Application of MOTS-c and its amino acid mutant polypeptides in wound healing

Abstract

This application discloses the use of MOTS-c and its amino acid mutant polypeptides in wound healing, wherein the amino acid sequence of the amino acid mutant polypeptide is: MRWQEMGYIFYPFKLR. Both can be used to prepare drugs for wound healing, and the amino acid mutant polypeptides are particularly effective. Compared with conventional wound healing treatments, this application's solution is less expensive and has a certain degree of 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 in response to injury, involving complex biological events including inflammation, proliferation, and remodeling. Effective wound healing is crucial for preventing infection and restoring tissue function and appearance. Traditionally, wound treatments have relied on physical barriers, antibiotics, and topical medications, but these approaches often have limitations, such as slow healing, inadequate pain management, or potential for drug resistance.

[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 leads to a decrease in the resistance of diabetic patients to infection, making them more susceptible to infection during wound healing, thereby delaying healing.

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

[0007] (4) Sustained inflammatory response: The inflammatory response in diabetic patients lasts longer during wound healing. 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 function of various cells (such as fibroblasts, endothelial cells and macrophages), inhibiting their proliferation, migration and differentiation abilities. 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 pain perception in the wound area, resulting in insufficient protection of the wound by the patient, increasing the risk of secondary damage 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.

[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 and alginate dressings, 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 that 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, it promotes blood circulation, reduces edema, and increases local cell proliferation in the wound.

[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 people.

[0024] (2) There are large individual differences in treatment effects: Due to the blood sugar control level, concomitant diseases and individual differences of diabetic patients, the effects of these treatments vary greatly. 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 clarifies 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 drug is particularly suitable for diabetic patients, but can also be applied to ordinary patients. The ordinary patients mentioned here (ordinary trauma patients) are relative to diabetic patients (diabetic patients who have also suffered trauma). The patients mentioned here can be humans or other mammals (such as mice, dogs, cats, pigs, rabbits, etc.).

[0029] In a third aspect, a wound healing drug, the active ingredient of which is the amino acid mutant polypeptide of the mitochondrial-derived peptide MOTS-c, is generally used as a topical medication. Alternatively, the topical medication further includes nano-CaCO3 and a hydrogel for loading the amino acid mutant polypeptide.

[0030] In a fourth aspect, a method for preparing a medicament 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 form a dispersion; an aqueous solution of CaCO3 nanoparticles is combined with the dispersion, horizontally stirred, and then centrifuged to produce nano-CaCO3 loaded with the amino acid mutant polypeptide; the mixture is then magnetically stirred, 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 through 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 drug is particularly suitable for diabetic patients, but can also be applied to ordinary patients. The ordinary patients mentioned here (ordinary trauma patients) are relative to diabetic patients (diabetic patients who have also suffered trauma). The patients mentioned here can be humans or other mammals (such as mice, dogs, cats, pigs, rabbits, etc.).

[0037] In a sixth aspect, a wound healing drug, the active ingredient of which is the mitochondrial-derived peptide MOTS-c, is typically used as a topical medication. Optionally, the topical medication also includes nano-CaCO3 and a hydrogel for loading the amino acid-mutated MOTS-c. Accordingly, the preparation method of the drug can also refer to the aforementioned preparation method.

[0038] The inventors of this 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 suitable for diabetic patients, but also for ordinary patients), wherein the amino acid mutant polypeptide has a more significant effect. Compared with existing wound healing treatment options, the solution of this application is lower in cost, more stable in effect, and has a certain degree of universality. Of course, medical staff can also combine the solution of this application with other solutions as appropriate. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a line graph showing the change in keratinocyte viability over time, showing the changing trend of keratinocyte viability (expressed as percentage) within 5 days under high glucose (HG) conditions and treatment with different concentrations of WT-MOTS-c. 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 in 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 showing the change in keratinocyte viability over time, showing the changing trend of keratinocyte viability (expressed as a percentage) over 5 days under HG conditions, under treatment with 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 increasing treatment concentration, keratinocyte viability showed different response trends. Statistical significance is indicated by asterisks: ** indicates p < 0.01.

