Hydrogel loaded with 10-shogaol and application thereof in treating diabetic wounds

Abstract

The invention discloses 10-shogaol-loaded hydrogel which is prepared by the following steps: (1) dissolving methacrylic anhydride gelatin in PBS (Phosphate Buffer Solution) to obtain a GelMA solution, and adding 10-shogaol into the GelMA solution to obtain a first mixed solution; (2) dissolving a photoinitiator in an organic solvent to obtain an organic solvent containing the photoinitiator; and (3) adding an organic solvent containing a photoinitiator into the first mixed solution, stirring in a dark place to obtain a second mixed solution, and then curing the second mixed solution under 300-400nm ultraviolet light to obtain the photoinitiator. The preparation method is simple, and the 10-shogaol-loaded hydrogel prepared through ultraviolet curing is high in transparency, smooth in surface and stable in water retention capacity, has the effects of inhibiting inflammation, resisting bacteria, promoting angiogenesis, promoting healing of diabetic wounds and the like aiming at the pathological characteristics of the diabetic wounds, and can be used for preparing the medicine for treating the diabetic wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and particularly relates to a hydrogel loaded with 10-shogaol and an application thereof in treating diabetic wounds. Background Art

[0002] Diabetes is a complex metabolic disease. Skin regeneration in diabetic wounds is often disrupted, preventing proper wound healing. Modern functional wound dressings, including nanofibers, hydrocolloids, and hydrogels, have demonstrated excellent therapeutic efficacy in diabetic wound repair. Among these, hydrogel soft materials, which combine physicochemical properties similar to those of the natural extracellular matrix (ECM) with multifunctional designability, offer a more ideal solution for diabetic wounds.

[0003] Generally speaking, the normal wound healing process mainly includes four overlapping but different stages: hemostasis, inflammation, proliferation and remodeling. However, diabetic wounds may be interrupted or delayed due to various harmful factors. Specific harmful factors include: (1) biochemical disorders (hyperglycemia, dyslipidemia and insulin resistance) leading to tissue damage that hinder normal wound healing; (2) immune cell abnormalities making inflammation difficult to suppress, and the persistent inflammatory phase will maintain the inflammatory state of the wound and hinder healing; (3) excessive oxidative stress and reduced tissue antioxidant capacity leading to redox imbalance; (4) the diabetic state leads to reduced angiogenesis, thereby hindering normal wound healing and the recovery of a healthy vascular system.

[0004] Based on the above harmful factors, it is concluded that the basic elements for constructing diabetic wound repair hydrogels include, in addition to the basic "plus points" hydrogel properties (such as adhesion, shape adaptability, etc.), the constructed system must also have the ability to load drugs, be antibacterial, anti-inflammatory, antioxidant, and promote angiogenesis. Summary of the Invention

[0005] The purpose of the present invention is to provide a hydrogel loaded with 10-shogaol and its application in treating diabetic wounds. Specifically, 10-shogaol is prepared into a drug for treating diabetic wounds to construct a drug-loaded hydrogel system and use it for diabetic wounds, thereby achieving the purpose of combining 10-shogaol with a hydrogel to prepare a drug for treating diabetic wounds.

[0006] According to a first aspect of the present invention, a hydrogel loaded with 10-shogaol is provided. The hydrogel loaded with 10-shogaol is prepared by the following steps:

[0007] (1) dissolving methacrylic anhydride-treated gelatin in PBS to obtain a GelMA solution, and adding 10-shogaol to the GelMA solution to obtain a first mixed solution;

[0008] (2) dissolving the photoinitiator in an organic solvent to obtain an organic solvent containing the photoinitiator;

[0009] (3) adding an organic solvent containing a photoinitiator to the first mixed solution, stirring in the dark to obtain a second mixed solution, and then curing the second mixed solution under 300-400 nm ultraviolet light to obtain a second mixed solution.

[0010] The present invention develops a hydrogel product for treating diabetic wounds based on 10-shogaol, which has a positive effect on improving the clinical application of hydrogels in diabetic wound treatment. The 10-shogaol-loaded hydrogel of the present invention is simple to prepare. The 10-shogaol-loaded hydrogel is prepared by ultraviolet light curing. The hydrogel has high transparency, a smooth surface, and stable water retention. 10-shogaol is a major active ingredient in ginger and belongs to the gingerol family of compounds. Its structural formula is as follows:

[0011]

[0012] In some embodiments, in step (1), the mass volume ratio of methacrylic anhydride gelatin in the GelMA solution is 0.9% to 2%.

[0013] In some embodiments, in step (1), the concentration of 10-shogaol in the first mixed solution is 30-100 μmol / L.

[0014] In some embodiments, in step (2), the mass volume ratio of the photoinitiator in the second mixed solution is 0.9% to 1.5%.

[0015] In some embodiments, in step (2), the organic solvent is anhydrous ethanol.

[0016] In some embodiments, in step (2), the photoinitiator is selected from at least one of lithium phenyl-2,4,6-trimethylbenzoylphosphonate (LAP) and 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone.

[0017] In some embodiments, methacrylic anhydride gelatin is prepared by the following steps:

[0018] (1) dissolving gelatin in PBS to obtain a third mixed solution;

[0019] (2) adding methacrylic anhydride dropwise to the third mixed solution for reaction, and then adding PBS to obtain a fourth mixed solution;

[0020] (3) dialyzing the fourth mixed solution with a dialysis bag until the mixed solution in the transparent bag becomes transparent;

[0021] (4) Freeze the mixed solution and then freeze-dry it in a vacuum environment to obtain the product.

[0022] Specifically, methacrylic anhydride gelatin is prepared by the following steps:

[0023] (1) dissolving 5-20 g of gelatin in 50-200 mL of PBS at 45-55° C. to obtain a third mixed solution;

[0024] (2) adding 10-30 mL of methacrylic anhydride dropwise to the third mixed solution, reacting for 3-4 hours, and then adding 300-500 mL of PBS to obtain a fourth mixed solution;

[0025] (3) dialyzing the fourth mixed solution using a dialysis bag for 5 to 7 days until the mixed solution in the transparent bag becomes transparent;

[0026] (4) Freeze the mixture at -30 to -20°C for 8 to 12 hours, and then freeze-dry it in a vacuum environment at a temperature of -50 to -60°C to obtain the product.

[0027] In some embodiments, in step (3), the dialysis treatment step includes: placing the fourth mixed solution into a dialysis bag, filling the dialysis bag with pure water, and replacing the pure water every day during the dialysis treatment. Specifically, the pure water is replaced four times a day on the first and second days of the dialysis treatment, three times a day on the third and fourth days of the dialysis treatment, and one to two times a day on the fifth to seventh days of the dialysis treatment.

[0028] According to a second aspect of the present invention, there is provided use of a hydrogel loaded with 10-shogaol in preparing a medicament for treating diabetic wounds.

[0029] The hydrogel loaded with 10-shogaol of the present invention can be used as a dressing for treating diabetic wounds. When applied to diabetic wounds, it can inhibit inflammation, resist bacteria, promote angiogenesis, and promote the healing of diabetic wounds.

