Sugar hydrogel loaded with SJMHE1 polypeptide, preparation method and application in wound healing promoting medicine

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and in particular relates to a glycogen hydrogel loaded with an SJMHE1 polypeptide, a preparation method thereof, and an application thereof in wound healing promoting drugs. Background Art

[0002] Skin injury repair is a dynamic and complex process that involves the close coordination of multiple cells and molecules to restore the balance after tissue damage. This repair process includes hemostasis, inflammation, cell proliferation and migration, and tissue remodeling. For successful wound healing, all of the above processes must occur in the appropriate order and timing. Bleeding caused by trauma leads to local vasoconstriction and platelet aggregation to form emboli, thereby activating the coagulation cascade and achieving hemostasis; the next stage is the inflammatory stage, in which inflammatory cells from the immune system enter the wound under the action of chemokines, mainly neutrophils, macrophages, and lymphocytes infiltrating in sequence. A large number of neutrophils not only play an important role in defending against invading microorganisms but also recruit monocyte-derived macrophages. Under the influence of local cytokines in the wound, monocyte-derived macrophages differentiate into mature wound macrophages. After phagocytizing tissue debris, microorganisms, and apoptotic cells, most mature macrophages undergo apoptosis, while a small number transform 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, contributing to the transition to the proliferative phase. During the proliferative phase, fibroblasts are recruited and begin to produce collagen, proteoglycans, fibronectin, and elastin, which constitute the extracellular matrix. In response to 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 in the dermal wound site. Disruption and / or dysregulation of the skin repair process can lead to wound non-healing. Furthermore, with the aging population, wound non-healing associated with conditions such as aging, atherosclerosis, and diabetes has become a major global public health concern.

[0003] Current wound healing therapies include various gels, bioactive peptides, growth factors, and herbal preparations. Despite extensive research, skin wound healing, particularly non-healing chronic wounds, remains an unmet clinical need due to challenges in assessment and wound management. Therefore, the development of novel approaches to skin wound healing is essential, as it holds significant medical significance in today's aging global population.

[0004] The immunomodulatory peptide SJMHE1, identified from Schistosoma japonicum, can inhibit delayed-type hypersensitivity (DTH), collagen-induced arthritis (CIA), asthma, acute and chronic colitis, allergic rhinitis, and psoriasis. However, peptide drugs are unstable and easily degraded by proteases in the circulation. Nanomaterials loaded with peptides can overcome these shortcomings 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 laborious synthesis and difficult-to-control manufacturing processes of most polymer nanomaterials hinder their scale-up and hinder their clinical and commercial application. Summary of the Invention

[0005] In response to the above technical problems, the present invention provides a glycogen hydrogel loaded with SJMHE1 polypeptide and its application in wound healing promoting drugs.

[0006] The present invention discloses a glycogen hydrogel loaded with SJMHE1 polypeptide for use in skin damage repair. Non-toxic, biodegradable glycogen (AG) has a natural hyperbranched dendritic nanostructure with a size range of 20-80 nm. The present invention involves amino-modifying natural glycogen with diethylenetriamine (DETA) to load it with SJMHE1 polypeptide, which is then mixed with polyvinyl alcohol (PVA) to produce a SJMHE1 polypeptide-loaded hydrogel (AG-SJMHE1-PVA gel). The SJMHE1 polypeptide-loaded glycogen hydrogel of the present invention can accelerate the repair of skin damage by promoting collagen deposition and angiogenesis. These natural glycogen nanoparticles are non-toxic, biodegradable, and have an easily controllable manufacturing process. They can be obtained on a large scale, avoiding the toxicity associated with the chemical components and organic solvents of most synthetic polymer nanomaterials, as well as the laborious synthesis and difficult-to-control manufacturing processes that hinder scale-up production and limit clinical and commercial applications.

[0007] Note that the inclusion of these objectives does not preclude the existence of other objectives. One embodiment of the present invention does not necessarily achieve all of the above objectives. Objectives other than the above objectives may be extracted from the description of the specification, drawings, and claims.

