Arginase 2 inhibiting peptides and their use in wound healing

Abstract

The application discloses arginase 2 inhibiting peptides and application thereof in wound healing. The arginase 2 inhibiting peptide has an amino acid sequence as shown in SEQ ID No. 1 or a mutant sequence thereof. It is found through research of the application at a cell level and in a mouse skin wound model that MUT4-short peptide and MUT5-short peptide can effectively improve the viability and migration ability of keratinocytes under high-sugar conditions in vitro and in a metabolic abnormality model in vivo, reduce the intracellular reactive oxygen level, and promote the repair process of skin wounds, wherein the MUT4-short peptide shows more significant in promoting the recovery of cell functions and accelerating wound healing, and has a good application prospect in promoting skin wound healing, especially in the treatment of difficult-to-heal wounds related to metabolic diseases such as diabetes.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to arginase 2 inhibitory peptide and its application in wound healing. Background Technology

[0002] Wound healing is the process by which the body repairs tissues and restores function after injury or tissue damage, through a series of highly coordinated biological processes. It typically includes phases such as inflammation, proliferation, and remodeling. This process relies on the synergistic action of multiple cell types (such as keratinocytes, fibroblasts, endothelial cells, and immune cells) and is finely regulated by the inflammatory response, metabolic state, and local microenvironment. Among these factors, the timely termination of the inflammatory response and the smooth transition to the proliferation phase are key factors determining whether a wound can heal normally.

[0003] Arginine metabolism plays a crucial role in inflammation regulation and tissue repair. Arginase 2 (ARG2), a key enzyme in arginine metabolism, is primarily located in mitochondria. Under pathological conditions such as high glucose, inflammation, and metabolic abnormalities, ARG2 expression levels can significantly increase. Elevated ARG2 expression can deplete arginine substrates, reduce nitric oxide production, exacerbate inflammatory responses and oxidative stress, thereby inhibiting the normal function of keratinocytes, fibroblasts, and endothelial cells, and hindering wound healing.

[0004] Currently, some studies have attempted to inhibit ARG2 through chemical small molecule compounds such as N-hydroxy-L-arginine (NOHA) and its derivative nor-NOHA, natural ARG2 inhibitors such as amino acid inhibitors (such as L-citrulline, L-ornithine and L-norvaline), plant-derived polyphenols (such as resveratrol and quercetin), traditional Chinese medicine and traditional Chinese medicine compound preparations, and gene or transcriptional regulation of ARG2 to promote wound healing. Although the above-mentioned ARG2 targeted inhibitors and regulation methods have shown some potential, they still generally have the following shortcomings: (1) Limited safety and stability: Many ARG2 inhibitors have problems such as metabolic instability, short half-life or potential toxicity; (2) High treatment cost: The preparation process of chemical small molecule and nucleic acid inhibitors is complicated, which is not conducive to long-term or large-scale application; (3) Insufficient adaptability to local application: Existing strategies are mostly not suitable for local and continuous administration of skin wounds, which is difficult to meet the actual needs of chronic wound healing treatment in diabetes. Therefore, developing a new ARG2 regulation strategy that is safe, low-cost, and suitable for local application to facilitate its use in skin wounds, especially in the treatment of chronic wounds requiring long-term or local medication, has significant research value and application prospects. Summary of the Invention

[0005] Therefore, embodiments of the present invention provide an arginase 2 inhibitory peptide and its application in wound healing.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] According to a first aspect of the present invention, the present invention provides an arginase 2 inhibitory peptide having an amino acid sequence as shown in SEQ ID No. 1 or a mutant sequence thereof.

[0008] Furthermore, the mutated sequence is a mutation occurring at one or more positions corresponding to the amino acid sequence shown in SEQ ID No. 1, or a nuclear localization signal sequence is added to the C-terminus.

[0009] Furthermore, it has any of the following amino acid sequences:

[0010] The first amino acid corresponding to the sequence shown in SEQ ID No. 1 is mutated to R;

[0011] The 7th amino acid corresponding to the sequence shown in SEQ ID No. 1 is mutated to K;

[0012] The 8th amino acid corresponding to the sequence shown in SEQ ID No. 1 is mutated to W;

[0013] The 14th amino acid corresponding to the sequence shown in SEQ ID No. 1 is mutated to R;

[0014] Add PKKKRKV to the end of the sequence shown in SEQ ID No.1.

[0015] According to a second aspect of the present invention, the present invention provides a pharmaceutical composition comprising the arginase 2 inhibitory peptide as described above.

[0016] Furthermore, it also includes one or more pharmaceutically acceptable carriers.

[0017] Furthermore, the method for preparing the pharmaceutical composition includes the following steps:

[0018] (1) The degummed silk fibroin, CaCl2 and formic acid are mixed to obtain a silk fibroin solution. The solution is induced to undergo structural transformation and physical cross-linking by solvent evaporation to obtain a three-dimensional network structure silk fibroin hydrogel.

[0019] (2) The inhibitory peptide as described in claim 1 is mixed with nanoporous CaCO3 in the presence of a solvent to obtain nano-CaCO3 loaded with the inhibitory peptide;

[0020] (3) After mixing the nano-CaCO3, CaCl2 and formic acid of the loaded inhibitory peptide obtained in step (2), the resulting mixture is coated on the surface of the silk fibroin hydrogel obtained in step (1) to obtain the inhibitory peptide-Ca gel, which is the drug composition.

[0021] The pharmaceutical composition provided by the present invention is a hydrogel formulation, which has the advantages of well-defined structure, strong targeting, low preparation cost, and suitability for local administration.

[0022] According to a third aspect of the present invention, the present invention provides the use of an arginase 2 inhibitory peptide as described in any of the preceding claims, or the use of a pharmaceutical composition as described in any of the preceding claims in the preparation of a medicament for promoting wound healing.

[0023] Furthermore, the wound is a common traumatic wound or a diabetic traumatic wound.

