Application of MOTS-c and amino acid mutant polypeptide thereof in wound healing

Abstract

The invention discloses MOTS-c and an application of an amino acid mutant polypeptide of the MOTS-c in wound healing. The amino acid sequence of the amino acid mutant polypeptide is MRWQEMGYIFYPFKLR. The amino acid mutant polypeptides can be used for preparing medicines for wound healing, and particularly, the amino acid mutant polypeptides have a more remarkable effect. Compared with a conventional wound healing treatment scheme, the scheme provided by the invention is lower in cost and has certain universality.

Description

Technical Field

[0001] The present application relates to the technical field of wound healing drugs, and in particular to the use of a bioactive peptide in wound healing. Background Art

[0002] Wound healing is a natural repair process of an organism in response to injury, involving complex biological events, including inflammation, proliferation, and remodeling phases. Effective wound healing is essential for preventing infection and restoring tissue function and appearance. Traditionally, wound treatment has relied on physical barriers, antibiotics, and topically applied drugs, but these approaches often have limitations, such as slow healing, inadequate pain management, or potential drug resistance issues.

[0003] For diabetic patients, wound healing also has the following special features:

[0004] (1) Microangiopathy: Diabetic patients often suffer from microangiopathy, which impairs blood circulation in the wound area and reduces the supply of oxygen and nutrients required for wound healing. In addition, microangiopathy may also cause local tissue hypoxia, affecting the wound healing process.

[0005] (2) Impaired immune function: A high blood sugar environment affects the function of white blood cells, including weakening their chemotaxis, phagocytosis, and bactericidal ability. This causes diabetic patients to have a decreased resistance to infection, making it more susceptible to infection during wound healing, thereby delaying healing.

[0006] (3) The role of insulin: Insulin is not only a key hormone for regulating blood sugar levels, but also directly participates in the wound healing process, including promoting cell proliferation, protein synthesis and migration. Diabetic patients may affect these processes due to insulin resistance or insufficient secretion, thereby affecting wound healing.

[0007] (4) Sustained inflammatory response: The inflammatory response lasts longer during the wound healing process in diabetic patients. Under normal circumstances, inflammation is a necessary response in the early stages of wound healing, but in diabetic patients, due to imbalanced regulation, the inflammatory response may be excessive and prolonged, which will hinder the subsequent healing stage.

[0008] (5) Effects of high-glucose environment on cell function: High blood sugar can directly affect the functions of various cells (such as fibroblasts, endothelial cells and macrophages), inhibiting their proliferation, migration and differentiation capabilities. The functions of these cells are crucial for wound healing.

[0009] (6) Impact of neuropathy: Diabetic patients often suffer from peripheral neuropathy, which may reduce the perception of pain in the wound area, resulting in insufficient protection of the wound by the patient, increasing the risk of secondary injury to the wound and further affecting the healing process.

[0010] For the treatment of wound healing in diabetic patients, the common treatment substances and methods are mainly divided into:

[0011] 1. Local treatment:

[0012] a. Antibiotics: used to prevent and treat wound infection. They can be applied topically or systemically. Choose the appropriate antibiotic based on the infection situation.

[0013] b. Growth factors: such as epidermal growth factor (EGF), fibroblast growth factor (FGF), etc., can act directly on the wound to promote cell proliferation and new blood vessel formation.

[0014] c. Moist dressings: such as hydrogels, alginate dressings, etc., can provide a moist environment for the wound, promote cell migration and proliferation, and help accelerate healing.

[0015] 2. Systemic treatment:

[0016] a. Blood sugar control: Maintaining blood sugar levels within the ideal range is the basis of treatment and can be achieved through oral medication or insulin therapy.

[0017] b. Nutritional support: Provide adequate nutrients such as protein, vitamin C and zinc to support the synthesis process required for wound repair.

[0018] c. Drugs to improve blood circulation: such as the use of vasodilators, antiplatelet drugs, etc., to improve local blood circulation and increase blood flow to the wound area.

[0019] 3. Physical therapy:

[0020] a. Negative pressure wound therapy (NPWT): By applying negative pressure on the wound, blood circulation is promoted, edema is reduced, and local cell proliferation in the wound is increased.

[0021] b. Hyperbaric oxygen therapy: By increasing the oxygen partial pressure in the blood, it increases the oxygenation of wound tissue and helps promote wound healing.

[0022] Although the above treatments play an important role in diabetic wound management, there are still many shortcomings:

[0023] (1) High cost of treatment: Some advanced treatments, such as growth factor therapy, negative pressure wound therapy, and hyperbaric oxygen therapy, are expensive, which may limit their application in a wide range of populations.

[0024] (2) There are large individual differences in treatment effects: Due to differences in blood sugar control levels, concomitant diseases and individual differences among diabetic patients, there are large differences in the effects of these treatments. Summary of the invention

[0025] This application proposes the use of MOTS-c and a specific amino acid mutant polypeptide thereof in wound healing, and defines various technical solutions. Specifically:

[0026] In the first aspect, an amino acid mutant polypeptide of a mitochondrial-derived peptide MOTS-c has an amino acid sequence of: MRWQEMGYIFYPFKLR.

