Use of polypeptides in the manufacture of products for preventing or treating skin damage diseases

Abstract

The invention provides the use of a polypeptide which is Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof in the manufacture of a product for the prevention or treatment of skin lesions.

Description

[Technical Field]

[0001] The present invention relates to the application of a polypeptide in the manufacture of a product for preventing or treating skin damage diseases, and the polypeptide of the present invention has a remarkable effect in preventing and / or treating skin damage diseases, particularly in preventing and / or treating chronic intractable wounds on the body surface, and acute and / or chronic skin diseases. [Background technology]

[0002] Human skin is composed of the epidermis, dermis, and subcutaneous tissue, and includes accessory organs (sweat glands, sebaceous glands), blood vessels, lymphatic vessels, nerves, and muscles. Skin is the outermost and largest organ of the human body, and it is the organ most frequently and most closely in contact with the outside world. It covers the surface of the human body, protecting the body from external aggression and regulating body temperature. It also has a sensory function and plays an important role in maintaining human health. Therefore, skin is considered the body's first line of defense. Because it covers the surface of the human body, it is susceptible to external damage, chemical irritation, and microbial infection. Skin lesions refer to visible or palpable abnormalities on the skin and mucous membranes. Skin lesions can have a double impact on patients, both mentally and physically.

[0003] In recent years, chronic intractable wound diseases on the body surface have attracted attention among diseases caused by skin injury. Chronic intractable wound diseases on the body surface, also known as intractable ulcers, are common intractable diseases. Local tissue loss, liquefaction, infection, and necrosis caused by various factors can lead to impaired endothelial progenitor cell proliferation, impaired vascular regeneration, insufficient wound perfusion, metabolic disorders, and delayed epithelial cell formation, disrupting the normal wound healing process and resulting in the formation of chronic ulcers.

[0004] Chronic, non-healing wounds on the body surface are common complications of diabetes, peripheral vascular disease, and radiation therapy. The International Society for Wound Healing defines a wound as one that fails to achieve anatomical and functional integrity through the normal, orderly, and timely repair process. Clinically, it often refers to wounds formed by various causes that have not healed even after more than one month of formal treatment and show no obvious tendency to heal. Wound healing proceeds through three phases: (1) the inflammatory phase, (2) the proliferative phase, and (3) the maturation and remodeling phase. Chronic wounds are characterized by prolonged inflammation, which inhibits the proliferation of vascular endothelial cells and fibroblasts and the deposition of collagen matrix.

[0005] Common chronic intractable wounds include diabetic foot ulcers, pressure ulcers, vascular ulcers, neurodystrophic ulcers, infectious ulcers, wound ulcers, autoimmune ulcers, cancer ulcers, and radiation ulcers. The incidence of these wounds increases with age, and the pathogenic mechanisms are complex and the course of the disease is relatively long.

[0006] Diabetic foot ulcers are primarily caused by nerve or vascular lesions due to underlying diabetes, leading to ischemic necrosis or infection of the skin tissue, or by pressure damage at supporting points, resulting in incomplete skin, exposing subcutaneous fat, muscle tissue, and even bone, resulting in ulcer formation. In mild cases, diabetic foot ulcers can cause itching, lack of sweating, dryness, pigmentation, mild foot pain, numbness, intermittent claudication, joint deformity, and superficial skin ulcers. In more moderate cases, infection can develop into relatively deep ulcers. In severe cases, bone can be affected, leading to fractures and osteonecrosis. In more severe cases, amputation may be necessary. Diabetic foot ulcers are a significant cause of disability and even death in diabetic patients, not only causing pain to patients but also imposing a significant economic burden. Currently, diabetic foot ulcers are often treated by controlling the underlying disease, but the treatment process is relatively slow, making localized ulcers prone to infection during the treatment process, leading to serious consequences.

[0007] Pressure ulcers occur when pressure on local tissues or impaired blood circulation causes local skin ischemia, hypoxia, and nutrient deficiency, resulting in the skin losing its normal function and tissue damage and necrosis. They often occur in bedridden elderly patients over bony prominences such as the sacrococcygeal region, occipital region, and feet. These areas have little soft tissue and low resistance to pressure, and prolonged immobilization and pressure can lead to ulcers. Pressure ulcers not only cause pain to patients and delay recovery, but in severe cases can be life-threatening due to secondary infection and sepsis.

[0008] Vascular ulcers are caused by varicose veins and vasculitis in the lower extremities. They are most commonly found in the distal legs and ankles and are a late complication of chronic lower extremity dysfunction. Severe obstruction of venous return causes elevated local venous pressure, resulting in edema, impaired oxygen diffusion, and nutrient deficiency in the skin. Patients often have a long history of primary venous disease. The wound is usually single and relatively superficial, with a dark base, rough skin around the wound, and obvious hyperpigmentation. The local skin temperature is very low. Regular wound dressing changes are ineffective, and even split skin grafting is unreliable.