[0030] In some embodiments, the drug is a drug having one or more of the following effects: a) inhibiting inflammation; b) antibacterial; c) promoting angiogenesis; d) promoting diabetic wound healing.

[0031] In some embodiments, a hydrogel loaded with 10-shogaol is used in the preparation of a dressing for promoting diabetic wound healing.

[0032] In some embodiments, the drug is a drug having one or more of the following effects: a) promoting collagen deposition in diabetic wound tissue; b) increasing vascular density in diabetic wound tissue; c) inhibiting inflammation in diabetic wound tissue; d) promoting repair of diabetic wound tissue.

[0033] In some embodiments, the drug is a drug that inhibits the expression level of at least one of Tnfa mRNA, Il1b mRNA, and Il6 mRNA.

[0034] In some embodiments, the drug is a drug that increases the expression level of at least one of Vegfa mRNA, CD31 mRNA, and Acta2 mRNA in diabetic wounds.

[0035] The beneficial effects of the present invention are:

[0036] The preparation method of the 10-shogaol-loaded hydrogel of the present invention is simple. The 10-shogaol-loaded hydrogel is prepared by ultraviolet curing. The hydrogel not only combines the biological activity of 10-shogaol with the performance of the hydrogel, but also has high transparency, a smooth surface, and stable water retention performance.

[0037] The 10-shogaol-loaded hydrogel of the present invention can be used to prepare a drug for treating diabetic wounds. The drug targets the pathological characteristics of diabetic wounds and has the effects of inhibiting inflammation, resisting bacteria, promoting angiogenesis, and promoting diabetic wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 (A) to (C) are the TEM characterization results of Sho-L@Gel of Example 3, Sho-H@Gel of Example 4, and GelMA hydrogel of Comparative Example 2; Figure 1 (D) Figure 1 (F) Figure 1 (H) Transparency test of Sho-L@Gel of Example 1, Sho-H@Gel of Example 2, and GelMA hydrogel of Comparative Example 1; Figure 1 (E) Figure 1 (G) Figure 1 (I) is a picture of the Sho-L@Gel of Example 3, the Sho-H@Gel of Example 4, and the GelMA hydrogel of Comparative Example 2;

[0039] Figure 2 (B) is the expansion coefficient evaluation result; Figure 2 (C) is the water retention performance evaluation result;

[0040] Figure 3 The mechanical properties test results of different hydrogels are shown in Figure 2. Figure 3 (D) is the stress-strain curve, Figure 3 (E) is a statistical graph of Young's modulus;

[0041] Figure 4 (A) is the result of cytoskeleton staining; Figure 4 (B) is the staining result of cell survival; Figure 4(C) shows the cell viability test results of RAW264.7; Figure 4 (D) is the result of cell viability experiment of HUVEC cells;

[0042] Figure 5 The hemolysis conditions and hemolysis rates of different concentrations of 10-shogaol;

[0043] Figure 6 (A) to (D) are the mRNA expression levels of Tnfa, Il6, Il1b, and Nos2, respectively;

[0044] Figure 7 (A) is a picture of the plate after 24 h of culture; Figure 7 (B) is the colony count;

[0045] Figure 8 (A) The wound healing process of each group; Figure 8 (B) Wound healing rate at different times; Figure 8 (C) shows the angiogenesis status of each group at 8 h; Figure 8 (D) is the result of node number calculation; Figure 8 (E) is the result of connection number calculation; Figure 8 (F) is the calculation result of the number of branches;

[0046] Figure 9 (A) Diabetic wound intervention and experimental process; Figure 9 (B) Pictures of the wounds of rats in each group at different times; Figure 9 (C) The wound areas of rats in each group at different times;

[0047] Figure 10 HE staining results, including panoramic images with a scale bar of 2000 μm and magnified images with a scale bar of 200 μm;

[0048] Figure 11 Masson staining results, including panoramic images with a scale bar of 800 μm and magnified images with a scale bar of 200 μm;

[0049] Figure 12 (A) Immunofluorescence staining results of inflammatory factors, including a panoramic image with a scale bar of 200 μm and a magnified image of 50 μm; Figure 12 (B) is the expression level of Tnfa mRNA, Il1b mRNA, and Il6 mRNA;

[0050] Figure 13 (A) Immunofluorescence staining results of VEGF and CD31, including the panoramic image with a scale bar of 200 μm and the magnified image of 50 μm; Figure 13 (B) Vegfa mRNA levels in diabetic wound tissues on day 3; Figure 13(C) CD31 mRNA levels in diabetic wound tissues on day 3; Figure 13 (D) Vegfa mRNA levels in diabetic wound tissues on day 8; Figure 13 (E) CD31 mRNA levels in diabetic wound tissues on day 8; Figure 13 (F) shows the immunofluorescence staining results of α-SMA, including a panoramic image with a scale bar of 200 μm and a magnified image with a scale bar of 50 μm. DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below with reference to the accompanying drawings, but the embodiments of the present invention are not limited thereto. The raw materials and reagents involved in the following examples can all be obtained from commercial channels.

[0052] 10-Zingiberol, molecular formula is C 21 H 32 O3, the structural formula is shown below:

[0053]

[0054] Example 1

[0055] This embodiment provides a method for preparing a hydrogel loaded with 10-shogaol, comprising the following steps:

[0056] 1. Preparation of methacrylic anhydride gelatin (GelMA)

[0057] (1) Weigh 10 g of gelatin into a 1 L round-bottom flask, add 100 mL of PBS, and then immerse the flask in a 50°C oil bath and stir with a magnetic stirrer to dissolve.

[0058] (2) 15 mL of methacrylic acid anhydride (MA; Sigma, 276685) was placed in a constant pressure funnel and added dropwise to the round-bottom flask through the funnel at a uniform rate. The reaction was continued for approximately 3 h.

[0059] (3) Add 400 mL of PBS to terminate the reaction, pour the mixture into the prepared dialysis bag (White Shark Bio, BS-QT-019), clamp the opening with a clamp, place the dialysis bag in a 5 L measuring cup, fill it with pure water, and dialyze at room temperature for 7 days, changing the water every day (4 times a day on the 1st and 2nd day, 3 times a day on the 3rd and 4th day, and 1-2 times a day on the 5th to 7th day);

[0060] (4) When the mixed solution in the dialysis bag becomes transparent, remove the dialysis bag and divide the mixed solution into 50 mL centrifuge tubes. Freeze at -20°C overnight and then freeze-dry in a vacuum freeze dryer at -60°C to obtain GelMA. Store the GelMA in a desiccator until use.

[0061] It should be noted that the dialysis bag needs to be boiled for 10 minutes to activate it before use, and tap water should be added to check for leaks.

