A glycogen hydrogel loaded with sjmhe1 polypeptide, preparation method and application in promoting wound healing drugs

Abstract

The application provides a glycogen hydrogel loaded with SJMHE1 polypeptide, a preparation method and application in a wound healing promoting medicine, and the SJMHE1 polypeptide is loaded on natural glycogen AG after amino modification of the natural glycogen AG by diethylenetriamine DETA, then the SJMHE1 polypeptide is mixed with polyvinyl alcohol PVA to obtain AG-SJMHE1 polypeptide-PVA gel, namely the hydrogel loaded with the SJMHE1 polypeptide. The glycogen hydrogel loaded with the SJMHE1 polypeptide can accelerate the repair of skin damage by promoting collagen deposition and angiogenesis. The natural glycogen nanoparticles are non-toxic, biodegradable, easy to control in the manufacturing process, and can be obtained on a large scale, the glycogen hydrogel loaded with the SJMHE1 polypeptide is low in cost, easy to prepare, and high in safety, has significant application potential, and provides a new direction for skin damage repair.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, and particularly relates to a glycogen hydrogel loaded with SJMHE1 polypeptide, its preparation method, and its application in wound healing drugs. Background Technology

[0002] Skin injury repair is a dynamic and complex process involving the close coordination of multiple cells and molecules to restore balance after tissue damage. This repair process includes hemostasis, inflammation, cell proliferation and migration, and tissue remodeling. For successful wound healing, all of these processes must occur in the appropriate sequence and timing. Bleeding caused by trauma leads to local vasoconstriction and platelet aggregation, forming an embolism and activating the coagulation cascade to achieve hemostasis. The next stage is the inflammatory stage, where inflammatory cells, under the influence of chemokines, enter the wound from the immune system, primarily neutrophils, macrophages, and lymphocytes infiltrating sequentially. A large number of neutrophils not only play an important defensive role against invading microorganisms but also recruit macrophages derived from monocytes. Under the influence of local cytokines at the wound site, macrophages derived from monocytes differentiate into mature wound macrophages. Most mature macrophages undergo apoptosis after phagocytizing tissue debris, microorganisms, and apoptotic cells, while a small portion transforms into M2 macrophages. M2 macrophages release growth factors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and TGF-β, which promote angiogenesis, epithelial regeneration, and collagen production, facilitating the transition to the proliferative phase. During the proliferative phase, fibroblasts are recruited and begin producing collagen, proteoglycans, fibronectin, and elastin, forming the extracellular matrix. Due to the presence of growth factors such as PDGF and TGF-β, fibroblasts also undergo proliferation, migration, and differentiation. Fibroblasts and endothelial cells are crucial for capillary growth and granulation tissue formation at the dermal wound site. Disruption and / or dysregulation of the skin repair process can lead to wound non-healing. Furthermore, with an aging population, wound non-healing associated with conditions such as aging, atherosclerosis, and diabetes has become a major global public health problem.

[0003] Current wound healing therapies include various gels, bioactive peptides, growth factors, and herbal preparations. Despite extensive research, skin wound healing, especially non-healing chronic wounds, remains unmet clinical needs due to difficulties in assessment and management. Therefore, the development of new skin wound healing methods is necessary, which has significant medical implications in today's aging global population.

[0004] The immunomodulatory peptide SJMHE1, identified from Schistosoma japonicum, can inhibit delayed-type hypersensitivity (DTH), collagen arthritis (CIA), asthma, acute and chronic colitis, allergic rhinitis, and psoriasis. However, peptide drugs are unstable and easily degraded by proteases in circulation. Nanomaterials loaded with peptides can overcome these drawbacks and have been widely used in various biomedical applications. However, the chemical components and organic solvents of most synthetic polymer nanomaterials are prone to toxicity; furthermore, the synthesis of most polymer nanomaterials is laborious, and the manufacturing process is difficult to control, hindering large-scale production and limiting clinical and commercial translation. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a glycogen hydrogel loaded with SJMHE1 peptide and its application in wound healing drugs.

