A tripeptide with the function of promoting HaCaT cell migration and barrier repair and application thereof

Abstract

The application discloses a tripeptide with the effects of promoting HaCaT cell migration and barrier repair and application of the tripeptide, and the amino acid sequence of the tripeptide is glycine-methionine-hydroxyproline. The tripeptide has the effects of promoting HaCaT cell migration and barrier repair, and can be applied to the preparation of cosmetics with the effects of skin tissue repair and / or skin barrier repair.

Description

Technical Field

[0001] This invention relates to a tripeptide, particularly a tripeptide that promotes HaCaT cell migration and barrier repair, and its applications. Background Technology

[0002] As the largest organ in the human body, the skin plays a crucial role in protecting against harmful external environmental stimuli, preventing excessive water loss, and maintaining internal homeostasis. Ultraviolet (UV) radiation, especially UVB, is one of the main exogenous factors causing skin barrier damage. Studies have shown that UVB irradiation leads to the dissolution of tight junctions between cells, loosening the connections between normally tightly packed epidermal cells and disrupting the structural integrity of the skin barrier. High doses of UVB irradiation also cause downregulation of tight junction-related proteins, resulting in impaired skin barrier function.

[0003] Epidermal growth factor (EGF) is an important endocrine cell growth factor in the human body that effectively promotes epidermal cell growth and enhances cellular metabolism. It is widely used in the medical field, with clinical indications including topical treatment of burns, trauma, and surgical wound healing, and accelerating the growth of transplanted epidermis. However, EGF has certain limitations. Due to its large molecular weight, it is difficult to be absorbed under normal skin barrier conditions. If the skin barrier function is compromised, it may lead to other potential safety issues. Based on considerations of efficacy and safety, EGF is prohibited from use as a cosmetic ingredient. Against this backdrop, exploring substances with superior bioactivity, absorption, and penetration properties has become an urgent need in the field of dermatological research.

[0004] Bioactive peptides are present in very small amounts in the human body, but possess extremely high bioactivity, exhibiting a variety of important biological effects and physiological functions. Among numerous anti-aging ingredients, peptides have become a research hotspot in the cosmetics field due to their excellent compatibility with human skin, well-defined targets, and gentle yet highly effective properties. Peptides play a vital role in skin care, including anti-aging, repair, and moisturizing, thanks to their unique chemical structure and bioactivity.

[0005] Peptides are molecules formed by two or more amino acids linked by peptide bonds. They can be classified into dipeptides, tripeptides, dodecapeptides, etc., according to the number of amino acid residues. Among them, tripeptides have shown excellent potential in skin care scenarios such as anti-wrinkle, whitening, and wound healing due to their small molecular structure, easy penetration into the skin surface, and multiple biological activities such as anti-oxidation, anti-inflammation, and promotion of collagen synthesis. This has laid the theoretical and applied foundation for the development of multifunctional peptide ingredients.

[0006] However, there are currently no literature reports on glycine-methionine-hydroxyproline tripeptide. Summary of the Invention

[0007] The purpose of this invention is to provide a tripeptide that promotes HaCaT cell migration and barrier repair, and its applications. The tripeptide of this invention has the effect of promoting HaCaT cell migration and barrier repair.

[0008] The technical solution of the present invention is a tripeptide that promotes HaCaT cell migration and barrier repair, wherein the amino acid sequence of the tripeptide is: glycine-methionine-hydroxyproline.

[0009] The aforementioned tripeptide that promotes HaCaT cell migration and barrier repair has a molecular weight of 319.

[0010] The aforementioned tripeptide that promotes HaCaT cell migration and barrier repair is prepared by solid-phase synthesis.

[0011] The present invention also provides the application of the above-mentioned tripeptide in the preparation of cosmetics with skin tissue repair and / or skin barrier repair functions.

[0012] In the aforementioned applications, the effective concentration of the tripeptide is not less than 40 ppm.

[0013] In the aforementioned applications, the effective concentration of the tripeptide is 40~120 ppm.

[0014] A cosmetic composition that promotes HaCaT cell migration and barrier repair, comprising the aforementioned tripeptide.

[0015] In the aforementioned cosmetic composition, the concentration of the tripeptide is not less than 40 ppm.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention employs a solid-phase synthesis method to prepare the tripeptide Gly-Met-Hyp. The preparation method features high purity, high safety, and convenient operation. The resulting tripeptide has a small molecular weight and strong absorption and permeability, providing strong technical support for the development of efficient and safe barrier repair products.