[0027] In a second aspect, the amino acid mutant polypeptide of the mitochondrial-derived peptide MOTS-c is used for preparing a drug for wound healing.

[0028] Optionally, the above-mentioned medicine is particularly suitable for diabetic patients, and can also be suitable for ordinary patients. The ordinary patients (ordinary trauma patients) mentioned here are relative to diabetic patients (diabetic patients who are also traumatized). The patients mentioned here can be humans or other mammals (such as mice, dogs, cats, pigs, rabbits, etc.).

[0029] In a third aspect, a medicine for wound healing, the active ingredient of which is the amino acid mutant polypeptide of the mitochondrial derived peptide MOTS-c, is generally used as an external medicine. Optionally, as an external medicine, it also includes nano-CaCO3 and hydrogel for loading the amino acid mutant polypeptide.

[0030] In a fourth aspect, a method for preparing a medicine for wound healing comprises:

[0031] Prepare CaCO3 nanoparticles and dry them for later use;

[0032] The amino acid mutant polypeptide of the mitochondrial derived peptide MOTS-c is dissolved in a phosphate buffer solution to prepare a dispersion; CaCO3 nanoparticles are prepared into an aqueous solution and combined with the dispersion, stirred horizontally, and then centrifuged to produce nano-CaCO3 loaded with the amino acid mutant polypeptide, and then stirred magnetically, centrifuged, and allowed to stand to obtain MOTS-c-Ca powder;

[0033] The MOTS-c-Ca powder is mixed with the FA-Ca solution, and the mixture is evenly coated on the surface of the silk fibroin to obtain the MOTS-c-Ca gel, which is a medicine for wound healing.

[0034] The above amino acid mutant polypeptide is obtained by introducing a point mutation into the coding gene sequence of the mitochondrial derived peptide MOTS-c, which can be achieved by existing molecular biological techniques.

[0035] Fifthly, the mitochondrial-derived peptide MOTS-c itself can also be used to prepare drugs for wound healing.

[0036] Optionally, the above-mentioned medicine is particularly suitable for diabetic patients, and can also be suitable for ordinary patients. The ordinary patients (ordinary trauma patients) mentioned here are relative to diabetic patients (diabetic patients who are also traumatized). The patients mentioned here can be humans or other mammals (such as mice, dogs, cats, pigs, rabbits, etc.).

[0037] In a sixth aspect, a medicine for wound healing, wherein the active ingredient is a mitochondrial derived peptide MOTS-c. It is usually used as an external medicine. Optionally, as an external medicine, it also includes nano-CaCO3 and a hydrogel for loading the amino acid mutant MOTS-c. Accordingly, the preparation method of the medicine can also refer to the aforementioned preparation method.

[0038] The inventors of the present application have found that the mitochondrial derived peptide MOTS-c and its aforementioned specific amino acid mutant polypeptide can be used to prepare medicines for wound healing (especially for diabetic patients, but also for ordinary patients), wherein the amino acid mutant polypeptide has a more significant effect. Compared with the existing wound healing treatment scheme, the scheme of the present application has lower cost, more stable effect, and has certain universality. Of course, medical staff can also combine the scheme of the present application with other schemes as appropriate. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a line graph of keratinocyte viability over time, showing the changing trend of keratinocyte viability (expressed as a percentage) under high glucose (HG) conditions within 5 days under different concentrations of WT-MOTS-c treatment. The horizontal axis is time (unit: day), and the vertical axis is the percentage of keratinocyte viability. Lines of different colors represent different treatment groups, where the control group (Con) is represented by green, the HG group is represented by pink, and the HG+WT-MOTS-c (50μM), HG+WT-MOTS-c (100μM) and HG+WT-MOTS-c (150μM) treatment groups are represented by red, orange and blue, respectively. With the increase of treatment concentration, cell viability showed different response trends. Statistical significance is indicated by asterisks: * indicates p<0.05, ** indicates p<0.01.

[0040] Figure 2This is a line graph of keratinocyte viability over time, showing the changing trend of keratinocyte viability (expressed as a percentage) within 5 days under HG conditions under different concentrations of mutant MOTS-c (MUT-MOTS-c). The horizontal axis is time (unit: day), and the vertical axis is the percentage of keratinocyte viability. The Con group is represented by green, the HG group is represented by pink, and the HG+MUT-MOTS-c (50μM), HG+MUT-MOTS-c (100μM) and HG+MUT-MOTS-c (150μM) treatment groups are represented by red, orange and blue, respectively. With the increase of treatment concentration, keratinocyte viability showed different response trends. Statistical significance is indicated by asterisks: ** indicates p<0.01.

