Application of a polypeptide in preparing a product for preventing or treating skin damage diseases

Abstract

Provided is a use of a polypeptide in preparing a product for preventing or treating skin damage diseases, wherein the polypeptide is Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof.

Description

Technical Field

[0001] The present invention relates to the use of a polypeptide in the preparation of a product for preventing or treating skin damage diseases. The polypeptide of the present invention has significant effects in preventing and / or treating skin damage diseases, especially in preventing and / or treating chronic difficult-to-heal wounds on the body surface, acute and / or chronic skin diseases. Background Art

[0002] Human skin is composed of the epidermis, dermis, and subcutaneous tissue, and contains accessory organs (sweat glands, sebaceous glands), as well as blood vessels, lymphatic vessels, nerves, and muscles. The skin is the outermost organ of the human body, and it is also the largest organ in the human body. It is also the one that has the most frequent and closest contact with the outside world. It covers the surface of the human body. In addition to protecting the body, regulating body temperature, and resisting external invasion, it also has sensory functions and plays an important role in protecting human health. Therefore, the skin is called the first line of defense of the human body. Because the skin covers the surface of the human body, it is easily damaged by external forces, chemical stimulation, microbial infection, etc. Skin damage refers to abnormal manifestations that can be seen or touched on the skin and mucous membranes. Skin damage can easily bring a double blow to patients both psychologically and physiologically.

[0003] In recent years, among the diseases caused by skin injuries, chronic and difficult-to-heal wounds on the body surface have received much attention. Chronic and difficult-to-heal wounds on the body surface, also known as refractory ulcers, are a common and difficult-to-heal disease. Local tissue defects, liquefaction, infection, and necrosis caused by various reasons lead to impaired proliferation of endothelial progenitor cells, resulting in impaired vascular regeneration, insufficient wound perfusion, metabolic disorders, and delayed epithelial cell formation. The normal healing process of the wound is disrupted, thus forming chronic ulcers.

[0004] Chronic and difficult-to-heal wounds on the body surface are common complications of diabetes, peripheral vascular disease, and radiotherapy. The International Wound Healing Society defines it as a wound that cannot achieve anatomical and functional integrity through a normal, orderly, and timely repair process. Clinically, it refers to wounds formed for various reasons that have not healed after more than one month of regular treatment and have no obvious healing tendency. Wound healing goes through three stages: ① Inflammation stage; ② Proliferation stage; ③ Maturation and reconstruction stage. Chronic wounds are hindered from prolonging the inflammatory period, which hinders the proliferation of vascular endothelial cells and fibroblasts and the deposition of collagen matrix.

[0005] Common chronic and difficult-to-heal wounds include diabetic foot ulcers, pressure ulcers, vascular ulcers, neurodystrophy ulcers, infectious ulcers, traumatic ulcers, autoimmune ulcers, cancerous ulcers, radiation ulcers, etc. Their incidence increases with age, the pathogenesis is complex, and the course of the disease is long.

[0006] Diabetic foot ulcers are mainly caused by the underlying disease of diabetes, which triggers nerve and blood vessel diseases, leading to ischemic necrosis of skin tissue or skin tissue infection, or damage due to pressure on the bearing point, which causes incomplete skin, exposing subcutaneous fat, muscle tissue, and even bones, forming ulcers. The harm caused by diabetic foot ulcers can cause itching, no sweating, dryness, pigmentation of the skin, slight pain in the feet, insensitivity, intermittent claudication, joint deformity, and skin surface ulcers.

[0007] Pressure ulcers are caused by local skin ischemia, hypoxia, and nutrient deficiency due to pressure on local tissues of the body, which results in damage and necrosis of tissues. Pressure ulcers often occur in the bony protrusions of bedridden elderly patients, such as the sacrum, occipital region, and heel region. These areas have little soft tissue and weak pressure resistance, and ulcers can occur if the body is fixed in position and compressed for a long time. Pressure ulcers not only bring pain to patients and delay recovery from the disease, but in severe cases, they can be life-threatening due to secondary infection and sepsis.

[0008] Vascular ulcers are lower limb ulcers caused by varicose veins and vasculitis of the lower limbs. They are more common in the distal calves and ankles and are late complications of chronic lower limb dysfunction. Venous return is severely obstructed, local venous pressure increases, and edema leads to oxygen diffusion disorders and skin nutrient deficiencies. Patients have a long history of venous primary disease. The wound surface is generally single and relatively superficial, with a dark wound base. The skin around the wound is rough and has obvious pigmentation. The local skin temperature is very low. Conventional wound dressing changes are very ineffective, and even if split-thickness skin grafts are performed, the efficacy is extremely unreliable.

[0009] Current research suggests that skin wound repair is a complex process involving the participation, high coordination, and mutual regulation of multiple factors, including keratinocytes, fibroblasts, vascular endothelial cells, inflammatory cells, extracellular matrix, cytokines, and growth factors. Currently, drugs commonly used for the prevention or treatment of skin injury diseases include recombinant human epidermal growth factor, etc. Although they have certain preventive and / or therapeutic effects, they still have disadvantages such as slow absorption, long treatment cycle, and unclear therapeutic effect. In particular, when applied to chronic, difficult-to-heal wounds on the body surface and acute and / or chronic skin diseases, the therapeutic effect is minimal. Summary of the invention

[0010] In order to overcome the deficiencies and defects of the prior art, the object of the present invention is to provide an application of a polypeptide in the preparation of a product for preventing or treating skin damage diseases.