[0041] Figure 3 This is a bar graph showing the effects of WT-MOTS-c and MUT-MOTS-c on keratinocyte viability after 4 days of HG treatment. 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 5Figure 1 shows a fluorescence image (a) of reactive oxygen species (ROS) levels in keratinocytes detected by the DCFH-DA probe, and a quantitative histogram (b) of its fluorescence intensity. (A) shows the ROS levels in four different treatment groups: the Con group, the HG group, the HG + WT-MOTS-c group, and the HG + MUT-MOTS-c group. (B) shows the quantitative analysis of ROS fluorescence intensity in each group, showing the fold change in fluorescence intensity relative to cell number. 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 fold expression of the proinflammatory cytokines IL-6, IL-1β, and TNF-α relative to the internal reference GAPDH. These graphs demonstrate the effects of WT-MOTS-c and MUT-MOTS-c treatment on the mRNA expression of the proinflammatory cytokines IL-6, IL-1β, and TNF-α under HG conditions. (a), (b), and (c) The bar graphs show the fold expression of IL-6, IL-1β, and TNF-α, respectively, relative to the internal reference GAPDH. The different treatment groups included Con, HG, HG + WT-MOTS-c, and HG + MUT-MOTS-c. 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 Graphs showing glucose tolerance (GTT) and insulin tolerance (ITT) tests in mice fed a high-fat diet (HFD). (a) shows the GTT curve, and (b) shows the ITT curve. The horizontal axis represents time (minutes), and the vertical axis represents 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 8Figures assessing the effects of different treatments on mouse skin wound healing show the effects of HFD treatment on the wound healing process in mice and the roles of WT-MOTS-c and MUT-MOTS-c in wound healing. (a) Images of wound healing at different time points (0, 1, 3, 5, 7, 9, and 12 days) for the Con, HFD, HFD + WT-MOTS-c, and HFD + MUT-MOTS-c groups. (b) Quantitative curve of wound healing rate, with the ordinate representing the percentage of wound size relative to the initial size and the abscissa representing time (days). Orange circles 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] Figure 9 Figure 1 shows histological analysis of mouse skin wound healing in different treatment groups (Con, HFD, HFD + WT-MOTS-c, and HFD + MUT-MOTS-c) 14 days after wound healing. H&E staining (upper row) and Masson staining (lower row) were used to assess skin tissue structure and collagen fiber formation at day 14. H&E staining was used to observe regeneration of the epidermis and dermis, and Masson staining was used to assess collagen fiber deposition.

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

[0049] Figure 11This is a diagram of a mouse skin wound healing experiment, showing the healing of mouse skin wounds under different treatment conditions. Among them, (a) shows the healing process of the wounds in each group of mice 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 the wound healing rate, where the ordinate represents the percentage of wound size relative to the initial size, and the abscissa represents time (days). In the 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 will be 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 (MDP) 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 by regulating mitochondrial metabolism and nuclear genome transcription. Studies have shown that MOTS-c regulates various important metabolic processes such as glucose metabolism, lipid metabolism, and bone metabolism through autocrine and paracrine pathways, via the AMPK pathway, the AKT pathway, the 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 biology 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 is even more effective in accelerating wound healing.

[0058] These two peptides 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 dry cocoon shells, take out the silkworm pupae, weigh a certain amount of dry cocoon shells, put them into a Na2CO3 solution with a bath ratio of 1:100, a temperature of 100℃, and a mass fraction of 0.2%. Boil for 40 minutes, then 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. 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 h.

[0062] (3) Pour the silk fibroin solution into the mold, place the chitosan non-woven fabric on the surface, and then cover the non-woven fabric surface with the same volume of silk fibroin solution to form a "sandwich" structure, and then move it 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. This mixture was then placed in a sealed container with 5 g of NH4HCO3 and incubated at 37°C for 24 hours. After 24 hours, the resulting nanoparticles were collected and washed three times with alcohol. The washed CaCO3 nanoparticles were then dried for subsequent use.

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

[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 effects of the present invention are 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 chemically synthesized with an HPLC purity of 97.217%. Dissolve the peptide powder in sterile water to prepare a 10 mg / mL solution, aliquot, and freeze for later use. Dilute with sterile water to the desired concentration before use.

[0074] (2) Experimental steps

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

[0076] Human keratinocytes in the logarithmic growth phase (purchased from Pronocell) were seeded in 96-well cell culture plates at a seeding density of 1×10 5 / mL, 100μL per well, placed in a carbon dioxide cell culture incubator, 37℃, 5% CO2 conventional 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 Biotech (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 the wells containing cells, CCK-8 solution, and drug solution

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

[0080] A(0 drug addition): absorbance of the well containing cells, CCK-8 solution but 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 plated in a 24-well plate in a cell culture incubator (37°C, 5% CO2). After growth to 90% confluence, the cells were starved for 12 hours with FBS-free medium to inhibit keratinocyte proliferation. The wound surface was scratched using a pipette tip (200 μL), and the isolated cells were washed three times with PBS. The remaining keratinocytes were then cultured with MOTS-c and mutant MOTS-c in basal medium (1% FBS) for 24 hours. Images of the injured area were taken immediately after the scratch and 24 hours later.