[0006] This invention discloses a glycogen hydrogel loaded with SJMHE1 peptide for skin damage repair. Non-toxic, biodegradable glycogen (AG) possesses a naturally occurring hyperbranched dendritic nanostructure with a size ranging from 20-80 nm. This invention modifies natural glycogen with divinyltriamine (DETA) to obtain an SJMHE1-loaded hydrogel (AG-SJMHE1-PVA gel) by amino-modifying the natural glycogen and then loading it with the SJMHE1 peptide. The SJMHE1-loaded glycogen hydrogel of this invention can accelerate skin damage repair by promoting collagen deposition and angiogenesis. These natural glycogen nanoparticles are non-toxic, biodegradable, and their manufacturing process is easily controlled and can be mass-produced. This avoids the toxicity of chemical components and organic solvents in most synthetic polymer nanomaterials, as well as the drawbacks of most polymer nanomaterials, such as the laborious synthesis, difficult-to-control manufacturing process, limited scale-up production, and limited clinical and commercial applications.

[0007] Note that the description of these objectives does not preclude the existence of other objectives. One aspect of the invention does not require achieving all of the above objectives. Objectives other than those described above can be extracted from the description, drawings, and claims.

[0008] The present invention achieves the above-mentioned technical objectives through the following technical means.

[0009] A glycogen hydrogel loaded with SJMHE1 peptide includes glycogen, SJMHE1 peptide and gel; wherein the glycogen is aminoglycogen obtained by aminoating natural glycogen, and the aminoglycogen loaded with SJMHE1 peptide is mixed with gel to obtain glycogen hydrogel loaded with SJMHE1 peptide.

[0010] The SJMHE1 polypeptide consists of 24 amino acids, and its amino acid sequence is as follows:

[0011] VPGGGTALLRCIPVLDTLSTKNED(Val ProGlyGlyGlyGlyThr AlaLeuLeuArgCys IleProValLeu Asp ThrLeuSerThr Lys AsnGlu Asp).

[0012] A method for preparing a glycogen hydrogel loaded with SJMHE1 peptide includes the following steps: modifying natural glycogen AG with divinyltriamine DETA by aminolation, loading it with SJMHE1 peptide, and then mixing it with polyvinyl alcohol PVA to obtain an aminoglycogen AG-SJMHE1 peptide-PVA gel, i.e., a hydrogel loaded with SJMHE1 peptide.

[0013] The preparation method of the glycogen hydrogel loaded with SJMHE1 peptide in the above scheme specifically includes the following steps:

[0014] Preparation of PVA gel: Polyvinyl alcohol (PVA) solution was mixed with glycerol and polyethylene glycol (PEG) 400 in a volume ratio of 10:1:1 and stirred evenly to obtain PVA gel.

[0015] Preparation of aminoglycogen: Glycogen and carbonyl diimidazole were dissolved in dimethyl sulfoxide. The solution was stirred under inert gas protection, and then diethylenetriamine was added to the solution. The ratio of glycogen, carbonyl diimidazole, dimethyl sulfoxide and diethylenetriamine was 20 mg: 50 mg: 1 ml: 50 mg. The mixture was stirred continuously, then dialyzed and lyophilized to obtain aminoglycogen (AG).

[0016] Preparation of aminoglycogen AG-SJMHE1 peptide-PVA gel: Aminoglycogen (AG) solution and SJMHE1 peptide solution were mixed at a weight ratio of 2:1, and the aminoglycogen AG-SJMHE1 peptide complex was added to the PVA gel on ice. The final concentration of SJMHE1 peptide was 0.2 mg / mL, and the prepared aminoglycogen AG-SJMHE1 peptide-PVA gel, i.e., glycogen hydrogel loaded with SJMHE1 peptide, was obtained.

[0017] Application of the glycogen hydrogel loaded with SJMHE1 peptide or the glycogen hydrogel loaded with SJMHE1 peptide obtained by the preparation method in wound healing drugs.

[0018] In the above scheme, the glycogen hydrogel loaded with SJMHE1 peptide accelerates the repair of skin damage by promoting collagen deposition and angiogenesis.

[0019] In the above scheme, the glycogen hydrogel loaded with SJMHE1 peptide can promote re-epithelialization of the wound and improve the wound healing rate.

[0020] In the above scheme, the glycogen hydrogel loaded with SJMHE1 peptide can promote early granulation tissue and angiogenesis at the wound site, and promote faster wound healing into the remodeling stage.

[0021] In the above scheme, the glycogen hydrogel loaded with SJMHE1 peptide can promote collagen deposition and type I collagen expression at the wound site in the later stage of wound healing.

[0022] In the above scheme, the glycogen hydrogel loaded with SJMHE1 peptide can promote the generation of hair follicles and accessory glands in the wound healing site.