[0009] Current research has shown that skin wound repair is a complex process in which multiple factors, such as keratinocytes, fibroblasts, vascular endothelial cells, inflammatory cells, extracellular matrix, cellular factors, and growth factors, are commonly involved, highly coordinated, and mutually regulated. Currently, commonly used drugs for the prevention or treatment of skin wound diseases include recombinant human epidermal growth factor, etc., which have certain preventive and / or therapeutic effects, but still suffer from the drawbacks of relatively slow absorption, relatively long treatment cycles, and unclear therapeutic effects, and have little therapeutic effect, especially when applied to chronic intractable wounds on the body surface and acute and / or chronic skin diseases. Summary of the Invention [Problem to be solved by the invention]

[0010] To overcome the shortcomings and deficiencies of the prior art, the object of the present invention is to provide an application of polypeptides in the manufacture of products for preventing or treating skin damage diseases. [Means for solving the problem]

[0011] According to a first aspect, the present invention provides the use of a polypeptide in the manufacture of a product for the prevention or treatment of skin damage diseases, wherein the polypeptide is Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof; or a method for the prevention or treatment of skin damage diseases, said method comprising topically administering to the site of skin damage a product comprising Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof; or a product comprising Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof for use in the prevention or treatment of skin damage diseases.

[0012] Further, skin damage diseases include wounds, ulcers, dermatitis, eczema, urticaria, polymorphous light eruption, herpes, acne, impetigo, melasma, vitiligo, lupus erythematosus, dermatomyositis, scleroderma, folliculitis, scabies, onychomycosis, chickenpox, infantile rash, warts, carbuncles, boils or ringworm. Furthermore, dermatitis includes atopic dermatitis, contact dermatitis, neurodermatitis, seborrheic dermatitis, hormone-dependent dermatitis or stasis dermatitis. Additionally, wounds include chronic non-healing wounds on the surface of the body. Furthermore, chronic non-healing wounds on the body surface include diabetic foot ulcers, pressure ulcers, vascular ulcers and infected ulcers.

[0013] Additionally, the product may include a drug, a skin care product, or a cosmetic product. Furthermore, the product is a topical formulation. Further, the dosage form of the topical preparation includes a solution, emulsion, gel, cream, spray, mask or dressing.

[0014] Furthermore, the polypeptide has a growth-promoting effect on human immortalized keratinocytes, human microvascular endothelial cells, fibroblasts, glial cells, and tissue and / or vascular regeneration, thereby healing wounds. Furthermore, the physiologically compatible salts refer to salt forms that are physiologically compatible (i.e., pharmacologically acceptable) and essentially non-toxic to individuals to whom the compounds of the present invention are administered. Physiologically compatible salts of the compounds of the present invention include conventional stoichiometric acid addition salts or base addition salts formed from suitable non-toxic organic or inorganic acids or inorganic bases.

[0015] The beneficial effects of applying the polypeptide provided by the present invention to the manufacture of products for preventing or treating skin wound diseases, particularly chronic intractable wounds on the body surface, are as follows:

[0016] Compared with recombinant human epidermal growth factor (REGF), which is commonly used in the treatment of skin wound diseases in the prior art, the polypeptides described in the present invention not only have a clear wound healing promoting effect on skin ulcers and wounds, but also, due to the short peptide chain of the present invention, have faster and better dermal absorption, excellent stability both in vivo and in vitro, and significant proliferation-promoting effects on human immortalized keratinocytes, human microvascular endothelial cells, fibroblasts, glial cells, and tissue and vascular regeneration, thereby exhibiting significant preventive and / or therapeutic effects on acute and / or chronic skin diseases and chronic intractable wounds on the body surface (diabetic foot ulcers, pressure ulcers, vascular ulcers, and infectious ulcers). The polypeptides described in the present invention can be used in the manufacture of products for preventing or treating skin wound diseases, and can induce dedifferentiation of cells near the wound. The dedifferentiated cells can regain stem cell morphology and cell division ability, forming new units for repairing the wound, thereby exerting positive therapeutic and preventive effects. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows the state of proliferation promotion of HaCaT cell line by polypeptides. [Figure 2] 1 shows the promotion of proliferation of HMEC-1 cell line by polypeptides. [Figure 3]1 shows the state of proliferation promotion of Balb-3T3 cell line by polypeptides. [Figure 4] 1 shows the state of proliferation promotion of the RSC96 cell line by polypeptides. [Figure 5] 1 shows the effect of polypeptides on zebrafish caudal fin regeneration. [Figure 6] 1 shows the effect of polypeptides on the area of ​​revascularization in zebrafish with microvascular loss. [Figure 7] 1 shows the effect of polypeptide-induced microvascular deletion on the number of regenerating branches in zebrafish. [Figure 8] 1 shows the rate of new granulation tissue formation by polypeptides in STZ-induced diabetic rats on day 3 after injury. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be described below in conjunction with specific tests, but is not intended to limit the scope of protection of the present invention. [Example]

[0019] Example 1: Chemical synthesis of polypeptides The polypeptide was synthesized using conventional solid-phase synthesis methods, and went through multiple cyclic processes of resin swelling, substitution, deprotection, washing, amino acid dissolution, amino acid activation and condensation process, washing, re-deprotection, and final degradation and side chain deprotection.