[0024] The embodiments of the present invention have the following advantages:

[0025] This invention, through cellular studies and mouse skin wound models, found that MUT4-short peptide and MUT5-short peptide, under in vitro high-glucose conditions and in vivo metabolic abnormality models, can effectively improve the vitality and migration ability of keratinocytes, reduce intracellular reactive oxygen species levels, and promote the repair process of skin wounds. Among them, MUT4-short peptide showed more significant effects in promoting cell function recovery and accelerating wound healing, and has good application prospects in promoting skin wound healing, especially in the treatment of difficult-to-heal wounds related to metabolic diseases such as diabetes. Attached Figure Description

[0026] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0027] Figure 1The line graph shows the change in keratinocyte viability over time under high glucose (HG) conditions, with different concentrations of WT-short peptides and different mutant short peptides (MUT-short peptides) as a percentage over 5 days. The horizontal axis represents treatment time (days), and the vertical axis represents the percentage of keratinocyte viability (%). Specifically, (a) represents the WT-short peptide treatment group; (b) represents the mutant MUT1-short peptide treatment group; (c) represents the mutant MUT2-short peptide treatment group; (d) represents the mutant MUT3-short peptide treatment group; (e) represents the mutant MUT4-short peptide treatment group; and (f) represents the mutant MUT5-short peptide treatment group. Different colored lines represent different treatment groups at different concentrations. The green curve represents the normal control group (CON), the red curve represents the high glucose treatment group (HG), the purple curve represents the treatment group treated with 50 μM WT-short peptide or corresponding mutant short peptide under HG conditions, the blue curve represents the treatment group treated with 100 μM WT-short peptide or corresponding mutant short peptide under HG conditions, and the orange curve represents the treatment group treated with 150 μM WT-short peptide or corresponding mutant short peptide under HG conditions. Cell viability showed different response trends with increasing treatment concentration. Statistical significance is indicated by asterisks: * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001.

[0028] Figure 2 The bar chart shows the effect of different short peptide treatments on keratinocyte viability after 4 days of culture under high glucose (HG) conditions. The vertical axis represents keratinocyte viability (%), and the horizontal axis represents different treatment groups, including the control group (Con), the high glucose group (HG), the HG+WT-short peptide (50 μM) treatment group, the HG+MUT1-short peptide (50 μM) treatment group, the HG+MUT2-short peptide (50 μM) treatment group, the HG+MUT3-short peptide (50 μM) treatment group, the HG+MUT4-short peptide (50 μM) treatment group, and the HG+MUT5-short peptide (50 μM) treatment group.

[0029] Figure 3The changes in ARG2 expression levels in keratinocytes under high glucose (HG) conditions are shown. (a) A bar chart of quantitative ARG2 mRNA expression levels, using qRT-PCR to detect the fold change in ARG2 expression relative to the GAPDH internal reference gene. The horizontal axis shows the different treatment groups: Con group and HG group, and the vertical axis represents the quantitative analysis bar chart of ARG2 mRNA relative to GAPDH. (b) A representative Western blot diagram of ARG2 protein, with β-actin as the internal reference protein; (c) A bar chart of quantitative analysis of ARG2 protein relative to β-actin using grayscale values. The horizontal axis represents the normal control group (Con) and the high glucose treatment group (HG), and the vertical axis represents the relative protein expression level of ARG2. Statistical significance is indicated by an asterisk: *** indicates p < 0.001.

[0030] Figure 4 This study investigated the effects of different short peptide treatments on ARG2 expression levels in keratinocytes. (a) is a quantitative bar chart of ARG2 mRNA expression levels, using qRT-PCR to detect the fold change in ARG2 expression relative to the GAPDH internal control gene; (b) is a representative Western blot chart of ARG2 protein expression, with β-actin as the internal control; and (c) is a bar chart of quantitative analysis of ARG2 protein expression relative to β-actin using grayscale values. Experimental groups included: control group (Con), WT-short peptide (50 μM) treatment group, MUT1-short peptide (50 μM) treatment group, MUT2-short peptide (50 μM) treatment group, MUT3-short peptide (50 μM) treatment group, MUT4-short peptide (50 μM) treatment group, and MUT5-short peptide (50 μM) treatment group. The vertical axis represents the relative expression level of ARG2, and the horizontal axis represents the different treatment groups. Statistical significance is indicated by an asterisk: ** indicates p < 0.01, *** indicates p < 0.001, and "ns" indicates no statistical significance.

[0031] Figure 5The scratch assay and bar charts of keratinocyte migration rate illustrate the effects of WT-short peptide and MUT1-5 short peptide treatments on keratinocyte migration ability under high glucose (HG) conditions. (a) shows representative images from the scratch assay, displaying cell migration at 0 h and 24 h for each treatment group. The red dashed lines in the figure indicate the scratch boundaries, used to assess the migration ability of keratinocytes to the scratched area. (b) is a bar chart of quantitative analysis of keratinocyte migration rate, with different colors distinguishing the experimental groups: green represents the control group (Con), red represents the high glucose group (HG), orange represents the HG+WT-short peptide treatment group, yellow represents the HG+MUT1-short peptide treatment group, gray represents the HG+MUT2-short peptide treatment group, purple represents the HG+MUT3-short peptide treatment group, pink represents the HG+MUT4-short peptide treatment group, and blue represents the HG+MUT5-short peptide (50 μM) treatment group. Statistical significance is indicated by an asterisk: * indicates p < 0.05, *** indicates p < 0.001, and "ns" indicates no statistical significance.

[0032] Figure 6 The fluorescence graph (a) and quantitative bar graph (b) of mitochondrial reactive oxygen species (MitoROS) levels in keratinocytes detected by the MitoSox probe are shown. (a) displays the MitoROS levels in eight different treatment groups: Con group, HG group, HG+WT-short peptide treatment group, HG+MUT1-short peptide treatment group, HG+MUT2-short peptide treatment group, HG+MUT3-short peptide treatment group, HG+MUT4-short peptide treatment group, and HG+MUT5-short peptide treatment group. (b) shows the quantitative analysis of MitoROS fluorescence intensity in each group, indicating the fold change in fluorescence intensity relative to cell number. Statistical significance is indicated by asterisks: *** indicates p < 0.001, and "ns" indicates no statistically significant difference.

