Application of polypeptide in preparation of medicine for treating wound healing difficulty

Abstract

The invention belongs to the technical field of medicines, and discloses application of polypeptide in preparation of a medicine for treating wound healing difficulty. The wounds comprise acute and chronic wounds caused by various reasons, and are selected from diabetic wounds and ulcers, traumatic wounds and ulcers, pressure wounds and ulcers, ischemic wounds and ulcers, venous wounds and ulcers, and arterial wounds and ulcers. The polypeptide has the beneficial effects that the polypeptide has the pharmacological effects of preventing and treating wound healing difficulty caused by various reasons, increasing wound healing speed and improving wound pathological injury. The polypeptide can be synthesized, is low in toxicity and has good application and development prospects. The traditional Chinese medicine composition can be used for preventing, relieving and treating wound healing difficulties caused by various reasons.

Description

Technical Field

[0001] The present invention relates to a polypeptide for use in the preparation of a method for preventing, alleviating, and / or treating wounds caused by various causes. The method involves using the compound alone or in combination with a medicament for promoting wound healing in the presence of a wound on a patient, the medicament being selected from the group consisting of diabetic wounds and ulcers, traumatic wounds and ulcers, pressure wounds and ulcers, ischemic wounds and ulcers, venous wounds and ulcers, and arterial wounds and ulcers, and belongs to the field of pharmaceutical technology. Background Art

[0002] Wound healing is a complex physiological process involving multiple factors and stages. Abnormalities at any stage of the healing process may lead to delayed wound healing. Chronic wounds are a disease state that mainly occurs in the combination of multiple chronic diseases and the aging process. They can seriously affect the quality of life and, in severe cases, lead to death. Clinically, it often refers to wounds that fail to heal as expected, or remain in a certain healing process for more than 4-6 weeks. The healing time is not absolute and depends on multiple factors such as wound size, cause, and individual general health status. Chronic wounds can generally be divided into five categories: venous, arterial, diabetic, wound, and pressure.

[0003] The high morbidity and cost of chronic wounds have become a major healthcare challenge, severely impacting patients and leading to prolonged hospitalizations, sepsis, and amputations. The cost of treating chronic wounds is substantial, with reports suggesting that chronic wound management represents a major cost to the healthcare system, costing approximately $10 to $20 billion annually in the United States alone (Fuentes et al., 2020, Sci Rep 2020;10:15064). Globally, the cost of wound treatment and management is even greater.

[0004] For the treatment of difficult-to-heal wounds caused by acute and chronic wounds, the first thing to do is to evaluate the cause of the wound, its depth, and whether it is infected. Currently, the treatment of wounds is mainly targeted at the cause, which mainly includes controlling infection and promoting healing. Treatments that promote healing include hyperbaric oxygen therapy, which promotes capillary regeneration and wound healing. Drug treatments mainly include the use of topical growth factors to promote tissue repair. In addition, treatment is carried out to target the cause of the wound, such as pressure ulcers that require pressure relief, and blood vessel obstruction that requires revascularization to promote blood circulation. Diabetic wounds are treated to relieve pressure and use dressings containing growth factors.

[0005] Peptides are compounds formed by linking α-amino acids in peptide chains and are also intermediate products of hydrolysis. Many active peptides are derived from inactive precursors through enzymatic processing and splicing. They share similar structures and retain unique biological activities. Studying the structure and function of active peptides will aid in the design and development of novel active peptides.

[0006] The polypeptide described in this invention is a polypeptide originally discovered in the tail of bony fish. In 1998, Coulouarn et al. first cloned human endogenous urotensin II (UⅡ), an 11-amino acid cyclic peptide with a disulfide-bonded cyclic hexapeptide structure at its C-terminus.

[0007] The present invention is based on new discoveries made through extensive animal and cell-based experimental research using the artificially synthesized endogenous polypeptide Urotensin II. The new invention primarily involves the preparation of drugs and drug combinations, providing clinically effective treatments for wounds and ulcers with various causes of difficulty healing, including diabetic wounds and ulcers, traumatic wounds and ulcers, pressure wounds and ulcers, ischemic wounds and ulcers, venous wounds and ulcers, and arterial wounds and ulcers. Currently, there are no reports on the direct or indirect therapeutic effects of Urotensin II in treating wounds and ulcers with various causes of difficulty healing, including diabetic wounds and ulcers, traumatic wounds and ulcers, pressure wounds and ulcers, ischemic wounds and ulcers, venous wounds and ulcers, and arterial wounds and ulcers. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to provide a new polypeptide for the preparation of a method for preventing, alleviating and / or treating wound healing difficulties caused by various reasons, wherein the wounds are selected from diabetic wounds and ulcers, traumatic wounds and ulcers, pressure wounds and ulcers, ischemic wounds and ulcers, venous wounds and ulcers, and arterial wounds and ulcers.

