mentioned SIRT protein promoter in the preparation of cosmetics or drugs that activate AMPK phosphorylation.

[0011] This invention also discloses the above-mentioned SIRT protein promoter in the preparation of NAD+ promoting agents. + Application in metabolic cosmetics or drugs.

[0012] This invention also discloses the application of the above-mentioned SIRT protein promoter in the preparation of cosmetics or pharmaceuticals that promote collagen production.

[0013] The present invention also discloses the application of the above-mentioned SIRT protein promoter in the preparation of anti-skin aging drugs or cosmetics.

[0014] The present invention also discloses the application of the above-mentioned SIRT protein promoter in the preparation of drugs or cosmetics that promote skin repair.

[0015] The present invention also discloses the application of the above-mentioned SIRT protein promoter in the preparation of drugs or cosmetics that promote skin firmness.

[0016] This invention also discloses the application of oligopeptide-6 in the preparation of SIRT protein promoters. The amino acid sequence of oligopeptide-6 is shown in SEQ ID No.1, and the concentration of oligopeptide-6 used is 1-100 μg / mL.

[0017] This invention, by using oligopeptide-6 as a SIRT protein promoter, has the following beneficial effects: oligopeptide-6 can activate the AMPK signaling pathway, upregulate NAMPT gene expression, and promote intracellular NAD+ expression. + Biosynthesis, and then through NAD + SIRT proteins are activated through a dependence mechanism. Experimental results show that oligopeptide-6 can significantly promote the production of various SIRT proteins, increasing the content of SIRT1 protein by 121.27-537.14%, SIRT2 protein by 47.11-62.74%, SIRT3 protein by 125.81-131.29%, and SIRT6 protein by 66.63-91.66%. Simultaneously, oligopeptide-6 can effectively promote collagen production, increasing the content of type I collagen by 67.84-139.11% and the expression level of type III collagen by 37.94-99.35%. This invention discloses the application of oligopeptide-6 as a SIRT protein promoter. Oligopeptide-6 exhibits significant effects in promoting SIRT protein expression and collagen production, showing promising application prospects. Attached Figure Description

[0018] Figure 1 This represents the results of relative cell activity.

[0019] Figure 2 Results for cell morphology.

[0020] Figure 3 The results represent cell migration and healing rates. BC represents the blank control group, and PC represents the positive control group.

[0021] Figure 4 The results represent cell adhesion and viability. BC represents the blank control group, and PC represents the positive control group.

[0022] Figure 5 For the Western blot results activating AMPK phosphorylation, BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group.

[0023] Figure 6 For the WB quantitative results of AMPK phosphorylation activation, BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group.

[0024] Figure 7The values ​​represent the mRNA expression level of the NAMPT gene. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group.

[0025] Figure 8 The ratio of NAD+ to NADH is given. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group.

[0026] Figure 9 The values ​​represent SIRT1 protein content. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group.

[0027] Figure 10 The values ​​represent SIRT2 protein content. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group.

[0028] Figure 11 The values ​​represent SIRT3 protein content. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group.

[0029] Figure 12 The values ​​represent SIRT6 protein content. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group.

[0030] Figure 13 The values ​​represent type I collagen content. BC represents the blank control group, and PC represents the positive control group.

[0031] Figure 14 The values ​​represent type III collagen content. BC represents the blank control group, and PC represents the positive control group. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] Example 1: A SIRT protein promoter includes oligopeptide-6, the sequence of which is H-Arg-Asp-Phe-Thr-Lys-Ala-Thr-Asn-Ile-Arg-Leu-Arg-Phe-Leu-Arg-OH, and the amino acid sequence is RDFTKATNIRLRFLR, as shown in SEQ ID No. 1.