[0020] Abbreviations: HBTU stands for benzotriazole-N,N,N',N'-tetramethylurea 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) etc. stands 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, and TIPS stands for triisopropylsilane.

[0021] The method for synthesis and purification of the polypeptide is as follows. Step 1, Preparation of fully protected peptide resin (1) Resin swelling: 2.0192 g (S = 0.73 mmol / g) of 2-chlorotriyl chloride resin was weighed and added to a synthesis tube equipped with a sieve plate, and the resin was swelled in 40 ml of dichloromethane for 30 minutes, followed by suction filtration to remove the dichloromethane. (2) Preparation of Fmoc-Asp(OtBu)-resin: Resin, Fmoc-Asp(OtBu)-OH, and DIPEA were weighed in a molar ratio of 1:1.5:1.65. Fmoc-Asp(OtBu)-OH and DIPEA were dissolved in 20 mL of dichloromethane and added to a synthesis tube. The mixture was shaken at room temperature with N2 bubbling for 1-3 hours, and then 2 mL of methanol was added directly to the reaction mixture for 30 minutes to block the mixture. The resin was then washed four times with 25 mL of dimethylformamide, and the resin was dried by suction.

[0022] (3) Removal of Fmoc protecting group: 20 ml of 20% piperidine-DMF (v / v) solution was added to the reactor, and the mixture was reacted for 20 minutes with N2 bubbling. The mixture was then suction dried, washed six times with 25 ml of dimethylformamide for 3 minutes each time, suction dried, and the Fmoc removal results were detected by the ninhydrin method. (4) Preactivation of amino acids: 4.38 mmol of Fmoc-protected amino acid, 5.26 mmol of HOBt, and 4.60 mmol of DIC were added to a 250 mL round-bottom flask and dissolved in 20 mL of 1:1 DCM-DMF (v / v). The mixture was preactivated for 30 to 60 min with stirring in an ice bath at -5 to 0°C.

[0023] (5) Amino acid coupling: The activated protected amino acid solution was poured into the reactor and supplemented with an appropriate amount of DCM cleaning agent. The reaction was allowed to proceed at room temperature with N2 bubbling for 1-3 hours. The ninhydrin method was used to determine whether the amino acid coupling was complete. If so, the resin was sucked dry. The resin was washed with dimethylformamide four times, 25 ml each time for 3 minutes, and then sucked dry. The dosage of each amino acid and condensation agent, and the specific reaction time are listed in Table 1. (6) After the first amino acid condensation was completed, steps (3) to (5) were repeated to extend the peptide chain in the order of amino acids until the coupling of the last amino acid was completed. (7) The resin peptide was washed six times with dichloromethane, 25 ml each time for 3 minutes each time, and then dried by suction.

[0024] [Table 1]

[0025] Step 2, cleavage and deprotection (1) 50 ml of cleavage agent (TFA:TIPS:H2O = 95:2.5:2.5, v / v) was added to the synthesis tube in step 1, and the mixture was reacted for 1.5 to 3 hours by bubbling with N2. (2) After the cleavage reaction was completed, the cleavage agent was suction filtered into a 250 ml round-bottom flask. After vacuum concentration to one-third of the original volume of the cleavage agent, 10 times the original volume of methyl tert-butyl ether was added and stirred for 30 minutes. The resulting mixed solvent was filtered and washed three times with 30 ml of methyl tert-butyl ether. The resulting crude peptide was placed in a sand-core funnel and dried with N2 in a fume hood to evaporate the solvent, resulting in a powdered crude peptide.

[0026] Step 3: Purification (salt exchange) and freeze-drying The crude peptide obtained in Step 2 was purified by HPLC using the following chromatographic parameters: Condition A. Specifically, the crude peptide obtained in Step 2 was dissolved in water and / or acetonitrile, filtered through a 0.45 μm filter membrane, injected, and gradient eluted with an acetonitrile-water mobile phase. The target peptide eluate was collected and finally concentrated by rotary evaporation.

[0027] Chromatography parameters Condition A: Chromatography column: YMC-Actus Triart C18 30*250mm, Eluent A: 0.1% (v / v) TFA / H2O, Eluent B: acetonitrile, Flow rate: 25ml / min, UV detection wavelength: 220nm.