[0009] In order to solve the technical problems of the present invention, the present invention provides the following technical solutions:

[0010] The present invention provides the use of the following polypeptide or polypeptide containing an active sequence in the preparation of a medicament for preventing, alleviating and / or treating wound healing difficulties caused by various reasons, wherein the wound is selected from diabetic wounds and ulcers, traumatic wounds and ulcers, pressure wounds and ulcers, ischemic wounds and ulcers, venous wounds and ulcers, and arterial wounds and ulcers, and the polypeptide sequence is:

[0011] Glu1-Thr2-Pro3-Asp4-Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10-Val11, wherein Cys5 and Cys10 are connected by a disulfide bond; the active sequence of the polypeptide is: Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10, wherein Cys5 and Cys10 are connected by a disulfide bond.

[0012] The present invention uses cells, normal animals, and diabetic animal models to detect

[0013] The effects of the Glu1-Thr2-Pro3-Asp4-Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10-Val11 peptide and other peptides containing the active sequence Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10 on wound healing, including cell proliferation, cell migration, wound size, and healing, were observed. It is determined that the Glu1-Thr2-Pro3-Asp4-Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10-Val11 polypeptide and other polypeptides containing the active sequence Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10 are useful in the preparation of a method for preventing, alleviating and / or treating a wound selected from diabetic wounds and ulcers, traumatic wounds and ulcers, pressure wounds and ulcers, ischemic wounds and ulcers, venous wounds and ulcers, and arterial wounds and ulcers.

[0014] The characteristics of the present invention are that a small amount of Glu1-Thr2-Pro3-Asp4-Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10-Val11 polypeptide and other polypeptides containing the active sequence Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10 can achieve therapeutic and preventive effects, and the medicine is safe and reliable.

[0015] The present invention also relates to the use of a pharmaceutical composition containing the polypeptide of the present invention as an active ingredient for preventing, alleviating, and / or treating a wound selected from diabetic wounds and ulcers, traumatic wounds and ulcers, pressure wounds and ulcers, ischemic wounds and ulcers, venous wounds and ulcers, and arterial wounds and ulcers. The pharmaceutical composition can be prepared according to methods known in the art. The polypeptide of the present invention can be combined with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants to form any dosage form suitable for human or animal use. The polypeptide of the present invention is typically present in a pharmaceutical composition at a concentration of 0.1-99.9% by weight.

[0016] The dosage of the polypeptide pharmaceutical composition of the present invention can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration, and the dosage form. Generally speaking, a suitable daily dosage range of the polypeptide of the present invention is 0.001-20 mg / kg body weight, preferably 0.1-200 μg / kg body weight. The above dosage can be administered as a single dosage unit or divided into several dosage units, depending on the physician's clinical experience and the dosage regimen including the use of other treatment methods. The polypeptide or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the polypeptide of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

[0017] The polypeptide of the present invention or a pharmaceutical composition containing the same can be administered in a unit dosage form, and the route of administration can be external or internal, such as wound application, wound spray, oral administration, intravenous injection, intramuscular injection, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc. The dosage form can be a liquid dosage form, a solid dosage form, or a semi-solid dosage form. Liquid dosage forms may be solutions (selected from true solutions and colloidal solutions), emulsions (selected from o / w, w / o, and multiple emulsions), suspensions, injections (selected from aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments; solid dosage forms may be tablets (selected from conventional tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (selected from hard capsules, soft capsules, and enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), and sprays; semisolid dosage forms may be ointments, gels, and pastes. The polypeptides of the present invention may be prepared into conventional preparations, sustained-release preparations, controlled-release preparations, targeted preparations, and various microparticle delivery systems.