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

[0009] A glycogen hydrogel loaded with an SJMHE1 polypeptide comprises glycogen, an SJMHE1 polypeptide and a gel; the glycogen is aminoglycogen obtained by amino-modifying natural glycogen, and the aminoglycogen is loaded with an SJMHE1 polypeptide and then mixed with the gel to obtain the glycogen hydrogel loaded with the SJMHE1 polypeptide.

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

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

[0012] A method for preparing the SJMHE1 polypeptide-loaded glycogen hydrogel comprises the following steps: amino-modifying natural glycogen AG with diethylenetriamine DETA, loading the SJMHE1 polypeptide, and then mixing with polyvinyl alcohol (PVA) to obtain aminoglycogen AG-SJMHE1 polypeptide-PVA gel, i.e., the SJMHE1 polypeptide-loaded hydrogel.

[0013] In the above scheme, the method for preparing the SJMHE1 polypeptide-loaded glycogen hydrogel specifically comprises 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 to obtain PVA gel;

[0015] Preparation of aminoglycogen: Glycogen and carbonyldiimidazole are dissolved in dimethyl sulfoxide, and the solution is stirred under inert gas. Diethylenetriamine is then added to the solution in a ratio of glycogen, carbonyldiimidazole, dimethyl sulfoxide, and diethylenetriamine of 20 mg:50 mg:1 ml:50 mg. Stirring is continued, and the mixture is then dialyzed and lyophilized to obtain aminoglycogen (AG).

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

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

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

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

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

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

[0022] In the above scheme, the glycogen hydrogel loaded with SJMHE1 polypeptide can promote the formation of skin hair follicles and skin accessory glands at the wound healing site.

[0023] In the above scheme, the glycogen hydrogel loaded with the 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 present invention has the following beneficial effects:

[0025] The present invention confirms through a series of experiments that glycogen-loaded SJMHE1 polypeptide hydrogel significantly promotes the re-epithelialization of mouse wounds and improves the wound healing rate; promotes the early granulation tissue and angiogenesis at the wound site, prompting wound healing to enter the remodeling stage faster; promotes collagen deposition and type I collagen expression at the wound site in the late stage of wound healing; promotes the formation of skin hair follicles and skin accessory glands at the wound healing site; compared with the positive control group of collagenase ointment, the glycogen-loaded SJMHE1 polypeptide hydrogel group promotes wound healing in the early stage of wound healing (0-7 days) The results showed that the hydrogels were more efficient and had a higher wound healing rate. Furthermore, the number of new blood vessels was increased on the seventh day after skin injury. Furthermore, the glycogen-loaded SJMHE1 peptide hydrogels were non-cytotoxic and had good blood compatibility. In vitro, SJMHE1 treatment promoted the expression of vascular endothelial growth factor A (VEGFA) mRNA and TGF-β1 protein in macrophages. Cultured macrophages treated with SJMHE1 (CM-SJMHE1) promoted 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 hydrogels are low-cost, easy to prepare, and highly safe, possessing 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 embodiment of the present invention does not necessarily have all of the above effects. Effects other than the above can be clearly seen and extracted from the description of the specification, drawings, claims, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1This is a graph showing the release rate detection and biosafety assessment of glycogen-loaded SJMHE1 polypeptide hydrogel according to one embodiment of the present invention, wherein Figure 1 (A) Image of the finished hydrogel; Figure 1 (B) Standard curve of peptide concentration and 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 showing the macroscopic wound healing rate of mice promoted by glycogen-loaded SJMHE1 polypeptide hydrogel according to one embodiment of the present invention, wherein Figure 2 (A) Macroscopic representation of mouse wound healing; Figure 2 (B) Wound healing rate of mice.