[0033] Figure 7Bar charts showing the fold change in mRNA expression of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α relative to the internal control GAPDH under HG conditions illustrate the effects of WT-short peptide and MUT1-5 short peptide treatments on the mRNA expression of these pro-inflammatory cytokines. (a), (b), and (c) show the fold change in IL-6, IL-1β, and TNF-α expression relative to the internal control GAPDH, respectively. Different treatment groups included Con, HG, HG+WT-short peptide treatment, HG+MUT1-short peptide, HG+MUT2-short peptide, HG+MUT3-short peptide, HG+MUT4-short peptide, and HG+MUT5-short peptide treatments. Statistical significance is indicated by asterisks: * indicates p < 0.05, *** indicates p < 0.001, and "ns" indicates no significant difference.

[0034] Figure 8 The effects of a high-fat diet (HFD) on body size and weight changes in mice were shown. (a) are representative photographs of mice in the normal control group (Con) and the high-fat diet group (HFD), clearly demonstrating a significant increase in body size in the HFD group. (b) is a line graph showing the change in mouse weight over time. The horizontal axis represents feeding time (weeks), and the vertical axis represents mouse weight (g). Blue dots represent the Con group, and pink squares represent the HFD group. Statistical significance is indicated by an asterisk: *** indicates p < 0.001.

[0035] Figure 9 Figures show the glucose tolerance test (GTT) and insulin tolerance test (ITT) in mice fed a high-fat diet (HFD). (a) is the GTT curve, and (b) is the ITT curve. The horizontal axis represents time (minutes), and the vertical axis represents blood glucose level (mg / dl). Blue dots represent the Con group, and pink squares represent the HFD group. Statistical significance is indicated by an asterisk: *** indicates p < 0.001.

[0036] Figure 10 The results of the assessment of skin wound healing in mice under different treatment conditions demonstrate the effects of different short peptide treatments on the skin wound healing process in mice under high-fat diet (HFD) induction.

[0037] (a) shows representative images of mouse skin wounds at different time points (0, 1, 3, 5, 7, 9, and 12 days) for each experimental group. The experimental groups were, in order, the control group (Con), the HFD group, the HFD+MUT4 short peptide treatment group, and the HFD+MUT5 short peptide treatment group. The continuous wound images visually demonstrate the effect of different treatments on the wound closure process. (b) is a quantitative analysis curve of the mouse skin wound healing rate. The vertical axis represents the percentage of wound area relative to the initial area, and the horizontal axis represents the healing time (days). Green dots represent the Con group, red dots represent the HFD group, orange dots represent the HFD+MUT4 short peptide treatment group, purple dots represent the HFD+MUT5 short peptide treatment group, and pink dots represent the HFD+BEC treatment group. Statistical significance is indicated by asterisks: ** indicates p < 0.01, *** indicates p < 0.001.

[0038] Figure 11 Histological analysis of mouse skin tissue 14 days after wound healing in different treatment groups (Con, HFD, HFD+MUT-4, HFD+MUT-5, HFD+BEC) is shown. The histological analysis of mouse skin wound healing under different treatment conditions is presented. H&E staining was used to assess the structure of skin tissue on day 14 to observe the regeneration of epidermis and dermis.

[0039] Figure 12 The bar chart shows the effects of MUT4, MUT5, and BEC treatments on the mRNA expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α under HFD conditions. The three bar charts show the fold increase of IL-6, IL-1β, and TNF-α expression relative to the internal control GAPDH. The experimental groups were divided into Con, HFD, HFD+MUT4-short peptide, HFD+MUT5-short peptide, and HFD+BEC treatment groups. Statistical significance is indicated by asterisks: ** indicates p < 0.01, *** indicates p < 0.001.

[0040] Figure 13The figures show the healing of mouse skin wounds under different treatment conditions. (a) shows the wound healing process at different time points (0, 1, 3, 5, 7, 9, and 12 days). The three experimental groups are the Con group, the MUT4 treatment group, and the BEC treatment group. (b) is a quantitative curve of wound healing rate, with the vertical axis representing the percentage of wound size relative to the initial size and the horizontal axis representing time (days). In the curve, green dots represent the Con group, red dots represent the BEC group, and orange dots represent the MUT4 treatment group. Statistical significance is indicated by asterisks: ** indicates p < 0.01, and "ns" indicates no significant difference.

[0041] Figure 14 Histological analysis of mouse skin 14 days after wound healing in different treatment groups (Con, BEC, and MUT4 treatment groups) is shown. The histological analysis of mouse skin wound healing under different treatment conditions is presented. H&E staining was used to assess the structure of skin tissue on day 14 to observe epidermal continuity, dermal structural reconstruction, and inflammatory cell infiltration. Detailed Implementation

[0042] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] Example 1: Obtaining the original peptide and mutant peptide

[0044] The original polypeptide involved in this application (amino acid sequence: MRWQEMGYIFYPFKLR, SEQ ID NO.1, denoted as WT-short peptide) is a small functional peptide encoded by mitochondria. Encoded by a small open reading frame in the mitochondrial genome, it participates in various biological processes such as cellular energy metabolism, oxidative stress regulation, and inflammatory responses. Previous studies have shown that this type of mitochondrial-derived polypeptide can positively regulate pathological states such as abnormal glucose metabolism, increased oxidative stress, and impaired tissue repair by modulating intracellular metabolism-related signaling pathways.