[0035] Experimental Example 1: Oligopeptide-6 was dissolved in dimethyl sulfoxide (DMSO) and then diluted with DMEM culture medium to obtain eight concentration gradients: 0.007813 mg / mL, 0.015625 mg / mL, 0.03125 mg / mL, 0.0625 mg / mL, 0.125 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL. These samples were used to detect its cytotoxic effect on fibroblasts. The specific steps were as follows: a zero-adjustment group, a solvent control group, a positive control group, and eight sample groups were set up, with three replicates for each group. In the zero-adjustment group, no fibroblasts were seeded, and 200 μL of DMEM culture medium was added. In the solvent control group, fibroblasts were seeded at a cell density of 8000 cells / well, and 200 μL of DMEM culture medium was added. In the positive control group, fibroblasts were seeded at a density of 8000 cells / well, and 200 μL of DMEM medium containing 10% dimethyl sulfoxide was added. In the eight sample groups, fibroblasts were seeded at a density of 8000 cells / well, and 200 μL of each of the eight concentration gradients of the sample was added sequentially. Each group was cultured at 37°C and 5% CO2 for 24 h. The supernatant was discarded, and 200 μL of 0.5 mg / mL thiazolyl blue (MTT) reagent was added. The cells were incubated at 37°C for 4 h. After incubation, the supernatant was discarded, and 150 μL of dimethyl sulfoxide was added. The optical density (OD) value was read at 490 nm. The relative cell viability was calculated based on the OD values ​​of each group. The formula for calculating relative cell viability is: Relative cell viability (%) = [(OD value of sample group - OD value of zeroing group) / (OD value of solvent control group - OD value of zeroing group)] × 100%. Cell morphology was observed under a microscope, and photographs were taken.

[0036] Table 1 Relative cell activity

[0037] Figure 1 This represents the results of relative cell activity. Figure 2 The results are for cell morphology. See Table 1 for the results. Figure 1 and Figure 2 As shown, oligopeptide-6 did not exhibit significant cytotoxicity within a mass concentration range not exceeding 0.25%.

[0038] Experimental Example 2: Constructing an oxidative stress injury model: Fibroblasts were subjected to 30 J / cm² stress. 2 The cells were irradiated with ultraviolet light for 24 hours, and then cultured at 37°C and 5% carbon dioxide for another 24 hours after the irradiation to obtain fibroblasts with oxidative stress injury.

[0039] Oligopeptide-6 was dissolved in dimethyl sulfoxide and then diluted with DMEM medium to final concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively, to obtain three concentration gradients for detecting its effect on cell migration. The specific steps are as follows: a blank control group, a positive control group, and three sample groups were set up, with three replicates for each group. The blank control group was prepared at 4 × 10⁻⁶ μg / mL. 4 Normal fibroblasts were seeded at a cell density of 4 × 10⁶ cells / well. The positive control group and the three sample groups were all seeded at this density. 4 Cells were seeded at a cell density per well to detect oxidative stress-damaged fibroblasts. Each group was cultured at 37°C and 5% CO2 for 24 h. Cells were streaked vertically into 24-well plates using a 200 μL pipette tip, washed three times with phosphate-buffered saline (PFS), and the streaked cells were removed. The cells were observed and photographed using an inverted microscope. The initial streak area was calculated using Image Pro Plus software and denoted as S0. Subsequently, each group underwent drug treatment: 200 μL of PFS was added to the blank control group; 200 μL of DMEM medium containing 10% fetal bovine serum was added to the positive control group; and 200 μL of oligopeptide-6 samples were added to the three sample groups at final concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively. After drug treatment, each group was cultured at 37°C and 5% CO2 for another 24 h. Cell migration was observed and photographed using an inverted microscope after culture, and the post-treatment streak area was calculated using Image Pro Plus software and denoted as S1. The healing rate is calculated using the following formula: Healing rate (%) = [(S0-S1) / S0] × 100%.

[0040] Figure 3 The results represent cell migration and healing rates. BC represents the blank control group, and PC represents the positive control group. Results are as follows: Figure 3As shown, the cell healing rate of the positive control group was significantly higher than that of the blank control group, indicating that the experimental system was stable and reliable. Compared with the blank control group, oligopeptide-6 at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL increased the cell healing rate by 21.29%, 40.46%, and 62.38%, respectively. This indicates that oligopeptide-6 has the ability to promote cell migration, which helps repair skin damage and shows a good repair effect. Compared with the positive control group, oligopeptide-6 at concentrations of 10 μg / mL and 100 μg / mL significantly improved the cell healing rate, indicating that oligopeptide-6 exhibits superior performance in promoting cell migration compared to traditional cell migration promoters.