[0011] In a first aspect, the present invention provides a use of a polypeptide in preparing a product for preventing or treating skin damage diseases, wherein the polypeptide is Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof; or provides a method for preventing or treating skin damage diseases, the method comprising topically applying a product comprising Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof to a skin damage site; or provides a product comprising Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof, which is used for preventing or treating skin damage diseases.

[0012] Furthermore, skin injury diseases include wounds, ulcers, dermatitis, eczema, urticaria, polymorphic light eruption, herpes, acne, impetigo, chloasma, vitiligo, lupus erythematosus, dermatomyositis, scleroderma, folliculitis, scabies, onychomycosis, varicella, exanthema infantum, warts, carbuncle, furuncle or ringworm.

[0013] Further, dermatitis includes atopic dermatitis, contact dermatitis, neurodermatitis, seborrheic dermatitis, hormone-dependent dermatitis or stasis dermatitis.

[0014] Furthermore, the wound surface includes chronic and difficult-to-heal wounds on the body surface.

[0015] Furthermore, chronic difficult-to-heal wounds on the body surface include diabetic foot ulcers, pressure ulcers, vascular ulcers and infectious ulcers.

[0016] Further, the product includes medicine, skin care product or cosmetics.

[0017] Furthermore, the product is an external preparation.

[0018] Furthermore, the dosage form of the external preparation includes solution, emulsion, gel, cream, spray, mask or dressing.

[0019] Furthermore, the polypeptide plays a role in wound healing by promoting proliferation of human immortalized keratinocytes, human microvascular endothelial cells, fibroblasts, glial cells and tissue and / or blood vessel regeneration.

[0020] Further, the physiologically compatible salt refers to a salt form that is physiologically compatible (i.e., pharmacologically acceptable) and substantially non-toxic to the individual to whom the compounds of the invention are administered. Physiologically compatible salts of the compounds of the invention include conventional and stoichiometric acid addition salts or base addition salts formed from suitable, non-toxic organic or inorganic acids or inorganic bases.

[0021] The beneficial effects of the polypeptide provided by the present invention in the preparation of products for preventing or treating skin damage diseases, especially chronic and difficult-to-heal wounds on the body surface:

[0022] The above-mentioned polypeptide of the present invention, compared with the recombinant human epidermal growth factor commonly used in the treatment of skin injury diseases in the prior art, not only has a significant wound healing effect on skin ulcers and injuries, but also because the peptide chain of the present invention is short, the skin absorption is faster and better, the in vivo and in vitro stability is good, and it has a significant proliferative effect on human immortalized keratinocytes, human microvascular endothelial cells, fibroblasts, glial cells, and tissue and vascular regeneration, and thus has a significant preventive and / or therapeutic effect on acute and / or chronic skin diseases, chronic difficult-to-heal wounds on the body surface (diabetic foot ulcers, pressure ulcers, vascular ulcers and infectious ulcers). The above-mentioned polypeptide of the present invention is suitable for preparing products for preventing or treating skin injury diseases, which can induce dedifferentiation of cells near the injury, and the dedifferentiated cells regain stem cell morphology and cell division potential, forming new units to repair the damaged wound, and can produce positive treatment and prevention effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The results show that the peptide promotes the proliferation of HaCaT cell lines;

[0024] Figure 2 The results show that the peptide promotes the proliferation of HMEC-1 cell line;

[0025] Figure 3 The results show that the polypeptide promotes the proliferation of Balb-3T3 cell line;

[0026] Figure 4 The results show that the peptide promotes the proliferation of RSC96 cell line;

[0027] Figure 5 The effect of the peptide on the regeneration of the zebrafish tail fin is shown;

[0028] Figure 6 The effect of the peptide on the area of ​​regenerated blood vessels in microvessel-deficient zebrafish is shown;

[0029] Figure 7 The effect of the peptide on the number of regenerating blood vessel branches in microvessel-deficient zebrafish is shown;

[0030] Figure 8 The results show the effect of peptides on the rate of new granulation tissue formation in STZ-induced diabetic rats on day 3 after injury. DETAILED DESCRIPTION

[0031] The following is an explanation of the present invention in combination with specific experiments, which is not intended to limit the scope of protection of the present invention.

[0032] Example 1: Chemical synthesis of polypeptides

[0033] The peptides were synthesized using conventional solid phase synthesis methods, which involved multiple cycles of resin swelling, substitution, deprotection, washing, amino acid dissolution, amino acid activation and condensation, washing, re-deprotection, and finally cleavage and side chain deprotection.

