Laminin polypeptide GF-13 and new application thereof

Abstract

The invention relates to the field of biological medicine and cosmetics, in particular to laminin polypeptide GF-13 and application thereof.

Description

Technical Field

[0001] The present application relates to the fields of biomedicine and cosmetics, and specifically to a laminin polypeptide GF-13 and a new use thereof. Background Art

[0002] The skin is the first semipermeable barrier between the human body and the environment, consisting of three layers: the dermis, epidermis, and dermal-epidermal junction (DEJ). The dermal-epidermal junction (DEJ) is a distinct boundary between the dermis and epidermis. It is a dense collagen network that forms an undulating dermal-epidermal junction structure and provides excellent structural support for the epidermis and dermis.

[0003] Numerous studies have shown that a healthy DEJ is crucial for skin homeostasis. Damage to the skin's basement membrane can affect skin homeostasis and health, and is closely linked to skin aging. Strengthening DEJ repair can improve epidermal-dermal communication and skin homeostasis, thereby reducing the effects of external stimuli on the skin and strengthening defenses against skin aging.

[0004] Laminin, a heterotrimer composed of α, β, and γ chains, is a multifunctional glycoprotein found in basement membranes. Integrins, dystroglycan, octosan, and several other cell surface molecules serve as cellular receptors for laminin. Globular domains located at the N- and C-termini of the laminin α chain are crucial for interaction with cellular receptors. Integrin α6β1 binds to most laminin isoforms. Integrin α3β1 interacts more specifically with laminins 5 and 10 / 11 than with other isoforms. Integrins α1β1, α2β1, and α7β1 have binding activity with laminins 1 and 2. The interaction of integrin α6β4 with laminin 5 forms hemidesmosomes in the skin. α-dystroglycan binds strongly to the laminin α1 and α2 chains and interacts modestly with the α5 chain.

[0005] Peptides targeting the epidermal-dermal junction (DEJ) have recently attracted significant attention in the fields of skin repair, anti-aging, and drug delivery. One research approach is to design peptides targeting laminin-5, based on its importance to the DEJ structure. This approach is driven by the fact that laminin-5 is crucial for epidermal cell attachment. Studies have shown that laminin-5 initiates the formation of hemidesmosomes, ensuring stable attachment of the epidermis to the dermis and accelerating the assembly of the basement membrane, which aids in the recovery of skin damage. An intact basement membrane is crucial for skin stability.

[0006] Based on the above reasons, the inventors designed a DEJ layer biomimetic polypeptide based on a similar principle. Summary of the Invention

[0007] The present invention relates to a bioactive peptide with anti-inflammatory and anti-aging effects. In a first aspect, the present invention relates to a bioactive peptide, whose amino acid sequence is GQVFHVAYVLIKF, as shown in SEQ ID NO: 1.

[0008] In some embodiments, modifications such as hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation can be performed on the amino acid side chain groups, amino terminus or carboxyl terminus of the bioactive peptide to obtain derivatives of the peptide without affecting its biological activity.

[0009] In a second aspect, the present invention relates to a pharmaceutical composition comprising the bioactive peptide and a pharmaceutically acceptable carrier.

[0010] In some embodiments, the pharmaceutically acceptable carrier is selected from isotonic saline, ethanol, phosphate-buffered saline, balanced salt solutions, and dimethyl sulfoxide (DMSO).

[0011] In other embodiments, the composition may be administered orally, by inhalation, rectally, vaginally, topically, nasally, ophthalmically, or parenterally.

[0012] In other embodiments, the compositions of the invention can be used in a variety of therapeutic applications, in particular in cosmetics and dermatology; they can take the form of cosmetic compositions for treating, protecting, caring for and removing make-up and / or cleansing the skin, lips and / or hair, as well as for applying make-up to the skin, lips, eyelashes and / or on the eyelashes.

[0013] In a third aspect, the present invention relates to the use of the above-mentioned bioactive peptides or pharmaceutical compositions in the preparation of medicines, cosmetics or skin care products with anti-aging, repairing and soothing effects.

[0014] In some embodiments, the present invention relates to the anti-aging effects of the bioactive peptide or pharmaceutical composition including: 1.

[0015] Protect the skin DEJ layer cells from collagen loss and increase collagen content, including type I collagen and type III collagen.

[0016] In some embodiments, the present invention relates to the repair efficacy of the bioactive peptide or the pharmaceutical composition comprising: promoting the repair ability of damaged skin cells.

[0017] In some embodiments, the soothing effect of the bioactive peptide or the pharmaceutical composition of the present invention includes: inhibiting inflammatory factors, including but not limited to: NO, IL-6.

[0018] In a fourth aspect, the present invention also relates to the use of the above-mentioned effective amount of bioactive peptides or related compositions in medicines, cosmetics or skin care products for accelerating wound healing and tissue regeneration;

[0019] In some embodiments, the aforementioned bioactive peptides or related compositions are generally used for skin and / or tissue regeneration.

