An oligopeptide for promoting wound healing and its application

Abstract

The present invention provides an oligopeptide for promoting wound healing and its application, belonging to the field of biomedical technology. The oligopeptide for promoting wound healing has an amino acid sequence as shown in SEQ ID NO.1. The oligopeptide can not only promote the proliferation and migration of NIH3T3 cells, but also accelerate the wound healing of mice. It has advantages in promoting tissue regeneration, collagen deposition, angiogenesis and reducing wound inflammation. In summary, the oligopeptide of the present invention can significantly promote wound healing.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to oligopeptides for promoting wound healing and applications thereof. Background Art

[0002] The skin is the main barrier that protects the internal tissues of the body from damage by harmful external substances. Wounds are damage to normal skin (tissue) caused by external factors such as surgery, external force, heat, electric current, chemicals, low temperature, and internal factors such as local blood supply disorders. Delayed or non-healing wound healing will not only seriously affect the patient's physical and mental health and quality of life, but also bring serious economic burdens to the patient's family and society. Once the skin is damaged, the body will immediately adjust to repair the damage. The human body's wound healing process mainly consists of four stages: hemostasis, inflammation, proliferation, and reconstruction. In recent years, studies have found that inflammatory factors, growth factors, immune cells, related signaling pathways, and microcirculation, oxygen supply, blood supply, and collagen formation of local wound tissues are all related to wound healing.

[0003] Pheretima is an animal-based Chinese medicinal material that contains a large amount of proteins, peptides and amino acids. Existing reports mostly focus on the research on the wound healing activity of worm extracts, but lack research on the monomer active ingredients. Therefore, it is of great significance to find the monomer components in worm extracts that have the activity of promoting wound healing. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide an oligopeptide and to provide the use of the compound in the preparation of a drug for promoting wound healing.

[0005] The present invention provides an oligopeptide for promoting wound healing, and its amino acid sequence is shown as SEQ ID NO.1.

[0006] The oligopeptide provided by the present invention is a heptapeptide named DL-1, and its amino acid sequence is Ile-Leu-Leu-Leu-Ala-His-Ile (ILLLAHI); its structural formula is shown in formula (I):

[0007]

[0008] The present invention provides the use of the oligopeptide in preparing drugs for treating trauma, surgery, burns and scalds, and in preparing medical cosmetic products.

[0009] The present invention also provides a method for preparing a medicine for treating trauma, surgery, burns and scalds, which contains the oligopeptide for promoting wound healing.

[0010] The present invention also provides a medical cosmetic product containing the oligopeptide for promoting wound healing.

[0011] A pharmaceutical composition, characterized by containing the oligopeptide and pharmaceutically acceptable excipients.

[0012] The oligopeptide of the present invention can significantly promote wound healing. Experiments have shown that the oligopeptide provided by the present invention can promote the proliferation and migration of mouse fibroblasts NIH3T3, and has an EC 50 =14.42 μM. The oligopeptide provided by the present invention can also promote the repair of full-thickness cortical injuries in mice, significantly reduce cellular inflammatory infiltration, increase the hydroxyproline content in wound skin tissue, thereby increasing the content of fibrin, and can promote the generation of new blood vessels. In short, the oligopeptide provided by the present invention has advantages in promoting tissue regeneration, collagen deposition, angiogenesis and reducing wound inflammation. Therefore, the oligopeptide has broad application prospects in beauty products, skin care products and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the HPLC chromatogram of oligopeptide DL-1.

[0014] Figure 2 This is the mass spectrum of oligopeptide DL-1.

[0015] Figure 3 This is a graph showing the effect of oligopeptide DL-1 on the proliferation activity of NIH3T3 cells.

[0016] Figure 4 The results of the effect of oligopeptide DL-1 on the migration ability of NIH3T3 cells are shown in Figure 1. (A) is a photograph of the effect of different concentrations of DL-1 on the migration of NIH3T3 cells; (B) is the migration rate of NIH3T3 cells after being cultured in different concentrations of DL-1 for different periods of time.

[0017] Figure 5 The results of the effect of oligopeptide DL-1 on the wound healing rate of mice. (A) is a macroscopic evaluation of mouse skin wounds after oligopeptide DL-1 treatment; (B) is a statistical graph of the healing rate of mouse skin wounds after oligopeptide DL-1 treatment.

[0018] Figure 6 HE staining of mouse skin wound tissue was used to observe pathological changes after 3, 7, and 11 days of treatment with oligopeptide DL-1.