[0045] Based on the amino acid sequence of the original polypeptide, the inventors used conventional molecular biology and polypeptide engineering techniques to perform site-directed mutagenesis or structural modifications at different key sites in the original polypeptide sequence, constructing a variety of mutant polypeptides, specifically including the following five forms:

[0046] MUT1-Short Peptide: The first amino acid of the original polypeptide was mutated from methionine (M) to arginine (R), that is, the amino acid sequence is RRWQEMGYIFYPFKLR, SEQ ID NO.2;

[0047] MUT2-Short Peptide: The 7th amino acid of the original polypeptide was mutated from glycine (G) to lysine (K), that is, the amino acid sequence is MRWQEMKYIFYPFKLR, SEQ ID NO.3;

[0048] MUT3-Short Peptide: The 8th amino acid of the original polypeptide was mutated from tyrosine (Y) to tryptophan (W), that is, the amino acid sequence is MRWQEMGWIFYPFKLR, SEQ ID NO.4;

[0049] MUT4-Short Peptide: The 14th amino acid of the original polypeptide was mutated from lysine (K) to arginine (R), that is, the amino acid sequence is MRWQEMGYIFYPFRLR, SEQ ID NO.5;

[0050] MUT5-Short Peptide: A nuclear localization signal sequence (NLS) is introduced at the C-terminus of the original polypeptide, namely the amino acid sequence MRWQEMGYIFYPFKLRPKKKRKV, SEQ ID NO.6;

[0051] All of the above-mentioned mutant peptides can be prepared using existing mature molecular biology methods or peptide chemical synthesis methods. This invention uses chemical synthesis to prepare the aforementioned short peptides. The purity of the products, as detected by high-performance liquid chromatography (HPLC), is greater than 95%. Structural characterization and identification by mass spectrometry confirm that the obtained products are the target peptides.

[0052] Example 2: Preparation of Inhibitory Peptide Formulation

[0053] The short peptides from Example 1 (i.e., WT-short peptide, MUT1-short peptide, MUT2-short peptide, MUT3-short peptide, MUT4-short peptide, and MUT5-short peptide) were prepared into hydrogels (i.e., short peptide-Ca composite silk fibroin hydrogel formulations). The preparation method includes the following steps.

[0054] (1) Preparation of silk fibroin hydrogel

[0055] 1.1 Degumming of Silk Fibroin: Dry silkworm cocoons were cut open and the inner pupae removed. An appropriate amount of cocoon shell was placed in a 0.2% sodium carbonate solution and heated at 100℃ for approximately 40 minutes at a liquid-to-solid ratio of approximately 1:100 to remove sericin components. After treatment, the cocoon shell was removed and thoroughly rinsed with deionized water until no obvious residue remained on the surface. It was then dried in a 60℃ constant temperature oven to obtain degummed silk fibroin.

[0056] 1.2 Preparation of silk fibroin solution: Calcium chloride was added to anhydrous formic acid to form a FA-Ca solution. Degummed silk fibroin was added in batches under continuous stirring to allow it to gradually dissolve. The mass ratio of CaCl2, silk fibroin, and formic acid was controlled at 5:8:85. The mixture was stirred at 25°C and approximately 500 rpm for about 2 hours until a homogeneous and transparent silk fibroin solution was formed.

[0057] 1.3 Construction of the hydrogel: The silk fibroin solution obtained in step 1.2 was injected into a pre-designed mold, and chitosan nonwoven fabric was laid on its surface as a reinforcing layer. Then, an equal volume of silk fibroin solution was covered to form a sandwich structure. Subsequently, it was placed in a ventilated environment at room temperature (25℃) for 24-48 h to allow the solvent to gradually evaporate and induce structural transformation of the silk fibroin, finally obtaining a silk fibroin hydrogel with a stable three-dimensional network structure that can be hydrated to form a hydrogel.

[0058] (2) Construction of nanoporous CaCO3 and loading of short peptides

[0059] 2.1 Preparation of nanoporous CaCO3: 0.5 g of CaCl2·2H2O and 0.1 g of dopamine were weighed and dissolved thoroughly in 50 mL of anhydrous ethanol and mixed well. Then, 0.42 g of NH4HCO3 were placed together in a closed reaction system and reacted at 37 °C for approximately 24 h, allowing calcium carbonate to gradually form porous nanoparticles. After the reaction was complete, the product was collected by centrifugation and repeatedly washed with anhydrous ethanol to remove unreacted substances. The product was then vacuum dried at 40 °C for 12 h to obtain nanoporous CaCO3 powder.

[0060] 2.2 Short peptide loading process: Short peptides were dissolved in phosphate buffer to prepare a uniform short peptide solution with a concentration of 1 mg / mL (PBS, pH 7.4). Subsequently, nanoporous CaCO3 powder was dispersed in deionized water at a material-to-liquid ratio of 10 mg:1 mL. The resulting mixture was then combined with the short peptide solution at a volume ratio of 1:1. The reaction was carried out under continuous shaking or magnetic stirring for approximately 1 h, allowing the short peptides to adsorb and enter the nanoporous structure. After the reaction, the loaded product was separated by centrifugation at approximately 10,000 rpm. The resulting precipitate was further dispersed at a low temperature of 4°C, centrifuged again, and allowed to stand at room temperature to remove residual solvent, finally obtaining the short peptide-loaded CaCO3 composite powder (denoted as short peptide-Ca).

[0061] (3) Preparation of short peptide-Ca composite silk fibroin hydrogel formulation

[0062] The short peptide-Ca obtained above was added to the FA-Ca solution prepared in step 1.2 and mixed thoroughly. Then it was uniformly coated on the surface of the silk fibroin hydrogel prepared in step 1.3 and allowed to stand at room temperature (25℃) for 12-24 h to form a stable composite hydrogel system, and finally a short peptide loaded hydrogel formulation that can be used for local wound treatment was obtained.

[0063] Experiment Example 1: In vitro cell experiments

[0064] (1) Treatment of wild-type and mutant short peptides

[0065] Dissolve WT-short peptide powder and MUT1-MUT5-short peptide powder separately in sterile water to prepare stock solutions with a mass concentration of 10 mg / mL. Aliquot and store at -20°C for later use. Before experimental use, dilute with sterile culture medium to the corresponding working concentration as needed.