[0041] Figure 3 The figure is a bar graph of keratinocyte viability, showing the effect of WT-MOTS-c and MUT-MOTS-c on keratinocyte viability after 4 days of treatment under HG conditions. The ordinate represents the percentage of keratinocyte viability, and the abscissa shows the different treatment groups: Con group, HG group, HG+WT-MOTS-c (50 μM) treatment group, and HG+MUT-MOTS-c (50 μM) treatment group. Statistical significance is indicated by asterisks: ** indicates p<0.01, *** indicates p<0.001.

[0042] Figure 4 The scratch test diagram and the bar graph of keratinocyte migration rate show the effect of WT-MOTS-c and MUT-MOTS-c treatment on the migration ability of keratinocytes under HG conditions. Among them, (a) shows the cell migration of each group at 0 hours and 24 hours. The red dotted line marks the position of the cell boundary, which is used to evaluate the migration of keratinocytes. (b) The migration rate of keratinocytes is quantified, and the experimental groups are distinguished by different colors: red represents the Con group, blue represents the HG group, orange represents the HG+WT-MOTS-c treatment group, and green represents the HG+MUT-MOTS-c treatment group. Statistical significance is indicated by asterisks: * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0043] Figure 5The fluorescence image (a) and the quantitative bar graph (b) of the fluorescence intensity of the reactive oxygen species (ROS) in keratinocytes detected by the DCFH-DA probe. Among them, (a) shows the ROS levels of 4 different treatment groups, namely the Con group, HG group, HG+WT-MOTS-c treatment group and HG+MUT-MOTS-c treatment group. (b) shows the quantitative analysis of the ROS fluorescence intensity of each group, showing the change multiple of the fluorescence intensity relative to the number of cells. Statistical significance is indicated by asterisks: * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0044] Figure 6 The bar graphs show the mRNA expression folds of proinflammatory cytokines IL-6, IL-1β, and TNF-α relative to the internal reference GAPDH, showing the effects of WT-MOTS-c and MUT-MOTS-c treatment on the mRNA expression of proinflammatory cytokines IL-6, IL-1β, and TNF-α under HG conditions. (a), (b), and (c) The three bar graphs show the expression folds of IL-6, IL-1β, and TNF-α relative to the internal reference GAPDH. The different treatment groups include Con, HG, HG+WT-MOTS-c treatment group, and HG+MUT-MOTS-c treatment group. Statistical significance is indicated by asterisks: * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and "ns" indicates no significant difference.

[0045] Figure 7 Glucose tolerance (GTT) and insulin tolerance (ITT) experiments of mice on a high-fat diet (HFD). (a) is the GTT curve and (b) is the ITT curve. The horizontal axis is time (minutes) and the vertical axis is blood glucose level (mg / dl). Orange dots represent the Con group and blue squares represent the HFD group. Statistical significance is indicated by asterisks: *** indicates p<0.001.

[0046] Figure 8Figure 1 shows the effect of different treatments on wound healing in mouse skin, showing the effect of HFD treatment on the wound healing process in mouse skin, and the role of WT-MOTS-c and MUT-MOTS-c in wound healing. Among them, (a) is the healing image of the wound at different time points (0, 1, 3, 5, 7, 9, 12 days), which is divided into Con, HFD, HFD+WT-MOTS-c treatment group and HFD+MUT-MOTS-c treatment group. (b) is the quantitative curve of wound healing rate, the ordinate represents the percentage of wound size relative to the initial size, and the abscissa is time (days). Orange dots represent the Con group, blue squares represent the HFD group, purple triangles represent the HFD+WT-MOTS-c group, and green inverted triangles represent the HFD+MUT-MOTS-c group. Statistical significance is indicated by asterisks: ** indicates p<0.01, *** indicates p<0.001.

[0047] Fig. 9 The figure shows the histological analysis of mouse skin wound healing in different treatment groups (Con, HFD, HFD+WT-MOTS-c, HFD+MUT-MOTS-c) after 14 days of wound healing. H&E staining (upper row) and Masson staining (lower row) were used to evaluate the structure of skin tissue and the formation of collagen fibers on day 14. H&E staining was used to observe the regeneration of the epidermis and dermis, and Masson staining was used to evaluate the deposition of collagen fibers.

[0048] Fig.10 The bar graphs show the mRNA expression of proinflammatory cytokines IL-6, IL-1β, and TNF-α, showing the effects of WT-MOTS-c and MUT-MOTS-c treatment on the mRNA expression levels of proinflammatory cytokines IL-6, IL-1β, and TNF-α under HFD conditions. The three bar graphs show the expression folds of IL-6, IL-1β, and TNF-α relative to the internal reference GAPDH. The experimental groups were Con, HFD, HFD+WT-MOTS-c treatment group, and HFD+MUT-MOTS-c treatment group. Statistical significance is indicated by asterisks: ** indicates p<0.01, *** indicates p<0.001.