[0062] 2. Preparation of 10-shogaol-loaded hydrogel

[0063] (1) GelMA was weighed and placed in a beaker, and an appropriate amount of PBS was added to the beaker. The GelMA was dissolved by heating and stirring at 50°C to prepare a GelMA solution with a mass volume ratio of 1%; 10-shogaol was then added to the GelMA solution so that the final concentration of 10-shogaol in the solution was 40 μmol / L, thereby obtaining a first mixed solution;

[0064] (2) dissolving a photoinitiator, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, in anhydrous ethanol to obtain a photoinitiator-anhydrous ethanol with a mass volume ratio of 10%, then adding the photoinitiator-anhydrous ethanol to the first mixed solution (the volume ratio of the photoinitiator-anhydrous ethanol to the first mixed solution is 1:10), stirring and mixing in the dark to obtain a second mixed solution;

[0065] (3) Using a pipette, transfer the second mixed solution to a hydrogel mold with a diameter of 1 cm and a depth of 0.5 mm or 8 mm. Place the mold in a fully functional UV curing chamber for curing to obtain a solid 10-shogaol-loaded hydrogel, designated Sho-L@Gel. Remove the Sho-L@Gel from the mold and store it in a refrigerator at 4°C, away from light, to maintain freshness.

[0066] Example 2

[0067] This embodiment provides a method for preparing a hydrogel loaded with 10-shogaol, comprising the following steps:

[0068] 1. Preparation of methacrylic anhydride gelatin (GelMA)

[0069] (1) Weigh 10 g of gelatin into a 1 L round-bottom flask, add 100 mL of PBS, and then immerse the flask in a 50°C oil bath and stir with a magnetic stirrer to dissolve.

[0070] (2) 15 mL of methacrylic anhydride was placed in a constant pressure funnel and added dropwise to the round-bottom flask through the funnel at a uniform rate. The reaction was continued for approximately 3 h.

[0071] (3) Add 400 mL of PBS to terminate the reaction, pour the mixture into the prepared dialysis bag, clamp the opening with a clamp, place the dialysis bag in a 5 L measuring cup, fill it with pure water, and dialyze at room temperature for 7 days, changing the water every day (4 times a day on the 1st and 2nd day, 3 times a day on the 3rd and 4th day, and 1-2 times a day on the 5th to 7th day);

[0072] (4) When the mixed solution in the dialysis bag becomes transparent, remove the dialysis bag and divide the mixed solution into 50 mL centrifuge tubes. Freeze at -20°C overnight and then freeze-dry in a vacuum freeze dryer at -60°C to obtain GelMA. Store the GelMA in a desiccator until use.

[0073] It should be noted that the dialysis bag needs to be boiled for 10 minutes to activate it before use, and tap water should be added to check for leaks.

[0074] 2. Preparation of 10-shogaol-loaded hydrogel

[0075] (1) GelMA was weighed and placed in a beaker, and an appropriate amount of PBS was added to the beaker. The GelMA was dissolved by heating and stirring at 50°C to prepare a 1% GelMA solution. 10-Zingiol was then added to the GelMA solution so that the final concentration of 10-Zingiol in the solution was 80 μmol / L, thereby obtaining a first mixed solution.

[0076] (2) dissolving a photoinitiator, 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone, in anhydrous ethanol to obtain a photoinitiator-anhydrous ethanol with a mass volume ratio of 10%, then adding the photoinitiator-anhydrous ethanol to the first mixed solution (the volume ratio of the photoinitiator-anhydrous ethanol to the first mixed solution is 1:10), stirring and mixing in the dark to obtain a second mixed solution;

[0077] (3) Using a pipette, the second mixed solution was transferred to a hydrogel mold with a diameter of 1 cm and a depth of 0.5 mm. The mold was placed in a fully functional UV curing chamber for curing to obtain a solid 10-shogaol-loaded hydrogel, designated Sho-H@Gel. The Sho-H@Gel was removed from the mold and placed in a 4°C refrigerator to protect from light and store temporarily.

[0078] Example 3

[0079] This example provides a method for preparing a hydrogel loaded with 10-shogaol. The difference from Example 1 is that the hydrogel mold used has a diameter of 1 cm and a depth of 8 mm, and is denoted as Sho-L@Gel.

[0080] Example 4

[0081] This example provides a method for preparing a hydrogel loaded with 10-shogaol. The difference from Example 2 is that the hydrogel mold used has a diameter of 1 cm and a depth of 8 mm, and is denoted as Sho-H@Gel.

[0082] Comparative Example 1

[0083] This comparative example provides a method for preparing GelMA hydrogel, comprising the following steps:

[0084] (1) Weigh 10 g of gelatin into a 1 L round-bottom flask, add 100 mL of PBS, and then immerse the flask in a 50°C oil bath and stir with a magnetic stirrer to dissolve.

[0085] (2) 15 mL of methacrylic anhydride was placed in a constant pressure funnel and added dropwise to the round-bottom flask through the funnel at a uniform rate. The reaction was continued for approximately 3 h.

[0086] (3) Add 400 mL of PBS to terminate the reaction, pour the mixture into the prepared dialysis bag, clamp the opening with a clamp, place the dialysis bag in a 5 L measuring cup, fill it with pure water, and dialyze at room temperature for 7 days, changing the water every day (4 times a day on the 1st and 2nd day, 3 times a day on the 3rd and 4th day, and 1-2 times a day on the 5th to 7th day);

[0087] (4) When the mixed solution in the dialysis bag becomes transparent, remove the dialysis bag and divide the mixed solution into 50 mL centrifuge tubes. Freeze at -20°C overnight and then freeze-dry in a vacuum freeze dryer at -60°C to obtain GelMA. Store the GelMA in a desiccator until use.

[0088] 2. Preparation of GelMA Hydrogel

[0089] (1) Weigh GelMA and place it in a beaker. Add an appropriate amount of PBS to the beaker and heat and stir at 50°C to dissolve the GelMA to prepare a 1% GelMA solution.

[0090] (2) dissolving the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone in anhydrous ethanol to obtain a photoinitiator-anhydrous ethanol with a mass volume ratio of 10%, then adding the photoinitiator-anhydrous ethanol to the GelMA solution (the volume ratio of the photoinitiator-anhydrous ethanol to the first mixed solution is 1:10), stirring and mixing in the dark to obtain a mixed solution;

[0091] (3) Use a pipette to transfer the mixed solution to a hydrogel mold with a diameter of 1 cm and a depth of 0.5 mm. Place the mold in a fully functional UV curing box for curing to obtain GelMA hydrogel. Remove the GelMA hydrogel from the mold and place it in a 4°C refrigerator to avoid light, keep it fresh, and store it temporarily.

[0092] Comparative Example 2

[0093] This comparative example provides a method for preparing GelMA hydrogel, which differs from comparative example 1 in that the diameter of the hydrogel mold used is 1 cm and the depth is 8 mm.

[0094] 1. Hydrogel Appearance and Characterization

[0095] The Sho-L@Gel of Example 1, the Sho-H@Gel of Example 2, and the GelMA hydrogel of Comparative Example 1 were placed on a paper printed with black words, and photographed to observe their transparency. Figure 1 As shown in (D), (F), and (H), it can be observed that the hydrogels loaded with 10-shogaol and GelMA at different concentrations at a depth of 0.5 mm have high transparency, and the black words on the paper can be clearly seen through the hydrogels.

