Deer blood peptide hydrogel and application thereof in preparation of medicine for diabetic skin injury

Abstract

The present application relates to a new use of deer blood peptide and a preparation method of deer blood peptide hydrogel, wherein the new use is the application of deer blood peptide in drugs or cosmetics for promoting skin repair of diabetes, and the preparation method comprises the following steps: (1) enzymolysis: preparing deer blood solution, adjusting pH, preheating, adding pepsin, digesting, adjusting pH with NaOH, adding trypsin, further reacting, heating to deactivate the enzyme after digestion, centrifuging to obtain supernatant of the enzymolysis solution, and filtering to obtain deer blood peptide; (2) separation and freeze-drying: preparing deer blood peptide solution, sequentially performing fractionation treatment with molecular cut-off to obtain corresponding components, performing desalting treatment, and freeze-drying to obtain deer blood peptide with different components; (3) preparation of deer blood peptide hydrogel: adding chitosan into glacial acetic acid solution to fully dissolve, adding β-GP solution to obtain chitosan / β-GP solution, adding sodium alginate powder to dissolve, then dropping CaCl2 solution to obtain blank hydrogel, and finally adding deer blood peptide to obtain deer blood peptide hydrogel. The discovery of the new use provides a new method and favorable basis for the treatment of chronic skin infectious diseases of diabetes.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to the preparation method of deer blood peptide hydrogel and its new medical applications. Background Technology

[0002] Deer antler blood (VB) is the blood obtained from harvesting antlers from red deer or sika deer. It has a long history of medicinal use, recorded in numerous herbal classics such as the *Shennong Bencao Jing*, *Mingyi Bielu*, *Bencao Gangmu*, and *Shennong Bencao Jing Baizhong Lu*. It is believed to have kidney-tonifying, aphrodisiac, bone-strengthening, and sperm-replenishing effects, and is hailed as a "celestial product." VB contains abundant protein, polypeptides, and amino acids. Traditional Chinese medicine believes its pharmacological activity is similar to that of deer antler, and it can combat fatigue, provide antioxidant benefits, enhance immunity, slow aging, lower blood pressure, fight tumors, and alleviate osteoporosis. Currently, polypeptide components, including deer antler peptides, exhibit excellent antioxidant and anti-inflammatory activity; however, reports on deer blood peptides (VBPs) in these areas are relatively scarce.

[0003] Skin is the largest, most exposed, most sensitive, and most vulnerable tissue in the human body, playing a crucial role in various processes such as preventing dehydration, defending against harmful substances and pathogens, initiating vitamin D synthesis, excretion, and thermoregulation. Due to its diverse causes, skin trauma has become a significant medical problem requiring urgent attention. In modern skin dressings, hydrogels have become ideal candidates for wound dressings due to their excellent hydrophilicity, extracellular matrix (ECM)-like structure, good biodegradability, biocompatibility, adhesion, and breathability. Studies have found that adding bioactive peptides to hydrogels can enhance their skin repair capabilities in multiple ways, including anti-inflammatory effects, antibacterial activity, promotion of cell proliferation, and induction of angiogenesis.

[0004] Currently, wound healing and angiogenesis in diabetic patients remain a major challenge in clinical medicine. Improper care can easily lead to gangrene, amputation, and even patient death. Traditional dressings, such as gauze, may adhere to newly formed granulation tissue, causing pain and affecting tissue integrity upon removal, and lack antibacterial, antioxidant, or other active functions. Among modern wound dressings in the form of films, nanofibers, hydrogels, and sponges, hydrogels have become an ideal candidate product due to their three-dimensional network structure, good biodegradability, biocompatibility, adhesion, and breathability, as well as their ability to maintain a moist environment necessary for cell migration. The purpose of this invention is to disclose the regulatory effect of CAVBPH on diabetes-related chronic skin wounds and skin microbiota.

[0005] The present invention crosslinks deer blood peptides with hydrogels to obtain hydrogels loaded with deer blood peptides. Through research on the regulation of skin wound microbiota by deer blood peptide hydrogels, it has been determined that deer blood peptides can regulate the structure of skin microbiota and improve the richness, diversity and uniformity of microbiota in damaged skin. Therefore, the first objective of the present invention is to provide a new use for deer blood peptides, specifically: the application of deer blood peptides in the preparation of drugs and / or cosmetics that regulate the structure of skin microbiota.

[0006] As a preferred embodiment of the present invention, the novel use of deer blood peptide is: the application of the deer blood peptide in the preparation of drugs and / or cosmetics that regulate the structure of damaged skin flora and improve the richness and diversity of damaged skin flora.

[0007] As a preferred embodiment of the present invention, the novel use of deer blood peptide is: the application of the deer blood peptide in the preparation of drugs and / or cosmetics that regulate the structure of damaged skin flora and improve the uniformity of damaged skin flora.

[0008] As a preferred embodiment of the present invention, when deer blood peptide is used to regulate the skin microbiome structure, the deer blood peptide is loaded in a hydrogel and then applied to the skin by coating.

[0009] In addition, this invention studies how deer blood peptide hydrogel promotes the repair process of diabetic wound skin by regulating the SIRT1 / NF-κB signaling pathway, and determines that deer blood peptide hydrogel promotes wound skin repair by activating the SIRT1 / NF-κB signaling pathway; therefore, the second objective of this invention is to provide a SIRT1 / NF-κB signaling pathway activator, which includes a hydrogel loaded with deer blood peptide.

[0010] This invention studies the effects of deer blood peptide hydrogel on VEGF, HIF-α, PCNA, CD31, and CD68 in diabetic wound tissue, and determines that deer blood peptide can promote the expression of growth factors in diabetic injured tissue and accelerate wound repair; therefore, the third objective of this invention is to provide a drug that promotes the expression of growth factors in diabetic injured tissue, the drug comprising a hydrogel loaded with deer blood peptide.

[0011] This invention studies the effect of deer blood peptide hydrogel on wound healing in diabetic mice, confirming that hydrogel samples containing deer blood peptide are beneficial for wound repair. Furthermore, histopathological analysis of mouse wound healing revealed that after treatment with deer blood peptide hydrogel, mouse fibroblasts were densely and regularly arranged, skin appendages such as sweat glands and hair follicles were being generated, lymphocytes were negligible, and collagen fibers showed deeper staining and a wider distribution. This further confirms that deer blood peptide hydrogel treatment can accelerate epidermal regeneration at the wound site, exhibiting a good promoting effect in the skin repair process. Therefore, the fourth objective of this invention is to provide a method for promoting skin wound repair, which involves applying a drug containing deer blood peptide hydrogel to the skin wound.

[0012] The fifth objective of this invention is to provide a method for preparing a hydrogel loaded with deer blood peptides, the method comprising the following steps:

[0013] (1) Deer blood enzymatic hydrolysis: Deer blood peptides were prepared by simulating the in vitro gastrointestinal digestion of deer blood powder using a two-step enzymatic hydrolysis method. A 20 mg / ml deer blood solution was prepared, and the pH was adjusted to 1.5-2.5 with 1-2 M HCl. After preheating at 37℃ for 10-30 min, pepsin was added at an enzyme-to-substrate ratio of 1:10-50. The solution was digested at 37℃ for 2-4 h at 120 rpm in a constant temperature shaker. The pH was then adjusted to 7.5-8.0 with 1-2 M NaOH, and trypsin was added at an enzyme / substrate ratio of 1:15-25, w / w. The reaction was further carried out for 4 h to simulate intestinal digestion. During the enzymatic hydrolysis, 1-2 M HCl or 1-2 M NaOH was used to maintain pH stability. After digestion, the enzyme was inactivated by heating in a boiling water bath for 15-30 min. The reaction mixture was cooled to room temperature and centrifuged at 8000 rpm for 30 min to obtain the supernatant of the hydrolysate. The supernatant was then filtered twice with double-layer slow filter paper to obtain deer blood peptides, which were then freeze-dried and stored at -20°C for later use.

[0014] (2) Separation and freeze-drying: Prepare a 50 mg / mL deer blood peptide solution, and pass it through ultrafiltration membranes with molecular cutoff of 10 kDa and 3 kDa in sequence for fractionation to obtain components F1, F2 and F3. Then, pass the three components through a 150 Da nanofiltration membrane for desalting, freeze-dry and store at -20℃ for later use.