[0018] In order to prepare the polypeptide of the present invention into a dosage form for external or internal use, various excipients known in the art can be widely used, selected from diluents, binders, wetting agents, disintegrants, lubricants, and glidants. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropyl alcohol, etc.; binders can be starch slurry, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinyl pyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, etc.; lubricants and glidants can be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc. Tablets can also be further prepared as coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets. To prepare the dosing unit as a capsule, the active ingredient compound of the present invention can be mixed with a diluent and a glidant, and the mixture can be directly placed in a hard or soft capsule. Alternatively, the active ingredient compound of the present invention can be first prepared into granules or pellets with a diluent, a binder, and a disintegrant, and then placed in a hard or soft capsule. The diluents, binders, wetting agents, disintegrants, and glidants used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention. To prepare the compound of the present invention as an injection, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of solubilizers, cosolvents, pH adjusters, and osmotic pressure regulators commonly used in the art can be added. Solubilizers or cosolvents may include poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc.; pH adjusters may include phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure regulators may include sodium chloride, mannitol, glucose, phosphates, acetates, etc. For the preparation of lyophilized powder injections, mannitol, glucose, etc. may also be added as support agents. Furthermore, colorants, preservatives, fragrances, flavorings, or other additives may be added to the pharmaceutical preparations as needed. To achieve the intended use and enhance the therapeutic effect, the drugs or pharmaceutical compositions of the present invention may be administered using any known method.

[0019] Beneficial technical effects

[0020] 1. The polypeptides of the present invention can be used to prevent, alleviate, and / or treat conditions selected from diabetic wounds, traumatic wounds, pressure wounds, ischemic wounds, venous wounds, and arterial wounds. This use is disclosed for the first time. This broadens the clinical application of the polypeptides, particularly for such diseases for which no clinically effective drugs exist. The polypeptides provide a drug option for clinical applications.

[0021] 2. Currently, the use of the polypeptides of the present invention in preventing, alleviating, and / or treating wounds selected from diabetic wounds, traumatic wounds, pressure wounds, ischemic wounds, venous wounds, and arterial wounds is the first disclosed application, both internationally and domestically. No related papers or patents have been published.

[0022] 3. For diabetic wounds, traumatic wounds, pressure wounds, ischemic wounds, venous wounds, and arterial wounds, no clinically effective medications have been found, either domestically or internationally, aside from skin grafting and topical growth factors. Growth factors have the disadvantages of being limited to topical use and unstable. Therefore, new, safe, and effective treatments are urgently needed.

[0023] 4. The polypeptide of the present invention can achieve therapeutic and preventive effects with a small dose and can be applied externally. It is safe and reliable and has significant advantages in its development as a drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Definition: Urotensin II (UII) is an active polypeptide (urotensin II); LPS is lipopolysaccharide.

[0025] Figure 1 Effects of UII on proliferation and migration of normal fibroblasts. Normal group: no treatment; drug administration group: different concentrations of UⅡ (10 -9 M, 10 -8 M, 10 -7 This study investigated the effects of UII administration on the proliferation and migration of normal fibroblasts. UII administration significantly promoted the proliferation and migration of fibroblasts, with statistically significant differences compared to the normal group. N = 3-6, mean ± SD. Compared to the high glucose group: *p < 0.05,

[0026] **p<0.01, ***p<0.001, ****p<0.0001.

[0027] Figure 2 Effects of UII on the proliferation and migration of fibroblasts induced by high glucose. Normal group: 25 mM glucose; high glucose group: 65 mM glucose; drug administration group: 65 mM glucose + different concentrations of UII (10 -9 M, 10 -8 M, 10 -7 M). In this experiment, high glucose treatment reduced fibroblast proliferation and inhibited fibroblast migration, whereas UII administration significantly promoted fibroblast proliferation and migration, with statistically significant differences compared to the high glucose model. N = 4-6, mean ± SD. Compared to the high glucose group: *p < 0.05, **p < 0.01,

[0028] ***p<0.001, ****p<0.0001.