[0029] Figure 3 This is a histological diagram showing that the glycogen-loaded polypeptide hydrogel according to one embodiment of the present invention promotes wound healing in mice, wherein: Figure 3 (A) Representative images of HE staining of the wounds of mice in each group on day 7 of skin injury; Figure 3 (B) Representative images of HE staining of the wounds of mice in each group on day 11 of skin injury; Figure 3 (C) Epidermal thickness of wound tissues of mice in each group on day 11; Figure 3 (D) Wound length of mice in each group on day 11.

[0030] Figure 4 This is a diagram showing that glycogen-loaded SJMHE1 polypeptide hydrogel promotes collagen deposition in mouse wound tissues according to one embodiment of the present invention, wherein Figure 4 (A) Representative images of Masson staining of wound tissues of mice in each group on day 11 of skin injury; Figure 4 (B) Representative images of Col1a1 immunohistochemistry in wound tissues of mice in each group on day 11 of skin injury; Figure 4 (C) Collagen deposition in wound tissue of mice in each group on day 11 of skin injury; Figure 4 (D) Relative expression of Col1a1 mRNA in the wound tissue of mice in each group on the 11th day after skin injury.

[0031] Figure 5 This is a diagram showing that glycogen-loaded SJMHE1 polypeptide hydrogel promotes early angiogenesis in wound healing according to one embodiment of the present invention, wherein Figure 5 (A) Representative images of CD31 immunohistochemistry in wound tissues of mice in each group on days 7 and 11; Figure 5 (B) The number of new blood vessels in each group of mice on the 7th day after wound injury; Figure 5 (C) The number of new blood vessels in each group of mice on the 11th day after wound injury.

[0032] Figure 6FIG is a diagram showing that macrophage conditioned medium treated with SJMHE1 promotes HUVEC and L929 cell migration according to one embodiment of the present invention, wherein Figure 6 (A) SJMHE1 treatment promoted VEGFA mRNA expression in macrophages; Figure 6 (B) SJMHE1 treatment promoted TGF-β1 protein expression in macrophages; Figure 6 (C) Representative images of HUVEC cell migration; Figure 6 (D) Statistical graph of HUVEC cell scratch healing rate; Figure 6 (E) Representative images of L929 cell migration; Figure 6 (F) Statistical graph of the number of L929 cells migrating into the scratch area. DETAILED DESCRIPTION

[0033] The following detailed description of the embodiments of the present invention is provided in conjunction with the accompanying drawings. This description is intended to illustrate the present invention and is not to be construed as limiting the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The reagents and materials used in the following examples are commercially available unless otherwise specified.

[0034] Example 1: Preparation of SJMHE1 polypeptide-loaded glycogen hydrogel and release rate detection

[0035] (1) Preparation of glycogen-loaded SJMHE1 polypeptide 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) and stirred to obtain a PVA gel. Then, 200 mg of glycogen and 500 mg of carbonyldiimidazole were dissolved in 10 mL of dimethyl sulfoxide. The solution was stirred under inert gas for 1 hour. 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. The aminoglycogen AG-SJMHE1 peptide complex was then added to the PVA gel to 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) Peptide release rate detection:

[0038] FITC-labeled SJMHE1 peptide was used instead of ordinary peptide, such as Figure 1 middle Figure 1As shown in (A), 2 mL of aminoglycogen AG-FITC-labeled SJMHE1 peptide-PVA gel 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 extracted from the centrifuge tube and the absorbance of the solution was measured using a microplate reader to calculate the concentration of the released peptide. After each aliquot was taken, 200 μl of fresh H2O was added to the test tube. The results are shown in Figure 2. Figure 1 As shown in (B), the fluorescence intensity is linearly related to the concentration of SJMHE1 polypeptide. The SJMHE1 polypeptide concentration of the unknown sample can be calculated by measuring the fluorescence intensity. The release rate of SJMHE1 polypeptide is more than 80% within 48 h. Figure 1 (C) shown.

