Application of Hepu pearl polypeptide Nacrein-LP20 in preparation of medicine for promoting wound healing

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to an application of a Pucurbita pepo polypeptide Nacrein-LP20 in preparation of a medicine for promoting wound healing and a preparation method of the Pucurbita pepo polypeptide Nacrein-LP20. The amino acid sequence of the Hepu pearl polypeptide Nacrein-LP20 is as shown in SEQ ID NO: 1 (Sequence Identity Number 1). According to the present invention, the Pucurbita pepo active polypeptide Nacrein-LP20 has characteristics of strong DPPH free radical and hydroxyl free radical removing ability and excellent antioxidant activity, can significantly promote HaCaT cell proliferation in the high glucose environment, and can promote migration and repair of HaCaT cells in the high glucose environment; the traditional Chinese medicine composition has a remarkable healing promoting effect on full-cortical resection wound surfaces of normal rats and diabetic rats.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to an application of Hepu pearl polypeptide Nacrein-LP20 in the preparation of a medicine for promoting wound healing. Background Art

[0002] The skin is the largest organ in the human body and has many important biological functions. As the first barrier of the human body, it can effectively protect internal organs and tissues from damage by harmful external factors, such as physical stimulation, chemical stimulation and invasion of microorganisms. However, in daily life, the skin will inevitably be traumatized by various factors such as external force, heat, electric current, chemicals, low temperature, surgery, etc., which will destroy the integrity of the skin and cause damage to its normal functions. In this case, microorganisms can easily invade the wound surface, cause serious infection, hinder the wound repair process, seriously affect the patient's quality of life, and even endanger the patient's life.

[0003] Wound repair is a complex, dynamic and orderly physiological process that requires a variety of tissues and cells to work together to replace, repair and rebuild missing cell structures and tissue layers. It can be roughly divided into three overlapping but different stages: inflammatory stage, proliferation stage and remodeling stage. The hemostasis stage is included in the inflammatory stage, which occurs about 24-48 hours after the formation of the wound. It is characterized by the formation of fibrin clots in the wound in an oxygen-deficient and ischemic environment, and a large number of bacteria, neutrophils and platelets in the wound; the second stage: the proliferation stage, which occurs about 2-10 days after the formation of the wound, is characterized by the migration, proliferation and differentiation of keratinocytes, fibroblasts and endothelial cells, which causes neovascularization, fibrosis, extracellular matrix (ECM) deposition and re-epithelialization, and eschar will form on the wound surface; the third stage: the remodeling stage, which begins 2-3 weeks after the formation of the wound, or lasts for a year or even longer. Its characteristics are that most fibroblasts and vascular endothelial cells gradually undergo apoptosis and regression, the granulation tissue rich in type III collagen is replaced by the stronger type I collagen, and the extracellular matrix is ​​gradually remodeled to form scar tissue.

[0004] However, the wound repair process will be affected by many factors, resulting in chronic difficult-to-heal wounds, which will increase the difficulty of treatment, prolong the treatment cycle, and increase the cost, seriously affecting the physical and mental health and quality of life of the people. According to the Notice of the General Office of the National Health Commission on Strengthening the Diagnosis and Treatment Management of Chronic Difficult-to-Heal Wounds (Ulcers) on the Body Surface in 2019, the main factors leading to chronic difficult-to-heal wounds include diabetic wounds, diabetic foot ulcers, pressure injuries (pressure sores, bedsores), vascular (arterial, venous) ulcers, cancerous ulcers, radiation ulcers, drug-induced skin ulcers, tuberculous ulcers, special bacterial infection wounds and tophi wounds. Therefore, the development of drugs that promote wound repair is of great significance for patients with trauma, burns, diabetic wounds and foot ulcers, chronic skin injuries, etc. Summary of the invention

[0005] The present invention finds that the Hepu pearl polypeptide Nacrein-LP20 can promote wound healing, can be used to prepare a medicine for promoting wound healing, and has an excellent wound healing effect.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a use of Hepu pearl polypeptide Nacrein-LP20 in the preparation of a medicament for promoting wound healing. The amino acid sequence of Hepu pearl polypeptide Nacrein-LP20 is shown in SEQ ID NO: 1.

[0008] Preferably, the above application includes: application of Hepu pearl polypeptide Nacrein-LP20 in the preparation of drugs with antioxidant activity.