[0096] The Sho-L@Gel of Example 3, the Sho-H@Gel of Example 4, and the GelMA hydrogel of Comparative Example 2 were placed on pure white paper, photographed, and their overall morphology and color were observed. Figure 1 As shown in (E), (G), and (I), it can be observed that the hydrogels at a depth of 8 mm all have smooth surfaces, regular cylindrical shapes, and bright colors.

[0097] The freeze-dried Sho-L@Gel of Example 3, Sho-H@Gel of Example 4, and GelMA hydrogel of Comparative Example 2 were carefully placed in liquid nitrogen for quick freezing and brittle fracture. The flat cross-sections were selected for scanning electron microscope (SEM) characterization at 400 times magnification. The results are as follows: Figure 1 As shown in (A) to (C), Sho-L@Gel, Sho-H@Gel, and GelMA hydrogels all have clear pore structures, which are conducive to the air permeability of the hydrogel and the absorption of wound exudate.

[0098] 2. Evaluation of hydrogel expansion coefficient and water retention performance

[0099] 1. Expansion coefficient evaluation: Take out the Sho-L@Gel of Example 3, Sho-H@Gel of Example 4, and GelMA hydrogel of Comparative Example 2 from a 4°C refrigerator, freeze them in a -80°C refrigerator overnight, and freeze-dry them in a vacuum freeze dryer the next day. Weigh the freeze-dried hydrogel and record it as W0. Then, place it in PBS at room temperature and weigh it after 2, 4, 6, 8, 10, 20, 30, 40, 50, and 60 hours, respectively, and record it as W t When weighing, use gauze to absorb the water on the surface of the hydrogel and put it back into PBS after weighing. Calculate the expansion coefficient according to the formula:

[0100] Expansion coefficient (%) = (W t -W0) / W0×100.

[0101] 2. Water retention performance evaluation: Fresh Sho-L@Gel, Sho-H@Gel and GelMA hydrogels were prepared according to the preparation methods of Example 3, Example 4 and Comparative Example 2, respectively, and weighed. The initial weight was recorded as W0. The hydrogels were placed on gauze and left at room temperature. The weights were weighed after 1, 6, 12, 24, 36, 48 and 60 hours, respectively, and recorded as W. t Calculate the water retention rate according to the formula:

[0102] Water retention rate (%) = 100-(W t -W0) / W0×100.

[0103] 3. Results

[0104] The expansion coefficient evaluation results are shown in Figure 2 (B), Sho-H@Gel has the largest expansion coefficient, while Sho-L@Gel and GelMA hydrogels have similar expansion coefficients, indicating that the increase of 10-shogaol concentration increases the expansion coefficient of hydrogels. Figure 2 (C) The water retention rates of Sho-L@Gel, Sho-H@Gel, and GelMA hydrogels are almost the same, indicating that the water retention properties of the hydrogels are relatively stable and are less affected by the concentration of 10-shogaol.

[0105] 3. Mechanical properties of hydrogels

[0106] Fresh Sho-L@Gel, Sho-H@Gel and GelMA hydrogels were prepared according to the preparation methods of Example 3, Example 4 and Comparative Example 2. The Young's modulus of the hydrogels was tested using an INSTRON 3400 series dual-column electronic tensile testing machine (Instron, 34TM-30).

[0107] Instrument operation: Replace the appropriate module according to the pressure range that the hydrogel can withstand, and adjust the safety ruler to 8mm to prevent the module from hitting the base; open the software, set the parameters such as hydrogel thickness, force (N), displacement (mm), module downward movement speed, and create a new experiment; place the sample in the center of the base, adjust the instrument to zero, operate the instrument to slowly move the module downward until the hydrogel ruptures, and export the data as a CSV file; draw a stress-strain curve with compressive strain (Strain) as the x-axis and compressive stress (Stress) as the y-axis; calculate the Young's modulus according to the formula Young's modulus (Kpa) = Stress / Strain × 100% (select the data corresponding to the linear range of Stress 10% to 20% for calculation).

[0108] Mechanical properties test results are shown in Figure 3 , Figure 3(D) shows the stress-strain curve, and (E) shows the Young's modulus. The results show that Sho-L@Gel, Sho-H@Gel, and GelMA hydrogels exhibit excellent mechanical properties. Specifically, GelMA hydrogels without 10-shogaol loading exhibited greater compressive strength than Sho-L@Gel and Sho-H@Gel, indicating that the addition of 10-shogaol reduces the hydrogel's compressive strength. As the 10-shogaol concentration increases, the hydrogel's mechanical properties decrease.

[0109] It should be noted that the 10-shogaol-loaded hydrogels of Examples 1 and 2 have a slow release effect. In order to achieve the same effect of the drug in the 10-shogaol-loaded hydrogel at the cellular level as that of the drug at the cellular level, the concentration of the 10-shogaol-loaded hydrogel in Example 1 (40 μmol / L) and the concentration of Example 2 (80 μmol / L) were reduced by 10 times, that is, 4 μmol / L and 8 μmol / L were selected as the concentrations of 10-shogaol in the following in vitro cell and antibacterial experiments, thereby realizing the observation and detection of the efficacy of the drug (10-shogaol) itself.

[0110] 4. Effects of 10-gingerol on the cytoskeleton and cell survival.

[0111] 1. Drug intervention

[0112] Human umbilical vein endothelial cells (HUVEC) were divided into Control group, Sho-L group and Sho-H group. The Control group served as the blank control group, and the Sho-L and Sho-H groups served as the drug-treated groups. The Sho-L group was added with 4 μmol / L 10-shogaol; the Sho-H group was added with 8 μmol / L 10-shogaol.

[0113] HUVECs were seeded in confocal dishes. When the density reached 60% to 70%, drugs were added according to the above groups for intervention for 12 hours or 24 hours.

[0114] 2. Cytoskeleton

[0115] Drug intervention was performed according to the above operation. After the end, the cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 for 5 minutes, blocked with 1% BSA for 30 minutes, stained with FITC-conjugated phalloidin (Yisheng Bio, 40735ES75; diluted 200 times with 1% BSA) in the dark for 30 minutes, and counterstained with Hoechst33342 (1:1000; ThermoFisher, 62249) for 20 minutes. Finally, the cells were washed with PBS and immersed in PBS, and photographed and recorded using a laser confocal oil immersion lens.

[0116] 3. Cell survival

[0117] Drug intervention was performed according to the above procedures. After the end, a buffer solution containing Calcein AM (1 / 1000; staining live cells; green) and PI (1 / 1000; staining dead cells; red) was prepared according to the Calcein / PI Cell Viability and Cytotoxicity Assay Kit (Biyuntian, C2015S). The cell culture medium was replaced and incubated in a cell culture incubator for 30 minutes. The cells were then washed with PBS and replaced with complete culture medium again. The cells were photographed and recorded using an inverted fluorescence microscope.

[0118] 4. Results

[0119] The results of cytoskeleton staining were as follows Figure 4 (A) shows the cell survival staining results. Figure 4 (B) shown.