[0015] (3) Preparation of deer blood peptide hydrogel: 200 mg of chitosan (CS) was dissolved in 10 mL of 0.1 M glacial acetic acid solution, and 2.5 mL of 55% (w / v) β-GP solution was added dropwise under stirring in an ice-water bath to obtain a CS / β-GP solution. Then, 100 mg of sodium alginate (SA) powder was added to the prepared solution and stirred continuously to dissolve it. Then, 0.2 mL of 0.5% CaCl2 solution (w / v) was added dropwise and stirred continuously to obtain a blank hydrogel (CAH) sol. Then, the deer blood peptide F3 powder obtained in step 2 of claim 5 was added to the CAH system and mixed evenly to a concentration of 0.1% to obtain the deer blood peptide hydrogel.

[0016] Advantages and beneficial effects of the present invention:

[0017] (1) This invention has demonstrated through experiments that deer blood peptides can regulate the structure of damaged skin flora, improve the richness, diversity and uniformity of damaged skin flora, and promote the growth of beneficial bacteria. The discovery of this new use provides a new method and favorable basis for the treatment of chronic skin diseases.

[0018] (2) This invention provides a novel SIRT1 / NF-κB signaling pathway activator, which offers a new method for the treatment of chronic skin diseases involving the SIRT1 / NF-κB signaling pathway.

[0019] (4) The preparation method of deer blood peptide hydrogel provided by the present invention is simple to operate, has low production cost and high yield. Attached Figure Description

[0020] Figure 1 The effect of enzymatic hydrolysis on the in vitro activity of vitamin B. The results included: (A) TCA-YSP; (B) SDS-PAGE analysis; (C) DPPH radical scavenging activity; (D) ABTS radical scavenging activity; (E) hydroxyl radical scavenging activity; (F) Fe2+ chelating activity; (G) LOX inhibitory activity; (H) XOD in vitro inhibitory activity; and (I) tyrosinase inhibitory activity. Different letters above the error lines represent significant differences between groups (p<0.05). All sample concentrations were 1 mg / mL.

[0021] Figure 2 Bioactivity evaluation of ultrafiltration components. The evaluation included: (A) DPPH radical scavenging activity; (B) ABTS radical scavenging activity; (C) hydroxyl radical scavenging activity; (D) Fe2+ chelating activity; (E) LOX in vitro inhibitory activity; (F) XOD inhibitory activity; and (G) tyrosinase inhibitory activity. Different letters above the error line represent significant differences between groups (p < 0.05). All sample concentrations were 1 mg / mL.

[0022] Figure 3 In vitro activity analysis of synthetic peptides. The results include: (A) hemolysis rate; (B) DPPH radical scavenging activity; (C) ABTS radical scavenging activity; (D) hydroxyl radical scavenging activity; (E) Fe2+ chelating activity; (F) LOX inhibitory activity; (G) XOD inhibitory activity; and (H) tyrosinase inhibitory activity. Different letters above the error line represent significant differences between groups (p < 0.05). All samples were at a concentration of 1 mg / mL.

[0023] Figure 4 Preparation of deer blood peptide hydrogel and its potential mechanism for promoting wound healing.

[0024] Figure 5 Characterization of deer blood peptide hydrogels. SEM (A), FTIR (B), pH (C), viscosity (D), water retention (E), swelling (F), and biodegradability (G) of CAH and CAPBPH, as well as the cumulative release of VBPs in CAVBPH (H). Scale bar = 50 μm.

[0025] Figure 6 In vitro activity analysis of hydrogel samples. This included (A) DPPH free radical scavenging activity, (B) ABTS free radical scavenging rate, (C) hemolytic activity analysis, (DF) antibacterial analysis of the hydrogel against Staphylococcus aureus and Escherichia coli, and (G) cell viability of CAH and CAVBPH.

[0026] Figure 7 H&E staining of pancreatic tissues from normal and diabetic mice. Scale bar = 200 μm.

[0027] Figure 8 Skin wound size (A) and wound healing rate (B) at 0, 3, 9, and 15 days post-injury in different treatment groups. *, **, or *** indicate p<0.001, p<0.01, and p<0.001, respectively.

[0028] Figure 9 Routine pathological staining of wound tissue. (A) H&E staining and Masson staining, scale bar = 100 μm; (B) Quantitative analysis of collagen deposition in each group. *, **, or *** indicate p < 0.001, p < 0.01, and p < 0.001, respectively.

[0029] Figure 10IHC staining of skin samples. (A) Representative images of CD31, PCNA, α-SMA, and CD68 (scale bar = 200 μm). Quantitative expression of CD31 (B), PCNA (C), α-SMA (D), and CD68 (E) proteins. *, **, or *** indicate p < 0.001, p < 0.01, and p < 0.001, respectively.

[0030] Figure 11 Effects of different treatments on proteins related to the PI3K / AKT / mTOR / HIF-1α signaling pathway. (A) Representative images of each protein band; (B) p-PI3K / PI3K; (C) p-AKT / AKT; (D) p-mTOR / mTOR; (E) HIF-1α / β-actin; (F) VEGFA / β-actin. *, **, or *** represent p<0.001, p<0.01, and p<0.001, respectively.

[0031] Figure 12 Effects of different treatments on proteins related to the SIRT1 / NF-κB signaling pathway in diabetic wounds. (A) Protein bands of SIRT1, NF-κB, TNF-α, and IL-1β; (B) SIRT1 / β-actin; (C) NF-κB / β-actin; (D) TNF-α / β-actin; (E) IL-1β / β-actin. *, **, or *** represent p<0.001, p<0.01, and p<0.001, respectively.

[0032] Figure 13 Analysis of skin microbiota at mouse wound sites. This included: (A) taxonomic annotation, (B) sparse curves, (C) alpha diversity analysis, (D) beta diversity analysis, (E) community abundance analysis at the phylum level, (F) community abundance analysis at the genus level, (G) Venn analysis of each group, (H) difference heatmap at the species level, and (I) random forest characteristic analysis. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments of the present invention are not limited thereto. For process parameters not specifically specified, conventional techniques can be referred to. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field.

[0034] Example 1: Extraction and Identification of Deer Blood Peptides

[0035] 1.1 Materials and Instruments

[0036] 1.1.1 Experimental Materials

[0037] Sika deer ( Cervus nippon Temminck) VB was purchased from Jilin Jinlu Pharmaceutical Co., Ltd. in 2020.

[0038] 1.1.2 Experimental Apparatus

[0039] Table 1 Experimental Instruments and Manufacturers

[0040]

[0041] 1.1.3 Experimental Reagents

[0042] Table 2. Experimental Reagents and Manufacturers

[0043]

[0044] 1.2 Experimental Methods

[0045] 1.2.1 Preparation of Deer Blood Peptide (VBH)

[0046] VBH was prepared by simulating in vitro gastrointestinal digestion of vitamin B using a two-step enzymatic hydrolysis method. A 20 mg / ml VB solution was prepared, and the pH was adjusted to 1.5 with 1 M HCl. After preheating at 37°C for 10 min, pepsin was added at an enzyme-to-substrate ratio of 1:50. The solution was digested at 37°C for 2 h at 120 rpm in a constant-temperature shaker. The pH was then adjusted to 7.5 with 1 M NaOH, and trypsin (enzyme / substrate = 1:25, w / w) was added for a further 4 h to simulate intestinal digestion. During the enzymatic hydrolysis, 1 M HCl or 1 M NaOH was used to maintain pH stability. After digestion, the enzyme was inactivated by heating in a boiling water bath for 15 min. The reaction mixture was cooled to room temperature and centrifuged at 8000 rpm for 30 min to obtain the supernatant. The supernatant was then filtered twice through double-layered slow-speed filter paper to obtain VBH, which was then freeze-dried and stored at -20°C for later use.

[0047] 1.2.2 Trichloroacetic acid-soluble peptide yield (TCA-YSP)

[0048] TCA-YSP represents the percentage of trichloroacetic acid-soluble peptides in the sample supernatant relative to the total VB protein content, used to evaluate protein hydrolysis. 1 mL of sample is mixed with 1 mL of 10% (w / v) trichloroacetic acid, incubated at 25°C for 30 min, then centrifuged at 8000 × g for 10 min. The peptide content in the supernatant is determined using a BCA kit, and TCA-YSP is calculated using the following formula:

[0049] TCA-YSP (%) = (A / B) × 100%

[0050] Where: A is the content of trichloroacetic acid-soluble peptides in the supernatant, and B is the content of total protein in the sample before digestion.

[0051] 1.2.3 SDS-PAGE

[0052] A 5% stacking gel and a 12% separating gel were prepared for SDS-PAGE analysis of deer blood and its hydrolysate.

[0053] 1.2.4 Ultrafiltration separation of deer blood peptide (VBH)

[0054] A 50 mg / mL VBH solution was prepared and fractionated by passing it sequentially through ultrafiltration membranes with molecular weight cutoffs (MW) of 10 kDa and 3 kDa to obtain components F1 (>10 kDa), F2 (3-10 kDa), and F3 (<3 kDa). The three components were then desalted by passing them through a 150 Da nanofiltration membrane, lyophilized, and stored at -20°C for later use.