[0023] In the above scheme, the glycogen hydrogel loaded with SJMHE1 polypeptide can promote the expression of vascular endothelial growth factor A (VEGFA) mRNA and transforming growth factor (TGF)-β1 protein in macrophages.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] This invention demonstrates through a series of experiments that glycogen-loaded SJMHE1 polypeptide hydrogel significantly promotes reepithelialization of mouse wounds, improves wound healing rate, promotes early granulation tissue and angiogenesis at the wound site, and accelerates the wound healing process into the remodeling stage. It also promotes collagen deposition and type I collagen expression at the wound site during the later stages of wound healing, and promotes the formation of hair follicles and accessory glands at the wound site. Compared to the positive control group of collagenase ointment, the glycogen-loaded SJMHE1 polypeptide hydrogel group promoted wound healing in the early stage (0-7 days). The process is faster and results in a higher wound healing rate; more new blood vessels are formed on day 7 after skin injury, and the glycogen-loaded SJMHE1 peptide hydrogel is non-cytotoxic and has good blood compatibility. In vitro, SJMHE1 treatment promotes the expression of vascular endothelial growth factor A (VEGFA) mRNA and TGF-β1 protein in macrophages, and SJMHE1-treated macrophage culture medium (CM-SJMHE1) promotes the migration of human umbilical vein endothelial cells (HUVEC cells) and mouse fibroblasts (L929 cells), thereby promoting wound healing in mice. The glycogen-loaded SJMHE1 peptide hydrogel is low in cost, easy to prepare, and has high safety, showing significant application potential and providing a new direction for skin injury repair.

[0026] Note that the description of these effects does not preclude the existence of other effects. One aspect of the invention does not necessarily have all the aforementioned effects. Effects other than those described above can be readily observed and extracted from the description, drawings, claims, etc. Attached Figure Description

[0027] Figure 1This is a graph illustrating the release rate detection and biosafety assessment of glycogen-loaded SJMHE1 peptide hydrogel according to an embodiment of the present invention. Figure 1 (A) Image of the finished hydrogel product; Figure 1 (B) Standard curve of peptide concentration versus fluorescence intensity; Figure 1 (C) Peptide release rate; Figure 1 (D) Cytotoxicity of hydrogels; Figure 1 (E) Hemolysis rate of hydrogel.

[0028] Figure 2 This is a graph illustrating the rate at which glycogen-loaded SJMHE1 peptide hydrogel promotes macroscopic wound healing in mice according to an embodiment of the present invention. Figure 2 (A) Macroscopic representation of wound healing in mice; Figure 2 (B) Wound healing rate in mice.

[0029] Figure 3 This is a histological image of a glycogen-loaded polypeptide hydrogel promoting wound healing in mice according to an embodiment of the present invention. Figure 3 (A) Representative HE staining images of skin lesions in mice on day 7 after skin injury; Figure 3 (B) Representative HE staining images of skin lesions in mice on day 11 after skin injury in each group; Figure 3 (C) Epidermal thickness of wound tissue in mice of each group on day 11; Figure 3 (D) Wound length of mice in each group on day 11.

[0030] Figure 4 This is an image showing how glycogen-loaded SJMHE1 peptide hydrogel promotes collagen deposition in mouse wound tissue according to an embodiment of the present invention. Figure 4 (A) Representative images of Masson staining of wound tissue from mice on day 11 after skin injury in each group; Figure 4 (B) Immunohistochemical representation of Col1a1 in wound tissue of mice on day 11 after skin injury in each group; Figure 4 (C) Collagen deposition in wound tissue of mice on day 11 after skin injury in each group; Figure 4 (D) Relative expression of Col1a1 mRNA in wound tissue of mice in each group on day 11 after skin injury.

[0031] Figure 5 This is an image illustrating how glycogen-loaded SJMHE1 polypeptide hydrogel promotes early angiogenesis in wound healing according to an embodiment of the present invention. Figure 5 (A) Immunohistochemical representations of CD31 in wound tissues of mice in each group on days 7 and 11; Figure 5 (B) Number of new blood vessels in each group of mice on day 7 after wound injury; Figure 5 (C) Number of new blood vessels in each group of mice on day 11 after wound injury.