[0120] from Figure 4 (A) It can be seen that compared with the blank control group, the Sho-L group and the Sho-H group had no significant damage to the HUVEC cytoskeleton after 12h and 24h intervention. Figure 4 (B) It can be seen that after 12h and 24h intervention, the Sho-L group and the Sho-H group had no significant effect on cell survival. The above results indicate that 10-gingerol has good safety for cells.

[0121] 5. Effect of 10-shogaol on cell viability

[0122] 1. Experimental methods

[0123] The cells were divided into control group, experimental group and control group. In the experimental group, 10-shogaol was added to complete culture medium to obtain final concentrations of 10-shogaol of 2, 4, 8, 16 and 32 μmol / L; 10-shogaol was not added to the control group, and neither 10-shogaol nor CCK-8 solution was added to the control group.

[0124] RAW264.7 and HUVEC cells were cultured at 1×10 4 and 5×10 4 The cells were seeded at a density of 10 cells / well in a 96-well culture plate and cultured for 24 hours; after 24 hours, the culture medium was discarded, the cells were washed with PBS, and 10-shogaol was added according to the above grouping and treated for 24 hours; the culture medium was discarded, the cells were washed with PBS, and 90 μL of complete culture medium and 10 μL of CCK-8 solution were added to each well, incubated in an incubator for 2 to 4 hours, and then the OD value at 450 nm was detected using a microplate reader (BioTek, SYNERGY H1).

[0125] Calculate cell viability according to the formula:

[0126] Cell viability (%) = (OD A -OD空白 ) / (OD 阴性 -OD 空白 )×100, where OD A OD value measured for the experimental group, OD 空白 OD value measured for the Control group, OD 阴性 is the OD value measured for the control group.

[0127] 2. Results

[0128] The results of the RAW264.7 cell viability experiment are as follows Figure 4 (C) As shown in the results of the cell viability experiment of HUVEC cells, Figure 4 As shown in (D), it can be seen that 10-shogaol at a concentration below 16 μmol / L has no obvious toxicity to RAW264.7 cells, and 10-shogaol at a concentration below 32 μmol / L has no obvious toxicity to HUVEC cells.

[0129] VI. Effect of 10-shogaol on hemolysis rate

[0130] 1. Experimental methods

[0131] Prepare 10 mL containing 1×10 4 Unit of sodium heparin PBS was added, and 2 mL of blood was collected from the rat's orbital cavity and dripped into it, and mixed by inverting. The diluted blood was centrifuged at 1000 rpm for 10 minutes to separate red blood cells (RBCs). Next, the RBCs were washed three times with PBS and finally resuspended in PBS. Then, 750 μL of diluted RBCs were transferred to a new 2 mL centrifuge tube, and an equal volume of 10-gingerol at different concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 μmol / L) was added, mixed, and incubated at 37°C for 3 hours. RBCs incubated with ultrapure water served as a positive control, and RBCs incubated with PBS served as a negative control. Finally, the treated centrifuge tube was centrifuged at 1000 rpm for 15 minutes, the supernatant was separated, and the OD value at 540 nm was measured using a microplate reader (BioTek, SYNERGY H1).

[0132] The formula for calculating the hemolysis rate is:

[0133] Hemolysis rate (%) = (A p -A b ) / (A t -A b )×100, where A p 、A t 、A b are the OD values of drug, positive control, and negative control, respectively.

[0134] 2. Results

[0135] The hemolysis and hemolysis rate of different concentrations of 10-gingerol are as follows Figure 5 As shown. Figure 5 It can be seen that the centrifuge tubes with different concentrations of 10-shogaol added are light in color and have no obvious hemolysis phenomenon, indicating that 10-shogaol has good biocompatibility.

[0136] VII. Regulation of 10-shogaol on inflammatory factors in macrophages

[0137] 1. Experimental methods

[0138] RAW264.7 cells were seeded in 12-well culture plates. When the cell density reached 60%-70%, fresh serum-free medium was replaced. The cells were divided into control, LPS, Sho-L, and Sho-H groups. The control and LPS groups were not treated, 4 μmol / L shogaol was added to the Sho-L group, and 8 μmol / L shogaol was added to the Sho-H group. The cells were cultured for 12 h. Then, serum-free medium was replaced, and 100 ng / mL LPS was added to the LPS, Sho-L, and Sho-H groups to stimulate the cells for 4 h. Finally, serum-free medium was replaced, and the control and LPS groups were not treated. The Sho-L and Sho-H groups were treated with the same drugs for a further 6 h, and then the cells were harvested. Total cellular RNA was extracted using an RNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd., DP419), and reverse transcribed into cDNA using the PrimeScript RT reagent Kit with gDNA Eraser Perfect Real Time (Takara, Japan). Using cDNA as a template, SYBR Green qPCR Mix (Hunan Aikerui Biological) and gene-specific primers were used to prepare the system, mixed well, and qRT-PCR was performed on an ABI ViiATM 7 System instrument (Thermo Life, ViiA7). -ΔΔCT Methods: Relative mRNA expression levels of genes were calculated, with α-Tublin as an internal reference. The specific primers used are shown in Table 1.

[0139] Table 1 Specific primers

[0140]

[0141]

[0142] 2. Results

[0143] The expression levels of inflammatory factors in RAW264.7 cells induced by LPS are shown in Figure 6(A)-(D). As can be seen from the figure, different concentrations of 10-shogaol can inhibit the LPS-induced upregulation of inflammatory factors Tnfa, Il1b, Il6, and Nos2 mRNA in RAW264.7 cells. Compared with the control group without LPS induction, the mRNA expression levels of Tnfa, Il1b, Il6, and Nos2 in the Sho-L group and the Sho-H group increased significantly. * P<0.05, which was statistically significant, indicating that LPS induction increased the expression level of inflammatory factors. Compared with the LPS group, the expression levels of Tnfa, Il1b, Il6, and Nos2 in the Sho-L and Sho-H groups were significantly reduced. # P<0.05 was statistically significant, indicating that 10-shogaol had good anti-inflammatory activity.

[0144] 8. Inhibitory Effect of 10-Zingiol on Staphylococcus aureus

[0145] 1. Experimental methods

[0146] S. aureus was provided by Professor Wu Jun's team from the Hong Kong University of Science and Technology (Guangzhou).

[0147] Activate the bacteria: Take out the bacterial solution from the -20℃ freezer, thaw it, add it to 20mL of sterilized LB liquid medium at a ratio of 1:200, and culture it on a shaker (37℃, 220rpm) for 4 hours; activate the cultured bacterial solution a second time, i.e., activate it in the same way at a ratio of 1:200. Then, dilute the activated bacterial solution with liquid LB medium to 1×10 6 times.

[0148] The diluted bacterial solution was treated and divided into control group, Sho-L group and Sho-H group. Among them, the control group did not add drugs; the Sho-L group added 10-shogaol to make the final concentration of 10-shogaol in the bacterial solution reach 4 μmol / L; the Sho-H group added 10-shogaol to make the final concentration of 10-shogaol in the bacterial solution reach 8 μmol / L.