[0041] Experimental Example 3: Cell adhesion plays a crucial role in many life processes in multicellular organisms, including morphogenesis, tissue maintenance, intercellular communication, and cell growth and differentiation. Therefore, it is necessary to evaluate the cell adhesion capacity of oligopeptide-6 by detecting cell adhesion patterns.

[0042] Constructing an oxidative stress injury model: Fibroblasts were subjected to 30 J / cm² stress. 2 The cells were irradiated with ultraviolet light for 24 hours, and then cultured at 37°C and 5% carbon dioxide for another 24 hours after the irradiation to obtain fibroblasts with oxidative stress injury.

[0043] Oligopeptide-6 was dissolved in pure water and then diluted with phosphate buffer to final concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL, respectively, to obtain three concentration gradients for detecting its effect on cell adhesion. The specific steps are as follows: A blank control group, a positive control group, and three sample groups were set up, with three replicates for each group. 20 μL of phosphate buffer was added to the blank control group; 20 μL of DMEM medium containing 10% fetal bovine serum was added to the positive control group; and 20 μL of oligopeptide-6 samples with final concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL were added to the three sample groups sequentially. Each group was incubated at room temperature for 1 h, and then cell-containing culture medium was added to each group. The blank control group was added with 180 μL of culture medium containing 5 × 10⁵ cells / mL. 4 DMEM medium containing 5 × 10⁶ cells / mL of normal fibroblasts was added to the positive control group and the three sample groups. 4Cells / mL of DMEM medium containing oxidative stress-damaged fibroblasts were incubated for 1 hour at 37°C with 5% carbon dioxide. Cells were then gently washed three times with phosphate buffer. Cell adhesion viability was then measured using a lactate dehydrogenase cytotoxicity assay kit (purchased from Shanghai Beyotime Biotechnology Co., Ltd.). The specific procedures were performed according to the kit's instructions. The cell adhesion viability of the control group was used as a baseline, and the improvement rate of cell adhesion viability in the positive control group and the three sample groups was statistically analyzed and calculated.

[0044] Figure 4 The results represent cell adhesion and viability. BC represents the blank control group, and PC represents the positive control group. The results are as follows: Figure 4 As shown, compared with the blank control group, the cell adhesion viability of the positive control group was significantly increased, indicating that the experimental system was stable and reliable. Compared with the blank control group, oligopeptide-6 at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL increased cell adhesion viability by 27.90%, 36.50%, and 50.10%, respectively. This indicates that oligopeptide-6 has the ability to promote cell adhesion. Compared with the positive control group, oligopeptide-6 at concentrations of 10 μg / mL and 100 μg / mL significantly improved cell adhesion viability, indicating that oligopeptide-6 has better cell adhesion promoting performance than traditional cell adhesion promoting substances.

[0045] Experiment Example 4: Constructing an oxidative stress injury model: Fibroblasts were subjected to 30 J / cm² stress. 2 The cells were irradiated with ultraviolet light for 24 hours, and then cultured at 37°C and 5% carbon dioxide for another 24 hours after the irradiation to obtain fibroblasts with oxidative stress injury.

[0046] Oligopeptide-6 was dissolved in dimethyl sulfoxide and then diluted with DMEM medium to a final concentration of 100 ppm to obtain a sample for detecting its effect on AMPK pathway activation. The specific steps are as follows: a blank control group, a negative control group, a positive control group, and a sample group were set up, with 3 replicates in each group.

[0047] In the blank control group, 1×10 5 Normal fibroblasts were seeded at a cell density of cells / well and cultured in DMEM medium until the cell plating rate reached 40%. The culture medium was then discarded, and 2 mL of fresh DMEM medium was added. The cells were incubated at 37°C and 5% carbon dioxide for 24 hours.