[0029] Figure 3 Effects of UII on proliferation and migration of fibroblasts induced by high fat. Normal group: no oleic acid; high fat group: 0.2 mM oleic acid; drug administration group: 0.2 mM oleic acid + different concentrations of UII (10 -9 M, 10 -8 M, 10 -7 M). In this experiment, high-fat treatment reduced fibroblast proliferation and inhibited fibroblast migration. However, UII administration significantly promoted fibroblast proliferation and migration, demonstrating statistically significant differences compared to the high-fat model. N = 3-6, mean ± SD. Compared with the high-fat group: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0030] Figure 4 Effects of UII on LPS-induced NO production in macrophages. Normal group: no LPS; Model group: 500ng / ml LPS; Drug group: 500ng / ml LPS + different concentrations of UII (10 -9 M, 10 -8 M, 10 -7 M). In this experiment, LPS treatment stimulated macrophage NO production, but UII administration significantly inhibited macrophage NO production, demonstrating statistically significant differences compared to the LPS model group. N = 3-6, mean ± SD. Compared with the LPS group: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0031] Figure 5 Effects of UII on the expression of proinflammatory cytokines TNFα, IL-1β, and IL-6 in macrophages induced by LPS. Normal group: no LPS; model group: 500ng / ml LPS; drug group: 500ng / ml LPS + 10 -7 M UII. In this experiment, LPS treatment significantly promoted the expression of inflammatory cytokines TNFα, IL-1β, and IL-6. UII administration effectively suppressed inflammatory cytokine expression, demonstrating statistically significant differences compared with the LPS model group. N = 6, mean ± SD. Compared with the LPS group: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0032] Figure 6 Effect of UII on the expression of anti-inflammatory cytokine IL-10 in macrophages induced by LPS. Normal group: no LPS; model group: 500ng / ml LPS; drug group: 500ng / ml LPS + 10-7 M UII. In this experiment, LPS treatment significantly inhibited the expression of the anti-inflammatory cytokine IL-10, while UII administration effectively promoted this expression, demonstrating statistically significant differences compared with the LPS model group. N = 3-6, mean ± SD. Compared with the LPS group: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0033] Figure 7 Effects of UII on the expression of surface markers CD80, CD86, and iNOS in M1 macrophages induced by LPS. Normal group: no LPS; model group: 500ng / ml LPS; drug group: 500ng / ml LPS+10 -7 M UⅡ. In this experiment, the expression of M1 macrophage surface markers CD80, CD86, and iNOS increased significantly after LPS treatment, while their expression decreased significantly after UII administration, showing statistically significant differences compared with the LPS model group. N = 3-6, mean ± SD. Compared with the LPS group: *p < 0.05, **p < 0.01, ***p < 0.001,

[0034] ****p<0.0001.

[0035] Figure 8 Effect of UII on the expression of Arg-1, CD163, and CD206 surface markers of M2 macrophages induced by LPS. Normal group: no LPS; model group: 500ng / ml LPS; drug group: 500ng / ml LPS + 10 -7 M UII. In this experiment, LPS treatment significantly decreased the expression of M2 macrophage surface markers Arg-1, CD163, and CD206. UII administration significantly increased their expression, demonstrating statistically significant differences compared with the LPS model group. N = 3-6, mean ± SD. Compared with the LPS group: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0036] Figure 9 Effects of UII on wounds in C57BL / 6 mice with type 1 diabetes. Normal control group: normal control mice; model group: type 1 diabetic mice; treatment group: type 1 diabetic mice administered different concentrations of UII (10 μg / kg, 30 μg / kg, 100 μg / kg). In this experiment, topical UII significantly promoted wound healing in animals with type 1 diabetes. N = 6-8, mean ± SD. Compared with normal animals: *p < 0.05, **p < 0.01, ****p < 0.0001.

[0037] Figure 10 Effects of UII on body weight in type 1 diabetic C57BL / 6 mice. Normal control group: normal control mice; model group: type 1 diabetic mice; treatment group: type 1 diabetic mice administered different concentrations of UII (10 μg / kg, 30 μg / kg, 100 μg / kg). Compared with the normal control group, the body weight of the diabetic animals decreased significantly. UII treatment showed no significant effect on the body weight of the diabetic animals after one week of treatment. N = 6-8, mean ± SD. Compared with normal animals: ****p < 0.0001. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the difficulty of wound healing caused by UII, which is selected from diabetic wounds, traumatic wounds, pressure wounds, ischemic wounds, venous wounds, and arterial wounds. The following examples illustrate the present invention in more detail and are not intended to limit the present invention in any way.

[0039] Experimental Example 1: Effects of UII on the proliferation and migration of normal fibroblasts

[0040] Experimental Principle

[0041] The proliferation and migration of fibroblasts can reflect the promotion of wound healing by drugs and their impact on the speed of wound healing.

[0042] Experimental methods

[0043] Cell proliferation: After digestion, cells were plated in 96-well plates at a density of 4,000 cells per well. The next day, the medium was replaced with serum-free medium and incubated with different doses of UII. CCK8 was added after 24, 48, and 72 hours of treatment, and the absorbance at OD 450 nM was measured.