[0019] Figure 7 Masson staining of mouse skin wound tissues after oligopeptide DL-1 treatment for 3, 7, and 11 days to observe fibrin formation. (A) is a Masson staining image of skin wounds; (B) is a statistical graph of collagen volume fractions.

[0020] Figure 8The changes in hydroxyproline content in mouse skin wound tissue after treatment with oligopeptide DL-1 for 3, 7, and 11 days.

[0021] Fig. 9 This is a diagram of the diagnostic results of CD31 and CD68 immunohistochemical staining in mouse skin wound tissue sections.

[0022] (A) is the immunohistochemical observation of CD 31; (B) is the immunohistochemical observation of CD 68. DETAILED DESCRIPTION

[0023] Example 1 Liquid phase and mass spectrum of oligopeptide DL-1

[0024] The amino acid sequence of oligopeptide DL-1 is as follows: ILLLAHI (SEQ ID NO. 1), and the molecular weight is 792.02 Da. Oligopeptide DL-1 was purchased from GenScript Biotech Co., Ltd.

[0025] Example 2 Effect of oligopeptide DL-1 on proliferation of mouse fibroblasts NIH3T3

[0026] The oligopeptide DL-1 was dissolved in DMEM medium at a final concentration of 10 μM and 50 μM, and the effect of oligopeptide DL-1 on the proliferation activity of mouse fibroblast NIH3T3 was detected by MTT method. The specific method is as follows: Mouse fibroblast NIH3T3 (Jiangsu KeyGen Biotechnology Co., Ltd.) was prepared into 5×10 4 Cell suspension of 100 μL of cell suspension was added to each test well of the 96-well plate, and the cells were cultured in a CO2 incubator for 4 hours to allow the cells to adhere to the wall. A blank group was set up in which the cell suspension was replaced with DMEM medium. After the cells adhered to the wall, the culture medium was discarded, and 10 μM and 50 μM oligopeptide DL-1 solutions (the oligopeptide DL-1 solution was obtained by dissolving the oligopeptide DL-1 in DMEM medium without serum FBS) were added to the test wells, 100 μL per well, and 5 replicate wells were set for each concentration of oligopeptide DL-1 solution. Different concentrations of oligopeptide solutions were also added to the blank group; a control group was set up, and the same volume of DMEM medium was added to replace the oligopeptide DL-1 solution, and the rest was the same as the test group. Then, the 96-well plate was cultured in a CO2 incubator for 48 hours, 10 μL of 5 mg / mL MTT (thiazolyl blue) solution was added to each well, and after 4 hours of culture in a CO2 incubator, the liquid in each well was discarded, and 100 μL of DMSO (dimethyl sulfoxide) was added, and the cells were shaken and cultured in a 37°C thermostat for 10 minutes, and the absorbance at 570 nm was measured using an ELISA reader. The cell proliferation activity was calculated according to cell proliferation activity = (OD test group - OD blank group) / (OD control group - OD blank group) × 100%.

[0027] result( Figure 3 ): 10 μM oligopeptide DL-1 solution had a significant effect on the proliferation of NIH3T3 cells (p<0.05), with a cell proliferation activity of 116.5±3.4%, and with the increase of the concentration of oligopeptide DL-1 solution, the cell proliferation activity gradually increased; when the concentration of oligopeptide DL-1 solution was 50 μM, the cell proliferation activity reached 137.9±5.6%. Example 3 Effect of oligopeptide DL-1 on the migration of mouse fibroblasts NIH3T3

[0028] Use a marker pen to draw even horizontal lines across the holes on the back of the 6-well plate with a ruler. Add about 5×10 5 cells, and the fusion rate reached 100% after overnight culture. Use the gun tip perpendicular to the cell plane to scratch the cell layer. After the scratching is completed, wash the cells twice with sterile PBS to wash away the cells that are not attached to the wall, that is, the cells that are scratched when streaking, so that the gap left after streaking is clearly visible, and then add 2mL of serum-free DMEM medium containing oligopeptide DL-1. The concentrations of oligopeptide DL-1 are 0μM (Control group), 10μM, 25μM and 50μM, respectively, and 3 replicates are set for each concentration. The cells were cultured in a 37℃ 5% CO2 incubator. After culturing for 0h, 24h, and 48h, the cell culture plates were taken out, observed under a microscope and photographed. Image J software was used to calculate the cell scratch area, and then the cell migration rate was calculated.

[0029] result( Figure 4 ): As an in vitro wound healing model, the cell scratch test can reflect the migration and repair ability of cells. Figure A shows that compared with the blank group, 50μM concentration of DL-1 can significantly reduce the wound area after 24 hours of treatment, and the cells in the wound area have obviously healed after 48 hours. Figure B shows that after 48 hours of DL-1 treatment, the migration ability of NIH3T3 cells can be significantly increased at concentrations of 25μM and 50μM.