[0034] Abbreviations: HBTU stands for benzotriazole-N, N, N′, N′-tetramethyluronium hexafluorophosphate; Methanol stands for methanol; Tert-Butyl methyl ether stands for methyl tert-butyl ether; Ethanol stands for ethanol; AA stands for amino acid; Cl-2-Cl-Resin stands for 2-chlorotrityl chloride resin; Fmoc-Aa(n) and the like stand for 9-fluorenylmethoxycarbonyl amino acid; DIPEA stands for N, N-diisopropylethylamine; DCM stands for dichloromethane; PIP stands for piperidine; DMF stands for N, N-dimethylformamide; HOBt stands for 1-hydroxybenzotriazole; DIC stands for N, N′-diisopropylcarbodiimide; TFA stands for trifluoroacetic acid; TIPS stands for triisopropylsilane.

[0035] The methods for peptide synthesis and purification are as follows:

[0036] Step 1. Preparation of fully protected peptide resin

[0037] (1) Resin swelling: Weigh 2.0192 g of 2-Chlorotrityl Chloride Resin (S = 0.73 mmol / g), add it into a synthesis tube with a sieve plate, swell it with 40 ml of dichloromethane for 30 min, and remove the dichloromethane by suction filtration.

[0038] (2) Preparation of Fmoc-Asp(OtBu)-resin: Weigh Fmoc-Asp(OtBu)-OH and DIPEA at a molar ratio of 1:1.5:1.65, dissolve in 20 ml of dichloromethane, and add to a synthesis tube. Bubble with N2 at room temperature for 1 to 3 hours, directly add 2 ml of methanol to the reaction solution to seal for 30 minutes. Then wash with dimethylformamide 4 times, 25 ml / time, and drain the resin.

[0039] (3) Removal of Fmoc protecting group: 20 ml of 20% piperidine-DMF (v / v) solution was added to the reactor, N2 was bubbled for 20 min, and then the mixture was drained; then the mixture was washed with dimethylformamide for 6 times, 25 ml / time, 3 min / time, and the removal of Fmoc was detected by the ninhydrin method.

[0040] (4) Amino acid preactivation: 4.38 mmol of Fmoc-protected amino acid, 5.26 mmol of HOBt and 460 mmol of DIC were added to a 250 ml round-bottom flask, dissolved in 20 ml of 1:1 DCM-DMF (v / v), and preactivated for 30 to 60 min under stirring at -5 to 0°C ice bath.

[0041] (5) Amino acid connection: Pour the activated protected amino acid solution into the reactor and add an appropriate amount of DCM to clean the utensils. Bubble N2 at room temperature for 1 to 3 hours. Use the ninhydrin method to check whether the amino acid connection is complete. If complete, drain. Wash the resin with dimethylformamide 4 times, 25 ml / time, 3 min / time, and drain. The amount of each amino acid, condensation agent and specific reaction time are shown in Table 1.

[0042] (6) After the first amino acid is condensed, steps (3) to (5) are repeated to extend the peptide chain in the order of amino acids until the last amino acid is coupled.

[0043] (7) The resin peptide was washed with dichloromethane 6 times, 25 ml / time, 3 min / time, and then dried.

[0044] Amino acid name AA / eq Amino acid dosage / g HOBt / g DIPEA / g DIC / g Fmoc-L-Leu-OH 4.38 1.55 0.71 0.57 0.58 Fmoc-L-Pro-OH 4.38 1.48 0.71 0.57 1.16 Fmoc-L-Val-OH 4.38 1.49 0.71 0.57 0.58 Fmoc-L-Pro-OH 4.38 1.48 0.71 0.57 1.16 <![CDATA[Fmoc-L-Glu(OtBu)-OH H2O]]> 4.38 1.94 0.71 0.57 1.16 <![CDATA[Fmoc-L-Ala-OH H2O]]> 4.38 1.44 0.71 0.57 1.16 <![CDATA[Fmoc-L-Ala-OH H2O]]> 4.38 1.44 0.71 0.57 1.16 Fmoc-L-Pro-OH 4.38 1.48 0.71 0.57 1.16

[0045] Step 2: Cutting and deprotection

[0046] (1) Add 50 ml of a cutting agent (TFA:TIPS:H2O=95:2.5:2.5, v / v) to the synthesis tube of step 1, and allow N2 to bubble for reaction for 1.5-3 hours.

[0047] (2) After the cleavage reaction is completed, the cutting agent is filtered into a 250 ml round-bottom flask. After vacuum concentration to one-third of the original volume of the cutting agent, 10 times the current volume of methyl tert-butyl ether is added and stirred for 30 minutes. The resulting mixed solvent is filtered and washed three times with 30 ml of methyl tert-butyl ether respectively, and the resulting crude peptide product is placed in a sand core funnel and blown dry with N2 in a fume hood to evaporate the solvent until the crude peptide is in powder form.

[0048] Step 3: Purification (salt exchange) and freeze-drying

[0049] The crude peptide obtained in step 2 was purified by HPLC using the following chromatographic parameter condition A. Specifically, the crude peptide obtained in step 2 was dissolved in water and / or acetonitrile and filtered through a 0.45 μm filter membrane; injected; gradient eluted with an acetonitrile-water mobile phase; the target peptide eluate was collected; and finally concentrated by rotary evaporation.