[0009] Preferably, the above application includes one or more combinations of the following applications:

[0010] (a) Application of Hepu Pearl polypeptide Nacrein-LP20 in the preparation of a drug for scavenging DPPH free radicals;

[0011] (b) Application of Hepu Pearl Polypeptide Nacrein-LP20 in the preparation of drugs for scavenging hydroxyl free radicals.

[0012] Preferably, the above application includes: application of Hepu Pearl Polypeptide Nacrein-LP20 in the preparation of a drug for promoting healing of diabetic wounds.

[0013] Preferably, the above application includes: application of the Hepu pearl polypeptide Nacrein-LP20 in the preparation of a drug for promoting the proliferation of human immortalized keratinocytes HaCaT under high sugar conditions.

[0014] Preferably, the above application includes: application of the Hepu Pearl polypeptide Nacrein-LP20 in the preparation of a drug for promoting HaCaT cell migration under high sugar conditions.

[0015] Preferably, the above application includes: application of the Hepu Pearl polypeptide Nacrein-LP20 in the preparation of a drug for promoting the repair of HaCaT cells under high sugar conditions.

[0016] Preferably, the dosage form of the drug in the above application includes tablets, injections, powders or patches.

[0017] The beneficial effects of the present invention include at least:

[0018] Hepu Pearl active polypeptide Nacrein-LP20 has a strong ability to scavenge DPPH free radicals and hydroxyl free radicals, and has excellent antioxidant activity. In a high sugar environment (such as a concentration of 25mM), it can significantly promote the proliferation of HaCaT cells, as well as their migration and repair in a high sugar environment; it has a significant effect on promoting the healing of full-thickness excision wounds in rats, and also has a significant effect on promoting the healing of full-thickness excision wounds in diabetic rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1a This is the structure diagram of Hepu pearl Nacrein-LP20 polypeptide;

[0020] Figure 1b This is the HPLC chromatogram of Hepu pearl Nacrein-LP20 peptide;

[0021] Figure 1c This is the mass spectrometry analysis of Hepu pearl Nacrein-LP20 polypeptide;

[0022] Figure 2 is the hemolytic activity of Hepu Pearl Nacrein-LP20 polypeptide, wherein 1 is the PBS treatment group; 2 is the 1% TritonX-100 treatment group; 3-12 are 1280μg / mL-2.5μg / mL Nacrein-LP20 polypeptide treatment groups;

[0023] Figure 3a The standard curve of standard substance Trolox;

[0024] Figure 3b The total antioxidant capacity of Hepu Pearl Nacrein-LP20 polypeptide;

[0025] Figure 3c The ability of Nacrein-LP20 peptide to scavenge hydroxyl radicals;

[0026] Figure 3dThe ability of Nacrein-LP20 peptide to scavenge DPPH free radicals;

[0027] Figure 4a The cell viability of human immortalized keratinocytes HaCaT treated with different concentrations of Nacrein-LP20 (10-1280 μg / mL) for 24 h in a low-glucose environment; ns means P>0.05 compared with the control group (Nacrein-LP concentration of 0 μg / mL);

[0028] Figure 4b The cell viability of human immortalized keratinocytes HaCaT treated with different concentrations of Nacrein-LP20 (10-1280 μg / mL) for 48 h in a low-glucose environment; ns means P>0.05 compared with the control group (Nacrein-LP concentration of 0 μg / mL);

[0029] Figure 4c In a high-glucose environment, the cell viability of human immortalized keratinocytes HaCaT after being treated with different concentrations of Nacrein-LP20 (10-1280 μg / mL) for 24 hours; * indicates P < 0.05 compared with the control group (Nacrein-LP concentration of 0 μg / mL); ns indicates P > 0.05 compared with the control group (Nacrein-LP concentration of 0 μg / mL);

[0030] Figure 4d The cell viability of human immortalized keratinocytes HaCaT treated with different concentrations of Nacrein-LP20 (10-1280 μg / mL) for 48 h in a high-glucose environment; * indicates P < 0.05 compared with the control group (Nacrein-LP concentration of 0 μg / mL); ns indicates P > 0.05 compared with the control group (Nacrein-LP concentration of 0 μg / mL);

[0031] Figure 5a A is the detection of the scratch repair activity of drugs on HaCaT cells in a high-glucose environment (n=3); * indicates P<0.05 compared with the control group. ns indicates P>0.05 compared with the control group;