[0028] [Table 2]

[0029] The product obtained in the above step was then salt-exchanged by HPLC using the following chromatography parameter condition B: 95% A1 + 5% B equilibrated chromatography column was used, followed by injection, then 95% A2 + 5% B equilibrated chromatography column was used, and gradient elution with A1 and B was performed. The target peptide eluate was collected, and finally concentrated by rotary evaporation and lyophilized to obtain the polypeptide. The structure of the polypeptide was determined by MS.1 This was confirmed by H-NMR.

[0030] Chromatography parameters Condition B: Chromatography column: YMC-Actus Triart C18 30*250mm Eluent A1: 0.1M acetic acid Eluent A2: 0.025M acetic acid + 0.1M ammonium acetate Eluent B: Acetonitrile Flow rate: 25ml / min UV detection wavelength: 220 nm

[0031] [Table 3]

[0032] Polypeptide 1 H-NMR is as follows: 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).

[0033] Polypeptide MS: 793.4, 397.3 (double charge). The amino acid sequence of the polypeptide is: Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu. The following materials were used in the examples of this application:

[0034] Polypeptides, unless otherwise specified, i.e., the amino acid sequence is Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu. Kinjin Peptide, trade name: Kinjin Peptide (GeneTime), generic name: Recombinant Human Epidermal Growth Factor Derivative for External Use (I), English name: Recombinant Human Epidermal Growth Factor Derivative for External Use, Liquid, manufacturer: Shenzhen Huashengyuan Gene Engineering Development Co., Ltd. Ingredients: The active ingredient is recombinant human epidermal growth factor (rhEGF), with 10% glycerin and 1.0% mannitol as protective agents. The rhEGF in Kinjin Peptide promotes the synthesis of DNA, RNA, and hydroxyproline during the skin and mucosal wound tissue repair process, accelerating the production of wound granulation tissue and epithelial cell proliferation, thereby shortening wound healing time. Kangfuxin Ye, manufactured by Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd., is a solution of dried extract of Periplaneta americana used for wounds such as wounds, trauma, ulcers, fistulas, burns, scalds, and bedsores.

[0035] Example 2: Healing effect of polypeptide on skin ulcers in db / db mice Experimental animals: diabetic modeling mice (db / db male mice), normal mice (m / m male mice), SPF grade, 8-10 weeks old, weighing 40-50g, Changzhou Cabins Laboratory Animal Co., Ltd., Laboratory Animal Production Permit Certificate Number: SCXK(Su)2016-0010, Laboratory Animal Use Permit Certificate Number: SYXK(Hu)2020-0038.

[0036] Experimental Method: After the blood glucose level of db / db mice reached the baseline (blood glucose ≥ 16.7 mmol / L after 4 hours of fasting), the grouping experiment was started. According to the random grouping method, the animals were divided into groups of 10 each, and were administered the following: normal control group, model control group, new recovery liquid group (40 μL / animal), and gold-in peptide group (40 IU / cm 2 ), target polypeptide group (30 μg / cm 2) was divided into 5 groups. After the mice were anesthetized by inhaling isoflurane, a circular wound with a diameter of about 12 mm was cut through the full thickness of the skin on the back of the mice with a cutter, reaching a depth to the fascia layer. Photographs of the wounds were taken, and the wound area was recorded as the baseline value (the day of the wound was designated as Day 0). From the next day (Day 1), the animals in each group were given drug treatment as needed, all applied topically at the skin wound site. 40 μL of physiological saline was administered to both the normal control group and the model control group. The recombinant human epidermal growth factor topical solution (produced by Shenzhen Huashengyuan Genetic Engineering Development Co., Ltd.) diluted with 40 μL of physiological saline was administered to the recombinant peptide group (40 IU / cm 2 ) and the corresponding concentration of polypeptide solution prepared with 40 μL of physiological saline was administered once a day to the target polypeptide group (30 μg / cm 2 ), and 40 μL of Kangfuxin solution (produced by Sichuan Good Doctor Panxi Pharmaceutical Co., Ltd.) was administered twice a day to the Kangfuxin solution group. Continuous administration was carried out daily until the wound was basically healed. The skin wounds were measured twice a week and photographed, and the wound area was calculated with Image-J software to observe the wound healing status of the animals in each group. Experimental results: The experimental results are shown in Table 4.

[0037]

Table 4

[0038] The research results in Table 4 showed that the polypeptide had a significant promoting effect on the skin wound healing of diabetic mice on the 14th day, and the effect was better than that of the Kangfuxin solution group, and the effect of the polypeptide group was equivalent to that of the recombinant peptide group.

[0039] Example 3 Healing effect of polypeptide on acute mechanical skin injury in rats Experimental animals: SD male rats, SPF grade, 6 weeks old, body weight 180 - 220 g, from Beijing Vital River Laboratory Animal Technology Co., Ltd., animal license number: SCXK(Beijing)2016 - 0006, certificate of conformity number: NO.110011210104746538.