[0066] (2) Experimental steps and results

[0067] 2.1 Evaluation of the proliferative activity of short peptides on keratinocytes

[0068] Human keratinocytes (HaCaT, purchased from Pronos) in the logarithmic growth phase were seeded into 96-well cell culture plates at a seeding density of 1 × 10⁻⁶. 5 Cells were cultured at a concentration of 100 μL / mL, with 100 μL of cell suspension added to each well. The cells were incubated at 37°C and 5% CO2 for 24 h to allow for full cell adhesion. Subsequently, 10 μL of different concentrations of wild-type or mutant short peptide treatment solutions were added to each well, with parallel wells for each treatment group. Cell viability was assessed after 24 h, 48 h, 72 h, and 96 h of further incubation. All short peptide solutions were sterilized by filtration through a 0.22 μm microporous membrane before use. For assays, the culture medium was discarded, and 10 μL of CCK-8 reagent (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was added to each well. The wells were incubated at 37°C and 5% CO2 for 2 h. The absorbance of each well was then measured at 450 nm using a microplate reader, and the experimental data were recorded. Four parallel samples were used for each group. The relative cell proliferation rate was calculated using the following formula:

[0069] Cell viability % = [A (drug-treated) - A (blank)] / [A (0-drug-treated) - A (blank)] * 100;

[0070] Wherein, A (drug addition): absorbance of pores containing cells, CCK-8 solution and short peptide solution;

[0071] A (blank): Absorbance of pores containing culture medium and CCK-8 solution but without cells;

[0072] A (0 drug added): Absorbance of pores containing cells, CCK-8 solution, but no short peptide solution.

[0073] like Figure 1 As shown, under high glucose (HG, 30 mM) conditions, the viability of keratinocytes decreased significantly with prolonged culture time, indicating that the high glucose environment had a significant inhibitory effect on keratinocytes. Compared with the control group (Con), the cell viability of the HG group was significantly reduced at all time points. After adding wild-type short peptides (WT-short peptides) under HG conditions, keratinocyte viability did not show significant improvement, and its trend was basically consistent with that of the HG group, suggesting that WT-short peptides did not significantly improve cell viability under HG conditions. Similarly, treatment with mutant short peptides MUT2-short peptides and MUT3-short peptides did not show significant recovery of keratinocyte viability, and the difference was not statistically significant compared with the HG group, indicating that the above mutation sites failed to effectively improve the high glucose-induced decrease in cell viability. In contrast, treatment with MUT1-short peptides had a certain alleviating effect on keratinocyte viability, with a slight increase in viability observed at some time points, but the overall improvement was limited, and the statistical significance was not significant or only weakly significant, suggesting that its cell viability-promoting effect was weak. Notably, MUT4-short peptide and MUT5-short peptide significantly improved keratinocyte viability under high glucose conditions. On day 4 of treatment, keratinocyte viability reached its peak in the groups treated with different concentrations of MUT4-short peptide and MUT5-short peptide, especially in the 50 μM treatment group, where the improvement in keratinocyte viability was most significant, showing a statistically significant difference compared to the HG group (***p < 0.001). Although keratinocyte viability was also improved in the 100 μM and 150 μM treatment groups, the effect of 50 μM was the most pronounced.

[0074] like Figure 2As shown, the experimental results indicated that after 4 days of treatment, HG significantly reduced keratinocyte viability, showing a significant difference compared to the Con group (***p < 0.001). Adding wild-type short peptides (WT-short peptide, 50 μM) to the HG group did not significantly improve keratinocyte viability, and the difference was not statistically significant compared to the HG group, suggesting that WT-short peptides have limited effect on high glucose-induced cell viability damage. Similarly, keratinocyte viability in the MUT2-short peptide and MUT3-short peptide treatment groups did not show significant recovery, and their levels were basically consistent with the HG group. In contrast, keratinocyte viability in the MUT1-short peptide treatment group was slightly improved compared to the HG group, but the overall improvement was small, showing only a slight improvement with weak statistical significance. Notably, keratinocyte viability in the MUT4-short peptide and MUT5-short peptide treatment groups was significantly higher than that in the HG group, and the difference was statistically significant (***p < 0.001). Among them, MUT4-short peptide and MUT5-short peptide showed the most significant cell viability restoration effect at the same drug concentration.

[0075] In summary, only short peptides with specific mutation sites (MUT4-short peptide and MUT5-short peptide) can significantly reverse the high glucose-induced decrease in keratinocyte viability, while WT-short peptide and other mutant short peptides have limited effects.

[0076] 2.2 Effects of short peptides on ARG2 mRNA expression in keratinocytes

[0077] Human keratinocytes in the logarithmic growth phase were seeded in 6-well culture plates and cultured at 37°C and 5% CO2 until the cells adhered and grew stably. Wild-type short peptides or different mutant short peptides were then added for treatment. After 24 hours of treatment, the culture medium was discarded, and cells were lysed using Trizol reagent to extract total RNA. The extracted total RNA was reverse transcribed to synthesize cDNA, and the mRNA expression level of ARG2 was detected by real-time quantitative PCR (qRT-PCR), with GAPDH used as an internal control gene. The relative expression level of ARG2 was measured using a 22 - The ΔΔCt method was used to calculate and evaluate the regulatory effect of different short peptide treatments on the transcriptional level of the ARG2 gene.

[0078] 2.3 Effects of short peptides on ARG2 protein expression in keratinocytes

[0079] Under the same treatment conditions as in 2.2, keratinocytes from each treatment group were collected, and total protein was extracted using cell lysis buffer. Protein concentration was determined using the BCA method. Equal volumes of protein samples were separated by SDS-PAGE electrophoresis and then transferred to a PVDF membrane. After transfer, the membrane was incubated with ARG2 primary antibody, with β-actin used as an internal control. After incubation with the appropriate secondary antibody and chemiluminescence imaging, image analysis software was used to quantitatively analyze the grayscale values ​​of the protein bands, thereby evaluating the regulatory effect of different short peptide treatments on ARG2 protein expression levels.

[0080] like Figure 3 As shown, the experimental results indicate that in keratinocytes cultured under high glucose (HG) conditions, the mRNA and protein expression levels of ARG2 were significantly increased, showing a significant difference compared to the control group (Con). Figure 3 (a), (b), (c))(***p < 0.001), suggesting that high glucose stimulation can induce abnormal upregulation of ARG2.