[0032] Figure 5b A is the healing rate of cell scratches at 20h; C is the healing rate of cell scratches at 40h; * indicates P<0.05 compared with the control group. ns indicates P>0.05 compared with the control group;

[0033] Figure 5c The healing rate of cell scratches after 40 hours;

[0034] Figure 6aRepresentative images of full-thickness excision wound healing in rats;

[0035] Figure 6b The wound healing curves of cortical excision in each group; * indicates P < 0.05 compared with the control group;

[0036] Figure 7a Representative images of H&E staining of each group on day 11 after surgery (the red double-ended arrows indicate the distance between the wound edges);

[0037] Figure 7b This is a quantitative diagram of wound tissue diameter on the 11th day after surgery; * indicates P < 0.05 compared with the control group or positive group;

[0038] Figure 8 These are representative images of Masson staining of full-thickness cortical excision wounds in rats of each group on day 11 after surgery;

[0039] Figure 9a It is the serum total cholesterol (TC) index of rats in each group on the 11th day after surgery;

[0040] Figure 9b It is the serum triglyceride (TG) index of rats in each group on the 11th day after surgery;

[0041] Fig.9c It is the serum uric acid (UA) index of rats in each group on the 11th day after surgery;

[0042] Figure 9d It is the serum glutamic oxalacetictransaminase (GOT) index of rats in each group on the 11th day after surgery.

[0043] Fig.10a Representative images of full-thickness excision wound healing in diabetic rats;

[0044] Fig.10b The full-thickness excision wound healing curves of each group; * indicates P < 0.05 compared with the control group; DETAILED DESCRIPTION

[0045] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0046] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0047] The embodiment of the present invention provides a use of Hepu pearl polypeptide Nacrein-LP20 in the preparation of a medicament for promoting wound healing. The amino acid sequence of Hepu pearl polypeptide Nacrein-LP20 is shown in SEQ ID NO: 1.

[0048] Specifically, the amino acid of SEQ ID NO: 1 is as follows: GENGHKHGCRVKKAKHLSRI.

[0049] It should be noted that the shells of Hepu pearl oysters can be used as raw materials for shell carving, the pearl oysters (meat) are edible, and the pearl layer (medicinal name: mother-of-pearl) and pearls that have been removed of impurities can be processed into pearl powder for medicinal purposes, which has a history of more than 2,000 years of medicinal use. This is recorded in "Shennong's Herbal Classic", "Compendium of Materia Medica", "Sea Drugs Herbal Classic", "Compendium of Materia Medica", etc. The 2020 edition of the Chinese Pharmacopoeia states: "Pearl is sweet, salty, and cold. It enters the heart and liver meridians. It calms the nerves and calms the nerves, improves eyesight and eliminates cataracts, detoxifies and regenerates muscles, moisturizes the skin and removes freckles. It is used for insomnia caused by palpitations, convulsions, epilepsy, red eyes, cataracts, sores that do not heal, and skin spots. This shows that pearl powder has had an excellent repair effect on wound damage since ancient times. Pearl polypeptide is the main active ingredient in pearl powder. In the present invention, it is found that a derivative peptide Nacrein-LP20 from the Hepu pearl matrix protein Nacrein has strong antioxidant activity, has a certain effect on promoting the proliferation of skin keratinocytes, can significantly promote the healing of full-thickness excision wounds, and shows excellent performance in wound repair. It can provide new ideas and methods for wound repair and the treatment of chronic and difficult-to-heal wounds.

[0050] In some specific examples, the above applications include: application of Hepu pearl polypeptide Nacrein-LP20 in the preparation of drugs with antioxidant activity.

[0051] In some specific examples, the above application includes one or more combinations of the following applications:

[0052] (a) Application of Hepu Pearl polypeptide Nacrein-LP20 in the preparation of a drug for scavenging DPPH free radicals;

[0053] (b) Application of Hepu Pearl Polypeptide Nacrein-LP20 in the preparation of drugs for scavenging hydroxyl free radicals.

[0054] It should be noted that the hemolytic activity of the active polypeptide Nacrein-LP20 of Hepu pearl in the present invention is almost negligible, and it has significant antioxidant activity in vitro, has a strong ability to scavenge DPPH free radicals and hydroxyl free radicals, and has certain application value in antioxidant.

[0055] In some specific examples, the above applications include: application of Hepu Pearl Polypeptide Nacrein-LP20 in the preparation of drugs for promoting healing of diabetic wounds.