[0040] Experimental method: SPF-grade SD rats were housed in clean, sterile cages. They were given water and food at regular intervals every day, and the bedding was changed. The temperature was maintained at 20-26°C and the humidity at 40-70%. They were then housed for one week to adapt to the environment. According to the random grouping method, the animals were divided into six groups: a model control group (physiological saline), a Kinin peptide group (40 IU / cm), and a control group (40 IU / cm). 2 , recombinant human epidermal growth factor topical solution, Shenzhen Huashengyuan Gene Engineering Development Co., Ltd.), target polypeptide group (8 μg / cm 2 The rats were divided into three groups: 1) a 1-cm thick circular full-thickness skin wound (1.5 cm diameter) and 2) a 1-cm thick circular full-thickness skin wound (1.5 cm diameter). The wound area was anesthetized with an intraperitoneal injection of 3% pentobarbital sodium. The area was first disinfected with iodophor and then topically disinfected with 75% alcohol. A 1.5 cm diameter circular full-thickness skin wound was created 4 cm below the neck, extending from the center of the line connecting the ear and back to the midline of the spine, down to the depth of the muscle layer. The surrounding skin was then secured with a rubber ring of the same size to form an animal model of acute mechanical injury. After modeling, the rats were exposed to the wound and housed in single cages. During drug replacement, the wound surface was first excised with iodophor, then washed with sterile saline and wiped dry. Each group of rats received an additional 40 μL of the corresponding drug solution, which was applied topically to the wound at regular intervals once daily. On days 0, 3, 7, 10, and 14 after administration, images of the wounds of the rats in each group were taken, and the wound area was calculated using image analysis software (Image J), ​​and the wound healing rate was calculated using the formula. Experimental Results: The experimental results are shown in Table 5.

[0041] [Table 5]

[0042] The results of Table 5 showed that the polypeptide group had a significant promoting effect on wound healing after mechanical skin injury in rats on the 14th day, and the effect was significantly superior to that of the Kinin peptide group.

[0043] Example 4 Promotion of HaCAT cell proliferation by polypeptide Experimental method: The concentration of human immortalized keratinocytes (HaCaT cells) was 1.0 × 10 5 ~5.0×10 5 The cells were subcultured at a concentration of 1000 / mL and then cultured at 37°C and 5% CO2 for 24-36 hours to detect biological activity. The cells were digested with 0.25% trypsin for 5 minutes, and then 1x the volume of 1640 whole blood medium was added to terminate the digestion. The cell suspension was collected and centrifuged at 1000 RPM for 3 minutes. The supernatant was discarded, and 2 mL of 1640 whole blood medium was added to resuspend the cells. 20 μL of the cell suspension was taken and stained with AOPI. The cell concentration in the suspension was then measured using a cell counter. A concentration of 5x10 cells was then obtained using 1640 medium with 10% serum. 4 The solution was prepared to a concentration of 1 / mL, and 100 μL / well, i.e., 5000 cells / well, was inoculated into a 96-well cell culture plate, and cultured overnight at 37° C. and 5% CO 2 .

[0044] After 24 hours, the original medium was discarded and 100 μL of polypeptide solutions prepared in 1% serum 1640 medium were added. At the same time, an EGF control group was set up, in which 100 μL of recombinant human epidermal growth factor (EGF) solution prepared in 1% serum 1640 medium was added to a final concentration of 100 ng / mL. An equal volume of 1% serum 1640 medium was added as a model control group. The cells were cultured at 37°C and 5% CO2 for 72 hours, and the proliferation of the HaCaT cell line was detected using the CCK8 reagent kit.

[0045] Experimental Results: The experimental results are shown in Figure 1 (*: P<0.05, **: P<0.01). The research results in Figure 1 show that after 72 hours of action, the polypeptide had a significant proliferation-promoting effect on HaCaT cells (human immortalized keratinocytes), which are epithelial cells, one of the main cell types involved in the wound repair process.

[0046] Example 5 Promotion of HMEC-1 cell proliferation by polypeptides Experimental method: Human microvascular endothelial cells (HMEC-1 cells) were cultured in 10% serum medium at 37°C and 5% CO2. The drug was replaced every 1-2 days, and the cell concentration was maintained at 4 × 10 6The cells were collected and passaged at 4 × 10 cells / mL in serum-free medium. 4 The solution was prepared to give a concentration of 1000 cells / mL, and inoculated into a 96-well cell culture plate at 100 μL / well, i.e., 4000 cells / well, and cultured at 37°C and 5% CO 2 until the cells attached to the wall. Polypeptides were dissolved in PBS to prepare a 1 mg / mL mother solution. Experimental groups were prepared with the mother solution in 0% serum medium at test concentrations. Recombinant human vascular endothelial growth factor (VEGF) (100 ng / mL) was prepared in the same manner and added as a positive control. An equal volume of 0% serum-free medium without drug was added to a blank control group, which was added to five parallel wells and incubated at 37°C and 5% CO2 for 48 hours. CCK-8 was used to detect cell proliferation. 10% CCK-8 in serum-free medium was added to each well. After 2 hours of incubation in an incubator, the absorbance at 450 nm was measured using a microplate reader.