[0081] like Figure 4 As shown, the experimental results indicate that: regarding the expression level of ARG2 mRNA ( Figure 4 (a) WT-short peptide (50 μM) treatment did not change ARG2 mRNA expression (ns). The ARG2 mRNA expression levels in the MUT1-short peptide, MUT2-short peptide, and MUT3-short peptide treatment groups also showed no significant change compared to the Con group. In contrast, the ARG2 mRNA expression levels in the MUT4-short peptide and MUT5-short peptide treatment groups were significantly reduced, with statistically significant differences compared to the Con group (***p < 0.001). Regarding ARG2 protein expression levels ( Figure 4 (b) and (c)) Western blot results showed that treatment with WT-short peptide, MUT1-short peptide, MUT2-short peptide, and MUT3-short peptide had no significant effect on ARG2 protein expression, and the difference was not statistically significant compared with the Con group. However, treatment with MUT4-short peptide and MUT5-short peptide significantly downregulated the expression level of ARG2 protein, and its expression level was significantly lower than that of the Con group (***p <0.001).

[0082] 2.4 Keratinocyte migration assay (scratch assay)

[0083] Human keratinocytes in the logarithmic growth phase were seeded into 24-well culture plates, with a cell density of approximately 1.5 × 10⁻⁶ cells per well. 5Cells were cultured at 37°C and 5% CO2 until approximately 90% confluence, then starved for 12 h in serum-free medium to reduce the impact of cell proliferation on migration. Subsequently, straight vertical scratches were made on the cell monolayer surface using a 200 μL pipette tip, and the cells were gently washed three times with PBS buffer to remove detached cells. Afterward, basal medium containing 1% FBS was added, followed by treatment with wild-type short peptides or different mutant short peptides. Images of the scratched areas were captured using an inverted microscope at 0 h and 24 h post-scratching, and the scratch closure was quantitatively analyzed using image analysis software to evaluate the effect of different short peptides on the migration ability of keratinocytes.

[0084] like Figure 5 As shown, the experimental results indicate that HG treatment significantly inhibited the migration ability of keratinocytes, with the migration rate after 24 hours being significantly lower than that of the control group (*p < 0.05). Under HG conditions, the addition of WT-short peptide, MUT2-short peptide, and MUT3-short peptide treatments did not significantly improve migration ability (ns), while MUT4-short peptide and MUT5-short peptide treatments significantly improved the HG-inhibited cell migration ability. This suggests that MUT4-short peptide and MUT5-short peptide have significant advantages in restoring keratinocyte migration inhibition induced by high glucose, and their effects far exceed those of the WT-short peptide, MUT2-short peptide, and MUT3-short peptide treatment groups.

[0085] 2.5 Detection of Mitochondrial Reactive Oxygen Species (MitoROS) Levels

[0086] MitoSOX provided by Abclonal TM The Red Mitochondrial Superoxide Anion Detection Kit was used to detect the level of reactive oxygen species (ROS) in mitochondria of keratinocytes. The specific procedure was performed according to the kit instructions. The simplified steps are as follows: After removing the cell culture medium, MitoSOX working solution was diluted proportionally with serum-free medium. An appropriate amount of working solution was added to the cells, and the cells were incubated at 37°C in the dark for approximately 10-20 minutes. After incubation, the cells were washed three times with serum-free medium to remove any unexploded dye. Subsequently, images were acquired and observed using a fluorescence microscope, and the MitoROS levels in cells from different treatment groups were compared and analyzed.

[0087] like Figure 6As shown, the experimental results indicate that under high glucose (HG) conditions, the generation of mitochondrial reactive oxygen species (MitoROS) in keratinocytes significantly increased, and the fluorescence intensity of MitoROS was significantly higher than that of the control group (Con) (***p< 0.001), suggesting that the high glucose environment induced severe mitochondrial oxidative stress. However, the addition of WT-short peptides under HG conditions did not significantly decrease intracellular MitoROS levels. Similarly, the MitoROS levels in the MUT1-short peptide, MUT2-short peptide, and MUT3-short peptide treatment groups remained at high levels, with no statistically significant difference compared to the HG group (ns), indicating that the above short peptides failed to effectively alleviate high glucose-induced oxidative damage. Notably, treatment with MUT4-short peptides and MUT5-short peptides significantly reduced the MitoROS levels in keratinocytes, with a substantial decrease in fluorescence intensity compared to the HG group, and the difference was statistically significant (***p< 0.001). These results indicate that MUT4-short peptide and MUT5-short peptide are significantly superior in reducing high glucose-induced MitoROS production, demonstrating their key protective role in maintaining mitochondrial redox homeostasis.

[0088] 2.6 Effects of short peptides on high-glucose-induced inflammatory responses in keratinocytes

[0089] The following groups were established: a control group (Con); a high-glucose group (HG); a high-glucose + wild-type short peptide treatment group (HG+WT); and a high-glucose + mutant short peptide treatment group (HG+MUT). The high-glucose model was constructed by adding glucose to the standard culture medium to a final concentration of 30 mM. After 24 h of cell culture, cells were lysed using Trizol reagent, and total RNA was extracted. cDNA was obtained via reverse transcription and then analyzed by quantitative real-time PCR (qRT-PCR) to determine the mRNA expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, thereby evaluating the regulatory effects of different short peptide treatments on high-glucose-induced inflammatory responses.

[0090] like Figure 7 As shown, the experimental results indicate that high glucose (HG) treatment significantly increased the expression levels of pro-inflammatory cytokines. Compared with the control group (Con), the HG group showed significantly higher levels of IL-6 (…). Figure 7 a) IL-1β Figure 7 b) and TNF-α ( Figure 7c) The mRNA expression levels of all groups showed a highly significant upregulation trend (***p < 0.001), indicating that the high glucose environment triggered an inflammatory response in keratinocytes. The WT-short peptide, MUT1-short peptide, MUT2-short peptide, and MUT3-short peptide treatment groups had no significant effect on IL-6 expression, showing no statistically significant difference compared to the HG group. However, MUT4-short peptide and MUT5-short peptide treatments significantly downregulated IL-6 mRNA expression levels, with their inhibitory effect being significantly superior to the WT group and other mutant groups (***p < 0.001). Regarding the control of IL-1β expression, the WT-short peptide and MUT1-short peptide, MUT2-short peptide, and MUT3-short peptide groups failed to effectively reverse the upregulation trend induced by high glucose. In contrast, the IL-1β expression levels of the MUT4-short peptide and MUT5-short peptide treatment groups were significantly reduced (***p < 0.001), exhibiting strong anti-inflammatory activity. The experimental results showed that the TNF-α levels in the MUT4-short peptide and MUT5-short peptide treatment groups were the lowest, significantly lower than those in the HG group and the WT-short peptide treatment group (***p < 0.001), while the improvement effects in the other groups were limited. These results confirm that MUT4-short peptide and MUT5-short peptide have significant advantages in inhibiting the expression of high glucose-induced pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α), and can effectively alleviate cellular inflammatory damage induced by a high glucose environment.