[0032] Figure 6This is a diagram illustrating the effect of SJMHE1-treated macrophage conditioned medium on the migration of HUVEC and L929 cells according to an embodiment of the present invention. Figure 6 (A) SJMHE1 treatment promotes VEGFA mRNA expression in macrophages; Figure 6 (B) SJMHE1 treatment promotes TGF-β1 protein expression in macrophages; Figure 6 (C) Representative diagram of HUVEC cell migration; Figure 6 (D) Statistical chart of HUVEC cell scratch healing rate; Figure 6 (E)L929 cell migration representation diagram; Figure 6 (F)Statistical chart of the number of migrating cells in the scratched area of ​​L929 cells. Detailed Implementation

[0033] The embodiments of the present invention are described in detail below with reference to the accompanying drawings. These embodiments are intended to explain the present invention and should not be construed as limiting it. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the reagents and materials used in the following embodiments are commercially available unless otherwise specified.

[0034] Example 1: Preparation and release rate detection of glycogen hydrogels loaded with SJMHE1 peptides

[0035] (1) Preparation of glycogen-loaded SJMHE1 peptide hydrogel:

[0036] First, a polyvinyl alcohol (PVA) solution (18% by weight) was mixed with glycerol and PEG 400 at a ratio of 10:1:1 (v / v / v). After thorough mixing, a PVA gel was obtained. 200 mg of glycogen and 500 mg of carbonyl diimidazole were dissolved in 10 mL of dimethyl sulfoxide. The solution was stirred for 1 hour under inert gas protection. Then, 500 mg of diethylenetriamine was added to the solution, and stirring continued for 24 hours. The mixture was then dialyzed for 3 days and lyophilized to obtain aminoglycogen (AG). The aminoglycogen AG solution was mixed with the SJMHE1 peptide solution at a weight ratio of 2:1 (AG / peptide) and incubated on ice for 1 hour. Subsequently, the aminoglycogen AG-SJMHE1 peptide complex was added to the PVA gel, with a final SJMHE1 peptide concentration of approximately 0.2 mg / mL. The prepared aminoglycogen AG-SJMHE1 peptide-PVA gel was sealed and stored at 4°C.

[0037] (2) Detection of peptide release rate:

[0038] Replace ordinary peptides with FITC-labeled SJMHE1 peptides, such as... Figure 1 middle Figure 1As shown in (A), 2 mL of AG-FITC-labeled SJMHE1 peptide-PVA gel containing aminoglycogen was added to a dialysis bag (3.5 kDa). The dialysis bag was then placed in a 50 mL centrifuge tube containing 35 mL of H2O. At regular intervals, an equal amount of H2O (200 μL) was drawn from the centrifuge tube, and the absorbance of the solution was measured using a microplate reader to calculate the concentration of the released peptide. For each aliquot of the sample, 200 μL of fresh H2O was added to the test tube. The results are as follows: Figure 1 As shown in (B), the fluorescence intensity is linearly related to the SJMHE1 peptide concentration. The SJMHE1 peptide concentration can be calculated by measuring the fluorescence intensity of an unknown sample. The SJMHE1 peptide release rate reaches over 80% within 48 hours. Figure 1 As shown in (C).

[0039] (3) Cytotoxicity

[0040] 0.2 g of hydrogel was immersed in 1 ml of culture medium and incubated at 37°C in a shaker for 24 hours. The hydrogel extract was collected and stored at 4°C. L929 cells were seeded at 5000 cells / well in 96-well plates and cultured for 12-24 hours to ensure cell adhesion. The hydrogel extract was added to the cell culture wells, with cells cultured in fresh culture medium as a control group, and incubated in an incubator for 24-48 hours. After incubation, the original culture medium was discarded, and 100 μL of fresh culture medium and 10 μL of CCK-8 reagent were added to each well. The 96-well plate was gently shaken and incubated at 37°C for 1-4 hours. The absorbance (OD value) of each well was measured at 450 nm using a microplate reader, and the cell viability was calculated using the formula: Cell viability (%) = [(Experimental group OD value - Blank group OD value) / (Control group OD value - Blank group OD value)] × 100%. The results are as follows. Figure 1 As shown in (D), the hydrogel extract did not inhibit cell proliferation after co-culturing with cells for 48 hours, indicating good cell compatibility.

[0041] (4) Blood compatibility

[0042] Whole blood was collected from mice and centrifuged at 1500 rpm for 10 minutes to remove plasma components. Red blood cells were collected and washed twice with sterile PBS to remove residual plasma and white blood cells. The washed red blood cells were resuspended in PBS to prepare a 5% red blood cell suspension for later use. Subsequently, 900 μl of the 5% red blood cell suspension and 100 μl of hydrogel were added to centrifuge tubes. An equal volume of Triton X-100 (0.1%) was added as a positive control, and PBS as a negative control. The mixtures were incubated at 37°C for 2 hours, centrifuged at 1500 rpm for 10 minutes, and 100 μL of the supernatant was transferred to each well of a 96-well microplate. The absorbance (OD value) was measured at 540 nm using a microplate reader to assess the degree of hemolysis. Results are as follows: Figure 1 As shown in (E), the glycogen-loaded peptide hydrogel does not induce significant hemolysis, with a hemolysis rate of less than 5%.