[0056] In some specific examples, the above applications include: application of Hepu Pearl polypeptide Nacrein-LP20 in the preparation of a drug for promoting the proliferation of human immortalized keratinocytes HaCaT under high sugar conditions.

[0057] In some specific examples, the above applications include: application of Hepu Pearl polypeptide Nacrein-LP20 in the preparation of drugs for promoting HaCaT cell migration under high sugar conditions.

[0058] In some specific examples, the above applications include: application of Hepu Pearl polypeptide Nacrein-LP20 in the preparation of drugs for promoting HaCaT cell repair under high sugar conditions.

[0059] It should be noted that the Hepu pearl active polypeptide Nacrein-LP20 provided by the present invention has no effect on the proliferation of human immortalized keratinocytes HaCaT at low sugar concentration (5.5mM), but can significantly promote the proliferation of HaCaT cells in a high sugar environment (for example, a concentration of 25mM), as well as promote their migration and repair in a high sugar environment.

[0060] In some specific examples, the dosage form of the drug in the above application includes tablets, injections, powders or patches.

[0061] It should be noted that the active polypeptide Nacrein-LP20 of Hepu pearl in the present invention can be added with pharmaceutically acceptable carriers to prepare different dosage forms, such as tablets, injections, powders or patches; the pharmaceutically acceptable carrier can be selected according to the type of dosage form, which is a well-known method in the art.

[0062] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0063] In the following examples, the statistical software IBM SPSS Statistics 19.0 was used for data analysis, and GraphPad Prism 8 software was used for statistical plotting. All experiments were repeated 3 times, and the quantitative results were expressed as mean ± standard deviation (X ± S). The quantitative numerical comparison between two groups was performed by independent sample t test, and the quantitative numerical comparison between multiple groups was performed by one-way analysis of variance. In all tests, P < 0.05 was considered to be significantly different and statistically significant, indicated by "*" or "#".

[0064] 1. Preparation and characterization of Nacrein-LP20 peptide

[0065] (I) Preparation of Hepu Pearl Nacrein-LP20 Peptide

[0066] Hepu Pearl Nacrein-LP20 polypeptide (SEQ ID NO.1: 'GENGHKHGCRVKKAKHLSRI') was synthesized by Wuhan Tiande Biotechnology Co., Ltd. (Wuhan, China) using a solid phase method with a purity of >95%. Nacrein-LP20 was dissolved in phosphate buffered saline (PBS) to prepare different concentrations for subsequent experiments.

[0067] (II) Characterization of Hepu Pearl Nacrein-LP20 Peptide

[0068] The structure, HPLC and MS analysis of Hepu Pearl Nacrein-LP20 peptide are shown in Figure 1a , Figure 1b and Figure 1c As shown. Liquid chromatography analysis was performed using an Agilent 1260II liquid chromatography system: a Symmetrix ODS-R column (4.6×250 mm, 5 μm) was used, the mobile phase was acetonitrile-water (60:40), the flow rate was 1.0 mL / min, the detection wavelength was 220 nm, the column temperature was 25°C, and the sample volume was 20 μL. Agilent single quadrupole mass spectrometer (Agilent G6125B LC / MSD) was used for mass spectrometry detection. The mass spectrometry conditions were: ionization mode ESI, probe bias voltage was +4.5 kv, nebulizer gas flow rate was 1.5 L / min, detector was 1.5 kv, nebulizer gas flow rate was 1.5 L / min, total flow rate was 0.2 mL / min, capillary electrophoresis-electrospray ionization source voltage was -20 v, the capillary temperature of the electrospray ionization source was 250°C, and the module temperature was 200°C.

[0069] 2. In vitro tests of Nacrein-LP20 peptide

[0070] (I) Hemolytic activity test of Hepu Pearl Nacrien-LP20 polypeptide

[0071] 2% (V / V) porcine red blood cells were used for hemolytic activity detection, specifically including: using PBS to dilute the Nacrein-LP20 polypeptide to concentrations of 2560μg / mL, 1280μg / mL, 640μg / mL, 320μg / mL, 160μg / mL, 80μg / mL, 40μg / mL, 20μg / mL, 10μg / mL and 5μg / mL respectively according to the doubling dilution method, taking 100μL of the polypeptide solution and adding it to a 96-well plate as an experimental group, and taking another 100μL of PBS and 1.0% (V / V) TritonX-100 as a negative control group. For the positive control and the negative control, 100 μL of 2% pig red blood cell suspension was added to each well, with 3 replicates per group, so that the final concentration of the polypeptide was 1280 μg / mL-2.5 μg / mL (i.e., 1280 μg / mL, 640 μg / mL, 320 μg / mL, 160 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL and 2.5 μg / mL), incubated in a 37°C incubator for 3 hours, centrifuged at 1500 rpm for 10 minutes, 150 μL of the supernatant was taken to a new 96-well plate, the absorbance was measured at 450 nm, and the hemolysis rate was calculated. Hemolysis rate = [OD value of experimental group] 450 - Negative control group OD 450 ] / [OD of positive control group 450 - Negative control group OD 450 ]×100%.