[0050] Chromatographic parameter conditions A:

[0051] Chromatographic column: YMC-Actus Triart C18 30*250mm;

[0052] Eluent A: 0.1% (v / v) TFA / H2O;

[0053] Eluent B: acetonitrile;

[0054] Flow rate: 25 ml / min;

[0055] UV detection wavelength 220nm;

[0056] Table 2 Gradient elution conditions

[0057]

[0058]

[0059] Then, the product obtained in the above steps was salt-exchanged using HPLC method with the following chromatographic parameter conditions B: 95% A1 + 5% B was used to balance the chromatographic column; then the sample was injected; then 95% A2 + 5% B was used to balance the chromatographic column; gradient elution was performed with A1 and B; the target peptide eluate was collected; finally, the eluted solution was concentrated by rotary evaporation and freeze-dried to obtain the polypeptide. The structure of the polypeptide was confirmed by MS, 1 H-NMR confirmed.

[0060] Chromatographic parameter condition B:

[0061] Chromatographic column: YMC-Actus Triart C18 30*250mm

[0062] Eluent A1: 0.1 M acetic acid

[0063] Eluent A2: 0.025M acetic acid + 0.1M ammonium acetate

[0064] Eluent B: acetonitrile

[0065] Flow rate 25ml / min

[0066] UV detection wavelength 220nm

[0067] Time min Eluent A1 (%) Eluent B (%) 0 95 5 5 95 5 35 70 30

[0068] Peptide 1 H-NMR is as follows:

[0069] 1H NMR (600MHz, DMSO) δ8.25 (s, 1H), 8.09 (d, J = 7.5Hz, 1H), 7.94 (d, J = 7.6Hz, 1H), 7.89 (d, J = 8.3Hz, 2 H), 4.53-4.46 (m, 1H), 4.39 (dd, J=8.3, 4.2Hz, 1H), 4.34 (dd, J=8.4, 3.8Hz, 1H), 4.31-4.19 (m, 3H) , 4.13 (dd, J=15.1, 7.7Hz, 1H), 3.71-3.49 (m, 5H), 2.94-2.77 (m, 2H), 2.33-2.20 (m, 2H), 2.06-1.7 7 (m, 13H, AcOH), 1.77-1.56 (m, 6H), 1.46 (t, J=7.3Hz, 2H), 1.25-1.11 (m, 6H), 0.95-0.76 (m, 12H).

[0070] MS of peptide: 793.4, 397.3 (doubly charged).

[0071] Amino acid sequence of the polypeptide: Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu.

[0072] The following substances were used in the examples of this application:

[0073] A polypeptide, unless otherwise indicated, is a polypeptide having an amino acid sequence of Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu.

[0074] GeneTime, also known as GeneTime, has a generic name: Recombinant Human Epidermal Growth Factor Derivative for External Use, Liquid (I), and is manufactured by Shenzhen Huashengyuan Gene Engineering Development Co., Ltd. Its ingredients: The active ingredient is recombinant human epidermal growth factor (rhEGF), with 10% glycerol and 1.0 mannitol as protective agents. The rhEGF in GeneTime promotes the synthesis of DNA, RNA and hydroxyproline in the repair process of skin and mucosal wound tissue, accelerates the formation of wound granulation tissue and epithelial cell proliferation, and thus shortens the healing time of the wound.

[0075] Kangfuxin Ye; Manufacturer: Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd.; It is a solution of dried extracts of American cockroaches; It is used for wounds such as wounds, trauma, ulcers, fistulas, burns, scalds, and bedsores.

[0076] Example 2 Effect of polypeptide on healing of skin ulcer in db / db mice

[0077] Experimental animals: diabetic model mice (db / db male mice), normal mice (m / m male mice), SPF grade, 8-10 weeks, body weight 40-50 g, Changzhou Cavens Laboratory Animal Co., Ltd., Experimental Animal Production License No.: SCXK (Su) 2016-0010, Experimental Animal Use License No.: SYXK (Shanghai) 2020-0038.

[0078] Experimental method: After the blood sugar of db / db mice reached the standard (blood sugar ≥ 16.7mmol / L after fasting for 4 hours), the group experiment was started. The animals were randomly divided into 5 groups, 10 in each group, namely normal control group, model control group, Kangfuxin liquid group (40μL / mouse), Jinyin peptide group (40IU / cm 2 ), test peptide group (30μg / cm 2 ). After the mice were anesthetized by inhalation of isoflurane, a circular wound with a diameter of about 12 mm was cut on the back of the mouse skin with a cutter, deep into the fascia layer. The wound was photographed and the wound area was recorded as the baseline value (the day of the wound was recorded as Day 0). The next day (Day 1), each group of animals was given drug treatment according to the appropriate treatment, and all were applied externally to the skin wound. The normal control group and the model control group were given 40 μL of normal saline, and the Jinyin peptide group (40 IU / cm 2 ) were given 40 μL of recombinant human epidermal growth factor solution diluted with normal saline (Shenzhen Huashengyuan Gene Engineering Development Co., Ltd.), and the test peptide group (30 μg / cm 2 ) were given 40 μL of peptide solution of corresponding concentration prepared with normal saline once a day; Kangfuxin liquid group was given 40 μL Kangfuxin liquid (Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd.) twice a day. The drug was given continuously every day until the wound was basically healed. The skin wound was measured and photographed twice a week, the wound area was calculated using Image-J software, and the wound healing of animals in each group was observed.