[0039] (3) Cytotoxicity

[0040] Soak 0.2g of hydrogel in 1ml of culture medium and incubate in a shaker at 37°C for 24 hours. Collect the hydrogel extract and store it at 4°C. L929 cells were seeded into a 96-well plate at 5000 cells / well and cultured for 12-24 hours to ensure cell adhesion. The hydrogel extract was added to the cell culture wells, and the cells cultured with fresh culture medium were used as the control group and incubated in an incubator for 24-48 hours. After the incubation, the original culture medium was discarded, 100μL of fresh culture medium and 10μL 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 a wavelength of 450nm using an enzyme reader, and the cell survival rate was calculated according to the formula: Cell survival rate (%) = [(OD value of the experimental group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)] × 100%. The results are as follows. Figure 1 As shown in (D), the hydrogel extract did not inhibit cell proliferation after co-culture with cells for 48 hours, indicating good cell compatibility.

[0041] (4) Blood compatibility

[0042] Whole blood was collected from mice and centrifuged at 1500rpm for 10 minutes to remove plasma components. The obtained 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 use. Subsequently, 900μl of 5% red blood cell suspension and 100μl of hydrogel were added to the centrifuge tube, and an equal amount of Triton X-100 (0.1%) was added as a positive control, and PBS was used as a negative control. After incubating each group of mixed solutions at 37°C for 2 hours, centrifuged at 1500rpm for 10 minutes, and 100μL of the supernatant was aspirated into a 96-well enzyme-labeled plate. The absorbance (OD value) was measured at a wavelength of 540nm using an enzyme-labeled instrument to evaluate the degree of hemolysis. The results are as follows. Figure 1 As shown in (E), glycogen-loaded polypeptide hydrogel did not induce significant hemolysis, with a hemolysis rate of less than 5%.

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

[0044] Female C57BL / 6J mice aged 6-8 weeks were fed a normal diet for 2 days. They were then anesthetized with isoflurane and hair was removed from their backs (approximately 2 × 2 cm) using an electric shaver and depilatory cream. The mice were then placed in lateral recumbency. The approximate locations of two raised bumps were marked on both sides, and the intersection of the line connecting the two bumps and the midline of the mouse's back was used as the final wound center. A skin punch was used, aligning the center of the wound with the intersection point, to create an 8 mm diameter circular wound. The mice were randomly divided into four groups: a full-thickness injury model group (untreated); a blank hydrogel group (50 μl of blank hydrogel was applied daily to the wound); a glycogen-loaded SJMHE1 peptide hydrogel group (SJMHE1-hydrogel group): 50 μl of glycogen-loaded SJMHE1 peptide hydrogel was applied daily to the wound); and a collagenase group (collagenase ointment was applied daily to the wound). The mice were maintained on a normal diet and a 12 / 12 h light / dark cycle.

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

[0046] To verify the healing effect of the glycogen-loaded peptide hydrogel, the size of the original wound on the back of the mice was recorded on the day of full-thickness skin injury surgery (day 0). The wound healing status of the mice's back was then observed and photographed on the 3rd, 7th, and 11th days of treatment. The area of ​​mouse skin damage was measured and statistically analyzed using Image J software to calculate the wound healing rate. The wound healing rate formula is as follows:

[0047] Wound healing rate (%) = 1-wound area 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 the 3rd and 7th days of treatment, the wound healing rate of mice in the group treated with glycogen-loaded peptide hydrogel was significantly increased compared with the other groups. Figure 2 (B) Compared with the model group, the polypeptide hydrogel group *** P<0.001; compared with the blank hydrogel group, ## P<0.01, ###P<0.001. On the 3rd day, the wound healing area of ​​mice treated with glycogen-loaded polypeptide hydrogel was approximately 63%, which was significantly higher than the wound healing areas of the model group (approximately 46%), blank hydrogel group (approximately 36%) and collagenase group (approximately 38%). On the 7th day, the wound healing area of ​​mice treated with glycogen-loaded polypeptide hydrogel was approximately 80%, which was significantly higher than the wound healing areas of the model group (approximately 64%), blank hydrogel group (approximately 68%) and collagenase group (approximately 65%). On the 11th day, the wound healing areas of mice in the model group and blank hydrogel group were approximately 90% and 87%, respectively, and some dry scabs could still be observed in the wounds. The wounds of mice in the glycogen-loaded polypeptide hydrogel group and collagenase group were almost 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 killed on the 7th and 11th days respectively. After killing the mice, skin tissue with a diameter of about 8 mm around the wound was quickly cut, and the surface impurities were rinsed with normal saline. The tissue blocks were then immersed in 4% paraformaldehyde tissue fixative to maintain the original tissue structure and prevent corruption. After fixation, the tissue was gradually dehydrated through gradient alcohol (70%, 80%, 90%, 95% and 100% ethanol), soaking for 1-2 hours at each level. After dehydration, xylene was used for two transparent treatments, each for 30 minutes, until the tissue became translucent. The transparent tissue was transferred to melted paraffin and cooled and solidified to form wax blocks. The slice thickness was adjusted to 3-5 microns, the cut tissue slices were placed on a glass slide, and stored at room temperature for use. Sections were deparaffinized by immersing them in environmentally friendly deparaffinization solution I for 20 minutes, followed by immersion in environmentally friendly deparaffinization solution II for 20 minutes. They were then treated with anhydrous ethanol I and anhydrous ethanol II for 5 minutes each to completely remove residual paraffin. The sections were then immersed in 75% ethanol for 5 minutes and rinsed with tap water to complete hydration. Hematoxylin and eosin (H&E) staining was then performed. Sections were immersed in hematoxylin solution for 3-5 minutes to stain nuclei. Excess stain was removed by rinsing with tap water. Sections were then briefly differentiated in differentiation solution to remove nonspecific binding. After rinsing with tap water, the sections were bluing solution to restore the bright blue color of the nuclei. Finally, the sections were rinsed thoroughly with running water. After nuclear staining, sections were dehydrated in 95% ethanol for 1 minute and then quickly immersed in eosin solution for 15 seconds. After staining, the sections were dehydrated and cleared by immersing them in 80%, 90%, 95%, and 100% ethanol, followed by two 5-minute cycles of xylene. After drying, neutral gum was added to cover the tissue area and the slides were sealed. The sealed sections were observed under a microscope, and images were collected and analyzed.

[0051] The results are as follows Figure 3As shown in Figure 2, on the 7th day after wound injury, the inflammatory cell infiltration in the wound tissue of mice in the glycogen-loaded polypeptide hydrogel group was less and the granulation tissue was thicker than that in the model group and the blank hydrogel group. Figure 3 As shown in (A). On the 11th day after wound injury, i.e., in the late stage of wound healing, mice in the glycogen-loaded polypeptide hydrogel group and the collagenase group completed re-epithelialization, with less inflammatory cell infiltration, as shown in Figure 3 As shown in (B), the wound length was significantly shorter than that of the model group and the blank hydrogel group; however, there were still blood scabs and more inflammatory cell infiltration in the wounds of the model group and the blank hydrogel group, as shown in Figure 3 (C) As shown; the thickness of the new 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 was closer to the thickness of the normal skin epidermis, but the statistical difference was not significant compared with the collagenase group. Figure 3 (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 the paraffin sections of mouse skin tissue were dewaxed and hydrated, they were immersed in Masson A solution for overnight incubation and then rinsed thoroughly with running tap water. The sections were then placed in a dye solution mixed with Masson B solution and Masson C solution in equal proportions for 1 minute. After rinsing with tap water, they were immediately immersed in differentiation solution for a short differentiation for a few seconds and then rinsed with tap water again. Thereafter, the sections were transferred to Masson D solution for staining for 6 minutes. After rinsing with tap water, they were directly immersed in Masson E solution for staining for 1 minute. After staining, there is no need to wash with water. After the sections are slightly drained, they are immersed in Masson F solution for staining for 2 to 30 seconds. They were then rinsed and differentiated with 1% acetic acid solution and dehydrated in two cylinders of anhydrous ethanol. The dehydrated sections need to be immersed in a third cylinder of anhydrous ethanol for 5 minutes, then transferred to xylene for transparent treatment for 5 minutes, and finally sealed with neutral gum. The samples that have completed the sealing are observed under a microscope and image acquisition and analysis are performed. The results are as follows Figure 4 middle Figure 4 (A)- Figure 4 As shown in (C), the amount of collagen deposition in the wound tissue of mice in the glycogen-loaded SJMHE1 polypeptide hydrogel group was greater than that in the blank group and the blank hydrogel group, and the collagen fibers were arranged more orderly, presenting a wavy or reticular structure; however, there was no significant difference in the amount of collagen deposition compared with the collagenase group. 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, added with 1 ml of Trizol, and lysed using a tissue homogenizer. The mixture was centrifuged at 12,000 g at 4°C for 5 minutes. The supernatant was aspirated, and 200 μl of chloroform was added. The mixture was shaken vigorously for 15 seconds, allowed to stand at 4°C for 5 minutes, and then centrifuged at 12,000 g at 4°C for 15 minutes. 400 μl of the upper aqueous phase was transferred to a fresh centrifuge tube, and an equal volume of pre-chilled isopropanol was added. The mixture was mixed by inversion, allowed to stand at 4°C for 10 minutes, and centrifuged at 12,000 g at 4°C for 10 minutes. The supernatant was discarded. The pellet was then washed with 1 ml of pre-chilled 75% ethanol, centrifuged at 12,000 g at 4°C for 5 minutes, and the supernatant discarded. The pellet was dried at room temperature for 2-5 minutes. The pellet was dissolved in an appropriate amount of RNase-free ddH2O, and the RNA concentration was determined. After reverse transcription, COL1a1 mRNA expression was detected by qRT-PCR.

