Application of Pearl Polypeptide Nacrein-LP20 from Hepu in the Preparation of Drugs for Promoting Wound Healing

Abstract

The present invention belongs to the field of biomedical technology, and particularly relates to the application of nacre polypeptide Nacrein-LP20 in the preparation of a drug for promoting wound healing. The amino acid sequence of nacre polypeptide Nacrein-LP20 is shown in SEQ ID NO: 1. Nacre active polypeptide Nacrein-LP20 has strong ability to scavenge DPPH free radicals and hydroxyl free radicals, has excellent antioxidant activity, can significantly promote the proliferation of HaCaT cells in a high-glucose environment, and promote its migration and repair in a high-glucose environment; it has a significant effect on promoting the healing of full-thickness excisional wounds in normal rats and diabetic rats.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of nacre polypeptide Nacrein-LP20 in the preparation of a drug for promoting wound healing. Background Art

[0002] The skin is the largest organ of the human body and has multiple important biological functions. As the first line of defense of the human body, it can effectively protect internal organs and tissues from damage by external harmful factors, such as physical stimuli, chemical stimuli, and microbial invasion. However, in daily life, the skin is inevitably traumatized by various factors such as external force, heat, electric current, chemical substances, low temperature, and surgical operations, which destroys the integrity of the skin and impairs its normal functions. In this case, microorganisms can easily invade the wound surface, causing severe infections, hindering the wound repair process, seriously affecting the quality of life of patients, and even endangering the lives of patients.

[0003] Wound repair is a complex, dynamic, and orderly physiological process that requires the highly coordinated action of multiple tissues and cells to replace, repair, and reconstruct the missing cell structures and tissue layers. It can be roughly divided into three overlapping but distinct stages: the inflammatory stage, the proliferative stage, and the remodeling stage. Among them, the hemostasis period is included in the inflammatory stage, which occurs about 24-48 hours after the formation of the wound. The characteristic of this stage is that a fibrin clot is formed in the wound under anoxic and ischemic conditions, and there are a large number of bacteria, neutrophils, and platelets in the wound; the second stage: the proliferative stage, occurs about 2-10 days after the formation of the wound. Its characteristics are the migration, proliferation, and differentiation of keratinocytes, fibroblasts, and endothelial cells, resulting in the formation of new blood vessels, fibroplasia, deposition of extracellular matrix (ECM), and re-epithelialization, and a scab will form on the wound surface; the third stage: the remodeling stage, begins 2-3 weeks after the formation of the wound, or lasts for one year or even longer. Its characteristics are that most fibroblasts and vascular endothelial cells gradually apoptose and disappear, the granulation tissue rich in type III collagen is replaced by stronger type I collagen, and the extracellular matrix remodeling is gradually completed, forming scar tissue.

[0004] However, during the wound repair process, it will be affected by various factors, leading to chronic non-healing wounds, which increase the treatment difficulty, prolong the treatment cycle, and raise 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 Non-healing Wounds (Ulcers) on the Body Surface in 2019, the main factors leading to chronic non-healing 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 gouty tophus wounds, etc. Therefore, the development of drugs to 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 discovers that the Hepu pearl polypeptide Nacrein-LP20 can promote wound healing and can be used to prepare drugs for promoting wound healing, with excellent wound healing effects.

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

[0007] On the one hand, the present invention provides an application of the Hepu pearl polypeptide Nacrein-LP20 in the preparation of drugs for promoting wound healing, and the amino acid sequence of the Hepu pearl polypeptide Nacrein-LP20 is as shown in SEQ ID NO: 1.

[0008] Preferably, the above application includes: the application of the 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) The application of the Hepu pearl polypeptide Nacrein-LP20 in the preparation of drugs for scavenging DPPH free radicals;

[0011] (b) The application of the Hepu pearl polypeptide Nacrein-LP20 in the preparation of drugs for scavenging hydroxyl free radicals.