[0030] Example 4 Oligopeptide DL-1 promotes wound healing in mice

[0031] After 27 ICR mice were anesthetized by intraperitoneal injection of 4% (w / v) chloral hydrate solution (0.1 mL / 10 g), the hair on the back of the mouse was shaved with a razor to expose the back skin, and the depilatory cream was applied to the exposed skin. After 10 minutes, the depilatory cream was wiped off with gauze soaked in warm water, and the skin on the back of the mouse was completely cleaned. The next day, the back skin of the mouse was pinched, and a sterilized corneal trephine with a diameter of 6 mm was pressed on the skin to create a circular wound with a diameter of 6 mm. The fascia on the surface of the skin was cut with sterilized ophthalmic scissors, and the skin wound was covered with sterilized gauze and fixed with medical tape. The wound was dried and retracted for 2 hours and then photographed (using a 9 mm diameter circular hole plate to assist in photography), and the area of ​​the mouse skin wound was calculated using Image J software, which was the initial wound area.

[0032] After the full-thickness skin wound model of mice was successfully established, the mice were randomly divided into 3 groups, with 9 mice in each group. They were blank group (Control), positive control group (FGF) and oligopeptide DL-1 group (DL-1). The Control group was smeared with 20 μL solvent solution (10% glycerol + 1% mannitol solution), the positive control group was smeared with 20 μL positive drug FGF (epidermal growth factor, concentration is 50 μM / mL), and the DL-1 group was smeared with 20 μL oligopeptide DL-1 solution with a concentration of 50 μM / mL.

[0033] Determination of wound healing rate: Observe the changes of wounds every day, fix the shooting position and light, take pictures of the mouse wounds on the 3rd, 7th and 11th days respectively, and calculate the wound area using Image J software, which is the unhealed wound area. The calculation formula is as follows:

[0034]

[0035] result( Figure 5 ): Figure A shows that the skin wounds of mice in each group gradually healed as the days went by. Compared with the control group, the wound area was significantly smaller on days 3, 7, and 11 after treatment with oligopeptide DL-1 and FGF. Figure B shows that after days 3, 7, and 11, the healing rate of wounds treated with oligopeptide DL-1 and FGF was significantly accelerated compared with the control group (P<0.05).

[0036] Example 5 Analysis of pathological sections of mouse skin wound tissue stained with hematoxylin and eosin (HE)

[0037] Hematoxylin and eosin (HE) is used to evaluate granulation tissue growth and skin maturity. The HE staining procedure is as follows:

[0038] (1) Dewaxing of sections: dewaxing with xylene twice, 20 min each time; dewaxing with anhydrous ethanol twice, 5 min each time; dewaxing with 75% ethanol for 5 min; and rinsing with tap water.

[0039] (2) Staining: stain with hematoxylin for 5 minutes, rinse with tap water; differentiate with differentiation solution, rinse with tap water; blue with bluing solution, rinse with running water; dehydrate with 85% and 95% gradient ethanol for 5 minutes, and stain with eosin for 5 minutes. (3) Dehydration and sealing: dehydrate with anhydrous ethanol 3 times, 5 minutes each time; dehydrate with xylene 2 times, 5 minutes each time, and seal with neutral gum.

[0040] (4) Observe the slices under a microscope, collect images, and analyze the pathological changes of the skin wound.

[0041] result( Figure 6 ): On the 3rd day, all groups showed a large number of inflammatory cell infiltrations, and the inflammatory cell infiltration in the DL-1 group was significantly weaker than that in the Control group. On the 7th day, new blood vessels were visible in the DL-1 group and new epidermis had formed. On the 11th day, the new epidermis in the DL-1 group was obviously advanced to the edge, and collagen fiber production was also relatively abundant.

[0042] Example 6 Masson staining of mouse skin wound tissue to observe fibrin formation Masson staining is used to evaluate collagen deposition. The steps of Masson staining are as follows:

[0043] (1) Dewaxing of sections: dewaxing with xylene twice, 20 min each time; dewaxing with anhydrous ethanol twice, 5 min each time; dewaxing with 75% ethanol for 5 min; and rinsing with tap water.

[0044] (2) Staining: stain with potassium dichromate overnight and rinse with tap water; stain with iron hematoxylin for 3 minutes and rinse with tap water; differentiate with differentiation solution and rinse with tap water; bluing solution and rinse with running water; stain with ponceau acid fuchsin for 10 minutes and rinse with tap water; stain with phosphomolybdic acid for 3 minutes and directly stain with aniline blue for 5 minutes.