[0055] 1.2.5 Protein content and amino acid composition analysis

[0056] Protein content: The protein content of the samples was determined using BSA as the standard protein and the BCA protein kit.

[0057] Amino acid composition: The sample was hydrolyzed with 6 M HCl at 110℃ for 22 h, and the content of each amino acid in the sample was determined using an L-8900 automatic amino acid analyzer.

[0058] 1.2.6 Nano LC-MS / MS Identification of Peptide Sequences

[0059] Peptide sequences possessing optimal bioactivity were analyzed using nano LC-MS / MS. First, DTT was added to the sample to a final concentration of 10 mM for 1 h, followed by reduction with IAA solution to a final concentration of 50 mM. Alkylation was then performed at room temperature in the dark for 40 min. Subsequently, desalting was carried out using a self-packed desalting column, and the solvent was evaporated at 45 °C. The peptide sample was resuspended in 20 μL of 0.1% FA and analyzed using an Ultimate 3000 high-performance liquid chromatography system equipped with an Orbitrap electrospray ion trap mass spectrometer.

[0060] The chromatographic conditions are as follows:

[0061] 1) Pre-column: 300 μm id × 5 mm, packed with Acclaim PepMap RPLC C18, 5 μm, 100Å,

[0062] 2) Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100 Å

[0063] 3) Injection volume 5 μL, column temperature 20℃.

[0064] 4) Mobile phase A: 0.1% FA;

[0065] 5) Mobile phase B: 0.1% FA, 80% ACN;

[0066] 6) Flow rate: 600 nL / min;

[0067] The gradient elution conditions are shown in Table 3.

[0068] Table 3 Liquid phase elution conditions

[0069]

[0070] Mass spectrometry conditions:

[0071] 1) Primary mass spectrometry parameters: Resolution: 70,000; AGC target: 3e6; Maximum IT: 40 ms

[0072] Scan range: 100 to 1800 m / z

[0073] 2) Secondary mass spectrometry parameters: Resolution: 17500; AGC target: 1e5; Maximum IT: 60 ms

[0074] TopN: 20; NCE / steppedNCE: 27

[0075] The raw MS / MS files were analyzed using XCalibur and Byonic software, and the peptide sequences were identified by comparison with the proteome of the species in the UniProt database.

[0076] 1.2.7 Preparation of Monopeptides

[0077] Monopeptides from deer blood peptides were prepared by dextran gel column chromatography. The molecular weight (MW) and purity (>95%) of the prepared monopeptides were analyzed by a Waters ZQ 2000 single quadrupole mass spectrometer and a Waters Alliance 2695 high-performance liquid chromatography system, respectively.

[0078] 1.2.8 Determination of antioxidant activity

[0079] A 1 mg / mL protein peptide solution and a positive control solution were prepared to evaluate the antioxidant capacity of the peptides. DPPH, ABTS, and hydroxyl radical scavenging capacity were assessed using GSH as a positive control, and ferrous ion (Fe) scavenging capacity was evaluated. 2+ The chelating ability was tested using EDTA-2Na as a positive control.

[0080] 1.2.8.1 DPPH free radical scavenging activity

[0081] Add the sample and an equal volume of 0.2 mM DPPH ethanol solution to a 96-well plate, react in the dark at room temperature for 30 min, and read the absorbance at 517 nm using a microplate reader. The formula for calculating the DPPH free radical scavenging rate is as follows:

[0082] DPPH free radical scavenging rate (%) = [1−(A 样品 -A 样品空白 ) / A 对照 ]×100%

[0083] In the formula, A 样品 A represents the absorbance of the peptide and DPPH solution. 对照 It is the mixed absorbance of DPPH and distilled water, A 样品空白 This represents the absorbance of the sample and ethanol.

[0084] 1.2.8.2 ABTS free radical scavenging activity

[0085] ABTS and potassium persulfate solutions with concentrations of 7.4 mM and 2.45 mM were prepared, respectively. After mixing at a 1:1 (v / v) ratio, the solutions were reacted in the dark at low temperature for 12 h. Before use, the solutions were further diluted with distilled water to a absorbance of 0.7 ± 0.02 at 734 nm. The test sample was mixed with the ABTS working solution at a 1:2 (v / v) ratio and incubated at room temperature in the dark for 10 min. The absorbance at 734 nm was then measured. The calculation formula is as follows:

[0086] ABTS free radical scavenging capacity (%) = [1−(A 样品 -A 样品空白 ) / A 对照 ]×100%

[0087] In the formula, A 样品 It is the mixed absorbance of the sample solution and the ABTS working solution, A 对照 A represents the absorbance of distilled water and ABTS working solution. 样品空白 This indicates the absorbance of the sample and distilled water.

[0088] 1.2.8.3 Hydroxyl radical scavenging ability

[0089] Equal volumes of 10 mM salicylic acid ethanol solution, 10 mM ferrous sulfate solution, and 8.8 mM H₂O₂ solution were added sequentially to the sample. The mixture was reacted in the dark at 37 °C for 30 min, centrifuged at 3000 × g for 5 min, and then the absorbance of the supernatant was measured at 510 nm (A). 样品 The formula for calculating the hydroxyl radical scavenging capacity is as follows:

[0090] Hydroxyl radical scavenging rate (%) = [1−(A 样品 -A 样品空白 ) / A 对照 ]×100%

[0091] In the formula, A 样品空白 Includes samples, salicylic acid, ferrous sulfate, and distilled water, A 对照 It contains distilled water, salicylic acid, ferrous sulfate, and H2O2.

[0092] 1.2.8.4 Fe 2+ Chelation ability

[0093] 100 μL of sample was mixed with 20 μL of FeCl2 solution (2 mM) and reacted at room temperature for 30 min. Then 100 μL of phenoxyazine (5 mM) was added, and the reaction was continued for 10 min. The absorbance was measured at 562 nm. 2+ The formula for calculating chelating capacity is as follows:

[0094] Fe 2+ Chelation capacity (%) = [1 − (A 样品 -A 样品空白 ) / A 对照 ]×100%

[0095] Among them, A 样品 The mixed absorbance of the representative sample, FeCl2, and phenoxyazine, A 对照 Including distilled water, FeCl2, and phenoxybenzamine, A 样品空白 It is the absorbance of the sample, FeCl2, and distilled water mixture.

[0096] 1.2.9 LOX Inhibitory Activity

[0097] First, mix 20 μL of sample, 200 μL of PBS (0.01 M, pH=7.0), and 20 µL of LOX solution (~100 U), and pre-incubate at room temperature for 5 min. Then, add 20 µL of linoleic acid borate buffer solution (3.2 mM, pH=9.0) to initiate the reaction for 3 min, and read the absorbance at 234 nm (A). 样品 Using diclofenac sodium as a positive control and calculating according to the following formula:

[0098] LOX inhibitory activity (%) = [1−(A 样品 -A 样品空白 ) / A 对照 ]×100%

[0099] In the formula, A 样品空白 Replace LOX with borate buffer, A 对照 Use distilled water instead of the sample.

[0100] 1.2.10 XOD Inhibitory Activity Assay

[0101] 50 μL of sample and 50 μL of XOD (0.02 U) were mixed thoroughly and pre-incubated at room temperature for 5 min. Then, 120 μL of xanthine (0.48 mM) was added and the reaction was carried out at 37°C for 20 min. Finally, 80 μL of hydrochloric acid (1 M) was added to terminate the reaction. Allopurinol was used as a positive control. The XOD inhibitory activity was calculated using the following formula:

[0102] XOD inhibitory activity (%) = [1−(A) 样品 -A 样品空白 ) / A 对照 ]×100%

[0103] Among them, A 样品 A 样品空白 and A 对照 The absorbance values ​​represent (sample + XOD + xanthine + hydrochloric acid), (sample + PBS + xanthine + hydrochloric acid), and (distilled water + XOD + xanthine + hydrochloric acid), respectively.

[0104] 1.2.11 Assessment of anti-tyrosinase activity

[0105] Mix 100 μL of sample with an equal volume of tyrosinase (125 U / mL), pre-incubate at room temperature for 15 min, add 100 μL of L-DOPA (10 mM), react for 10 min, and then measure the absorbance (A) at 492 nm. 样品 Kojic acid was used as a positive control, and the formula for calculating its inhibitory activity is as follows:

[0106] Tyrosinase inhibitory activity (%) = [1−(A 样品 -A 样品空白 ) / A 对照 ]×100%

[0107] In the formula, A 样品空白 Replace tyrosinase with PBS, A 对照 Replace the sample solution in the system with distilled water.