[0012] Preferably, the above application includes: the application of the Hepu pearl polypeptide Nacrein-LP20 in the preparation of drugs for promoting diabetic wound healing.

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

[0014] Preferably, the above application includes: the application of nacrein polypeptide Nacrein-LP20 in the preparation of a drug for promoting the migration of HaCaT cells under high glucose conditions.

[0015] Preferably, the above application includes: the application of nacrein polypeptide Nacrein-LP20 in the preparation of a drug for promoting the repair of HaCaT cells under high glucose 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 at least include:

[0018] The nacrein active polypeptide Nacrein-LP20 has strong abilities to scavenge DPPH free radicals and hydroxyl free radicals, and has excellent antioxidant activity. Under high glucose environments (such as a concentration of 25 mM), it can significantly promote the proliferation of HaCaT cells, as well as promote their migration and repair under high glucose environments; it has a significant effect on promoting the healing of the full-thickness skin excision wounds of rats, and also has a significant effect on promoting the healing of the full-thickness skin excision wounds of diabetic rats. Description of the Drawings

[0019] Figure 1a It is the polypeptide structure diagram of nacrein Nacrein-LP20;

[0020] Figure 1b It is the high performance liquid chromatography diagram of nacrein Nacrein-LP20 polypeptide;

[0021] Figure 1c It is the mass spectrometry analysis diagram of nacrein Nacrein-LP20 polypeptide;

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

[0023] Figure 3a It is the standard curve of the standard product Trolox;

[0024] Figure 3b It is the total antioxidant capacity of nacrein Nacrein-LP20 polypeptide;

[0025] Figure 3c It is the ability of Nacrein-LP20 polypeptide to scavenge hydroxyl free radicals;

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

[0027] Figure 4a Under low-glucose conditions, after treating human immortalized keratinocytes HaCaT with different concentrations of Nacrein-LP20 (10 - 1280 μg / mL) for 24 h, the cell viability; ns indicates P > 0.05 compared with the control group (Nacrein-LP concentration is 0 μg / mL);

[0028] Figure 4b Under low-glucose conditions, after treating human immortalized keratinocytes HaCaT with different concentrations of Nacrein-LP20 (10 - 1280 μg / mL) for 48 h, the cell viability; ns indicates P > 0.05 compared with the control group (Nacrein-LP concentration is 0 μg / mL);

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

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

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

[0032] Figure 5b The healing rate of the 20-h cell scratch; C is the healing rate of the 40-h cell scratch; * 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 the 40-h cell scratch;

[0034] Figure 6aIt is a representative diagram of the wound healing of the total cortex resection in rats;

[0035] Figure 6b They are the wound healing curves of the cortex resection in each group; * indicates P < 0.05 compared with the control group;

[0036] Figure 7a They are the representative images of H&E staining on the 11th day after surgery in each group (the red double-ended arrow indicates the distance between the wound edges);

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

[0038] Figure 8 They are the representative images of Masson staining of the total cortex resection wounds of rats in each group on the 11th day after surgery;

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

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

[0041] Figure 9c They are the indexes of uric acid (UA) in the serum of rats in each group on the 11th day after surgery;

[0042] Figure 9d They are the indexes of glutamic oxalacetic transaminase (GOT) in the serum of rats in each group on the 11th day after surgery.

[0043] Figure 10a It is a representative diagram of the wound healing of the total cortex resection in diabetic rats;

[0044] Figure 10b They are the wound healing curves of the total cortex resection in each group; * indicates P < 0.05 compared with the control group; Detailed implementation manners

[0045] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation manners based on the above invention content still fall within the protection scope of the present invention.

[0046] The terms used in this document are only for describing specific embodiments and are not intended to limit the present disclosure. Unless there is an obviously different meaning in the context, the expressions in the singular form include the plural form. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the existence 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 may exist or can be added. As used herein, depending on the circumstances, " / " can be interpreted as "and" or "or".