[0047] Experimental Results: The experimental results are shown in Figure 2 (*: P<0.05, **: P<0.01). The research results in Figure 2 showed that the polypeptide had a significant growth-promoting effect on HMEC-1 cells (human microvascular endothelial cells), which are endothelial cells, one of the main cell types involved in the wound repair process.

[0048] Example 6 Promotion of Balb-3T3 cell proliferation by polypeptides Experimental method: Mouse embryonic fibroblasts (Balb-3T3 cells) were cultured in 10% serum medium at 37°C and 5% CO2. Drugs were replaced every 1-2 days, and the cell concentration was maintained at 4 x 10 6 The cells were collected and passaged at 3 × 10 cells / mL in 2.5% FBS maintenance medium. 4 The solution was prepared to give a concentration of 1000 cells / mL, and inoculated into a 96-well cell culture plate at 100 μL / well, i.e., 3000 cells / well, and cultured at 37°C and 5% CO 2 until the cells attached to the wall.

[0049] Polypeptides were dissolved in 2.5% FBS maintenance medium to prepare a 1 mg / mL mother solution. Experimental groups were prepared with the mother solution in 2.5% FBS maintenance medium at test concentrations. Recombinant human basic fibroblast growth factor (FGF) (50 ng / mL) and recombinant human platelet-derived growth factor (PDGF-BB) (30 ng / mL) were prepared in the same manner and added as positive controls. Equal volumes of 2.5% FBS maintenance medium were added to blank controls. Five parallel wells were added and incubated at 37°C and 5% CO2 for 48 hours. CCK-8 was used to detect cell proliferation. 10% CCK-8 in serum-free medium was added to each well. After 2 hours of incubation in an incubator, the absorbance at 450 nm was measured using a microplate reader.

[0050] Experimental Results: The experimental results are shown in Figure 3 (*: P<0.05, **: P<0.01). The research results in Figure 3 showed that the polypeptide had a significant proliferation-promoting effect on Balb-3T3 cells (mouse embryonic fibroblasts), which are fibroblasts, one of the main cell types involved in the wound repair process.

[0051] Example 7 Effect of polypeptides on RSC96 cell proliferation Experimental method: Rat Schwann cells (RSC96 cells) were cultured at a concentration of 1.0 × 10 5 ~5.0×10 5 The cells were then subcultured at a concentration of 5 × 10 / mL and incubated at 37°C and 5% CO2 for 24–36 hours to detect biological activity. The cells were digested with trypsin, collected, and resuspended in serum-free medium at a concentration of 5 × 10 4 The cells were inoculated into a 96-well cell culture plate at 100 μL / well, i.e., 8000 cells / well, and cultured overnight at 37° C. and 5% CO 2 .

[0052] Polypeptides were dissolved in PBS to prepare a 400 μg / ml mother solution. The test concentrations were adjusted to the mother solution in 0% serum medium. An equal volume of 0% serum-free medium without drug was added to the control group, which was added to four parallel wells. The wells were incubated at 37°C and 5% CO2 for 48 hours. CCK-8 was used to detect cell proliferation. The old medium was removed, and serum-free medium containing 10% CCK-8 was added to each well. After incubation in an incubator for 2 hours, the absorbance at 450 nm was measured using a microplate reader. Experimental Results: The experimental results are shown in Figure 4 (*: P<0.05, **: P<0.01). The research results in Figure 4 showed that the polypeptide had a significant proliferation-promoting effect on RSC96 cells.

[0053] Example 8 Promotion of tissue regeneration in zebrafish by polypeptides Experimental animals: Wild-type AB strain zebrafish, 3 days postfertilization (dpf), were maintained in fish culture water at 28°C (water quality: reverse osmosis water supplemented with 200 mg of instant sea salt per liter, conductivity 450–550 μS / cm, pH 6.5–8.5, hardness 50–100 mg / L CaCO3). They were bred and provided by the Fish Breeding Center of Hangzhou Huante Biotechnology Co., Ltd., under the Laboratory Animal Use Permit Certificate No. SYXK (Zhejiang) 2012-0171. Animal husbandry practices met the requirements of the international AAALAC certification (certificate number: 001458).

[0054] Experimental Method: Wild-type AB zebrafish at 3 dpf were randomly selected, and their caudal fins were removed to establish a zebrafish caudal fin injury model. Model zebrafish were randomly distributed into 6-well plates, with 30 zebrafish per well (experimental group). Different concentrations of polypeptide were dissolved in water and administered (final concentrations of 250, 500, and 1000 μg / mL). A normal control group (zebrafish with intact caudal fins) and a model control group were also established. Each well contained 3 mL of polypeptide. After 3 days of treatment at 28°C, 10 zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Data were collected and analyzed using advanced image processing software NIS-Elements D 3.20. The regenerated area of ​​the zebrafish caudal fin was analyzed, and statistical analysis of the parameters was used to evaluate the effect of the samples on promoting tissue regeneration. Experimental Results: The experimental results are shown in Figure 5 (compared to the model control group, *: P<0.05, **: P<0.01). The research results showed that the polypeptide had a significant promoting effect on zebrafish tissue regeneration.

