A polypeptide and its application in the preparation of products that promote skin wound repair

Abstract

This invention provides a polypeptide and its application in the preparation of products that promote skin wound repair. The polypeptide of this invention, with the amino acid sequence shown in SEQ ID NO:1, not only possesses excellent anti-inflammatory and antioxidant activities, making it suitable for the preparation of anti-inflammatory and antioxidant products, but also effectively promotes the migration of skin fibroblasts and accelerates wound healing, making it suitable for the preparation of products that promote skin wound repair.

Description

Technical Field

[0001] This invention belongs to the fields of biopharmaceutical manufacturing, gene-engineered drugs, and vaccine manufacturing technology. More specifically, it relates to a polypeptide and its application in the preparation of products that promote skin wound repair. Background Technology

[0002] Currently, the common clinical methods to promote skin wound repair include: (1) Traditional dressings: such as gauze and bandages, but they are prone to sticking to the wound and may cause secondary damage when changing dressings; (2) Growth factor drugs: such as recombinant human epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but excessive use may increase the risk of tumor development; (3) Cell therapy: such as stem cell transplantation and skin transplantation, but cell therapy has problems such as limited sources and immune rejection, and is complicated to operate and expensive, making it difficult to be widely used in clinical practice; (4) Physical therapy: such as laser therapy and ultrasound therapy, but requires professional equipment and technical support, and improper operation may aggravate wound damage.

[0003] Peptides have advantages such as simple structure, few side effects, easy synthesis and modification, and low cost, and are expected to become a new generation of bioactive materials to promote skin wound repair. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention aims to provide a polypeptide with an amino acid sequence as shown in SEQ ID NO:1, which not only possesses excellent anti-inflammatory and antioxidant activities, making it suitable for preparing anti-inflammatory and antioxidant products, but also effectively promotes the migration of skin fibroblasts and accelerates wound healing, making it suitable for preparing products that promote skin wound repair.

[0005] The primary objective of this invention is to provide a polypeptide that promotes the repair of skin wounds.

[0006] A second objective of this invention is to provide related biomaterials for the aforementioned polypeptides.

[0007] A third objective of this invention is to provide the application of the aforementioned polypeptide or related biomaterials in the preparation of products that promote the repair of skin wounds.

[0008] A fourth objective of this invention is to provide the application of the above-mentioned polypeptides or related biomaterials in the preparation of anti-inflammatory products.

[0009] The fifth objective of this invention is to provide the application of the above-mentioned polypeptides or related biomaterials in the preparation of antioxidant products.

[0010] The sixth objective of this invention is to provide a product that promotes the repair of skin wounds.

[0011] The seventh objective of this invention is to provide an anti-inflammatory product.

[0012] The eighth objective of this invention is to provide an antioxidant product.

[0013] The above-mentioned objective of this invention is achieved through the following technical solution:

[0014] This invention provides a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 that promotes skin wound repair, and related biomaterials thereof, wherein the related biomaterials include nucleic acid molecules capable of expressing the polypeptide, or recombinant plasmids, recombinant vectors, recombinant cells, recombinant bacteria or expression cassettes containing the nucleic acid molecules.

[0015] The polypeptides of this invention not only possess excellent anti-inflammatory and antioxidant activities, making them suitable for preparing anti-inflammatory and antioxidant products, but also effectively promote the migration of skin fibroblasts and accelerate wound healing, making them suitable for preparing products that promote skin wound repair. Therefore, the application of the aforementioned polypeptides or related biomaterials in the preparation of products that promote skin wound repair, anti-inflammatory products, and antioxidant products, as well as any product containing the aforementioned polypeptides or related biomaterials that promotes skin wound repair, anti-inflammatory products, or antioxidant products, should all be within the scope of protection of this invention.

[0016] Preferably, the product is a drug.

[0017] More preferably, the drug is one of the following: oral liquid, suppository, liniment, powder inhaler, aerosol, tincture, sterile powder for injection, or injection.

[0018] More preferably, the drug further contains excipients.