[0048] In the negative control group, 1×10 5Cells were seeded at a cell density of oxidative stress-damaged fibroblasts and cultured in DMEM medium until the cell plate formation rate reached 40%. The culture medium was then discarded, and 2 mL of fresh DMEM medium was added. The cells were incubated at 37°C and 5% carbon dioxide for 24 h.

[0049] In the positive control group, 1×10 5 Cells were seeded at a cell density of oxidative stress-damaged fibroblasts and cultured in DMEM medium until the cell plating rate reached 40%. The culture medium was then discarded, and 2 mL of DMEM medium containing 300 nmol / L quercetin was added. The cells were then incubated at 37°C and 5% carbon dioxide for 24 h.

[0050] In the sample group, at 1×10 5 Cells were seeded at a cell density of oxidative stress-damaged fibroblasts and cultured in DMEM medium until the cell platelet formation rate reached 40%. The culture medium was then discarded, and 2 mL of DMEM medium containing 100 ppm oligopeptide-6 was added. The cells were incubated at 37°C and 5% carbon dioxide for 24 h.

[0051] After processing, each group was centrifuged, and proteins were extracted using a protein extraction kit purchased from Beyotime Biotechnology Co., Ltd. RNA was also extracted using an RNA extraction kit purchased from Beyotime Biotechnology Co., Ltd. Western blotting was used to detect the protein levels of phosphorylated AMPK (p-AMPK) and total AMPK, and the ratio of p-AMPK to total AMPK protein levels was calculated to assess the degree of AMPK signaling activation.

[0052] NAMPT is NAD + The rate-limiting enzyme in the synthetic pathway, whose expression is upregulated, is linked to AMPK activation and NAD. +The intermediate steps of horizontal upflow. The mRNA expression level of nicotinamide phosphoribosyltransferase (NAMPT) in each group was detected by qRT-PCR, with β-actin as an internal reference gene. The upstream primer for NAMPT was 5'-AGGGCTTTGTCATTCCCAGAG-3', its amino acid sequence is shown in SEQ ID No. 2; the downstream primer for NAMPT was 5'-CTGTGATTGGATACCAGGACTGA-3', its amino acid sequence is shown in SEQ ID No. 3. The upstream primer for β-actin was 5'-TGGCACCCAGCACAATGAA-3', its amino acid sequence is shown in SEQ ID No. 4; the downstream primer for β-actin was 5'-CTAAGTCATAGTCCGCCTAGAAGCA-3', its amino acid sequence is shown in SEQ ID No. 5.

[0053] NAD + The NAD / NADH ratio is a key indicator of cellular energy status and redox balance, and a direct regulator of SIRT protein activity. Utilizing NAD... + The NADH assay kit was used to detect intracellular oxidized nicotinamide adenine dinucleotide (NAD) in each group. + The ratio of NADH to the reduced form.

[0054] Figure 5 For the Western blot results activating AMPK phosphorylation, BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group. Figure 6 To determine the Western blot (WB) results for AMPK phosphorylation activation, BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group. Results are as follows: Figure 5 and Figure 6 As shown, compared with the blank control group and the negative control group, the average p-AMPK / AMPK relative OD value of the positive control group was significantly increased, indicating that the experimental system was stable and reliable. Compared with the negative control group, the p-AMPK / AMPK value of oligopeptide-6 treated with oxidative stress-damaged fibroblasts at a concentration of 100 ppm was significantly increased by 147.46%, indicating that oligopeptide-6 can directly activate AMPK.

[0055] Figure 7 The mRNA expression level of the NAMPT gene is shown in the figure. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group. Results are as follows: Figure 7As shown, compared with the blank control group and the negative control group, the amplification fold of the positive control group was significantly increased, indicating that the experimental system was stable and reliable. Compared with the negative control group, oligopeptide-6 at a concentration of 100 ppm treated oxidative stress-damaged fibroblasts, which upregulated NAMPT gene expression by 113.33%, indicating that oligopeptide-6 can promote the upregulation of NAMPT gene expression.