[0057] The paraffin sections of the mouse wound skin were dewaxed and hydrated before antigen retrieval. After the repair was completed, they were cooled naturally and washed with PBS three times, each time for 5 minutes. The sections were then immersed in a 3% hydrogen peroxide solution and incubated in the dark at room temperature for 25 minutes to block endogenous peroxidase. They were washed again with PBS three times and blocked with 5% BSA antigen blocking solution for 60 minutes. Anti-COL1a1 primary antibody was added and incubated overnight at 4°C. After washing with PBS three times, drying, HRP-labeled secondary antibody was added, incubated at room temperature for 60 minutes, and washed again with PBS three times. After coloring with freshly prepared DAB coloring solution, the reaction was immediately terminated by rinsing with tap water. Hematoxylin counterstaining was then performed. After dehydration and clearing, the sections were sealed with sealing glue after drying. The sections were observed under a microscope and image acquisition and analysis were performed. The results are shown in the figure below. Figure 4 As shown in (D), the expression of COL1a1 mRNA and protein in the wound tissues of mice in the glycogen-loaded SJMHE1 polypeptide hydrogel group was increased compared with the blank group and the blank hydrogel group, but the difference was not significant compared with the collagenase group. Figure 4 middle, * P<0.05, ** P<0.01, *** P<0.001.