[0072] Test results such as Figure 2 As shown, column 1 is a negative control with no hemolytic activity; column 2 is a positive control with obvious hemolysis; columns 3-12 are Hepu Pearl Nacrein-LP20 peptide groups with a final concentration of 1280μg / mL-2.5μg / mL, and their hemolytic activity is almost negligible.

[0073] (II) Antioxidant activity test of Hepu pearl Nacrein-LP20 peptide

[0074] The total antioxidant capacity of Hepu Pearl Nacrein-LP20 polypeptide was detected using the Biyuntian Total Antioxidant Capacity Test Kit (ABTS Rapid Method) (Product No.: S0121). The hydroxyl radical test kit (A018-1-1) and DPPH free radical scavenging capacity kit (A153-1-1) of Nanjing Jiancheng Bioengineering Institute were used to detect the scavenging hydroxyl and DPPH free radicals of Hepu Pearl Nacrein-LP20 polypeptide, and the positive control was set to the same concentration of vitamin C.

[0075] Test results such as Figures 3a to 3d As shown, the standard curve equation of standard Trolox is y=1.2593x-0.0868, R2 =0.9911 (see Figure 3a ); Therefore, according to the standard curve equation, the total antioxidant capacity of the sample to be tested can be calculated. When the concentration of Hepu Pearl Nacrein-LP20 polypeptide is 0.25 mg / mL, its total antioxidant capacity is equivalent to 1.7861 mMTrolox (see Figure 3b ); and Hepu Pearl Nacrein-LP20 polypeptide has a significant ability to scavenge hydroxyl free radicals and DPPH free radicals, which is almost equivalent to vitamin C. Its IC 50 151.36 μg / mL and 1.39 μg / mL respectively (see Figure 3c and Figure 3d ), indicating that Hepu pearl Nacrein-LP20 polypeptide has strong antioxidant activity in vitro.

[0076] (III) Hepu Pearl Nacrein-LP20 Peptide CCK-8 Detection

[0077] Human immortalized keratinocytes HaCaT (iCell-h066, Biopharmaceuticals (Shanghai) Co., Ltd.) (STR identified) were cultured in a 37°C 5% CO2 incubator (MCO-18AIC (UV), Sanyo Co., Ltd., Japan) using DMEM (low glucose) complete medium (containing 10% FBS, 1% penicillin-streptomycin) (C11885500BT, Gibco).

[0078] HaCaT cells were seeded into 96-well plates at 1 × 10 cells per well. 5 cells, and used DMEM (low glucose) (C11885500BT, Gibco) or DMEM (high glucose) complete medium (C11995500BT, Gibco) to dissolve the Hepu Pearl Nacrein-LP20 polypeptide into a 1 mg / mL stock solution. The cells were treated with different concentrations of Nacrein-LP20 polypeptide (0, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, 320 μg / mL, 640 μg / mL and 1280 μg / mL)) for 24 h and 48 h, respectively. The 96-well plate cells to be tested were replaced with the same culture medium, 100 μL per well, and 10 μL CCK8 reagent (BS350B, Biosharp, China) was added to each well, and the cells were incubated in an incubator for 4 h. A full-function microplate reader (Synergy H1, BioTek, USA) was used to detect the absorbance of each well at a wavelength of 450 nm. Relative cell proliferation rate = (absorbance of the experimental group - absorbance of the blank control) / (absorbance of the control group - absorbance of the blank control) × 100%.

[0079] The test results are shown in Figure 4. In low-glucose conditions, there was no significant change in the survival rate of cells treated with 10 μg / mL to 1280 μg / mL Nacrein-LP20 peptide after 24h and 48h compared with the control group (see Figure 4a and Figure 4b ); Under high glucose conditions, compared with the control group, the survival rate of cells treated with 10μg / mL to 640μg / mL Nacrein-LP20 peptide was significantly increased after 24h and 48h, while the survival rate of cells treated with 1280μg / mL Nacrein-LP20 peptide did not change significantly after 24h and 48h (see Figure 4c and Figure 4d ).