[0019] More preferably, the excipients are vegetable oils (such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and / or cocoa butter, used as solvents or carriers to help the drug components dissolve or disperse better), wetting agents (such as sodium lauryl sulfate, used to help the drug components disperse and dissolve better), binders (such as gelatin, used to help the drug components bind together better), cellulose and its derivatives (such as sodium carboxymethyl cellulose, ethyl cellulose, and / or methyl cellulose, used to improve the compressibility and formability of the drug, and also to control the release rate of the drug), emulsifiers (such as Tween and / or polyoxyethylene castor oil, used to reduce interfacial tension, promote emulsification of the dispersed phase, and maintain the stability of the emulsion), flavoring agents (used to improve the taste of the drug and improve patient compliance), antioxidants (used to prevent the drug from deteriorating due to oxidation during storage or use and extend the shelf life of the drug), sugars (such as lactose, glucose, and / or sucrose, used to help the drug components disperse and dissolve better, while providing necessary energy support), and polyols (such as propylene glycol, glycerin, sorbitol, and mannose). Alcohols and / or polyethylene glycol, etc., used to improve drug stability, prevent drug dehydration or deterioration during storage, and also to improve drug taste and solubility; stabilizers (increase the physical stability of drugs, preventing deterioration or degradation during storage or transportation); solid lubricants (such as stearic acid and / or magnesium stearate, used to reduce friction and ensure successful drug manufacturing); starches (such as corn starch and / or potato starch, used to help tablets disintegrate rapidly after administration and release drug components, thereby improving drug bioavailability); isotonic salt solutions (used to adjust the osmotic pressure of drugs to meet human physiological requirements, thereby reducing irritation and adverse reactions to the human body); lubricants (such as talc, used to reduce drug friction); colorants (used to improve the appearance and color of drugs, making them easier to identify and distinguish); tablet compressors (facilitating the compression of loose granular substances into solid tablets, making it convenient for patients to take and carry, and also helping to improve drug stability and bioavailability); alginic acid (maintaining the physical stability of drugs and preventing drug particles from settling or agglomerating). The type of excipient can be selected based on the need to improve drug stability, activity, and / or bioavailability.

[0020] The present invention has the following beneficial effects:

[0021] The polypeptide of this invention, whose amino acid sequence is shown in SEQ ID NO:1, not only has excellent anti-inflammatory and antioxidant activities, making it suitable for the preparation of anti-inflammatory and antioxidant products, but also can effectively promote the migration of skin fibroblasts and accelerate wound healing, making it suitable for the preparation of products that promote skin wound repair. Attached Figure Description

[0022] Figure 1This is a graph showing the results of electrospray ionization mass spectrometry (ESI-MS) identification of the peptide.

[0023] Figure 2 This is a graph showing the purity analysis results of the peptide using reversed-phase high-performance liquid chromatography (RP-HPLC).

[0024] Figure 3 The figure shows the results of detecting the effect of peptides on LPS-induced IL-6 secretion in RAW264.7 macrophages.

[0025] Figure 4 The figure shows the results of detecting the effect of peptides on ROS levels in LPS-induced RAW264.7 macrophages.

[0026] Figure 5 This is a graph showing the results of the detection of the effect of peptides on the scratch healing ability of human skin fibroblasts (HSF). Detailed Implementation

[0027] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0028] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0029] Example 1: Synthesis and Identification of Polypeptides

[0030] I. Synthesis of Polypeptides

[0031] The polypeptide with the amino acid sequence shown in SEQ ID NO:1 was synthesized by Sangon Biotech (Shanghai) Co., Ltd. using solid-phase synthesis (Fmoc method). SEQ ID NO:1: NSPDAPLPR.

[0032] II. Analysis and Identification of Polypeptides

[0033] (1) Mass spectrometry identification

[0034] The molecular weight of the peptide was determined and its structure confirmed using electrospray ionization mass spectrometry (ESI-MS). The mass spectrometry conditions were as follows:

[0035] Ion source: ESI source, positive ion mode;

[0036] Mobile phase A: contains 1% ( v / v Aqueous solution of formic acid;

[0037] Mobile phase B: contains 1% ( v / v Formic acid in acetonitrile solution;

[0038] Flow rate: 0.2 mL / min;

[0039] Scan range: m / z 0~2000 Da;

[0040] Sample loading volume: 1 μL.