[0043] Example 2: Construction and grouping of a mouse model of full-scale cortical injury

[0044] Female C57BL / 6J mice aged 6-8 weeks were acclimatized to a normal diet for 2 days. Mice were then anesthetized with isoflurane, and the hair on their backs (approximately 2×2 cm) was removed using an electric shaver and depilatory cream. The mice were then placed in a lateral recumbent position, and the approximate locations of two raised areas were marked by palpation on both sides. The intersection of the line connecting these two raised areas and the midline of the mouse's back was taken as the final wound center. Using a skin piercing device, a circular wound with a diameter of 8 mm was created, aligned with the intersection. Mice were randomly divided into four groups: a full-scale dermal injury model group (no wound treatment); a blank hydrogel group (50 μL of blank hydrogel applied daily to the wound); a glycogen-loaded SJMHE1 peptide hydrogel group (50 μL of glycogen-loaded SJMHE1 peptide hydrogel applied daily to the wound); and a collagenase group (collagenase ointment applied daily to the wound). Mice were fed a normal diet, with a 12 / 12 h light / dark cycle.

[0045] Example 3: Assessment of macroscopic wound healing rate in mice

[0046] To verify the healing effect of glycogen-loaded peptide hydrogel, the size of the original wound on the back of mice was recorded on the day of full-thickness dermal injury surgery (day 0). Subsequently, the wound healing was observed and photographed on days 3, 7, and 11 of treatment. The area of ​​skin lesions was measured and statistically analyzed using ImageJ software to calculate the wound healing rate. The formula for the wound healing rate is as follows:

[0047] Wound healing rate (%) = 1 - (Area of ​​damage on the day of injury / Original wound area) × 100%

[0048] like Figure 2 middle Figure 2 (A) and Figure 2 As shown in (B), on days 3 and 7 of treatment, the wound healing rate of mice treated with glycogen-loaded peptide hydrogel was significantly increased compared with other groups. Figure 2 (B) Compared with the model group, the polypeptide hydrogel group in (B) *** P<0.001; Compared with the blank hydrogel group, the peptide hydrogel group, ## P<0.01, ###P < 0.001. On day 3, the wound healing area of ​​mice treated with glycogen-loaded peptide hydrogel reached approximately 63%, significantly higher than that of the model group (approximately 46%), the blank hydrogel group (approximately 36%), and the collagenase group (approximately 38%). On day 7, the wound healing area of ​​mice treated with glycogen-loaded peptide hydrogel reached approximately 80%, significantly higher than that of the model group (approximately 64%), the blank hydrogel group (approximately 68%), and the collagenase group (approximately 65%). On day 11, the wound healing areas of mice in the model group and the blank hydrogel group were approximately 90% and 87%, respectively, with some dried scabs still observed at the wound site. In contrast, the wounds of mice in the glycogen-loaded peptide hydrogel group and the collagenase group were almost completely healed, with healing areas of approximately 98% and 96%, respectively.