[0049] Fig.11It is a mouse skin wound healing experiment diagram, showing the healing of mouse skin wounds under different treatment conditions. Among them, (a) shows the healing process of mouse wounds in each group at different time points (0, 1, 3, 5, 7, 9, and 12 days). The four experimental groups are Con group, human epidermal growth factor (EGF) treatment group, WT-MOTS-c and MUT-MOTS-c treatment group. (b) is a quantitative curve of wound healing rate, the ordinate represents the percentage of wound size relative to the initial size, and the abscissa is time (days). In the curve graph, orange dots represent Con, blue squares represent EGF group, purple triangles represent MOTS-c treatment group, and green inverted triangles represent MUT-MOTS-c treatment group. Statistical significance is indicated by asterisks: ** indicates p<0.01, *** indicates p<0.001, and "ns" indicates no significant difference. DETAILED DESCRIPTION

[0050] The present application is further described below in detail through specific embodiments in conjunction with the accompanying drawings.

[0051] In the description of the present application: unless otherwise specified, expressions such as “include”, “comprising”, “having”, etc. also mean “not limited to” (certain units, components, materials, steps, etc.).

[0052] MOTS-c (mitochondrial open reading frame of the 12S rRNA-c) is a mitochondrial-derived peptide (MDPs) composed of 16 amino acids. It is encoded by small open reading frames (sORFs) in the mitochondrial genome and participates in the regulation of various metabolic homeostasis of the body by regulating mitochondrial metabolism and nuclear genome transcription. Studies have shown that MOTS-c regulates many important metabolic processes such as glucose metabolism, lipid metabolism and bone metabolism through autocrine and paracrine pathways, AMPK pathway, AKT pathway, oxidative stress pathway and inflammation-related pathways. The discovery of MOTS-c provides a new strategy for the treatment of metabolic diseases such as diabetes and obesity.

[0053] The original MOTS-c sequence is as follows:

[0054] MRWQEMGYIFYPRKLR

[0055] The inventors of the present application used conventional molecular biological techniques to introduce a specific point mutation into the original MOTS-c sequence (the arginine at the fourth to last position was changed to phenylalanine), and obtained the following mutant MOTS-c sequence:

[0056] MRWQEMGYIFYPFKLR.

[0057] The study found that the normal peptide MOTS-c can accelerate the healing of skin wounds in diabetic mice and reduce the levels of inflammatory factors and ROS. The mutant peptide MOTS-c has a more significant effect in accelerating wound healing.

[0058] These two polypeptides can also be used in the form of hydrogel drugs for wound healing. The specific preparation method is as follows:

[0059] 1. Preparation of silk fibroin hydrogel:

[0060] (1) Degumming: Cut the dried cocoon shells, take out the silkworm pupae, weigh a certain amount of dried cocoon shells, put them into a Na2CO3 solution with a bath ratio of 1:100, a temperature of 100°C, and a mass fraction of 0.2%, boil them for 40 minutes, take them out, wash them repeatedly with deionized water, dry them in an oven, and check the degree of degumming until the degumming is completed.

[0061] (2) Subsequently, to prepare the silk fibroin solution, CaCl2 was first added to HCOOH, and then the degummed silk fibroin (SF) was added strand by strand to the FA-Ca solution (mass ratio of CaCl2: SF: HCOOH = 5:8:85) under magnetic stirring. The resulting mixture was stirred at 500 rpm for 2 hours.

[0062] (3) Pour the silk fibroin solution into the mold, place the chitosan nonwoven fabric on the surface, and then cover the surface of the nonwoven fabric with the same volume of silk fibroin solution to form a "sandwich" structure, which is then moved to a fume hood until the formic acid is completely volatilized to obtain a hydrogel.

[0063] 2. Preparation of nanoporous CaCO3:

[0064] (1) Preparation of Nano-CaCO3: 200 mg of CaCl2-2H2O and 4 mg of dopamine were uniformly mixed with 100 mL of anhydrous ethanol. The mixture was then placed in a sealed container together with 5 g of NH4HCO3 and placed at 37°C for 24 hours. After 24 hours, the obtained nanoparticles were collected and washed three times with alcohol, and then the washed CaCO3 nanoparticles were dried for subsequent use.

[0065] (2) Coating: First, 0.1 g of MOTS-c was dissolved in a phosphate buffer solution to prepare a dispersion, and 0.7 g of the washed nanoporous CaCO3 was combined with a 10 ug / mL MOTS-c dispersion, stirred horizontally for 1 hour, and then centrifuged at 10,000 rpm for 10 minutes to produce MOTS-c-loaded nano-CaCO3. The mixture was magnetically stirred at 0°C for 30 minutes. Subsequently, the mixture was centrifuged at 10,000 rpm for 5 minutes, and the MOTS-c-Ca powder was obtained at room temperature after the alcohol evaporated.

[0066] (3) Preparation of MOTS-c-Ca gel:

[0067] The MOTS-c-Ca powder obtained in step (2) is mixed with the FA-Ca solution and evenly coated on the surface of the hydrogel to obtain the MOTS-c-Ca gel.

[0068] The effect of the present application scheme is verified by cell experiments and mouse experiments.