[0047] An embodiment of the present invention provides an application of nacre polypeptide Nacrein-LP20 in the preparation of a drug for promoting wound healing. The amino acid sequence of nacre polypeptide Nacrein-LP20 is shown as SEQ ID NO: 1.

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

[0049] It should be noted that the shells of Pinctada fucata can be used as raw materials for shell carving, the meat of Pinctada fucata can be eaten, the nacre layer (medicinal name: mother-of-pearl) and pearls after removing impurities can be processed into pearl powder for medicinal use, and it has a medicinal history of more than two thousand years. This is recorded in "Shennong Ben Cao Jing", "Ben Cao Jing Ji Zhu", "Hai Yao Ben Cao", "Ben Cao Gang Mu", etc. The 2020 edition of the Chinese Pharmacopoeia records: "Pearl, sweet, salty, cold. It belongs to the heart and liver meridians. Soothes the nerves and relieves convulsions, clears the eyes and removes nebulae, detoxifies and promotes granulation, and moistens the skin and removes freckles. It is used for palpitations and insomnia, infantile convulsions and epilepsy, red eyes and nebulae, sores that do not heal, and skin freckles." This shows that pearl powder has excellent repair effects on wound injuries since ancient times. Pearl polypeptide is the main active substance of pearl powder. In the present invention, it is found that a derivative peptide Nacrein-LP20 from nacre matrix protein Nacrein has strong antioxidant activity, has a certain promoting effect on the proliferation of skin keratinocytes, can significantly promote the healing of full-thickness excision wounds, and shows excellent performance in wound repair, which can provide new ideas and methods for wound repair and the treatment of chronic non-healing wounds.

[0050] In some specific examples, the above application includes: the application of nacre polypeptide Nacrein-LP20 in the preparation of a drug with antioxidant activity.

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

[0052] (a) The application of nacre polypeptide Nacrein-LP20 in the preparation of a drug for scavenging DPPH free radicals;

[0053] (b) Application of nacrein-LP20, a pearl polypeptide from Hepu, in the preparation of a drug for scavenging hydroxyl radicals.

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

[0055] In some specific examples, the above application includes: Application of nacrein-LP20, a pearl polypeptide from Hepu, in the preparation of a drug for promoting the healing of diabetic wounds.

[0056] In some specific examples, the above application includes: Application of nacrein-LP20, a pearl polypeptide from Hepu, in the preparation of a drug for promoting the proliferation of human immortalized keratinocytes HaCaT under high glucose conditions.

[0057] In some specific examples, the above application includes: Application of nacrein-LP20, a pearl polypeptide from Hepu, in the preparation of a drug for promoting the migration of HaCaT cells under high glucose conditions.

[0058] In some specific examples, the above application includes: Application of nacrein-LP20, a pearl polypeptide from Hepu, in the preparation of a drug for promoting the repair of HaCaT cells under high glucose conditions.

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

[0060] In some specific examples, the dosage forms of the drugs in the above application include tablets, injections, powders or patches.

[0061] It should be noted that the nacrein-LP20, an active pearl polypeptide in the present invention, can be prepared into different dosage forms by adding pharmaceutically acceptable carriers, such as tablets, injections, powders or patches; the pharmaceutically acceptable carriers can be selected according to the type of dosage form, which is a well-known means in the art.

[0062] To better understand the present invention, the following further clarifies the content of the present invention in combination with specific examples, but the content of the present invention is not limited to the following examples only.

[0063] In the following examples, IBM SPSS Statistics 19.0 statistical software was used for data analysis, and GraphPad Prism 8 software was used for statistical graphing. All experiments were repeated 3 times, and the quantitative results were expressed as mean ± standard deviation (X±S). The independent samples t-test was used for the comparison of quantitative values between two groups, and one-way ANOVA was used for the comparison of quantitative values among multiple groups. In all tests, P<0.05 was considered to indicate significant differences and statistical significance, which was represented by "*" or "#".