[0049] Example 4: Hematoxylin-eosin (HE) staining of mouse wound skin tissue

[0050] Mice were sacrificed on days 7 and 11. Immediately after sacrifice, skin tissue approximately 8 mm in diameter around the wound was harvested and rinsed with physiological saline to remove surface impurities. The tissue blocks were then immersed in 4% paraformaldehyde fixative to maintain the original tissue structure and prevent putrefaction. After fixation, the tissue was progressively dehydrated by passing it through a gradient of alcohols (70%, 80%, 90%, 95%, and 100% ethanol), each soaking for 1-2 hours. After dehydration, the tissue was cleared twice with xylene for 30 minutes each time until it became translucent. The cleared tissue was then transferred to molten paraffin to cool and solidify, forming a wax block. The section thickness was adjusted to 3-5 micrometers, and the prepared tissue sections were placed on glass slides and stored at room temperature for later use. The sections were dewaxed by immersing them sequentially in environmentally friendly dewaxing solution I for 20 minutes, then in environmentally friendly dewaxing solution II for 20 minutes each, followed by treatment with anhydrous ethanol I and anhydrous ethanol II for 5 minutes each to completely remove residual paraffin. They were then transferred to 75% ethanol for 5 minutes and finally rinsed with tap water to complete hydration. Next, hematoxylin-eosin (H&E) staining was performed. The sections were immersed in hematoxylin staining solution for 3-5 minutes to stain the cell nuclei, rinsed with tap water to remove excess staining solution, and then briefly differentiated with differentiation solution to remove non-specific binding. After rinsing with tap water, a blue-reversion solution was used to restore the bright blue color of the cell nuclei, and finally, the sections were rinsed thoroughly with running water. After nuclear staining, the sections were first dehydrated in 95% ethanol for 1 minute, then quickly immersed in eosin staining solution for 15 seconds. After staining, the sections underwent dehydration and clearing: they were sequentially immersed in 80%, 90%, 95%, and 100% ethanol for dehydration, then placed in xylene twice, 5 minutes each time. After drying, apply neutral resin to cover the tissue area and mount the slide. Observe the mounted slide under a microscope, acquire images, and analyze them.

[0051] The results are as follows Figure 3As shown, on day 7 of wound injury, compared with the model group and the blank hydrogel group, the glycogen-loaded peptide hydrogel group mice had less inflammatory cell infiltration and thicker granulation tissue in the wound tissue. Figure 3 As shown in (A). On day 11 of wound injury, the later stage of wound healing, mice in the glycogen-loaded polypeptide hydrogel group and the collagenase group completed reepithelialization with less inflammatory cell infiltration, as... Figure 3 As shown in (B), the wound length was significantly shorter in the model group and the blank hydrogel group; while the wounds in the model group and the blank hydrogel group still showed blood crusts and a large amount of inflammatory cell infiltration, such as Figure 3 As shown in (C); and the thickness of newly formed epidermis in the wound tissue of mice in the polypeptide hydrogel group was significantly lower than that in the model group and the blank hydrogel group, and closer to the thickness of normal skin epidermis, but the difference from the collagenase group was not statistically significant. Figure 3 As shown in (D); Figure 3 middle, * P<0.05, ** P<0.01, *** P<0.001.

[0052] Example 5: Collagen deposition and gene expression in mouse wound skin tissue

[0053] (1) Masson staining to assess collagen deposition

[0054] After dewaxing and hydrating paraffin sections of mouse skin tissue, they were incubated overnight in Masson A solution, followed by thorough rinsing with running tap water. The sections were then immersed in a mixture of equal parts Masson B and Masson C solutions for 1 min, rinsed with tap water, and immediately immersed in differentiation solution for a few seconds, followed by rinsing again with tap water. The sections were then transferred to Masson D solution for staining for 6 min, rinsed with tap water, and directly immersed in Masson E solution for staining for 1 min. After staining, no further rinsing was required; the sections were allowed to drain slightly before being immersed in Masson F solution for staining for 2-30 seconds. They were then rinsed with 1% acetic acid solution for differentiation and subsequently dehydrated using two separate baths of anhydrous ethanol. The dehydrated sections were then immersed in a third bath of anhydrous ethanol for 5 min, followed by clearing in xylene for 5 min, and finally mounted with neutral resin. The mounted samples were observed under a microscope, and images were acquired and analyzed. Results are as follows: Figure 4 middle Figure 4 (A)- Figure 4 As shown in (C), mice in the glycogen-loaded SJMHE1 peptide hydrogel group showed increased collagen deposition in wound tissue compared to the control group and the control hydrogel group, with collagen fibers arranged in a more orderly, wavy, or reticular structure; however, the difference in collagen deposition compared to the collagenase group was not significant. Figure 4 middle, * P<0.05, ** P<0.01,*** P<0.001.

[0055] (2) Expression level of type I collagen (COL1a1)

[0056] Mouse wound skin tissue was minced and lysed with 1 ml Trizol using a tissue homogenizer. The homogenate was centrifuged at 12000 g, 4°C for 5 min, and the supernatant was collected. Then, 200 μl of chloroform was added, the mixture was vigorously vortexed for 15 s, incubated at 4°C for 5 min, and centrifuged at 12000 g, 4°C for 15 min. 400 μl of the upper aqueous phase was transferred to a new centrifuge tube, and an equal volume of pre-chilled isopropanol was added. The mixture was inverted and mixed thoroughly, incubated at 4°C for 10 min, and centrifuged at 12000 g, 4°C for 10 min. The supernatant was discarded. The precipitate was washed with 1 ml of pre-chilled 75% ethanol, centrifuged at 12000 g, 4°C for 5 min, and the supernatant was discarded. The precipitate was dried at room temperature for 2-5 min. An appropriate amount of RNase-free ddH2O was added to dissolve the precipitate, and the RNA concentration was measured. After reverse transcription, qRT-PCR was used to detect COL1a1 mRNA expression.