[0069] 1. Cell experiment description

[0070] (1) Preparation of peptide MOTS-c and mutant peptide MOTS-c

[0071] Peptide MOTS-c amino acid sequence: MRWQEMGYIFYPRKLR

[0072] Mutant polypeptide MOTS-c amino acid sequence: MRWQEMGYIFYPFKLR

[0073] Both peptides were synthesized by chemical methods, with a HPLC purity of 97.217%. The peptide powders were dissolved in sterile water to prepare 10 mg / mL solutions, which were packaged and frozen for later use. When used, they were diluted with sterile water to the corresponding concentration.

[0074] (2) Experimental procedures

[0075] 2.1) Evaluation of the cell proliferation activity of mutant peptide MOTS-c

[0076] Human keratinocytes in the logarithmic growth phase (purchased from Pronocell) were inoculated in a 96-well cell culture plate at a density of 1×10 5 / mL, 100μL per well, placed in a carbon dioxide cell culture incubator, 37℃, 5% CO2 for routine culture for 24h. Then add 10μL of the test substance at different concentrations and continue to culture for 24h, 48h, 72h, and 96h. Each solution is sterilized by filtering with a 0.22μm filter membrane. After the culture is completed, the culture medium is discarded, and 10μL of CCK-8 solution (purchased from Sangon Biotechnology (Shanghai) Co., Ltd.) is added to each well, placed in a cell culture incubator at 37℃, 5% CO2, and continued to incubate for 2h before taking out. Use an enzyme reader to read the absorbance of the 96-well plate at 450nm, record the measurement results, and then obtain the relative proliferation rate according to the following formula:

[0077] Cell viability % = [A(drug added) - A(blank)] / [A(0 drug added) - A(blank)] * 100

[0078] A (drug added): absorbance of wells with cells, CCK-8 solution, and drug solution

[0079] A (blank): absorbance of wells with culture medium and CCK-8 solution but no cells

[0080] A(0 drug addition): absorbance of the well with cells, CCK-8 solution and no drug solution

[0081] There were 4 parallel samples in each group.

[0082] 2.2) Cell migration assay (scratch assay)

[0083] Keratinocytes in the logarithmic phase were seeded in a 24-well plate, with 1.5 × 10 5 Cells were cultured in a 24-well plate in a cell culture incubator (37°C, 5% CO2). When the cells grew to 90% confluence, they were starved for 12 hours with a medium without FBS to inhibit keratinocyte proliferation. The wound was scratched with a pipette tip (200 μL), the separated cells were washed 3 times with PBS, and the remaining keratinocytes were cultured with MOTS-c and mutant MOTS-c prepared with basal medium (1% FBS) for 24 hours. The injured area was photographed immediately after the scratch and 24 hours later.

[0084] 2.3) ROS content detection

[0085] The ROS content was detected using a reactive oxygen species detection kit purchased from Bio-Tech Biotech Co., Ltd., and the experiment was performed according to the instructions attached to the kit. The steps are as follows: DCFH-DA was diluted with serum-free culture medium at 1:1000 to a final concentration of 10 μM. The cell culture medium was removed and an appropriate volume of diluted DCFH-DA was added. Incubate in a cell culture incubator at 37°C for 20 minutes. Wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells, and then detect using a fluorescence microscope.

[0086] 2.4) Mutant MOTS-c inhibits the production of pro-inflammatory cytokines in keratinocytes induced by high glucose

[0087] A control group (Con), a high glucose group (HG), a high glucose + MOTS-c treatment group (HG + WT-MOTS-c) and a high glucose + mutant MOTS-c treatment group (HG + MUT-MOTS-c) were set up, where HG was based on Con with glucose added at a concentration of 30 mM. After 24 hours of culture, the collected keratinocytes were lysed by Trizol, total RNA was extracted, reverse transcribed and qRT-PCR experiments were performed to detect the mRNA levels of inflammatory factors IL-6, IL-1β and TNF-α.

[0088] (3) Experimental results

[0089] like Figure 1 As shown in the figure, the experimental results show that under HG (30mM) conditions, the viability of keratinocytes decreased over time. The treatment of different concentrations of WT-MOTS-c showed a concentration-dependent effect in inhibiting the decrease in keratinocyte viability. On the 4th day of treatment, the viability of keratinocytes in the groups treated with different concentrations of WT-MOTS-c reached its peak, especially in the 50μM treatment group, where the viability of keratinocytes increased most significantly, which was significantly different from the HG group (**p<0.01). Although the viability of keratinocytes in the 100μM and 150μM treatment groups also increased, the effect of 50μM was the most prominent.

[0090] like Figure 2 As shown in the experimental results, under HG conditions, treatment with different concentrations of MUT-MOTS-c significantly improved the decline in keratinocyte viability and was significantly higher than that of WT-MOTS-c ( Figure 1) treated with MUT-MOTS-c had a more significant effect on improving cell viability. Especially in the 50μM treatment group, cell viability reached its peak on the 4th day, showing a significant upward trend (**p<0.01). In addition, the keratinocyte viability of the 100μM and 150μM MUT-MOTS-c treatment groups also showed a significant increase. Overall, MUT-MOTS-c can more effectively reverse the decrease in cell viability caused by HG at different concentrations, and the effect is particularly significant on the 4th day.