[0080] (IV) Hepu Pearl Nacrein-LP20 Peptide Scratch Test

[0081] The HaCaT cells in the logarithmic growth phase were collected and resuspended in DMEM (high glucose) complete medium at a rate of 5 × 10 5 Inoculate in 6-well plates, the final total volume of culture medium in each well is 2mL, and culture in a 37℃, 5% CO2 incubator for 24h, use a 200μL pipette tip to scratch, suck off the culture medium, rinse the cells 3 times with PBS, remove the scratched cells, set up a control group (serum-free high-glucose culture medium), a positive group (EGF diluted to 10ng / mL with serum-free high-glucose culture medium), and an experimental group (80μg / mL, 160μg / mL, 320μg / mL, and 640μg / mL of Hepu Pearl Nacrein-LP20 polypeptide). Take pictures under a microscope at 0, 20h, and 40h to observe the results.

[0082] The results are as follows Figure 5a , Figure 5b and Figure 5c As shown, compared with the control group, the scratch repair ability of HaCaT cells increased in a concentration-dependent and time-dependent manner after treatment with Hepu Pearl Nacrein-LP20 polypeptide (160 μg / mL, 320 μg / mL and 640 μg / mL).

[0083] 3. Animal Experiments of Nacrein-LP20 Peptide

[0084] The preparation method and drug administration of the full-thickness excision wound model of normal rats are as follows: (1) Experimental grouping: 21 SPF-grade SD male rats (180g-200g) were divided into 7 groups. All experimental procedures and animal care were carried out in accordance with the regulations of the Animal Ethics Committee of Guangxi University of Chinese Medicine. The rat tails were numbered and grouped into a control group (normal saline), a positive control group (Kangfuxin solution), and an experimental group: 80μg / mL-1280μg / mL Hepu Pearl Nacrein-LP20 peptide solution; (2) Model preparation: The rats were anesthetized by inhaling isoflurane through an anesthesia machine, and the back hair was shaved with an electric clipper and removed with Veet depilatory cream. Circular wounds (wound size: 2×2 cm) were prepared on both sides of the rat's back (preferably 2 cm on each side of the animal's spine and 2 cm below the shoulder blade). The wounds were cleaned with 75% alcohol and disinfected with iodine to keep them clean. (3) Treatment method and observation: After the wound preparation was completed, the drug was administered immediately, once a day, with a volume of 100 μL each time. The wounds were photographed and recorded every other day. The drug was administered continuously for 11 days.

[0085] The preparation method and drug administration of the full-thickness excision wound model of diabetic rats are as follows: (1) Establishment of diabetic rat model: After SD rats were adaptively fed for one week (body weight was 170-250g), they were fasted but not watered for 12 hours, and 1% streptozotocin was injected intraperitoneally at a dose of 65mg / kg. The rats were observed and blood sugar was measured daily. After 7 days, the blood sugar concentration was greater than 16.7mmol / L, which was considered a successful model. (2) The wound model was established as above. A normal group, a diabetic model group, and an experimental group (160, 320, and 640μg / mL Nacrein-LP20 polypeptide) were set up, with 3 rats in each group. 100μL of drug was administered daily, and the wound size was recorded every other day.

[0086] 1. Evaluation of wound healing ability

[0087] The full-thickness excision wound model was used to evaluate the effect of Hepu Pearl Nacrein-LP20 peptide on the healing ability of rat skin wounds. Figure 6a and Figure 6b As shown, compared with the normal saline control group, the positive group Kangfuxin solution and the Hepu Pearl Nacrein-LP20 polypeptide treatment group could significantly promote the healing of full-thickness excision wounds in rats.

[0088] in, Figure 6aThe representative images after drug treatment show that scabs began to form in each group on the 3rd day, and on the 3rd day, the 640μg / mL Nacrein-LP20 treatment group significantly reduced the wound area compared with the saline control group. Over time, both the positive group Kangfuxin solution and the Nacrein-LP20 treatment group showed a significant effect on promoting skin wound contraction, and the wound closure rate was significantly higher than that of the control group. ImageJ was used to monitor and quantify the wound at 0, 3, 5, 7, 9 and 11 days after injury ( Figure 6b ), the statistical results showed that at 11 days after surgery, the wound healing rates of the positive group Kangfuxin liquid and the 160μg / mL, 320μg / mL, and 640μg / mL Nacrein-LP20 treatment groups were 81.26%±3.33%, 79.28%±1.33%, 83.34%±0.82%, and 90.65±2.20%, respectively, while the wound closure rate of the control group was only 48.24%±3.27% ( Figure 6b )(P<0.05).