[0108] 1.2.12 Hemolysis rate

[0109] First, a 2% human erythrocyte suspension (HRBC) was prepared. 500 µL of HRBC was incubated with the sample solution at 37 °C for 1 h, and the absorbance of the supernatant was measured at 540 nm after centrifugation. HRBC and Triton X-100 (1% v / v) were used as positive controls, and HRBC and physiological saline were used as negative controls.

[0110] 1.2.13 Statistical Analysis

[0111] All experiments were conducted in triplicate, and the data were analyzed using Duncan's one-way ANOVA with IBM SPSS Statistics 23.0 software. p <0.05 indicates a significant difference between the two groups. Graphs were plotted using software such as GraphPad Prism 5.

[0112] 1.3 Experimental Results

[0113] 1.3.1 Preparation and Activity Analysis of VBH

[0114] like Figure 1 As shown in Figure A, the TCA-YSP of VBH was 33.07%, an increase of 31.81% compared to before digestion. SDS-PAGE results indicated that after enzymatic digestion, the bands of the original proteins in VB, including ~70 kDa, ~55 kDa, ~25 kDa, and ~12 kDa, were weakened or disappeared. Figure 1 B). Therefore, these proteins were successfully hydrolyzed. Subsequently, scavenging was primarily carried out by DPPH, ABTS, and hydroxyl radicals, as well as Fe... 2+ The antioxidant activity of the peptides was analyzed using chelation capacity as an indicator; the results are detailed below. Figure 1 At a concentration of 1 mg / mL, GSH and VBH showed scavenging rates of 94.18% and 55.29% for DPPH free radicals, respectively, significantly higher than VB (21.28%). p <0.05). The ABTS radical scavenging abilities of VB, VBH, and GSH were 74.16%, 95.77%, and 99.93%, respectively, while their hydroxyl radical scavenging abilities were 28.48%, 32.99%, and 34.80%, respectively. Figure 1 (D and E). Figure 1 As shown in F, Fe of VBH 2+ The chelating ability increased by 21.37% compared to VB. At a concentration of 1 mg / mL, the LOX, XOD, and tyrosinase inhibitory activities of VBH were 21.13%, 35.37%, and 45.64%, respectively, significantly higher than before enzymatic hydrolysis. p <0.05, Figure 1 GI).

[0115] 1.3.2 Separation of VBH

[0116] SDS-PAGE showed that the intensity of the F1 protein band was higher than that of VBH at ~55 kDa, and no obvious protein bands were observed in F2 and F3 in the 10-180 kDa range. Figure 1 B). The DPPH radical scavenging activities of the three components were ranked as F2 > F3 > F1. p <0.05, Figure 2 A). For example Figure 2 As shown in Figure B, except for F1, the ABTS scavenging rates of the other components all exceeded 95%. Additionally, F3 exhibited high hydroxyl radical scavenging and Fe... 2+ The chelating capacity was significantly higher than that of its parent hydrolysate and other components. p <0.05, Figure 2 (C and D). For example... Figure 2 As shown in EG, F3 exhibited inhibitory activities of 22.36%, 37.23%, and 83.58% against LOX, XOD, and tyrosinase, respectively.

[0117] Table 4 shows the types and contents of hydrolyzed amino acids in F3, of which 8 are essential amino acids (65.92%) and 9 are non-essential amino acids (34.08%). The four most abundant amino acids are lysine (19.51%), valine (13.41%), leucine (13.25%), and glutamic acid (10.15%), accounting for 56.73% of the total amino acids.

[0118] Table 4 Amino acid composition of component F3

[0119]

[0120] 1.3.3 Peptide sequence identification and activity analysis

[0121] A total of 372 peptides were identified in F3, mainly derived from parental proteins such as Adult beta-globin, Toll-like receptor 8, Tyrosine-protein kinase receptor, MHC class II antigen, and Serum albumin. Among these, 97.78% of the peptides had a molecular weight (MW) of <1 kDa, while the remaining peptides had MWs between 1 kDa and 3 kDa. A score >200 and an intensity >10 were selected. 7 The sequences (27 in total) were further analyzed. The bioactivity, antioxidant, anti-inflammatory and antibacterial properties of these peptides were predicted using online tools, and the results are shown in Table 5.

[0122] Table 5. Computer predictions for identifying peptide activity

[0123]

[0124]

[0125] Therefore, based on the online prediction results, EHF, FPH, LFP, FSAL, VGYP, LSQKFPK, HHGGEFTPV, and LKECCDKPV were selected to observe their in vitro biological characteristics.

[0126] 1.3.4 Bioactivity analysis of peptides

[0127] In in vitro hemolysis experiments, the hemolysis rate of all peptides at a concentration of 1 mg / mL was less than 1%. Figure 3 A). LKECCDKPV exhibited the highest DPPH (89.55%) and ABTS (89.55%) scavenging capacity among all peptides, with activity comparable to GSH. p >0.05, Figure 3 (B and C). For example... Figure 3 As shown in Figure D, the hydroxyl radical scavenging abilities of these peptides are, in descending order: FPH (52.50%), LFP (51.66%), GSH (32.35%), EHF (25.99%), HHGGEFTPV (25.53%), and LKECCDKPV (22.94%). However, the Fe... 2+ The chelating capacity was all below 15%. Figure 3 E).

[0128] The inhibitory activity of these peptides was then tested. Figure 3 FH). Peptides FSAL and LFP exhibited 11.61% and 10.22% LOX enzyme inhibitory activity, respectively, while the remaining peptides were all below 10% (FH). Figure 3 F). For example Figure 3 As shown in G, the top four peptides with strong inhibitory activity against XOD at 1 mg / mL were EHF (82.19%), VGYP (73.26%), LKECCDKPV (64.69%), and FSAL (51.82%). Furthermore, LKECCDKPV exhibited significantly higher tyrosinase inhibitory activity than the other peptides. p <0.05, Figure 3 H).

[0129] This invention marks the first time that multiple bioactive peptides have been isolated from vitamin B (VB). Simulating in vitro digestion of VB, we discovered that VBH possesses high antioxidant activity, LOX, XOD, and tyrosinase inhibitory activity, and is rich in essential amino acids, meeting human needs. This indicates that VB, as a valuable protein resource, can be absorbed orally and contributes to regulating the homeostasis of biological systems. Furthermore, we identified 27 high-confidence peptides from F3, and combined online prediction, solid-phase synthesis, and activity analysis to demonstrate the biological functions of some of these peptides. These results provide scientific data for the application of VB and its derived peptides in the medical and health fields.

[0130] Example 2: Preparation and characterization of deer blood peptide hydrogel (CAVBPH)

[0131] 1.1 Instruments and Materials

[0132] 1.1.1 Experimental Apparatus

[0133] Table 6 Experimental Instruments and Manufacturers

[0134]

[0135] 1.1.2 Experimental Reagents

[0136] Table 7 Experimental Instruments and Manufacturers

[0137]

[0138] 1.2 Experimental Methods

[0139] 1.2.1 Preparation of VBPs

[0140] Component F3, or VBPs, with MW < 3 kDa was prepared using the enzymatic hydrolysis and ultrafiltration techniques described in Example 1, and then lyophilized and stored at -20°C for later use.

[0141] 1.2.2 Preparation of CAVBPH

[0142] 200 mg of CS was dissolved in 10 mL of glacial acetic acid solution (0.1 M), and 2.5 mL of 55% (w / v) β-GP solution was added dropwise while stirring in an ice-water bath to obtain a CS / β-GP solution. Then, 100 mg of SA powder was added to the prepared solution and stirred continuously until dissolved. Next, 0.2 mL of CaCl2 solution (0.5%, w / v) was added dropwise while stirring continuously to obtain CAH sol. To prepare CAVBPH, VBP powder was added to the CAH system and mixed thoroughly to a concentration of 0.1% (w / v).

[0143] The gelation time of CAH and CAVBPH at 37°C was determined using an inverted centrifuge tube method. The results showed that the blank hydrogel gelled at 37°C in 5 minutes, while the deer blood peptide hydrogel gelled in 2 minutes, indicating that the prepared hydrogels are temperature-sensitive. All solutions were freshly prepared and stored at 4°C for use.

[0144] 1.2.3 Characterization of hydrogels

[0145] 1.2.3.1 Microscopic characteristics

[0146] The microstructure of the gold-sprayed gel cross section was observed using scanning electron microscopy (SEM) at 15 kV.