[0058] Example 6: Counting of new blood vessels in mouse wound skin tissue

[0059] The paraffin sections of the mouse wound skin were dewaxed and hydrated, and then naturally cooled after antigen repair. They were washed with PBS three times, each for 5 minutes. The sections were then immersed in a 3% hydrogen peroxide solution and incubated in the dark at room temperature for 25 minutes to block endogenous peroxidase. They were washed again with PBS three times and blocked with 5% BSA antigen blocking solution for 60 minutes. Anti-CD31 primary antibody was added and incubated overnight at 4°C. After washing with PBS three times, HRP-labeled secondary antibody was added after drying, and the sections were incubated at room temperature for 60 minutes. The sections were washed again with PBS three times, and then color was developed with freshly prepared DAB coloring solution. The reaction was immediately terminated by rinsing with tap water. Hematoxylin was then used for counterstaining. After dehydration and clearing, the sections were sealed with neutral gum after drying. The sections were observed under a microscope, and images were collected and the number of new blood vessels was counted. The results are as follows Figure 5 As shown in the figure, on the 7th day, the number of new blood vessels in the skin wounds of mice in the glycogen-loaded SJMHE1 polypeptide hydrogel group was significantly higher than that in the model group, and was also higher than that in the blank hydrogel group and the collagenase group, but the statistical difference was not significant. On the 11th day, the number of new blood vessels in the skin wounds of mice in the glycogen-loaded SJMHE1 polypeptide hydrogel group was significantly lower than that in the blank hydrogel group, and was also lower than that in the model group and the collagenase group, but the statistical difference was not significant. Figure 5 As shown in (A). In the glycogen-loaded SJMHE1 polypeptide hydrogel treatment group, the number of new blood vessels in the wound tissue of mice decreased significantly from the 7th to the 11th day after skin injury, while in the model group and the blank hydrogel group, the number of new blood vessels still showed an upward trend from the 7th to the 11th day. This may indicate that the glycogen-loaded polypeptide hydrogel treatment accelerated the process from the proliferation stage to the remodeling stage of wound healing, as shown in Figure 2. Figure 5 (B) and Figure 5 (C) * P<0.05.

[0060] Example 7:

[0061] Mouse macrophage RAW264.7 cells were seeded in 6-well plates. The control group (Control) was cultured in complete medium (DMEM + 10% FBS) for 24 hours, and the experimental group was treated with an equal amount of medium containing 1 μg / ml SJMHE1 polypeptide for 24 hours. Macrophage RNA was extracted using the same method as in Example 5. After reverse transcription, qRT-PCR was used to detect the expression of VEGFA mRNA. The results are shown in Figure 5. Figure 6 As shown in Figure 2, VEGFA mRNA expression in macrophages increased after SJMHE1 treatment. Figure 6 (A) shown.

[0062] Macrophage proteins treated in the same manner as above were extracted using RAPI lysis buffer, and equal amounts of protein samples were taken and mixed with protein loading buffer. Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and after electrophoresis separation, they were transferred to PVDF membranes. Blocked with 5% skim milk at room temperature for 1 hour, washed with TBST, and incubated with primary antibodies TGF-β1 and β-actin at 4°C overnight. After thorough washing with TBST, incubated with horseradish peroxidase (HRP)-labeled secondary antibodies at room temperature for 1 hour and washed again. ECL chemiluminescence reagent was used for color development, and the signal was captured by the chemiluminescence imaging system. The results are shown in Figure 2. Figure 6 As shown in (B), TGF-β1 expression in macrophages was increased after SJMHE1 treatment.

[0063] Macrophage culture medium treated in the same manner as above was collected, centrifuged at 2000 rpm for 10 minutes, and filtered through a 0.22 μm filter to obtain culture medium for the control group (CM-Control) and the peptide-treated group (CM-SJMHE1). Logarithmically growing mouse fibroblasts L929 and human umbilical vein endothelial cells (HUVEC) were seeded in 6-well plates and cultured to 95% confluence. A linear scratch was then made using a sterile pipette tip. After washing with PBS, conditioned medium consisting of a 1:2 mixture of CM-Control or CM-SJMHE1 and basal DMEM medium was added to each well and incubated at 37°C in a 5% CO2 incubator. The fixed positions were photographed under an inverted microscope at 0 and 24 hours for HUVEC and at 0, 24, 48, and 72 hours for L929. L929 cells migrate irregularly, and the number of migrating cells within the scratch area represents the wound closure rate. ImageJ software was used to quantify the HUVEC cell scratch healing rate (%) = (initial scratch area - scratch area at a specified time point) / initial scratch area × 100%. Figure 6 (C)- Figure 6 As shown in (F), the scratch healing rate and the number of migrating cells 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 narrative method of the specification is only for the sake of 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 series of detailed descriptions listed above are only specific descriptions of feasible embodiments of the present invention. They are not intended to limit the scope of protection of the present invention. Any equivalent embodiments or changes that do not deviate from the technical spirit of the present invention should be included in the scope of protection of the present invention.