[0018] This tripeptide has been experimentally proven to significantly promote cell migration, helping to accelerate the repair and regeneration of damaged skin tissue. Simultaneously, it promotes the expression of skin barrier-related genes, such as FLG and AQP3, effectively repairing and strengthening skin barrier function. Furthermore, the bioactivity of this tripeptide in promoting cell migration and the expression of skin barrier-related genes is superior to that of an equivalent amount of epidermal growth factor. Attached Figure Description

[0019] Figure 1 The present invention relates to the effects of the tripeptide and epidermal growth factor on keratinocyte scratches.

[0020] Figure 2 The present invention relates to the effects of the tripeptide and epidermal growth factor on the migration of keratinocytes.

[0021] Figure 3 This is a bar chart showing the effect of the tripeptide and epidermal growth factor of the present invention on the relative expression level of the skin barrier-related factor FLG. In the figure, t-test analysis was used. Compared with the BC group, the significance is indicated by *, where * indicates P-value < 0.05; compared with the NC group, the significance is indicated by #, where # indicates P-value < 0.05, ## indicates P-value < 0.01, and ### indicates P-value < 0.001.

[0022] Figure 4 This is a bar chart showing the effect of the tripeptide and epidermal growth factor of the present invention on the relative expression level of the skin barrier-related factor AQP3. In the figure, t-test analysis was used. Compared with the BC group, significance is indicated by *, and ** indicates P-value < 0.01; compared with the NC group, significance is indicated by #, ## indicates P-value < 0.01, ### indicates P-value < 0.001, and #### indicates P-value < 0.0001. Detailed Implementation

[0023] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0024] Example:

[0025] A tripeptide that promotes HaCaT cell migration and barrier repair, with the amino acid sequence: glycine-methionine-hydroxyproline, abbreviated as Gly-Met-Hyp.

[0026] The above-mentioned tripeptide was prepared as follows: using natural amino acids glycine, methionine, and hydroxyproline as starting materials, a fluorene methoxycarbonyl (Fmoc) N-terminal protection strategy was adopted, and the corresponding amino acids were sequentially linked according to the resin solid-phase synthesis method. During the process, the Fmoc- protecting groups were removed sequentially. Then, the peptide was cleaved to obtain the crude product, which was then purified by column chromatography to obtain the tripeptide: glycine-methionine-hydroxyproline (GMH).

[0027] The molecular weight of the tripeptide was determined using liquid chromatography-mass spectrometry (LC-MS) and was 319.12.

[0028] Experimental example:

[0029] 1. Skin tissue repair experiment:

[0030] 1.1 Experimental Grouping:

[0031] The study included a blank control group, a *Gynostemma pentaphyllum* group, and a sample group, with three replicates for each group. The blank control group (BC: serum-free culture medium only); the *Gynostemma pentaphyllum* group (EGF: 40, 80, and 120 ppm samples); and the sample group (GMH: 40, 80, and 120 ppm samples).

[0032] 1.2 Experimental Methods:

[0033] Human immortalized keratinocytes in good growth condition were used at a density of 1×10⁻⁶ cells per well. 6 Cells were seeded in 6-well plates and cultured at 37°C in a 5% CO2 incubator. After 24 h of culture, two perpendicular lines were drawn using a 10 μL pipette tip, aligned with a ruler. Cells were washed three times with PBS to remove the drawn cells. Then, 2 mL of different concentrations of sample were added to each well. Serum-free DMEM medium served as the blank control. The sample groups were treated with 40 ppm, 80 ppm, and 120 ppm GMH solution, respectively, while the *Gynostemma pentaphyllum* group was treated with 40 ppm, 80 ppm, and 120 ppm commercially available EGF standard solution, respectively. Cells were stimulated for 48 h. Observations and photographs were taken under an inverted microscope at 0 h, 24 h, and 48 h post-treatment. Data analysis was performed using ImageJ software to calculate the area of ​​the scratched region and cell migration rate.

[0034] The specific design scheme is shown in Table 1.

[0035] Table 1. Cell scratch assay design

[0036]

[0037] 1.3 Experimental Results:

[0038] The skin tissue repair experimental results of the tripeptide are as follows: Figure 1 , 2 As shown. Figure 2 In the table, E4, E8, and E12 represent the *Gynostemma pentaphyllum* groups with administration concentrations of 40 ppm, 80 ppm, and 120 ppm, respectively, while G4, G8, and G12 represent the sample groups with administration concentrations of 40 ppm, 80 ppm, and 120 ppm, respectively.