[0089] The above results show that Hepu Pearl Nacrein-LP20 peptide can promote skin wound healing, among which the low and medium concentrations are equivalent to the positive group Kangfuxin liquid in efficacy, and the high concentration group is even better than the Kangfuxin liquid treatment group in terms of wound repair.

[0090] (ii) Hematoxylin and eosin (H&E) and Masson staining

[0091] On the 11th day after surgery, the rats underwent wound histological analysis, including: 11 days after surgery, wound tissue and surrounding normal skin areas (a total of 2 cm in size) were obtained, fixed with 4% formaldehyde, dehydrated with gradient ethanol, transparentized with xylene, and embedded in paraffin; the tissue specimens were stained with H&E and Masson; the tissue sections were photographed using a PANNORAMIC panoramic slice scanner; CaseViewer 2.4 scanning software was used to select the target area of ​​skin tissue for 10x imaging, and the tissue was allowed to fill the entire field of view as much as possible during imaging to ensure that the background light of each photo was consistent; after imaging, Image-Pro Plus 6.0 analysis software was used to measure the width of the damaged area in each slice using millimeters (mm) as the standard unit.

[0092] (1) HE staining

[0093] HE staining was further used to evaluate the effect of Hepu Pearl Nacrein-LP20 peptide on the healing of full-thickness excision wounds in rats. Figure 7aThe results shown in the figure show that compared with the control group, the positive group and the Hepu Pearl Nacrein-LP20 polypeptide treatment group significantly promoted wound healing, and the wound diameter was significantly shortened; on the 11th day, the wound diameter of the positive group Kangfuxin liquid was 5.10±0.20mm, and the wound diameters of the 160μg / mL, 320μg / mL and 640μg / mL Nacrein-LP20 polypeptide treatment groups were 5.57±0.40mm, 3.71±0.10mm, and 4.86±0.33mm, respectively. Among them, the wound diameters of the 320 and 640μg / mL Nacrein-LP20 polypeptide treatment groups were smaller than those of the positive group, while the wound diameter of the normal saline control group was 9.30±0.41mm. It can be seen that the drug treatment group can significantly promote wound healing in rats, and at a certain concentration, the Hepu Pearl Nacrein-LP20 polypeptide has a better effect on promoting wound healing than the positive control group Kangfuxin liquid (see Figure 7b ).

[0094] (2) Masson staining

[0095] Collagen, as an important component of the extracellular matrix (ECM), is necessary for filling wounds, improving tensile strength, and forming angiogenesis matrix framework; Masson staining was used to detect the regeneration and deposition of collagen fibers in wound tissue. Figure 8 As shown, on the 11th day, the collagen production and deposition in the positive group Kangfuxin liquid and Hepu Pearl Nacrein-LP20 polypeptide treatment group were more intensive and organized than those in the normal saline control group, indicating that the relative expression of collagen in the drug treatment group was significantly higher than that in the control group.

[0096] (III) Safety Testing

[0097] The serum of rats in each group was collected 11 days after surgery, and the serum biochemical indicators, including total cholesterol (TC), triglyceride (TG), uric acid (UA) and glutamic oxalacetic transaminase (GOT), were detected using biochemical kits. The samples were processed according to the instructions of the kit and measured using a full-function microplate reader (Synergy H1, BioTek, USA). On the 11th day, the safety of serum biochemical indicators of rats in each group was evaluated. Total cholesterol and triglycerides are important indicators of the development of hyperlipidemia into severe atherosclerosis and are basic blood lipid detection indicators for vascular risk assessment.

[0098] For total cholesterol, the test results are as follows Figure 9aAs shown, compared with the normal group, except for the 320μg / mL Hepu Pearl Nacrein-LP20 peptide treatment group, the other groups were significantly increased. However, compared with the normal saline control group, there was no significant difference between the low and high concentrations of Hepu Pearl peptide treatment groups. The medium concentration treatment group could also significantly reduce the TC level.