Claims

1. A glycogen hydrogel loaded with SJMHE1 polypeptide, characterized in that: The invention comprises glycogen, SJMHE1 polypeptide and gel; the glycogen is aminoglycogen obtained by amino-aminoing natural glycogen; the aminoglycogen is loaded with SJMHE1 polypeptide and then mixed with gel to obtain glycogen hydrogel loaded with SJMHE1 polypeptide.

2. A method for preparing the SJMHE1 polypeptide-loaded glycogen hydrogel according to claim 1, characterized in that: The following steps are involved: Natural glycogen AG was amino-modified by diethylenetriamine DETA and loaded with SJMHE1 polypeptide, and then mixed with polyvinyl alcohol PVA to obtain aminoglycogen AG-SJMHE1 polypeptide-PVA gel, that is, a hydrogel loaded with SJMHE1 polypeptide.

3. The method for preparing the SJMHE1 polypeptide-loaded glycogen hydrogel according to claim 2, characterized in that: The specific steps include: 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 to obtain PVA gel; Preparation of aminoglycogen: Glycogen and carbonyldiimidazole are dissolved in dimethyl sulfoxide, and the solution is stirred under inert gas. Then, diethylenetriamine is added to the solution in a ratio of glycogen, carbonyldiimidazole, dimethyl sulfoxide, and diethylenetriamine of 20 mg:50 mg:1 ml:50 mg. Stirring is continued, and the mixture is dialyzed and lyophilized to obtain aminoglycogen AG. Preparation of aminoglycogen AG-SJMHE1 polypeptide-PVA gel: Mix the aminoglycogen AG solution and the SJMHE1 polypeptide solution at a weight ratio of 2:1, and add the aminoglycogen AG-SJMHE1 polypeptide complex to the PVA gel on ice to obtain the prepared aminoglycogen AG-SJMHE1 polypeptide-PVA gel.

4. Use of the SJMHE1 polypeptide-loaded glycogen hydrogel according to claim 1 or the SJMHE1 polypeptide-loaded glycogen hydrogel obtained by the preparation method according to any one of claims 2 or 3 in a wound healing promoting drug.

5. Use of the SJMHE1 polypeptide-loaded glycogen hydrogel in a wound healing promoting drug according to claim 4, characterized in that: The SJMHE1 polypeptide-loaded glycogen hydrogel accelerates the repair of skin damage by promoting collagen deposition and angiogenesis.

6. Use of the SJMHE1 polypeptide-loaded glycogen hydrogel in a wound healing promoting drug according to claim 4, characterized in that: The SJMHE1 polypeptide-loaded glycogen hydrogel can promote re-epithelialization of the wound surface and improve the wound healing rate.

7. Use of the SJMHE1 polypeptide-loaded glycogen hydrogel in a wound healing promoting drug according to claim 4, characterized in that: The SJMHE1 polypeptide-loaded glycogen hydrogel can promote early granulation tissue and angiogenesis at the wound site, prompting wound healing to enter the remodeling stage faster.

8. Use of the SJMHE1 polypeptide-loaded glycogen hydrogel in a wound healing promoting drug according to claim 4, characterized in that: The SJMHE1 polypeptide-loaded glycogen hydrogel can promote collagen deposition and the expression of type I collagen at the wound site in the late stage of wound healing.

9. Use of the SJMHE1 polypeptide-loaded glycogen hydrogel in a wound healing promoting drug according to claim 4, characterized in that: The glycogen hydrogel loaded with the SJMHE1 polypeptide can promote the formation of skin hair follicles and skin accessory glands at the wound healing site.

10. Use of the SJMHE1 polypeptide-loaded glycogen hydrogel in a wound healing promoting drug according to claim 4, characterized in that: The glycogen hydrogel loaded with the SJMHE1 polypeptide can promote the expression of vascular endothelial growth factor A (VEGFA) mRNA and transforming growth factor-β1 protein in macrophages.

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