[0044] Cell migration: After digestion, cells are plated into a 96-well plate, with the appropriate number of cells adhering to the plate and covering the bottom. The next day, use a 10 μL pipette tip perpendicular to the plate to create scratches, ensuring that each scratch is of uniform width. Aspirate the cell culture medium and wash the plate twice with an equal amount of Hanks' solution to remove cell debris from the scratches. Add 2 mL of serum-free culture medium containing different drug treatments and photograph the plate. Place the plate in an incubator and culture. Take photos under a microscope after 12, 24, and 48 hours. Analyze the experimental results based on the collected image data.

[0045] Experimental results

[0046] In this experiment, UⅡ administration significantly promoted the proliferation and migration of fibroblasts, with statistically significant differences compared to the control group. UⅡ promoted the proliferation and migration of fibroblasts, indicating that the drug can promote the healing of in vitro wound models. The results are shown in Tables 1-2 and Figure 1 .

[0047] Table 1 Effects of UII on the proliferation of normal fibroblasts.

[0048]

[0049] Table 2 Effects of UII on the migration of normal fibroblasts.

[0050]

[0051] Experimental Example 2: Effects of UII on high glucose-induced fibroblast proliferation and migration

[0052] Experimental Principle

[0053] The proliferation and migration of fibroblasts can reflect the promotion of wound healing by drugs and their impact on the speed of wound healing.

[0054] Experimental methods

[0055] Cell proliferation: After digestion, cells were plated in 96-well plates at a density of 4,000 cells per well. The next day, the cells were replaced with serum-free medium containing varying glucose concentrations and incubated with various doses of UII. After 24 hours, CCK8 was added, and the absorbance at OD 450 nM was measured.

[0056] Cell migration: After digestion, cells were plated into a 96-well plate, with the appropriate number of cells adhering to the wall and covering the bottom of the plate. The next day, use a 10 μL pipette tip perpendicular to the plate to create cell scratches, ensuring that each scratch is of uniform width. Aspirate the cell culture medium and wash the plate twice with an equal amount of Hanks' solution to remove cell debris from the scratches. Add 2 mL of serum-free culture medium containing different drug treatments and different glucose concentrations, and photograph and record. Place the culture plate in an incubator. Take photos under a microscope after 12, 24, and 48 hours. Analyze the experimental results based on the collected image data.

[0057] Experimental results

[0058] In this experiment, high glucose treatment reduced the proliferation and migration of fibroblasts, while UⅡ administration significantly promoted the proliferation and migration of fibroblasts, with statistically significant differences compared to the high glucose model. UⅡ can promote the proliferation and migration of fibroblasts in a dose-dependent manner, indicating that the drug can promote the healing of in vitro wound models. The results are shown in Tables 3-4 and Figure 2 .

[0059] Table 3 Effects of UII on high glucose-induced fibroblast proliferation.

[0060]

[0061] Table 4 Effects of UII on high glucose-induced fibroblast migration.

[0062]

[0063]

[0064] Experimental Example 3: Effects of UII on high-fat-induced fibroblast proliferation and migration

[0065] Experimental Principle

[0066] The proliferation and migration of fibroblasts can reflect the promotion of wound healing by drugs and their impact on the speed of wound healing.

[0067] Experimental methods

[0068] Cell proliferation: After digestion, cells were plated in 96-well plates at a density of 4,000 cells per well. The next day, the cells were replaced with serum-free medium containing varying oleic acid concentrations and incubated with various doses of UII. After 24 hours, CCK8 was added, and the absorbance at OD 450 nM was measured.

[0069] Cell migration: After digestion, cells were plated into a 96-well plate, with the appropriate number of cells adhering to the wall and covering the bottom of the plate. The next day, use a 10 μL pipette tip perpendicular to the plate to create scratches, ensuring that each scratch is of uniform width. Aspirate the cell culture medium and wash the plate twice with Hanks' solution equal to the original inoculated cell volume to remove cell debris generated by the scratches. Add 2 mL of serum-free culture medium containing different drug treatments and different oleic acid concentrations, and photograph and record. Place the culture plate in an incubator and culture. Take photos under a microscope after 12, 24, and 48 hours. Analyze the experimental results based on the collected image data.