[0099] For triglycerides, the test results are as follows Figure 9b As shown, there was no significant difference between the normal saline group and the normal group. Compared with the normal group and the normal saline group, the Hepu Pearl polypeptide treatment group was able to significantly reduce the triglyceride content. The results showed that compared with the normal saline group, the Hepu Pearl Nacrein-LP20 polypeptide treatment group did not cause obesity and cardiovascular abnormalities in rats.

[0100] In addition, serum uric acid test results such as Fig.9c As shown, compared with the normal group, each group had a decrease (P<0.05), and compared with the normal saline group, there was no significant difference in the Hepu Pearl Peptide treatment group (P>0.05), indicating that peptide treatment did not lead to the occurrence of diseases such as hyperuricemia or gout.

[0101] The liver contains aspartate aminotransferase, which is an indicator of liver function test in clinical medicine to determine whether the liver is damaged. Figure 9d As shown, there was no significant difference in serum GOT levels among the rats in each group (P>0.05), indicating that the liver cell membrane was intact and there was no significant difference in serum transaminase activity.

[0102] In addition, during the experiment, the rats treated with the peptide did not suffer from liver damage, that is, the Hepu Pearl Nacrein-LP20 peptide did not produce any obvious toxic side effects on the rats during the experiment, indicating that it is safe and effective for the treatment of wound healing.

[0103] 4. Evaluation of diabetic wound healing ability

[0104] The full-thickness excision wound model was used to evaluate the effect of Hepu Pearl Nacrein-LP20 peptide on the healing ability of skin wounds in diabetic rats. Fig.10a and Fig.10b As shown, compared with the diabetic model group, the Hepu Pearl Nacrein-LP20 peptide treatment group was able to significantly promote the healing of full-thickness excision wounds in rats.

[0105] in, Fig.10aThe representative images after drug treatment show that the scabs in the drug-treated group and the normal group began to form on the 3rd day, and on the 3rd day, the wound area in the drug-treated group was significantly reduced compared with the model group. As time went on, the drug-treated group showed a significant effect of promoting skin wound contraction, and the wound closure rate was significantly higher than that in the model group. ImageJ was used to monitor and quantify the wound on days 0, 3, 5, 7, 9, 11, and 13 after injury ( Fig.10b ), the statistical results showed that at 13 days after surgery, the wound healing rates of the 160μg / mL, 320μg / mL, and 640μg / mL Nacrein-LP20 treatment groups were 98.48%±0.45%, 94.55%±4.68%, and 97.53%±0.15%, respectively, while the wound closure rate of the model group was only 87.35%±6.00% ( Fig.10b )(P<0.05).

[0106] The above results show that Hepu Pearl Nacrein-LP20 peptide can promote the healing of diabetic skin wounds.

[0107] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. Application of Hepu Pearl polypeptide Nacrein-LP20 in the preparation of a drug for promoting wound healing. The amino acid sequence of Hepu Pearl polypeptide Nacrein-LP20 is shown in SEQ ID NO:

1.

2. The use according to claim 1, characterized in that: Applications include: application of Hepu pearl polypeptide Nacrein-LP20 in the preparation of drugs with antioxidant activity.

3. The use according to claim 1, characterized in that: Applications include one or a combination of the following: (a) Application of Hepu Pearl polypeptide Nacrein-LP20 in the preparation of a drug for scavenging DPPH free radicals; (b) Application of Hepu Pearl Polypeptide Nacrein-LP20 in the preparation of drugs for scavenging hydroxyl free radicals.

4. The use according to claim 1, characterized in that: Applications include: application of Hepu Pearl Polypeptide Nacrein-LP20 in the preparation of drugs for promoting healing of diabetic wounds.

5. The use according to claim 1, characterized in that: Applications include: Use of Hepu Pearl Polypeptide Nacrein-LP20 in the preparation of drugs for promoting the proliferation of human immortalized keratinocytes HaCaT under high sugar conditions.

6. The use according to claim 1, characterized in that: Applications include: Use of Hepu Pearl Peptide Nacrein-LP20 in the preparation of drugs for promoting HaCaT cell migration under high sugar conditions.

7. The use according to claim 1, characterized in that: Applications include: Use of Hepu Pearl Polypeptide Nacrein-LP20 in the preparation of drugs for promoting HaCaT cell repair under high sugar conditions.

8. The use according to any one of claims 1 to 7, characterized in that: The medicine comes in tablet, injection, powder or patch form.

Images