[0147] 1.2.3.2 Infrared Analysis

[0148] Powders of CS, β-GP, SA, VBPs, CAH, and CAVBPH were mixed with KBr at a mass ratio of 1:100, and the mixtures were analyzed by FTIR spectrometry at 4000-500 cm⁻¹. -1 They were scanned within a certain wavenumber range to detect their characteristic functional groups.

[0149] 1.2.3.3 pH and viscosity determination

[0150] The pH values ​​of CAH and CAVBPH at 37°C were determined using a pH meter. Similarly, the viscosity of the samples was measured at 37°C using an NDJ-8S digital viscometer with rotor #3 set to 1.5 rpm.

[0151] 1.2.3.4 Water retention test

[0152] The hydrogel sample with an initial weight of W0 was placed in a 37℃ constant temperature oven for 48 hours, and its weight was recorded as W at intervals. t Weight retention rate (WRR) is calculated using the following formula:

[0153] WRR=W t / W0×100%

[0154] 1.2.3.5 Swelling Test

[0155] A certain mass of lyophilized hydrogel (W0) was immersed in PBS (pH=7.4) at 37℃ for 48 h. During this period, the sample was removed at fixed time intervals, excess surface moisture was removed, and the weight was recorded as W1. The swelling ratio (SR) is calculated as follows:

[0156] SR = (W1 - W0) / W0 × 100%

[0157] 1.2.3.6 In vitro enzymatic biodegradation

[0158] The hydrogel sample (W0) was placed in PBS containing 1 mg / mL lysozyme and incubated at 37°C and 100 rpm for 14 days. During this period, the hydrogel was removed at the set time intervals, and excess surface moisture was removed with filter paper. The sample was then quickly weighed (W0). t The solvent was replaced with freshly prepared solution every 48 hours to maintain lysozyme activity. The residual weight (WR) of the hydrogel was calculated using the following formula:

[0159] WR = W t / W0×100%

[0160] 1.2.3.7 In vitro release of VBPs

[0161] To evaluate the release behavior of VBPs in CAVBPH, a certain mass of sample was completely immersed in 20 mL of PBS and continuously shaken at 100 rpm at 37°C for 10 days. At different time points, 1 mL of supernatant was taken out, and 1 mL of fresh PBS was added simultaneously to maintain a constant volume. The release of VBPs was determined using the BCA method.

[0162] 1.2.4 In vitro activity of hydrogels

[0163] 1.2.4.1 In vitro antioxidant

[0164] First, solutions of different concentrations (1 and 5 mg / L) were prepared by immersing the lyophilized hydrogel in PBS for 24 h. Simultaneously, 1 mg / mL VBPs and GSH solutions were prepared as positive controls. As shown in Example 1, the antioxidant activity was evaluated using the DPPH and ABTS free radical scavenging methods.

[0165] 1.2.4.2 In vitro hemolysis

[0166] The hemolysis rate of the hydrogel was determined according to Example 1.

[0167] 1.2.4.3 Antibacterial activity

[0168] The effects of hydrogels on *Escherichia coli* were evaluated based on time-dependent co-culture. E. coil Gram-negative bacteria) and Staphylococcus aureus ( S. aureus Inhibitory activity against Gram-positive bacteria. First, all tested strains were cultured in Luria-Bertani (LB) liquid medium at 37°C with shaking until the logarithmic growth phase, and then diluted with physiological saline to a concentration of approximately 10. 5-6CFU / mL. 0.5 mL of bacterial culture was mixed with 20 mL of LB liquid medium containing 0.4 g of UV-sterilized hydrogel and incubated at 37°C and 100 rpm. The absorbance of the culture at 600 nm was measured periodically. The control group did not contain hydrogel samples. Subsequently, the bacterial cultures from each group were appropriately diluted with physiological saline and inoculated onto LB solid medium, incubated at 37°C for 12 h, and the colony count was observed to evaluate the antibacterial activity of CAH and CAVBPH.

[0169] 1.2.4.4 Cytotoxicity

[0170] The UV-sterilized hydrogel was immersed in the culture medium and incubated at 37°C for 24 h to obtain the hydrogel extract. HaCaT cells were cultured at 0.5 × 10⁻⁶ cells / mL. 4 Cells were seeded at a density per well in 96-well plates. After 24 h of cell adhesion, the culture medium was replaced with hydrogel extraction medium and cultured for another 24 h. The viability of HaCaT cells was determined using the MTT assay to assess the cytotoxicity of the hydrogel.

[0171] 1.2.5 Statistical Analysis

[0172] The experiment was conducted in triplicate, and the data are expressed as mean ± standard deviation (SD). ANOVA was performed using IBM SPSS Statistics 23.0 software. p A value <0.05 was considered statistically significant. GraphPad Prism 5 and Adobe Illustrator CS6 software were used to create the graphs.

[0173] 1.3 Experimental Results

[0174] 1.3.1 Characterization of hydrogels

[0175] Thermosensitive composite hydrogel preparation scheme as follows Figure 4 As shown. In the CS structure, the -OH groups at C3 and C6, as well as the partially protonable -NH3 group at the C2 position. + The group readily forms hydrogen bonds with water molecules, increasing the polymer's water solubility. Introducing β-GP into a CS solution and heating at 37°C promotes the formation of hydrogen bonds in the -PO4 group within the β-GP structure. 3- With -NH3 in the CS molecule + Through electrostatic interactions, and furthermore, CS, β-GP, and H2O more readily form stable hydrogen bonds, thus forming a CS-based thermosensitive hydrogel network. Simultaneously, Ca is introduced into the system. 2+ It can react with the -COO of the two guluronic acid segments in the SA molecule. - It forms four coordinate bonds with -OH, thereby realizing SA / Ca 2+Networks enhance the viscosity and gelation effect of gel systems.

[0176] The microstructures of CAH and CAVBPH were observed using SEM, such as... Figure 5 As shown in Figure A, both CAH and CAVBPH possess porous, interconnected network structures. Comparing images of CAH and CAVBPH at the same magnification reveals that the introduction of VBPs increases the number of pores in the hydrogel. The porous structure of the hydrogel not only facilitates moisture retention and the creation of a moist environment but also provides more oxygen to the wound bed. Furthermore, the pores in CAVBPH also facilitate the release of VBPs, enabling them to interact with the wound more quickly.

[0177] The IR spectra of CS, β-GP, SA, VBPs, CAH and CAVBPH are as follows: Figure 5 As shown in B. For CS, 1655, 1597, 1383cm -1 The absorption peaks at these locations represent amide I (CO stretching vibration), primary amine (NH bending vibration), and CH bending vibration, respectively. For β-GP, PO4 3- The symmetrical and asymmetric stretching vibration peaks appear at 978 cm⁻¹, respectively. -1 and 1080 cm -1 Nearby. PO4 3- The in-plane bending vibrations of the functional group are located at 800-500 cm. -1 In the IR spectrum of SA, 1634 and 1420 cm⁻¹ -1 The characteristic absorption peaks are attributed to the symmetric and asymmetric stretching vibrations of the carboxyl group (C=O), with the CO stretching vibration peak located at 1032 cm⁻¹. -1 In the infrared spectrum of VBPs, 1629 and 1583 cm⁻¹ -1 The absorption peaks are attributed to the characteristic absorption bands of amides I and II, and the bending vibrations of CH and OH appear at 1456 and 1404 cm⁻¹, respectively. -1 1111 cm -1 The absorption peak is caused by the stretching vibration of CO. For CAH and CAVBPH, PO4 3- Symmetric and asymmetric stretching vibration peaks (973 and 1081 cm⁻¹) -1 ) and the NH vibration absorption peak (1568 cm⁻¹) -1 The decrease indicates that part of the -NH3 in CS is reduced. + PO4 with β-GP 3- An electrostatic interaction occurred between them. (The text abruptly ends here, likely due to an incomplete sentence or a formatting error.) 2+ After crosslinking, the OH deformation vibrations, C=O and CO stretching vibrations associated with SA in the hydrogel were significantly weakened. The NH deformation vibration of CS (1634 cm⁻¹) was also reduced. -1) and the C=O stretching vibration of SA (1597 cm) -1 The reduction or disappearance of peak intensity may be due to the -COO in the SA structure. - and the -NH3 of the CS molecule + This is due to the interaction between them.

[0178] The pH value of a wound is generally between slightly acidic and neutral (5.4-7.4), playing an important role directly or indirectly in wound healing. For example... Figure 5 As shown in Figure C, the pH values ​​of CAH and CAVBPH are approximately 6.8. The viscosities of CAH and CAVBPH are 73.49 ± 0.51 Pa·s and 76.75 ± 0.01 Pa·s, respectively. Figure 5 D).