[0070] Experimental results

[0071] In this experiment, high-fat treatment can reduce the proliferation and migration of fibroblasts, while UⅡ administration can significantly promote the proliferation and migration of fibroblasts, with statistically significant differences compared with the high-fat model. UⅡ can effectively promote the proliferation and migration of fibroblasts, indicating that the drug can promote the healing of in vitro wound models. The results are shown in Tables 5-6 and Figure 3 .

[0072] Table 5 Effects of UII on high-fat-induced fibroblast proliferation.

[0073]

[0074] Table 6 Effects of UII on high-fat-induced fibroblast migration.

[0075]

[0076] Experimental Example 4: Effect of UII on LPS-induced NO production in macrophages

[0077] Experimental Principle

[0078] Inflammation-related diseases often manifest as excessive inflammatory responses. Macrophages are one of the key cells involved in responding to inflammation. The anti-inflammatory effects of drugs on macrophages can reflect the drug's ability to prevent or treat inflammation-related diseases. NO production can reflect the level of cellular inflammation; greater NO production indicates a higher level of cellular inflammation.

[0079] Experimental methods

[0080] After digestion, cells were plated in 96-well plates at a density of 20,000 cells per well. The next day, serum-free culture medium was replaced with either lipopolysaccharide (LPS)-containing or lipopolysaccharide-containing medium. Different concentrations of UⅡ were added to the treatment groups. After 24 hours, NO levels in the supernatant were measured using a NO assay.

[0081] Experimental results

[0082] In this experiment, LPS treatment increased the production of NO in macrophages, while UⅡ administration significantly inhibited the production of NO in macrophages, with statistically significant differences compared with the model group. The results are shown in Table 7 and Figure 4 .

[0083] Table 7 Effects of UII on LPS-induced NO production in macrophages.

[0084]

[0085] Experimental Example 5: Effect of UII on LPS-induced expression of proinflammatory cytokines TNFα, IL-1β, and IL-6 in macrophages

[0086] Experimental Principle

[0087] Inflammation-related diseases often manifest as excessive inflammatory responses. Macrophages are one of the key cells that respond to inflammation. A drug's anti-inflammatory effect on macrophages can reflect its ability to prevent or treat inflammation-related diseases. The production of proinflammatory cytokines can reflect the level of cellular inflammation; more proinflammatory cytokines are produced, indicating a higher level of cellular inflammation.

[0088] Experimental methods

[0089] After cell digestion, the cells were plated in six-well plates with 60,000 cells per well. The next day, the medium was replaced with serum-free medium containing or without lipopolysaccharide (LPS) concentration. The drug-treated group was added with 10 -7 M UⅡ. After 24 hours, RNA was extracted from the cells and the expression levels of TNF-α, IL-1β, and IL-6 were detected.

[0090] Experimental results

[0091] In this experiment, LPS treatment significantly increased the expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 at the RNA level in macrophages, while UⅡ administration significantly inhibited their expression, with statistically significant differences compared with the model group. The results are shown in Table 8 and Figure 5 .

[0092] Table 8 Effects of UII on LPS-induced macrophage proinflammatory cytokines TNFα, IL-1β, and IL-6 production

[0093]

[0094] Experimental Example 6: Effect of UII on LPS-induced expression of anti-inflammatory cytokine IL-10 in macrophages

[0095] Experimental Principle

[0096] Inflammatory diseases often manifest as excessive inflammatory responses. Macrophages are one of the key cells that respond to inflammation. A drug's anti-inflammatory effect on macrophages can reflect the drug's ability to prevent or treat inflammatory diseases. The production of anti-inflammatory cytokines can reflect the level of cellular inflammation; higher levels of anti-inflammatory cytokines indicate lower levels of cellular inflammation.

[0097] Experimental methods

[0098] After cell digestion, the cells were plated in six-well plates with 60,000 cells per well. The next day, the medium was replaced with serum-free medium containing or without lipopolysaccharide (LPS) concentration. The drug-treated group was added with 10 -7 M UⅡ. After 24 hours, RNA was extracted from the cells and the expression level of IL-10 was detected.

[0099] Experimental results

[0100] In this experiment, LPS treatment significantly reduced the expression of anti-inflammatory cytokine IL-10 at the RNA level in macrophages, while UⅡ administration significantly promoted its expression, with statistically significant differences compared with the model group. The results are shown in Table 9 and Figure 6 .