[0149] After the bacterial solution was treated, it was mixed and then dispensed into 96-well plates, 200 μL per well, 3 replicates per group, and placed in a 37°C biochemical incubator for 12 hours. Then, the bacterial solution in the plate was further diluted to 1×10 6 Dilution times, transfer 100 μL to a plate containing LB solid medium using a pipette, spread evenly, and return to a 37°C biochemical incubator for inversion culture for 24 hours. Finally, take a photo and count the colonies, and calculate the number of colonies in the original 96-well plate based on the dilution times.

[0150] 2. Results

[0151] See the plate picture after 24h of culture. Figure 7 (A), colony counts are shown in Figure 7 (B). From Figure 7 (A) It can be seen that the number of Staphylococcus aureus in the plates treated with different concentrations of 10-shogaol was significantly reduced. Figure 7 (B) It can be seen that after counting the number of colonies, the number of colonies in the plates of the Sho-L and Sho-H groups was significantly lower than that of the Control group. * P<0.05 was statistically significant, indicating that 10-gingerol had good antibacterial activity.

[0152] IX. Regulatory Effects of 10-Shogaol on Endothelial Wound Healing and Angiogenesis

[0153] 1. Assessment of vascular endothelial wound healing

[0154] HUVEC cells were seeded in 6-well plates and divided into control, Sho-L, and Sho-H groups. The control group served as a blank control group, while the Sho-L and Sho-H groups served as drug-treated groups. When the density reached 100%, a 200 μL yellow pipette tip was used to move vertically across the bottom of the well plate to form a scratch (wound), which was then photographed and recorded. The wound area was calculated using ImageJ and recorded as A0, with the time being recorded as 0 h. Subsequently, 4 μmol / L 10-shogaol was added to the Sho-L group and 8 μmol / L 10-shogaol was added to the Sho-H group. The scratches were photographed and recorded at 12, 24, 36, and 48 h, respectively, and the wound area was recorded as A t ; Summarize the wound healing status and calculate the wound healing rate according to the following formula:

[0155] Wound healing rate (%) = A t / A0×100.

[0156] 2. Angiogenesis Assessment

[0157] Matrigel (Corning, 354277) was taken out from -20℃ and thawed on ice. After thawing, 10μL was added to the wells of μ-Plate96Well 3D ibiTreat (ibidi, 89646), spread flat, and placed at 37℃ for 30 minutes to solidify. Subsequently, HUVEC cell suspension was prepared and diluted. 10-shogaol was added to the Sho-L group to make the final concentration of 10-shogaol in the cell suspension 4μmol / L, and 10-shogaol was added to the Sho-H group to make the final concentration of 10-shogaol in the cell suspension 8μmol / L. 100μL of cell suspension (containing 10 4The cells were added to the solidified matrix gel and carefully placed back into the incubator. After 8 h, photos were taken and recorded, and angiogenesis indicators, including the number of nodes (Nb nodes), the number of connections (Nb junctions), and the number of branches (Nbbranches), were analyzed using ImageJ.

[0158] 3. Results

[0159] The wound healing process of each group is shown in Figure 8 (A), Wound healing rates at different times are shown in Figure 8 (B) As can be seen from the figure, the wounds of the Sho-L and Sho-H groups were completely healed at 48 hours, while the wounds of the Control group had not yet healed, indicating that 10-shogaol can promote the healing of vascular endothelial cell wounds.

[0160] The vascular status of each group at 8 hours is shown in Figure 8 (C) It can be seen that the vascular density of the Sho-L and Sho-H groups is higher than that of the Control group. The calculation results of the number of nodes, connections and branches are shown in Figure 2. Figure 8 As shown in (D) to (F), it can be seen that the number of nodes, connections, and branches in the Sho-L and Sho-H groups were significantly increased compared with the Control group, indicating that 10-gingerol can significantly promote angiogenesis, which lays the foundation for its treatment of diabetic wounds.

[0161] 10. Effect of 10-shogaol-loaded hydrogel on diabetic wound healing

[0162] 1. Establishment of diabetic wound model in rats

[0163] Wild-type male Sprague-Dawley rats (8 weeks old, 200 ± 20 g) were purchased from Guangdong Weitonglihua Company and housed in an SPF environment at the Animal Experimental Center of Guangdong Academy of Chinese Medicine with a 12-h light-dark cycle. Formal experiments began after one week of adaptive feeding. The license number is SYXK (Yue) 2018-0094, and the ethics number is 2023017.

[0164] Preparation of streptozotocin (STZ): first prepare citric acid buffer (weigh 2.1 g powder and dissolve it in 100 mL ultrapure water), then prepare trisodium citrate buffer (weigh 2.94 g powder and dissolve it in 100 mL ultrapure water), mix the citric acid buffer and trisodium citrate buffer in a volume ratio of 1:1.32 to obtain a 0.1 mol / L buffer, weigh STZ powder and dissolve it in it to make the final STZ concentration of 10 mg / mL, filter the filtrate through a 0.22 μm filter, and store it in a 4°C refrigerator away from light for no more than 12 h.

[0165] SD rats were fasted for 12 hours and then intraperitoneally injected with the STZ solution at a concentration of 55 mg / kg. Blood glucose was measured 5 days later. A blood glucose concentration >16.7 mmol / L indicated successful establishment of a type 1 diabetic rat model. The diabetic rats were anesthetized, and the hair on their backs was removed. A 1 cm diameter circle was drawn on the left or right side of the spine. The skin was carefully cut along the trajectory of the circle with scissors to create a diabetic wound.

[0166] 2. Diabetic Wound Intervention

[0167] Rats with diabetic wounds were randomly divided into the control group, the GelMA group, the Sho-L@Gel group, and the Sho-H@Gel group, with 5 rats in each group. The control group served as the blank control group, and the GelMA group, the Sho-L@Gel group, and the Sho-H@Gel group served as the drug-treated groups.

[0168] According to the above grouping, rats in each group were anesthetized. After the rats fell into a coma, the hydrogels taken out from the 4°C refrigerator were carefully applied to the wound sites of the rats in the drug-treated group with tweezers. The hydrogel in the GelMA group was the GelMA hydrogel prepared in Comparative Example 1, the hydrogel in the Sho-L@Gel group was the Sho-L@Gel prepared in Example 1, and the hydrogel in the Sho-H@Gel group was the Sho-H@Gel prepared in Example 2. The control group was not treated, and this time was recorded as day 0. The drug-treated groups were subsequently treated with hydrogel patches on days 2, 4, 6, 8, 10, 12, and 14. Diabetic wound intervention and experimental process are shown in [1]. Figure 9 (A).

[0169] 3. Effects of 10-gingerol-loaded hydrogel on diabetic wounds

[0170] The wounds of rats in each group were photographed on days 0, 2, 6, 8, and 12. Figure 9 (B) shows the area of the wound, and the results are as follows Figure 9 (C) is shown. Figure 9 (B) It can be observed that the wound areas of the rats in the Sho-L@Gel and Sho-H@Gel groups were smaller than those in the Control and GelMA groups on days 2, 6, 8, and 12 after administration, indicating that the wounds of the rats healed faster. Figure 9 (C) shows the wound area of rats at different time points. It can be seen that the wound area of rats in the Sho-L@Gel group and Sho-H@Gel group at different time points was significantly lower than that in the GelMA group. * P<0.05 was statistically significant, indicating that the hydrogel loaded with 10-gingerol significantly promoted the healing of diabetic wounds in rats.