[0091] like Figure 3 As shown, the experimental results showed that: after 4 days of treatment, HG significantly reduced the viability of keratinocytes, which was significantly different from Con (***p<0.001). Both WT-MOTS-c (50μM) and MUT-MOTS-c (50μM) treatments significantly improved the HG-induced decrease in keratinocyte viability. In particular, the MUT-MOTS-c treatment group showed a stronger effect on promoting keratinocyte viability than the WT-MOTS-c treatment group (**p<0.01). The overall results showed that MUT-MOTS-c showed a stronger effect than WT-MOTS-c in improving HG-induced keratinocyte viability damage, further verifying the superiority of MUT-MOTS-c in keratinocyte protection.

[0092] like Figure 4 As shown, the experimental results show that HG treatment significantly inhibited the migration ability of keratinocytes, and the migration rate after 24 hours was significantly lower than that of the control group (*p<0.05). In contrast, both WT-MOTS-c and MUT-MOTS-c treatments significantly promoted the migration of keratinocytes, among which the migration rate of the HG+MUT-MOTS-c treatment group was the highest, significantly higher than that of the HG group (***p<0.001). In particular, at the 24-hour time point, the migration rate of keratinocytes in the MUT-MOTS-c group was close to 80%, significantly better than that of the WT-MOTS-c group (*p<0.05), indicating that MUT-MOTS-c has a more significant improvement effect on the inhibition of keratinocyte migration caused by high glucose than WT-MOTS-c.

[0093] like Figure 5As shown in the figure, the experimental results showed that HG significantly increased the generation of ROS in keratinocytes, and the fluorescence intensity increased significantly compared with the control group (**p<0.01). In comparison, the WT-MOTS-c treatment group significantly reduced the level of ROS (***p<0.001). The MUT-MOTS-c treatment group further significantly reduced the level of ROS (***p<0.001) and was significantly lower than the WT-MOTS-c treatment group (*p<0.05). MUT-MOTS-c was more effective than WT-MOTS-c in reducing HG-induced ROS generation, showing its superiority in antioxidant protection.

[0094] like Figure 6 As shown in the figure, the experimental results showed that HG treatment significantly increased the expression levels of proinflammatory cytokines IL-6, IL-1β and TNF-α, which were significantly different from those in the Con group (*p<0.05, **p<0.01). Compared with the HG group, both WT-MOTS-c and MUT-MOTS-c treatments significantly reduced the expression of these proinflammatory factors. (a) showed that the expression of IL-6 in the HG group was significantly increased, WT-MOTS-c treatment significantly reduced the expression of IL-6 (*p<0.05), and MUT-MOTS-c treatment further significantly reduced the level of IL-6 (*p<0.05). (b) showed that HG significantly upregulated the expression of IL-1β, the WT-MOTS-c treatment group showed a certain downward trend but no significant difference (ns), while the MUT-MOTS-c treatment group significantly reduced the expression of IL-1β (**p<0.01). (c) showed that the expression of TNF-α in the HG group was significantly increased (*p<0.05), while both WT-MOTS-c and MUT-MOTS-c treatments significantly reduced the expression of TNF-α (***p<0.001), among which the TNF-α level in the MUT-MOTS-c treatment group was the lowest and more significant than that in WT-MOTS-c (*p<0.05). In general, the experimental results showed that MUT-MOTS-c had a more significant effect than WT-MOTS-c in inhibiting the expression of HG-induced proinflammatory cytokines IL-6, IL-1β and TNF-α, indicating the superiority of MUT-MOTS-c in inhibiting inflammation.

[0095] 2. Mouse Experiment Description

[0096] (1) Experimental procedures

[0097] Diabetic mouse modeling: 5 mice were fed with a high-fat diet for a total of 8 weeks.

[0098] Preparation of diabetic mouse wound injury model: 5 mice fed with high-fat diet and successfully established model were selected as diabetic group and 5 mice fed with normal diet were selected as control group, and a circular full-thickness skin wound with a diameter of 5 mm was created on the back.

[0099] Administration method: The polypeptide hydrogel precursor material was dissolved in sterile water for injection and injected into the wound site until gelation occurred. The dosage was 200 μL per mouse. After hydrogel treatment, the wounds of the mice were photographed and observed every day. The wound area was measured using Image-Pro Plus software.

[0100] About 12 days after administration, the wound was basically healed. Healing rate = healed / (healed + unhealed) × 100%.

[0101] Glucose tolerance test (GTT):

[0102] The mice were fasted for 12 h, and blood was collected by tail vein puncture. The first drop of blood was discarded and fasting blood glucose (G0) was measured with a blood glucose meter. 1 g / (kg·bw) glucose solution (w / v 20%) was gavaged, and the blood glucose values ​​of each group at 30, 60, 90 and 120 minutes after gavage were measured and recorded.