[0079] Experimental results: The experimental results are shown in Table 4:

[0080] Table 4 Wound healing rate (%, Mean ± SD, n = 10)

[0081]

[0082] Note: Compared with the model control group, *: P < 0.05; **: P < 0.01;

[0083] The results in Table 4 show that the polypeptide significantly promoted the healing of skin wounds in diabetic mice on the 14th day, and the effect was better than that of the Kangfuxin liquid group, and the effect of the polypeptide group was equivalent to that of the Jinyin peptide group.

[0084] Example 3 Effect of polypeptide on healing of acute mechanical skin injury in rats

[0085] Experimental animals: SD male rats, SPF grade, 6 weeks old, weighing 180-220 g, Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; Animal License No.: SCXK (Beijing) 2016-0006; Certificate No.: NO.110011210104746538.

[0086] Experimental method: SPF SD rats were kept in clean and sterilized cages, and water, feed and bedding were changed regularly every day. The temperature was kept at 20-26℃ and the humidity was 40%-70%. The rats were kept for one week to adapt to the environment. The animals were randomly divided into three groups: model control group (normal saline), Jinyin peptide group (40IU / cm 2 , recombinant human epidermal growth factor solution for external use, Shenzhen Huashengyuan Gene Engineering Development Co., Ltd.), test peptide group (8μg / cm 2 ), 6 rats in each group. After the rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital, the hair at 1 cm from the edge of the wound was cut off, the wound area was first disinfected with iodine, and then the wound area was locally disinfected with 75% alcohol. A circular full-thickness skin wound with a diameter of 1.5 cm was made from the middle of the ear line to the lower back 4 cm near the neck side with the spine as the midline, deep to the muscle layer. The surrounding skin was fixed with a rubber ring of the same size to form an acute mechanical injury animal model. After modeling, the wounds of the rats were exposed and kept in a single cage. When changing the dressing, the wounds were first cleaned with iodine, and then the wounds were rinsed and dried with sterile saline. Rats in each group were given 40 μL of the corresponding drug solution, and the wounds were topically applied once a day. On the 0th, 3rd, 7th, 10th, and 14th days of administration, the wound images of each group of rats were taken, and the wound area was calculated using image analysis software (ImageJ), and the wound healing rate was calculated according to the formula.

[0087] Experimental results: The experimental results are shown in Table 5:

[0088] Table 5 Wound healing rate (%, Mean ± SD, n = 6)

[0089]

[0090] Note: Compared with the model control group, *: P < 0.05; **: P < 0.01;

[0091] The results in Table 5 show that the polypeptide group showed a significant effect in promoting wound healing after mechanical injury of rat skin on the 14th day, and the effect was significantly better than that of the Jinyin peptide group.

[0092] Example 4: Promoting effect of polypeptide on proliferation of HaCAT cells

[0093] Experimental method: The concentration of human immortalized keratinocytes (HaCaT cells) was adjusted to 1.0×10 5 ~5.0×10 5 / mL for subculture, and culture at 37°C, 5% CO2 for 24 to 36 hours for biological activity detection. Digest the cells with 0.25% trypsin for 5 minutes, add 1640 whole blood medium with a volume of more than 1 times the trypsin volume to terminate the digestion, collect the cell suspension, centrifuge at 1000RPM for 3 minutes, discard the supernatant, add 2mL of 1640 whole blood medium to resuspend the cells, take 20uL of the cell suspension, stain with AOPI, and then use a cell counter to detect the concentration of cells in the suspension, and use 10% serum concentration of 1640 medium to make a concentration of 5×10 4 / mL was inoculated into a 96-well cell culture plate, 100 μL per well, i.e. 5000 cells / well, and cultured overnight at 37°C, 5% CO2.

[0094] After 24 hours, the original culture medium was discarded, and 100 μL of different concentrations of peptide solutions prepared with 1% serum concentration of 1640 culture medium were added; at the same time, an EGF control group was set up, that is, 100 μL of recombinant human epidermal growth factor (EGF) solution prepared with 1% serum concentration of 1640 culture medium was added, with a final concentration of 100 ng / mL; a model control group was added, that is, an equal volume of 1% serum concentration of 1640 culture medium was added. The cells were cultured at 37°C and 5% CO2 for 72 hours, and the proliferation of HaCaT cells was detected using a CCK8 kit.

[0095] Experimental results: The experimental results are shown in Figure 1 As shown (*: P < 0.05; **: P < 0.01). Figure 1 The results of the study showed that after 72 hours of action, the peptide had a significant proliferation-promoting effect on HaCaT cells (human immortalized keratinocytes), one of the main cell types involved in the wound repair process.

[0096] Example 5: Promoting effect of polypeptide on HMEC-1 cell proliferation

[0097] Experimental method: Human microvascular endothelial cells (HMEC-1 cells) were cultured in 10% serum medium at 37°C and 5% CO2. The medium was changed every 1-2 days and the cell concentration was controlled to be 4×10 6 Cells were collected and diluted to 4×10 cells / mL using serum-free medium. 4 cells / mL; inoculated into a 96-well cell culture plate, 100uL per well, i.e. 4000 cells / well, and cultured at 37°C, 5% CO2 until the cells adhered to the wall.