[0041] The results are as follows Figure 1 As shown, the molecular ion peak of the polypeptide has an m / z of 484.10 (double charge), which is consistent with its theoretical molecular weight (967.07 Da).

[0042] (2) Purity analysis

[0043] The purity of the peptides was analyzed using reversed-phase high-performance liquid chromatography (RP-HPLC). The chromatographic conditions were as follows:

[0044] Column: NanoChrom Chromcore™ 120 C18 (4.6 mm × 250 mm, 5 μm);

[0045] Mobile phase A: contains 0.1% ( v / v Aqueous solution of trifluoroacetic acid;

[0046] Mobile phase B: contains 0.1% ( v / v A trifluoroacetic acid solution in acetonitrile;

[0047] Flow rate: 1.0 mL / min;

[0048] Detection wavelength: 214 nm;

[0049] Sample loading volume: 30 μL.

[0050] The results are as follows Figure 2 As shown, the main peak area accounts for ≥95%, and there are very few impurity peaks, indicating that the purity of the peptide meets the requirements.

[0051] The above results confirm that the polypeptide synthesized in this embodiment is the polypeptide with the amino acid sequence shown in SEQ ID NO:1, and the purity meets the requirements for subsequent experiments.

[0052] Example 2: Determination of the anti-inflammatory activity of the peptide

[0053] I. Solution Preparation

[0054] Peptide solution: The peptide obtained in Example 1 was dissolved in DMEM basal medium to achieve final concentrations of 50, 100, and 200 μg / mL. After sterilization by filtration through a 0.22 μm filter membrane, it was ready for use.

[0055] II. Methods for Assaying Anti-inflammatory Activity

[0056] RAW264.7 mouse macrophages in the logarithmic growth phase were harvested and processed at a concentration of 1×10⁻⁶ cells / mL.5 Cells were seeded at a density of 100 μL / mL into 96-well plates and incubated at 37 °C with 5% CO2 until the cells adhered. After discarding the supernatant, the cells were grouped as follows:

[0057] (1) Blank control group (NC group): 100 μL of DMEM basal medium was added and incubated at 37 °C for 1 h;

[0058] (2) Model group (LPS group): 100 μL of DMEM basal medium was added and incubated at 37 °C for 1 h. Then LPS was added to make the final concentration 1 μg / mL.

[0059] (3) Sample group: Add 100 μL of peptide solution, incubate at 37 °C for 1 h, and then add LPS to make the final concentration 1 μg / mL.

[0060] After incubation at 37 °C for 24 h, the supernatant of each group was collected, and the IL-6 content was determined according to the instructions of the mouse IL-6 ELISA kit.

[0061] III. Results of Anti-inflammatory Activity Assay

[0062] The results are as follows Figure 3 As shown in the figure, compared with the blank control group, LPS stimulation significantly increased the IL-6 secretion level of RAW264.7 cells, while the sample groups with the peptide obtained in Example 1 (50-200 μg / mL) significantly alleviated the increase in IL-6 secretion level induced by LPS stimulation in a dose-dependent manner. This indicates that the peptide of the present invention has excellent anti-inflammatory activity and is suitable for the preparation of anti-inflammatory products.

[0063] Example 3: Determination of the antioxidant activity of peptides

[0064] I. Solution Preparation

[0065] Peptide solution: The peptide obtained in Example 1 was dissolved in DMEM basal medium to achieve final concentrations of 50, 100, and 200 μg / mL. After sterilization by filtration through a 0.22 μm filter membrane, it was ready for use.

[0066] II. Methods for Determining Antioxidant Activity

[0067] RAW264.7 mouse macrophages in the logarithmic growth phase were harvested and processed at a concentration of 1×10⁻⁶ cells / mL. 5 Cells were seeded at a density of 100 μL / mL into 96-well plates and incubated at 37 °C with 5% CO2 until the cells adhered. After discarding the supernatant, the cells were grouped as follows:

[0068] (1) Blank control group (NC group): 100 μL of DMEM basal medium was added and incubated at 37 °C for 1 h;

[0069] (2) Model group (LPS group): 100 μL of DMEM basal medium was added and incubated at 37 °C for 1 h. Then LPS was added to make the final concentration 1 μg / mL.