[0101] Table 9 Effects of UII on LPS-induced production of anti-inflammatory cytokine IL-10 in macrophages

[0102]

[0103] Experimental Example 7: Effect of UII on the expression of LPS-induced M1 macrophage surface markers CD80, CD86, and iNOS

[0104] Experimental Principle

[0105] Inflammation-related diseases often manifest as excessive inflammatory responses. Macrophages are one of the key cells that respond to inflammation. The anti-inflammatory effect of a drug on macrophages can reflect the drug's ability to prevent or treat inflammation-related diseases. Macrophages typically have two phenotypes: M1 and M2. M1 macrophages are typical activated macrophages that secrete proinflammatory factors to trigger inflammation and clear necrotic cells and bacteria. M2 macrophages are alternatively activated macrophages that secrete anti-inflammatory factors, prompting the inflammatory response to transition from a pro-inflammatory phase to an anti-inflammatory phase. The expression of M1 macrophage surface markers can reflect the number of M1 macrophages, and thus the overall level of inflammation in the cells. Higher expression of M1 macrophage surface markers indicates a greater number of M1 macrophages and a higher level of inflammation.

[0106] Experimental methods

[0107] After cell digestion, the cells were plated in six-well plates with 60,000 cells per well. The next day, the medium was replaced with serum-free medium containing or without lipopolysaccharide (LPS) concentration. The drug-treated group was added with 10 -7 M UⅡ. After 24 hours, RNA was extracted from the cells and the expression levels of CD80, CD86, and iNOS were detected.

[0108] Experimental results

[0109] In this experiment, LPS treatment significantly increased the expression of M1 macrophage surface markers CD80, CD86, and iNOS at the RNA level, while UⅡ administration significantly reduced their expression, with statistically significant differences compared with the model group. The results are shown in Table 10 and Figure 7 .

[0110] Table 10 Effects of UII on the expression of LPS-induced M1 macrophage surface markers CD80, CD86, and iNOS

[0111]

[0112] Experimental Example 8: Effect of UII on the expression of Arg-1, CD163, and CD206 surface markers of M2 macrophages induced by LPS

[0113] Experimental Principle

[0114] Inflammatory diseases often manifest as excessive inflammatory responses. Macrophages are one of the key cells that respond to inflammation. The anti-inflammatory effect of a drug on macrophages can reflect the drug's ability to prevent or treat inflammatory diseases. Macrophages typically have two phenotypes: M1 and M2. M1 macrophages are typically activated macrophages that secrete proinflammatory factors to trigger inflammation and clear necrotic cells and bacteria. M2 macrophages are alternatively activated macrophages that secrete anti-inflammatory factors, prompting the inflammatory response to transition from a pro-inflammatory phase to an anti-inflammatory phase. The expression of M2 macrophage surface markers can reflect the number of M2 macrophages, and thus the overall level of inflammation in the cells. Higher expression of M2 macrophage surface markers indicates a greater number of M2 macrophages and a lower level of inflammation.

[0115] Experimental methods

[0116] After cell digestion, the cells were plated in six-well plates with 60,000 cells per well. The next day, the medium was replaced with serum-free medium containing or without lipopolysaccharide (LPS) concentration. The drug-treated group was added with 10 -7 M UⅡ. After 24 hours, RNA was extracted from the cells and the expression levels of Arg-1, CD163, and CD206 were detected.

[0117] Experimental results

[0118] In this experiment, LPS treatment significantly reduced the expression of M2 macrophage surface markers Arg-1, CD163, and CD206 at the RNA level, while UⅡ administration significantly increased their expression, with statistically significant differences compared with the model group. The results are shown in Table 11 and Figure 8 .

[0119] Table 11 Effects of UII on the expression of Arg-1, CD163, and CD206 surface markers of M2 macrophages induced by LPS

[0120]

[0121] Experimental Example 9: UII promotes wound healing in normal mice

[0122] Experimental Principle

[0123] The effect of UII on normal wound healing was evaluated by observing the healing of wounds of the same size in different groups. The effect on normal wounds can be used to preliminarily determine whether the drug's effect is disease-specific, so the drug's effect on normal wound healing was evaluated.

[0124] Experimental methods

[0125] Fourteen C57BL / 6 mice, weighing 17-20 g, were randomly divided into two groups of seven. One group received UII 30 μg / kg topical medication, and the other a normal control group, which received the same volume of saline. The animals' skin was depilated and punctured, and the next day, the drug was administered. UII was administered daily for one week, with the drug prepared fresh before each administration. Wound conditions were photographed and recorded.