[0084] 2.3) ROS content detection

[0085] ROS levels were measured using a reactive oxygen species detection kit purchased from Beyotime Biotechnology Co., Ltd. The assay was performed according to the included instructions. The following steps were performed: DCFH-DA was diluted 1:1000 in serum-free culture medium to a final concentration of 10 μM. The cell culture medium was removed and an appropriate volume of the diluted DCFH-DA was added. The cells were incubated in a 37°C cell culture incubator for 20 minutes. The cells were washed three times with serum-free culture medium to fully remove the DCFH-DA that had not entered the cells, and then examined 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. HG was based on Con supplemented with glucose at a concentration of 30 mM. After 24 hours of culture, the collected keratinocytes were lysed using Trizol, and total RNA was extracted, reverse transcribed, and subjected to qRT-PCR to measure the mRNA levels of the 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. Treatment with different concentrations of WT-MOTS-c showed a concentration-dependent effect in inhibiting the decline in keratinocyte viability. On the fourth 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 keratinocyte viability was most significantly improved, with a significant difference compared to 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 decreased viability of keratinocytes and was significantly better than that of WT-MOTS-c ( Figure 1) treatment was even more significant. In particular, in the 50μM treatment group, cell viability peaked on day 4, demonstrating a significant upward trend (**p<0.01). Furthermore, keratinocyte viability was also significantly enhanced in the 100μM and 150μM MUT-MOTS-c treatment groups. Overall, MUT-MOTS-c, at various concentrations, was more effective in reversing the HG-induced decrease in cell viability, with the effect being particularly pronounced on day 4.

[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 in 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 in the figure, 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 treatment significantly promoted the migration of keratinocytes, with the HG+MUT-MOTS-c treatment group having the highest migration rate, significantly higher than that of the HG group (***p<0.001). In particular, at the 24-hour time point, the keratinocyte migration rate 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 than WT-MOTS-c on the high glucose-induced inhibition of keratinocyte migration.

[0093] like Figure 5As shown in the figure, the experimental results showed that HG significantly increased ROS production in keratinocytes, with fluorescence intensity significantly increasing compared to the control group (**p<0.01). In comparison, the WT-MOTS-c treatment group significantly reduced ROS levels (***p<0.001). The MUT-MOTS-c treatment group further significantly reduced ROS levels (***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 production, demonstrating its superior antioxidant protection.

[0094] like Figure 6 As shown in the figure, the experimental results show that HG treatment significantly increased the expression levels of proinflammatory cytokines IL-6, IL-1β, and TNF-α, with significant differences compared with the Con group (*p<0.05, **p<0.01). Compared with the HG group, both WT-MOTS-c and MUT-MOTS-c treatment significantly reduced the expression of these proinflammatory cytokines. (a) shows that IL-6 expression was significantly increased in the HG group, while WT-MOTS-c treatment significantly reduced IL-6 expression (*p<0.05), and MUT-MOTS-c treatment further significantly reduced IL-6 levels (*p<0.05). (b) shows that HG significantly upregulated IL-1β expression, while WT-MOTS-c treatment showed a slight downward trend but no significant difference (ns), while MUT-MOTS-c treatment significantly reduced IL-1β expression (**p<0.01). (c) shows that TNF-α expression was significantly increased in the HG group (*p<0.05), while both WT-MOTS-c and MUT-MOTS-c treatment significantly reduced TNF-α expression (***p<0.001). The MUT-MOTS-c treatment group had the lowest TNF-α level, which was significantly higher than that of WT-MOTS-c (*p<0.05). Overall, these results indicate that MUT-MOTS-c is more effective than WT-MOTS-c in inhibiting HG-induced expression of the proinflammatory cytokines IL-6, IL-1β, and TNF-α, demonstrating the superiority of MUT-MOTS-c in suppressing inflammation.

[0095] 2. Mouse Experiment Description

[0096] (1) Experimental steps

[0097] Diabetic mouse model: 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 circular full-thickness skin wounds with a diameter of 5 mm were created on the back.

[0099] Administration: Dissolve the peptide hydrogel precursor in sterile water for injection and inject into the wound site until gelation occurs. A dosage of 200 μL per mouse was administered. Following hydrogel treatment, the wounds of the mice were photographed and observed daily. 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) x 100%.

[0101] Glucose tolerance test (GTT):

[0102] 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 glucometer. 1 g / (kg·bw) glucose solution (w / v 20%) was gavaged, and the blood glucose values ​​of each group were measured and recorded 30, 60, 90, and 120 minutes after gavage.

[0103] Insulin tolerance test (ITT):

[0104] Mice need to fast for 4-6 hours before the experiment, but 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). The required insulin dose is calculated 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 fast to reduce stress response. Blood samples are collected at 30, 60, 90 and 120 minutes after the injection of insulin, and the blood glucose level is measured. At each time point, the blood glucose concentration of the mouse is quickly measured 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 pathological changes in wound skin tissue. Skin tissue was fixed in 4% paraformaldehyde for 24 hours, then dehydrated with graded ethanol, embedded in paraffin, and cut into 5-μm-thick sections. H&E staining was performed, and the pathological morphology and structure of the skin tissue were observed under an optical microscope. Masson staining was performed, and collagen fibers appeared bright green, while muscle fibers appeared red. Bright green (collagen fibers) was considered positive, reflecting collagen content and observing fibrosis. H&E-stained and Masson-stained sections were imaged using an optical microscope.