[0091] Experiment Example 2: In vivo animal experiments

[0092] This experimental case investigated the biological function of short peptides on diabetic wounds.

[0093] (1) Experimental steps

[0094] Establishment of a diabetic mouse model: Healthy male mice were randomly divided into a high-fat diet group and a normal diet group. The high-fat diet group was fed a high-fat diet for 8 weeks to establish a metabolic abnormality model; the normal diet group was fed a standard diet as a control. The model establishment was assessed by changes in body weight and blood glucose levels, and mice that successfully established the model were selected for subsequent experiments.

[0095] Construction of the skin wound model: Under anesthesia, the backs of mice that had successfully established the model and normal control mice were prepared and disinfected. Then, a circular full-thickness skin defect with a diameter of about 5 mm was prepared in the central area of ​​the back to establish the skin wound model.

[0096] (2) Experimental steps and results

[0097] 2.1 Glucose Tolerance Test (GTT)

[0098] Mice were fasted for 12 hours prior to the experiment, but had free access to water. Fasting blood glucose levels were measured via tail vein sampling, and the basal blood glucose value (G0) was recorded after discarding the first drop of blood. Subsequently, mice were administered 20% (w / v) glucose solution by gavage at a dose of 1 g / (kg·bw). Tail vein blood samples were collected at 30, 60, 90, and 120 min after gavage, and blood glucose changes at each time point were measured and recorded using a glucometer to evaluate the mice's glucose metabolism capacity.

[0099] 2.2 Insulin Tolerance Test (ITT)

[0100] Mice were fasted for 4-6 hours prior to the experiment, but had free access to water. First, the basal blood glucose level in the tail vein was measured and recorded as 0 min. Then, the insulin dosage was calculated based on the mouse's body weight, and the insulin solution was administered via intraperitoneal injection. Blood samples were collected from the tail vein at 30, 60, 90, and 120 min after injection, and blood glucose levels were measured using a glucometer. Changes in blood glucose were recorded to assess the mice's sensitivity to insulin.

[0101] like Figure 9 As shown, the experimental results indicate that in the glucose tolerance test (GTT), the blood glucose levels of mice in the high-fat diet (HFD) group were significantly higher than those in the control group (Con) at all time points, reaching a peak at approximately 20 minutes after glucose loading. Although blood glucose gradually decreased thereafter, it remained significantly higher than that in the control group at 120 minutes (***p < 0.001), suggesting that HFD treatment led to a decrease in glucose clearance capacity in mice, indicating significant glucose tolerance impairment. In the insulin tolerance test (ITT), the blood glucose levels of mice in the control group decreased rapidly after insulin injection and remained at a low level, while the decrease in blood glucose levels in the HFD group was significantly smaller (***p < 0.001), and gradually recovered to near basal levels in the later stages, indicating a reduced responsiveness to insulin. These results demonstrate that a high-fat diet significantly impairs glucose tolerance and insulin sensitivity in mice, leading to disordered glucose metabolism, and providing a stable metabolic abnormality model basis for subsequent research on diabetes-related wound healing.

[0102] 2.3 Administration method and wound assessment

[0103] The short peptide-Ca composite silk fibroin hydrogels prepared in Example 2 were dissolved in sterile water for injection (the final concentration of silk fibroin was 3% (w / v)). After thorough mixing, the solutions were evenly applied to the wound areas on the backs of mice to allow for in-situ gelation. The dosage was 200 μL per mouse. Wounds were photographed daily after administration, and the wound area was measured and analyzed using Image-Pro Plus software. After approximately 14 days of continuous observation, most wounds had essentially closed. The wound healing rate was calculated using the following formula: Healing rate (%) = Healed area / (Healed area + Unhealed area) × 100%.

[0104] like Figure 10 As shown, the experimental results indicate that in the mouse wound healing experiment, HFD significantly delayed wound healing compared to the control group (Con), especially during days 5 to 12, when the wound healing rate of the HFD group was significantly lower than that of the Con group (***p < 0.001). Treatment group comparison: Treatment with MUT4-short peptide, MUT5-short peptide, and the positive control BEC all significantly improved the HFD-induced delay in wound healing. In particular, the MUT4-short peptide group showed the most significant wound healing promotion effect throughout the experiment. By day 12, the wound in the MUT4-short peptide group was almost completely healed, with a significantly higher healing rate than the HFD group (***p < 0.001). The wound healing effect of the MUT5-short peptide group was also significantly better than that of the HFD group (**p < 0.01), but slightly lower than that of the MUT4-short peptide group. Overall, HFD significantly inhibited wound healing in mice, while treatment with MUT4-short peptide and MUT5-short peptide effectively reversed this adverse effect, with MUT4-short peptide showing a more significant effect in promoting wound healing.

[0105] 2.4 Hematoxylin & Eosin (H&E) staining

[0106] To evaluate wound tissue repair, H&E staining analysis was performed on the skin tissue at the healing site. Wound tissue was taken and fixed in 4% paraformaldehyde for 24 h, dehydrated with graded ethanol, embedded in paraffin, and then cut into tissue sections approximately 5 μm thick. H&E staining was performed according to standard methods. After staining, images were observed and acquired under an optical microscope to assess epidermal regeneration, dermal structure recovery, and inflammatory infiltration.