[0055] Example 9 Promotion of vascular regeneration in zebrafish by polypeptides Experimental animals: vascular green fluorescent transgenic zebrafish, 1 day postfertilization (dpf), were reared in fish culture water at 28°C (water quality: reverse osmosis water supplemented with 200 mg of instant sea salt per liter, conductivity 450-550 μS / cm, pH 6.5-8.5, hardness 50-100 mg / L CaCO3). They were bred and provided by the Fish Breeding Center of Hangzhou Huante Biotechnology Co., Ltd., and were licensed under the Laboratory Animal Use Permit Certificate No. SYXK (Zhejiang) 2012-0171. Animal husbandry practices met the requirements of the international AAALAC certification (certificate No. 001458).

[0056] Experimental Method: 1-dpf vascular green fluorescent transgenic zebrafish were randomly selected and placed in a 6-well plate with 30 fish per well (each experimental group). The normal control group was treated with standard diluent water, while the remaining experimental groups were treated with 60 nM simvastatin dissolved in water for 3 hours to establish a zebrafish microvascular defect model. Each well contained 3 mL of simvastatin. After 3 hours, the simvastatin induction was terminated. The positive control group's aqueous solution was then replaced with 5.90 μg / mL astragaloside IV, and the remaining groups' aqueous solutions were replaced with standard diluent water. Each well contained 3 mL of simvastatin. The polypeptide was intravenously injected into the test drug groups at different doses (12.5, 25.0, and 50.0 mg / mL) in a 10 nL injection volume. After treatment at 28°C for 2 days, 10 zebrafish from each experimental group were randomly selected and placed under a fluorescence microscope to take photographs. The data was analyzed using the advanced image processing software NIS-Elements D 3.20, and the area of ​​the intestinal vascular system and the number of intestinal vascular branches were collected. The statistical analysis results of these indicators were used to evaluate the effect of the samples on promoting vascular regeneration.

[0057] Experimental Results: The experimental results are shown in Figures 6 and 7 (compared to the model control group, *: P<0.05, **: P<0.01). The research results showed that the polypeptide significantly increased the area of ​​the intestinal vascular system and the number of intestinal vascular branches in zebrafish, and had the effect of promoting vascular regeneration.

[0058] Example 10: Healing effect of polypeptide on skin ulcers in streptozotocin (STZ)-induced diabetic rats Experimental animals: SPF-grade, healthy male SD rats (180–200 g body weight) were fed a high-fat, high-carbohydrate diet for 2 weeks, then fasted for 6 h. Each rat was then administered an STZ solution (50 mg kg -1 A single intraperitoneal injection of ) was considered a successful model of type 2 diabetes in rats if two random blood glucose levels were >16.7 mmol / L 48 h later. Experimental animals were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. with the Experimental Animal Quality Qualification Certificate No. SCXK(Kyoto)2017-0033 and Experimental Animal Use Permit Certificate: SCXK(Kyoto)2017-0033.

[0059] Experimental method: 90 diabetic rats were selected, 15 rats per group, and each group was administered a model control group (physiological saline), a high-dose polypeptide group (30 μg / cm 2 ), medium dose polypeptide group (10 μg / cm 2 ), low-dose polypeptide group (3 μg / cm 2 ), Kinin peptide group (40IU / cm 2 , recombinant human epidermal growth factor topical solution, Shenzhen Huashengyuan Gene Engineering Development Co., Ltd.), Kangfu New Solution Group (36μL / cm 2 The rats were randomly divided into six groups, each receiving a 2% sodium pentobarbital solution (0.2 mL / 100 g) intraperitoneally. After anesthesia, two 2 cm incisions were made on each side of the spinal column. 2 Full-thickness skin defect wounds were created, and a 2.3cm diameter rubber ring was placed around the outer edge of the wound. After hemostasis, topical drug treatment was administered to the wounds according to group. The recovery solution group received the treatment twice daily, while the remaining groups received it once daily, with a total volume of 72μL per wound for 14 consecutive days. After administration, the wounds were covered with sterile Vaseline gauze (5cm x 5cm) and then wrapped in multiple layers of sterile gauze. After wound surgery, the wounds were photographed, and the wound area was recorded as the baseline value (the day of wounding was designated Day 0). During the treatment period, the wound area was photographed and measured three times weekly, and the granulation tissue growth and healing status of the wounds in each group were observed daily.