[0057] Paraffin sections of mouse wound skin were dewaxed and hydrated before antigen retrieval. After retrieval, the sections were allowed to cool naturally and washed three times with PBS for 5 minutes each time. The sections were then incubated in 3% hydrogen peroxide solution at room temperature in the dark for 25 minutes to block endogenous peroxidase. They were washed three times again with PBS and blocked with 5% BSA antigen blocking solution for 60 minutes. Anti-COL1a1 primary antibody was added, and the sections were incubated overnight at 4°C. After washing three times with PBS and drying, HRP-labeled secondary antibody was added, and the sections were incubated at room temperature for 60 minutes. The sections were washed three times again with PBS, and after developing the color with freshly prepared DAB chromogenic solution, the reaction was immediately stopped by rinsing with tap water. Hematoxylin counterstaining was then performed. After dehydration, clearing, and slight drying, the sections were mounted with mounting adhesive. The sections were observed under a microscope, and images were acquired and analyzed. Results are as follows: Figure 4 As shown in (D), mice in the glycogen-loaded SJMHE1 peptide hydrogel group showed increased COL1a1 mRNA and protein expression in wound tissue compared to the blank group and the blank hydrogel group, but the difference was not significant compared to the collagenase group. Figure 4 middle, * P<0.05, ** P<0.01, *** P<0.001.

[0058] Example 6: Counting of neovascularization in mouse wound skin tissue

[0059] Paraffin sections of mouse wound skin were dewaxed and hydrated, then subjected to antigen retrieval and naturally cooled. They were washed three times with PBS for 5 minutes each time. The sections were then incubated in 3% hydrogen peroxide solution at room temperature in the dark for 25 minutes to block endogenous peroxidase. After washing three times with PBS, they were blocked with 5% BSA antigen blocking solution for 60 minutes. Anti-CD31 primary antibody was added, and the sections were incubated overnight at 4°C. After washing three times with PBS, the sections were dried and then incubated with HRP-labeled secondary antibody at room temperature for 60 minutes. After washing three times with PBS, the sections were developed with freshly prepared DAB chromogenic solution, and immediately rinsed with tap water to terminate the reaction. Hematoxylin counterstaining was then performed. After dehydration, clearing, and slight drying, the sections were mounted with neutral resin. The sections were observed under a microscope, images were acquired, and the number of new blood vessels was counted. Results are as follows: Figure 5 As shown, on day 7, the number of new blood vessels in the skin wounds of mice in the glycogen-loaded SJMHE1 peptide hydrogel group was significantly higher than that in the model group, and also higher than that in the blank hydrogel group and the collagenase group, but the difference was not statistically significant. On day 11, the number of new blood vessels in the skin wounds of mice in the glycogen-loaded SJMHE1 peptide hydrogel group was significantly lower than that in the blank hydrogel group, and also lower than that in the model group and the collagenase group, but the difference was not statistically significant. Figure 5 As shown in (A), the number of new blood vessels in the mouse wound tissue of mice treated with glycogen-loaded SJMHE1 peptide hydrogels decreased significantly from day 7 to day 11 after skin injury, while the number of new blood vessels in the model group and the blank hydrogel group continued to increase from day 7 to day 11. This may indicate that glycogen-loaded peptide hydrogel treatment accelerates the process from the proliferative phase to the remodeling phase of wound healing, as shown in (A). Figure 5 (B) and Figure 5 As shown in (C) * P<0.05.

[0060] Example 7:

[0061] Mouse macrophages RAW264.7 were seeded in 6-well plates. The control group was cultured in complete medium (DMEM + 10% FBS) for 24 hours, while the experimental group was treated in an equal volume of medium containing 1 μg / ml SJMHE1 peptide for 24 hours. Macrophage RNA was extracted using the same method as in Example 5, and after reverse transcription, VEGFA mRNA expression was detected by qRT-PCR. The results are as follows. Figure 6 As shown, SJMHE1 treatment increased VEGFA mRNA expression in macrophages, such as... Figure 6 As shown in (A).