[0064] I. Preparation and Characterization of Nacrein-LP20 Polypeptide

[0065] (I) Preparation of Nacrein-LP20 Polypeptide from Pinctada fucata

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

[0067] (II) Characterization of Nacrein-LP20 Polypeptide from Pinctada fucata

[0068] The structure, high-performance liquid chromatography, and mass spectrometry analysis of the Nacrein-LP20 polypeptide from Pinctada fucata are shown respectively in Figure 1a 、 Figure 1b and Figure 1c . Liquid chromatography analysis was performed using an Agilent 1260II liquid chromatography system: using a Symmetrix ODS-R chromatographic column (4.6×250mm, 5μm), 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 injection volume was 20 μL. Mass spectrometry detection was performed using an Agilent single quadrupole mass spectrometer (Agilent G6125B LC / MSD). The mass spectrometry conditions were as follows: ionization mode ESI, probe bias voltage +4.5 kv, nebulizer gas flow rate 1.5 L / min, detector 1.5 kv, nebulizer gas flow rate 1.5 L / min, total flow rate 0.2 mL / min, capillary electrophoresis-electrospray ionization source voltage -20 v, capillary temperature of the electrospray ion source 250°C, and module temperature 200°C.

[0069] II. In Vitro Related Tests of Nacrein-LP20 Polypeptide

[0070] (I) Detection of Hemolytic Activity of Nacrien-LP20 Polypeptide from Pinctada fucata

[0071] The hemolytic activity was detected using 2% (V / V) porcine erythrocytes, specifically including: diluting the Nacrein-LP20 polypeptide to concentrations of 2560 μg / mL, 1280 μg / mL, 640 μg / mL, 320 μg / mL, 160 μg / mL, 80 μg / mL, 40 μg / mL, 20 μg / mL, 10 μg / mL, and 5 μg / mL respectively by the serial dilution method with PBS. 100 μL of the polypeptide solution was taken and added to a 96-well plate as the experimental group. Another 100 μL of PBS and 1.0% (V / V) Triton X-100 were used as the negative control and positive control respectively. 100 μL of the 2% porcine erythrocyte suspension was added to each well, with 3 replicates in each 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). It was incubated in a 37 °C incubator for 3 h, centrifuged at 1500 rpm for 10 min, 150 μL of the supernatant was taken and transferred to a new 96-well plate, and the absorbance was measured at 450 nm, and the hemolysis rate was calculated. Hemolysis rate = [OD of the experimental group 450 - OD of the negative control group 450 / [OD of the positive control group 450 - OD of the negative control group 450 ×100%.

[0072] The detection results are as Figure 2 shown. Column 1 is the negative control with no hemolytic activity; column 2 is the positive control with obvious hemolysis; columns 3 - 12 are the groups of Nacrein-LP20 polypeptide from Pinctada fucata with final concentrations of 1280 μg / mL - 2.5 μg / mL, and their hemolytic activity is almost negligible.

[0073] (2) Detection of the antioxidant activity of Nacrein-LP20 polypeptide from Pinctada fucata

[0074] The total antioxidant capacity of Nacrein-LP20 polypeptide from Pinctada fucata was detected using the Total Antioxidant Capacity Assay Kit (ABTS rapid method) from Beyotime (Product No.: S0121); the ability of Nacrein-LP20 polypeptide from Pinctada fucata to scavenge hydroxyl radicals and DPPH radicals was detected using the Hydroxyl Radical Assay Kit (A018-1-1) and DPPH Radical Scavenging Capacity Assay Kit (A153-1-1) from Nanjing Jiancheng Bioengineering Institute, and the positive control was set as vitamin C at the same concentration.