[0103] Insulin tolerance test (ITT):

[0104] Mice need to fast for 4-6 hours before the experiment, but they can drink water freely. Before injecting insulin, use a blood glucose meter to measure and record the basal blood glucose level. The blood glucose value at this time is taken as 0 minutes (basal value). Calculate the required insulin dose according to the weight of the mouse. Use a syringe to inject the insulin solution into the mouse by intraperitoneal injection. The injection should be gentle and quick to reduce stress response. Blood samples are collected at 30, 60, 90 and 120 minutes after the injection of insulin, and blood glucose levels are measured. At each time point, the blood glucose concentration of the mouse is quickly determined and recorded using a blood glucose meter.

[0105] Peptide MOTS-c administration method:

[0106] The silk fibroin hydrogel precursor material was dissolved in sterile water for injection and injected into the wound site until it formed a gel. The dosage was 200 μL per mouse. After hydrogel treatment, the wounds of the mice were photographed and observed every day. The wound area was measured using Image-Pro Plus software. About two weeks after administration, the wound was basically healed. Healing rate = healing / (healing + non-healing) × 100%. The results showed that the multifunctional hydrogel can significantly promote the healing rate of diabetic wounds (for detailed data, please see Figure 8 ).

[0107] Hematoxylin-eosin (H&E) staining and Masson staining:

[0108] H&E staining was used to observe the pathological changes of wound skin tissue. The skin tissue was fixed in 4% paraformaldehyde for 24 hours, then dehydrated with gradient ethanol, embedded in paraffin, cut into 5 μm thick sections, and stained with H&E. The pathological morphology and structure of the skin tissue were observed under an optical microscope. Masson staining was further performed, and collagen fibers were bright green and muscle fibers were red. Bright green (collagen fibers) was positive, reflecting the collagen content, and fibrosis was observed. H&E stained sections and Masson stained sections were taken using an optical microscope.

[0109] Tissue qPCR detection:

[0110] Cut the tissue into small pieces and put them into a clean round-bottom EP tube on ice. Add 500 μL Trizol lysis buffer, add one large grinding bead and two small grinding beads, and grind on an automatic grinder until the tissue is completely ground and evenly formed into a suspension. Perform qPCR experiments according to the cell experiment method to detect the expression of inflammatory factors IL-6, IL-1β and TNF-α mRNA in skin tissue.

[0111] (2) Experimental results

[0112] like Figure 7 As shown, the experimental results showed that in the GTT experiment, the blood glucose level of mice on a high-fat diet (HFD) was significantly higher than that of the Con group (***p<0.001), and reached a peak about 20 minutes after the injection of glucose, and then gradually decreased, but until 120 minutes, the blood glucose level of the HFD group was still higher than that of the Con group. This shows that HFD mice have significant impaired glucose tolerance. In the ITT, the blood glucose of mice in the HFD group decreased significantly less than that of the Con group after insulin injection (***p<0.001), showing a trend of decreased insulin sensitivity. The blood glucose of mice in the Con group decreased significantly after insulin injection and remained at a low level, while the blood glucose of mice in the HFD group decreased only slightly and returned to near the initial level at 120 minutes. These results show that HFD significantly impaired the glucose tolerance and insulin sensitivity of mice, leading to dysfunction of glucose metabolism.

[0113] like Figure 8As shown in the figure, the experimental results showed that compared with Con, HFD significantly delayed wound healing, especially from day 5 to day 12, the wound healing rate of the HFD group was significantly lower than that of the Con group (***p<0.001). Both WT-MOTS-c and MUT-MOTS-c treatment significantly improved the delayed wound healing induced by HFD, especially the MUT-MOTS-c group showed the most significant wound healing promotion effect during the entire experimental period. On day 12, the wounds in the MUT-MOTS-c group were almost completely healed, and the healing rate was significantly higher than that of the other groups (***p<0.001). The wound healing effect of the WT-MOTS-c group was also significantly better than that of the HFD group (**p<0.01), but slightly lower than that of the MUT-MOTS-c group. Overall, HFD significantly inhibited the wound healing ability of mice, while the treatment of WT-MOTS-c and MUT-MOTS-c could effectively reverse this adverse effect, especially the effect of MUT-MOTS-c in promoting wound healing was more significant.