[0098] The polypeptide was dissolved in PBS solution to prepare a mother solution with a concentration of 1 mg / mL. The mother solution of the polypeptide was prepared with 0% serum culture medium to the test concentration as the experimental group. The same method was used to prepare recombinant human vascular endothelial growth factor VEGF (100 ng / mL) as a positive control. The blank control group was added with an equal amount of 0% serum-free medium without drugs, added to 5 replicates, and incubated at 37°C, 5% CO2 for 48 hours. CCK-8 was used to detect cell proliferation: serum-free culture medium containing 10% CCK-8 was added to each well, and the absorbance was measured at 450nm using an enzyme reader after incubation in the incubator for 2 hours.

[0099] Experimental results: The experimental results are shown in Figure 2 As shown (*: P < 0.05; **: P < 0.01). Figure 2 The results of the study showed that the peptide had a significant proliferative effect on endothelial cells (HMEC-1 cells (human microvascular endothelial cells), one of the main cell types involved in the wound repair process.

[0100] Example 6: Effect of polypeptide on the proliferation of Balb-3T3 cells

[0101] Experimental method: Mouse embryonic fibroblasts (Balb-3T3 cells) were cultured in 10% serum medium at 37°C and 5% CO2. The medium was changed every 1-2 days and the cell concentration was controlled to be 4×10 6 Cells were collected and maintained in 2.5% FBS medium at 3 × 10 4 cells / mL; inoculated into a 96-well cell culture plate, 100uL per well, i.e. 3000 cells / well, and cultured at 37°C, 5% CO2 until the cells adhered to the wall.

[0102] The polypeptide was dissolved in 2.5% FBS maintenance medium to prepare a mother solution with a concentration of 1 mg / mL. The polypeptide mother solution was prepared with 2.5% FBS maintenance medium to the test concentration as the experimental group. The same method was used to prepare recombinant human basic fibroblast growth factor FGF (50 ng / mL), and recombinant human platelet-derived growth factor PDGF-BB (30 ng / mL) was added as a positive control. The blank control group was added with an equal amount of 2.5% FBS maintenance medium, added to 5 replicates, and incubated at 37°C, 5% CO2 for 48 hours. CCK-8 was used to detect cell proliferation: serum-free medium containing 10% CCK-8 was added to each well, and the absorbance was measured at 450nm using an enzyme reader after incubation in the incubator for 2 hours.

[0103] Experimental results: The experimental results are shown in Figure 3 As shown (*: P < 0.05; **: P < 0.01). Figure 3The results of the study showed that the peptide had a significant proliferative effect on fibroblasts, one of the main cell types involved in the wound repair process: Balb-3T3 cells (mouse embryonic fibroblasts).

[0104] Example 7 Effect of polypeptide on RSC96 cell proliferation

[0105] Experimental method: The concentration of rat Schwann cells (RSC96 cells) was adjusted to 1.0×10 5 ~5.0×10 5 / mL for subculture and culture at 37°C, 5% CO2 for 24 to 36 hours for biological activity detection. Digest and collect cells with trypsin and use serum-free medium to make a concentration of 5×10 4 / mL was inoculated into a 96-well cell culture plate, 100 μL per well, i.e. 8000 cells / well, and cultured overnight at 37°C, 5% CO2.

[0106] The polypeptide was dissolved in PBS solution to prepare a mother solution with a concentration of 400ug / ml. The polypeptide mother solution was prepared with 0% serum culture medium to the test concentration as the experimental group. The control group was added with an equal amount of 0% serum-free culture medium, added to 4 replicates, and incubated at 37°C, 5% CO2 for 48 hours. CCK-8 was used to detect cell proliferation: the old culture medium was removed, and serum-free culture medium containing 10% CCK-8 was added to each well. After incubation in the incubator for 2 hours, the absorbance was measured at 450nm using a microplate reader.

[0107] Experimental results: The experimental results are shown in Figure 4 As shown (*: P < 0.05; **: P < 0.01). Figure 4 The results showed that the peptide had a significant proliferation-promoting effect on RSC96 cells.

[0108] Example 8: Effect of polypeptide on promoting tissue regeneration in zebrafish

[0109] Experimental animals: Wild-type AB strain zebrafish, raised in 28°C fish water (water quality: 200 mg instant sea salt per 1L reverse osmosis water, conductivity 450-550 μS / cm; pH 6.5-8.5; hardness 50-100 mg / LCaCO3), zebrafish 3 days post fertilization (3 dpf). Produced by Hangzhou Huante Biotechnology Co., Ltd. Fish Breeding Center, experimental animal use license number: SYXK (Zhejiang) 2012-0171, breeding management in accordance with the requirements of the international AAALAC certification (certification number: 001458).