[0075] The detection results are as Figures 3a to 3d shown. The standard curve equation of the standard product 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 measured can be calculated. When the concentration of Nacrein-LP20 polypeptide from Hepu pearl is 0.25 mg / mL, its total antioxidant capacity is equivalent to 1.7861 mM Trolox (see Figure 3b ); Moreover, Nacrein-LP20 polypeptide from Hepu pearl has significant ability to scavenge hydroxyl radicals and DPPH radicals, almost comparable to vitamin C, and its IC 50 values are 151.36 μg / mL and 1.39 μg / mL respectively (see Figure 3c and Figure 3d ), indicating that Nacrein-LP20 polypeptide from Hepu pearl has strong antioxidant activity in vitro.

[0076] (III) CCK-8 assay of Nacrein-LP20 polypeptide from Hepu pearl

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

[0078] HaCaT cells were seeded into 96-well plates at 1×10 5 cells per well. The Nacrein-LP20 polypeptide from Hepu pearl was dissolved in DMEM (low glucose) (C11885500BT, Gibco) or DMEM (high glucose) complete medium (C11995500BT, Gibco) to prepare a stock solution of 1 mg / mL. 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 cells in the 96-well plates to be measured were replaced with the same medium, 100 μL per well, and 10 μL of CCK8 reagent (BS350B, Biosharp, China) was added to each well. The plates were incubated in the incubator for 4 h, and the absorbance of each well was measured at 450 nm using a multifunctional microplate reader (Synergy H1, BioTek, USA). The 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 detection results are shown in Figure 4. Under low-glucose conditions, compared with the control group, there were no significant changes in the survival rate of cells treated with Nacrein-LP20 polypeptide at 10 μg / mL to 1280 μg / mL for 24 h and 48 h (see Figure 4a and Figure 4b ); under high-glucose conditions, compared with the control group, the survival rate of cells treated with Nacrein-LP20 polypeptide at 10 μg / mL to 640 μg / mL for 24 h and 48 h was significantly increased, and there was no significant change in the survival rate of cells treated with 1280 μg / mL of Nacrein-LP20 polypeptide for 24 h and 48 h (see Figure 4c and Figure 4d ).

[0080] (IV) Scratch experiment of Nacrein-LP20 polypeptide from Pinctada fucata

[0081] Collect the above-mentioned HaCaT cells in the logarithmic growth phase, resuspend them with DMEM (high-glucose) complete medium, inoculate them into a 6-well plate at 5×10 5 cells per well, and the final total volume of the medium in each well is 2 mL. After culturing in a 37°C, 5% CO2 incubator for 24 h, use a 200 μL pipette tip to make a scratch, aspirate the medium, wash the cells 3 times with PBS to remove the scratched cells, and set up a control group (serum-free high-glucose medium), a positive group (EGF diluted to 10 ng / mL with serum-free high-glucose medium), and an experimental group (80 μg / mL, 160 μg / mL, 320 μg / mL, and 640 μg / mL of Nacrein-LP20 polypeptide from Pinctada fucata). Take microscope photos at 0, 20 h, and 40 h to observe the results.

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

[0083] III. Animal experiment of Nacrein-LP20 polypeptide

[0084] The preparation method and drug administration of the full-thickness skin excision wound model for normal rats are as follows: (1) Experimental grouping: 21 SPF-grade male SD rats (180 g - 200 g) 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 Liquid), and experimental groups: Nacrein-LP20 polypeptide solutions with concentrations of 80 μg / mL - 1280 μg / mL; (2) Model preparation: The rats were anesthetized by inhaling isoflurane with an anesthesia machine. The back hair was shaved off with an electric clipper and then completely removed with Veet hair removal cream. Circular wounds (wound size: 2 × 2 cm) were prepared on both sides of the rat's back (2 cm on each side of the spinal column on the back of the animal, 2 cm below the scapula). The wounds were cleaned with 75% alcohol and disinfected with iodine tincture to keep the wounds clean; (3) Treatment method and observation: After the wound preparation was completed, drugs were administered immediately, once a day, with a volume of 100 μL each time. The wounds were photographed every other day and the drugs were continuously administered for 11 days.