[0056] Figure 8 For NAD + The ratio of BC to NADH, where BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group. Results are as follows: Figure 8 As shown, compared with the blank control group and the negative control group, the amplification fold of the positive control group was significantly increased, indicating that the experimental system is stable and reliable. Compared with the negative control group, oligopeptide-6 at a concentration of 100 ppm regulates NAD+. + The NADH ratio increased by 33.17%, indicating that oligopeptide-6 can enhance NAD+ uptake. + The / NADH ratio provides a cofactor for SIRT.

[0057] Therefore, oligopeptide-6 can promote NAD by directly activating AMPK and regulating the expression of its downstream target gene NAMPT. + The biosynthesis of NAD+ is enhanced. Increased NAD+ levels increase intracellular NAD+ biosynthesis. + The ratio of NADH to SIRT protein provides an essential cofactor for the SIRT protein family.

[0058] Experimental Example 5: Constructing an oxidative stress injury model: Fibroblasts were subjected to 30 J / cm² stress. 2 The cells were irradiated with ultraviolet light for 24 hours, and then cultured at 37°C and 5% carbon dioxide for another 24 hours after the irradiation to obtain fibroblasts with oxidative stress injury.

[0059] Oligopeptide-6 was dissolved in dimethyl sulfoxide and then diluted with DMEM medium to a final concentration of 1 μg / mL, 10 μg / mL and 100 μg / mL to obtain three concentration gradients of oligopeptide-6 solutions, which were used to detect its effect on SIRT protein expression. The specific steps are as follows: a blank control group, a negative control group, a positive control group and three sample groups were set up, and three replicates were set up for each group.

[0060] In the blank control group, cells were cultured in DMEM medium until the cell plate-laying rate reached 40%. The culture medium was then discarded, and 2 mL of fresh DMEM medium was added. The cells were incubated at 37°C and 5% carbon dioxide for 24 h without hydrogen peroxide treatment.

[0061] In the negative control group, 1×10 5Cells were seeded at a cell density of oxidative stress-damaged fibroblasts and cultured in DMEM medium until the cell plate formation rate reached 40%. The culture medium was then discarded, and 2 mL of fresh DMEM medium was added. The cells were incubated at 37°C and 5% carbon dioxide for 24 h. Then, 200 μL of 500 μmol / L hydrogen peroxide solution was added to stimulate the cells for 2 h.

[0062] In the positive control group, 1×10 5 Cells were seeded at a cell density of oxidative stress-damaged fibroblasts and cultured in DMEM medium until the cell plate formation rate reached 40%. The culture medium was then discarded, and 2 mL of DMEM medium containing 100 μg / mL vitamin C (VC) and 7 μg / mL vitamin E (VE) was added. The cells were incubated at 37°C and 5% carbon dioxide for 24 h, followed by stimulation with 200 μL of 500 μmol / L hydrogen peroxide solution for 2 h.

[0063] In the three sample groups, with 1×10 5 Cells were seeded at a cell density per well to induce oxidative stress-damaged fibroblasts. After culturing in DMEM medium until the cell plate-laying rate reached 40%, the culture medium was discarded, and 2 mL of oligopeptide-6 solution at three different concentration gradients was added sequentially. The cells were incubated at 37°C and 5% carbon dioxide for 24 h, followed by stimulation with 200 μL of 500 μmol / L hydrogen peroxide solution for 2 h.

[0064] After each group was treated, the levels of SIRT1, SIRT2, SIRT3, and SIRT6 proteins in each group were detected using Sirtuin 1 ELISA kits, Sirtuin 2 ELISA kits, Sirtuin 3 ELISA kits, and Sirtuin 6 ELISA kits, respectively. All Sirtuin 1 ELISA kits, Sirtuin 2 ELISA kits, Sirtuin 3 ELISA kits, and Sirtuin 6 ELISA kits were purchased from NOVUS.

[0065] Figure 9The values ​​represent SIRT1 protein levels. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group. Compared with the negative control group, the SIRT1 protein level in the positive control group was significantly increased, indicating the effectiveness of the experimental system. Compared with the negative control group, treatment of oxidative stress-damaged fibroblasts with oligopeptide-6 at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL increased SIRT1 protein levels by 121.27%, 340.66%, and 537.14%, respectively. This indicates that oligopeptide-6 can promote SIRT1 protein production. Compared with the positive control group, the SIRT1 levels at all three concentrations of oligopeptide-6 were significantly increased, indicating that oligopeptide-6 is more effective in promoting SIRT1 production than traditional SIRT protein promoters.