[0179] A properly humid environment can promote the healing of skin wounds. Figure 5 E shows the weight change curve of the hydrogel over time. In the first 6 hours, the weights of both CAH and CAVBPH decreased rapidly, then decreased slightly from 10 to 48 hours before stabilizing. Overall, the initial water content of the hydrogel was above 85%, effectively maintaining a moist environment around the wound. Good absorbency is equivalent to the skin dressing's ability to absorb wound secretions; compared to CAH, CAVBPH has a faster water absorption capacity, reaching its maximum SR (residue transfer ratio) within the first hour due to its rich porous structure. Figure 5 F) indicates that the hydrogel scaffold can rapidly absorb exudate and prevent it from accumulating excessively on the wound surface.

[0180] The biodegradability of biopolymer materials is one of the most important characteristics affecting their medical applications. The weight of CAH and CAVBPH gradually decreased with increasing incubation time. Figure 5 (G). The rapid degradation in the first 12 hours was caused by β-GP, which was released from the hydrogel during immersion, reducing the degree of cross-linking. After 14 days of cultivation, the weights of CAH and CAVBPH decreased from 100% to 56.85±8.42% and 59.26±2.70%, respectively, demonstrating certain biodegradability and resistance to hydrolysis.

[0181] The cumulative release behavior of VBPs from CAVBPH is as follows: Figure 5 As shown in Figure H, VBPs were rapidly released within the first 48 hours, with approximately 50% of VBPs released in the first 24 hours. After 10 days of incubation in the culture medium, a total of 82.04 ± 1.10% of VBPs were released. Therefore, the CAVBPH hydrogel can meet the requirements for sustained VBP release.

[0182] 1.3.2 In vitro bioactivity

[0183] Skin abrasions generate a large number of free radicals, leading to DNA breaks, lipid peroxidation, and inactivation of related enzymes. Increasing research indicates that skin dressings with antioxidant activity improve the therapeutic effect of tissue repair. CAVBPH at 1 mg / mL and 5 mg / mL showed significantly higher scavenging abilities against DPPH and ABTS free radicals than the CAH group. p <0.05), indicating that the antioxidant capacity of the hydrogel is related to the introduction of VBPs. Figure 6 (A and B).

[0184] In this study, the supernatant of both the hydrogel and VBP-treated groups was colorless and transparent, while the positive control group was bright red. Figure 6 C). Quantitative results showed that the hemolysis rates of CAH, CAVBPH, and VBPs were all below 5%, indicating that they would hardly cause hemolysis. Therefore, these materials are biosafe and can be used for skin care.

[0185] CAH and CAVBPH S. aureus (32 h) and E. coli (24 h) antibacterial activity such as Figure 6 As shown in DF. Compared with the control group, CAH and CAVBPH had... S. aureus and E. coli The growth and proliferation of bacteria are significantly inhibited, partly due to the inherent antibacterial effect of CS. The addition of VBPs further enhances the long-lasting antibacterial performance of CS hydrogel, thereby reducing the chance of bacteria invading skin wounds.

[0186] Cytotoxicity is an important indicator for the clinical application of hydrogel dressings. For example... Figure 6 As shown in Figure G, cell viability remained almost unchanged when the hydrogel concentration increased from 10 μg / mL to 100 μg / mL. At a concentration of 1000 μg / mL, the viability of HaCaT cells treated with CAH and CAVBPH were 81.29% and 86.74%, respectively. According to GB / T 16886.5-2003 (ISO 10993-5:1999), samples with a cell viability exceeding 75% are generally considered non-cytotoxic. Based on these results, the toxicity of CAH and CAVBPH is negligible.

[0187] Example 3: Study on the effect of deer blood peptide hydrogel on diabetic chronic skin wounds

[0188] 1.1 Instruments and Materials

[0189] 1.1.1 Experimental Apparatus

[0190] Table 8 Experimental Instruments and Manufacturers

[0191]

[0192] 1.1.2 Experimental Reagents

[0193] Table 9. Experimental Reagents and Manufacturers

[0194]

[0195]

[0196] 1.2 Experimental Methods

[0197] 1.2.1 Induction of type 2 diabetes (T2D) and establishment of a skin wound model

[0198] One hundred healthy male ICR mice, weighing 18-20 g, were purchased from Changchun Yisi Laboratory Animal Technology Co., Ltd. (Certificate No.: SCXK-2020-0001). After one week of acclimatization (22±2℃, 60±5% humidity, diurnal light and shadow), except for the normal control group (NC) which was fed a regular diet, the other mice were fed a high-sugar, high-fat diet for five consecutive weeks to induce insulin resistance. For the following three days, the mice were first fasted for 12 hours but allowed free water. Then, T2D was induced by intraperitoneal injection of STZ (pH=4.3, 0.1 M citrate buffer) at doses of 80 mg / kg body weight / day, 70 mg / kg body weight / day, and 60 mg / kg body weight / day, respectively. The NC group received only citrate buffer solution. One week after the third STZ injection, fasting blood glucose (FBG) in the tail vein was measured. Mice with FBG values ​​≥11.1 mM and exhibiting clinically typical polydipsia, polyphagia, polyuria, and weight loss were selected as T2D mice and randomly divided into a diabetic model group (T2D), a CAH treatment group (CAH), and a CAVBPH treatment group (CAVBPH) for wound experiments.

[0199] Diabetic mice were anesthetized by intraperitoneal injection of a 5% chloral hydrate solution (0.1 mL / 10 g body weight). The hair on their backs was shaved with an electric shaver, and a circular full-thickness excision wound with a diameter of 1 cm was induced on the back. The drug dosage was 0.2 mL / wound. Postoperatively, the NC and T2D groups received only PBS treatment for the wound, while the CAH and CAVBPH groups received CAH and CAVBPH hydrogel treatments, respectively. The wound area was measured on days 0, 3, 9, and 15 to calculate the wound healing rate. The formula for calculating the wound area is:

[0200] Wound area = unhealed area / initial area×100%

[0201] 1.2.2 General pathological and IHC staining analysis

[0202] Fifteen days after surgery, skin tissue was taken from the wound and fixed in 10% formalin solution for H&E, Masson and IHC staining.

[0203] 1.2.3 Western blot analysis

[0204] Total protein was extracted from skin wound tissue by lysing it with RIPA lysis buffer (pre-cooled in an ice-water bath) containing protease inhibitors and phosphatase inhibitors, and the protein concentration in the lysis supernatant was measured using a BCA kit. 1 / 4 volume of 5× loading buffer was added to each lysis supernatant, and the samples were denatured by boiling in a water bath for 10 min. Sample proteins were separated by 10% SDS-PAGE electrophoresis and transferred to a PVDF membrane. After blocking with 5% BSA for 2 h, the membrane was incubated overnight at 4°C with primary antibodies PI3K, AKT, mTOR, p-PI3K, p-AKT, p-mTOR, SIRT1, NF-κB, IL-1β, TNF-α, and β-actin. The membrane was washed with TBST and then incubated at room temperature for 1 h with secondary antibody. Protein bands were detected using enhanced chemiluminescence (ECL), and quantification was performed using ImageJ software.

[0205] 1.2.4 16S rRNA sequencing analysis of skin microbiota

[0206] On day 15, sterile cotton swabs were moistened with medical saline and gently wiped repeatedly on the newly formed skin around the mouse wounds to collect bacterial flora samples from each group (n=10). The samples were temporarily stored in sterile centrifuge tubes at -80℃ and used for analysis within one week. Using Silva as the reference database, 16S V3V4 amplification and sequencing were performed by Shanghai Paisenno Biotechnology Co., Ltd., and the results were analyzed using a gene cloud platform.

[0207] 1.2.5 Statistical Analysis

[0208] The experiment was conducted in triplicate, and the data are expressed as mean ± SD. ANOVA was performed using IBM SPSS Statistics 23.0 software. p A value <0.05 was considered statistically significant. GraphPad Prism 5 and Adobe Illustrator CS6 software were used to create the graphs.

[0209] 1.3 Results

[0210] 1.3.1 T2D Model Validation

[0211] Throughout the animal experiments, the FBG level in diabetic mice remained above 11.1 mM and exhibited typical symptoms of polyuria, polydipsia, polyphagia, and weight loss. Compared to the NC group mice, H&E staining of pancreatic tissue in diabetic mice confirmed the pathological changes following intraperitoneal injection of STZ. Figure 7 As shown, the area and number of pancreatic islets in diabetic mice are reduced, acinar cells are swollen, and pancreatic β cells are damaged. These results prove that the T2D model has been successfully established.