[0085] The preparation method and drug administration of the full-thickness skin excision wound model for diabetic rats are as follows: (1) Establishment of diabetic rat model: After one week of adaptive feeding of SD rats (body weight 170 - 250 g), they were fasted but allowed to drink water for 12 h, and then intraperitoneally injected with 1% streptozotocin at a dose of 65 mg / kg. The rats' states were observed daily and their blood glucose levels were measured. If the blood glucose concentration was greater than 16.7 mmol / L after 7 days, the model was considered successfully established. (2) The establishment of the wound model was the same as above. A normal group, a diabetic model group, and experimental groups (160, 320, and 640 μg / mL Nacrein-LP20 polypeptide) were set up, with 3 rats in each group. 100 μL of drugs were administered every day, and the wound size was photographed every other day.

[0086] (I) Evaluation of wound healing ability

[0087] The full-thickness skin excision wound model was used to evaluate the effect of Nacrein-LP20 polypeptide from Heping pearl on the wound healing ability of rat skin. The results are as Figure 6a and Figure 6b shown. Compared with the normal saline control group, both the positive group of Kangfuxin Liquid and the Nacrein-LP20 polypeptide treatment group of Heping pearl could significantly promote the healing of the full-thickness skin excision wounds in rats.

[0088] Among them, Figure 6aRepresentative images after drug treatment show that scabs began to form in each group on day 3. Furthermore, the 640 μg / mL Nacrein-LP20 treatment group significantly reduced the wound area compared to the saline control group on day 3. Over time, both the positive Kangfuxin solution and Nacrein-LP20 treatment groups showed a significant effect in promoting skin wound contraction, with wound closure rates significantly higher than those in the control group. ImageJ was used to monitor and quantify wound closure rates on days 0, 3, 5, 7, 9, and 11 after injury ( Figure 6b ), the statistical results showed that 11 days after surgery, the wound healing rates of the positive group Kangfuxin solution 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. The low and medium concentrations are as effective as the positive group Kangfuxin liquid, while the high concentration group is even better than the Kangfuxin liquid treatment group in wound repair.

[0090] (2) Hematoxylin and eosin (H&E) and Masson staining

[0091] On the 11th day after surgery, the rats underwent wound histological analysis, which included the following steps: 11 days after surgery, wound tissue and surrounding normal skin (a total of 2 cm in size) were collected, fixed with 4% formaldehyde, dehydrated with gradient ethanol, transparentized with xylene, and embedded in paraffin; tissue specimens were stained with H&E and Masson staining; 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 imaged as much as possible to fill the entire field of view during imaging to ensure consistent background light in each photo; 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 indicate that compared with the control group, both the positive group and the groups treated with nacrein-LP20 polypeptide from Pinctada fucata martensii significantly promoted wound healing, and the wound diameter was significantly shortened. On the 11th day, the wound diameter of the positive group treated with Kangfuxin Liquid was 5.10 ± 0.20 mm, and the wound diameters of the groups treated with 160 μg / mL, 320 μg / mL, and 640 μg / mL nacrein-LP20 polypeptide were 5.57 ± 0.40 mm, 3.71 ± 0.10 mm, and 4.86 ± 0.33 mm respectively. Among them, the wound diameters of the groups treated with 320 and 640 μg / mL nacrein-LP20 polypeptide were smaller than that of the positive group, while the wound diameter of the normal saline control group was 9.30 ± 0.41 mm. It can be seen that the drug treatment groups can significantly promote the wound healing of rats, and at a certain concentration, the effect of nacrein-LP20 polypeptide from Pinctada fucata martensii in promoting wound healing is better than that of the positive control group, Kangfuxin Liquid (see Figure 7b ).