[0062] Macrophage proteins, treated in the same manner as described above, were extracted using RAPI lysis buffer. An equal volume of protein sample was mixed with protein loading buffer. Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the separated samples were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour, washed with TBST, and incubated overnight at 4°C with primary antibodies TGF-β1 and β-actin. After thorough washing with TBST, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour, followed by another wash. The results were analyzed using ECL chemiluminescence reagent, and the signal was captured using a chemiluminescence imaging system. The results are shown below. Figure 6 As shown in (B), SJMHE1 treatment increased TGF-β1 expression in macrophages.

[0063] The culture medium of macrophages treated in the same manner as above was collected, centrifuged at 2000 rpm for 10 min, and filtered through a 0.22 μm filter membrane to obtain the control group culture medium (CM-Control) and the peptide treatment group culture medium (CM-SJMHE1). Logarithmic growth phase mouse fibroblasts L929 and human umbilical vein endothelial cells (HUVECs) were seeded in 6-well plates and cultured to 95% confluence. Straight scratches were then made using a sterile pipette tip. After washing with PBS, conditioned medium consisting of CM-Control or CM-SJMHE1 and basal DMEM culture medium at a 1:2 ratio was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator. Images were taken at 0 h and 24 h (HUVECs) and 0 h, 24 h, 48 h, and 72 h (L929s) under an inverted microscope to maintain their positions. L929 cell migration was irregular; the number of cells migrating within the scratched area represented the scratch closure rate. ImageJ software quantifies the scratch healing rate (%) of HUVEC cells = (initial scratch area - scratch area at specified time point) / initial scratch area × 100%. Results are as follows: Figure 6 (C)- Figure 6 As shown in (F), the scratch healing rate and the number of cells migrating in the scratch area of ​​the CM-SIMHE1 group were significantly higher than those of the CM-control group, indicating that the macrophage conditioned medium treated with SJMHE1 significantly promoted the migration of HUVEC vascular endothelial cells and L929 fibroblasts. Figure 6 middle * P<0.05, ** P<0.01, *** P<0.001.

[0064] It should be understood that although this specification is described according to various embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

[0065] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A glycogen hydrogel loaded with SJMHE1 peptide, characterized in that, The product comprises aminoglycogen, SJMHE1 peptide, and polyvinyl alcohol (PVA) gel; the aminoglycogen is obtained by amination of natural glycogen with divinyltriamine (DETA); the aminoglycogen and SJMHE1 peptide are combined in a weight ratio of 2:1 to form an aminoglycogen AG-SJMHE1 peptide complex, which is distributed in the PVA gel.

2. A method for preparing the glycogen hydrogel loaded with SJMHE1 peptide as described in claim 1, characterized in that, Includes the following steps: Preparation of PVA gel: Polyvinyl alcohol solution was mixed with glycerol and polyethylene glycol 400 in a volume ratio of 10:1:1 and stirred evenly to obtain PVA gel. Preparation of aminoglycogen: Natural glycogen and carbonyl diimidazole were dissolved in dimethyl sulfoxide. The solution was stirred under inert gas protection, and then diethylenetriamine was added to the solution. The ratio of natural glycogen, carbonyl diimidazole, dimethyl sulfoxide and diethylenetriamine was 20 mg: 50 mg: 1 ml: 50 mg. The mixture was stirred continuously, then dialyzed and lyophilized to obtain aminoglycogen AG. Preparation of aminoglycogen AG-SJMHE1 peptide-PVA gel: The aminoglycogen AG solution and the SJMHE1 peptide solution were mixed at a weight ratio of 2:1, and the aminoglycogen AG-SJMHE1 peptide complex was added to the PVA gel on ice to obtain the prepared glycogen hydrogel loaded with SJMHE1 peptide.

3. The application of the glycogen hydrogel loaded with SJMHE1 peptide according to claim 1 or the glycogen hydrogel loaded with SJMHE1 peptide obtained by the method of claim 2 in the preparation of a drug for promoting wound healing.

4. The application of the glycogen hydrogel loaded with SJMHE1 peptide according to claim 3 in the preparation of a wound-healing drug, characterized in that, The drug promotes wound healing through one or more of the following mechanisms: (a) Promotes macrophage polarization to M2 type, inhibits the expression of inflammatory factors, and induces macrophage secretion of TGF-β1 and VEGFA; (b) Promotes the migration of fibroblasts and endothelial cells; (c) Promotes collagen deposition and type I collagen expression; (d) Promotes angiogenesis.

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