[0020] In some embodiments, accelerating wound healing comprises increasing skin collagen expression.

[0021] In a fifth aspect, another aspect of the present invention is the use of the bioactive peptides or related compositions in the preparation of medicines or cosmetics or skin care products for treating and / or preventing skin aging phenomena and / or improving the appearance of the skin.

[0022] In a sixth aspect, the present invention relates to the dosage of the bioactive peptide. In some embodiments, the relevant dosage includes: 1-25 ppm.

[0023] In a seventh aspect, the present invention relates to the use of a bioactive peptide in preparing any of the following products:

[0024] ① Anti-aging products;

[0025] ② Products with repairing and soothing effects;

[0026] ③ Products that prevent or improve the loss of collagen in the DEJ layer of the skin;

[0027] ④Inflammatory factor inhibition products;

[0028] ⑤Products that increase collagen content;

[0029] ⑥ Products that improve the ability of skin cells to repair damage;

[0030] The amino acid sequence of the bioactive peptide is shown in SEQ ID NO: 1.

[0031] In some embodiments, the product is a pharmaceutical or a cosmetic or skin care product.

[0032] In some embodiments, the collagen includes: type I collagen and type III collagen.

[0033] In some embodiments, the inflammatory factor comprises NO, IL-4, IL-6, IL-13, IL-31, COX-2, TNF-α, IL-1β, IL-8, MCP-1, MMP-1, MMP-2, or TGF-β. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1: Experimental results of GF-13 peptide increasing the content of type I collagen in UVA-damaged HFF-1 cells;

[0035] Figure 2 : Results of keratinocyte scratch test;

[0036] Figure 3 : Experimental results of GF-13 peptide reducing LPS-induced inflammatory factors;

[0037] Figure 4 : Experiment on the effect of GF-13 on repairing DEJ layer cells (HSF cell model);

[0038] Figure 5 : Experiment on the effect of GF-13 on repairing DEJ layer cells (HaCaT cell model); DETAILED DESCRIPTION

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0040] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0041] Example 1: Results of UVA-induced Col I repair experiments in HFF-1 cells

[0042] Experimental materials: HFF-1 cells (UVA: 40mJ / cm 2 )

[0043] Experimental method: After UVA induction of cells, the test sample was added and the supernatant was collected after 24 hours to detect the Col I content of cells by ELISA.

[0044] Test group: GF-13 (10ppm and 25ppm);

[0045] GF-13:GQVFHVAYVLIKF,SEQ ID NO:1

[0046] The above polypeptides were prepared by conventional chemical synthesis.

[0047] Effect calculation formula:

[0048] Increase rate (%) = (T / C-1) * 100%

[0049] Where: T—average value of type I collagen content of the test substance (sample group)

[0050] C—Average value of type I collagen content in negative group (Model, induction only)

[0051] The blank group was the untreated cell group, and the negative control was the UVA-induced group. The positive control was 100 ng / mL TGF-β1; the cell treatment was to induce HFF-1 fibroblasts with UVA for 24 hours, and then use ELISA to detect the content of Col I (induction intensity: 40 mJ / cm 2 ).

[0052] Experimental results (see Figure 1 ):10ppm and 25ppm of GF-13 can significantly increase the type I collagen content in UVA-damaged HFF-1 cells.

[0053] In summary, GF-13 has the effect of enhancing UVA-induced photoaging of skin fibroblasts to a certain extent.

[0054] Example 2: Keratinocyte scratch test

[0055] Experimental Method: HaCaT Cell Scratch Repair Model

[0056] Test groups: 1ppm, 5ppm and 10ppm GF-13

[0057] Positive control: 100 ng / mL TGF-β1

[0058] The specific steps are as follows:

[0059] The blank group was an untreated cell group, and the positive control was 100 ng / mL TGF-β1. Cell treatment consisted of scratching keratinocytes with a pipette tip and then culturing them for 24 hours. An inverted microscope was used to take pictures and measure the scratch distance.

[0060] The experimental results show that (see Figure 2 ):1ppm, 5ppm and 10ppm of GF-13 can significantly improve the repair ability of HaCaT cells.

[0061] In summary, GF-13 can enhance the repair effect of skin keratinocytes to a certain extent.

[0062] Example 3: Cell Soothing Test

[0063] Experimental Methods: LPS-induced RAW264.7 inflammatory cell model

[0064] Test groups: 1ppm, 5ppm, 10ppm GF-13

[0065] Positive control: 10 ppm DEX (dexamethasone)

[0066] The specific steps are as follows:

[0067] Effect calculation formula:

[0068] Downward adjustment rate (%) = (1-T / C) * 100%

[0069] Where: T—average value of inflammatory factor content of the test substance (sample group)

[0070] C—Average value of inflammatory factor content in negative group (Model, induction only)

[0071] The blank group was untreated, and the negative control was the LPS-induced group. The positive control was 10 μg / mL DEX (dexamethasone). The treated cells were RAW264.7 macrophages induced with LPS for 24 hours, followed by ELISA analysis of NO and TNF-α levels (induction concentration: 200 ng / mL).