[0060] The experimental results are shown in Table 6 and Figure 8 (note: *: P<0.05, **: P<0.01 compared with the model control group). As can be seen from Table 6, on day 10, the healing rates of the low-dose and medium-dose polypeptide groups were slightly higher than those of the model control group. Both low- and medium-dose polypeptides tended to promote wound healing in diabetic rats. Throughout the wound healing process, the high-dose polypeptide group had a higher healing rate. On day 10, the healing rate was significantly higher than that of the model control group and slightly higher than that of the Kinin peptide group. From day 7 to day 12, the healing rate was slightly higher than that of the Kangfu Xinliqiu group. This indicates that the high-dose polypeptide group significantly promoted wound healing in diabetic rats and was slightly more effective than Kinin peptide in this experiment. As can be seen from Figure 8, the promotion of new granulation tissue growth rates in the low-, medium-, and high-dose polypeptide groups all showed significant differences compared to the model control group. Therefore, polypeptides have the effect of promoting new granulation tissue growth and wound healing in STZ-induced diabetic rat wounds.

[0061] [Table 6]

[0062] Example 11 Healing effect of polypeptide on SD rat total cortical defect wound Experimental animals: SD male rats, SPF grade, 6 weeks old, weighing 180-220g, Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., Laboratory Animal Quality Approval Certificate No.: NO. 110011220109610345, Laboratory Animal Use Permit Certificate: SCXK(Kyoto)2021-0011. Experimental method: SD rats were randomly divided into groups according to weight, with eight animals per group. The rats were divided into two groups: a solvent group, a polypeptide group (50 μg / cm 2 ), comparative polypeptide group 1 (41.90 μg / cm 2 ), comparative polypeptide group 2 (52.66 μg / cm 2 The amino acid sequence of comparative polypeptide 1 was Glu-Pro-Val-Pro-Leu, and the amino acid sequence of comparative polypeptide 2 was Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu-Val-Lys-Gln-Glu.

[0063] Each experimental rat was anesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.2 mL / 100 g), and then a 2 cm incision was made on each side of the dorsal spine. 2 Total cortical defect wounds were created in each group. After hemostasis, the corresponding medication was administered topically to the wound according to group. Each group received a single daily dose of 72 μL per wound for 14 consecutive days. After administration, the wound was covered with sterile Vaseline gauze (5 cm x 5 cm) and then wrapped in multiple layers of sterile gauze. After wound surgery, the wound was photographed and the wound area was recorded as the baseline value (the day of wounding was designated Day 0). During the administration period, the wound area was photographed and measured three times weekly to observe the healing status. Experimental Results: The experimental results are shown in Table 7.

[0064] [Table 7]

[0065] The results of the study showed that the polypeptide group of the present invention (50 μg / cm ) was significantly higher than the solvent group. 2 ) showed a high and significantly higher healing rate on the 3rd to 10th day after surgery, and the healing effect of the polypeptide of the present invention was superior to that of the comparative polypeptide 1 group and the comparative polypeptide 2 group.

[0066] In summary, compared with recombinant human epidermal growth factor (REGF), which is commonly used in the treatment of skin wounds in the prior art, the polypeptides described in the present invention not only have a clear wound healing-promoting effect on skin ulcers and wounds, particularly chronic, intractable wounds on the body surface, such as diabetic foot ulcers, pressure ulcers, vascular ulcers, and infectious ulcers, as well as acute and / or chronic skin diseases and diabetic foot ulcers, but also have a shorter peptide chain, which allows for faster and better dermal absorption, excellent stability both in vitro and in vivo, and significant proliferation-promoting effects on human immortalized keratinocytes, human microvascular endothelial cells, fibroblasts, glial cells, and tissue and vascular regeneration. The polypeptides described in the present invention can be used to manufacture products for preventing or treating skin wounds, and can achieve positive therapeutic and repair effects.

[0067] Although the present invention discloses the above examples, the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the present invention are equivalent substitution methods, and all are included in the scope of the claims of the present invention.

Claims

1. Use of a polypeptide in the manufacture of a product for preventing or treating a skin damage disease, comprising: The polypeptide is Pro-Ala-Ala-Glu-Pro-Val-Pro-Leu or a physiologically compatible salt thereof, and the skin wound disease is a chronic intractable wound on the body surface.

2. The chronic intractable wound on the body surface includes a diabetic foot ulcer, a pressure ulcer, a vascular ulcer, or an infected ulcer; 2. The use according to claim 1.

3. The product comprises a drug, a skin care product, or a cosmetic product.

2. The use according to claim 1.

4. The product is a topical formulation, 2. The use according to claim 1.

5. The topical formulation includes a solution, emulsion, gel, emulsion, cream, gel, spray, mask or dressing; 5. The use according to claim 4.

6. The polypeptide has a growth-promoting effect on human immortalized keratinocytes, human microvascular endothelial cells, fibroblasts, glial cells, and tissue and / or vascular regeneration, thereby healing wounds.

5. The use according to claim 4.

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