[0171] 4. Effects of 10-gingerol-loaded hydrogel on diabetic wound tissue pathology

[0172] The effect of 10-gingerol-loaded hydrogel on diabetic wound tissue pathology was evaluated by HE staining.

[0173] HE staining: On the 3rd, 8th, and 14th days, the diabetic wound tissues of rats in each group were cut, flattened and placed in embedding boxes, and then fixed with 4% paraformaldehyde (>24 h) for histopathological staining. The sections were sliced at a thickness of 4 μm, and the slides were picked up and dried. The sections were stained using a HE staining kit (Biyuntian Biotechnology, C0105M), sealed with neutral resin, and then images were collected and analyzed using a digital slide scanner (PanoVIEW VS200).

[0174] HE staining results are shown in Figure 10 Panoramic and magnified images show that diabetic wound tissue becomes denser over time. The Sho-H@Gel group showed the best recovery on days 3, 8, and 14, with hair follicles beginning to form on day 14. The Sho-L@Gel group showed second-best recovery after the Sho-H@Gel group, with wound recovery significantly faster than the Control and GelMA groups. These results demonstrate that 10-gingerol-loaded hydrogels can significantly promote pathological repair of diabetic wound tissue.

[0175] 5. Effect of 10-gingerol-loaded hydrogel on collagen deposition in diabetic wounds

[0176] The effect of 10-gingerol-loaded hydrogel on collagen deposition in diabetic wounds was evaluated by Masson staining.

[0177] Masson staining: On the 3rd, 8th, and 14th days, the diabetic wound tissues of rats in each group were cut, flattened and placed in embedding boxes, and then fixed with 4% paraformaldehyde (>24h) for histopathological staining. The sections were sliced at a thickness of 4μm, and the slides were picked up and dried. M staining was performed using a Masson staining kit (Solebo, G1340), and the sections were sealed with neutral resin. Then, images were collected and analyzed using a digital slide scanner (PanoVIEW VS200).

[0178] Masson staining results are shown in Figure 11 From the panoramic and magnified images, it can be found that compared with the Control and GelMA groups, the deposition of collagen in the diabetic wound tissue of the Sho-L@Gel and Sho-H@Gel groups on days 8 and 14 was more obvious, and the formation of hair follicles could be observed. The above results indicate that the hydrogel loaded with 10-gingerol can significantly promote the deposition of collagen in diabetic wounds and is beneficial to the healing of diabetic wound tissue.

[0179] 6. Inhibitory effect of 10-gingerol-loaded hydrogel on inflammation of diabetic wound tissue in rats

[0180] The anti-inflammatory effect of 10-gingerol-loaded hydrogel was evaluated by immunofluorescence staining and qRT-PCR experiments.

[0181] Immunofluorescence staining: Diabetic wound tissue sections from each group on day 3 were dewaxed using a fully automatic multifunctional stainer (Leica, ST5020). Sodium citrate antigen retrieval solution (Boster, AR0024; each package of powder was dissolved in 2 L of ultrapure water) was prepared and the dewaxed tissue sections were immersed in water. The sections were placed in a microwave oven, boiled on high heat, then switched to medium heat for 15 min, cooled naturally to room temperature, and then washed three times with PBS for 5 min each. Immunohistochemistry circle pen was used to draw circles around the tissue, 5% BSA was added for blocking for 30 min, and primary antibody dilution was added for incubation at 4°C overnight. The next day, the sections were washed with PBS three times for 5 min each, and fluorescent secondary antibody dilution was added for incubation at room temperature in the dark for 1 h. The PBS washing step was repeated, and the cell nuclei were counterstained with Hoechst33342 solution for 20 min. The sections were mounted with anti-fluorescence decay mounting medium (Solabo, S2100). Finally, images were acquired using a digital slide scanner. The primary antibodies used were inflammatory factors IL-1β (1:800; Seville, GB11113), TNF-α (1:100; Santa Cruz, sc-12744), and MPO (1:300; Wuhan Tri-Tac-3 Biotechnology Co., Ltd., 66177-1-Ig). The secondary antibodies used were donkey anti-rabbit Cy3 (1:200; Biolegend, 406402) or goat anti-mouse Cy3 (1:200; Biolegend, 405309).

[0182] qRT-PCR Experiment: Wound tissue was harvested on days 3, 8, and 14 and stored at -80°C. Before the experiment, mung bean-sized cutaneous wound tissue was removed from the -80°C incubation area and total RNA was extracted using an RNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd., DP419). Reverse transcription, qRT-PCR, and relative gene expression were then calculated according to the protocol described for "10-Zingiol Regulates Inflammatory Factors in Macrophages." β-actin was used as an internal control. The specific primers used are listed in Table 2.

[0183] Table 2 Specific primers for qRT-PCR

[0184]

[0185] Immunofluorescence staining results of inflammatory factors Figure 12As shown in (A), it can be seen that on the third day, the fluorescence signals of inflammatory factors IL-1β, TNF-α, and MPO in the skin tissue of the Sho-L@Gel group and the Sho-H@Gel group were significantly less than those in the Control group and the GelMA group, indicating that the hydrogel loaded with 10-gingerol can inhibit the expression levels of inflammatory factors IL-1β, TNF-α and the inflammatory mediator myeloperoxidase MPO.

[0186] The expression levels of Tnfa mRNA, Il1b mRNA, and Il6 mRNA are shown in Figure 12 (B) It can be seen that compared with the GelMA group, the mRNA expression levels of inflammatory factors Tnfa, Il1b, and Il6 in the Sho-L@Gel group and the Sho-H@Gel group were significantly reduced. * P<0.05 was statistically significant, indicating that the hydrogel loaded with 10-gingerol significantly inhibited the expression of inflammatory factor mRNA and promoted tissue repair by inhibiting inflammation of diabetic wound tissue.

[0187] 7. Effects of 10-gingerol-loaded hydrogel on vascular density and angiogenesis in diabetic wound tissue

[0188] Immunofluorescence staining and qRT-PCR experiments were used to evaluate the effects of 10-gingerol-loaded hydrogel on vascular density and angiogenesis in diabetic wound tissue.