[0110] Experimental methods: 3dpf wild-type AB strain zebrafish were randomly selected, and the tail fin of the zebrafish was removed to establish a zebrafish tail fin injury model. The model zebrafish were randomly assigned to a 6-well plate, and 30 zebrafish were placed in each well (each experimental group). Different concentrations of peptides (final concentrations of 250, 500, and 1000 μg / mL) were dissolved in water, and a normal control group (zebrafish tail fin was not damaged) and a model control group were set up at the same time, with a capacity of 3mL per well. After being placed at 28°C for 3 days, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software, and the regeneration area of ​​the zebrafish tail fin was analyzed. The statistical analysis results of this indicator were used to evaluate the efficacy of the sample in promoting tissue regeneration.

[0111] Experimental results: The experimental results are shown in Figure 5 As shown (compared with the model control group, *: P < 0.05, **: P < 0.01). The results of the study showed that the peptide has a significant effect on promoting tissue regeneration in zebrafish.

[0112] Example 9 Effect of polypeptide on promoting angiogenesis in zebrafish

[0113] Experimental animals: Transgenic zebrafish with vascular green fluorescence, raised in 28°C fish water (water quality: 200 mg instant sea salt per 1L reverse osmosis water, conductivity 450-550 μS / cm; pH 6.5-8.5; hardness 50-100 mg / L CaCO3), zebrafish 1 day after fertilization (1 dpf). Produced by the fish breeding center of Hangzhou Huante Biotechnology Co., Ltd., the experimental animal use license number is: SYXK (Zhejiang) 2012-0171, and the breeding management meets the requirements of the international AAALAC certification (certification number: 001458).

[0114] Experimental method: 1dpf vascular green fluorescent transgenic zebrafish were randomly selected in 6-well plates, with 30 fish in each well (each experimental group). The normal control group was treated with standard dilution water, and the other experimental groups were given 60nM simvastatin in water for 3h to establish the zebrafish microvascular deficiency model, with a volume of 3mL per well. After 3h, simvastatin induction was terminated. Then the aqueous solution of the positive control group was replaced with 5.90μg / mL astragaloside IV, and the aqueous solutions of the other groups were replaced with standard dilution water, with a volume of 3mL per well. The test drug group was intravenously injected with different doses of peptides, that is, the concentrations were 12.5, 25.0, and 50.0mg / mL, respectively, and the injection volume was 10nL. After being placed at 28℃ for 2 days, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. The data were analyzed and collected using NIS-Elements D 3.20 advanced image processing software, and the intestinal vascular area and the number of intestinal vascular branches were analyzed. The statistical analysis results of this index were used to evaluate the efficacy of the sample in promoting angiogenesis.

[0115] Experimental results: The experimental results are shown in Figure 6 and Figure 7 As shown (compared with the model control group, *: P < 0.05, **: P < 0.01). The results of the study showed that the peptide can significantly promote the increase of the area of ​​the subintestinal blood vessels in zebrafish and the increase of the number of subintestinal blood vessel branches, and has the effect of promoting angiogenesis.

[0116] Example 10 Effect of polypeptide on healing of skin ulcers in diabetic rats induced by streptozotocin (STZ)

[0117] Experimental animals: SPF-grade healthy male SD rats (weight 180-200 g) were fed with a high-fat, high-sugar diet for 2 weeks, fasted for 6 h, and each rat was injected intraperitoneally with a single rapid injection of STZ solution (50 mg kg -1 ), 2 random blood glucose values ​​after 48 hours were >16.7mmol / L, and the type II diabetic rat model was considered to be successfully established. Experimental animals were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd., with the laboratory animal quality certificate number SCXK (Beijing) 2017-0033; laboratory animal use license: SCXK (Beijing) 2017-0033.

[0118] Experimental method: 90 diabetic rats were randomly divided into 6 groups, 15 rats in each group, including model control group (normal saline), high-dose peptide group (30 μg / cm 2 ), medium-dose peptide group (10 μg / cm 2 ), low-dose peptide group (3 μg / cm 2 ), Jinyin peptide group (40IU / cm 2, recombinant human epidermal growth factor topical solution, Shenzhen Huashengyuan Gene Engineering Development Co., Ltd.), Kangfuxin liquid group (36μL / cm 2 Each experimental rat was anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.2 mL / 100 g) and a 2 cm diameter puncture was prepared on both sides of the dorsal spine. 2 The full-thickness skin defect wound was sutured with a rubber ring with a diameter of 2.3 cm at the outer edge of the wound. After the wound hemostasis, local drug treatment was performed according to the group. Except for the Kangfuxin liquid group, which was administered twice a day, the other groups were administered once a day, and the administration volume was 72 μL / wound each time for 14 consecutive days. After the administration, the wound was covered with sterile vaseline gauze (5cm×5cm) and multi-layer sterile gauze. After the wound surgery, the wound was photographed and the wound area was recorded as the baseline value (the day of the trauma was recorded as Day 0). The wound area was photographed and measured 3 times a week during the administration period, and the growth and healing of granulation tissue in the wound of each group were observed every day.

[0119] Experimental results: See Table 6 and Figure 8 (Note: compared with the model control group, *: P < 0.05; **: P < 0.01).