[0094] (2) Masson staining results

[0095] Collagen, as an important component of the extracellular matrix (ECM), is necessary for filling wounds, increasing tensile strength, and forming the matrix framework for angiogenesis. Masson staining was used to detect the regeneration and deposition of collagen fibers in the wound tissue. The results are as Figure 8 shown. On the 11th day, the collagen production and deposition in the positive group treated with Kangfuxin Liquid and the groups treated with nacrein-LP20 polypeptide from Pinctada fucata martensii were denser and more organized compared with the normal saline control group, indicating that the relative expression level of collagen in the drug treatment groups was significantly higher than that in the control group.

[0096] (3) Safety detection

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

[0098] For the total cholesterol content, the detection results are as Figure 9aAs shown, compared with the normal group, except for the group treated with 320 μg / mL of Heping pearl Nacrein-LP20 polypeptide in the wound group, which showed no significant difference, the others all increased significantly. However, compared with the normal saline control group, there was no significant difference in the low- and high-concentration Heping pearl polypeptide treatment groups, and the medium-concentration treatment group could significantly reduce the TC level.

[0099] For triglycerides, the test results are as Figure 9b 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 Heping pearl polypeptide treatment groups could significantly reduce the triglyceride content. The results showed that compared with the normal saline group, the Heping pearl Nacrein-LP20 polypeptide treatment group did not cause obesity and cardiovascular abnormalities in rats.

[0100] In addition, the test results of serum uric acid are as Figure 9c shown. Compared with the normal group, each group decreased (P<0.05). Compared with the normal saline group, there was no significant difference in the Heping pearl polypeptide treatment group (P>0.05), indicating that the polypeptide treatment did not lead to the occurrence of diseases such as hyperuricemia or gout.

[0101] The liver contains glutamic oxaloacetic transaminase, which is an index for liver function examination in medical clinics and is used to judge whether the liver is damaged. The test results are as Figure 9d shown. There was no significant difference in the serum GOT levels of rats in each group (P>0.05), indicating that the liver cell membrane was intact and there was no significant difference in the serum transaminase activity.

[0102] In addition, during the experiment, the rats treated with the polypeptide did not have liver damage, that is, the Heping pearl Nacrein-LP20 polypeptide did not cause any obvious toxic and side effects on the rats during the experiment, indicating that it is safe and effective for treating wound healing.

[0103] (IV) Evaluation of the ability of diabetic wound healing

[0104] The full-thickness excision wound model was used to evaluate the effect of Heping pearl Nacrein-LP20 polypeptide on the skin wound healing ability of diabetic rats. The results are as Figure 10a and Figure 10b shown. Compared with the diabetic model group, the Heping pearl Nacrein-LP20 polypeptide treatment groups could significantly promote the healing of the full-thickness excision wounds in rats.

[0105] Among them, Figure 10aRepresentative images after drug treatment showed that scabbing began in the drug-administered group and the normal group on the 3rd day. On the 3rd day, compared with the model group, the wound area in the drug-administered group was significantly reduced. Over time, the drug-administered group showed an obvious effect of promoting skin wound contraction, and the wound closure rate was significantly higher than that of the model group. Monitoring and quantification were performed using ImageJ at 0, 3, 5, 7, 9, 11, and 13 days after injury( Figure 10b ). Statistical results showed that on the 13th day after surgery, the wound healing rates of the Nacrein-LP20 treatment groups at 160 μg / mL, 320 μg / mL, and 640 μg / mL were 98.48% ± 0.45%, 94.55% ± 4.68%, and 97.53% ± 0.15%, respectively, while the wound closure rate of the model group only reached 87.35% ± 6.00%( Figure 10b )(P < 0.05).

[0106] The above results indicate that the Nacrein-LP20 polypeptide from Pinctada fucata 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 solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Application of nacre polypeptide Nacrein-LP20 in the preparation of a drug for promoting wound healing, wherein the amino acid sequence of nacre polypeptide Nacrein-LP20 is shown as SEQ ID NO:

1.

2. The application according to claim 1, wherein The application includes: application of nacre polypeptide Nacrein-LP20 in the preparation of a drug for promoting diabetic wound healing.

Images