[0109] Tissue qPCR detection:

[0110] Cut the tissue into small pieces and place them in a clean, round-bottomed EP tube on ice. Add 500 μL of Trizol lysis buffer, one large grinding bead and two small grinding beads, and grind the tissue in an automatic grinder until the tissue is completely ground and a uniform suspension forms. qPCR was performed according to the cell-based assay protocol to measure the mRNA expression of the inflammatory factors IL-6, IL-1β, and TNF-α in the 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 high-fat diet (HFD) mice was significantly higher than that of the Con group (***p<0.001), and reached a peak about 20 minutes after glucose injection, 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 the HFD mice had significant impaired glucose tolerance. In the ITT, the blood glucose level 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 level of mice in the Con group decreased significantly after insulin injection and remained at a low level, while the blood glucose level 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 results, compared with Con, HFD significantly delayed wound healing. Specifically, from days 5 to 12, the wound healing rate in the HFD group was significantly lower than that in the Con group (***p < 0.001). Both WT-MOTS-c and MUT-MOTS-c treatment significantly ameliorated the HFD-induced delayed wound healing, with the MUT-MOTS-c group showing the most significant wound healing-promoting effect throughout the experimental period. By day 12, the wounds in the MUT-MOTS-c group were almost completely healed, with a significantly higher healing rate than the other groups (***p < 0.001). The wound healing effect in the WT-MOTS-c group was also significantly better than that in the HFD group (**p < 0.01), but slightly lower than that in the MUT-MOTS-c group. Overall, HFD significantly inhibited the wound healing ability of mice, while treatment with WT-MOTS-c and MUT-MOTS-c effectively reversed this adverse effect, with MUT-MOTS-c showing a more pronounced effect in promoting wound healing.

[0114] like Figure 9 As shown, the experimental results showed that on day 14, wound healing was significantly delayed in the HFD group. H&E staining revealed severe damage to skin tissue structure, a thin epidermis, and a lack of complete tissue repair in the dermis. In contrast, the Con group showed an intact epidermis and a thicker dermis, indicating that the wound had largely healed. In the HFD + WT-MOTS-c group, WT-MOTS-c treatment partially ameliorated the HFD-induced delayed wound healing. H&E staining revealed improved repair of the epidermis and dermis. Masson staining revealed increased collagen fiber deposition in this group, but at a lower density, indicating incomplete tissue repair. In the HFD + MUT-MOTS-c group, MUT-MOTS-c significantly promoted wound healing. H&E staining revealed a relatively intact epidermis and significant repair of the dermis. Masson staining revealed abundant collagen fiber deposition in this group, with a density significantly higher than in the other treatment groups, indicating more complete tissue regeneration and collagen fiber reconstruction. HFD significantly inhibited skin wound healing, 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 Figure 10As shown, the experimental results show that HFD significantly upregulated the expression levels of proinflammatory cytokines IL-6, IL-1β, and TNF-α, and there were significant differences 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 IL-6 expression was significantly higher in HFD than in the control group (***p<0.001). WT-MOTS-c treatment reduced IL-6 levels (**p<0.01), and MUT-MOTS-c treatment further significantly reduced IL-6 expression (***p<0.001). (b) shows that HFD significantly upregulated IL-1β expression (***p<0.001), while WT-MOTS-c treatment significantly reduced IL-1β levels (**p<0.01), and MUT-MOTS-c treatment further decreased IL-1β expression (***p<0.001). (c) shows that compared with Con, HFD significantly increased TNF-α expression (***p<0.001), while WT-MOTS-c treatment significantly reduced TNF-α levels (**p<0.01), with the MUT-MOTS-c treatment group having the lowest TNF-α level (***p<0.001).

[0116] The scheme of this application is also applicable to normal mice (non-HFD), that is, a control group is established for normal mice, and conventional EGF treatment and MOTS-c treatment and MUT-MOTS-c treatment proposed in this application are performed, and the experiment is carried out according to the aforementioned administration method. Figure 11 As shown in the figure, the experimental results showed that under non-HFD conditions, the wound healing rate of mice in the Con group was the slowest, especially from the 5th to the 12th day, the wound healing rate was significantly lower than that of the 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 speed and significant wound healing effect. 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 in normal 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 according to 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 according to claim 1 is dissolved in a phosphate buffer solution to prepare a dispersion; an aqueous solution of CaCO3 nanoparticles is combined with the dispersion, horizontally stirred, and then centrifuged to produce nano-CaCO3 loaded with the amino acid mutant polypeptide, which is then magnetically stirred, 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.

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