[0066] Figure 10 The values ​​represent SIRT2 protein content. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group. Compared with the negative control group, the SIRT2 protein content in the positive control group was significantly increased, indicating the effectiveness of the experimental system. Compared with the negative control group, treatment of oxidative stress-damaged fibroblasts with oligopeptide-6 at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL increased SIRT2 protein content by 47.11%, 51.87%, and 62.74%, respectively. This indicates that oligopeptide-6 can promote SIRT2 protein production. Compared with the positive control group, the SIRT2 content of oligopeptide-6 at all three concentrations was significantly increased, indicating that oligopeptide-6 is more effective in promoting SIRT2 production than traditional SIRT protein promoters.

[0067] Figure 11 The values ​​represent SIRT3 protein content. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group. Compared with the negative control group, the SIRT3 protein content in the positive control group was significantly increased, indicating the effectiveness of the experimental system. Compared with the negative control group, treatment of oxidative stress-damaged fibroblasts with oligopeptide-6 at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL increased SIRT3 protein content by 127.48%, 131.29%, and 125.81%, respectively. This indicates that oligopeptide-6 can promote SIRT3 protein production. Compared with the positive control group, the SIRT3 content of oligopeptide-6 at all three concentrations was significantly increased, indicating that oligopeptide-6 is more effective in promoting SIRT3 production than traditional SIRT protein promoters.

[0068] Figure 12The values ​​represent SIRT6 protein levels. BC represents the blank control group, NC represents the negative control group, and PC represents the positive control group. Compared with the negative control group, the SIRT6 protein level in the positive control group was significantly increased, indicating the effectiveness of the experimental system. Compared with the negative control group, treatment of oxidative stress-damaged fibroblasts with oligopeptide-6 at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL increased SIRT6 protein levels by 66.63%, 91.66%, and 119.39%, respectively, showing a dose-dependent increasing trend. This indicates that oligopeptide-6 can promote SIRT6 protein production. Compared with the positive control group, the SIRT6 levels at all three concentrations of oligopeptide-6 were significantly increased, indicating that oligopeptide-6 has a superior regulatory effect compared to traditional SIRT protein promoters.

[0069] Therefore, oligopeptide-6 can promote SIRT protein expression, and its promoting effect is dose-dependent. Compared with traditional SIRT protein promoters, oligopeptide-6 exhibits a superior promoting effect.

[0070] Experimental Example 6: Constructing an oxidative stress injury model: Fibroblasts were subjected to 30 J / cm² stress. 2 The cells were irradiated with ultraviolet light for 24 hours, and then cultured at 37°C and 5% carbon dioxide for another 24 hours after the irradiation to obtain fibroblasts with oxidative stress injury.

[0071] Oligopeptide-6 was dissolved in dimethyl sulfoxide and then diluted with DMEM medium to a final concentration of 1 μg / mL, 10 μg / mL and 100 μg / mL to obtain three concentration gradients of oligopeptide-6 solutions, which were used to detect its effect on SIRT protein expression. The specific steps are as follows: a blank control group, a negative control group, a positive control group and three sample groups were set up, and three replicates were set up for each group.

[0072] In the blank control group, cells were cultured in DMEM medium until the cell plate-laying rate reached 40%. The culture medium was then discarded, and 2 mL of fresh DMEM medium was added. The cells were incubated at 37°C and 5% carbon dioxide for 24 h.

[0073] In the positive control group, 1×10 5 Cells were seeded at a cell density of oxidative stress-damaged fibroblasts and cultured in DMEM medium until the cell plating rate reached 40%. The culture medium was then discarded, and 2 mL of DMEM medium containing 100 μg / mL palmitoyl tripeptide-1 was added. The cells were then incubated at 37°C and 5% carbon dioxide for 24 h.