[0045] (3) Differentiation: Differentiation with 1% glacial acetic acid and dehydration with anhydrous ethanol.

[0046] (4) Transparent sealing: Place the sections in anhydrous ethanol for 5 min, in xylene for 5 min, and seal with neutral gum.

[0047] (5) Observe the sections under a microscope, collect images, and analyze the level of collagen deposition in skin wounds.

[0048] result( Figure 7 ): Compared with the Control group, the collagen fibers increased significantly on the 7th and 11th days after DL-1 treatment (Figure B), and the collagen fibers were more orderly and dense (Figure A).

[0049] Example 7 Changes in Hydroxyproline Content in Mouse Skin Wound Tissue

[0050] The tissues at the wound surface of mice were collected in sterilized EP tubes and stored at -80℃ for later use. Take an appropriate amount of tissue, cut it into pieces in dry ice, add physiological saline, and homogenize it in an ice-water bath to make a tissue homogenate with a mass concentration of 10%. Centrifuge at 3000rpm / min for 30min, collect the supernatant, and store it in aliquots at -80℃ for later use. Take the supernatant of the tissue homogenate to be tested, thaw it in an ice-water bath, and determine the content of Hyp in the sample according to the instructions of the Elisa kit.

[0051] result( Figure 8 ): The hydroxyproline content in the wound tissue increased with the increase of healing days. On the 7th day after modeling, the hydroxyproline content in the DL-1 group was 5.41 μg / mg, and on the 11th day, the hydroxyproline content in the wound tissue of the DL-1 group was 6.91 μg / mg, both significantly (P<0.05) higher than that of the Control group. Hydroxyproline is an important source of collagen fibril protein, and the content of hydroxyproline reflects the content of collagen fibril protein. This result is consistent with the results of Example 6.

[0052] Example 8 CD31 and CD68 Immunohistochemical Staining Diagnosis in Mouse Skin Wound Tissue Sections The sections were routinely dewaxed and washed with water, and the sections were placed in an antigen retrieval buffer (pH 6.0) for antigen retrieval. After natural cooling, they were placed in a phosphate buffer solution PBS (pH 7.4) and washed three times for 5 min each time; the sections were placed in a 3% hydrogen peroxide solution, incubated at room temperature in the dark for 25 min, and washed three times with PBS (pH 7.4), each for 5 min; 3% BSA was dripped into the tissue circle to evenly cover the tissue, and blocked at room temperature for 30 min; the blocking solution was aspirated, and the primary antibody (CD31, CD68 polyclonal antibody) was added, and incubated at 4°C overnight; PBS was washed three times for 5 min each time; the secondary antibody (HRP labeled) was added, and incubated at room temperature for 50 min; PBS was rinsed three times for 5 min each time; DAB color developing solution was added, and positive results were shown as brown-yellow hematoxylin counterstaining, dehydrated to transparency, sealed, and examined under a microscope. Three fields of view were randomly selected for each group and each slice to take pictures. When taking pictures, the tissue was made to fill the entire field of view as much as possible to ensure that the background light was consistent. The immunohistochemical images (field of view diameter 0.9 mm) magnified 200 times by an optical microscope were used as the observation objects. The MVD counting method was used to calculate the CD31 content. The average number of blood vessels in the three fields of view was counted as the MVD value (n / mm 2 ).

[0053] result( Fig. 9): Figure A shows that the content of CD31 can reflect the degree of neovascularization. Compared with the Control group, the content of CD31 in the treatment group increased significantly on the 3rd and 7th days (Table 1), and showed a decreasing trend on the 11th day. Figure B shows that CD 68 is a macrophage marker that can reflect the degree of inflammation. The degree of macrophage infiltration in the wound surface of the treatment group was significantly lower than that of the Control group.

[0054] Table 1 is a statistical table of CD31 content in mouse skin tissue after oligopeptide DL-1 treatment for 3, 7, and 11 days

[0055]

[0056] Note: If the letters between two numbers in the same row are completely different, there is a significant difference (P<0.05).

Claims

1. An oligopeptide for promoting wound healing, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

1.

2. Use of the oligopeptide according to claim 1 in the preparation of a drug for promoting wound healing, wherein the wound is caused by trauma, surgery, burns and scalds.

3. Use of the oligopeptide according to claim 1 in the preparation of medical cosmetic products.

4. A pharmaceutical composition, characterized in that Contains the oligopeptide according to claim 1 and pharmaceutically acceptable excipients.

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