[0212] 1.3.2 Internal wound healing effect

[0213] During the experiment, no obvious infections or other complications were found in any group, and the wound size in all groups gradually decreased over time. Figure 8 A). Compared to the NC group, the T2D group experienced slower wound healing throughout the recovery process due to diabetes, and tissue fluid exudation persisted on day 9. These conditions significantly improved after treatment with CAH and CAVBPH. p <0.05). By day 15, the wounds in the CAVBPH group (96.55%) and the NC group (88.77%) had essentially healed, with most areas covered by hair, while the healing rates in the CAH and T2D groups were 84.23% and 63.81%, respectively. Figure 8 B).

[0214] 1.3.2 Histological assessment

[0215] H&E staining showed that the fibroblasts in the CAVBPH and NC groups were densely and regularly arranged, with skin appendages such as sweat glands and hair follicles being generated and lymphocytes being negligible; while in the T2D group, the epithelialization process was not yet complete, there was less granulation tissue, and infiltrative lymphocytes were still present, indicating a persistent inflammatory response. Figure 9 A) Evaluate ECM deposition and maturation using Masson staining and quantitative analysis. For example... Figure 9 As shown in Figure A, at 15 days post-surgery, collagen fibers in the CAVBPH and NC groups showed deeper staining and a wider distribution. Quantitative analysis revealed that the collagen content in both the CAVBPH and NC groups was significantly higher than that in the other two groups. p <0.05, Figure 9 B).

[0216] 1.3.3 IHC Analysis

[0217] IHC staining was used to detect CD31, PCNA, α-SMA, and CD68 levels in the wound, and to assess angiogenesis, cell proliferation, ECM secretion, and inflammatory response. Combining staining images and quantitative analysis, we found that the expression of CD31, PCNA, and α-SMA proteins was significantly inhibited in the T2D group compared to the NC group. p<0.001). The above conditions improved after hydrogel treatment, and the expression levels of the above three proteins in the CAVBPH group were similar to those in the NC group, while being higher than those in the CAH group. Figure 10 These phenomena indicate that CAVBPH improves the restricted angiogenesis in diabetic wounds, enhances skin cell proliferation and ECM regeneration, thereby promoting wound healing. In the subsequent CD68 staining results, except for the T2D group, no visible positive expression was found in the NC, CAH, and CAVBPH groups. Quantitative analysis was consistent with the image observation results, namely, the positive expression level of CD68 in the T2D group was significantly higher than that in the other three groups. p <0.001, Figure 10 (A and E), therefore CAH and CAVBPH can significantly alleviate persistent inflammation at the site of diabetic wounds ( p <0.001).

[0218] 1.3.4 Western blot analysis

[0219] like Figure 11 As shown, on day 15, the expression of p-PI3K / PI3K, p-AKT / AKT, p-mTOR / mTOR, HIF-1α, and VEGFA in the wound tissue of the CAVBP group was significantly higher than that of the T2D group. p <0.01). We found that CAH hydrogel activated the expression of PI3K / AKT / mTOR / HIF-1α-related proteins in diabetic wounds to some extent, but CAVBPH showed a more significant effect, thereby promoting angiogenesis in the wound healing process. p <0.05).

[0220] The high-sugar environment at the site of a diabetic wound easily induces bacterial infection and chronic inflammation. Compared with the non-nosocomial (NC) group, the expression of SIRT1 protein was significantly reduced in the T2D group. p <0.05), while the expression levels of NF-κB, TNF-α, and IL-1β were significantly increased ( p <0.01, Figure 12 Following CAVBPH hydrogel treatment, SIRT1 expression was significantly upregulated. p <0.05), and downregulated the expression of NF-κB, TNF-α, and IL-1β proteins ( p <0.001). Furthermore, compared to the blank hydrogel, i.e., the CAH group, CAVBPH showed more significant upregulation of SIRT1 and downregulation of NF-κB, TNF-α, and IL-1β. p <0.05).

[0221] 1.3.5 Skin microbiome analysis

[0222] like Figure 13 As shown in Figure A, the sequencing information for all samples can be found in the Silva database, and unclassified sequences are negligible, indicating that the sample quality is acceptable. Furthermore, the sparse curves of each group gradually flatten with increasing flattening depth, eventually approaching saturation, indicating that the sequencing depth is reasonable and also reflects the true microbial situation in the samples. Figure 13 B).

[0223] The alpha diversity of the samples was assessed using the observed species and Simpson index, and the results are as follows: Figure 13 As shown in Figure C, the Observed species index represents the species richness in the sample, while the Simpson index is used to characterize diversity. We found that the Observed species index of the CAVBPH and NC groups was higher than that of the T2D and CAH groups. p =0.065); the Simpson index of the T2D group was lower than that of the NC group, while the diversity of the bacterial flora in the samples improved after treatment with CAH and CAVBPH, with a significant difference in Simpson index between the CAVBPH and T2D groups ( p =0.0041). Therefore, the community diversity and richness of group T2D changed significantly, and this situation recovered somewhat after CAVBPH intervention. Meanwhile, principal coordinate analysis (PCoA) was used to compare community differences between groups to reflect beta diversity. Figure 13 As shown in Figure D, the NC and T2D groups were clearly divided into two regions. There was some overlap between the CAH and T2D group samples. However, compared to the T2D and CAH groups, the CAVBPH group samples were more concentrated and closer to the NC group. This result indicates that CAVBPH treatment improved the skin microbiota dysbiosis induced by T2D at the wound site.

[0224] Further analysis revealed differences in the skin microbiota composition among the four groups of mice, and identified the dominant microbiota at the phylum level in each group as follows: Firmicutes , Proteobacteria and Actinobacteria ( Figure 13 E). Among them, Firmicutes The proportions in the NC, T2D, CAH, and CAVBPH groups were 34.05%, 80.22%, 70.52%, and 46.96%, respectively; among the four groups Proteobacteria The contents were 62.63%, 16.13%, 28.21%, and 48.79%, respectively; while Actinobacteria They accounted for 1.46%, 0.90%, 0.84%, and 3.47% in the NC, T2D, CAH, and CAVBPH groups, respectively. Furthermore, BacteroidetesIt also accounted for a certain proportion in the NC and CAVBPH groups (0.85% and 0.30%, respectively), while it was 0.09% and 0.11% in the T2D and CAH groups, respectively. Therefore, we can conclude that, at the gate level, compared with ordinary wound skin, T2D causes more damage to the skin wound. Firmicutes The relative abundance of [something] increased, while the abundance of [something] decreased. Proteobacteria , Actinobacteria and Bacteroidetes CAVBPH treatment improved the dysbiosis, bringing the four major bacterial phyla on the skin surface closer to normal levels. Furthermore, the relative abundance of microorganisms at the genus level also differed among the four groups. Figure 13 F): Compared with the NC group, the T2D group Staphylococcus The relative abundance increased. Vulcaniibacterium , Cupriavidus , Pseudomonas , Lactobacillus , Anoxybacillus and Chelatococcus The relative abundance was reduced; CAH and CAVBPH treatments improved this abnormality, with CAVBPH treatment making the microbiota composition more similar to the NC group. Furthermore, CAVBPH significantly increased abundance compared to the other groups. Weissella (20.66%) and Corynebacterium_1 (2.71%) level.

[0225] Subsequently, Venn analysis was performed on the taxonomic units (OTUs) of each group, and the results are as follows: Figure 13 As shown in G, the NC, T2D, CAH, and CAVBPH groups contained 3989, 3627, 3110, and 3879 OUTs, respectively. We speculate that the CAVBPH intervention may be related to the recovery of OUT numbers. The T2D and CAH groups contained 1186 overlapping OTUs, followed by the CAH and CAVBPH groups (1142), the T2D and CAVBPH groups (966), the NC and CAVBPH groups (935), and the NC and CAH groups (730). The NC and T2D groups had the fewest overlapping OTUs, with only 670. In addition, there were 350 OTUs shared by all four groups.

[0226] Figure 13 H represents the relative expression abundance of the top 20 bacterial species across the four groups at the species level. The T2D and CAH groups were clustered together, while the CAVBPH group was adjacent to the NC group in the cluster. Compared to the NC group, the T2D group... Clostridiales_ bacterium , Staphylococcus_lentus , Streptococcus_hyointestinalis , Dorea_sp. , Dechloromonas_sp. , Lactobacillus_reuteri , Pseudogracilibacillus_auburnensis , Lactobacillus_intestinalis , Streptococcus_danieliae , Pseudomonas_stutzeri , Lactobacillus_hominis , Chelatococcus_sp. and Acinetobacter_lwoffii The relative abundance was lowered. Staphylococcus_sciuri The CAVBPH level was increased, effectively alleviating and reversing this condition; furthermore, compared to the other three groups, Corynebacterium_ammoniagenes , Corynebacterium_glutamicum , Corynebacterium_stationis , Weissella_paramesenteroides and Leuconostoc_citreum The relative abundance in the CAVBPH group increased significantly.