[0074] In the three sample groups, with 1×10 5Cells were seeded at a cell density of oxidative stress-damaged fibroblasts and cultured in DMEM medium until the cell plating rate reached 40%. The culture medium was then discarded, and 2 mL of oligopeptide-6 solution at three different concentration gradients was added sequentially. The cells were then incubated at 37°C and 5% carbon dioxide for 24 h.

[0075] After each group was processed, the supernatant was collected, and the content of type I collagen and type III collagen in each group was measured using a type I collagen ELISA kit and a type III collagen ELISA kit, respectively. Both the type I and type III collagen ELISA kits were purchased from Abcam.

[0076] Figure 13 The values ​​represent type I collagen content. BC represents the blank control group, and PC represents the positive control group. Results are as follows: Figure 13 As shown, compared with the blank control group, the positive control group showed a significant increase in type I collagen content, indicating the effectiveness of the experimental system. Compared with the blank control group, treatment of oxidative stress-damaged fibroblasts with oligopeptide-6 at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL increased type I collagen content by 67.84%, 105.66%, and 139.11%, respectively, showing a dose-dependent increasing trend. This indicates that oligopeptide-6 can promote type I collagen synthesis. Compared with the positive control group, the type I collagen content of oligopeptide-6 was significantly increased at all three concentrations, indicating that oligopeptide-6 has better performance in promoting type I collagen synthesis and has anti-wrinkle and firming effects.

[0077] Figure 14 The values ​​represent type III collagen content. BC represents the blank control group, and PC represents the positive control group. Results are as follows: Figure 14 As shown, compared with the blank control group, the positive control group showed a significant increase in type III collagen content, indicating the effectiveness of the experimental system. Compared with the blank control group, treatment of oxidative stress-damaged fibroblasts with oligopeptide-6 at concentrations of 1 μg / mL, 10 μg / mL, and 100 μg / mL increased type III collagen content by 37.94%, 50.68%, and 99.35%, respectively, showing a dose-dependent increasing trend. This indicates that oligopeptide-6 can promote type III collagen synthesis. Compared with the positive control group, type III collagen levels were significantly increased at all three concentrations of oligopeptide-6, indicating that oligopeptide-6 has better performance in promoting type III collagen synthesis and possesses anti-wrinkle and firming effects.

[0078] Therefore, it can be seen that oligopeptide-6 has a better ability to promote collagen synthesis and has good anti-wrinkle and firming effects.

[0079] The conventional operations in the operation steps of this invention are well known to those skilled in the art and will not be described in detail here.

[0080] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any changes and modifications made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A SIRT protein promoter, characterized in that, include: Oligopeptide-6; the amino acid sequence of said oligopeptide-6 is shown in SEQ ID No.

1.

2. The SIRT protein promoter according to claim 1, characterized in that, The SIRT protein includes at least one of SIRT1, SIRT2, SIRT3, and SIRT6 proteins.

3. The SIRT protein promoter according to claim 1, characterized in that, The concentration of the oligopeptide-6 used is 1-100 μg / mL.

4. The use of the SIRT protein promoter according to any one of claims 1-3 in the preparation of cosmetics or pharmaceuticals that activate AMPK phosphorylation.

5. The SIRT protein promoter according to any one of claims 1-3 in the preparation of NAD+ promoters + Application in metabolic cosmetics or drugs.

6. The use of the SIRT protein promoter according to any one of claims 1-3 in the preparation of cosmetics or pharmaceuticals that promote collagen production.

7. The use of the SIRT protein promoter according to any one of claims 1-3 in the preparation of anti-skin aging drugs or cosmetics.

8. The use of the SIRT protein promoter according to any one of claims 1-3 in the preparation of a medicament or cosmetic for promoting skin repair.

9. The use of the SIRT protein promoter according to any one of claims 1-3 in the preparation of a medicament or cosmetic for promoting skin firmness.

10. The application of oligopeptide-6 in the preparation of SIRT protein promoters, characterized in that, The amino acid sequence of the oligopeptide-6 is shown in SEQ ID No. 1, and the concentration of the oligopeptide-6 used is 1-100 μg / mL.

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