[0126] Experimental results

[0127] UII administration significantly promoted wound healing in normal animals. During the experimental period, no infection or other abnormalities were observed in any group, except for wound modeling. Results are shown in Table 12.

[0128] Table 12. Effects of drugs on wounds in normal C57BL / 6 mice.

[0129]

[0130] Experimental Example 10: Effect of UⅡ on wound healing in diabetic mice

[0131] Experimental Principle

[0132] The efficacy of UⅡ in improving wound healing in diabetic patients was evaluated by observing the healing of wounds of equal size in different groups. The nitrosourea in the streptozotocin structure is cytotoxic, and the deoxyglucose moiety facilitates its entry into pancreatic beta cells. STZ damages pancreatic beta cells through free radicals such as CH3, impairing intracellular insulin synthesis and leading to insulin deficiency. It can be used to induce animal models of both type 1 and type 2 diabetes.

[0133] Experimental methods

[0134] C57BL / 6 mice were intraperitoneally injected with streptozotocin (150 mg / kg). One week later, blood glucose was measured using a Roche blood glucose meter. Mice with blood glucose levels above 15 mmol / L were considered diabetic for the experiment. The animals were divided into three groups: normal group, model group, UII low-dose 10 μg / kg group, medium-dose 30 μg / kg group, and high-dose 100 μg / kg group, with 10 mice in each group. After anesthesia, the animals were hairless, and wounds were punctured on the back every other day. UII was applied topically for one week. During this period, mice were fed a normal, full-body diet. Wound healing was recorded daily by photographing the mice.

[0135] Experimental results

[0136] During the experiment, no wound infection occurred in any group of animals, and no abnormal symptoms other than diabetes and wounds were observed in the animals. External application of UⅡ significantly promoted wound healing in diabetic animals. Compared with the model control, the wound area decreased faster, and no other significant changes were observed except for the shrinkage of the wound surface. The results are shown in Table 13 and Figure 9 .

[0137] Table 13. Effects of UⅡ on wound healing in diabetic animals.

[0138]

[0139] Experimental Example 11: Effect of UⅡ on the Body Weight of Diabetic Mice

[0140] Experimental methods

[0141] Animal treatment is described in Example 4. Body weight is an important indicator of animal energy utilization balance and growth. In this experiment, the activity status, hair growth, and other general conditions of the animals were observed and recorded daily, and the animal weight was monitored daily.

[0142] Experimental results

[0143] In this experiment, the body weight of the diabetic group was significantly lower than that of the normal control group, and UⅡ had no significant effect on the body weight of the animals. Figure 10 .

[0144] Table 14. Effects of drugs on body weight in diabetic animals.

[0145]

Claims

1. Use of the polypeptide shown below or a polypeptide sequence containing the active sequence shown below in the preparation of a method for treating and / or preventing wound healing difficulties, wherein the polypeptide sequence is: Glu1-Thr2-Pro3-Asp4-Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10-Val11, where Cys5 and Cys10 is a disulfide bond connection; the active sequence of the active sequence polypeptide is: Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10, wherein Cys5 and Cys10 are connected by a disulfide bond.

2. The use according to claim 1, characterized in that The wounds described are acute and chronic wounds.

3. The use according to claim 1, characterized in that The wound is selected from diabetic wounds and ulcers, traumatic wounds and ulcers, pressure wounds and ulcers, ischemic wounds and ulcers, venous wounds and ulcers, and arterial wounds and ulcers.

4. The use according to claim 2, wherein the chronic wound refers to delayed healing wounds and ulcers.

5. The use according to claim 3, characterized in that The diabetes is selected from type 1 or type 2 diabetes.

6. Use of a pharmaceutical composition in the preparation of a drug for preventing and / or treating difficulty in wound healing, characterized in that: The pharmaceutical composition contains an effective dose of the following polypeptide or active sequence polypeptide and a pharmaceutically acceptable carrier; the polypeptide sequence is: Glu1-Thr2-Pro3-Asp4-Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10-Val11, where Cys5 and Cys10 is a disulfide bond connection; the active sequence of the active sequence polypeptide is: Cys5-Phe6-Trp7-Lys8-Tyr9-Cys10, wherein Cys5 and Cys10 are connected by a disulfide bond.

7. Use according to claim 5, characterized in that The pharmaceutical composition contains not only the polypeptide or active sequence polypeptide described below and a pharmaceutically acceptable carrier as pharmaceutical active ingredients, but also other active ingredients.

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