[0227] Figure 13 In section I, random forest analysis and difference tests were applied to identify the marker bacterial genera that had the greatest impact on inter-group differences. The results showed that... Staphylococcus , Jeotgalicoccus , Weissella , Ruminococcaceae _UCG-013 and Candidatus_Stoquefichus They were the most sensitive to changes among the four groups, indicating that they are a bacterial genus with a significant impact on skin wound healing in T2D mice, but administration of CAVBPH exacerbated the effect. Weissella The upward adjustment.

Claims

1. An activator of the SIRT1 / NF-κB signaling pathway, characterized in that, The activator comprises a hydrogel loaded with deer antler blood peptides; the preparation method of the hydrogel loaded with deer antler blood peptides includes the following steps: (1) Enzymatic hydrolysis of deer antler blood: Deer antler blood peptides were prepared by simulating the in vitro gastrointestinal digestion of deer antler blood powder using a two-step enzymatic hydrolysis method. A 20 mg / ml deer antler blood solution was prepared, and the pH was adjusted to 1.5-2.5 with 1-2 M HCl. After preheating at 37℃ for 10-30 min, pepsin was added at an enzyme-to-substrate ratio of 1:10-50. The solution was digested at 37℃ for 2-4 h at 120 rpm in a constant temperature shaker. The pH was then adjusted to 7.5-8.0 with 1-2 M NaOH, and trypsin was added at an enzyme / substrate ratio of 1:15-25, w / w. The reaction was further carried out for 4 h to simulate intestinal digestion. During the enzymatic hydrolysis, 1-2 M HCl or 1-2 M NaOH was used to maintain pH stability. After digestion, the enzyme was inactivated by heating in a boiling water bath for 15-30 min. The reaction mixture was cooled to room temperature and centrifuged at 8000 rpm for 30 minutes. The supernatant of the enzymatic hydrolysate was obtained by filtration of the supernatant twice with double-layer slow filter paper to obtain deer antler blood peptides, which were then freeze-dried and stored at -20℃ for later use. (2) Separation and freeze-drying: Prepare a 50 mg / mL deer antler blood peptide solution, and pass it through ultrafiltration membranes with molecular cutoff of 10 kDa and 3 kDa in sequence for fractionation to obtain components F1, F2 and F3, where component F3 < 3 kDa. The three components are then desalted by passing them through a 150 Da nanofiltration membrane, freeze-dried and stored at -20℃ for later use. (3) Preparation of deer antler blood peptide hydrogel: 200 mg of chitosan was added to 10 mL of 0.1 M glacial acetic acid solution and dissolved completely. 2.5 mL of 55% β-GP solution was added dropwise under the condition of stirring in an ice-water bath to obtain chitosan / β-GP solution. Then, 100 mg of sodium alginate powder was added to the prepared solution and stirred continuously to dissolve it. Then, 0.2 mL of 0.5% CaCl2 solution was added dropwise and stirred continuously to obtain blank hydrogel sol. Then, the F3 powder component of the obtained deer antler blood peptide was added to the blank hydrogel system and mixed evenly to make its concentration 0.1% to obtain deer antler blood peptide hydrogel, abbreviated as CAVBPH.

2. A drug that promotes the expression of growth factors in diabetic damaged tissues, characterized in that, The drug comprises a hydrogel loaded with deer antler blood peptides, and the preparation method of the hydrogel loaded with deer antler blood peptides includes the following steps: (1) Enzymatic hydrolysis of deer antler blood: Deer antler blood peptides were prepared by simulating the in vitro gastrointestinal digestion of deer antler blood powder using a two-step enzymatic hydrolysis method. A 20 mg / ml deer antler blood solution was prepared, and the pH was adjusted to 1.5-2.5 with 1-2 M HCl. After preheating at 37℃ for 10-30 min, pepsin was added at an enzyme-to-substrate ratio of 1:10-50. The solution was digested at 37℃ for 2-4 h at 120 rpm in a constant temperature shaker. The pH was then adjusted to 7.5-8.0 with 1-2 M NaOH, and trypsin was added at an enzyme / substrate ratio of 1:15-25, w / w. The reaction was further carried out for 4 h to simulate intestinal digestion. During the enzymatic hydrolysis, 1-2 M HCl or 1-2 M NaOH was used to maintain pH stability. After digestion, the enzyme was inactivated by heating in a boiling water bath for 15-30 min. The reaction mixture was cooled to room temperature and centrifuged at 8000 rpm for 30 minutes. The supernatant of the enzymatic hydrolysate was obtained by filtration of the supernatant twice with double-layer slow filter paper to obtain deer antler blood peptides, which were then freeze-dried and stored at -20℃ for later use. (2) Separation and freeze-drying: Prepare a 50 mg / mL deer antler blood peptide solution, and pass it through ultrafiltration membranes with molecular cutoff of 10 kDa and 3 kDa in sequence for fractionation to obtain components F1, F2 and F3, where component F3 < 3 kDa. The three components are then desalted by passing them through a 150 Da nanofiltration membrane, freeze-dried and stored at -20℃ for later use. (3) Preparation of deer antler blood peptide hydrogel: 200 mg of chitosan was added to 10 mL of 0.1 M glacial acetic acid solution and dissolved completely. 2.5 mL of 55% β-GP solution was added dropwise under the condition of stirring in an ice-water bath to obtain chitosan / β-GP solution. Then, 100 mg of sodium alginate powder was added to the prepared solution and stirred continuously to dissolve it. Then, 0.2 mL of 0.5% CaCl2 solution was added dropwise and stirred continuously to obtain blank hydrogel sol. Then, the F3 powder component of the obtained deer antler blood peptide was added to the blank hydrogel system and mixed evenly to make its concentration 0.1% to obtain deer antler blood peptide hydrogel, abbreviated as CAVBPH.

3. A method for preparing a hydrogel loaded with deer antler blood peptides, characterized in that: Includes the following steps: (1) Enzymatic hydrolysis of deer antler blood: Deer antler blood peptides were prepared by simulating the in vitro gastrointestinal digestion of deer antler blood powder using a two-step enzymatic hydrolysis method. A 20 mg / ml deer antler blood solution was prepared, and the pH was adjusted to 1.5-2.5 with 1-2 M HCl. After preheating at 37℃ for 10-30 min, pepsin was added at an enzyme-to-substrate ratio of 1:10-50. The solution was digested at 37℃ for 2-4 h at 120 rpm in a constant temperature shaker. The pH was then adjusted to 7.5-8.0 with 1-2 M NaOH, and trypsin was added at an enzyme / substrate ratio of 1:15-25, w / w. The reaction was further carried out for 4 h to simulate intestinal digestion. During the enzymatic hydrolysis, 1-2 M HCl or 1-2 M NaOH was used to maintain pH stability. After digestion, the enzyme was inactivated by heating in a boiling water bath for 15-30 min. The reaction mixture was cooled to room temperature and centrifuged at 8000 rpm for 30 minutes. The supernatant of the enzymatic hydrolysate was obtained by filtration of the supernatant twice with double-layer slow filter paper to obtain deer antler blood peptides, which were then freeze-dried and stored at -20℃ for later use. (2) Separation and freeze-drying: Prepare a 50 mg / mL deer antler blood peptide solution, and pass it through ultrafiltration membranes with molecular cutoff of 10 kDa and 3 kDa in sequence for fractionation to obtain components F1, F2 and F3, where component F3 < 3 kDa. The three components are then desalted by passing them through a 150 Da nanofiltration membrane, freeze-dried and stored at -20℃ for later use. (3) Preparation of deer antler blood peptide hydrogel: 200 mg of chitosan was added to 10 mL of 0.1 M glacial acetic acid solution and dissolved completely. 2.5 mL of 55% β-GP solution was added dropwise under the condition of stirring in an ice-water bath to obtain chitosan / β-GP solution. Then, 100 mg of sodium alginate powder was added to the prepared solution and stirred continuously to dissolve it. Then, 0.2 mL of 0.5% CaCl2 solution was added dropwise and stirred continuously to obtain blank hydrogel sol. Then, the F3 powder component of the obtained deer antler blood peptide was added to the blank hydrogel system and mixed evenly to make its concentration 0.1% to obtain deer antler blood peptide hydrogel, abbreviated as CAVBPH.

4. The method for preparing a hydrogel loaded with deer antler blood peptides according to claim 3, characterized in that, At 37°C, the blank hydrogel gelled in 5 minutes, while the deer antler blood peptide hydrogel gelled in 2 minutes.

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