[0072] The experimental results show that (see Figure 3 ):1ppm, 5ppm and 10ppm of GF-13 can significantly inhibit the LPS-induced inflammatory factor content in RAW264.7 cells.

[0073] In summary, GF-13 has a soothing effect to a certain extent.

[0074] Example 4: DEJ layer repair test (HSF cell model)

[0075] Experimental method: 40mJ / cm2 UVA induced HSF cell model

[0076] Test groups: 1ppm, 5ppm, 10ppm GF-13

[0077] The specific steps are as follows:

[0078] Effect calculation formula:

[0079] Increase rate (%) = (T / C-1) * 100%

[0080] Where: T—average value of type I collagen content of the test substance (sample group)

[0081] C—Average value of type I collagen content in negative group (Model, induction only)

[0082] The blank group was the untreated cell group, and the negative control group was the UVA-induced group. Cell treatment was to induce HSF fibroblasts with UVA and then culture them for 24 hours. The content of collagen and matrix metalloproteinases was detected by ELISA (induction intensity: 40 mJ / cm 2 ).

[0083] The experimental results show that (see Figure 4 Under the conditions of this experiment, compared to the negative control group, 1ppm, 5ppm, and 10ppm GF-13 significantly promoted the upregulation of type III and type IV collagen in HSF cells after UVA induction and inhibited the release of matrix metalloproteinases (MMP-1) and MMP-2. Furthermore, 1ppm GF-13 significantly promoted the release of type I collagen.

[0084] In summary, GF-13 can inhibit UVA-induced cellular photoaging to a certain extent by releasing DEJ layer-related collagen and inhibiting the release of matrix metalloproteinases.

[0085] Example 5: DEJ layer repair test (HaCaT cell model)

[0086] Experimental method: 40mJ / cm2 UVB-induced HaCaT cell model

[0087] Test groups: 1ppm, 5ppm, 10ppm GF-13

[0088] The specific steps are as follows:

[0089] Effect calculation formula:

[0090] Increase rate (%) = (T / C-1) * 100%

[0091] Where: T—average value of type I collagen content of the test substance (sample group)

[0092] C—Average value of type I collagen content in negative group (Model, induction only)

[0093] The blank group was the untreated cell group, and the negative control group was the UVA-induced group. Cell treatment was to induce keratinocytes HaCaT with UVB and then culture them for 24 hours. The content of type IV collagen and type XVII collagen was detected by ELISA (induction intensity: 40 mJ / cm 2 ).

[0094] The experimental results show that ( Figure 5 ): Under the experimental conditions, compared with the negative control group, 1ppm, 5ppm and 10ppm GF-13 all significantly promoted UVB-induced upregulation of type IV collagen in HaCaT cells and had an increasing trend for type XVII collagen.

[0095] In summary, GF-13 can inhibit UVB-induced photoaging of keratinocytes by releasing DEJ layer-related collagen to a certain extent.

[0096] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A bioactive peptide, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The bioactive peptide according to claim 1 can be modified by hydroxylation, carboxylation, carbonylation, methylation, acetylation, phosphorylation, esterification or glycosylation on the amino acid side chain groups, amino terminus or carboxyl terminus of the bioactive peptide to obtain a derivative of the peptide without affecting its biological activity.

3. A pharmaceutical composition comprising the bioactive peptide according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable carrier is selected from isotonic saline, ethanol, phosphate-buffered saline, balanced salt solution and dimethyl sulfoxide (DMSO).

5. Use of a bioactive peptide in the preparation of any of the following products: ① Anti-aging products; ② Products with repairing and soothing effects; ③ Products that prevent or improve the loss of collagen in the DEJ layer of the skin; ④Inflammatory factor inhibition products; ⑤Products that increase collagen content; ⑥ Products that improve the ability of skin cells to repair damage; The amino acid sequence of the bioactive peptide is shown in SEQ ID NO:

1.

6. The use according to claim 5, wherein the product is a medicine, a cosmetic or a skin care product.

7. The use according to any one of claims 5 to 6, wherein the collagen comprises: Collagen type I, collagen type III and collagen XVII.

8. The use according to any one of claims 5 to 7, wherein the inflammatory factors include NO, IL-4, IL-6, IL-13, IL-31, COX-2, TNF-α, IL-1β, IL-8, MCP-1, MMP-1, MMP-2 or TGF-β.

9. The use according to any one of claims 5 to 8, wherein the concentration of the bioactive peptide is 1-25 ppm.

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