[0070] (3) Sample group: Add 100 μL of peptide solution, incubate at 37 °C for 1 h, and then add LPS to make the final concentration 1 μg / mL.

[0071] After incubating at 37 °C for 24 h, the culture medium was discarded, and 100 μL of DMEM basal medium containing 10 μM DCFH-DA was added. After incubating at 37 °C for 30 min, the supernatant was discarded, and the samples were washed twice with PBS (pH=7.4). The fluorescence intensity was measured using an ELISA reader (excitation wavelength 488 nm, emission wavelength 525 nm).

[0072] III. Results of Antioxidant Activity Measurement

[0073] The results are as follows Figure 4 As shown in the figure, compared with the blank control group, LPS stimulation significantly increased the ROS level of RAW264.7 cells, while the sample groups with the peptide obtained in Example 1 (50-200 μg / mL) significantly alleviated the increase in ROS level caused by LPS stimulation in a dose-dependent manner. This indicates that the peptide of the present invention has excellent antioxidant activity and is suitable for the preparation of antioxidant products.

[0074] Example 4: Assay of the skin wound repair activity of peptides

[0075] I. Solution Preparation

[0076] Peptide solution: The peptide obtained in Example 1 was dissolved in DMEM basal medium to a final concentration of 100 μg / mL, and then filtered through a 0.22 μm filter membrane for sterilization before use.

[0077] II. Methods for Assessing the Activity of Promoting Skin Wound Repair

[0078] Human skin fibroblasts (HSF) in the logarithmic growth phase were harvested at a concentration of 5 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured at 37 °C until 80% confluence. Then, a sterile 200 μL pipette tip was used to make a vertical incision at the bottom of the well (simulating a physical wound). After washing three times with PBS (pH=7.4) to remove exfoliated cells, the cells were grouped as follows:

[0079] (1) Blank control group (control group): 2 mL of DMEM basal culture medium was added;

[0080] (2) Sample group: Add 2 mL of peptide solution.

[0081] Each group was incubated at 37 °C for 24 h, and photographs were taken at the same location using a fluorescence microscope at 0, 12, and 24 h.

[0082] III. Results of the assay for promoting skin wound repair activity

[0083] The results are as follows Figure 5 As shown in the figure, compared with the blank control group, the HSF cells in the sample group treated with 100 μg / mL peptide showed significantly faster scratch healing speed and significantly increased cell migration rate within 12 h and 24 h. This indicates that the peptide of the present invention can effectively promote the migration of skin fibroblasts and accelerate wound healing, and is suitable for the preparation of products that promote skin wound repair.

[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A polypeptide that promotes skin wound repair, characterized in that, Its amino acid sequence is shown in SEQ ID NO:

1.

2. A biomaterial related to the polypeptide of claim 1, characterized in that, Includes nucleic acid molecules capable of expressing the polypeptide of claim 1, or recombinant plasmids, recombinant vectors, recombinant cells, recombinant bacteria or expression cassettes containing said nucleic acid molecules.

3. The use of the polypeptide of claim 1 or the related biomaterial of claim 2 in the preparation of a medicament for promoting skin wound repair.

4. The use of the polypeptide of claim 1 or the related biomaterial of claim 2 in the preparation of anti-inflammatory products.

5. The use of the polypeptide of claim 1 or the related biomaterial of claim 2 in the preparation of antioxidant products.

6. The application according to any one of claims 4 to 5, characterized in that, The product is a medicine.

7. A drug for promoting skin wound repair, characterized in that, Contains the polypeptide of claim 1 or the related biomaterial of claim 2.

8. An anti-inflammatory product, characterized in that, Contains the polypeptide of claim 1 or the related biomaterial of claim 2.

9. An antioxidant product, characterized in that, Contains the polypeptide of claim 1 or the related biomaterial of claim 2.

10. The product according to any one of claims 8 to 9, characterized in that, The product is a medicine.

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