[0189] Immunofluorescence staining: Diabetic wound tissue sections were obtained from each group on days 3 and 8 and dewaxed using a fully automated multifunctional stainer. Sodium citrate antigen retrieval solution was prepared and used to immerse the dewaxed tissue sections in water. The sections were then microwaved, brought to a boil on high, then reduced to medium heat for 15 minutes. The sections were then cooled to room temperature and washed three times with PBS for 5 minutes each. Immunohistochemistry circles were drawn around the tissue using a pen. 5% BSA was added for 30 minutes and the sections were incubated with primary antibody dilution overnight at 4°C. The next day, the sections were washed three times with PBS for 5 minutes each. Fluorescent secondary antibody dilution was added and incubated at room temperature in the dark for 1 hour. The PBS wash step was repeated, and cell nuclei were counterstained with Hoechst 33342 solution for 20 minutes. The sections were then mounted with anti-fluorescence decay mounting media. Finally, images were acquired using a digital slide scanner. Sections from day 3 were stained for vascular endothelial growth factor (VEGF), while sections from day 8 were stained for the endothelial cell marker CD31 and the smooth muscle cell marker α-SMA. The primary antibodies used were VEGF (1:100; Santa Cruz, sc-7269), CD31 (1:500; Seville, GB113151), and α-SMA (1:500; Seville, GB111364). The secondary antibodies used were donkey anti-rabbit Cy3 (1:200; Biolegend, 406402) or FITC (1:200; Biolegend, 406403), and goat anti-mouse FITC (1:200; Biolegend, 405305).

[0190] qRT-PCR Experiment: Wound tissue was harvested on days 3, 8, and 14 and stored at -80°C. Before the experiment, mung bean-sized cutaneous wound tissue was removed from the -80°C chamber and total RNA was extracted using an RNA extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd., DP419). Reverse transcription, qRT-PCR, and relative gene expression were then calculated according to the protocol described in "10-Zingiol Regulates Inflammatory Factors in Macrophages." β-actin was used as an internal control. The specific primers used are listed in Table 3.

[0191] Table 3 Specific primers

[0192]

[0193] The results of immunofluorescence staining of vascular endothelial growth factor VEGF and endothelial cell marker CD31 are shown in Figure 13(A). As can be seen from the figure, for vascular endothelial growth factor VEGF, the Sho-H@Gel group had the highest fluorescence signal, followed by the Sho-L@Gel group, and their fluorescence signals were significantly higher than those of the Control group and the GelMA group. For the vascular endothelial cell marker CD31, the fluorescence signal distribution of the Sho-H@Gel group and the Sho-L@Gel group was similar, and both were significantly higher than those of the Control and GelMA groups. The immunofluorescence staining results of the vascular smooth muscle cell marker α-SMA (encoding gene is Acta2) are shown in Figure 2. Figure 13 As shown in (F), the fluorescence signals of the Sho-H@Gel and Sho-L@Gel groups were higher than those of the Control and GelMA groups. These results indicate that 10-shogaol-loaded hydrogels can enhance the expression of VEGF, CD31, and α-SMA in diabetic wounds.

[0194] On the third day, the Vegfa mRNA level in diabetic wound tissue was Figure 13 (B), CD31 mRNA levels are shown in Figure 13 (C) It can be seen that the Vegfa mRNA level and CD31 mRNA level in the diabetic wound tissue of the Sho-H@Gel group and the Sho-L@Gel group on the 3rd day were significantly higher than those in the other two groups. Figure 13 (D), CD31 mRNA levels are shown in Figure 13 (E) It can be found that the Vegfa mRNA levels and CD31 mRNA levels in the diabetic wound tissue of the Sho-H@Gel group and the Sho-L@Gel group were significantly higher than those in the other two groups on day 8. The Vegfa mRNA levels and CD31 mRNA levels in the Sho-H@Gel group and the Sho-L@Gel group were significantly higher than those in the GelMA group. * P<0.05, statistically significant. Acta2 mRNA levels are shown in Figure 13 (G) As shown in the figure, the Acta2 mRNA levels in the Sho-H@Gel group and the Sho-L@Gel group were significantly higher than those in the Control group and the GelMA group. * P < 0.05, statistically significant. These results indicate that 10-shogaol-loaded hydrogels can increase the expression of Vegfa mRNA, CD31 mRNA, and Acta2 mRNA in diabetic wounds, thereby increasing the expression of VEGF, CD31, and α-SMA. Therefore, 10-shogaol-loaded hydrogels can increase vascular density in diabetic wound tissue, promote angiogenesis, and thus promote wound healing.

[0195] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A hydrogel loaded with 10-shogaol, characterized in that: Prepared by the following steps: (1) dissolving methacrylic anhydride-treated gelatin in PBS to obtain a GelMA solution, and adding 10-shogaol to the GelMA solution to obtain a first mixed solution; (2) dissolving the photoinitiator in an organic solvent to obtain an organic solvent containing the photoinitiator; (3) adding an organic solvent containing a photoinitiator to the first mixed solution, stirring in the dark to obtain a second mixed solution, and then curing the second mixed solution under 300-400 nm ultraviolet light to obtain a second mixed solution.

2. The hydrogel loaded with 10-shogaol according to claim 1, characterized in that In step (1), the mass volume ratio of methacrylic anhydride gelatin in the GelMA solution is 0.9% to 2%, and the concentration of 10-shogaol in the first mixed solution is 30 to 100 μmol / L.

3. The hydrogel loaded with 10-shogaol according to claim 1, characterized in that In step (2), the mass volume ratio of the photoinitiator in the second mixed solution is 0.9% to 1.5%.

4. The hydrogel loaded with 10-shogaol according to claim 1, characterized in that Methacrylic anhydride gelatin is prepared by the following steps: (1) dissolving gelatin in PBS to obtain a third mixed solution; (2) adding methacrylic anhydride dropwise to the third mixed solution for reaction, and then adding PBS to obtain a fourth mixed solution; (3) dialyzing the fourth mixed solution with a dialysis bag until the mixed solution in the transparent bag becomes transparent; (4) Freeze the mixed solution and then freeze-dry it in a vacuum environment to obtain the product.

5. Use of the 10-shogaol-loaded hydrogel according to any one of claims 1 to 4 in the preparation of a drug for treating diabetic wounds.

6. Use of the 10-shogaol loaded hydrogel according to claim 5 in preparing a drug for treating diabetic wounds, characterized in that: The drug has one or more of the following effects: a) inhibiting inflammation; b) antibacterial; c) promoting angiogenesis; d) promoting diabetic wound healing.

7. Use of the 10-shogaol loaded hydrogel according to claim 6 in preparing a drug for treating diabetic wounds, characterized in that: Application of the 10-shogaol-loaded hydrogel in preparing a dressing for promoting diabetic wound healing.

8. Use of the 10-shogaol loaded hydrogel according to claim 6 or 7 in preparing a drug for treating diabetic wounds, characterized in that: The drug has one or more of the following effects: a) promoting collagen deposition in diabetic wound tissue; b) increasing blood vessel density in diabetic wound tissue; c) inhibiting inflammation in diabetic wound tissue; and d) promoting repair of diabetic wound tissue.

9. Use of the 10-shogaol loaded hydrogel according to claim 8 in preparing a drug for treating diabetic wounds, characterized in that: The drug is a drug that inhibits the expression level of at least one of Tnfa mRNA, Il1b mRNA, and Il6 mRNA.

10. Use of the 10-shogaol loaded hydrogel according to claim 8 in preparing a drug for treating diabetic wounds, characterized in that: The medicine is a medicine for increasing the expression level of at least one substance among Vegfa mRNA, CD31 mRNA and Acta2 mRNA in diabetic wounds.

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