[0039] Figure 1 and Figure 2 The experimental results showed that, compared with the blank control group BC, HaCaT cells treated with 40, 80, and 120 ppm of the tripeptide or the same concentration of the positive control EGF grew well. They stretched and spread on the scratched surface, promoting cell migration. Furthermore, the tripeptide Gly-Met-Hyp exhibited superior biological activity and migration rate compared to EGF.

[0040] 2. Barrier damage repair experiment:

[0041] 2.1 Experimental Grouping:

[0042] The study included a blank control group, a negative control group, a ginseng-containing group, and a sample group, with three replicates for each group. The blank control group (BC: culture medium only, no UVB irradiation); the negative control group (NC: culture medium only, UVB irradiation); the ginseng-containing group (EGF: 40, 80, and 120 ppm samples, UVB irradiation); and the sample group (GMH: 40, 80, and 120 ppm samples, UVB irradiation).

[0043] 2.2 Experimental Methods:

[0044] Log-phase human immortalized keratinocytes were seeded into 6-well plates at a density of 3.5 × 10⁻⁶. 5 When the cell density reached 40%–60%, cells were divided into groups for drug administration. 2 mL of culture medium was added to each well of the blank control group and negative control group, while 2 mL of culture medium containing the corresponding concentration of the sample group and ginseng group was added to each well. After pre-incubation in a CO2 incubator for 3 hours, the wells were removed, the culture medium was discarded, and an appropriate amount of PBS was added to cover the cell surface. The negative control group, ginseng group, and sample group were exposed to UVB irradiation at a dose of 40 mJ. After irradiation, the cells were cultured in a CO2 incubator for another 24 hours. Total RNA was extracted from each group, and the expression levels of FLG and AQP3 genes in the cells were detected according to the real-time quantitative PCR instructions. The internal reference gene was β-actin, and its sequence is shown in Table 2.

[0045] The specific design scheme is shown in Table 3.

[0046] Table 2 Gene Primer Sequences

[0047]

[0048] Table 3. Experimental Design Scheme for Barrier Repair

[0049]

[0050] 2.3 Experimental Results:

[0051] The relative expression levels of barrier-related factor mRNAs are shown in Table 4. Figure 3 and Figure 4 As shown, Figure 3 and Figure 4 In the table, E4, E8, and E12 represent the *Gynostemma pentaphyllum* groups with administration concentrations of 40 ppm, 80 ppm, and 120 ppm, respectively, while G4, G8, and G12 represent the sample groups with administration concentrations of 40 ppm, 80 ppm, and 120 ppm, respectively.

[0052] Table 4. Relative expression levels of barrier-related factor mRNAs

[0053]

[0054] Based on the results of the barrier repair experiment, compared with the blank control group (BC), the relative expression levels of FLG and AQP3 mRNA in cells after UVB irradiation were significantly decreased, indicating that UVB has a damaging effect on the skin barrier. Compared with the negative control group (NC), the relative expression levels of FLG and AQP3 mRNA in different doses of the tripeptide Gly-Met-Hyp group and the EGF group were significantly increased, effectively repairing UVB-induced skin barrier damage. Furthermore, it was demonstrated that the tripeptide Gly-Met-Hyp, at all concentrations, was superior to EGF at the same concentration in enhancing UVB-induced intracellular FLG and AQP3 mRNA expression.

[0055] It should be understood that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Those skilled in the art can modify the technical solutions described in the above embodiments, or make equivalent substitutions for some of the technical features; and all such modifications and substitutions should fall within the protection scope of the appended claims of the present invention.

Claims

1. The application of a tripeptide with the function of promoting HaCaT cell migration and barrier repair in the preparation of cosmetics with skin tissue repair and / or skin barrier repair functions, characterized in that: The amino acid sequence of the tripeptide is: glycine-methionine-hydroxyproline.

2. The application according to claim 1, characterized in that: The tripeptide has a molecular weight of 319.

3. The application according to claim 1, characterized in that: The tripeptide was prepared by solid-phase synthesis.

4. The application according to claim 1, characterized in that: The effective concentration of the tripeptide is not less than 40 ppm.

5. The application according to claim 4, characterized in that: The effective concentration of the tripeptide is 40~120ppm.

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