[0107] like Figure 11As shown, the experimental results indicate that, by observing the repair of skin structure through H&E staining of wound tissue on day 14, the wound healing of mice in the HFD group was significantly delayed. Staining revealed severe damage to the tissue structure, with a thin epidermis and a lack of complete repair in the dermis. In contrast, the Con group showed an intact epidermis and a thicker dermis, indicating that the wound had basically healed. In the HFD+MUT5-short peptide and HFD+BEC treatment groups, the treatment with MUT5-short peptide and BEC partially improved the delayed wound healing caused by HFD, and H&E staining showed some improvement in the repair of the epidermis and dermis. In the HFD+MUT4-short peptide treatment group, MUT4-short peptide significantly promoted wound healing. H&E staining showed that the epidermis in this group was relatively intact, and the dermis was also significantly repaired, with the tissue structure tending towards normal.

[0108] 2.5 Tissue qPCR detection

[0109] Skin tissue from the wound site was minced and placed in pre-chilled EP tubes. 500 μL of Trizol lysis buffer was added, and grinding beads were added for mechanical homogenization until the tissue was fully lysed to form a homogeneous suspension. Total RNA was then extracted using the same method as in cell experiments. cDNA was obtained through reverse transcription and then detected by quantitative real-time PCR (qRT-PCR) to analyze the mRNA expression levels of inflammation-related factors IL-6, IL-1β, and TNF-α in the tissue, in order to evaluate the regulatory effects of different treatments on the local inflammatory response in the wound.

[0110] like Figure 12As shown, the experimental results indicate that a high-fat environment (HFD) induces a severe chronic inflammatory response in wound tissue. HFD treatment significantly upregulated the mRNA expression levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, showing a highly significant difference compared to the Con group (***p < 0.001). Under HFD conditions, treatments with MUT4-short peptide, MUT5-short peptide, and BEC significantly reduced the expression of these pro-inflammatory factors, with MUT4-short peptide showing the most significant effect. (a) shows that the IL-6 level was extremely high in the HFD group, and IL-6 expression was further significantly reduced after treatment with MUT5-short peptide and BEC (***p < 0.001), while MUT4-short peptide treatment further significantly reduced IL-6 expression. (b) This indicates that HFD significantly upregulated IL-1β expression, while IL-1β levels decreased in the MUT5-short peptide and BEC treatment groups (***p < 0.001), and IL-1β expression further decreased in the MUT4-short peptide treatment group, demonstrating extremely strong anti-inflammatory activity. (c) This indicates that compared with the HFD group, TNF-α levels were significantly reduced in the MUT5-short peptide and BEC treatment groups, with the MUT4-short peptide treatment group showing the lowest TNF-α level (***p < 0.001), significantly better than the expression level under HFD alone.

[0111] The above results indicate that MUT4- and MUT5-peptides, under in vitro high-glucose conditions and in vivo metabolic abnormality models, can improve the viability and migration ability of keratinocytes, reduce intracellular reactive oxygen species levels, and promote the repair process of skin wounds. Compared with the unmutated original peptides, the aforementioned MUT4-peptides show more significant effects in promoting cell function recovery and accelerating wound healing, especially under metabolic disorder conditions, where their advantages are more pronounced.

[0112] Experiment Example 3: In vivo animal experiments

[0113] This experimental case investigated the biological functions of short peptides on common wounds.

[0114] In this experiment, normal mice (non-HFD model) were used to set up a control group (Con), a BEC treatment group, and a MUT4-short peptide treatment group, and local administration and observation were carried out in accordance with the administration method in Experiment Example 2.

[0115] like Figure 13As shown, the experimental results indicate that under non-HFD conditions, the wound healing rate of mice in the control group was relatively slow, especially from day 5 to day 12, with a significantly lower wound closure rate than that of the treatment groups. Compared with the control group, both BEC treatment and MUT4-short peptide treatment significantly promoted wound healing, showing a faster wound closure trend from day 5 and maintaining high healing efficiency in the later stages. Further statistical analysis showed no significant difference in wound healing rate between the BEC treatment group and the MUT4-short peptide treatment group. Figure 14 This indicates that MUT4-short peptide has a promoting effect comparable to BEC in wound repair in normal mice.

[0116] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An arginase 2 inhibitory peptide, characterized in that, It has the amino acid sequence shown in SEQ ID No. 1 or a mutated sequence thereof.

2. The arginase 2 inhibitory peptide according to claim 1, characterized in that, The mutated sequence is a mutation occurring at one or more positions corresponding to the amino acid sequence shown in SEQ ID No. 1, or a nuclear localization signal sequence is added to the C-terminus.

3. The arginase 2 inhibitory peptide according to claim 1, characterized in that, It has any of the following amino acid sequences: The first amino acid corresponding to the sequence shown in SEQ ID No. 1 is mutated to R; The 7th amino acid corresponding to the sequence shown in SEQ ID No. 1 is mutated to K; The 8th amino acid corresponding to the sequence shown in SEQ ID No. 1 is mutated to W; The 14th amino acid corresponding to the sequence shown in SEQ ID No. 1 is mutated to R; Add PKKKRKV to the end of the sequence shown in SEQ ID No.

1.

4. A pharmaceutical composition, characterized in that, It contains the arginase 2 inhibitory peptide as described in claim 1.

5. The pharmaceutical composition according to claim 4, characterized in that, It also includes one or more pharmaceutically acceptable carriers.

6. The pharmaceutical composition according to claim 4, characterized in that, The preparation method of the pharmaceutical composition includes the following steps: (1) The degummed silk fibroin, CaCl2 and formic acid are mixed to obtain a silk fibroin solution. The solution is induced to undergo structural transformation and physical cross-linking by solvent evaporation to obtain a three-dimensional network structure silk fibroin hydrogel. (2) The inhibitory peptide as described in claim 1 is mixed with nanoporous CaCO3 in the presence of a solvent to obtain nano-CaCO3 loaded with the inhibitory peptide; (3) After mixing the nano-CaCO3, CaCl2 and formic acid of the loaded inhibitory peptide obtained in step (2), the resulting mixture is coated on the surface of the silk fibroin hydrogel obtained in step (1) to obtain the inhibitory peptide-Ca gel, which is the drug composition.

7. The use of the arginase 2 inhibitory peptide according to any one of claims 1-3, or the use of the pharmaceutical composition according to any one of claims 4-6 in the preparation of a medicament for promoting wound healing.

8. The application according to claim 7, characterized in that, The wound is either a common traumatic wound or a diabetic traumatic wound.

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