[0120] As can be seen from Table 6, on Day 10, the healing rates of the low-dose and medium-dose peptide groups were slightly higher than those of the model control group, indicating that low- and medium-dose peptides have a tendency to promote healing of wounds in diabetic rats; throughout the wound healing process, the healing rate of the high-dose peptide group was relatively high, significantly higher than the model control group on Day 10, and slightly higher than the Jinyin peptide group. On Day 7-Day 12, the healing rate was slightly higher than the Kangfuxin liquid group, indicating that the high-dose peptide group has a significant effect on promoting healing of wounds in diabetic rats, and the effect is slightly better than Jinyin peptide in this experiment. Figure 8 It can be seen that the growth rate of new granulation tissue promoted by the low, medium and high doses of peptides is significantly different from that of the model control group. Therefore, the peptide has the effect of promoting the growth of new granulation tissue and wound healing in STZ-induced diabetic rats.

[0121] Table 6 Wound healing rate of diabetic rats in each group at different days after injury (%, Mean ± SD, n = 15)

[0122]

[0123] Example 11 Effect of polypeptide on healing of full-thickness cortical defect in SD rats

[0124] Experimental animals: SD male rats, SPF grade, 6 weeks old, weighing 180-220 g, Beijing Weitonglihua Experimental Animal Technology Co., Ltd.; Experimental Animal Quality Certificate Number: NO.110011220109610345; Experimental Animal Use License: SCXK (Beijing) 2021-0011.

[0125] Experimental methods: SD rats were randomly divided into 8 groups according to their body weight, with 8 rats in each group; vehicle group, peptide group (50 μg / cm 2 ), control peptide group 1 (41.90 μg / cm 2 ), control peptide group 2 (52.66 μg / cm 2 ). The amino acid sequence of comparative polypeptide 1 is: Glu-Pro-Val-Pro-Leu; the amino acid sequence of comparative polypeptide 2 is: Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu-Val-Lys-Gln-Glu.

[0126] Each experimental rat was anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.2 mL / 100 g). 2 The wound was a full-thickness defect. After the wound hemostasis, the corresponding drugs were given locally according to the groups. Each group was given once a day, and the volume of administration was 72 μL / wound each time for 14 consecutive days. After the administration, the wound was covered with sterile vaseline gauze (5cm×5cm) and multi-layer sterile gauze. After the wound surgery, the wound was photographed and the wound area was recorded as the baseline value (the day of the injury was recorded as Day 0). During the medication period, the wound area was photographed and measured 3 times a week and the healing condition was observed.

[0127] Experimental results: The experimental results are shown in Table 7:

[0128] Day 0 Day 3 Day 5 Day 7 Day 10 Solvent group 0.00±0.00 11.01±23.19 38.94±18.95 61.81±11.97 87.77±5.65 Peptide Group 0.00±0.00 29.70±19.21* 52.21±11.54* 74.12±5.95*** 94.55±2.40*** Comparative peptide group 1 0.00±0.00 21.13±17.43 40.18±17.43 68.84±7.64 88.90±8.40 Comparative peptide group 2 0.00±0.00 17.21±22.78 50.08±16.55 65.26±14.46 90.34±3.04

[0129] Note: Compared with the vehicle group, *: P < 0.05; **: P < 0.01;

[0130] The results showed that compared with the vehicle group, the peptide group (50 μg / cm 2 ) The healing rate was high and significantly different on the 3rd to 10th day after surgery; and the healing effect of the polypeptide of the present invention was better than that of the comparison polypeptide group 1 and the comparison polypeptide group 2.

[0131] In summary, the above-mentioned polypeptide of the present invention, compared with the recombinant human epidermal growth factor commonly used in the treatment of skin injury diseases in the prior art, not only has a significant wound healing effect on skin ulcers and injuries, especially diabetic foot ulcers, pressure ulcers, vascular ulcers and infectious ulcers, etc., chronic and difficult to heal wounds on the body surface, acute and / or chronic skin diseases, diabetic foot ulcers, etc., and due to the short peptide chain of the present invention, the skin absorption is faster and better, the stability in vivo and in vitro is good, and it has a significant pro-proliferation effect on human immortalized keratinocytes, human microvascular endothelial cells, fibroblasts, glial cells, and tissue and vascular regeneration. The above-mentioned polypeptide of the present invention is suitable for preparing products for preventing or treating skin injury diseases, and can produce positive treatment and repair effects.

[0132] Although the present invention discloses the above embodiments, the implementation of the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, simplifications that do not deviate from the present invention should be considered as equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a polypeptide in the preparation of a drug for preventing or treating skin damage diseases, characterized in that: The polypeptide is Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof, wherein the skin injury disease is a chronic, difficult-to-heal wound on the body surface.

2. The use according to claim 1, characterized in that: The chronic difficult-to-heal wounds on the body surface include diabetic foot ulcers, pressure ulcers, vascular ulcers or infectious ulcers.

3. The use according to claim 1, characterized in that: The medicine is an external preparation.

4. The use according to claim 3, characterized in that: The topical preparations include solutions, emulsions, gels, creams, sprays, masks or dressings.

5. The use according to claim 3, characterized in that: The polypeptide plays a role in wound healing by promoting proliferation of human immortalized keratinocytes, human microvascular endothelial cells, fibroblasts, glial cells and tissue and / or blood vessel regeneration.

Images