[0114] like Fig. 9 As shown in the figure, the experimental results showed that on the 14th day, the wound healing of mice in the HFD group was significantly delayed, and H&E staining showed that the skin tissue structure was severely damaged, the epidermis was thin, and the dermis lacked complete tissue repair. In contrast, the Con group showed an intact epidermis and a thicker dermis, indicating that the wound had basically healed. In the HFD+WT-MOTS-c treatment group, WT-MOTS-c treatment partially improved the delayed wound healing caused by HFD, and H&E staining showed that the repair of the epidermis and dermis was improved. Masson staining showed that the collagen fiber deposition in this group increased, but the density was low, indicating that the tissue repair was incomplete. In the HFD+MUT-MOTS-c treatment group, MUT-MOTS-c significantly promoted wound healing, and H&E staining showed that the epidermis of this group was relatively intact, and the dermis was also significantly repaired. Masson staining showed that there was a large amount of collagen fiber deposition in this group, and the density was significantly higher than that of other treatment groups, indicating that tissue regeneration and collagen fiber reconstruction were more complete. HFD significantly inhibited the healing of skin wounds, while WT-MOTS-c and MUT-MOTS-c improved this situation to varying degrees, especially MUT-MOTS-c, which was more effective in promoting tissue regeneration and collagen fiber deposition.

[0115] like Fig.10As shown, the experimental results showed that HFD significantly upregulated the expression levels of proinflammatory cytokines IL-6, IL-1β and TNF-α, and there was a significant difference compared with Con (***p<0.001). Under HFD conditions, both WT-MOTS-c and MUT-MOTS-c treatment significantly reduced the expression of these proinflammatory cytokines, especially MUT-MOTS-c. (a) shows that the expression of IL-6 in HFD was significantly higher than that in the control group (***p<0.001), the IL-6 level was reduced after WT-MOTS-c treatment (**p<0.01), and MUT-MOTS-c treatment further significantly reduced the expression of IL-6 (***p<0.001). (b) showed that HFD significantly upregulated the expression of IL-1β (***p<0.001), the WT-MOTS-c treatment group significantly reduced the IL-1β level (**p<0.01), and the MUT-MOTS-c treatment group further decreased the IL-1β expression (***p<0.001). (c) showed that compared with Con, the HFD significantly increased the expression of TNF-α (***p<0.001), the WT-MOTS-c treatment group significantly reduced the TNF-α level (**p<0.01), and the MUT-MOTS-c treatment group had the lowest TNF-α level (***p<0.001).

[0116] The scheme of the present application is also applicable to ordinary mice (non-HFD), that is, a control group is established for ordinary mice, and conventional EGF treatment and MOTS-c treatment and MUT-MOTS-c treatment proposed in the present application are performed, and the experiment is carried out according to the aforementioned administration method. Fig.11 As shown, the experimental results showed that under non-HFD conditions, the wound healing rate of mice in the Con group was the slowest, especially during the 5th to 12th day, when the wound healing rate was significantly lower than that of other treatment groups. Compared with the Con group, EGF, MOTS-c and MUT-MOTS-c treatments all significantly accelerated wound healing. Starting from the 5th day, the EGF and MUT-MOTS-c treatment groups showed accelerated healing rates and significant wound healing effects. Further analysis showed that there was no significant statistical difference in healing effect between the EGF treatment group and the MUT-MOTS-c treatment group, indicating that MUT-MOTS-c showed similar therapeutic effects as EGF in wound healing of ordinary mice.

Claims

1. An amino acid mutant polypeptide of a mitochondrial-derived peptide MOTS-c, characterized in that: Its amino acid sequence is: MRWQEMGYIFYPFKLR.

2. Use of the amino acid mutant polypeptide of the mitochondrial-derived peptide MOTS-c according to claim 1 in the preparation of a medicament for wound healing.

3. The use according to claim 2, characterized in that: The trauma patients suitable for the medicine are general patients or diabetic patients.

4. A medicine for wound healing, characterized in that: The active ingredient of the medicine is the amino acid mutant polypeptide of the mitochondrial-derived peptide MOTS-c as described in claim 1.

5. The medicament for wound healing according to claim 4, characterized in that: As an external medicine, it also includes nano-CaCO3 and hydrogel for loading the amino acid mutant polypeptide.

6. A method for preparing a medicine for wound healing, characterized in that: include: Prepare CaCO3 nanoparticles and dry them for later use; The amino acid mutant polypeptide of the mitochondrial-derived peptide MOTS-c of claim 1 is dissolved in a phosphate buffer solution to prepare a dispersion; CaCO3 nanoparticles are prepared into an aqueous solution and combined with the dispersion, stirred horizontally, and then centrifuged to produce nano-CaCO3 loaded with the amino acid mutant polypeptide, and then stirred magnetically, centrifuged, and allowed to stand to obtain MOTS-c-Ca powder; The MOTS-c-Ca powder is mixed with the FA-Ca solution, and the mixture is evenly coated on the surface of the silk fibroin to obtain the MOTS-c-Ca gel, which is a medicine for wound healing.

7. Use of mitochondrial derived peptide MOTS-c in the preparation of medicines for wound healing.

8. The use according to claim 7, characterized in that The trauma patients suitable for the medicine are general patients or diabetic patients.

9. A medicine for wound healing, characterized in that: The active ingredient of the drug is the mitochondrial-derived peptide MOTS-c.

10. The medicament for wound healing according to claim 9, characterized in that: As an external medicine, it also includes nano-CaCO3 and hydrogel for loading the mitochondrial derived peptide MOTS-c.

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